newcastle disease virus and its uses

专利摘要:
NEWCASTLE DISEASE VIRUS AND ITS USES The invention relates to the chimeric Newcastle disease virus genetically modified to express an agonist of an immune cell co-stimulating signal and compositions comprising such viruses. The invention also relates to a chimeric Newcastle disease virus genetically modified to express an antagonist of an inhibitory signal from an immune cell and compositions comprising such viruses. Newcastle disease chimeric virus and compositions are useful in the treatment of cancer. In addition, the invention provides methods of treating cancer comprising administering the Newcastle disease virus in combination with a co-stimulating signal agonist to an immune cell and / or an inhibiting signal antagonist of an immune cell.
公开号:BR112015021414B1
申请号:R112015021414-2
申请日:2014-03-04
公开日:2020-11-10
发明作者:Peter Palese；Adolfo Garcia-Sastre；Dmitriy Zamarin；James Allison；Jedd D Wolchok
申请人:Icahn School Of Medicine At Mount Sinai；Memorial Sloan Kettering Cancer Center；
IPC主号:

专利说明:

[1] This order claims priority to US Provisional Order No. 61 / 782,994, filed on March 14, 2013, which is hereby incorporated by reference in its entirety.
[2] This invention was made, in part, with government support under concessions n— 5T32CA009207-35 and HHSN26620070010C granted by the National Institutes of Health. The government has some rights in the invention. 1. INTRODUCTION
[3] Chimeric Newcastle disease viruses engineered to express an agonist of an immune cell co-stimulating signal and compositions comprising such viruses are described herein. Also described herein are chimeric Newcastle disease viruses engineered to express an antagonist of an immune cell inhibitory signal and compositions comprising such viruses. Newcastle disease chimeric viruses and compositions are useful in the treatment of cancer. In addition, methods for the treatment of cancer comprising administering the Newcastle disease virus, in combination with an immune cell co-stimulating signal agonist and / or an immune cell inhibitory signal antagonist described herein. 2, BACKGROUND
[4] Newcastle Disease Virus (NDV) belongs to the family Paramyxoviridae, genus Avulavirus, which has been shown to infect a number of bird species (Alexander, DJ (1988). Newcastle disease, Newcastle disease virus - an avian paramyxovirus. Kluwer Academic Publishers: Dordrecht, The Netherlands, pp 1-22). NDV has a single-stranded RNA of the genome in the negative sense and does not undergo recombination with the host genome or other viruses Alexander, DJ (1988). Newcastle disease, Newcastle disease virus - an avian paramyxovirus. Kluwer Academic Publishers: Dordrecht, The Netherlands, pp 1-22). The genomic RNA contains genes on the order of 3'-NP-P-M-F-HN-L-5 ', described in greater detail below. Two additional proteins, V and W, are produced by NDV from the P gene by alternative mRNAs that are generated by RNA editing. The genomic RNA also contains a command sequence at the 3 'end.
[5] The structural elements of virion include the envelope virus, which is a derivative of the lipid bilayer of the cell's plasma membrane. The glycoprotein, hemagglutinin-neuraminidase (HN) protrudes from the envelope, allowing the virus to contain both hemagglutinin (eg, binding / Fusogenic receptors) and neuraminidase activities. The fusion glycoprotein (F), which also interacts with the viral membrane, is initially produced as an inactive precursor, then cleaved post-translationally to produce two disulfide-linked polypeptides. The active protein F is involved in the penetration of NDV into host cells by facilitating the fusion of the viral envelope with the plasma membrane of the host cell. The matrix protein (M) is involved with the viral assembly, and interacts with both the viral membrane and the nucleocapisid proteins.
[6] The subunit of the main protein of the nucleocapisid is the nucleocapisid protein (NP) that confers helical symmetry in the capsid. In association with the nucleocapisid are proteins P and L. Phosphoprotein (P), which is subjected to phosphorylation, is of the opinion that it plays a regulatory role in transcription, and may also be involved in methylation, phosphorylation and polyadenylation. The L gene, which codes for an RNA-dependent RNA polymerase, is required for the synthesis of viral RNA together with protein P. Protein G, which accepts almost half the coding capacity of the viral genome, is the largest of viral proteins, and plays an important role in both transcription and replication. Protein V has been shown to inhibit alpha-interferon and to contribute to the virulence of the NDV strain (Huang et al. (2003). Newcastle disease virus protein V is related to viral pathogenesis and functions as an interferon antagonist alpha, Journal of Virology 77: 8676-8685).
[7] The natural occurrence of NDV has been reported to be an effective oncolytic agent in a variety of animal tumor models (Sinkovics, JG, and Horvath, JC (2000). Newcastle disease virus (NDV): brief history of its oncolytic strains J. Clin Virollβ: 1-15; Zamarin et al., 2009; Mol Ther 17: 697; Elankumaran et al., 2010; J Virol 84: 3835; Schirrmacher et al., 2009; Methods Mol Biol 542: 565; Bart et al., 1973; Nat New Biol 245: 229). Naturally occurring NDV strains have been used in several clinical trials against human cancers ((Sinkovics, JG, and Horvath, JC (2000). Newcastle disease virus (NDV): brief history of its oncolytic strains.J Clin Virollδ: 1-15; Lorence et al. (2007). Phase 1 clinical experience using intravenous administration of PV701, an oncolytic Newcastle disease virus. Curr Cancer Drug Targets' !: 157-167; Hotte et al. (2007). An optimized clinical regimen for the oncolytic virus PV701. Clin Cancer Resl3: 977-985; Freeman et al. (2006). Phase I / II trial of intravenous NDV-HUJ oncolytic virus in recurrent glioblastoma multiforme. Mol Therl3: 221-228; Pecora et al. (2002). Phase I trial of intravenous administration of PV701, an oncolytic virus, in patients with advanced solid cancers. J Clin Oncol20: 2251-2266; Csatary et al. (2004). MTH-68 / H oncolytic viral treatment in human high-grade gliomas. J NeurooncolGI: 83-93). However, the success of naturally occurring NDV strains in these clinical trials for advanced human cancers was only marginal (Hotte et al. (2007). An optimized clinical regimen for the oncolytic virus PV701. Clin Cancer Resl3: 977-985; Freeman et al. (2006). Phase I / II trial of intravenous NDV-HUJ oncolytic virus in recurrent glioblastoma multiforme. Mol Therl3: 221-228; Pecora et al. (2002). Phase I trial of intravenous administration of PV701, an oncolytic virus, in patients with advanced solid cancers (J Clin 0ncol2Q: 2251-2266). As such, there remains a need for NDV-based therapies useful in cancer treatment, especially for advanced cancer. 3. SUMMARY
[8] In one aspect, Newcastle disease chimeric viruses (NDVs) engineered to express an agonist of an immune cell co-stimulating signal and / or an antagonist of an immune cell inhibitory signal are shown here. In a specific embodiment, chimeric NDVs are presented in this document, comprising a packaged genome that encodes an agonist of an immune cell co-stimulating signal, in which the agonist is expressed. In a specific embodiment, chimeric NDVs comprising a packaged genome encoding an antagonist of an inhibitory signal from an immune cell, in which the antagonist is expressed are presented.
[9] In another embodiment, chimeric NDVs comprising a packaged genome encoding an agonist of an immune cell co-stimulating signal and a mutated F protein that makes NDV highly fusogenic, are presented in this document, in which the agonist and the mutated F protein are expressed. In another embodiment, chimeric NDVs comprising a packaged genome encoding an agonist of an immune cell co-stimulating signal and a mutated F protein with a mutated cleavage site are presented in which the agonist and the mutated F protein are presented. cast. In a specific embodiment, the chimeric NDVs that express the mutated F protein increased fusogenic activity in relation to the corresponding virus expressing the homologous F protein without the mutations at the cleavage site. In another specific embodiment, the modified F protein is incorporated into the virion.
[10] In another embodiment, chimeric NDVs are presented herein, comprising a packaged genome that encodes an antagonist of an immune cell inhibitory signal and a mutated F protein that makes NDV highly fusogenic, in which the antagonist and the mutated F protein are expressed. In another embodiment, chimeric NDVs are presented herein, comprising a packaged genome that encodes an immune cell inhibitor signal antagonist and a mutated F protein with a mutated cleavage site, where the antagonist and the mutated F protein are cast. In a specific embodiment, the chimeric NDVs that express the mutated F protein increased the fusogenic activity in relation to the corresponding virus that expresses the homologous F protein without the mutations at the cleavage site. In another specific embodiment, the modified F protein is incorporated into the virion.
[11] In another embodiment, chimeric NDVs, comprising a packaged genome encoding an agonist of an immune cell co-stimulating signal and a cytokine (eg, interleukin (IL-2)), are shown in this document. agonist and cytokine are expressed. In another embodiment, chimeric NDVs are presented in this document, comprising a packaged genome that encodes an immune cell co-stimulating signal agonist, a cytokine (eg, IL-2) and a mutated F protein that causes the NDV is highly fusogenic, in which the agonist, the cytokine and the mutated F protein are expressed. In another form, chimeric NDVs are presented herein, comprising a packaged genome that encodes an immune cell co-stimulating signal agonist, a cytokine (eg, IL-2) and a mutated F protein with a mutated cleavage site , in which of the agonist, the cytokine and the mutated F protein are expressed. In a specific embodiment, chimeric NDVs that express the F protein mutated with the mutated cleavage site are highly fusogenic. In another specific embodiment, the mutated F protein is incorporated into the virion.
[12] In another embodiment, chimeric NDVs are presented herein, comprising a packaged genome encoding an antagonist of an immune cell inhibitory signal from an immune cell and a cytokine (for example, IL-2), where the antagonist and the cytokine are expressed. In another embodiment, chimeric NDVs are presented herein, comprising a packaged genome that encodes an antagonist of an immune cell inhibitory signal, a cytokine (for example, IL-2) and a mutated F protein that causes the NDV is highly fusogenic, in which the antagonist, the cytokine and the mutated F protein are expressed. In another embodiment, chimeric NDVs are presented herein, comprising a packaged genome that encodes an antagonist of an immune cell inhibitory signal, a cytokine (eg, IL-2) and a mutated F protein with a cleavage site mutated, in which the antagonist, the cytokine and the mutated F protein are expressed. In a specific embodiment, chimeric NDVs that express the F protein mutated with the mutated cleavage site are highly fusogenic. In another specific embodiment, the mutated F protein is incorporated into the virion.
[13] In a specific embodiment, the agonist of a co-stimulating signal from an immune cell is an agonist of a co-stimulating receptor expressed by an immune cell. Specific examples of co-stimulating receptors include glucocorticoid-induced tumor necrosis factor (GITR) receptor, inducible T cell co-stimulator (ICOS or CD278), 0X40 (CD134), CD27, CD28, 4-1BB (CD137), CD40, CD226, cytotoxic molecule and regulatory T cells (CRTAM), exterminator receptor 3 (DR3), lymphotoxin-beta receptor (LTBR), transmembrane activator and CAML interactor (TACI), B cell activation factor receptor (BAFFR), and the B cell maturation protein (BCMA). In a specific embodiment, the agonist of a co-stimulatory receptor expressed by an immune cell is an antibody (or an antigen-binding fragment) or ligand that specifically binds to the co-stimulatory receptor. In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a sc-Fv. In a specific embodiment, the antibody is a bispecific antibody that binds to two receptors on an immune cell. In one embodiment, the bispecific antibody binds to a receptor in an immune cell and to another receptor in a cancer cell. In specific embodiments, the antibody is a human or humanized antibody. In certain embodiments, the linker or antibody is a chimeric protein comprising an NDV F protein or fragment thereof, or NDV HN protein or fragment thereof. Methods for generating these chimeric proteins are known in the art. See, for example, in the publication of United States patent application No. 2012-0122185, the disclosure of which is hereby incorporated by reference in its entirety. See also Park et al, PNAS., 2006; 103 :. 8203-8 and Murawski et. al., J Virol 2010; 84: 1110-1123, the disclosures of which are hereby incorporated by reference in their entirety. In certain embodiments, the ligand or antibody is expressed as a chimeric F protein or NDV F-fusion protein, wherein the chimeric F protein or NDV F-fusion protein comprises the transmembrane and cytoplasmic domains or fragments of the F glycoprotein of the NDV and the extracellular domain comprises the ligand or antibody. In some embodiments, the ligand is expressed as a chimeric HN protein or NDV HN fusion protein, wherein the chimeric HN protein or NDV HN fusion protein comprises the extracellular and transmembrane domains or fragments of the NDV HN glycoprotein and comprises an extracellular domain of the linker or antibody. In a specific embodiment, the linker or antibody is expressed as a chimeric protein, as described in Section 7, Example 2, below.
[14] In a specific embodiment, the antagonist of an inhibitory signal from an immune cell is an antagonist of an inhibitory receptor expressed by an immune cell. Specific examples of inhibitory receptors include immunoglobulin-like receptor associated with cytotoxic T lymphocytes associated with antigen 4 (CTLA-4 or CD52) programmed death proteins (PD1 or CD279), T and B lymphocyte attenuator (BTLA), receptors similar to immunoglobulin from killer cells (KIR), lymphocyte activation gene 3 (LAG3), or protein 3 T cell membrane (TIM3), A2a adenosine receptor (A2aR), T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), receptor leukocyte-associated immunoglobulin (LAIR1), and CD160. In a specific embodiment, the inhibitor receptor antagonist expressed by an immune cell is an antibody (or an antigen binding fragment) that specifically binds to the co-stimulatory receptor. In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a sc-Fv. In specific embodiments, the antibody is a human or humanized antibody. In another specific embodiment, the inhibitory receptor antagonist is a soluble receptor or an antibody (or an antigen binding fragment) that specifically binds to an inhibitory receptor ligand. In certain embodiments, the antibody is a chimeric protein comprising an NDV F protein or fragment thereof, or NDV HN protein or fragment thereof. See, for example, the publication of the North American patent application No. 2012-0122185, Park et al, PNAS., 2006; 103: 8203-8, and Murawski et al, J. Virol 2010; 84: 1110-1123, each of which is incorporated herein by reference in its entirety. In certain embodiments, the antibody is expressed as a chimeric protein or the NDV-F fusion protein, wherein the chimeric F protein or the NDV F fusion protein comprises the transmembrane and cytoplasmic domains or fragments of the F glycoprotein of NDV and the extracellular domain comprises the antibody. In some embodiments, the antibody is expressed as a chimeric HN protein or NDV HN fusion protein, wherein the chimeric HN protein or NDV HN fusion protein comprises the transmembrane and intracellular domains or fragments of the NDV HN glycoprotein and the extracellular domain comprises the antibody.
[15] In another aspect, the methods for propagating NDVs described here are presented here (for example, chimeric NDVs described here). The NDVs described herein (for example, chimeric NDVs described herein) can be propagated in any cell, tissue, organ or subject susceptible to an NDV infection. In one embodiment, the NDVs described herein (for example, chimeric NDVs described herein) can be propagated in a cell line. In another embodiment, the NDVs described herein (for example, chimeric NDVs described herein) can be propagated in cancer cells. In another embodiment, the NDVs described here (for example, chimeric NDVs described here) can be propagated in an embryonic egg. In certain embodiments, isolated cells, tissues or organs infected with an NDV described herein are presented here (for example, a chimeric NDV described herein). See, for example, Section 5.4, below, for examples of cells, animals and eggs to infect with an NDV described here (for example, a chimeric NDV described here). In specific embodiments, shown here, cancer cells are infected with an isolated NDV described here (for example, a chimeric NDV described here). In certain embodiments, cell lines infected with an NDV described herein are presented here (for example, a chimeric NDV described here). In other embodiments, embryonic eggs infected with NDV described here are presented here (for example, a chimeric NDV described here).
[16] In another aspect, compositions comprising an NDV described herein are presented here (for example, a chimeric NDV described here). In a specific embodiment, pharmaceutical compositions comprising an NDV described herein (for example, a chimeric NDV described herein) and a pharmaceutically acceptable carrier are disclosed herein. In another embodiment, pharmaceutical compositions comprising cancer cells infected with an NDV described herein (for example, a chimeric NDV described herein), and a pharmaceutically acceptable carrier are disclosed herein. In specific embodiments, the cancer cells were treated with gamma radiation before incorporation into the pharmaceutical composition. In specific embodiments, cancer cells were treated with gamma radiation before infection with NDV (eg, chimeric NDV). In other specific embodiments, cancer cells were treated with gamma radiation after infection with NDV (for example, chimeric NDV). In another embodiment, pharmaceutical compositions comprising protein concentrate from lysed NDV-infected cancer cells (e.g., cancer cells infected with chimeric NDV), and a pharmaceutically acceptable carrier are shown herein.
[17] In another aspect, methods for producing pharmaceutical compositions comprising an NDV described herein are presented here (for example, a chimeric NDV described here). In one embodiment, a method for producing a pharmaceutical composition comprising: (a) propagating an NDV described herein (for example, a chimeric NDV described here) in a cell line that is susceptible to an NDV infection; and (b) collecting the viral progeny, in which the virus is grown in sufficient quantities and under sufficient conditions for the virus to be free from contamination, so that the viral progeny is suitable for formulation in a pharmaceutical composition. In another embodiment, a method for producing a pharmaceutical composition includes: (a) propagating an NDV described herein (for example, a chimeric NDV described here) in an embryonic egg; and (b) collecting the viral progeny, in which the virus is grown in sufficient quantities and under sufficient conditions for the virus to be free from contamination, so that the viral progeny is suitable for formulation in a pharmaceutical composition.
[18] In another aspect, methods for treating cancer using a chimeric NDV described herein (for example, a chimeric NDV described in Section 5.2, infra), or a composition comprising a chimeric NDV are presented here. In a specific embodiment, a method for the treatment of cancer which comprises the infection of a cancer cell in a subject with a chimeric NDV described herein (for example, a chimeric NDV described in Section 5.2, below), or its composition. In another embodiment, a method for the treatment of cancer that comprises administering to a subject in need thereof, a chimeric NDV described herein (for example, a chimeric NDV described in Section 5.2, below), or its composition. In specific embodiments, an effective amount of a chimeric NDV described herein (for example, a chimeric NDV described in Section 5.2, below) or a composition comprising an effective amount of a chimeric NDV presently described, is administered to a subject to treat the cancer. In specific embodiments, the chimeric NDV comprises a genome, the genome comprising an agonist of an immune cell co-stimulating signal (for example, an agonist of an immune cell co-stimulating receptor) and / or an antagonist of an inhibitory signal from an immune cell. immune cell (for example, an inhibitory receptor antagonist of an immune cell). In certain embodiments, the NDV genome also comprises a mutated F protein. In certain embodiments, two or more chimeric NDVs are administered to a subject to treat cancer.
[19] In another embodiment, a method for the treatment of cancer that comprises administering to a subject in need of the cancer cells infected with a chimeric NDV described here (for example, a chimeric NDV described in Section 5.2, below), or its composition. In specific embodiments, cancer cells were treated with gamma radiation prior to administration to the subject or incorporation into the composition. In another embodiment, a method for treating cancer that comprises administering to a subject in need of it a protein concentrate or plasma membrane fragments from cancer cells infected with chimeric NDV (for example, a chimeric NDV described in Section 5.2, below), or its composition. In specific embodiments, the chimeric NDV comprises a genome, the genome comprising an agonist of an immune cell co-stimulating signal (for example, an agonist of an immune cell co-stimulating receptor) and / or an antagonist of a signal inhibitor of an immune cell. immune cell (for example, an inhibitory receptor antagonist of an immune cell). In certain embodiments, the NDV genome also comprises a mutated F protein.
[20] In another aspect, methods for treating cancer using an NDV described here (for example, a chimeric NDV as described in Section 5.2, below) or a composition comprising such NDV in combination with one or more therapies are presented herein. . In one embodiment, methods for treating cancer are presented herein comprising administering to a subject an NDV described herein (for example, a chimeric NDV, as described in Section 5.2.1, infra) and one or more other therapies. In another embodiment, methods for treating cancer are presented herein comprising administering to an individual an effective amount of an NDV described herein or a composition comprising an effective amount of an NDV described herein, and one or more other therapies. NDV and one or more other therapies can be administered, at the same time or sequentially, to the subject. In certain embodiments, NDV and one or more other therapies are administered in the same composition. In other embodiments, NDV and one or more other therapies are administered in different compositions. NDV and one or more other therapies can be administered by the same or different routes of administration to the subject.
[21] Any type or strain of NDV can be used in a combination therapy described herein, including, but not limited to naturally occurring strains, variants or mutants, mutagenized recombinant viruses, and / or genetically modified viruses. In a specific modality, NDV used in combination with one or more therapies is a naturally occurring strain. In another embodiment, the NDV used in combination with one or more other therapies is a chimeric NDV. In a specific embodiment, the chimeric NDV comprises a packaged genome, the genome comprises a cytokine (for example, IL-2, IL-7, IL-15, IL-17, or IL-21). In specific embodiments, the cytokine is expressed by cells infected with chimeric NDV. In a specific embodiment, the chimeric NDV comprises a packaged genome, the genome comprising a tumor antigen. In specific modalities, the tumor antigen is expressed by cells infected with chimeric NDV. In a specific embodiment, the chimeric NDV comprises a packaged genome, the genome comprising a pro-apoptotic molecule or an anti-apoptotic molecule. In specific embodiments, the pro-apoptotic molecule or anti-apoptotic molecule is expressed by cells infected with chimeric NDV.
[22] In another specific embodiment, the chimeric NDV comprises a packaged genome, the genome comprising an agonist of an immune cell co-stimulating signal (for example, an agonist of an immune cell co-stimulating receptor) and / or an antagonist an inhibitory signal from an immune cell (for example, an antagonist to an inhibitory receptor on an immune cell). In specific modalities, the agonist and / or antagonist are expressed by cells infected with chimeric NDV. In certain embodiments, the NDV genome also comprises a mutated F protein. In certain embodiments, one or more therapies used in combination with an NDV described here is one or more other therapies described in Section 5.6.4 below. In particular embodiments, the one or more therapies used in combination with an NDV described here is an agonist of an immune cell co-stimulating signal and / or an antagonist of an immune cell inhibiting signal (see, for example, Section 5.6. 4.1, below). See, for example, Section 5.2.1, below, for examples of agonists of an immune cell co-stimulating signal and antagonists of an immune cell inhibitory signal. In a specific embodiment, the antagonist of an immune cell inhibitory signal is the anti-CTLA-4 antibody described in Sections 6 and 7, infra. In another specific embodiment, the antagonist of an immune cell inhibitory signal is an anti-PD-1 antibody or an anti-PD-Ll antibody described in Section 7, infra. In another specific embodiment, the agonist of an immune cell co-stimulating signal is the ICOS ligand described in Sections 6 and 7, below. 3.1 TERMINOLOGY
[23] The terms "about" or "approximately" when used in conjunction with a number refer to any number within 1, 5 or 10% of the mentioned number.
[24] As used herein, the term "agonist" refers to a molecule that binds to another molecule and induces a biological reaction. In a specific embodiment, an agonist is a molecule that binds to a receptor in a cell and triggers one or more signal transduction pathways. For example, an agonist includes an antibody or ligand that binds to a receptor in a cell and induces one or more signal transduction pathways. In certain embodiments, the antibody or ligand binds to a receptor in a cell and induces one or more signal transduction pathways. In other modalities, the agonist facilitates the interaction of the native ligand with the native receptor.
[25] As used herein, the term "antagonist" refers to a molecule that inhibits the action of another molecule without causing a biological response of its own. In a specific embodiment, an antagonist is a molecule that binds to a receptor in a cell and blocks or attenuates the biological activity of an agonist. For example, an antagonist includes an antibody or ligand that binds to a receptor on a cell and blocks or attenuates the binding of the native ligand to the cell, without inducing one or more signal transduction pathways. Another example of an antagonist includes an antibody or a soluble receptor that competes with the native receptor on cells to bind to the native ligand, and thus blocks or attenuates one or more induced signal transduction pathways when the native receptor binds to the native ligand.
[26] As used herein, the terms "antibody" and "antibodies" refer to molecules that contain an antigen-binding site, for example, immunoglobulins. Antibodies include, but are not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, single domain antibodies, camelized antibodies, single chain Fv (scFv), antibodies single-stranded, Fab fragments, F (ab ') fragments, bispecific disulfide-bound (sdFv), intrabodies and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies and anti-Id antibodies to antibodies ), and fragments that bind to the epitope of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules. The immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA and IgY), class (for example, IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass. In a specific embodiment, an antibody is a human or humanized antibody. In another specific embodiment, an antibody is a monoclonal antibody or scFv. In certain embodiments, an antibody is a human or humanized monoclonal antibody or scFv. In other specific embodiments, the antibody is a bispecific antibody. In certain embodiments, the bispecific antibody specifically binds to a co-stimulatory receptor on an immune cell or an inhibitory receptor on an immune, and a receptor on a cancer cell. In some embodiments, the bispecific antibody specifically binds to two immune cell receptors, for example, two co-stimulator receptors on immune cells, two inhibitory receptors on cells of the recipient's immune system, or a co-stimulator on immune cells and an inhibitory receptor on immune cells.
[27] As used herein, the term "derivative" in the context of proteins or polypeptides refers to: (a) a polypeptide that is at least 40%, 45%, 50%, 55%, 60%, 65 %, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, that is, 40% to 65%, 50% to 90%, 65% to 90%, 70 % to 90%, 75% to 95%, 80% to 95%, or 85% to 99% identical to a native polypeptide; (b) a polypeptide encoded by a nucleic acid sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% , 95%, 98%, or 99%, that is, 40% to 65%, 50% to 90%, 65% to 90%, 70% to 90%, 75% to 95%, 80% to 95%, or 85% to 99% identical to a nucleic acid sequence encoding a native polypeptide; (c) a polypeptide containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, ou2a5 , 2 to 10, 5 to 10, 5 to 15, 5 to 20, 10 to 15, or mutations of amino acids 15 to 20 (i.e., additions, deletions and / or substitutions) relative to a native polypeptide; (d) a polypeptide encoded by the nucleic acid sequence that can hybridize under conditions of high, moderate, or typical stringency of hybridization with a nucleic acid sequence encoding a native polypeptide; (e) a polypeptide encoded by a nucleic acid sequence that can hybridize under conditions of high, moderate, or typical stringency of hybridization with a nucleic acid sequence that encodes a fragment of a native polypeptide of at least 10 contiguous amino acids 12 contiguous amino acids, at least 15 contiguous amino acids, at least 20 contiguous amino acids, at least 30 contiguous amino acids, at least 40 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, at least 100 contiguous amino acids, at least 125 amino acids contiguous, at least 150 contiguous amino acids, or 10 to 20, 20 to 50, 25 to 75, 25 to 100, 25 to 150, 50 to 75, 50 to 100, 75 to 100, 50 to 150, 75 to 150, from 100 to 150, or 100 to 200 contiguous amino acids; or (f) a fragment of a native polypeptide. Derivatives also include a polypeptide comprising the naturally occurring mature amino acid sequence of a mammalian polypeptide and a heterologous amino acid sequence of the signal peptide. In addition, derivatives include polypeptides that have been chemically modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, by derivatization known protective / blocking groups, proteolytic cleavage, binding to a cell linker or other protein portion, etc. In addition, derivatives include polypeptides that comprise one or more non-classic amino acids. In one embodiment, a derivative is isolated. In specific embodiments, a derivative retains one or more functions of the native polypeptide from which it was derived.
[28] The percentage of identity can be determined using any method known to a person skilled in the art. In a specific embodiment, the percentage of identity is determined using the "best fit" or "Gap" Program of the Sequence Analysis Software Package (Version 10; Genetics Computer Group, Inc., University of Wisconsin Biotechnology Center, Madison, Wisconsin). Information regarding hybridization conditions (for example, high, moderate, typical, and stringency conditions) has been previously published, for example, in the publication of US patent application No. US 2005/0048549 (for example, 72-73 ).
[29] As used herein, the term "fragment" is the context of a fragment of a protein agent (for example, a protein) refers to a fragment that is 8 or more contiguous amino acids, 10 or more contiguous amino acids, 15 or more contiguous amino acids, of 20 or more contiguous amino acids, 25 or more contiguous amino acids, 50 or more contiguous amino acids, 75 or more contiguous amino acids, 100 or more contiguous amino acids, 150 or more contiguous amino acids, 200 or more contiguous amino acids, or in the range from 10 to 300 contiguous amino acids, 10 to 200 contiguous amino acids, 10 to 250 contiguous amino acids, 10 to 150 contiguous amino acids, from 10 to 100 contiguous amino acids, from 10 to 50 contiguous amino acids, 50 to 100 contiguous amino acids, from 50 to 150 contiguous amino acids, from 50 to 200 contiguous amino acids, 50 and 250 contiguous amino acids, from 50 to 300 contiguous amino acids, from 25 to 50 contiguous amino acids, from 25 to 75 contiguous amino acids, from 25 to 100 contiguous amino acids, or 75 to 100 contiguous amino acids of a proteinaceous agent. In a specific embodiment, a fragment of a protein agent retains one or more functions of the protein agent - in other words, it is a functional fragment. For example, a fragment of a protein agent retains the ability to interact with another protein and / or to induce, increase or activate one or more signal transduction pathways.
[30] As used herein, the term "functional fragment", in the context of a protein agent, refers to a portion of a protein agent that retains one or more activities or functions of the proteinaceous agent. For example, a functional fragment of an inhibitory receptor may retain the ability to bind one or more of its ligands. A functional fragment of a ligand from a co-stimulatory receptor can retain the ability to bind to the receptor and / or induce, increase or activate one or more signal transduction pathways mediated by binding to its ligand co-stimulatory receptor.
[31] As used herein, the term "heterologous" refers to an entity not found in nature to be associated with (for example, encoded by and / or expressed by the genome of) a naturally occurring NDV.
[32] As used herein, the term "elderly human" refers to a human being 65 years or older.
[33] As used herein, the term "human adult" refers to a human being who is 18 years or older.
[34] As used herein, the term "young human" refers to a human being who is 1 year to 18 years old.
[35] As used herein, the term "human child" refers to a human being who is 1 year to 3 years old.
[36] As used herein, the term "human infant" refers to a newborn human being 1 year old.
[37] In certain embodiments, the terms "highly fusogenic" and "increased fusogenic activity", and the like, as used herein, refer to an increase in the ability of NDV to form syncytia that involve a large number of cells. In a specific embodiment, cells infected with an NDV described here that is engineered to express a mutated F protein have an increased ability to form syncytia relative to cells infected with the parental virus from which the virus is derived, which the parental virus has an unmutated F protein. In another specific embodiment, about 10% to about 25%, about 25% to about 50%, about 25% to about 75%, about 50% to about 75%, about 50 % to about 95%, or about 75% to about 99% or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of the cells infected with an NDV described here that are engineered to express a mutated F protein form of syncytia in relation to the number of syncytial-forming cells that are infected with the parental virus from the chimeric virus is derived having an unmutated F protein. In certain embodiments, syncytia are quantified microscopically by counting the number of nuclei by syncytia after a certain period of time (for example, about 8 hours to about 12 hours, about 12 hours to about 24 hours, about 24 hours to about 36 hours, or about 36 hours to about 48 hours).
[38] As used herein, the term "interferon antagonist" refers to an agent that reduces or inhibits the cellular immune response to interferon. In one embodiment, an interferon antagonist is a proteinaceous agent that reduces or inhibits the interferon cellular immune response. In a specific embodiment, an interferon antagonist is a viral protein or polypeptide that reduces or inhibits the cellular interferon response.
[39] In a specific embodiment, an interferon antagonist is an agent that reduces or inhibits interferon expression and / or activity. In one embodiment, the interferon antagonist reduces or inhibits the type I IFN expression and / or activity. In another mode, the interferon antagonist reduces or inhibits the type II IFN expression and / or activity. In another embodiment, the interferon antagonist reduces or inhibits the expression and / or activity of type III IFN. In a specific embodiment, the interferon antagonist reduces or inhibits the expression and / or activity of any IFN-α, IFN-β or both. In another specific embodiment, the interferon antagonist reduces or inhibits IFN-y expression and / or activity. In another embodiment, the interferon antagonist reduces or inhibits the expression and / or activity of one, two or all of IFN-α, IFN-β, and IFN-y.
[40] In certain embodiments, the expression and / or activity of IFN-α, IFN-β and / or IFN-y in an embryonic egg or cell is reduced from about 1 to about 100 times, about 5 to about 80 times, about 20 to about 80 times, about 1 to about 10 times, about 1 to about 5 times, about 40 to about 80 times, or 1, 2, 3, 4, 5, 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 times by an interferon antagonist in relation to expression and / or the activity of IFN-α, IFN-β, and / or IFN-Y θm an embryonated control egg or a cell not expressing or not contacting an interferon antagonist, as measured by the techniques described or known herein by an expert in the art. In other embodiments, the expression and / or activity of IFN-α, IFN-β and / or IFN-y θm in an embryonated egg or cell is reduced by at least 20% to 25%, at least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, at least 80% to 85%, at least 85% to 90%, at least 90% 95%, at least 95% to 99% or 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80 %, 85%, 90%, 95%, or 99% by an interferon antagonist in relation to the expression and / or activity of IFN-α, IFN-β, and / or IFN-y in an embryonated control egg or a cell that does not express or contact an antagonist, such as interferon as measured by the techniques described herein or known to a person skilled in the art.
[41] As used herein, the phrases "IFN deficient systems" or "IFN deficient substrates" refer to systems, for example, cells, cell lines and animals, such as mice, chickens, turkeys, rabbits, mice, horses , etc., which do not produce one, two or more types of IFN, or do not produce any type of IFN, or produce low levels of one, two or more types of IFN, or produce low levels of any IFN (ie , a reduction in any IFN expression of 5 to 10%, 10 to 20%, 20 to 30%, 30 to 40%, 40 to 50%, 50 to 60%, 60 to 70%, 70 to 80%, 80 to 90 or more, when compared to competent IFN systems under the same conditions), do not respond or respond less efficiently to one, two or more types of IFN, or do not respond to any type of IFN, have a delayed response of one , two or more types of IFN, and / or are deficient in the activity of antiviral genes induced by one, two or more types of IFN, or induced by any type of IFN.
[42] As used herein, the terms "immunospecifically bind" "immunospecifically bind", "specifically bind", and "specifically recognize" are analogous terms in the context of antibodies and refer to molecules that specifically bind to an antigen (e.g., immune complex or epitope), as understood by one skilled in the art. A molecule that specifically binds to an antigen can bind to other peptides or polypeptides with less affinity, as determined by, for example, immunoassays (for example, ELISA), surface plasmon resonance (for example, BIAcore®), a KINEX assay (using, for example, a KinExA 3000 instrument (Instruments Sapidyne, Boise, ID)), or other assays known in the art. In a specific embodiment, molecules that specifically bind to an antigen that binds to the antigen with a dissociation constant (ie, Ka) that is at least 2 logs, 2.5 logs, 3 logs, 3.5 logs, 4 logs or greater than Ka when the molecules bind to another antigen. In another specific modality, molecules that specifically bind to an antigen do not cross-react with other proteins.
[43] As used herein, the term "monoclonal antibody" is a term in the art and generally refers to an antibody obtained from a population of homogeneous or substantially homogeneous antibodies, and each monoclonal antibody typically recognizes a single epitope ( for example, epitope conformation) on the antigen.
[44] As used herein, the phrase "multiplicity or infection" or "MOI" is the average number of viruses per infected cell. MOI is determined by dividing the number of virus added (ml added x Pfu) by the number of cells added (ml added x cells / ml).
[45] As used herein, the term "natural ligand" refers to any naturally occurring ligand that binds to a naturally occurring receptor. In a specific embodiment, the linker is a mammalian linker. In another specific embodiment, the ligand is a human ligand.
[46] As used herein, the term "native polypeptide" in the context of proteins or polypeptides refers to any naturally occurring amino acid sequence, including immature or precursor and mature forms of a protein. In a specific embodiment, the native polypeptide is a human protein or polypeptide.
[47] As used herein, the term "native receptor" refers to any naturally occurring receptor that binds to a naturally occurring ligand. In a specific embodiment, the receptor is a mammalian receptor. In another specific embodiment, the recipient is a human recipient.
[48] As used herein, the terms "subject" or "patient" are used interchangeably. As used herein, the terms "subject" and "subjects" refer to an animal. In some embodiments, the individual is a mammal, including a non-primate (for example, a camel, donkey, zebra, cow, horse, cat, dog, rat, and mouse) and a primate (for example, a monkey, chimpanzee, and a human being). In some embodiments, the individual is a non-human mammal. In certain embodiments, the individual is a pet (eg, dog or cat) or farm animal (eg, a horse, a pig or a cow). In other modalities, the subject is a human being. In certain embodiments, the mammal (for example, humans) is 0 to 6 months old, 6 to 12 months old, 1 to 5 years old, 5 to 10 years old, 10 to 15 years old, 15 to 20 years old. age, 2 0 to 2 5 years old, 25 to 30 years, 30 to 35 years, 35 to 40 years, 40 to 45 years, 45 to 50 years, 50 to 55 years, 55 to 60 years, 60 to 65 years old, 65 to 70 years, 70 to 75 years, 75 to 80 years, 80 to 85 years, 85 to 90 years, 90 to 95 years of age or 95 to 100 years of age. In specific modalities, the subject is an animal that is not a bird.
[49] As used herein, the terms "treat" and "treatment" in the context of administering a therapy refer to a treatment / therapy from which a subject receives a beneficial effect, such as reduction, reduction, attenuation , decrease, stabilization, remission, suppression, inhibition or stopping the development or progression of cancer, or its symptom. In certain modalities, the treatment / therapy that a subject receives results from at least one or more of the following effects: (i) the reduction or improvement in the severity of the cancer and / or a symptom associated with it; (ii) reducing the duration of a symptom associated with cancer; (iii) preventing the recurrence of a symptom associated with cancer; (iv) regression of the cancer and / or a symptom associated with it; (v) reducing a subject's hospitalization; (Vi) the reduction in the length of hospital stay; (vii) increasing a subject's survival; (viii) inhibition of cancer progression and / or a symptom associated with it; (ix) the increase or improvement in the therapeutic effect of another therapy; (x) the reduction or elimination in the cancer cell population; (xi) a reduction in the growth of a tumor or neoplasm; (xii) a reduction in the size of the tumor; (xiii) a reduction in the formation of a tumor; (xiv) the eradication, removal or control of primary, regional and / or metastatic cancer; (xv) a decrease in the number or size of metastases; (xvi) the reduction in mortality; (xvii) an increase in the cancer-free survival rate of patients; (xviii) an increase in relapse-free survival; (cix) an increase in the number of patients in remission; (Xx) a decrease in the hospitalization rate; (xxi) the size of the tumor is maintained and does not increase in size or increase the size of the tumor by less than 5% or 10% after administration of a therapy, as measured by conventional methods available to a person skilled in the art, such as such as magnetic resonance, X-ray, and computed tomography; (xxii) the prevention of the development or appearance of cancer and / or a symptom associated with it; (xxiii) an increase in the length of remission in patients; (xxiv) the reduction in the number of symptoms associated with cancer; (xxv) an increase in the symptom-free survival of cancer patients; and / or (xxvi) limitation or reduction in metastasis. In some modalities, the treatment / therapy that a subject receives does not cure the cancer, but prevents the progression or worsening of the disease. In certain modalities, the treatment / therapy that a subject receives does not prevent the onset / development of cancer, but it can prevent the onset of cancer symptoms.
[50] As used herein, the term "in combination" in the context of administering therapy or therapies to a subject, refers to the use of more than one therapy. The use of the term "in combination" does not restrict the order in which therapies are administered to a subject. A first therapy can be administered before (for example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours , 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequently to (for example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, one week, two weeks, three weeks, 4 weeks, five weeks, 6 weeks, 8 weeks, or 12 weeks after) administering a second therapy to a subject.
[51] As used herein, the terms "therapies" and "therapy" can refer to any protocol (s), method (s), and / or agent (s) that can be used in the treatment of cancer. In certain embodiments, the terms "therapies" and "therapy" refer to biological therapy, supportive therapy, hormonal therapy, chemotherapy, immunotherapy and / or other therapies, useful in the treatment of cancer. In a specific embodiment, a therapy includes adjuvant therapy. For example, using therapy in conjunction with drug therapy, biological therapy, surgery and / or supportive therapy. In certain embodiments, the term "therapy" refers to a chimeric NDV described herein. In other embodiments, the term "therapy" refers to an agent that is not a chimeric NDV 4. BRIEF DESCRIPTION OF THE DRAWINGS
[52] Figure 1. NDV infection positively regulates the expression of MHC I, MHC II, and ICAM-1 on the surface of infected cells in vitro, B16-F10 (24 hours post-infection).
[53] Figures 2A-2E. NDV intratumor treatment leads to infiltration with macrophages, NK cells, CD8 and CD4 effector cells and decreases the frequency of Tregs. A) General outline of the study. B) total CD45 + infiltrates. C) Total immune cell infiltrates. D) Graphs of cytometry points representing the flow of the CD4 subsets FoxP3 + and FoxP3-. E) Teff / Treg and CD8 / Treg rates.
[54] Figures 3A-3C. NDV therapy has favorable effects on the tumor microenvironment of distant tumors. A) Graphs of flow cytometry points representative of the CD4 subsets FoxP3 + and FoxP3-. B) Absolute numbers of CD4, Treg, and CD8 effector cells per gram of tumor. C) Tef / Treg and CD8 / Treg rates.
[55] Figures 4A-4C. Lymphocytes that infiltrate distant tumors positively regulate activation, lytic, and proliferation markers. Representative graphs of expression in CD4 effector cells (left) and corresponding percentages in CD4, CD8, Tregs (right) effectors are shown for A) CD44, B) Granzima B, and C) Ki-67.
[56] Figures 5A-5D. NDV monotherapy slows the growth of distant tumors and provides some protection against subsequent tumor provocation. Tumors of the bilateral flanks were established, as described in Figure 2A and the animals were treated and followed for survival. A) The growth of tumors on the right side (treated). B) The growth of tumors on the left side (untreated). C) Overall survival. The numbers in the boxes indicate the percentage of animals free of tumors. D) Survival in animals cured of melanoma B16-F10 by NDV challenged again on day 75 with melanoma cells B16-F10. Representative results from two different experiments with 10 mice per group.
[57] Figures 6A-6B. lymphocytes that infiltrate tumors from treated and untreated tumors up-regulate CTLA-4 in response to NDV therapy. A) Plots of points representative of CTLA-4 expression in effectors CD8, CD4, and Tregs in tumors (treated) on the right. B) Plots of points representative of CTLA-4 expression in effectors CD8, CD4, and Tregs in left (untreated) tumors.
[58] Figure 7A-7C. Combination therapy with NDV and CTLA-4 improves blocking the anti-tumor effect in injected and distant tumors. Bilateral side B16 tumors were established and the animals were treated as described in Figure 2A, with or without anti-CTLA-4 9H10. A) Growth of treated tumors. B) Growth of distant tumors. The numbers in the boxes represent the percentage of tumor-free mice. C) Long-term survival. Representative results from two different experiments, with 10 mice per group.
[59] Figure 8. Combination therapy with NDV and anti-CTLA-4 is systemically effective against non-permissible virus TRAMP prostate tumors. TRAMP tumors on the left (day 3) and right (day 12) were established and the animals were treated with NDV as described in Figure 2A, with or without systemic anti-CTLA-4 antibody. Growth of tumors on the left side (uninjected) is shown. The numbers in the boxes indicate the percentage of animals free of tumors.
[60] Figure 9A-9C. NDV infection upregulates DP-L1 expression in B16-F10 tumors. A) Surface expression of PD-L1 in B16-F10 cells infected with NDV for 24 hours. B) Surface expression of PD-L1 in B16-F10 cells infected with NDV UV inactivated supernatant from infected B16-F10 cells. C) Positive regulation of PD-L1 on the surface of tumor cells isolated from injected tumors and distant from treated animals, as in Figure 2A (Graphs of cytometry points representative of left panels 2, averages calculated on the right panel - calculated averages of five rats per group).
[61] Figures 10A-10F. Combination therapy with NDV and anti-DP-1 is systemically effective against B16 melanoma and results in increased T cell infiltration with positive regulation of activation markers. A) Overall survival. The animals were treated as described in Figure 2A, with or without anti-DP-1 antibody. B) Absolute numbers of CD45, CD3, CD8, CD4 and CD4 effector cells in tumors. C) Relative percentage of regulatory T cells in tumor infiltrating lymphocytes. D-E) Lymphocytes that infiltrate distant tumors were isolated and stained for expression of ICOS (D) and Granzima B (E). F) Lymphocytes that infiltrate tumors were restimulated with lysed tumor-loaded dendritic cells and evaluated for expression of the IFN range by staining intracellular cytokines.
[62] Figure 11. Combination therapy with NDV and CTLA-4 induces positive regulation of CD4 ICOS effector cells and in distant tumors and tumor-draining lymph nodes (TDLN).
[63] Figures 12A-12D. Generation and in vitro evaluation of NDV-ICOSL virus. A) Scheme of viral genomic construction. B) Expression of ICOSL on the surface of infected B16-F10 cells for 24 hours (representative histogram, to the left and average of 3 samples per group, to the right). C) NDV cytolytic activity in infected B16-F10 cells determined by the LDH assay. D) Replication of recombinant NDV in B16-F10 cells.
[64] Figures 13A-13C. Combination therapy with NDV-mICOSL and anti-CTLA-4 protects mice from the challenge of the contralateral tumor and results in the animals' long-term survival. The animals were challenged with a larger dose of the tumor and were treated with NDV, as described in Figure 2A, with or without systemic anti-CTLA-4 antibody. Growth of tumors on the left side (uninjected) is shown. B) Long-term survival. The numbers in the boxes indicate the percentage of animals protected from tumors. Data set from 3 different experiments of 5 to 10 rats per group. C) Rats treated with combination therapy develop vitiligo at the posterior sites of the tumor, but not systemically.
[65] Figure 14A-14B. Combination therapy with NDV-mICOSL and anti-CTLA-4 protects mice from the challenge of the contralateral tumor and results in the long-term survival of the animals in the CT26 colon carcinoma model. The animals were challenged with a larger dose of the tumor and treated with NDV, as described in Figure 2A, with or without systemic anti-CTLA-4 antibody. Growth of tumors on the left side (uninjected) is shown. The numbers in the boxes indicate the percentage of animals protected from tumors. B) Long-term survival. Representative experiment with 5 to 10 rats per group (A) pooled data from two different experiments of 5 to 10 rats per group (B).
[66] Figures 15A-15C. NDV treatment leads to distant B16 tumor that infiltrates with macrophages, NK cells, CD8 and CD4 effector cells and decreases the frequency of Tregs. A) Total infiltrates CD45 +, CD3 +, CD8 +, CD4 + FoxP3- (Tef), and CD4 + FoxP3 + (Treg). B) Tef / Treg and CD8 / Treg Rates. C) Total cellular infiltrates of macrophages, NK and NKT.
[67] Figure 16A-16B. Lymphocytes that infiltrate distant B16 tumors positively regulate activation, lytic, and proliferation markers. Representative Ki-67, Granzima B (GRB) and ICOS expression plots (A) and the corresponding percentages in effectors CD4 and CD8 (B).
[68] Figure 17. Lymphocytes that infiltrate tumors from treated animals that secrete IFN-gamma in response to stimulation with DC's loaded with B16-F10 cell lysates. Graphs of representative points are shown.
[69] Figures 18A-18B. Animals cured by combination therapy are protected from another tumor challenge. A) Melanoma B16-F10, day 120 new challenge with 1 x 105 cells. B) CT26 colon carcinoma, day 90 new challenge with 1 x 106 cells. Representative results from two different experiments with 10 mice per group.
[70] Figure 19A-19B. Recombinant chimeric protein ICOSL-F is efficiently expressed on the surface. A) Schematic representation of the chimeric protein. B) Expression of the chimeric ICOSL-Ftm fusion protein on the surface of transfected cells.
[71] Figure 20A-20D. NDV infection is restricted to the injected tumor. A) Recombinant NDV-Fluc was administered intratumorally (IT) or intravenously (IV) in Balb / C animals with CT26 tumors and images were acquired over the next 72 hours. B) NDV-Fluc was administered to C57BL / 6 rats with bilateral B16-F10 melanoma tumors and the animals were monitored for 120 hours. Representative luminescence images are shown. C) Quantification of luminescence from the tumor site normalized to the luminescence background. D) The area under the curve (AUC) calculated from the data in panel (C). The data show representative results from 1 of 3 independent experiments with 3 to 5 rats / group. *** P <0.001.
[72] Figure 21A-21F. NDV infection increases leukocyte infiltration in tumors injected with viruses. The animals were treated according to the scheme described in figure 22A. Tumors were excised on day 15, and TILs were scored and analyzed by flow cytometry. A) Graphs of cytometry points representing percentages of CD45 + and CD3 + cells that infiltrate the tumor. B) Absolute numbers of CD45 + cells / tumor. C) Absolute numbers of innate immune cells / g tumor. D) Graphs representative of the percentages of CD4 + FoxP3 + (Treg) and CD4 + FoxP3- (T conv) cells. E) Absolute numbers of conventional and regulatory cells CD4 + and CD8 + / g tumor cells. F) Tconv / Treg and CD8 + / Treg rates calculated for tumors. The data represent the cumulative results of three independent experiments with 3 to 5 rats / group. The mean +/- SEM is shown. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.
[73] Figure 22A-22M. NDV increases lymphocytes that infiltrate distant tumors and slows tumor growth. A) treatment schedule. B) Graphs of cytometry points representing the percentages of CD3 + CD45 + cells that infiltrate the tumor. C) Absolute numbers of CD45 + / g tumor cells. D) Absolute numbers of innate immune cells / tumor. E) Tumor sections from distant tumors were stained with H&E (upper panels) or labeled for CD3 and FoxP3 (lower panels) and analyzed by microscopy. Areas indicated by arrows indicate areas of necrosis and inflammatory infiltrate. The scale bars represent 200 pm. F) Graphs of cytometry points representing percentages of CD4 + FoxP3- (Tconv) and CD4 + FoxP3 + cells (Treg). G) Absolute numbers of conventional cells regulating CD4 + and CD8 + cells / g tumor calculated from flow cytometry. H) Relative percentages of Tregs outside of CD45 + cells. I) Tconv / Treg and CD8 + / Treg rates calculated. (J, K) Positive regulation of ICOS, Ggranzima B, and Ki-67 in Tconv that infiltrate the tumor (J) and CD8 + cells (K). L) Growth of distant and injected NDV tumors. M) Overall animal survival. The data represent the cumulative results of 3 (BK) or 2 independent experiments (L-M) with n = 3 to 5 per group. The mean +/- SEM is shown. * P <0.05, ** P <0.01, *** P <0.001, **** p <o, 0001.
[74] Figures 23A-23E. NDV therapy increases lymphocytes that infiltrate distant tumors in a bilateral foot melanoma model. Animals with melanoma tumors were treated according to the schedule described in Figure 22A. Distant tumors were excised on day 15 and TILs were scored and analyzed by flow cytometry. A) Graphs of cytometry points representing the percentages of CD45 + and CD3 + cells infiltrating the tumor. B) Graphs of cytometry points representing percentages of CD4 + Foxp3 + and CD4 + FoxP3- cells. C) Absolute numbers of conventional and regulatory cells CD4 + and CD8 + / g tumor. D, E) Positive regulation of ICOS, Granzima B, and Ki-67 in lymphocytes that infiltrate CD8 + (D) and Tconv (E) tumor. The data show representative results from 1 of 2 independent experiments with 5 rats / group. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.
[75] Figure 24A-24I. NDV induces infiltration of specific lymphocytes from adoptively transferred tumors and facilitates tumor inflammation. A) treatment schedule. B) Representative images of illuminescence of animals treated with NDV and TRP1-FLUC lymphocytes adopted adoptively. C) Average luminescence quantification of tumor sites. D) The area under the curve (AUC) calculated from the data in panel (C). E) Absolute number of PMEL lymphocytes from distant tumors calculated from flow cytometry. F) Graphs of cytometry points representing the percentages of CD3 + CD45 + cells that infiltrate tumors distant from animals treated by the treatment regime in panel (A). G) Experimental scheme for the transfer of serum from animals treated intratumorally with a single injection of NDV or PBS. H) Cytometry point plots of the percentages of CD3 + CD45 + cells that infiltrate tumors injected into the serum. I) Absolute numbers of the set of cells indicated in the tumors injected into the serum, calculated from flow cytometry. Data for B-E represent 1 of 3 experiments with n = 4-5 per group. The data gathered from panels G-I represent data from two independent experiments with n = 5 per group. The mean +/- SEM is shown. * P <0.05, ** P <0.01, *** p <0.001, **** P <0.0001.
[76] Figure 25. Intratumoral NDV provides protection against the redefined tumor. Animals cured of B16-F10 melanoma received NDV injections on day 75 with 1x105 B16-F10 melanoma cells, monitored for tumor growth, and sacrificed when the tumors reached 100mm3. The overall survival of the animal is shown. The data show the cumulative results of 1 of 2 independent experiments with 10 rats / group. **** P <0.0001.
[77] Figure 26A-26B. CD8 + lymphocytes that infiltrate tumors up-regulate CTLA-4 in response to NDV therapy. Plots of representative points (left) and cumulative results (right) of CTLA-4 expression in CD8 + cells in those treated with NDV (A), and distant tumors (B). Representative results from 1 of 3 experiments, with three mice per group. * P <0.05.
[78] Figure 27A-27K. NDV and CTLA-4 block synergy to reject local and distant tumors. A) treatment schedule. B) Growth of B16-F10 tumors treated with viruses (right side). C) Growth of distant B16-F10 tumors (left side). D) Long-term survival in model B16-F10. E), Surviving animals were injected with 1x105 B16-F10 cells on the right side on day 90 and left for survival. The data represent the cumulative results of three experiments with n = 6-11 per group. F) Growth of TRAMP C2 tumors far left and (right side) treated with viruses. G) Long-term survival in the TRAMP C2 model. H) In vitro sensitivity of B16-F10 cells and TRAMP C2 cells for NDV-mediated lysis in different multiplicities of infection (MOI 's). I-K) Positive regulation of MHC I, MHC II, CD80, and CD86 in NDV-infected B16-F10 and TRAMP C2 cells. Flow cytometric points representative of B16-F10 (I) cells Average calculated fluorescence intensity (MFI) by B16-F10 (J) and TRAMP C2 (K) cells are shown. Average +/- SEM is shown. The data represent the results of 1 of 3 (B-E), or 1 of 2 (F, G) independent experiments with n = 5 to 10 per group. * P <0.05, ** P <0.01, *** p <0.001, **** P <0.0001.
[79] Figure 28A-28E. Systemic antitumor effect is restricted to the type of tumor injected. A) Animals were injected on the right side with B16-F10 melanoma, MC38 colon carcinoma, or PBS, and on the left side with B16-F10 cells and treated as described in the scheme. B, C) Growth of distant tumors (B) and overall survival (C) of animals that received B16-F10 on the right side or nothing on this side. The data show representative results from 1 of 2 independent experiments with 5 to 10 rats / group. D, E) Growth of distant tumors (D) and overall survival (E) of animals that received B16-F10 or MC38 tumors on the right side. Data represent results from 1 of 2 independent experiments with n = 10 per group. ** P <0.01, **** p <0.0001.
[80] Figure 29A-29E. Combination therapy with NDV and anti-CTLA-4 increases the infiltration of the tumor with cells of the adaptive and innate immune system. The animals were treated with the combination therapy, as described in Figure 27A. Tumors were harvested on day 15 and analyzed to infiltrate immune cells using flow cytometry. A) Absolute numbers of CD45 + / g tumor cells. B) Absolute numbers of CD11b + and NK 1.1+ cells / g tumor. C) Absolute numbers of conventional and regulatory cells CD4 + and CD8 + cells / g tumor. D) Relative percentages of Tregs outside of CD45 + cells. E) Tconv / Treg calculated and CD8 + / Treg rates. The data represent cumulative results from four independent experiments with 3-5 rats / group. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.
[81] Figure 30A-30J. Combination therapy with NDV and CTLA-4 blockade induces inflammatory changes in distant tumors. The animals were treated by the scheme in Figure 27A. The tumors were harvested on day 15 and analyzed for infiltration of immune cells. A) Tumor sections from distant tumors were stained with H&E (upper panels) or by CD3 and FoxP3 (lower panels) and analyzed by light and fluorescence microscopy, respectively. Areas indicated by arrows indicate necrosis and inflammatory infiltrates. The scale bars represent 200 pm. B) Absolute number of infiltrating tumor CD45 + and CD3 + cells / g tumor calculated from flow cytometry. C) Flow cytometry points representing the percentage of CD4 + and CD8 + cells that infiltrate the tumor, selected from the CD45 + population. D) absolute numbers of Tconv, Treg and CD8 + cells per gram of tumor. E) Relative percentages of Tregs outside of CD45 + cells that infiltrate the tumor. F) Tconv / Treg and CD8 + / Treg rates calculated. G-I) Positive regulation of ICOS, Granzima B, and Ki-67 in CD8 + that infiltrates Tconv tumors and lymphocytes. Points representing flow cytometry (upper panels) and cumulative results (lower panels) are shown. J) TILs were restimulated with DC's pulsed with B16-F10 tumor lysates, and IFNy production was determined by staining intracellular cytokines. Points representing flow cytometry (left panel) and accumulated results (right panel) are shown. The data represent the cumulative results of 5 (A-I) or 2 (J) independent experiments with n = 3-5 per group. Average +/- SEM is shown. * P <0.05, ** P <0.01, *** P <0.001, **** p <0.0001.
[82] Figure 31. Antibodies to CD8, CD4, and NK1.1 deplete cells of interest in vivo. Destroying antibodies were injected as discussed in Materials and Methods in Section 7.1, below. Blood samples were collected on day 5 and processed by flow cytometry of CD4 +, CD8 + cells and NK cells with non-cross-reactive antibodies. Positive staining is represented by the horizontal bars. Representative graphs of 1 of 2 independent experiments with five rats per group are shown.
[83] Figure 32A-32F. The antitumor activity of the NDV combination therapy depends on CD8 + and NK cells and type I and type II interferons. A-C) Animals were treated, as described in Fig. 27A, with or without depleting antibodies for CD4 +, CD8 +, NK, or IFNy cells. A) Growth of injected tumors. B) Growth of distant tumors. C) Long-term survival. D-F) RIFNA - / - or C57BL / 6 (BL / 6) age matched mice were treated as described in Fig. 3A and monitored for tumor growth. D) Growth of injected tumors. E) Growth of distant tumors. F) Long-term survival. The data for all panels represent the cumulative results of two independent experiments with n = 3-10 per group. * P <0.05, ** P <0.01, *** P <0.001, **** p <0.0001.
[84] Figure 33A-33B. NDV therapy leads to positive regulation of PD-L1 in tumors and leukocytes that infiltrate tumors. THE). PD-L1 expression in infected B16-F10 cells in vitro (left panel), and in vivo in injected viruses and distant tumors. Left, histograms representing flow cytometry, right, mean median fluorescence intensity (MFI) of PD-LI expression in tumor B16-F10 cells. B) Expression of DP-L1 on the surface of leukocytes that infiltrate tumors, isolated from distant tumors. Left: flow cytometry representative histograms, right: MFI calculated mean for each subset of cells.
[85] Figure 34A-34D. Combination therapy of NDV with DP-1 blocking antibodies leads to an increase in antitumor efficacy in that of the B16 melanoma model on the bilateral side. A) Treatment schedule. B) Tumor growth (NDV-injected) on the right side. C) Growth of the tumor (distant) left side. D) Overall survival.
[86] Figure 35A-35D. Combination therapy of NDV with PD-L1 blocking antibodies leads to improved antitumor efficacy in the B16 melanoma model on the bilateral side. A) Treatment schedule. B) Tumor growth (NDV- injected) on the right side. C) Growth of the tumor (distant) left side. D) Overall survival.
[87] Figure 36A-36E. Combination therapy with NDV and anti-DP-1 therapy results in an increase in distant tumor infiltration with the effector, but not with the regulatory T cell. A) Cytometric points representing the percentages of CD4 + and CD8 + cells in tumors. B) Cytometric points representing the percentages of Tconv (CD4 + FoxP3-) and Treg cells (CD4 + FoxP3 +). C) Absolute numbers of subsets of T cells per gram of tumor, calculated from flow cytometry. D) Relative percentages of CD4 + T cell Tregs. E) Calculated Tconv / Treg and CD8 / Treg rates.
[88] Figure 37A-37B. TILs from distant tumors in animals treated with the combination of NDV and anti-DP-1 therapy positively regulate proliferation and lytic markers. A) Cytometric points representing the percentages of Tconv and CD8 lymphocytes positive for Granzima B and Ki67. B) Percentages of Tconv and CD8 + T cells positive for Granzyme B and Ki67.
[89] Figure 38A-38C. NDV induces the infiltration of the immune tumor and the positive regulation of ICOS in CD4 and CD8 cells in the injected viruses and distant tumors. A) Treatment schedule. B) Expression of ICOS in CD4 + FoxP3- and CD8 + cells that infiltrate tumors isolated from tumors injected with NDV on the right side). Representative flow cytometry points (top) and median fluorescence intensities (MFI) (bottom) are shown. C) Expression of ICOS in CD4 + FoxP3- and CD8 + cells isolated from distant tumors (left side). Representative flow cytometry points (top) and median fluorescence intensities (MFI) (bottom) are shown.
[90] Figure 39A-39D. Generation and in vitro evaluation of the NDV-ICOSL virus. A) Scheme of viral genomic construction. B) Expression of ICOSL on the surface of infected B16-F10 cells for 24 hours (representative histogram on the left and mean of 3 samples per group on the right). C) NDV cytolytic activity in infected B16-F10 cells determined by the LDH assay. D) Replication of recombinant NDV in B16-F10 cells.
[91] Figure 40A-40F. NDV-ICOSL causes delayed growth of distant tumors and induces better infiltration of tumor lymphocytes. Bilateral B16-F10 tumors were established as before and the animals were treated with four intratumoral injections of the virus indicated to the right of the tumor. A) Growth of tumors injected with viruses. B) Growth of distant tumors. C) Overall survival. D) Absolute numbers of leukocytes that infiltrate tumors on the right (tumors with virus injection). E) Absolute numbers of leukocytes that infiltrate tumors on the left (distant tumors). F) Relative concentration of Tregs in distant tumors.
[92] Figure 41A-41E. Combination therapy of NDV-ICOSL and CTLA-4 blocks results in the rejection of distant and injected tumors of the model B16-F10 and protects against tumor redefining. A) Treatment schedule. B) Growth of tumors (right) injected by viruses. C) Growth of distant tumors (left). D) Overall survival. E) Animals surviving on day 90 were redefied on the right side with 2x105 B16-F10 cells and left for survival.
[93] Figure 42A-42E. Combination therapy of NDV-ICOSL and CTLA-4 results from the block in the rejection of distant and injected tumors of the CT26 model. A) Treatment schedule. B) Growth of tumors (right) injected by viruses. C) Growth of distant tumors (left). D) Overall survival. E) Surviving animals on day 90 were re-challenged on the right side with 1 x 106 CT26 cells and left for survival.
[94] Figure 43A-43J. Combination therapy of NDV-ICOSL and anti-CTLA-4 leads to better infiltration of the tumor with cells of the adaptive and innate immune system. The animals with bilateral tumors B16-F10 were treated according to the schedule described in figure 41A. On day 15 the animals were sacrificed and the distant tumors were processed for Til's analysis. A) Points of flow cytometry representative of CD3 + and CD45 + cells taking up the entire population of tumor cells. The absolute number of CD45 + (B), CD11b + (C) and NK1.1 + cells that infiltrate the tumor, and (D) per gram of tumor was calculated from flow cytometry. E) Absolute numbers of infiltrating tumors, CD3 +, CD8 +, CD4 + FoxP3- (CD4eff), and CD4 + FoxP3 + (Treg) per gram of tumor. F) Relative percentage of Tregs of all CD45 + cells. G) Calculated effector / Treg rates. H, I, J) relative percentages of CD8 + and CD4 + effector cells that infiltrate positive tumors for ICOS, granzyme B, and Ki67, respectively.
[95] Figure 44A-44C. Schematic diagram for additionally generated recombinant NDV viruses expressing chimeric and native immunostimulatory proteins. A) Diagram of chimeric proteins of the TNF receptor superfamily (GITRL, OX40L, 4-1BBL, CD40L) fused to the transmembrane and intracellular region of the HN of HV NDV at the N terminal (top panel). The bottom panel shows the chimeric protein diagram of the immunoglobulin receptor superfamily, with extracellular anti-CD28scfv and ICOSL domains fused to the transmembrane and intracellular region of F at the C terminal. B) Length of extracellular and intracellular transmembrane domains (HN and F ) of each of the chimeric proteins described. C) Schematic diagram of the transgene insertion site and list of all recombinant NDVs that express immunostimulating ligands generated by this strategy.
[96] Figure 45A-45C. Confirmation of saving of the NDV recombinant. A) Hemagglutination test showing positive hemagglutinating activity in the wells for NDV-HN-GITRL, NDV-aCD28scfv-F, NDV-HN-OX40L, NDV-HN-CD40L, NDV-IL15, and NDV-IL21. B) Primers for confirming the insertion of the gene in viruses rescued by RT-PCR. C) RT-PCR in RNA isolated from rescued viruses.
[97] Figure 46. B16-F10 cells infected with recombinant NDVs express the ligands on the surface. B16-F10 cells were infected with the NDV's recombinant in MOI of 2 and were analyzed for surface ligand expression by flow cytometry 18 hours later. The points of the representative flow cytometry are shown.
[98] Figure 47. NDV-HN-4-1BBL induces increased distant immune tumor infiltration. The animals with bilateral B16 melanoma tumors were treated intratumorally on a single side with the virus as previously indicated. After three treatments, the animals were sacrificed and lymphocytes that infiltrate tumors from distant tumors were analyzed by flow cytometry. The total number of CD3, CD4 + FoxP3 + (Treg), CD4 + FoxP3- (Tconv), CD8, NK, and CD11b + cells that infiltrate tumors per gram of tumor is shown. 5. DETAILED DESCRIPTION
[99] In one aspect, Newcastle disease chimeric viruses (NDVs) engineered to express an agonist of an immune cell co-stimulatory signal and / or an antagonist of an immune cell inhibitory signal are shown here. In a specific embodiment, chimeric NDVs are presented in this document, comprising a packaged genome that encodes an agonist of an immune cell co-stimulating signal, in which the agonist is expressed. In a specific embodiment, chimeric NDVs are presented herein, comprising a packaged genome that encodes an antagonist of an immune cell inhibitory signal, in which the antagonist is expressed.
[100] In another aspect, the methods for propagating NDVs described herein are presented here (for example, chimeric NDVs described here). The NDVs described herein (for example, chimeric NDVs described herein) can be propagated in any cell, subject, tissue, organ or animal susceptible to an NDV infection.
[101] In another aspect, compositions comprising an NDV described herein are presented here (for example, a chimeric NDV described here). In a specific embodiment, pharmaceutical compositions comprising an NDV described herein (for example, a chimeric NDV described herein) and a pharmaceutically acceptable carrier are disclosed herein. In another embodiment, pharmaceutical compositions comprising cancer cells infected with an NDV described herein (for example, a chimeric NDV described herein), and a pharmaceutically acceptable carrier are disclosed herein. In another embodiment, pharmaceutical compositions comprising protein concentrate from lysed NDV-infected cancer cells (e.g., cancer cells infected with chimeric NDV), and a pharmaceutically acceptable carrier are presented herein.
[102] In another aspect, methods for producing pharmaceutical compositions comprising an NDV described herein are presented here (for example, a chimeric NDV described here). In one embodiment, a method for producing a pharmaceutical composition comprising: (a) propagating an NDV described herein (for example, a chimeric NDV described here) in a cell line that is susceptible to an NDV infection; and (b) collecting the viral progeny, in which the virus is grown in sufficient quantities and under sufficient conditions for the virus to be free from contamination, so that the viral progeny is suitable for formulation in a pharmaceutical composition. In another embodiment, a method for producing a pharmaceutical composition includes: (a) propagating an NDV described herein (for example, a chimeric NDV described here) in an embryonic egg; and (b) collecting the viral progeny, in which the virus is grown in sufficient quantities and under sufficient conditions for the virus to be free from contamination, so that the viral progeny is suitable for formulation in a pharmaceutical composition.
[103] In another aspect, methods for treating cancer using a chimeric NDV described herein (for example, a chimeric NDV described in Section 5.2, below), or a composition comprising a chimeric NDV are presented here. In a specific embodiment, a method for the treatment of cancer which comprises the infection of a cancer cell in a subject with a chimeric NDV described herein (for example, a chimeric NDV described in Section 5.2, below), or its composition. In another embodiment, a method for the treatment of cancer that comprises administering to a subject in need thereof, a chimeric NDV described herein (for example, a chimeric NDV described in Section 5.2, below), or its composition. In specific embodiments, an effective amount of a chimeric NDV described herein (for example, a chimeric NDV described in Section 5.2, below) or a composition comprising an effective amount of a chimeric NDV, presently described, is administered to a subject to treat cancer. In specific embodiments, the chimeric NDV comprises a packaged genome, the genome comprising an agonist of an immune cell co-stimulating signal (for example, an agonist of an immune cell co-stimulating receptor) and / or an antagonist of an inhibitory signal from an immune cell (for example, an immune cell inhibitory receptor antagonist), in which the agonist and / or antagonist are expressed by NDV. In certain embodiments, the NDV genome also comprises a mutated F protein. In certain embodiments, two or more chimeric NDVs are administered to a subject to treat cancer.
[104] In another embodiment, a method for the treatment of cancer that comprises administering to a subject in need of it, cancer cells infected with a chimeric NDV described here (for example, a chimeric NDV described in Section 5.2, below), or its composition. In specific embodiments, cancer cells were treated with gamma radiation prior to administration to the subject or incorporation into the composition. In another embodiment, a method for treating cancer that comprises administering to a subject in need of it, a protein concentrate or plasma membrane fragments of cancer cells infected with chimeric NDV (for example, a chimeric NDV described in Section 5.2, below), or its composition. In specific embodiments, the chimeric NDV comprises a packaged genome, the genome comprising an agonist of an immune cell co-stimulating signal (for example, an agonist of an immune cell co-stimulating receptor) and / or an antagonist of an inhibitory signal from an immune cell (for example, an immune cell inhibitory receptor antagonist), in which the agonist and / or antagonist are expressed by NDV. In certain embodiments, the NDV genome also comprises a mutated F protein, which is expressed by NDV.
[105] In another aspect, methods for treating cancer using an NDV described here (for example, a chimeric NDV, as described in Section 5.2, below) or a composition comprising such NDV in combination with one or more are presented herein. more other therapies. In one embodiment, methods for treating cancer are presented herein comprising administering to a subject an NDV described herein (for example, a chimeric NDV, as described in Section 5.2, below) and one or more other therapies. In another embodiment, methods for treating cancer are presented herein comprising administering to an individual an effective amount of an NDV described herein or a composition comprising an effective amount of an NDV described herein, and one or more other therapies. NDV and one or more other therapies can be administered to the subject at the same time or sequentially. In certain embodiments, NDV and one or more other therapies are administered in the same composition. In other embodiments, NDV and one or more other therapies are administered in different compositions. NDV and one or more other therapies can be administered by the same or different routes of administration to the subject.
[106] Any type or strain of NDV can be used in a combination therapy described herein, including, but not limited to, naturally occurring strains, variants or mutants, mutagenized recombinant viruses, and / or genetically modified viruses. In a specific modality, NDV used in combination with one or more other therapies is a naturally occurring strain. In another embodiment, the NDV used in combination with one or more other therapies is a chimeric NDV. In a specific embodiment, the chimeric NDV comprises a packaged genome, the genome comprises a cytokine (for example, IL-2, IL-7, IL-15, IL-17 or IL-21). In specific embodiments, the chimeric NDV comprises a packaged genome, the genome that comprises a tumor antigen. In specific modalities, the tumor antigen is expressed by cells infected with chimeric NDV. In another specific embodiment, the chimeric NDV comprises a packaged genome, the genome comprising a pro-apoptotic molecule (for example, Bax, Bak, Bad, BID, Bcl-xS, Bim, Noxa, Puma, AIF, FasL, and TRAIL or an anti-apoptotic molecule (for example, Bcl-2, Bcl-xL, Mcl-1, and XIAP. In specific embodiments, the pro-apoptotic molecule or anti-apoptotic molecule is expressed by cells infected with chimeric NDV. another specific embodiment, the chimeric NDV comprises a packaged genome, the genome comprising an agonist of an immune cell co-stimulating signal (for example, an agonist of an immune cell co-stimulating receptor) and / or an antagonist of an inhibitory signal an immune cell (for example, an inhibitor receptor for an immune cell antagonist). In specific embodiments, the agonist and / or antagonist are expressed by cells infected with chimeric NDV. In certain embodiments, the NDV genome also comprises a protein Mutated F, a tumor antigen, a heterologous interferon antagonist, a pro-apoptotic molecule and / or an anti-apoptotic molecule. In certain embodiments, the one or more therapies used in combination with an NDV described herein is one or more other therapies described in Section 5.6.4 below. In particular embodiments, the one or more therapies used in combination with an NDV described herein is an agonist of an immune cell co-stimulating signal and / or an antagonist of an immune cell inhibiting signal. See, for example, Section 5.2.1, below, for examples of agonists of an immune cell co-stimulating signal and antagonists of an immune cell inhibitory signal. In a specific embodiment, the antagonist of an immune cell inhibitory signal is the anti-CTLA-4 antibody described in Section 6, infra. In another specific embodiment, the agonist of an immune cell co-stimulating signal is the ICOS ligand described in Section 6, below. 5.1 NEWCASTLE DISEASE VIRUS
[107] Any type or strain of NDV can be used in a combination therapy described herein, including, but not limited to, naturally occurring strains, variants or mutants, mutagenized recombinant viruses, and / or genetically modified viruses. In a specific modality, the NDV used in a combination therapy presented here is a naturally occurring strain. In certain embodiments, NDV is a lytic strain. In other modalities, the NDV used in a combination therapy presented here is a non-lytic strain. In certain embodiments, the NDV used in a combination therapy described here is the lentogenic strain. In some modalities, NDV is a mesogenic strain. In other modalities, the NDV used in a combination therapy presented here is a velogenic strain. Specific examples of NDV strains include, but are not limited to, the 73-T strain, the NDU HUJ strain, the Ulster strain, the MTH-68 strain, the Italien strain, the Hickman strain, the PV701 strain, the Hitchner BI strain (see, for example, Genbank No. AF309418 or NC_002617), La Sota strain (see, for example, Genbank No. AY845400), YG97 strain, MET95 strain, Roakin strain, and F48E9 strain. In a specific embodiment, the NDV used in a combination therapy described here, which is the Hitchner BI strain. In another specific embodiment, the NDV used in a combination therapy presented here is a BI strain as identified by Genbank AF309418 or NC_002617. In another specific embodiment, the NDV used in a combination therapy described herein, is the NDV identified by ATCC No. VR2239. In another specific modality, the NDV used in a combination therapy described here, is the La Sota strain.
[108] In specific embodiments, the NDV used in a combination therapy described here are not from pathogenic birds, as assessed by a technique known to a specialist. In certain specific modalities, the NDV used in a combination therapy is non-pathogenic, as assessed by intracranial injection of day-old chicks with the virus, and the development of disease and death occurring over 8 days. In some embodiments, the NDV used in a combination therapy described here has an intracranial pathogenicity index of less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2 or less than 0.1. In certain embodiments, the NDV used in a combination therapy described here has an intracranial pathogenicity index of zero.
[109] In certain embodiments, the NDV used in a combination therapy presented here is a mesogenic strain that has been genetically modified so as not to be considered a pathogen in birds as assessed by techniques known to a person skilled in the art. In certain embodiments, the NDV used in a combination therapy presented here is a velogenic strain that has been genetically modified so that it is not a pathogen considered in birds as assessed by techniques known to a person skilled in the art.
[110] In certain embodiments, the NDV used in a combination therapy described herein expresses a mutated F protein. In a specific embodiment, the NDV used in a combination therapy expresses a mutated F protein is highly fusogenic and capable of forming syncytia. In another specific embodiment, the mutated F protein is incorporated into the virion.
[111] In one embodiment, an NDV genome used in a combination therapy presented here is manipulated to express a mutated F protein with a mutated cleavage site. In a specific embodiment, the NDV used in a combination therapy presented here is manipulated to express a mutated F protein in which the F protein cleavage site is mutated to produce a polybasic amino acid sequence, which allows the protein to be cleaved by intracellular proteases, which makes the virus more effective at entering cells and at forming syncytia. In another specific embodiment, the NDV used in a combination therapy presented here is manipulated to express a mutated F protein in which the F protein cleavage site is replaced by one containing one or two supplementary arginine residues, allowing the site mutant cleavage is activated by ubiquitously expressed proteases of the furin family. Specific examples of such NDVs that express a mutated F protein include, but are not limited to, rNDV / F2aa and rNDV / F3aa. For a description of mutations introduced into an NDV F protein to produce a mutant F protein with a mutated cleavage site, see, for example, Park et al. (2006) Engineered viral vaccine constructs with dual specificity: avian influenza and Newcastle disease. PNAS USA 103: 8203-2808, which is incorporated herein by reference in its entirety. In some embodiments, the NDV used in a combination therapy presented here is manipulated to express an F protein mutated with the L289A mutation of the amino acid. In specific embodiments, the mutated F mutated protein L289A has one, two or three arginine residues at the dividing site. In certain embodiments, the mutated F protein is of a different type or strain of NDV than the structure of NDV. In some embodiments, the mutated F protein is in addition to the structure of the NDV F protein. In specific embodiments, the mutated F protein replaces the NDV F protein structure.
[112] In certain embodiments, the NDV used in a combination therapy presented here is attenuated so that the NDV remains, at least partially, infectious and can replicate in vivo, but only generate low titers, resulting in subclinical levels of infection. that are non-pathogenic (see, for example, Khattar et al, 2009, J. Virol 83: 7779-7782). In a specific embodiment, NDV is attenuated by the deletion of protein V, such attenuated NDVs may be especially suitable for modalities in which the virus is administered to a subject, in order to act as an immunogen, for example, a live vaccine. Viruses can be mitigated by any method known in the art.
[113] In certain embodiments, the NDV used in a combination therapy described herein does not comprise a sequence encoding the NDV V protein. In other embodiments, the NDV used in a combination therapy described herein expresses a mutated V protein. See, for example, Elankumaran et al. , 2010, J. Virol. 84 (8): 3835-3484, which is incorporated herein by reference, for examples of mutated V proteins. In certain embodiments, a strain of mesogenic or velogenic NDV used in a combination therapy described herein expresses a mutated V protein, as described by Elankumaran et al., 2010, J. Virol. 84 (8): 3835-3844.
[114] In certain embodiments, the NDV used in a combination therapy presented here is an NDV disclosed in US patents 7,442,379, US 6,451,323, or US 6,146,642, which are incorporated herein by reference in their entirety. In specific embodiments, the NDV used in a combination therapy presented here is genetically modified to encode and express a heterologous peptide or protein. In certain embodiments, the NDV used in a combination therapy described herein is a chimeric NDV known to a person skilled in the art, or a chimeric NDV described here (see, for example, Section 5.2, below). In some embodiments, the NDV used in a combination therapy described here is a chimeric NDV comprising a genome engineered to express a tumor antigen (see examples of tumor antigens below). In certain embodiments, the NDV used in a combination therapy described herein is a chimeric NDV comprising a genome engineered to express heterologous interferon antagonists (see examples of heterologous interferon antagonists below). In some embodiments, the NDV used in a combination therapy described herein is a chimeric NDV described in the publication of U.S. Patent Application No. 2012/0058141, which is incorporated herein by reference in its entirety. In certain embodiments, the NDV used in a combination therapy described herein is a chimeric NDV described in U.S. Patent Application Publication No. 2012/0122185, which is incorporated herein by reference in its entirety. In some embodiments, the NDV used in a combination therapy described herein is a chimeric NDV comprising a genome engineered to express a cytokine, such as, for example, IL-2, IL-7, IL-9, IL-15, IL -17, IL-21, IL-22, IFN-gamma, GM-CSF and TNF-alpha. In some embodiments, the NDV used in a combination therapy described herein is a chimeric NDV comprising a genome engineered to express IL-2, IL-15, or IL-21. In a specific embodiment, the NDV used in a combination therapy described herein is a chimeric NDV comprising a genome engineered to express a cytokine, as described in Section 7, Example 2, below. 5.2 NEWCASTLE CHEMICAL VIRUSES
[115] In one aspect, chimeric NDVs are described in this document, comprising a genome engineered to express an agonist of a co-stimulatory signal and / or an antagonist of an inhibitory signal from an immune cell, such as, for example, a T lymphocyte or natural killer cell (NK). In some embodiments, the agonist and / or antagonist is incorporated into the virion. In a specific embodiment, an NDV genome is manipulated to express an agonist of an immune cell co-stimulating signal, such as, for example, a T lymphocyte or an NK cell. In another specific embodiment, an NDV genome is engineered to express an antagonist of an immune cell inhibitory signal, such as, for example, a T lymphocyte or an NK cell. In other words, NDV serves as a "structure" that is manipulated to express an agonist of a co-stimulatory signal and / or an antagonist of an inhibitory signal from an immune cell, such as, for example, a T-lymphocyte or natural killer cell ( NK). Specific examples of costimulatory signal agonists, as well as specific examples of inhibitory signal antagonists are provided below.
[116] In another aspect, chimeric NDVs are described in this document, comprising a genome engineered to express an agonist of a co-stimulatory signal and / or an antagonist of an immune cell inhibitory signal, such as, for example, a T lymphocyte or an NK cell, and a mutated F protein. In one embodiment, an NDV genome is engineered to express an agonist of an immune cell co-stimulating signal, such as, for example, a T lymphocyte or an NK cell, and a mutated F protein. In another embodiment, an NDV genome is engineered to express an immune cell inhibitory signal antagonist, such as, for example, a T lymphocyte or an NK cell, and a mutated F protein. In a specific embodiment, the mutated F protein is highly fusogenic and capable of forming syncytia. In another specific embodiment, the mutated F protein is incorporated into the virion. In certain embodiments, an NDV genome engineered to express a co-stimulating signal agonist and / or an immune cell inhibitory signal antagonist, such as, for example, a T lymphocyte or an NK cell, comprises a sequence of coding of NDV protein V.
[117] In one embodiment, an NDV genome is manipulated to express an agonist of a co-stimulatory signal and / or an antagonist of an inhibitory signal from an immune cell, such as, for example, a T lymphocyte or an NK cell, and a F protein mutated with a mutated cleavage site. In a specific embodiment, NDV is engineered to express a mutated F protein in which the F protein cleavage site is mutated to produce a sequence of polybasic amino acids, which allows the protein to be cleaved by intracellular proteases, making it most effective virus to enter cells and provide the formation of syncytia. In another specific embodiment, NDV is engineered to express a mutated F protein in which the F protein cleavage site is replaced by one containing one or two supplementary arginine residues, allowing the mutant cleavage site to be activated by proteases ubiquitously furin family. Specific examples of such NDVs that express a mutated F protein include, but are not limited to, rNDV / F2aa and rNDV / F3aa. For a description of mutations introduced into an NDV F protein to produce a mutant F protein with a mutated cleavage site, see, for example, Park et al. (2006) Engineered viral vaccine constructs with dual specificity: avian influenza and Newcastle disease. PNAS USA 103: 8203- 2808, which is incorporated herein by reference in its entirety. In some embodiments, the chimeric NDV is engineered to express an F protein mutated with the L289A amino acid mutation. In certain embodiments, the mutated F protein is of a different type or strain of NDV than the structure of NDV. In specific modalities of the mutated F L289A protein it has one, two or three arginine residues at the cleavage site. In some embodiments, the mutated F protein is in addition to the structure of the NDV F protein. In specific embodiments, the mutated F protein replaces the NDV F protein structure. In specific embodiments, the mutated F protein is incorporated into the virion.
[118] In some embodiments, an NDV genome engineered to express an agonist of a co-stimulatory signal and / or an antagonist of an inhibitory signal from an immune cell, such as, for example, a T lymphocyte or an NK cell, comprises a coding sequence for the mutated NDV V protein, as described by Elankumaran et al. , 2010, J. Virol. 84 (8): 3835-3844. In other embodiments, an NDV genome engineered to express a co-stimulating signal agonist and / or an immune cell inhibiting signal antagonist, such as, for example, a T lymphocyte or an NK cell, does not comprise a sequence of NDV protein V coding. In certain embodiments, the parent structure of the chimeric NDV is a mesogenic velogen or the NDV strain that is engineered to express a mutated V protein, as described by Elankumaran et al. , 2010, J. Virol. 84 (8): 3835-3844.
[119] In another aspect, chimeric NDVs are described in this document, comprising a genome engineered to express an agonist of a co-stimulatory signal and / or an antagonist of an inhibitory signal from an immune cell, such as, for example, a T lymphocyte or an NK cell, and a cytokine. In a specific embodiment, an NDV genome is engineered to express an agonist of an immune cell co-stimulating signal, such as, for example, a T lymphocyte or an NK cell, and a cytokine. In a specific embodiment, an NDV genome is engineered to express an antagonist of an immune cell inhibitory signal, such as, for example, a T lymphocyte or an NK cell, and a cytokine. Specific examples of cytokines include, but are not limited to, interleukin (IL) -2, IL-7, IL-9, IL-15, IL-17, IL-21, IL-22, interferon (IFN) gamma, GM- CSF, and tumor necrosis factor (TNF-alpha).
[120] In another aspect, chimeric NDVs are described in this document, comprising a genome engineered to express an agonist of a co-stimulatory signal and / or an antagonist of an immune cell inhibitory signal, such as, for example, a T lymphocyte or an NK cell, a mutated F protein, and a cytokine (e.g., IL-2, IL-7, IL-9, IL-15, IL-17, IL-21, IL-22, IFN-gamma, GM- CSF and TNF-alpha). In a specific embodiment, the mutated F protein is highly fusogenic. In a specific embodiment, the mutated F protein has a mutant cleavage site (as described herein). In some embodiments, the mutated F protein comprises the mutation of the amino acid L289A. In some embodiments, the chimeric NDV is engineered to express an F protein mutated with the L289A amino acid mutation. In certain embodiments, the mutated F protein is of a different type or strain of NDV than the structure of NDV. In specific embodiments, the mutated F protein L289A has one, two or three arginine residues at the cleavage site. In some embodiments, the mutated F protein is in addition to the NDV F protein structure. In specific embodiments, the mutated F protein replaces the NDV F protein structure. In specific embodiments, the mutated F protein is incorporated into the virion.
[121] In certain respects, chimeric NDVs comprising a genome engineered to express a cytokine such as, for example, IL-7, IL-15, IL-21 or another cytokine described herein or known to a person skilled in the art are provided herein in art. See, for example, Section 7 for examples of chimeric NDVs engineered to express cytokines, as well as methods of producing such chimeric NDVs.
[122] In another aspect, chimeric NDVs comprising a genome engineered to express (i) an agonist of a co-stimulatory signal and / or an antagonist of an inhibitory signal from an immune cell, and (ii) a tumor antigen are described in this document. . In a specific embodiment, an NDV genome is manipulated to express an agonist of an immune cell co-stimulating signal, such as, for example, a T lymphocyte or an NK cell, and a tumor antigen. In a specific embodiment, an NDV genome is engineered to express an antagonist of an immune cell inhibitory signal, such as, for example, a T lymphocyte or an NK cell, and a tumor antigen.
[123] Tumor antigens include tumor-associated antigens and tumor-specific antigens. Specific examples of tumor antigens include, but are not limited to, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, N-acetylglucosaminyltransferase V, p-15, gplOO, MART-l / MelanA, TRP- 1 (gp75), tyrosinase, cyclin kinase 4 dependent, β-catenin, MUM-1, CDK4, HER-2 / neu, human papillomavirus E6, human papillomavirus E7, CD20, carcinoembryonic antigen (CEA), growth factor receptor epidermal, MUC-1, caspase-8, CD5, mucin-1, Lewisx, CA-125, pl85HER2, IL-2R, Fap a, tenascin, antigens associated with a metalloproteinase, and CAMPATH-1. Other examples include, but are not limited to, KS 1/4 pan-carcinoma antigen, ovarian carcinoma antigen (CA125), prostate acid phosphate, prostate specific antigen, p97 melanoma antigen, gp75 melanoma antigen, melanoma antigen high molecular weight (HMW-MAA), specific prostate membrane antigen, CEA, polymorphic epithelial mucin antigen, milk fat globule antigen, antigens associated with colorectal tumors (such as: CEA, TAG-72, CO17- 1A, GICA 19-9, CTA-1 and LEA), Burkitt's lymphoma antigen 38.13, CD20, CD33 B lymphoma antigen, melanoma-specific antigens (such as GD2 ganglioside, GD3 ganglioside, GM2 ganglioside, GM3 ganglioside), antigen tumor-specific cell surface (TSTA) (such as virus-induced tumor antigens, including T-DNA antigen from tumor viruses and envelope antigens from RNA tumor virus), oncofetal-alpha antigen fetoprotein, such as, Colon CEA, oncofetal bladder tumor antigen, differentiation antigen (such as L6 and L20 human lung carcinoma antigen), fibrosarcoma antigens, GP37 T cell leukemia antigen, neoglycoprotein, sphingolipids, breast cancer antigens (as EGFR (epidermal growth factor receptor), HER2 antigen (pl85.sup.HER2) and HER2 neu epitope) polymorphic, epithelial mucin (PEM), malignant human lymphocyte antigen APO-1, differentiation antigen (such as, antigen I found in fetal erythrocytes, primary endoderm, antigen I found in adult erythrocytes, preimplantation embryos, I (Ma) found in gastric adenocarcinomas, M18, M39 found in mammary epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9, Myl, VIM-D5, D.sub.156-22 found in colorectal cancer, TRA-1-85 (blood group H), C14 found in colon adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in gastric cancer, hapten Y, Le.sup.y found in embryonic carcinoma cells, TL5 (blood group A), EGF receptor found in A431 cells, El series (blood group B) found in pancreatic cancer, FC10.2 found in embryonic carcinoma cells, gastric adenocarcinoma antigen, CO-514 (Lea blood group) found in adenocarcinoma, NS-10 found in adenocarcinomas, CO-43 (Leb blood group), G49 found in the A431 cell EGF receptor, MH2 (ALeb / Ley blood group) found in colon adenocarcinoma, 19.9 found in colon cancer, gastric cancer mucins, T5A7 found in myeloid cells, R24 found in melanoma, 4.2, GD3, Dl.l, OFA-1, GM2, OFA-2, GD2, and Ml: 22: 25: 8 found in embryonic carcinoma cells, and SSEA-3 and SSEA-4 found in 4 to 8 cells in embryonic stage), peptide T cell receptor derived from cutaneous T cell lymphoma, protein C- reactive (CRP), cancer antigen 50 (CA-50), cancer antigen 15-3 (CA15-3) assoc with breast cancer, cancer antigen 19 (CA-19) and cancer antigen 242 associated with gastrointestinal cancers, antigen associated with carcinoma (CEA), chromogranin A, epithelial mucin antigen (MC5), epithelium specific antigen human (EIA), Lewis antigen (a), melanoma antigen, antigens associated with 100, 25, and 150, antigen associated with mucin-like carcinoma, proteins related to multiple drug resistance (MRPm6), proteins related to resistance to multiple drugs (MRP41), Neu oncogene protein (C-erbB-2), specific neuronal enolase (NSE), P-glycoprotein (product of the mdr 1 gene), antigen related to multiple drug resistance, pl70, antigen related to resistance to multiple drugs, prostate specific antigen (PSA), CD56, and MACN.
[124] In another aspect, chimeric NDVs comprising a genome engineered to express an agonist of a co-stimulatory signal and / or an antagonist of an inhibitory signal from an immune cell, such as, for example, a T lymphocyte or a NK cell, a mutated F protein, and a tumor antigen. In a specific embodiment, the mutated F protein is highly fusogenic. In a specific embodiment, the mutated F protein has a mutant cleavage site (as described herein). In some embodiments, the mutated F protein comprises the mutation of the amino acid L289A. In some embodiments, the chimeric NDV is engineered to express an F protein mutated with the L289A amino acid mutation. In certain embodiments, the mutated F protein is of a different type or strain of NDV than the structure of NDV. In specific modalities of the mutated F protein L289A it has one, two or three arginine residues at the cleavage site. In some embodiments, the mutated F protein is in addition to the NDV F protein structure. In specific embodiments, the mutated F protein replaces the NDV F protein structure. In specific embodiments, the mutated F protein is incorporated into the virion.
[125] In another aspect, chimeric NDVs are described in this document, comprising a genome engineered to express (i) an agonist of a signal co-stimulator and / or an antagonist of an inhibitory signal of an immune cell, and (ii) an antagonist of heterologous interferon. In a specific embodiment, an NDV genome is engineered to express an agonist of an immune cell co-stimulating signal, such as, for example, a T lymphocyte or an NK cell, and a heterologous interferon antagonist. In a specific embodiment, an NDV genome is engineered to express an antagonist of an immune cell inhibitory signal, such as, for example, a T lymphocyte or an NK cell, and a heterologous interferon antagonist. See, for example, US Patent Application Publication No. 2012-0058141, which is incorporated herein by reference, for examples of chimeric NDV engineered to express heterologous interferon antagonists.
[126] Interferon antagonists can be identified using any technique known to a person skilled in the art, including, for example, the techniques described in US patents 6,635,416; US 7,060,430; and US 7,442,527; which are hereby incorporated by reference in their entirety. In a specific embodiment, the heterologous interferon antagonist is a viral protein. Such viral proteins can be obtained from or derived from any virus and the virus can infect any species (for example, the virus can infect humans or non-human mammals). Exemplary heterologous interferon antagonists include, but are not limited to, Nipah W virus protein, Nipah V protein, Ebola virus VP35 protein, vaccinia virus E3L protein, influenza virus NS1 protein, respiratory syncytial virus (RSV) protein, protein ICP34.5 of herpes simplex virus (HSV) type 1, protease hepatitis C virus NS3-4, dominant negative cellular proteins that block response induction or innate immunity (for example, STAT1, MyD88, IKK and TBK ), and cellular regulators of the innate immune response (for example, SOCS proteins, Pias proteins, CYLD proteins, IkB protein, Atg5 protein, Pinl protein, IRAK-M protein, and UBP43). See, for example, the publication of the American patent application No. 2012-0058141, which is incorporated herein by reference in its entirety, for additional information on heterologous interferon antagonists.
[127] In another aspect, chimeric NDVs are described in this document, comprising a genome engineered to express an agonist of a co-stimulatory signal and / or an antagonist of an immune cell inhibitory signal, such as, for example, a T lymphocyte or an NK cell, a mutated F protein, and a heterologous interferon antagonist. In a specific embodiment, the mutated F protein is highly fusogenic. In a specific embodiment, the mutated F protein has a mutant dividing site (as described herein). In some embodiments, the mutated F protein comprises the mutation of the amino acid L289A. In some embodiments, the chimeric NDV is engineered to express an F protein mutated with the L289A amino acid mutation. In certain embodiments, the mutated F protein is of a different type or strain of NDV than the structure of NDV. In specific modalities of the mutated F protein L289A it has one, two or three arginine residues at the cleavage site. In some embodiments, the mutated F protein is in addition to the NDV F protein structure. In specific embodiments, the mutated F protein replaces the NDV F protein structure. In specific embodiments, the mutated F protein is incorporated into the virion.
[128] In another aspect, chimeric NDVs comprising a genome engineered to express an agonist of a co-stimulatory signal and / or an antagonist of an inhibitory signal from an immune cell, such as, for example, a T lymphocyte or a NK cell, and a pro-apoptotic molecule. In a specific embodiment, an NDV genome is manipulated to express an agonist of an immune cell co-stimulating signal, such as, for example, a T lymphocyte or an NK cell, and a pro-apoptotic molecule. In a specific embodiment, an NDV genome is engineered to express an antagonist of an immune cell inhibitory signal, such as, for example, a T lymphocyte or an NK cell, and a pro-apoptotic molecule. Specific examples of propoptotic molecules include, but are not limited to Bax, Bak, Bad, BID, Bcl-xS, Bim, Noxas, Puma, FIA, FasL, and TRAIL.
[129] In another aspect, chimeric NDVs are described in this document, comprising a genome engineered to express an agonist of a co-stimulatory signal and / or an antagonist of an immune cell inhibitory signal, such as, for example, a T lymphocyte or an NK cell, a mutated F protein, and a pro-apoptotic molecule. In a specific embodiment, the mutated F protein is highly fusogenic. In a specific embodiment, the mutated F protein has a mutant cleavage site (as described herein). In some embodiments, the mutated F protein comprises the mutation of the amino acid L289A. In some embodiments, the chimeric NDV is engineered to express an F protein mutated with the L289A amino acid mutation. In certain embodiments, the mutated F protein is of a different type or strain of NDV than the structure of NDV. In specific modalities of the mutated F protein L289A it has one, two or three arginine residues at the cleavage site. In some embodiments, the mutated F protein is in addition to the NDV F protein structure. In specific embodiments, the mutated F protein replaces the NDV F protein structure. In specific embodiments, the mutated F protein is incorporated into the virion.
[130] In another aspect, chimeric NDVs are described in this document, comprising a genome engineered to express an agonist of a co-stimulatory signal and / or an antagonist of an immune cell inhibitory signal, such as, for example, a T lymphocyte or an NK cell, and an anti-apoptotic molecule. In a specific embodiment, an NDV genome is manipulated to express an agonist of an immune cell co-stimulating signal, such as, for example, a T lymphocyte or an NK cell, and an anti-apoptotic molecule. In a specific embodiment, an NDV genome is manipulated to express an antagonist of an immune cell inhibitory signal, such as, for example, a T lymphocyte or an NK cell, and an anti-apoptotic molecule. Specific examples of anti-apoptotic molecules include, but are not limited to, Bcl-2, Bcl-xL, Mcl-1, and XIAP.
[131] In another aspect, chimeric NDVs comprising a genome engineered to express an agonist of a co-stimulatory signal and / or an antagonist of an inhibitory signal from an immune cell, such as, for example, a T lymphocyte or a NK cell, a mutated F protein, and an anti-apoptotic molecule. In a specific embodiment, the mutated F protein is highly fusogenic. In a specific embodiment, the mutated F protein has a mutant cleavage site (as described herein). In some embodiments, the mutated F protein comprises the mutation of the amino acid L289A. In some embodiments, the chimeric NDV is engineered to express an F protein mutated with the L289A amino acid mutation. In certain embodiments, the mutated F protein is of a different type or strain of NDV than the structure of NDV. In specific modalities of the mutated F protein L289A it has one, two or three arginine residues at the cleavage site. In some embodiments, the mutated F protein is in addition to the NDV F protein structure. In specific embodiments, the mutated F protein replaces the NDV F protein structure. In specific embodiments, the mutated F protein is incorporated into the virion.
[132] In certain respects, provided herein are chimeric NDVs comprising a genome engineered to express a pro-apoptotic molecule. In certain respects, provided herein are chimeric NDVs comprising a genome engineered to express an anti-apoptotic molecule. Examples of pro-apoptotic molecules and anti-apoptotic molecules are provided herein.
[133] Any type or strain of NDV can be used as a structure that is manipulated to express an agonist of an immune cell co-stimulating signal and / or an antagonist of an immune cell inhibiting signal, such as, for example, a T lymphocyte or an NK cell, and in certain embodiments, is modified to express a cytokine, tumor antigen, heterologous interferon antagonists, pro-apoptotic molecule, anti-apoptotic molecule and / or mutated F protein, including, but not limited to limiting mutagenized recombinant viruses, and / or genetically modified viruses to naturally occurring strains, variants or mutants. In a specific modality, the NDV used in a combination therapy presented here is a naturally occurring strain. In certain embodiments, the NDV that serves as the genetic engineering framework is a lytic strain. In other modalities, the NDV that serves as the genetic engineering framework is a non-lytic strain. In certain modalities, the NDV that serves as the basis for genetic engineering is the lentogenic strain. In some modalities, the NDV that serves as the basis for genetic engineering is the mesogenic strain. In other modalities, the NDV that serves as the genetic engineering framework is a velogenic strain. Specific examples of VDN strains include, but are not limited to, strain 73-T, strain Ulster, strain MTH-68, strain Italien, strain Hickman, strain PV701, strain Hitchner Bl, strain La Sota, strain YG97, strain MET95 and strain F48E9. In a specific modality, the VDN that serves as a framework for genetic engineering is the Bl Hitchner strain. In another specific modality, the VDN that serves as a framework for genetic engineering is the La Sota strain.
[134] In certain embodiments, the attenuation, or further attenuation, of chimeric NDV is desired so that the chimeric NDV remains, at least partially, infectious and can replicate in vivo, but only generate low titers, resulting in subclinical levels of infection that are non-pathogenic (see, for example, Khattar et al, 2009, J. Virol 83: 7779-7782.). In a specific embodiment, NDV is attenuated by deletion of protein V, such attenuated chimeric NDVs may be especially suitable for modalities in which the virus is administered to a subject, in order to act as an immunogen, for example, a live vaccine. Viruses can be mitigated by any method known in the art.
[135] In certain embodiments, a chimeric NDV described herein, expresses one, two, three, or more, or all of the following, and a suicidal gene: (1) an agonist of a co-stimulating signal from an immune cell; (2) an immune cell inhibitory signal antagonist; (3) a cytokine; (4) a tumor antigen; (5) a heterologous interferon antagonist; (6) a pro-apoptotic molecule; (7) an anti-apoptotic molecule; and / or (8) a mutated F protein. In specific modalities, in addition to expressing an agonist of an immune cell co-stimulating signal and / or an antagonist of an immune cell inhibiting signal, such as, for example, a T lymphocyte or an NK cell, and in certain modalities, a mutated F protein and a cytokine, a chimeric NDV is engineered to express a suicide gene (eg, thymidine kinase) or another molecule that inhibits NDV replication or function (a gene that makes NDV sensitive to an antibiotic or an anti-viral agent). In some embodiments, in addition to expressing an immune cell co-stimulating signal agonist and / or an immune cell inhibiting signal antagonist, such as, for example, a T lymphocyte or an NK cell, and in certain embodiments, a mutated F protein and a cytokine, a chimeric NDV is engineered to encode tissue-specific microRNA (miRNA) target sites (e.g., sites targeted by miR-21, miR-184, miR-133a / 133b, miR-137, and / or miR-193a microRNAs).
[136] In certain embodiments, the tropism of the chimeric NDV is altered. In a specific embodiment, the tropism of the virus is altered by a modification of the protein F cleavage site to be recognized by specific proteases for specific tissues or tumors, such as matrix metalloproteases (MMP) and urokinase. In other modalities, the tropism of the virus is altered by the introduction of tissue-specific miRNA target sites. In certain embodiments, the NDV NH protein is mutated to recognize the specific tumor receptor.
[137] In certain embodiments, one or more of the following procedures are expressed by a chimeric NDV, such as a chimeric fusion protein or protein: (1) an agonist of an immune cell co-stimulating signal; (2) an immune cell inhibitory signal antagonist; (3) a cytokine; (4) a tumor antigen; (5) a heterologous interferon antagonist; (6) a propoptotic molecule; (7) an anti-apoptotic molecule; and / or (8) a mutated F protein. In specific embodiments, the chimeric fusion protein or protein comprises the transmembrane and cytoplasmic domains or fragments of the NDV F or NDV HN protein and an extracellular domain comprising one of the molecules referred to above. See US patent application No. 2012-0122185 for a description of such chimeric proteins or fusion proteins, and the publication of international application WO 2007/064802, which are incorporated herein by reference.
[138] In embodiments of this invention, the co-stimulatory signal agonist and / or the immune cell inhibitory signal antagonist can be inserted into the genome of the NDV structure between two transcription units. In a specific embodiment, the co-stimulating signal agonist and / or the inhibitory signal antagonist of an immune cell is inserted into the genome of the NDV structure between the M and P transcription units or between the HN and L. transcription units. according to other modalities here, the cytokine, tumor antigen, heterologous interferon antagonists, pro-apoptotic molecule, anti-apoptotic molecule and / or mutated F protein are inserted into the genome of the NDV structure between two or more transcription units (for example, between transcription units M and P or between transcription units HN and L). 5.2.1 IMMUNE CELL STIMULATING AGENTS
[139] The chimeric NDVs described herein can be modified to express any co-stimulating signal agonist and / or any immune cell inhibitory signal antagonist, such as, for example, a T lymphocyte, NK cells or cells that exhibit the antigen (for example, a dendritic cell or macrophage), known to a person skilled in the art. In specific embodiments, the agonist and / or antagonist is an agonist of a human co-stimulating signal from an immune cell and / or an antagonist of a human inhibitory signal from an immune cell. In certain embodiments, the co-stimulating signal agonist is an agonist of a co-stimulating molecule (for example, co-stimulating the receptor) found in immune cells, such as, for example, T lymphocytes (for example, CD4 + or CD8 + T lymphocytes) , NK cells and / or antigen presenting cells (for example, dendritic cells or macrophages). Specific examples of co-stimulatory molecules include the glucocorticoid-induced tumor necrosis factor receptor (GITR), inducible T cell co-stimulator (ICOS or CD278), 0X40 (CD134), CD27, CD28, 4-1BB (CD137), CD40, lymphotoxin alpha (alf LT), LIGHT (similar to lymphotoxinae, exhibits inducible expression, and competes with the herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes), CD226, regulatory and cytotoxic T cell molecule (CRTAM ), exterminator receptor 3 (DR3), lymphotoxin-beta receptor (LTBR), transmembrane activator and CAML interactor (TACI), B cell activation factor receptor (BAFFR), and B cell maturation protein (BCMA) . In specific embodiments, the agonist is an agonist for a human co-stimulating receptor on an immune cell. In certain embodiments, the co-stimulating receptor agonist is not an ICOS agonist. In some embodiments, the antagonist is an antagonist of an inhibitory molecule (eg, inhibitor receptor) found in immune cells, such as, for example, T lymphocytes (eg, CD4 + or CD8 + T lymphocytes), NK cells and / or antigen the cells they present (for example, dendritic cells or macrophages). Specific examples of inhibitory molecules include, cytotoxic T lymphocytes associated with antigen 4 (CTLA-4 or CD52), programmed cell death protein 1 (PD-1), B and T lymphocyte attenuator (BTLA), receptors similar to exterminating cell immunoglobulin (KIR), lymphocyte activation gene 3 (LAG3), or protein 3 T cell membrane (TIM3), CD160, A2a adenosine receptor (A2aR), T cell immunoreceptor with immunoglobulin and ITIM domains ( TIGIT), an immunoglobulin-like receptor associated with a leukocyte (LAIR1), and CD160. In specific embodiments, the antagonist is an antagonist of a human inhibitory receptor on an immune cell.
[140] In a specific embodiment, a co-stimulatory receptor agonist is an antibody or antigen-binding fragment that specifically binds to the co-stimulatory receptor. Specific examples of costimulatory receptors include GITR, ICOS, 0X40, CD27, CD28, 4-1BB, CD40, LT alpha, LIGHT, CD226, CRTAM, DR3, LTBR, TACI, BAFFR, and BCMA. In certain specific embodiments, the antibody is a monoclonal antibody. In other specific embodiments, the antibody is a sc-Fv. In a specific embodiment, the antibody is a bispecific antibody that binds to two receptors on an immune cell. In other embodiments, the bispecific antibody binds to a receptor in an immune cell and to another receptor in a cancer cell. In specific embodiments, the antibody is a human or humanized antibody. In some embodiments, the antibody is expressed as a chimeric protein with NDV F protein or its fragment, or NDV HN protein or its fragment. See, for example, the publication of the American patent application No. 2012/0122185, which is incorporated herein by reference for a description on generation of chimeric F or chimeric HN proteins. In a specific embodiment, the chimeric protein is the chimeric F protein described in Sections 6 and / or 7, below. The techniques described below for generating the chimeric ICOSL-F protein and the chimeric CD28-F protein can be used to produce other chimeric F proteins or chimeric HN proteins.
[141] In another embodiment, the co-stimulatory receptor agonist is a co-stimulatory receptor ligand. In certain embodiments, the ligand is a fragment of native ligand. Specific examples of native ligands include ICOSL, B7RP1, CD137L, OX40L, CD70, herpes virus entry mediator (HVEM), CD80 and CD86. The nucleotide sequences encoding the native ligands, as well as the amino acid sequences of the native ligands are known in the art. For example, the B7RP1 nucleotide and amino acid sequences (also known as ICOSL; human GenBank: NM_015259.4, NP_056074.1 murine: NM_015790.3, NP_056605.1), CD137L (human GenBank: NM_003811.3, NP_003802 .1, murine: NM_009404.3, NP_033430.1), OX40L (human GenBank: NM_003326.3, NP_003317.1, murine: NM_009452.2, NP_033478.1), CD70 (human GenBank: NM_001252.3, NP_001243.1 , murine: NM_011617.2, AAD00274.1), CD80 (human GenBank: NM_005191.3, NP_005182.1, murine: NM_009855.2, NP_033985.3), and CD86 (human GenBank: NM_005191.3, CAG46642.1, murine: NM_019388.3, NP_062261.3) can be found on GenBank. In other embodiments, the ligand is a derivative of a native ligand. In some embodiments, the linker is a fusion protein that comprises at least a portion of the native linker or a derivative of the native linker that specifically binds to the co-stimulatory receptor, and a heterologous amino acid sequence. In specific embodiments, the fusion protein comprises at least a portion of the native ligand or a derivative of the native ligand that specifically binds to the co-stimulatory receptor, and the Fe portion of an immunoglobulin or its fragment. An example of a fusion linker protein is a 4-1BB linker fused to the Fc portion of the immunoglobulin (described by Meseck M et al., J Immunother. 2011 34: 175-82) In some embodiments, the linker is expressed as a protein chimeric with NDV F protein or fragment, or NDV HN protein or fragment. In a specific embodiment, the protein is the chimeric HN protein described in Section 7, below. The techniques described below for generating the chimeric HN-GITRL, chimeric HN-OX40-L, chimeric HN-4-1BBL, chimeric and / or HN-CD40L can be used to produce other chimeric F proteins or chimeric HN proteins.
[142] In another embodiment, an inhibitor receptor antagonist is an antibody (or its antigen binding fragment) or a soluble receptor that specifically binds to the native ligand for the inhibitory receptor and blocks the native ligand from binding to the receptor and transduce an inhibitory signal. Specific examples of native ligands for inhibitory receptors include PDL-1, PDL-2, B7-H3, B7-H4, HVEM, Gal9 and adenosine. Specific examples of inhibitory receptors that bind to a native ligand include CTLA-4, DP-1, BTLA, KIR, LAG3, TIM3, and A2aR.
[143] In specific embodiments, an inhibitor receptor antagonist is a soluble receptor that specifically binds to the native ligand for the inhibitory receptor and blocks the native ligand from binding to the inhibitory receptor and transduces an inhibitory signal or signals. In certain embodiments, the soluble receptor is a fragment of a native inhibitory receptor or a fragment of a derivative of a native inhibitory receptor that specifically binds to the native ligand (for example, the extracellular domain of a native inhibitory receptor or a derivative of a inhibitory receptor). In some embodiments, the soluble receptor is a fusion protein comprising at least a portion of the native inhibitory receptor or a derivative of the native inhibitory receptor (e.g., the extracellular domain of the native inhibitor receptor or a derivative of the native inhibitory receptor), and a heterologous amino acid sequence. In specific embodiments, the fusion protein comprises at least a portion of the native inhibitory receptor or a derivative of the native inhibitory receptor, and the Fe portion of an immunoglobulin or its fragment. An example of a soluble receptor fusion protein is a LAG3-Ig fusion protein (described by Huard B et al., Eur J Immunol. 1995 25: 2718-21).
[144] In specific embodiments, an inhibitor receptor antagonist is an antibody (or its antigen binding fragment) that specifically binds to the native ligand for the inhibitory receptor and blocks the native ligand from binding to the inhibitory receptor and transduces a inhibitory signal. In certain specific embodiments, the antibody is a monoclonal antibody. In other specific embodiments, the antibody is a scFv. In particular embodiments, the antibody is a human or humanized antibody. A specific example of an antibody to the linker is the anti-PD-Ll inhibitor antibody (Iwai Y, et al. PNAS 2002; 99: 12293-12297).
[145] In another embodiment, the antagonist of an inhibitory receptor is an antibody (or its antigen-binding fragment) or a ligand that binds to the inhibitory receptor, but does not transduce an inhibitory signal. Specific examples of inhibitory receptors include CTLA-4, PD1, BTLA, KIR, LAG3, TIM3, and A2aR. In certain specific embodiments, the antibody is a monoclonal antibody. In other specific embodiments, the antibody is a scFv. In particular embodiments, the antibody is a human or humanized antibody. A specific example of an antibody to the inhibitory receptor is anti-CTLA-4 (Leach DR, et al. Science 1996; 271: 1734-1736). Another example of an antibody to the inhibitory receptor is the anti-DP-1 antibody (Topalian, SL, NEJM 2012; 28: 3167-75).
[146] In certain embodiments, a chimeric NDV described herein is designed for a CTLA-4 antagonist, such as, for example, ipilimumab or tremelimumab. In certain embodiments, a chimeric NDV described herein is designed for a PD1 antagonist, such as, for example, MDX-1106 (BMS-936558), MK3475, CT-011, AMP-224, or MDX-1105. In certain embodiments, a chimeric NDV described herein is engineered to express an LAG3 antagonist, such as, for example, IMP321. In certain embodiments, a chimeric NDV described herein is engineered to express an antibody (for example, a monoclonal antibody or its antigen-binding fragment, or scFv) that binds to B7-H3, such as, for example, MGA271. In specific embodiments, a chimeric NDV described herein is engineered to express an immune cell co-stimulating signal agonist and / or an immune cell inhibitory signal antagonist described in Section 6 and / or Section 7, infra. In specific embodiments, NDV described here is designed to express anti-CD28 scvFv, ICOSL, CD40L, OX40L, CD137L, GITRL, and / or CD70.
[147] In certain embodiments, a co-stimulating signal agonist induces an immune cell (for example, selectively) induces one or more of the signal transduction pathways induced by the binding of a co-stimulating receptor to its ligand. In specific embodiments, a co-stimulating receptor agonist induces one or more of the signal transduction pathways induced by binding the co-stimulating receptor to one or more of its ligands by at least 25%, 30%, 40%, 50%, 60% , 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in the range between 25% to 50%, 25% to 75%, 50% to 75%, 50% to 95%, 75% to 95%, or 75% to 100% with respect to one or more signal transduction pathways induced by binding the co-stimulatory receptor to one or more of its ligands in the absence of the agonist. In specific embodiments, a co-stimulator receptor agonist: (i) induces one or more of the signal transduction pathways induced by binding the co-stimulator receptor to a particular ligand by at least 25%, 30%, 40%, 50% , 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in the range between 25% to 50%, 25% to 75%, 50% to 75%, 50% to 95%, 75% to 95%, or 75% to 100% with respect to one or more signal transduction pathways induced by binding of the co-stimulatory receptor to the specific ligand in the absence of the agonist; and (ii) does not induce or induce one or more of the signal transduction pathways induced by binding the co-stimulatory receptor to one or more of other ligands by at least 20%, 15%, 10%, 5%, or 2% , or in the range of 2% to 5%, 2% to 10%, 5% to 10%, 5% to 15%, 5% to 20%, 10% to 15%, or 15% to 20% in relation to one or more signal transduction pathways induced by binding the co-stimulatory receptor to such one or more other ligands in the absence of the agonist.
[148] In certain embodiments, an agonist of a co-stimulating signal from an active immune cell or improves (for example, selectively active or increases) one or more of the signal transduction pathways induced by the binding of a co-stimulating receptor to its ligand. In specific embodiments, an agonist of a co-stimulating receptor activates or enhances one or more of the signal transduction pathways induced by the binding of the co-stimulating receptor to one or more of its ligands by at least 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in the range between 25% to 50%, 25% to 75%, 50% and 75%, 50% to 95 %, 75% to 95%, or 75% to 100% with respect to one or more signal transduction pathways induced by binding the co-stimulatory receptor to one or more of its ligands in the absence of the agonist. In specific embodiments, a co-stimulatory receptor agonist: (i) a co-stimulatory signal agonist activates or improves one or more of the signal transduction pathways induced by binding the co-stimulatory receptor to a particular ligand by at least 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in the range between 25% to 50%, 25% to 75%, 50 % to 75%, 50% to 95%, 75% to 95%, or 75% to 100% in relation to one or more signal transduction pathways induced by the binding of the co-stimulatory receptor to the specific ligand in the absence of the agonist; and (ii) not activating or increasing, or activates or improves one or more of the signal transduction pathways induced by binding the co-stimulatory receptor to one or more other ligands by at least 20%, 15%, 10%, 5%, or 2%, or in the range of 2% to 5%, 2% to 10%, 5% to 10%, 5% to 15%, 5% to 20%, 10% to 15%, or 15% to 20% in in relation to one or more signal transduction pathways induced by binding the co-stimulatory receptor to such one or more other ligands in the absence of the agonist.
[149] In some embodiments, an antagonist of an immune cell inhibitory signal (for example, selectively) inhibits or reduces one or more of the signal transduction pathways induced by the binding of an inhibitory receptor to its ligand. In specific embodiments, an inhibitory receptor antagonist inhibits or reduces one or more of the signal transduction pathways induced by binding the inhibitory receptor to one or more of its ligands by at least 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in the range between 25% to 50%, 25% to 75%, 50% to 75%, 50% to 95 %, 75% to 95%, or 75% to 100% with respect to one or more signal transduction pathways induced by the binding of the inhibitory receptor to one or more of its ligands in the absence of the antagonist. In specific embodiments, an inhibitory receptor antagonist: (i) inhibits or reduces one or more of the signal transduction pathways induced by binding of the inhibitory receptor to a particular ligand by at least 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in the range between 25% to 50%, 25% to 75%, 50% and 75%, 50 % to 95%, 75% to 95%, or 75% to 100% with respect to one or more signal transduction pathways induced by binding of the inhibitory receptor to a particular ligand, in the absence of the antagonist; and (ii) not inhibiting or reducing, or inhibiting or reducing one or more of the signal transduction pathways induced by the binding of the inhibitory receptor to one or more of other ligands by at least 20%, 15%, 10%, 5 %, or 2%, or in the range of 2% to 5%, 2% to 10%, 5% to 10%, 5% to 15%, 5% to 20%, 10% to 15%, or 15% to 20% relative to one or more signal transduction pathways induced by inhibitory receptor binding such as one or more other ligands in the absence of the antagonist.
[150] In specific embodiments, an agonist of an immune cell co-stimulating signal and / or an antagonist of an immune cell inhibiting signal induces, activates and / or enhances one or more immune activities, responses or functions. The one or more immune activities, responses or functions can be in the form of, for example, an antibody response (humoral response) or a cellular immune response, for example, cytokine secretion (for example, interferon-gamma), the activity helper or cell cytotoxicity. In one embodiment, the expression of an immune cell activation marker (for example, CD44, granzyme, or Ki-67), the expression of a co-stimulating receptor on immune cells (for example, ICOS, CD28, 0X40, or CD27) , expression of a ligand for a co-stimulating receptor (for example, B7HRP1, CD80, CD86, OX40L, or CD70), secretion of cytokine, infiltration of immune cells (for example, T lymphocytes, B lymphocytes and / or NK cells) to an tumor, antibody production, effector function, T cell activation, T cell differentiation, T cell proliferation, B cell differentiation, B cell proliferation, and / or NK cell proliferation is induced, activated and / or improved following contact with an agonist of an immune cell co-stimulating signal and / or an antagonist of an immune cell inhibiting signal. In another embodiment, infiltration and proliferation of the tumor derived from myeloid suppressor cells (MDSC), tumor infiltration Treg, activation and proliferation, peripheral blood MDSC and Treg count are inhibited after contact with an agonist of an immune cell co-stimulating signal and / or an antagonist of an inhibitory signal from an immune cell. 5.3 CONSTRUCTION OF NDVs
[151] The NDVs described here can be generated using the reverse genetics technique. The technique of reverse genetics involves the preparation of synthetic recombinant viral RNAs that contain the non-coding regions of the negative stranded RNA, which are essential for recognition by viral polymerases and for the packaging signals necessary to generate a mature virion. Recombinant RNAs are synthesized from a recombinant DNA model and reconstituted in vitro with purified viral polymerase complex to form recombinant ribonucleoproteins (RNPs) that can be used to transfect cells. More effective transfection is achieved if viral polymerase proteins are present during the transcription of synthetic RNAs, either in vitro or in vivo. Synthetic recombinant RNPs can be rescued in infectious virus particles. The prior art is described in US patent 5,166,057 issued on November 24, 1992; US patent 5,854,037 issued December 29, 1998; in US patent 6,146,642 issued on 14 November 14, 2000; in the publication of the European patent application EP 0702085A1, published on February 20, 1996; US patent application serial number US 09 / 152,845; in PCT international publications WO97 / 12032 published on April 3, 1997; WO96 / 34625 published November 7, 1996; in the publication of the European patent application EP A780475; WO 99/02657 published January 21, 1999; WO 98/53078 published November 26, 1998; WO 98/02530 published on January 22, 1998; WO 99/15672 published on April 1, 1999; WO 98/13501 published on April 2, 1998; WO 97/06270 published on February 20, 1997; and EPO 780 475A1 published on June 25, 1997, each of which is incorporated herein by reference in its entirety.
[152] The helper-free plasmid technology can also be used to produce an NDV described herein. Briefly, a complete NDV cDNA (for example, the Hitchner Bl strain) is constructed, inserted into a plasmid vector and modified to contain a single restriction site between two transcription units (for example, the P and M NDV genes; or NDV HN and the L genes). A nucleotide sequence that encodes a heterologous amino acid sequence (for example, a nucleotide sequence that encodes a co-stimulatory signal agonist and / or an antagonist of an immune cell inhibitory signal) can be inserted into the viral genome at the single location restriction. Alternatively, a nucleotide sequence that encodes a heterologous amino acid sequence (for example, a nucleotide sequence that encodes a co-stimulatory signal agonist and / or an antagonist of an immune cell inhibitory signal) can be manipulated in a NDV transcription so that insertion does not affect the virus's ability to infect and replicate. The single segment is positioned between the T7 promoter and the hepatitis delta virus ribozyme to produce an exact negative T7 polymerase transcript. Plasmid and expression vectors comprising the necessary viral proteins are transfected into cells that lead to the production of recombinant viral particles (see, for example, International Publication No. WO 01/04333; US Patent Nos. 7,442,379, 6,146,642, 6,649,372 , 6,544,785 and 7,384,774; Swayne et al. (2003). Avian Dis. 47: 1047-1050; and Swayne et al. (2001). J. Virol. 11868-11873, each of which is incorporated herein by reference in its wholeness.
[153] Techniques for producing a chimeric NDV that express an antibody are known in the art. See, for example, Puhler et al., Gene Ther. 15 (5): 371-283 (2008) for the generation of a recombinant NDV that expresses a complete IgG from two transgenes.
[154] Bicistronic techniques for producing multiple proteins from a single mRNA are known to a person skilled in the art. Bicistronic techniques allow the engineering of multiple protein coding sequences into a single mRNA through the use of IRES sequences. The IRES sequences direct the internal recruitment of ribosomes to the RNA molecule and allow translation downstream independently of the helmet (cap). In short, a protein coding region is inserted into the open reading phase (ORF) of a second protein. The insert is flanked by an IRES and any untranslated signal sequences necessary for the appropriate expression and / or function. The insert should not degrade ORF, transcriptional or polyadenylation promoters of the second protein (see, for example, García-Sastre et al., 1994, J. Virol. 68: 6254-6261 and García-Sastre et al., 1994 Dev. Stand 82: 237-246, each of which is incorporated herein by reference in its entirety). 5.4 PROPAGATION OF NDVs
[155] The NDVs described here (for example, chimeric NDVs) can be propagated on any substrate that allows the virus to grow in titrations that allow the uses of the viruses described here. In one embodiment, the substrate allows the NDVs described here (for example, the chimeric NDVs) to grow in titrations comparable to the values determined for the corresponding wild-type viruses.
[156] The NDVs described here (for example, chimeric NDVs) can grow on cells (for example, poultry cells, chicken cells, etc.) that are susceptible to virus infection, embryonated eggs (for example, chicken or quail eggs) or animals (for example, birds). Such methods are well known to those skilled in the art. In a specific embodiment, the NDVs described here (for example, chimeric NDVs) can be propagated in cancer cells, for example, carcinoma cells (for example, breast cancer cells and prostate cancer cells), sarcoma cells , leukemia cells, lymphoma cells, and tumor cell germ cells (for example, testicular cancer cells and ovarian cancer cells). In another specific embodiment, the NDVs described herein (for example, the chimeric NDVs) can be propagated in cell lines, for example, cancer cell lines, such as HeLa cells, MCF-7 cells, THP-1 cells, U87 cells , DU145 cells, LNCaP cells, and T47D cells. In certain embodiments, cells or cell lines (for example, cancer cells or cancer cell lines) are obtained and / or derived from humans. In another embodiment, the NDVs described here (for example, chimeric NDVs) are propagated in chicken cells or embryonated eggs. Representative chicken cells include, but are not limited to, chicken embryo fibroblasts and chicken embryo kidney cells. In a specific embodiment, the NDVs described here (for example, chimeric NDVs) are propagated in Vero cells. In another specific embodiment, the NDVs described here (for example, chimeric NDVs) are propagated in cancer cells according to the methods described in Section 6 and / or Section 7, below. In another specific embodiment, the NDVs described here (for example, chimeric NDVs) are propagated in chicken eggs or quail eggs. In certain embodiments, an NDV virus described here (for example, a chimeric NDV) is first propagated in embryonated eggs and then propagated in cells (for example, a cell line).
[157] The NDVs described herein (for example, chimeric NDVs) can be propagated in embryonated eggs, for example, from 6 to 14 days old, 6 to 12 days old, 6 to 10 days old, 6 to 9 days old, 6 to 8 days old, or 10 to 12 days old. Immature or new embryonated eggs can be used to propagate the NDVs described here (for example, chimeric NDVs). Immature embryonated eggs include eggs that are eggs less than 10 days old, for example, eggs 6 to 9 days old or 6 to 8 days old that are deficient in IFN. Immature embryonated eggs also cover eggs that are artificially immature until, but less than 10 days old, as a result of changes in growing conditions, for example, changes in incubation temperatures; drug treatment; or any other alteration that results in an egg with a delayed development, in such a way that the IFN system is not fully developed, in comparison with eggs of 10-12 days of age. The NDVs described herein (for example, chimeric NDVs) can be propagated at different locations in the embryonated egg, for example, in the allantoic cavity. For a detailed discussion of the growth and spread of the virus, see, for example, US patent US 6,852,522 and US 7,494,808, both of which are incorporated herein by reference in their entirety.
[158] For virus isolation, the NDVs described herein (for example, chimeric NDVs) can be removed from cell culture and separated from cell components, typically through well-known clarification processes, for example, such as centrifugation in gradient and column chromatography, and can be further purified as desired using procedures well known to those skilled in the art, for example, plate assays. 5.5 COMPOSITIONS AND ROUTES OF ADMINISTRATION
[159] This document covers the use of an NDV described herein (for example, chimeric NDVs) in compositions. The use of plasma membrane fragments from infected NDV cells or whole cancer cells infected with NDV in compositions is also covered herein. In a specific embodiment, the compositions are pharmaceutical compositions, such as immunogenic formulations (for example, vaccine formulations). The compositions can be used in cancer treatment methods.
[160] In one embodiment, a pharmaceutical composition comprises an NDV described herein (for example, chimeric NDVs), in a mixture with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic or prophylactic agents, as described in Section 5.6.4, below. In a specific embodiment, a pharmaceutical composition comprising an effective amount of an NDV described herein (for example, the chimeric NDVs), and, optionally, one or more additional prophylactic therapeutic agents, in a pharmaceutically acceptable carrier. In some embodiments, NDV (for example, a chimeric NDV) is the only active ingredient included in the pharmaceutical composition.
[161] In another embodiment, a pharmaceutical composition (for example, an anti-tumor vaccine) comprises a protein concentrate or a preparation of plasma membrane fragments from NDV-infected cancer cells, in a mixture with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic or prophylactic agents, as described in Section 5.6.4, below. In another embodiment, a pharmaceutical composition (for example, a whole cell vaccine) comprises cancer cells infected with NDV, in a mixture with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic or prophylactic agents, as described in Section 5.6.4, below.
[162] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of a state or federal government or that is listed in the United States Pharmacopoeia or another pharmacopoeia generally recognized for use in animals, more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, glycol, water , ethanol and similar substances. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The formulation must adjust to the mode of administration.
[163] In a specific embodiment, pharmaceutical compositions are formulated to serve the intended route of administration for a subject. For example, the pharmaceutical composition can be formulated to suit parenteral, intravenous, intraarterial, intrapleural, inhalation, intraperitoneal, oral, intradermal, colorectal, intraperitoneal, intracranial, and intratumoral administration. In a specific embodiment, the pharmaceutical composition can be formulated for intravenous, intraarterial, oral, intraperitoneal, intranasal, intratracheal, intrapleural, intracranial, subcutaneous, intramuscular, topical, pulmonary, or intratumoral administration. 5.6 USE OF ANTI-CANCER AND OTHER USES
[164] In one aspect, a chimeric NDV described here (for example, a chimeric NDV described in Section 5.2, supra), can be used in the treatment of cancer. In one embodiment, methods for treating cancer are provided here, which comprise administering to a subject in need of it, a chimeric NDV described herein (for example, a chimeric NDV described in Section 5.2, supra), or its composition. In a specific embodiment, a method for treating cancer is provided herein, comprising administering to a subject in need thereof, an effective amount of a chimeric NDV described herein (for example, a chimeric NDV described in Section 5.2, supra) or its composition.
[165] In specific embodiments, a chimeric NDV engineered to express an agonist of an immune cell co-stimulating signal, or its composition is administered to a subject to treat cancer. In other specific embodiments, a chimeric NDV engineered to express an antagonist of an immune cell inhibitory signal, or its composition, is administered to a subject to treat cancer. In certain embodiments, a chimeric NDV engineered to express an agonist of an immune cell co-stimulating signal and a mutated F protein or composition thereof is administered to a subject to treat cancer. In certain embodiments, a chimeric NDV engineered to express an antagonist of an immune cell inhibitory signal and a mutated F protein or composition thereof is administered to a subject to treat cancer.
[166] A chimeric NDV (for example, a chimeric NDV described in Section 5.2, supra) described herein or its composition, an anti-tumor vaccine, or a whole cell cancer vaccine used in a method for treating cancer may be used like any line of therapy (for example, a first, second, third, fourth or fifth therapeutic line).
[167] In certain embodiments, a chimeric NDV described here (for example, a chimeric NDV described in Section 5.2, supra), is the only active ingredient administered to treat cancer. In specific embodiments, a chimeric NDV described here (for example, a chimeric NDV described in Section 5.2, supra), is the only active ingredient in a composition administered to treat cancer.
[168] Chimeric NDV (for example, a chimeric NDV described in Section 5.2, supra), or its composition can be administered locally or systemically to a subject. For example, chimeric NDV (eg, a chimeric NDV described in Section 5.2, supra), or its composition can be administered parenterally (eg, intravenously, intraarterially, or subcutaneously), intratumorally, intrapleurally, intranasally, intraperitoneally, intracranially, orally, rectally, by inhalation, intramuscularly, topically or intradermally, to a subject. In a specific modality, the chimeric NDV is administered through the hepatic artery, for example, by injection of the hepatic artery, which can be performed by interventional radiology or by placing an arterial infusion pump. In another specific modality, chimeric NDV is administered intraoperatively, by laparoscopy, or by endoscopy. In a specific modality, the intraperitoneal administration of the chimeric NDV is performed by direct injection, through the infusion catheter, or injection during laparoscopy.
[169] In certain embodiments, the methods described here include treating cancer for which there is no treatment. In some embodiments, a chimeric NDV described herein (for example, a chimeric NDV described in Section 5.2, supra), or its composition is administered to a subject to treat cancer as an alternative to other conventional therapies.
[170] In one embodiment, a method for the treatment of cancer is provided herein, comprising administering to a subject in need thereof, a chimeric NDV described herein (for example, a chimeric NDV described in Section 5.2, supra) or a composition of them and one or more additional therapies, as described in Section 5.6.4, below. In a particular embodiment, one or more therapies are administered to an individual in combination with a chimeric NDV described herein (for example, a chimeric NDV described in Section 5.2, supra), or its composition to treat cancer. In a specific modality, additional therapies are currently used, have been used or are known to be useful in the treatment of cancer. In another embodiment, a chimeric NDV described herein (for example, a chimeric NDV described in Section 5.2, supra), or its composition is administered to a subject in combination with supportive therapy, pain relief therapy, or other therapy that does not have a therapeutic effect on cancer. In a specific embodiment, the one or more additional therapies administered in combination with a chimeric NDV described herein (for example, a chimeric NDV described in Section 5.2, supra) is one or more of the therapies described in Section 5.6.4.1, infra. In certain embodiments, a chimeric NDV described herein (for example, a chimeric NDV described in Section 5.2, supra) and one or more additional therapies are administered in the same composition. In other embodiments, a chimeric NDV and one or more additional therapies are administered in different compositions.
[171] In certain embodiments, two, three or more NDVs (including one, two or more chimeric NDVs described herein, such as one, two, or more of the chimeric NDVs described in Section 5.2, supra) are administered to a subject to treat cancer. The second or more chimeric NDVs used according to methods described herein, which comprise the administration of two, three or more NDVs to a subject to treat cancer may be naturally occurring or modified chimeric NDVs or chimeric NDVs that have been manipulated to express the heterologous amino acid sequence (for example, a cytokine). The first and second chimeric NDVs can be part of the same pharmaceutical composition or different pharmaceutical compositions. In certain embodiments, the first chimeric NDV and the second chimeric NDV are administered by the same route of administration (for example, both are administered intravenously or intratumorally). In other embodiments, the first chimeric NDV and the second chimeric NDV are administered by different routes of administration (for example, one is administered intratumorally and the other is administered intravenously).
[172] In specific embodiments, a first chimeric NDV engineered to express an agonist of a co-stimulating signal from an immune cell is administered to a patient to treat cancer in combination with a second chimeric NDV engineered to express an antagonist of an inhibitory signal from an immune cell. In other specific embodiments, a first chimeric NDV engineered to express an agonist of an immune cell co-stimulating signal and / or an antagonist of an immune inhibitory signal is administered in combination with a second chimeric NDV engineered to express one, two or more of the following: a cytokine (eg, IL-2), a heterologous interferon antagonist, a tumor antigen, a pro-apoptotic molecule, and / or an anti-apoptotic molecule. In a specific embodiment, the first chimeric NDV, the second chimeric NDV, or both express a mutated F protein that increases the fusogenic activity of the chimeric NDV. In another specific embodiment, the first chimeric NDV, the second chimeric NDV, or both express an F protein mutated with a mutation at the cleavage site (as described herein).
[173] In specific embodiments, a first composition (for example, a pharmaceutical composition) containing a first chimeric NDV engineered to express an agonist of an immune cell co-stimulating signal is administered to a patient to treat cancer in combination with a second composition (e.g., a pharmaceutical composition), which comprises a second chimeric NDV engineered to express an antagonist of an inhibitory signal from an immune cell. In other specific embodiments, a first composition (e.g., a pharmaceutical composition) containing a first chimeric NDV engineered to express an immune cell co-stimulating signal agonist and / or an immune inhibitory signal antagonist is administered in combination with a second composition (for example, a pharmaceutical composition), comprising a second chimeric NDV engineered to express one, two or more of the following: a cytokine (for example, IL-2), a heterologous interferon antagonist, a tumor antigen , a pro-apoptotic molecule, and / or an anti-apoptotic molecule. In a specific embodiment, the first chimeric NDV, the second chimeric NDV, or both express a mutated F protein that increases the fusogenic activity of chimeric NDV. In another specific embodiment, the first chimeric NDV, the second chimeric NDV, or both express an F protein mutated with a mutation at the cleavage site (as described herein).
[174] In another aspect, an NDV described here (for example, an NDV described in Section 5.1, supra), can be used in combination with one or more therapies, such as described here in Section 5.6.4, below (for example, Section 5.6.4.1, infra), in the treatment of cancer. In one embodiment, methods for the treatment of cancer are provided herein, comprising administering to a subject in need of it, an NDV described here (for example, an NDV described in Section 5.1, supra), or its composition and one or more additional therapies, as described here in Section 5.6.4, below. (For example, Section 5.6.4.1). In a specific embodiment, a method for the treatment of cancer is provided herein, comprising administering to a subject in need thereof an effective amount of an NDV described herein (for example, an NDV described in Section 5.1, supra), or its composition and an effective amount of one or more therapies, as described in Section 5.6.4, below. (For example, Section 5.6.4.1). In certain embodiments, an NDV described here (for example, an NDV described in Section 5.1, supra) and one or more additional therapies, as described in Section 5.6.4, below (for example, Section 5.6.4.1), are administered under the same composition. In other embodiments, an NDV (for example, an NDV described in Section 5.1, supra) and one or more additional therapies are administered in different compositions.
[175] NDV used in combination with one or more other therapies can be administered systemically or locally. For example, NDV or its composition can be administered parenterally (for example, intravenously, intraarterially, or subcutaneously), intratumor, intrapleurally, intranasally, intraperitoneally, intracranially, orally, rectally, by inhalation, intramuscularly, topically or intradermally, to a subject. In a specific modality, NDV is administered through the hepatic artery, for example, by injection into the hepatic artery, which can be performed by interventional radiology or by placing an arterial infusion pump. In another specific modality, NDV is administered intraoperatively, by laparoscopy, or by endoscopy. In a specific modality, the intraperitoneal administration of NDV is performed by direct injection, through the infusion catheter, or injection during laparoscopy.
[176] An NDV (for example, an NDV described in Section 5.1, supra) described herein or its composition, an anti-tumor vaccine, or a whole cell cancer vaccine in combination with one or more therapies, such as described in Section 5.6 .4, infra, can be used as any therapy line (for example, a first, second, third and fourth or fifth therapy, line) for the treatment of cancer, in accordance with a method described herein.
[177] In another aspect, entire cancer cells infected with the chimeric NDV described here (for example, a chimeric NDV described in Section 5.2, supra) can be used to treat cancer. In a specific embodiment, a chimeric NDV described here (for example, a chimeric NDV described in Section 5.2, supra), can be brought into contact with a cancer cell or a population of cancer cells and the cancer cells or population of cancer cells infected can be administered to a subject to treat cancer. In one embodiment, cancer cells are subjected to gamma radiation prior to infection with a chimeric NDV described here (for example, a chimeric NDV described in Section 5.2, supra). In another embodiment, cancer cells are subjected to gamma radiation after infection with a chimeric NDV described here (for example, a chimeric NDV described in Section 5.2, supra). In a particular embodiment, the cancer cells are treated before being administered to a subject so that the cancer cells cannot multiply in the subject. In a specific embodiment, cancer cells cannot multiply in the subject and the virus cannot infect the subject. In one embodiment, cancer cells are subjected to gamma radiation prior to administration to the subject. In another embodiment, cancer cells are sonicated before being administered to a subject. In another embodiment, cancer cells are treated with mitomycin C before being administered to a subject. In another embodiment, cancer cells are treated by freezing and thawing before being administered to a subject. In another embodiment, cancer cells are treated with heat treatment before being administered to a subject. Cancer cells can be administered locally or systemically to a subject. For example, cancer cells can be administered parenterally (for example, intravenously or subcutaneously), intratumor, intranasally, orally, by inhalation, intrapleurally, topically or intradermally, to a subject. In a specific embodiment, cancer cells are administered intratumorally or through the skin (for example, intradermally) of a subject. Cancer cells can be used autologously or allogeneously. In a specific embodiment, the structure of the chimeric NDV is a non-lytic strain. Cancer cells can be administered to an individual alone or in combination with additional therapy. Cancer cells are preferably in a pharmaceutical composition. In certain embodiments, cancer cells are administered in combination with one or more therapies, as described in Section 5.6.4, below. In certain embodiments, cancer cells and one or more additional therapies are administered in the same composition. In other embodiments, cancer cells and one or more additional therapies are administered in different compositions.
[178] In another aspect, entire cancer cells infected with NDV described here (for example, an NDV described in Section 5.1, supra) can be used in combination with one or more additional therapies described here in Section 5.6.4, below in the treatment of cancer. In one embodiment, methods for the treatment of cancer are provided herein, comprising administering to a subject in need thereof, entire cancer cells infected with NDV described herein (for example, an NDV described in Section 5.1, supra), in combination with one or more additional therapies described here in Section 5.6.4, below. In a specific embodiment, a method for the treatment of cancer is provided herein, comprising administering to a subject in need thereof an effective amount of whole cancer cells infected with NDV described herein (for example, an NDV described in Section 5.1, supra) , in combination with an effective amount of one or more additional therapies described in Section 5.6.4, below. In certain embodiments, the entire cancer cells infected with NDV described here (for example, an NDV described in Section 5.1, supra) and one or more additional therapies described in Section 5.6.4, below, are administered in the same composition. In other embodiments, the entire cancer cells infected with NDV described here (for example, an NDV described in Section 5.1, supra) and one or more additional therapies are administered in different compositions.
[179] In another aspect, a plasma membrane preparation or protein concentrate from lysed cancer cells infected with a chimeric NDV (for example, a chimeric NDV described in Section 5.2, supra) can be used to treat cancer. In one embodiment, a plasma membrane preparation comprising fragments of cancer cells infected with the chimeric NDV described herein can be used to treat cancer. In another embodiment, a protein concentrate from cancer cells infected with chimeric NDV described herein can be used to treat cancer. Techniques known to a person skilled in the art can be used to produce the protein concentrate or prepare the plasma membrane. In a specific embodiment, a chimeric NDV described here (for example, a chimeric NDV described in Section 5.2, supra), can be brought into contact with a cancer cell or a population of cancer cells and the infected cancer cells or population of cancer cells can be ligated using techniques known to a person skilled in the art to obtain protein concentrate or plasma membrane fragments from NDV-infected cancer cells, and protein concentrate or plasma membrane fragments from NDV-infected cancer cells can be administered to a subject to treat cancer. Fragments of the plasma membrane or protein concentrate can be administered locally or systemically to a subject. For example, fragments of the plasma membrane or protein concentrate can be administered parenterally, intratumoral, intranasal, intrapleural, orally, by inhalation, topically or intradermally, to a subject. In a specific embodiment, such a preparation of the plasma membrane or protein concentrate is administered intratumorally or through the skin (for example, intradermally) of a subject. The cancer cells used to produce the protein concentrate or plasma membrane preparation can be autologous or allogeneic. In a specific embodiment, the structure of the chimeric NDV is a lytic strain. The preparation of the plasma membrane or protein concentrate can be administered to a subject alone or in combination with additional therapy. The preparation of the plasma membrane or protein concentrate is preferably in a pharmaceutical composition. In certain embodiments, the preparation of protein concentrate or plasma membrane is administered in combination with one or more therapies, such as those described in Section 5.6.4, infra (for example, Section 5.6.4.1) In certain embodiments, the preparation plasma membrane or protein concentrate and one or more additional therapies are administered in the same composition. In other embodiments, the protein concentrate or plasma membrane preparation and one or more additional therapies are administered in different compositions.
[180] In another aspect, a plasma membrane or protein concentrate preparation from lysed cancer cells infected with NDV (for example, an NDV described in Section 5.1, supra) can be used in combination with one or more therapies, such as described here in Section 5.6.4, infra (for example, Section 5.6.4.1), in the treatment of cancer. In one embodiment, methods for treating cancer are provided here, which comprise administering to a subject in need of it, a plasma membrane or protein concentrate preparation from lysed cancer cells infected with NDV one (e.g., one NDV described in Section 5.1, supra), in combination with one or more therapies, as described here in Section 5.6.4, below. (For example, Section 5.6.4.1). In a specific embodiment, a method for the treatment of cancer is provided here, comprising administering to a subject in need thereof, an effective amount of a plasma membrane preparation or protein concentrate from lysed cancer cells infected with NDV ( for example, an NDV described in Section 5.1, supra), in combination with an effective amount of one or more therapies, as described in Section 5.6.4, below. (For example, Section 5.6.4.1). In certain embodiments, the plasma membrane or protein concentrate preparation and one or more other therapies, as described in Section 5.6.4, infra, are administered in the same composition. In other embodiments, the protein concentrate or plasma membrane preparation and one or more additional therapies are administered in different compositions.
[181] In another aspect, the chimeric NDVs described herein (for example, a chimeric NDV described in Section 5.2, supra), can be used to produce antibodies that can be used in diagnostic immunoassays, passive immunotherapy, and the generation of antibodies anti-idiotypic. For example, a chimeric NDV described here (for example, a chimeric NDV described in Section 5.2, supra), can be administered to an individual (for example, a mouse, rat, pig, horse, donkey, bird or human) to generate antibodies that can then be isolated and used, for example, in diagnostic assays, passive immunotherapy and generation of anti-idiotypic antibodies. In certain embodiments, an NDV described here (for example, an NDV described in Section 5.1 or 5.2, supra), is administered to a muck (for example, a mouse, rat, pig, horse, donkey, bird, or human) in combination with one or more additional therapies, as described in Section 5.6.4, infra, for antibodies generated that can then be isolated and used, for example, in diagnostic assays, passive immunotherapy and generation of anti-idiotypic antibodies. The generated antibodies can be isolated by standard techniques known in the art (for example, immunoaffinity chromatography, centrifugation, precipitation, etc.) and used in diagnostic immunoassays, passive immunotherapy and generation of anti-idiotypic antibodies.
[182] In certain embodiments, antibodies isolated from subjects administered a chimeric NDV described herein (for example, a chimeric NDV described in Section 5.2, supra), or isolated from individuals administered an NDV described here (for example , an NDV described in Section 5.1, or 5.2, supra), in combination with one or more therapies, such as those described in Section 5.6.4, below, are used to evaluate the expression of NDV proteins, a heterologous peptide or protein expressed by a chimeric NDV, or both. Any immunoassay system known in the art can be used for this proposal, including, but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assays), sandwich immunoassays , precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays and immunoelectrophoresis assays, just to name a few. 5.6.1 PATIENT POPULATION
[183] In some embodiments, an NDV (for example, a chimeric NDV) described herein or its composition, an antitumor vaccine described here, or a whole cell vaccine described here, or a combination therapy described here is administered to an individual suffering from cancer. In other embodiments, an NDV (for example, a chimeric NDV) described herein or its composition, an antitumor vaccine described herein, or a whole cell vaccine described here, or a combination therapy described herein is administered to a subject with a predisposition or susceptible to cancer. In some embodiments, an NDV (for example, a chimeric NDV) or one of its compositions, an anti-tumor vaccine described herein, or a whole cell vaccine described here, or a combination therapy described herein is administered to a subject diagnosed with cancer . Specific examples of the types of cancer are described here. In one embodiment, the subject has metastatic cancer. In another modality, the subject has stage 1, stage 2, stage 3, or stage 4 of cancer. In another mode, the subject is in remission. In yet another modality, the subject has a recurrence of cancer.
[184] In certain embodiments, an NDV (for example, a chimeric NDV) or its composition, an NDV, or a whole cell vaccine described herein, or a combination therapy described here, is administered to a human being who is 0 6 months old, 6 to 12 months old, 6 to 18 months old, 18 to 36 months, 1 to 5 years, 5 to 10 years, 10 to 15 years, 15 to 20 years, 20 to 25 years, 25 to 30 years old, from 3 to 35 years old, 35 to 40 years old, 40 to 45 years old, 45 to 50 years old, 50 to 55 years old, 55 to 60 years old, 60 to 65 years old, 65 to 70 years old , 70 to 75 years, 75 to 80 years, 80 to 85 years, 85 to 90 years, 90 to 95 years of age or 95 to 100 years of age. In some embodiments, an NDV (for example, a chimeric NDV) or its composition, an anti-tumor vaccine described herein, or a whole cell vaccine described herein, or a combination therapy described herein is administered to a human infant. In other embodiments, an NDV (e.g., a chimeric NDV) or its composition, an anti-tumor vaccine described herein, or a whole cell vaccine described herein, or a combination therapy described herein is administered to a human child. In other embodiments, an NDV (e.g., a chimeric NDV) or its composition, an anti-tumor vaccine described herein, or a whole cell vaccine described herein, or a combination therapy described herein is administered to a human child. In other embodiments, an NDV (e.g., a chimeric NDV) or its composition, an anti-tumor vaccine described herein, or a whole cell vaccine described herein, or a combination therapy described herein, is administered to an adult human. In still other embodiments, an NDV (e.g., a chimeric NDV) or its composition, an anti-tumor vaccine described herein, or a whole cell vaccine described herein, or a combination therapy described herein is administered to an elderly human being.
[185] In certain embodiments, an NDV (for example, a chimeric NDV) or its composition, an anti-tumor vaccine described herein, or a whole cell vaccine described herein, or a combination therapy described herein, is administered to a subject with a immunocompromised state or in an immunodepressed state or at risk of being immunocompromised or immunosuppressed. In certain embodiments, an NDV (for example, a chimeric NDV) or its composition, an anti-tumor vaccine described herein, or a whole cell vaccine described herein, or a combination therapy described herein is administered to a subject who receives or recovers immunosuppressive therapy. In certain embodiments, an NDV (for example, a chimeric NDV) or its composition, an anti-tumor vaccine described herein, or a whole cell vaccine described herein, or a combination therapy described herein is administered to a subject who has or is in risk of getting cancer. In certain modalities, the subject has been, is or will be subjected to surgery, chemotherapy and / or radiation therapy. In certain modalities, the patient underwent surgery to remove the tumor or neoplasm. In specific embodiments, the patient is administered an NDV (for example, a chimeric NDV) or its composition, an anti-tumor vaccine described here, or a whole cell vaccine described here, or a combination therapy described here followed by surgery to remove a tumor or neoplasm. In another embodiment, the patient is administered an NDV (for example, a chimeric NDV) or its composition, an anti-tumor vaccine described here, or a whole cell vaccine described here, or a combination therapy described here before undergoing surgery to remove a tumor or neoplasm. In certain embodiments, an NDV (for example, a chimeric NDV) or its composition, an antitumor vaccine described herein, or a whole cell vaccine described herein, or a combination therapy described herein is administered to a subject who has had, has, or you will have a tissue transplant, organ transplant or transfusion.
[186] In some embodiments, an NDV (for example, a chimeric NDV) or its composition, an anti-tumor vaccine described here, or a whole cell vaccine described here, or a combination therapy described here is administered to a patient who showed refractory to other therapies other than chimeric NDV or its composition, anti-tumor vaccine, whole cells, or a combination therapy, but not that these therapies are. In a specific embodiment, an NDV (for example, a chimeric NDV) or its composition, an anti-tumor vaccine described here, or a whole cell vaccine described here, or a combination therapy described here is administered to a patient who has shown to be refractory chemotherapy. In one embodiment, a cancer that is refractory to therapy means that at least a significant part of the cancer cells have not died or their cell division has not been interrupted. The determination of whether cancer cells are refractory can be made in vivo or in vitro by any method known in the art to test the effect of a cancer cell therapy, using the meanings in the "refractory" art in such a context. In a given modality, a refractory patient is a patient refractory to standard therapy. In certain modalities, a cancer patient is refractory to therapy in which the tumor or neoplasm has not been significantly eradicated and / or the symptoms have not been significantly alleviated. The determination of whether a patient is refractory can be done in vivo or in vitro by any method known in the art to test the effectiveness of a cancer treatment, using meanings accepted in the art of "refractory" in such a context.
[187] In certain embodiments, the patient to be treated according to the methods described here is a patient already treated with antibiotics, antivirals, antifungals, or other biological therapy / anti-cancer therapy or immunotherapy. Among these patients are refractory patients, and patients who are too young for conventional therapies. In some embodiments, the subject to be administered an NDV (for example, a chimeric NDV), an antitumor vaccine described herein, or a whole cell vaccine described herein, or a combination therapy described herein did not receive therapy prior to administration of the chimeric NDV or composition, antitumor vaccine, or whole cell vaccine, or combination therapy.
[188] In some embodiments, an NDV (for example, a chimeric NDV) or its composition, an anti-tumor vaccine described here, or a whole cell vaccine described here, or a combination therapy described here is administered to a patient to prevent the appearance of cancer in a patient at risk of developing cancer. In some embodiments, the compounds are administered to patients who are susceptible to adverse reactions to conventional therapies.
[189] In some embodiments, the subject to be administered an NDV (for example, a chimeric NDV) or its composition, an antitumor vaccine described herein, or a whole cell vaccine described here, or a combination therapy described here that has not received previous therapy. In other embodiments, an NDV (for example, a chimeric NDV) or its composition, an anti-tumor vaccine described herein, or a whole cell vaccine described herein, or a combination therapy described herein is administered to a subject who has received treatment prior to administration of NDV (for example, a chimeric NDV) or composition, antitumor vaccine, whole cell vaccine, or combination therapy. In some embodiments, the subject administered an NDV (for example, a chimeric NDV) or its composition, an anti-tumor vaccine described here, or a whole cell vaccine described here, or a combination therapy described here experienced adverse side effects from a previous treatment or previous therapy, the administration was interrupted due to unacceptable levels of toxicity to the subject. 5.6.2 DOSAGE & FREQUENCY
[190] The amount of an NDV or its composition, an anti-tumor vaccine, or a whole cell vaccine that will be effective in treating cancer will depend on the nature of the cancer, the route of administration, the general state of health of the subject, etc. and it must be decided according to the judgment of a doctor. Conventional clinical techniques, such as in vitro assays, can optionally be used to help identify optimal dosage ranges. However, suitable dosage ranges for an NDV for administration are generally about 102.5 x 102, 103, 5 x 103, 104, 5 x 104, 105, 5 x 105, 106, 5 x 10 6, 107, 5 x 107, 108, 5 x 108, 1 x 109, 5 x 109, 1 x 10 10, 5 x 10 10, 1 x 10 11, 5 x 10 11or 1012 pfu, and more preferably about 104 to about 1012, 106 to 1012, 108 to 1012, 109 to 1012 or 109 to 1011, and can be administered to a subject once, twice, three, four or more times at intervals as often as needed. Dosage ranges for antitumor vaccines for administration can include 0.001 mg, 0.005 mg, 0.01 mg, 0.05 mg, 0.1 mg, 0.5 mg, 1.0 mg, 2.0 mg, 3.0 mg, 4.0 mg, 5.0 mg, 10.0 mg, 0.001 mg to 10.0 mg, 0.01 mg to 1.0 mg, 0.1 mg to 1 mg, and 0.1 mg to 5 , 0 mg, and can be administered to a subject once, twice, three or more times at intervals as many times as necessary. The dosage ranges of whole cell vaccines for administration may include 102, 5 x 102, 103, 5 x 103, 104, 5 x 104, 105, 5 x 105, 106, 5 x 10s, 107, 5 x 107, 108 , 5 x 108, 1 x 109, 5 x 109, 1 x 10 10, 5 x 10 10, 1 x 10 11, 5 x 10 11 or 10 12 cells, and can be administered to a subject once, two, three or more times at intervals as often as necessary. In certain embodiments, dosages similar to those currently used in clinical trials for NDV, antitumor vaccines or whole cell vaccines are administered to a subject. Effective doses can be extrapolated from the dose response curves derived from in vitro or animal model test systems.
[191] In certain embodiments, an NDV (for example, a chimeric NDV) or its composition is administered to a subject as a single dose followed by a second dose of 1 to 6 weeks, 1 to 5 weeks, 1 to 4 weeks, 1 to 3 weeks, 1 to 2 weeks later. According to these modalities, booster inoculations can be administered to the subject at intervals of 6 to 12 months after the second inoculation. In certain embodiments, an anti-tumor vaccine or a whole cell vaccine is administered to a patient as a single dose followed by a second dose of 1 to 6 weeks, 1 to 5 weeks, 1 to 4 weeks, 1 to 3 weeks, 1 to 2 weeks later.
[192] In certain embodiments, the administration of the same NDV (for example, chimeric NDV) or its composition, antitumor vaccine, or whole cell vaccine can be repeated and the administrations can be separated by at least 1 day, 2 days, 3 days, 5 days, 6 days, 7 days, 10 days, 14 days, 15 days, 21 days, 28 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least six months. In other modalities, the administration of the same NDV (for example, an NDV) or its composition, anti-tumor vaccine, or whole cell vaccine can be repeated and the administrations can be separated for 1 to 14 days, 1 to 7 days, 7 to 7 days. 14 days, 1 to 30 days, 15 to 30 days, 15 to 45 days, 15 to 75 days, 15 to 90 days, 1 to 3 months, 3 to 6 months, 3 to 12 months, or 6 to 12 months. In some embodiments, a first NDV (for example, a first chimeric NDV) or its composition is administered to a subject, followed by the administration of a second NDV (for example, a second chimeric NDV) or its composition. In certain embodiments, the first and second NDVs (for example, the first and second chimeric NDVs) or their compositions can be separated by at least 1 day, 2 days, 3 days, 5 days, 6 days, 7 days, 10 days, 14 days, 15 days, 21 days, 28 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least six months. In other embodiments, the first and second NDVs (for example, the first and second chimeric NDVs) or their compositions can be separated by 1 to 14 days, 1 to 7 days, 7 to 14 days, 1 to 30 days, 15 to 30 days, 15 to 45 days, 15 to 75 days, 15 to 90 days, 1 to 3 months, 3 to 6 months, 3 to 12 months, or 6 to 12 months.
[193] In certain embodiments, an NDV or its composition, or antitumor vaccine or whole cell vaccine is administered to a subject in combination with one or more therapies, such as a therapy described in Section 5.6.4, below. The dosage of the other one or more additional therapies will depend on several factors, including, for example, the therapy, the nature of the cancer, the route of administration, the general state of health of the subject, etc., and must be decided according to the a doctor's judgment. In specific embodiments, the dose of the other therapy and / or the frequency of administration of the therapy recommended for therapy for use as a single agent is used according to the methods disclosed herein. In other embodiments, the dose of the other therapy is a lower dose and / or less frequent administration of the therapy than recommended for therapy for use as a single agent is used according to the methods disclosed herein. Recommended doses for approved therapies can be found in Physician's Desk Reference.
[194] In certain embodiments, an NDV or its composition, or antitumor vaccine or whole cell vaccine is administered to a subject simultaneously with the administration of one or more additional therapies. In other embodiments, an NDV or its composition, or anti-tumor vaccine or whole cell vaccine is administered to a subject every 3 to 7 days, 1 to 6 weeks, 1 to 5 weeks, 1 to 4 weeks, 2 to 4 weeks, 1 to 3 weeks, or 1 to 2 weeks and one or more additional therapies (as described in Section 5.6.4, below) is administered every 3 to 7 days, 1 to 6 weeks, 1 to 5 weeks, 1 to 4 weeks, 1 to 3 weeks, or 1 to 2 weeks. In certain embodiments, an NDV or its composition, or anti-tumor vaccine or whole cell vaccine is administered to a subject every 1 to 2 weeks and one or more additional therapies (as described in Section 5.6.4, below) are administered to every 2 to 4 weeks. In some embodiments, an NDV or its composition, or anti-tumor vaccine or whole cell vaccine is administered to a subject each week and one or more additional therapies (as described in Section 5.6.4, below) are administered every 2 weeks. . 5.6.3 TYPES OF CANCER
[195] Specific examples of cancers that can be treated according to the methods described here include, but are not limited to: leukemias, such as, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, such as myeloblastic , promyelocytic, myelomonocytic, monocytic and leukemias erythroleukemia and myelodysplastic syndrome; chronic leukemias, such as, but not limited to, chronic myelocytic (granulocytic), chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas, such as, but not limited to, Hodgkin's disease, non-Hodgkin's disease; multiple myelomas, such as, but not limited to, latent multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, placancer cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas, such as, but not limited to, bone sarcomas, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, bone fibrosarcoma, chordoma, periosteal sarcoma, soft tissue sarcomas, angiosarcoma (hemangiosarcoma) fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemoma, rhabdomyosarcoma, synovial sarcoma; brain tumors, such as, but not limited to, glioma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, nonglial tumor, glioblastoma multiforme, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, linoblastoma; breast cancer, including, but not limited to, ductal carcinoma, adenocarcinoma, lobular (cancer cell), intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease and cancer inflammatory breast; adrenal cancer, such as, but not limited to, pheochromocytomas and adrenocortical carcinoma; thyroid cancer, such as, but not limited to, papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer, such as, but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumors; pituitary cancers, such as, but limited to Cushing's disease, tumors that secrete prolactin, acromegaly, diabetes insipidus; eye cancers, such as, but not limited to, eye melanoma such as iris melanoma, choroidal melanoma, and ciliary body melanoma and retinoblastoma; vaginal cancers, such as squamous cell carcinoma, adenocarcinoma and melanoma; vulvar cancer, such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma and Paget's disease; cervical cancers, such as, but not limited to, squamous cell carcinoma and adenocarcinoma; uterine cancers such as, but not limited to, endometrial carcinoma and uterine sarcoma; ovarian cancers, such as, but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancers such as, but not limited to, squamous cancer, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and small cell carcinoma (cancer cell); stomach cancers, such as, but not limited to adenocarcinoma, fungoid (polypoid), ulcerated, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers; liver cancers, such as, but not limited to, hepatocellular carcinoma and hepatoblastoma; gallbladder cancer, such as adenocarcinoma; cholangiocarcinomas, such as, but not limited to, papillary, nodular and diffuse; lung cancers, such as non-cancerous lung cancer, squamous cell carcinoma (squamous cell carcinoma), adenocarcinoma, large cell carcinoma and cancer cell lung cancer; testicular cancers, such as, but not limited to, germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonic carcinoma, teratoma carcinoma, choriocarcinoma (yolk sac tumor), prostate cancers, such as, but not limited to, prostatic intraepithelial neoplasms, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; cancers of the penis; oral cancers, such as, but not limited to, squamous cell carcinoma; basal cancers; cancers of the salivary glands, such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharyngeal cancers, such as, but not limited to, squamous cell and verrucous cancer; skin cancers, such as, but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, malignant melanoma lentigo, acrolentiginous melanoma; kidney cancers, such as, but not limited to, renal cell carcinoma, adenocarcinoma, hypernefroma, fibrosarcoma, transitional cell cancer (renal pelvis and ureter); Wilms' tumor; bladder cancers, such as, but not limited to, transitional cell carcinoma, squamous cell cancer, carcinosarcoma adenocarcinoma. In addition, cancers include myxosarcoma, osteosarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, carcinoma of the sweat glands, carcinoma and gland carcinoma, see Fishman et al., 1985, Medicine, 2d Ed., JB Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books USA, Inc., United States of America).
[196] In a specific embodiment, the chimeric NDVs described here or their compositions, an antitumor vaccine described here, the whole cell vaccine described here, or combination therapy described here are useful in the treatment of a variety of cancers and proliferative diseases abnormal including (but not limited to) the following: carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin; including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B cell lymphoma, T cell lymphoma, Burkitt's lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myeloid leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid cancer and teratocarcinoma.
[197] In some modalities, cancers associated with aberrations in apoptosis are treated according to the methods described here. Such cancers can include, but are not limited to, follicular lymphomas, carcinomas with p53 mutations, hormone-dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis, and myelodysplastic syndromes. In specific modalities, malignancy or dysproliferative changes (such as metaplasias and dysplasias) or hyperproliferative disorders of the skin, lung, liver, bone, brain, stomach, colon, breast, prostate, bladder, kidney, pancreas, ovary, and / or uterus are treated according to the methods described here. In other specific embodiments, a sarcoma or melanoma is treated in accordance with the methods described here.
[198] In a specific modality, the cancer to be treated according to the methods described here is leukemia, lymphoma or myeloma (for example, myeloma, multiple). Specific examples of leukemias and other blood-borne cancers that can be treated according to the methods described here include, but are not limited to "ALL" acute lymphoblastic leukemia, acute B-cell lymphoblastic leukemia, acute T-lymphoblastic leukemia, leukemia acute myeloblastic "AML", acute promyelocytic leukemia "APL", monoblastic acute leukemia, acute erythroleukemic leukemia, acute megakarioblastic leukemia, acute myelomonocytic leukemia, chronic non-lymphocytic leukemia, acute undifferentiated leukemia, "chronic myelocytic leukemia" ", and hair cell leukemia.
[199] Specific examples of lymphomas that can be treated according to the methods described here include, but are not limited to, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and polycythemia vera .
[200] In another embodiment, the cancer to be treated according to the methods described here is a solid tumor. Examples of solid tumors that can be treated according to the methods described here include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendo- teliosscoma, mesothelioma, sinus telco Ewing's disease, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer, throat cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatic cell carcinoma, hepatic carcinoma choriocarcinoma, seminoma, embryonic carcinoma, Wilms' tumor, cervical cancer, ut cancer erine, testicular cancer, cancerous cell lung carcinoma, bladder carcinoma, lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligendomaloma, oxygendomal, skin, melanoma, neuroblastoma, and retinoblastoma. In another embodiment, the cancer to be treated according to the methods described here is a metastatic one. In another embodiment, the cancer to be treated according to the methods described here is malignant.
[201] In a specific modality, the cancer to be treated according to the methods described here is a cancer that has a poor prognosis and / or has a poor response to conventional therapies, such as chemotherapy and radiation. In another specific embodiment, the cancer to be treated according to the methods described here is malignant melanoma, malignant glioma, renal cell carcinoma, pancreatic adenocarcinoma, malignant pleural mesothelioma, lung adenocarcinoma, small cell lung cancer, carcinoma squamous cell lung cancer, anaplastic thyroid cancer and squamous cell cancer of the neck or head. In another specific modality, the cancer to be treated according to the methods described here is a type of cancer described in Section 6 and / or Section 7, below. 5.64 ADDITIONAL THERAPIES
[202] Additional therapies that can be used in combination with an NDV described herein or its composition, an anti-tumor vaccine, or a whole cell vaccine for the treatment of cancer include, but are not limited to small molecules, synthetic drugs, peptides ( including cyclic peptides), polypeptides, proteins, nucleic acids (for example, DNA and RNA nucleotides, including, but not limited to antisense nucleotide sequences, triple helices, RNAi, and nucleotide sequences encoding biologically active proteins, polypeptides or peptides), antibodies, natural or synthetic inorganic molecules, mimetic agents and natural or synthetic organic molecules. In a specific embodiment, the additional therapy is a chemotherapeutic agent.
[203] In some embodiments, an NDV described in this document or its composition, an antitumor vaccine, or a whole cell vaccine is used in combination with radiation therapy that includes the use of x-rays, gamma rays and other sources radiation to destroy cancer cells. In specific modalities, radiation therapy is administered as radiation with an external beam or teletherapy, in which the radiation is directed from a remote source. In other modalities, radiation therapy is administered as internal therapy or brachytherapy, in which a radioactive source is placed inside the body close to cancer cells and / or a tumor mass.
[204] In certain embodiments, an NDV described in this document or its composition, an anti-tumor vaccine, or a whole cell cancer vaccine is used in combination with adoptive T cell therapy. In a specific embodiment, the T cells used in the adoptive T-cell therapy are tumor infiltrating lymphocytes that have been isolated from a subject and a particular T cell or clone has been expanded for their use. In some embodiments, T cells used in adoptive T cell therapy are T cells taken from a patient's blood after receiving a cancer vaccine and expanded in vitro before use. In another specific modality, T cells used in adoptive T cell therapy are T cells that have been influenced to recognize and attack tumors in a potent way. In another specific embodiment, the T cells used in adoptive T cell therapy were genetically modified to express the T cell specific tumor antigen receptor or a chimeric antigen receptor (CAR). In a specific embodiment, the adopted T-cell therapy used is analogous to that described in Section 7, below.
[205] In certain embodiments, an NDV described in this document or its composition, an anti-tumor vaccine, or a whole cell cancer vaccine is used in combination with a cytokine. In a specific embodiment, an NDV described in this document or its composition, an anti-tumor vaccine, or a full-cell cancer vaccine is used in combination with interferon (for example, IFN-y)
[206] Currently available cancer therapies and their dosages, routes of administration and recommended use are known in the art and have been described in the literature, such as Physician's Desk Reference (67th ed., 2013).
[207] Specific examples of anticancer agents that can be used in combination with an NDV described herein or its composition include: hormonal agents (for example, aromatase inhibitor, selective estrogen receptor modulator (SERM) and estrogen receptor antagonist) , chemotherapeutic agents (for example, blocking microtubules, anti-metabolite, topoisomerase inhibitor, and DNA cross-linking agent or damaging agent), anti-angiogenic agents (for example, VEGF antagonist, receptor antagonists, integrin antagonist, vascular targeting agent (VTA) / vascular interrupting agent (VDA)), radiation therapy and conventional surgery.
[208] Examples of hormonal agents that can be used in combination with an NDV described herein or its composition include aromatase inhibitors, SERM, and non-limiting estrogen receptor antagonists. Hormonal agents that are aromatase inhibitors can be steroidal or non-steroidal. Examples of non-limiting hormonal non-steroidal agents include letrozole, anastrozole, aminoglutetimide, fadrozole, and vorozole. Non-limiting examples of steroidal hormonal agents include aromasin (exemestane), formestane, and testolactone. Examples of hormonal agents that are non-limiting SERMs include tamoxifen (branded / marketed as Nolvadex®), afimoxifene, arzoxifene, bazedoxifene, clomiphene, Femarelle, lasofoxifene, ormeloxifene, raloxifene, and toremifene. Non-limiting examples of hormonal agents that are estrogen receptor antagonists include fulvestrant. Other hormonal agents include, but are not limited to, abiraterone and telaprisan.
[209] Examples of chemotherapeutic agents that can be used in combination with an NDV described herein or its composition, an antitumor vaccine, or a full cell vaccine include microtubule disassembly blocker, antimetabolite, topoisomerase inhibitor, and crosslinking agent or non-limiting DNA damaging agent. Chemotherapeutic agents that are microtubule disassembly blockers include, but are not limited to, taxanes (for example, paclitaxel (branded / marketed as TAXOL®), docetaxel, Abraxane, larotaxel, ortataxel, and tesetaxel); epothilones (for example, ixabepilone); and vinca alkaloids (for example, vinorelbine, vinblastine, vindesine, and vincristine (branded / marketed as ONCOVIN®)).
[210] Chemotherapeutic agents that are antimetabolites include, but are not limited to, folate antimetabolites (eg, methotrexate, aminopterin, pemetrexed, raltitrexed); antimetabolites (for example, purine, cladribine, clofarabine, fludarabine, mercaptopurine, pentostatin, thioguanine); antimetabolites (for example, pyrimidine, 5-fluorouracil, capecitabine, gemcitabine (GEMZAR), cytarabine, decitabine, floxuridine, tegafur); and deoxyribonucleotide antimetabolites (e.g., hydroxyurea).
[211] Chemotherapeutic agents that are topoisomerase inhibitors include, but are not limited to, Class I (Camptotheca) topoisomerase inhibitors (for example, topotecan (branded / marketed as HYCAMTIN) irinotecan, rubitecan, and belotecan); class II (Podophyllum) topoisomerase inhibitors (for example, etoposide or VP-16, and teniposide); anthracyclines (for example, doxorubicin, epirubicin, Doxil, aclarubicin, amrubicin, daunorubicin, idarubicin, pyrarubicin, valrubicin, and zorubicin); anthracenodiones and (for example, mitoxantrone, and pixantrone).
[212] Chemotherapeutic agents that are DNA cross-linking agents (or agents that damage DNA) include, but are not limited to, alkylating agents (eg cyclophosphamide, mecloretamine, ifosfamide (branded / marketed as IFEX °), trophosphamide, chlorambucil, melphalan, prednimustine, bendamustine, uramustine, estramustine, carmustine (branded / marketed as BiCNU °), lomustine, semustine, fotemustine, nimustine, ranimustine, streptozocine, busulfan, nanosulfan, carbohydrates, nanosine, '-triethylene thiophosphoramide, triaziquone, triethylene melamine); alkylating agents (for example, as carboplatin (branded / marketed as PARAPLATIN), cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, satraplatin, picoplatin); non-classical DNA crosslinkers (for example, procarbazine, dacarbazine, temozolomide (branded / marketed as TEMODAR®), altretamine, mitobronitol); and intercalating agents (for example, actinomycin, bleomycin, mitomycin, and plicamycin). 5.6.4.1 IMMUNE MODULATORS
[213] In specific embodiments, an NDV described herein (for example, a chimeric NDV protein) or its composition, an anti-tumor vaccine, or a whole cell vaccine is administered to a subject in combination with one or more of the following: any agonist of a co-stimulating signal from an immune cell (such as, for example, a T lymphocyte, NK cells or cells that present the antigen (for example, a dendritic cell or macrophage) and / or any antagonist of a cell inhibitory signal immune (such as, for example, a T lymphocyte, NK cells or cells that present the antigen (for example, a dendritic cell or macrophage), known to a person skilled in the art. In particular embodiments, an NDV described here (for example , a chimeric NDV) or a composition thereof, an antitumor vaccine, or a whole cell vaccine is administered to a subject in combination with one or more of the agonists of a immune cell co-stimulating signal described in Section 5.2.1, supra. In some embodiments, an NDV described here (for example, a chimeric NDV) or its composition, an antitumor vaccine, or a whole cell vaccine is administered to a subject in combination with one or more antagonists of a cell inhibitory signal. immune system described in Section 5.2.1, supra. In certain embodiments, an NDV described here (for example, a chimeric NDV protein) or its composition, an anti-tumor vaccine, or a whole cell vaccine is administered to a subject in combination with one or more of the agonists of a co-stimulating signal from an immune cell and / or one or more of the antagonists of an immune cell inhibitory signal described in Section 6 and / or Section 7, infra (for example, an anti-CTLA-4 antibody, an ICOS-G, an antibody anti-PD-1, or an anti-PD-Ll antibody) 5.7 BIOLOGICAL TESTS Viral assays in Vitro
[214] Viral assays include those that measure altered viral replication (as determined, for example, by plaque formation) or the production of viral proteins (as determined, for example, by western blot analysis) or viral RNAs ( as determined, for example, by RT-PCR or Northern blot analysis) in cells cultured in vitro using methods that are well known in the art.
[215] Growth of the NDVs described herein can be assessed by any method known in the art or described herein (for example, in cell culture (eg, chicken embryo kidney cell cultures or chicken embryonic fibroblast cultures (CEF Viral titration can be determined by inoculating serial dilutions of an NDV described here in cell cultures (eg, CEF, MDCK, EFK-2, Vero cells, primary human umbilical vein cells (HUVEC), human epithelial cell line H292 or HeLa cells), chicken embryos, or live animals (for example, birds) .After incubating the virus for a specified time, the virus is isolated using conventional methods. be performed using PCR applied to viral supernatants (Quinn & Trevor, 1997; Morgan et al., 1990), hemagglutination tests, infectious doses for tissue culture (TCID50) or infectious doses eggs (DIE 50). An exemplary method of assessing viral titration is described in Section 6 and Section 7, below.
[216] The incorporation of nucleotide sequences encoding a heterologous peptide or protein (for example, a cytokine, a mutated F protein, a mutated V protein, or a myRNA target site in the genome of a chimeric NDV described herein can be evaluated by any method known in the art or described herein (for example, in cell culture, an animal model or viral culture in embryonated eggs). For example, cell culture viral particles of embryo egg allantoic fluid can be purified by centrifugation using a sucrose pad and subsequently analyzed for expression of the fusion protein by Westerning blotting methods well known in the art.
[217] Immunofluorescence-based approaches can also be used to detect the virus and assess viral growth. These approaches are well known to those skilled in the art, for example, fluorescence microscopy and flow cytometry (see Section 6, and Section 7, below). Antibody assays
[218] The antibodies generated by the NDVs described herein can be characterized in a variety of ways well known to a person skilled in the art (eg, ELISA, display of surface plasmon resonance (BIAcore), Western blot, immunofluorescence, immunostaining and / or microneutralization tests). In particular, the antibodies generated by the chimeric NDVs described herein can be tested for the ability to specifically bind to a virus antigen or a heterologous peptide or protein. Such an assay can be performed in solution (for example, Houghten, 1992, Bio / Techniques 13: 412 421), on beads (Lam, 1991, Nature 354: 82 84), on chips (Fodor, 1993, Nature 364: 555 556 ), on bacteria (US Patent No. 5,223,409), on spores (US Patent Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci. USA 89: 1865 1869 ) or on phage (Scott and Smith, 1990, Science 249: 386 390; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA 87: 6378 6382; and Felici, 1991, J. Mol. Biol. 222 : 301 310) (each of these references is incorporated herein in its entirety by reference).
[219] The antibodies generated by the chimeric NDVs described herein that have been identified to specifically bind to a virus antigen or a heterologous peptide or protein can be assayed for their specificity for said heterologous virus or peptide antigen or protein. Antibodies can be assayed for specific binding to a virus antigen or a heterologous peptide or protein and for their cross-reactivity with other antigens by any method known in the art. Immunoassays that can be used to analyze specific binding and cross-reactivity include, but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme-linked immunosorbent assay), immunoassays in " sandwich ", immunoprecipitation assays, precipitation reactions, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, just to name a few. Such assays are routine and well known in the art (see, for example, Ausubel et al., Eds., 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated as reference here in its entirety).
[220] The affinity of binding of an antibody to an antigen and the rate of dissociation of an antibody-antigen interaction can be determined by competitive binding assays. Alternatively, a surface plasmon resonance assay (eg, BIAcore kinetic analysis) or KinExA assay (Blake, et al., Analytical Biochem., 1999, 272: 123-134) can be used to determine the binding and rate of dissociation of antibodies to an antigen from the chimeric NDVs described herein. IFN assays
[221] IFN induction and NDV release described herein can be determined using techniques described herein or known to those skilled in the art. For example, the amount of IFN induced in cells after infection with an NDV described herein, can be determined using an immunological assay (for example, in ELISA or Western Blot assay) to measure IFN expression or to measure the expression of a protein whose expression is induced by IFN. Alternatively, the amount of IFN induced can be measured at the RNA level by assays, such as Northern blot and quantitative RT-PCR, known to a person skilled in the art. In specific embodiments, the amount of IFN released can be measured using an ELISPOT assay. (See, for example, the methods described in Section 6, and Section 7, below). In addition, cytokine induction and release can be determined by, for example, an immunoassay or ELISPOT assay at the protein level and / or quantitative RT-PCR or Northern blots at the RNA level. See Section 6 and / or Section 7, below, for assays to measure cytokine induction and release. Activation marker assays
[222] Techniques for evaluating the expression of the activation marker, co-stimulating molecule, ligand, or inhibitory molecule by cells of the immune system are known to a person skilled in the art. For example, the expression of an activation marker, co-stimulating molecule, ligand, or an inhibitory molecule by an immune cell (for example, T lymphocytes or NK cells) can be assessed by flow cytometry. In a specific embodiment, the techniques described in Section 6 and / or Section 7, below, are used to evaluate the expression of an activation marker, co-stimulating molecule, ligand, or inhibitory molecule by an immune cell. Immune cell infiltration assay
[223] Techniques for assessing the infiltration of immune cells are known to a person skilled in the art. In a specific embodiment, the techniques described in Section 6 and / or Section 7, below, are used to assess the infiltration of immune cells. Toxicity Studies
[224] In some embodiments, the NDVs described here or their compositions, antitumor vaccines described here, the whole cell vaccines described here, or combination therapies described here, are tested for cytotoxicity in mammalian cell lines, preferably human ( see, for example, the cytotoxicity test described in Section 6 and / or Section 7, below). In certain embodiments, cytotoxicity is assessed in one or more of the following non-limiting cell line examples: U937 cells, a human monocyte cell line; peripheral peripheral blood mononuclear primary cells (PBMC); Huh7, a human hepatoblastoma cell line; HL60, HT1080 cells, HEK 293T and 293H cells, MLPC cells, human embryonic kidney cell line; human melanoma cell lines, such as SkMel2, SKMEL-119 and SKMEL-197; THP-1, monocytic cells; a HeLa cell line; and neuroblastoma cell lines, such as MC-IXC, SK-N-MC and SK-N-MC and SK-N-DZ, SH-SY5Y and BE (2) -C. In certain embodiments, cytotoxicity is assessed in different cancer cells. In some embodiments, the ToxLite assay is used to assess cytotoxicity.
[225] Many assays well known in the art can be used to assess the viability of cells or cell lines after infection with an NDV described herein or its composition, or treatment with an anti-tumor vaccine described herein, a whole cell vaccine described here , or a combination therapy described herein and thereby determine the cytotoxicity of NDV or its composition, anti-tumor vaccine, whole cell vaccine, or combination therapy. For example, cell proliferation can be analyzed by measuring the incorporation of Bromodesoxyuridine (BrdU), incorporation of thymidine (3H), by direct cell count, or detecting changes in the transcription, translation or activity of genes known as proto-oncogenes (by example, fos, myc) or cell cycle markers (Rb, cdc2, cyclin A, DI, D2, D3, E, etc.). The levels of such protein and mRNA and activity can be determined by any method well known in the art. For example, the protein can be quantified by immunodiagnostic methods known as ELISA, Western blotting or immunoprecipitation using antibodies, including commercially available antibodies. The mRNA can be quantified using methods that are well known and routine in the art, for example, using northern analysis, RNase protection or polymerase chain reaction in connection with reverse transcription. Cell viability can be assessed using trypan blue staining or other cell viability or death markers known in the art. In a specific embodiment, the level of cellular ATP is measured to determine cell viability. In preferred embodiments, an NDV described herein or its composition, anti-tumor vaccine, whole cell vaccine, or combination therapy kills cancer cells, but does not kill normal (i.e., non-cancer) cells. In one embodiment, an NDV described herein or its composition, anti-tumor vaccine, therapy of whole cell vaccines, or combination preferably kills cancer cells, but does not kill normal (i.e., non-cancer) cells.
[226] In specific modalities, cell viability is measured over three-day and seven-day periods using an assay standard in the art, such as the CellTiter-Glo Assay Kit (Promega) that measures intracellular ATP levels. A reduction in cellular ATP is indicative of a cytotoxic effect. In another specific modality, cell viability can be measured in the neutral red absorption test. In other modalities, visual observation of morphological changes may include enlargement, granularity, cells with irregular edges, a membranous appearance, roundness, separation of the well surface, or other modifications.
[227] The NDVs described herein or their compositions, antitumor vaccines, whole cell vaccines or combination therapies can be tested for toxicity in vivo in animal models (see, for example, the animal models described in Section 6 and / or in Section 7, below). For example, the animal models described herein, and / or others known in the art, used to test the effects of compounds on cancer can also be used to determine the in vivo toxicity of the NDVs described herein or their compositions, antitumor vaccines, whole cells vaccines, or combination therapies. For example, animals are administered a range of pfu from an NDV described herein (for example, a chimeric NDV described in Section 5.2, infra). Subsequently, animals are monitored over time for lethality, weight loss or failure to gain weight and / or levels of serum markers that may be indicative of tissue damage (eg, creatine phosphokinase level as an indicator of damage general tissue level, level of glutamic oxalic acid transaminase or pyruvic acid transaminase as indicators of possible liver damage). These in vivo assays can also be adapted to test the toxicity of the mode of administration and / or regimen in addition to dosages.
[228] The toxicity and / or effectiveness of an NDV described herein or its composition, an anti-tumor vaccine described here, a whole cell vaccine described here, or a combination therapy described here can be determined by standard pharmaceutical procedures in experimental cultures cells or animals, for example, to determine LD50 (lethal dose for 50% of the population) and ED50 (therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the LD5o / ED5o.S ratio. Therapies that exhibit high therapeutic indexes are preferred. Although therapies that exhibit toxic side effects can be used, care must be taken to design a delivery system that directs these therapies to the site of the affected tissue in order to minimize the potential damage to non-cancerous cells and thereby reduce the effects collateral.
[229] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage therapies for use in subjects. The dosage of such agents is preferably within a range of circulating concentrations that include EDS0 with little or no toxicity. The dosage can vary within this range depending on the dosage form employed and the route of administration used. For any therapy described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the chimeric NDV that achieves one half of maximum symptom inhibition) as determined in cell culture. Such information can be used to more accurately determine the useful doses in the subject. Plasma levels can be measured, for example, by high performance liquid chromatography. Anticancer study
[230] The NDVs described here or their compositions, antitumor vaccines described here, whole cell vaccines described here, combination therapies described here can be tested for biological activity using animal models for cancer. Such animal model systems include, but are not limited to, mice, mice, chickens, cows, monkeys, pigs, dogs, rabbits, etc. In a specific embodiment, the anti-cancer activity of an NDV described herein or combination therapy is tested in a rat model system. Such model systems are widely used and well known to those skilled in the art, such as the SCID mouse model or transgenic mice.
[231] The anti-cancer activity of an NDV described herein or its composition, anti-tumor vaccine described herein, the whole cell vaccine described here, or a combination therapy described herein can be determined by administering NDV or its composition, anti-tumor vaccine, whole cell vaccine, or combination therapy for an animal model and verify that NDV or its composition, antitumor vaccine, whole cell vaccine, or combination therapy is effective in reducing the severity of cancer, reducing the symptoms of cancer, reducing metastasis of cancer, and / or reducing the size of a tumor in that animal model (see, for example, Section 6 and / or Section 7, below). Examples of animal models for cancer in general include, include, but are not limited to, pet tumors that occur spontaneously (see, for example, Vail & MacEwen, 2000, Cancer Invest 18 (8): 781-92). Examples of animal models for lung cancer include, but are not limited to, animal models of lung cancer described by Zhang and Roth (1994, in vivo 8 (5): 755-69) and a transgenic mouse model with the function of interrupted p53 (see, for example, Morris et al., 1998, J La State Med Soc 150 (4): 17 9-85). An example of an animal model of breast cancer I have included, but is not limited to, a transgenic mouse that expresses about cyclin Dl (see, for example, Hosokawa et al., 2001, Transgenic Res 10 (5): 471-8). An example of an animal model for colon cancer includes, but is not limited to, TCR be p53 double knockout rat (see, for example, Kado et al., 2001, Cancer Res. 61 (6): 2395-8) . Examples of animal models for pancreatic cancer include, but are not limited to, a metastatic model of murine pancreatic adenocarcinoma Panc02 (see, for example, Wang et al., 2001, Int. J. Pancreatol. 29 (1): 37 - 46) and nu-nu mice generated in subcutaneous pancreatic tumors (see, for example, Ghaneh et al., 2001, Gene Ther. 8 (3): 199-208). Examples of animal models for non-Hodgkin's lymphoma include, but are not limited to, a mouse severe combined immunodeficiency ("SCID") (see, for example, Bryant et al., 2000, Lab Invest 80 (4): 553 -73) and a transgenic IgHmu-HOXll mouse (see, for example, Hough et al., 1998, Proc. Natl. Acad. Sci. USA 95 (23): 13853-8). An example of an animal model for esophageal cancer includes, but is not limited to, a mouse transgenic for human papillomavirus type 16 oncogene E7 (see, for example, Berber et al., 1996, J. Virol. 70 (3): 1873-81). Examples of animal models for colorectal carcinomas include, but are not limited to, Ape rat models (see, for example, Fodde & Smits, 2001, Trends Mol Med 7 (8): 369 73 and Kuraguchi et al., 2000). In a specific embodiment, the animal models for cancer described in Section 6 and / or Section 7, below, are used to evaluate the effectiveness of an NDV or its composition, an antitumor, a whole cell vaccine, or a drug therapy. combination. 6. EXAMPLE 1
[232] This example demonstrates the therapeutic effectiveness of NDV therapy in combination with checkpoint modulators that are immune-stimulating in the treatment of cancer. 6.1 MATERIALS & METHODS Rats
[233] Balb / C mice (6 to 8 weeks old), and WT C57BL / 6 mice were purchased from Jackson Laboratory. All rats were kept in microisolator cages and treated according to NH and the merican Association of Laboratory Animal Care regulations. All rat procedures and experiments for this study were approved by the Memorial Sloan-Kettering Cancer Center Institutional Animal Care and Use Committee. Cell line
[234] Murine cancer cell lines for melanoma (B16-F10) and colon carcinoma (CT26 and MC38) were maintained in RPMI medium supplemented with 10% fetal calf serum and penicillin with streptomycin. The TRAMP-C2 cell line of murine prostate cancer was maintained in DMEM medium supplemented with 5% fetal calf serum (FCS; Mediatech, Inc.), 5% IV Serum IV (BD Biosciences) from HEPES, 2- ME, pen / strep, L-glut, 5 pg / ml insulin (Sigma), and 10 nmol / L DHT (Sigma). Antibodies.
[235] Therapeutic anti-CTLA-4 (clone 9H10), anti-DP-1 (clone RMP1-14), and anti-PD-L1 monoclonal antibodies were produced by BioXcell. The antibodies used for flow cytometry were purchased from eBioscience, BioLegend, Invitrogen, and BD Pharmingen. Viruses and Cloning
[236] The recombinant lentogenic LaSota NDV strain was used for all experiments. To generate NDV virus expressing murine ICOSL, a DNA fragment encoding murine ICOSL flanked by the appropriate NDV-specific RNA transcription signals was inserted into the SacII site created between the P and M genes of pT7NDV / LS. The viruses were rescued from cDNA, using methods previously described and sequenced by reverse transcription PCR for insertion fidelity. Virus titers were determined by serial dilution and immunofluorescence in Vero cells. Recombinant ICOSL-F fusion construct was generated by PCR amplification of the ICOSL DNA encoding the extracellular domain (amino acids 1-277) with MluI and EcoRI flanking of restriction sites, and NDV F DNA encoding the F transmembrane and domains intracellular (amino acids 501-554) with MluI and Xhol flanking restriction sites. The resulting DNA fragments were pooled in the pCAGGS vector using 3 part ligation. In vitro infection experiences
[237] For the evaluation of the positive regulation of the surface of MHC-I, MHC-II, and ICAM-1 by NDV, and for the evaluation of the surface expression of the ICOSL transgene of the NDV-ICOSL virus, B16-F10 cells were infected in 6 well plates in MOI 2 in triplicate. Twenty-four hours later, the cells were harvested by mechanical scraping and processed for surface marking and quantification by flow cytometry. For virus growth curve experiments, B16-F10 cells were incubated at room temperature with the virus in 6-well culture plates in the indicated MOIs in a total volume of 100 pl. One hour after incubation, the infection medium was aspirated and the cells were incubated at 37 ° C in 1 ml of DMEM with 10% allantoic chick fluid. After 24, 48, and 72 hours, supernatants were collected and virus titers were determined as above. For in vitro cytotoxicity experiments, infections were made in a similar way. 24, 48, 72, and 96 hours after infection the cells were washed and incubated with 1% Triton X-100 at 37 ° C for 30 minutes. LDH activity in lysates was determined using the Promega CytoTox 96 assay kit, according to the manufacturer's instructions. Survival experiments to the tumor challenge.
[238] Bilateral tumor models have been established to monitor therapeutic efficacy in both injected and systemic tumors. Treatment schedules and cell doses have been established for each tumor model to achieve 10 to 20% tumor reduction by NDV or anti-CTLA-4 / anti-DP-1, as isolated agents. For experiments evaluating combination therapy of wild-type NDV (NDV-WT) with immune blocking of the signaling pathway, B16F10 tumors were implanted by injecting 2 x 105 B16F10 cells on the right side i.d. on day 0 and 5 x 104 cells on the left side on day 4. On days 7, 10, 13 and 16 the rats were treated with 4 intratumoral injections of 2 x 107 pfu of NDV in PBS in a total volume of 100 pl. At the same time, on days 7, 10, 13 and 16 the rats received four i.p. anti-CTLA-4 antibody (100 pg) or anti-DP-1 antibody (250 pg). Control groups received a corresponding dose of antibody isotype i.p. and intratumoral injection of PBS. Tumor size and incidence were monitored over time by measuring with a calibrator.
[239] For the TRAMP-C2 model, 5 x 105 cells were implanted on the right side on day 0 and 5 x 105 cells were implanted on the left side on day 8. The treatment was performed on days 11, 14, 17, 20 and similarly above above.
[240] For experiments evaluating recombinant NDV that expresses ICOSL (NDV-ICOSL), B16F10 tumors were implanted by injection of 2 x 105 B16F10 cells on the right side i.d. on day 0 and 1 x 105 cells on the left side on day 4. The treatment was carried out as above.
[241] For the CT26 model, the tumors were implanted by injecting 1 x 10 CT2 6 cells on the right side i.d. on day 0 and 1 x 10 cells on the left side on day 2. The treatment was carried out as described above on days 6, 9 and 12. Isolation of lymphocytes that infiltrate tumors
[242] B16F10 tumors were implanted by injection of 2 x 105 B16F10 cells on the right side i.d. on day 0 and 2 x 105 cells on the left side on day 4. On day 7, 10 and 13 the rats were treated with three intratumoral injections of 2 x 107 pfu of NDV, and 100 pg of i.p. anti-CTLA-4 or 250 µg i.p. anti-PD-1, where specified. On day 15, the rats were sacrificed by CO2 inhalation. Tumors and lymph nodes draining the tumor were removed with forceps and surgical scissors and weighed. The tumors of each group were pricked with scissors, before incubation with 1.67 Wunsch U / mL of Liberase and 0.2 mg / mL of DNase for 30 minutes at 37 ° C. The tumors were homogenized by repeated pipetting and filtered through a 70pm nylon filter. The cell suspensions were washed once with RPMI and purified on a Ficoll gradient to eliminate dead cells. Tumor-draining lymph node cells were isolated by trituration of the lymph nodes through a 70pm nylon filter. Flow Cytometry
[243] Cells isolated from tumors or tumor draining lymph nodes were processed for labeling with several panels of stained antibodies CD45, CD3, CD4, CD8, CD44, PD-1, ICOS, CDllc, CD19, NK1.1, CDllb , F4 / 80, Ly6C and Ly6G. Fixable viability dye eFluor780 (eBioscience) was used to distinguish living cells. The cells were also permeabilized with FoxP3 fixative and permeabilization kit (eBioscience) and stained with Ki-67, FoxP3, Granzima B, CTLA-4 and IFN gamma. The data were acquired using the LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Treestar). DC Purification and Charging
[244] The spleens of untreated rats were isolated and digested with 1.67 Wunsch U / mL Liberase and 0.2 mg / mL DNase for 30 minutes at 37 ° C. The resulting cell suspensions were filtered through a 70pm nylon filter and washed once with complete RPMI. CDllc + dendritic cells were purified by positive selection using Miltenyi magnetic beads. Isolated dendritic cells were cultured overnight with recombinant GM-CSF and tumor lysates B16-F10 and purified in Ficoll gradient. Analysis of cytokine production
[245] Tumor cell suspensions or tumor-draining lymph nodes were pooled and enriched for T cells using a Miltenyi T cell purification kit. Isolated T cells were counted and co-cultured for 8 hours with dendritic cells loaded with B16-F10 cells lysed from tumor cells in the presence of 20 U / ml IL-2 (R and D), in addition to Brefeldin A (BD Bioscience) . After restimulation, lymphocytes were processed by flow cytometry as above. Statistic
[246] The data were analyzed using the Student's t test, and P <0.05 was considered statistically significant. 6.2 RESULTS
[247] To characterize the antitumor immune response induced by Newcastle disease virus (NDV) infection, the expression of the MHC I and MHC II molecules, as well as ICAM-1 on the surface of the infected cells in vitro were evaluated. As shown in Figure 1, NDV infection in B16 melanoma cells induces positive regulation of MHC class I and II molecules, as well as the adhesion molecule ICAM-1, which are considered to be important for lymphocyte recruitment specific tumor and activation of the antitumor immune response. Then, the anti-tumor immune response induced by the NDV infection in vivo was evaluated in a murine melanoma model and an established 2-sided model that allowed the monitoring of responses both in tumors injected and in viruses, as well as in distant tumors that did not. receive the virus. As shown in Figure 2, virus-infected tumors show dramatic infiltration with immune cells, such as NK cells, macrophages, and CD8 and CD4 cells, but not in regulatory T cells. Since this part of the immune response may be a response to the virus, rather than the tumor, the immune response to contralateral tumors was assessed (Figure 3). Interestingly, these tumors demonstrated a similar degree of increase in CD8 and CD4 effector cells, but not the infiltrated t reg. Analysis of these cells revealed that they positively regulate activation, proliferation, and lytic markers (Figure 4). NDV monotherapy was effective in controlling treated tumors (Figure 5A), but only slightly slower the growth of contralateral tumors (Figure 5B). The mice that shrunk the tumors, however, showed some degree of protection against additional tumor challenge (Figure 5D), which suggests that NDV therapy may induce lasting immunity.
[248] Next, it was assessed whether additional mechanisms that could be targeted to increase the antitumor effect generated by NDV. The characterization of the lymphocytes that infiltrate tumors from both injected and uninjected NDV tumors revealed positive regulation of the inhibitory CTLA-4 receptor in lymphocytes (Figure 6). Then it was assessed whether inhibition of the CTLA-4 receptor could result in better therapeutic efficacy for NDV. Surprisingly, combination therapy resulted in the rejection of bilateral tumors in most animals, an effect that was not seen with treatment alone (Figure 7). This effect was present even when the TRAMP adenocarcinoma of the prostate model was used, which is not susceptible to viral infection (Figure 8), suggesting that minimal viral replication and the resulting inflammatory response were sufficient for the generation of protective antitumor immunity.
[249] To determine whether the labeling of other immune signaling pathways in combination with NDV therapy can be beneficial, the effect on the DP-1 - PD-L1 pathway following NDV infection has been assessed. As shown in Figure 9, tumor cells infected with NDV both in vitro and in vivo positively regulated the expression of the inhibitory PD-L1 ligand on the cell surface. This effect was not only the result of direct virus infection, but was also seen when uninfected cells were treated with UV-inactivated supernatants from virus infected cells (Figure 9B) and in uninfected, contralateral tumors ( Figure 9C). This led to tests of combination therapy with NDV and anti-DP-1 antibody. Similar to CTLA-4 blockade, NDV therapy in combination with anti-DP-1 in the aggressive B16 melanoma model resulted in healing in most animals , an effect that was associated with increased infiltration of tumors with activated effector lymphocytes (Figure 10).
[250] Throughout the studies, the therapeutic effectiveness of a combination therapy decreased when the challenge of the larger tumor was used. Then, activation markers that could predict a better response and could be targeted to further improve therapeutic efficacy were evaluated. The analysis of lymphocytes isolated from tumors and the positive regulation of lymph nodes that drain tumors identified from the co-stimulatory molecule ICOS as one of the activation markers in the treated animals (Figure 11). ICOS positive regulation has previously been shown to be associated with more durable therapeutic responses and increased survival in patients treated with anti-CTLA-4 for malignant melanoma therapy. It was assessed whether the intratumor expression of the ICOS ligand (ICOSL) could further boost the therapeutic response of combination therapy. Using the gene inversion system for NDV, NDV expressing murine ICOSL (NDV-ICOSL) were generated. In vitro characterization of the virus revealed that it had similar replicative and lytic properties for the parental NDV strain (Figure 12). When tested in vivo, however, a challenge with a larger B16 tumor, NDV-ICOSL demonstrated a significant advantage over the parental NDV virus when used in combination with CTLA-4 block, with long-term survival in most treated animals (Figure 13 ). This effect was not limited to melanoma B16 and was demonstrated for CT2 6 colon carcinoma in the Balb / C mouse strain, suggesting that this therapeutic strategy can be translated to different types of tumors (Figure 14). Analysis of B16 tumors of the treated animals showed significant infiltration with different subtypes of immune cells with positive regulation of activation markers (Figures 15 and 16). These lymphocytes were tumor specific and demonstrated IFN gamma secretion in response to stimulation with dendritic cells loaded with tumor lysates (Figure 17). Finally, animals that were healed from their BI6 or CT26 tumors were again challenged with tumor cells and demonstrated complete protection against the new tumor challenge (Figure 18).
[251] To further improve the expression of ICOSL in the tumor and to incorporate the ligand into the virion, a chimeric protein consisting of the extracellular domain of ICOSL (amino acids 1-277) and the intracellular domains and the transmembrane of the NDV F protein (amino acids 501-554) were generated (Figure 19A). The transfection of the resulting construct in B16-F10 cells resulted in an increased expression of the chimeric ICOLS F ligand on the surface of the transfected cells, when compared with the transfected native ICOSL, suggesting that the regulatory mechanisms that regulate the transport of NDV F protein to the surface it can be used to increase the surface expression of immune stimulating ligands (Figure 19B).
[252] In general, these studies demonstrate that: 1) the combination of NDV with antibodies regulating the immune signaling pathway can be used as a strategy to circumvent the limitation of both oncolytic virus therapy and antibody therapy; and 2) the expression of immunostimulatory ligands by NDV can further improve the therapeutic efficacy of the virus, especially when used in combination with immunoregulatory antibodies. These conclusions have clinical application. 7. EXAMPLE 2
[253] This example demonstrates the antitumor immune responses induced by oncolytic NDV and the antitumor responses induced by NDV in combination with CTLA-4 blockade. 7.1 MATERIALS & METHODS Rats
[254] C57BL / 6J and Balb / C mice were purchased by Jackson Laboratory. IFNAR - / - mice in C57BL / 6J background was a kind gift from Dr. Eric Pamer. Transgenic mice Trp-1 TCR and Pmel-1 have been reported (Overwijk et al, 2003, J. Exp. Med, 198: 568, Muransky et al, 2008, Blood 112: 362) and N. Restifo (National Cancer Institute , Bethesda, MD). TRP1 mice were crossed with luciferase: CD2 mice provided by Patrick Hwu at the MD Anderson Cancer Center (Houston, TX) to create TRP1 luciferase * mice (TRP1-FLUC). All rats were kept in micro-insulated cages and treated according to the American NIH and American Association of Laboratory Animal Care regulations. All procedures and experiments on mice for this study were approved by the Memorial Sloan-Kettering Cancer Center Institutional Animal Care and Use Committee. Cell line
[255] Murine cancer cell lines for melanoma (B16-F10) and colon carcinoma (CT26 and MC3 8) were maintained in RPMI medium supplemented with 10% fetal calf serum and penicillin with streptomycin. The TRAMP-C2 cell line of murine prostate cancer was maintained in DMEM medium supplemented with 5% fetal calf serum (FCS; Mediatech, Inc.), 5% IV Serum IV (BD Biosciences) from HEPES, 2- ME, pen / strep, L-glut, 5 pg / ml insulin (Sigma), and 10 nmol / L DHT (Sigma). Antibodies.
[256] Therapeutic monoclonal antibodies anti-CTLA-4 (clone 9H10), anti-DP-1 (clone RMP1-14), anti-PD-L1 (clone 9G2), anti-CD8 (clone 2.43), anti-CD4 ( clone GK1.5), anti-IFN-gamma (XMG1.2 clone), and anti-NKl.l (clone PK136) were produced by BioXcell. The antibodies used for flow cytometry were purchased from eBioscience, BioLegend, Invitrogen, and BD Pharmingen. Viruses and cloning
[257] The recombinant lentogenic LaSota NDV strain was used for all experiments. To generate NDV virus expressing murine ICOSL, a DNA fragment encoding murine ICOSL flanked by the appropriate NDV-specific RNA transcription signals was inserted into the SacII site created between the P and M genes of pT7NDV / LS. The viruses were rescued from cDNA, using methods previously described and sequenced by reverse transcription PCR for insertion fidelity. Virus titers were determined by serial dilution and immunofluorescence in Vero cells. Recombinant ICOSL-F fusion construct was generated by PCR amplification of the ICOSL DNA encoding the extracellular domain (amino acids 1-277) flanking MluI and EcoRI of restriction sites, and the NDV F DNA encoding the F transmembrane and intracellular domains (amino acids 501-554) with MluI and Xhol flanking restriction sites. The resulting DNA fragments were pooled in the pCAGGS vector using 3 part ligation. The CD28scfv-F fusion construct was generated by PCR amplification of the hamster's anti-CD28scfv cDNA encoding MluI and EcoRI flanking of restriction sites, and the NDV F DNA encoding the F transmembrane and intracellular domains (amino acids 501- 554) flanking MluI and Xhol restriction sites. The resulting DNA fragments were pooled in the pCAGGS vector using 3-part ligation and then subcloned into the pNDV vector between the P and M genes. To generate the recombinant viruses that express other chimeric proteins (HN-GITRL, HN-4- 1BBL, HN-CD40L, OX40L-HN), the cDNA encoding the extracellular domain of each gene (Figure 44) was amplified with the specific gene primers with EcoRI and flanking the Mlu I and EcoRI restriction sites, and the transmembrane domain and intracellular HN protein was amplified with the specific primers with the restriction sites flanking MluI and Xhol. The chimeric genes were complete, they were assembled in the vector pCAGGS, using 3 binding parts and then subcloned into the NDV vector between the P and M. genes. The details of each chimeric construction are shown in Figure 44. To generate the recombinant NDV that encodes murine IL-2, IL-15 and IL-21, the cDNA for each gene was amplified with specific gene primers that flank the restriction sites with SacII and then cloned into the pNDV between the P and M genes. The viruses were rescued from cDNA using methods described above and sequenced by reverse transcription PCR for insertion fidelity. Virus titers were determined by serial dilution and immunofluorescence in Vero cells. In vitro infection experiments
[258] For cell surface labeling, cells were infected in 6-well plates in MOI 2 (B16-F10) or MOI 5 (TRAMP C2), in triplicate. Twenty-four hours later, the cells were harvested by scraping and processed for surface marking and quantification by flow cytometry. For in vitro cytotoxicity experiments, cells were infected in the indicated MOI's and incubated at 37 ° C in serum-free medium in the presence of 250 ng / ml of trypsin TPCK. At 24, 48, 72, and 96 hours after infection, the cells were washed and incubated with 1% Triton X-100 at 37 ° C for 30 minutes. LDH activity in lysates was determined using the Promega CytoTox 96 assay kit, according to the manufacturer's instructions. Survival experiences to the tumor challenge
[259] Bilateral tumor models have been established to monitor therapeutic efficacy in both injected and systemic tumors. Treatment schedules and cell doses have been established for each tumor model to achieve 10 to 20% tumor shrinkage by NDV or anti-CTLA-4 as isolated agents. For experiments evaluating the combination therapy of NDV with anti-CTLA-4 antibody, B16F10 tumors were implanted by injecting 2 x 105 B16F10 cells on the right side intradermal i.d. on day 0 and 5 x 104 cells on the left side on day 4. On days 7, 9, 11 and 13, the rats were treated with intratumoral injections of 2 x 107 pfu of NDV in PBS in a total volume of 100 pl. At the same time, on days 7, 9, 11 and 13 the rats received intraperitoneal injections i.p. of anti-CTLA-4 antibody (100 pg) or anti-DP-1 antibody (250 pg) or anti-Ll-PD antibody (250 pg). Control groups received a corresponding dose of the i.p. and intratumoral injection of PBS. The animals were sacrificed when showing signs of suffering or when the total tumor volume reached 100,000 mm3. For the depletion of immune cells, rats were injected i.p. 500 pg of monoclonal antibodies for CD8 +, CD4 +, NK1.1 or IFN-y, one day before and two days after tumor inoculation, followed by injection of 250 pg every 5 days throughout the experiment. For the TRAMP-C2 model, 1 x 10 6 cells were implanted on the right side on day 0 and 5 x 105 cells were implanted on the left side on day 4. The treatment was performed on days 7, 10, 13, and 16 in the same way as above . For the CT26 model, the tumors were implanted by injecting 1 x 106 CT26 cells on the right side i.d. on day 0 and 1 x 106 cells on the left side on day 2. The treatment was performed as described above on days 6, 9 and 12. For experiments evaluating recombinant NDV that expresses NDV ICOSL, 4-1BBL, OX40L, CD40L, GITRL, anti-CD28scfv, IL-2, IL-15 and IL-21 (NDV-transgene), B16F10 tumors are implanted by injection of 2 x 105 B16F10 cells on the right side id on day 0 and 1 x 105 cells on the left side on day 4. On days 7, 9, 11, and 13, the mice are treated with intratumoral injections of 2 x 107 pfu of NDV in PBS in a total volume of 100 pl. At the same time, on days 7, 9, 11 and 13 the rats received intraperitoneal (ip) injections of anti-CTLA-4 antibody (100 pg), 1-anti-PD antibody (250 pg), or anti -LI-PD (250 pg). Isolation of Pmel and Trpl lymphocytes and adoptive transfer
[260] Spleens and lymph nodes from transgenic rats were isolated and ground through 70 pm nylon filters. CD4 + and CD8 + cells were purified by positive selection using Miltenyi magnetic beads
[261] Isolated TRP1 or PMEL cells were injected into recipient animals through the tail vein in the schedule indicated at 2.5 x 104 cells per mouse and 1 x 106 cells per mouse, respectively. Serum transfer experiments
[262] Groups of tumor-bearing rats were treated with a single intratumor injection of NDV or PBS. On day 4, blood was collected by terminal bleeding and the serum was isolated by centrifugation. The sera were collected from each group and treated with UV in Stratalinker 1800 with six pulses of light of 300 mJ / cm2 UV to inactivate any virus that may be potentially present. 100 pl of undiluted serum was injected intratumorally without prior treatment in rats with B16-F10 tumors for a total of 3 injections administered on alternate days. The tumors were removed three days after the last injection and processed for the isolation of lymphocytes that infiltrate the tumor, as described below. Bioluminescence image
[263] The rats were photographed every 2 to 3 days, starting on day 6. The rats were injected retro-orbitally with 50 µl 40 mg / mL D-luciferin (Caliper Life Sciences) in PBS and immediately photographed using the system IVIS (Caliper Life Sciences). Grayscale photographic images and color images of bioluminescence were overlaid using The Living Sciences, version 4.0 (Caliper Life Sciences) software overlay. A region of interest (ROI) was manually selected on the tumor and the ROI area was kept constant. Isolation of lymphocytes that infiltrate the tumor
[264] B16-F10 tumors were implanted by injecting 2 x 105 B16-F10 cells on the right side i.d. on day 0 and 2 x 105 cells on the left side on day 4. On days 7, 9 and 11 the rats were treated with intratumoral injections of 2 x 107 pfu of NDV, and anti-CTLA-4 or anti-DP-1 antibodies ip where specified. Few animals died of the tumor burden (always in untreated control groups) or animals that completely extinguished the tumors (always in treatment groups) were not used for the analysis. On day 15, the rats were sacrificed and the tumors and lymph nodes that drain tumors were removed using forceps and surgical scissors and weighed. The tumors of each group were pricked with scissors, before incubation with 1.67 Wunsch U / mL of Liberase and 0.2 mg / mL of DNase for 30 minutes at 37 ° C. The tumors were homogenized by repeated pipetting and filtered through a 70 pm nylon filter. The cell suspensions were washed once with complete RPMI medium and purified on a Ficoll gradient to eliminate dead cells. The lymph nodes cells that drain tumor were isolated by grinding the lymph nodes through a 70 pm nylon filter. Flow cytometry
[265] Cells isolated from tumors or tumor-draining lymph nodes were processed for surface staining with various antibody panels staining for CD45, CD3, CD4, CD8, CD44, ICOS, CDllc, CD19, NK1.1, CDllb, F4 / 80, and Ly6C Ly6G. The fixable viability dye eFluor506 (eBioscience) was used to distinguish living cells. The cells were also permeabilized with FoxP3 fixation and permeabilization kit (eBioscience) and stained by Ki-67, FoxP3, Granzima B, CTLA-4 and IFNy. The data were acquired using the LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Treestar). DC purification and charging
[266] The spleens of untreated rats were isolated and digested with 1.67 Wunsch U / mL Liberase and 0.2 mg / mL DNase for 30 minutes at 37 ° C. The resulting cell suspensions were filtered through a 70pm nylon filter and washed once with complete RPMI. CD11c + DC were purified by positive selection using Miltenyi magnetic beads. Isolated DC's were grown overnight with recombinant B16-F10 and GM-CSF tumor lysates and purified in Ficoll gradient. Analysis of cytokine production
[267] Tumor cell suspensions or tumor-draining lymph nodes were pooled and enriched with T cells using a Miltenyi T cell purification kit. Isolated T cells were counted and co-cultured for 8 hours with DC's loaded with B16-F10 tumor cell lysates in the presence of 20 U / ml IL-2 (R and D) plus Brefeldin A (BD Bioscience). After re-stimulation, lymphocytes were processed by flow cytometry, as above. Immunofluorescence and microscopy
[268] The tumors were dissected from the rats, washed in PBS, fixed in 4% paraformaldehyde and processed for inclusion in paraffin according to the protocols described above. The sections were cut using a microtome, mounted on slides, and processed for staining with hematoxylin and eosin (H&E) or with an anti-CD3 and anti-Foxp3 antibody. The slides were analyzed in a Zeiss Axio 2 wide-field microscope using a 10x and 20x objective. Statistic
[269] The data were analyzed by Student's T test (for comparisons of 2 groups) and ANOVA if necessary. Survival data were analyzed by the Log-Rank (Mantel-Cox) Test. Front and back P <0.05 was considered statistically significant (P <0.05 (*), P <0.01 (**), P <0.001 (***), P <0.0001 (““) ). 7.2 RESULTS
[270] NDV replication is restricted to the site where the tumor was injected
[271] Viral distribution kinetics with intratumor and systemic administration of NDV have been characterized. Intratumor injection of recombinant NDV expressing reporter firefly luciferase (NDV-Fluc) resulted in the luciferase signal sustained in the tumor on the injected side, while systemic administration of the virus did not result in any detectable luciferase signal in the tumor (Figure 20A). As limited systemic virus delivery was unlikely to induce sufficient tumor lysis and immune response, intratumoral injection of NDV was explored as a means to elicit an anti-tumor immune response that could potentially overcome the limitations of systemic OV therapy. As such, for further studies of metastatic disease it was modeled using the tumor model B16-F10 using both sides (Figure 22A). Administration of NDV-Fluc to the right side tumor resulted in viral replication within the injected tumor, with the luciferase signal detectable for up to 96 hours (Figure 20B-D). No virus was detected in the contralateral region (left side) of the tumor by luminescent image (Figure 20B-D), through passage in embryonated eggs, or by RT-PCR. This system thus allowed for the characterization of immune responses both in tumors with viruses injected with distant ones, which were not directly affected by NDV. NDV therapy increases the infiltration of lymphocytes into local and distant tumors and slows tumor growth
[272] Analysis of tumors with injected viruses revealed an inflammatory response, as evidenced by increased infiltration with cells expressing common CD45 leukocyte antigen (Figures 21A-B). Immune infiltrates were characterized by an increase in the innate immune compartment, including myeloid cells, NK cells and NKT cells (figure 21C), and the adaptive compartment, including conventional CD8 + and CD4 + FoxP3- (Tconv) cells, which lead to an increase of CD8 and Tconv for regulatory T cell (Treg) rates (p = 0.0131 and p = 0.0006, respectively) (Figures 21D-21F). Notably, the analysis of contralateral tumors revealed a similar increase in inflammatory infiltrates (Figure 22B, C), characterized by the increasing numbers of both innate immune cells (Figure 22D) and effector T cells (figure 22E, G). Notably, although there were no major changes in the absolute number of Tregs (Figure 22G), there was a substantial decrease in their relative percentages (Figure 22E, F, H), with a significant improvement in CD8 and Tconv for Treg rates (p = 0.002 and p = 0.0021, respectively) (Figure 221). Effector T cells isolated from distal tumors expressed increased activation, proliferation, and lytic markers ICOS, Ki-67 and Granzyme B, respectively (Figure 1J, K). As before, the viral RNA or virus was unable to be isolated from distant tumors, suggesting that the changes observed in the distant tumor microenvironment did not occur due to viral infection. To further exclude the possibility of undetectable local viral spread, tumors were implanted in other distant sites, such as bilateral posterior feet, which generated similar results (Figure 23).
[273] Consistent with the observed inflammatory effect, intratumoral administration of NDV resulted in growth retardation, not only of the injected one, but also of the contralateral tumors, resulting in prolonged survival of the animal (Figure IL, M). To determine whether this effect was transient and whether durable anti-tumor protection was possible, rats with single-side B16-F10 tumors were treated intratumorally with NDV, and long-term survivors were injected with B16-F10 cells on the opposite side. Most animals demonstrated delayed growth of the tumor, and 30% of the animals completely rejected the re-challenged cells, suggesting that intratumor therapy with NDV may indeed induce protective antitumor memory responses (Figure 25). NDV induces tumor infiltration and the expansion of tumor-specific lymphocytes
[274] To determine whether the immune antitumor response was dependent on the type of tumor injected with NDV or a result of non-specific inflammation, generated by NDV infection, the experiment was carried out with heterologous tumors (colon carcinoma MC38 and melanoma B16- F10) implanted on opposite sides (Figure 24A). To accompany tumor-specific lymphocytes, the transgenic T-cell receptor congenically labeled luciferase-labeled CD8 + cells (Pmel) or CD4 + cells (TRP1) that recognize the differentiation of the Melanoma gplOO (Pmel) and TRP1 (TRP1) antigen Muranski et al., 2008, Blood, 112: 362; Overwijk et al., 2003, J Exp Med, 198: 569). The bioluminescence image was used to measure the kinetic distribution and expansion of the adopted TRP1 cells. The transfer of TRP1 cells in tumor-bearing animals treated with PBS failed to result in the accumulation of TRP1 in the tumors, highlighting the highly immunosuppressive nature of the tumor microenvironment in this model (Figure 24B-D). The injection of NDV into B16-F10 tumors resulted in a significant increase in the luciferase signal in the injected tumors (Figure 24B-D), which indicates the expansion of TRP1 T cells (area under the curve (AUC) p = 0.0084). Notably, similar expansion was seen in the contralateral tumor, albeit at a delay (p = 0.0009) (Figure 24B-D). In contrast, the injection of NDV into MC38 tumors failed to induce substantial TRP1 infiltration for the injected MC38 tumors or distant B16-F10 tumors (Figure 24B-D), which suggests that the infiltration of distant tumor specific lymphocytes is likely to be dependent of the identity of the injected tumor antigen. Likewise, the intratumoral injection of NDV resulted in an increase in the infiltration of PMEL cells in distant tumors, which was more noticeable when the tumor injected was B16-F10 cells, instead of MC38 (Figure 24E).
[275] Interestingly, although the infiltration of distant B16-F10 tumors with adoptively transferred lymphocytes was dependent on the identity of the injected tumor, distant tumors did not demonstrate increased immune infiltration, even when the first tumor injected was MC38 (Figure 24F), suggesting that a non-specific inflammatory response component can also play a role. In fact, the serum of animals treated with NDV, treated with UV irradiation to inactivate any potential virus, tumor-induced leukocyte infiltration when injected intratumorally into rats with B16-F10 tumor without treatment (Figure 24G, H), with the most of the increase observed in the CD8 and NK compartments (p = 0.0089 and p = 0.0443, respectively) (Figure 241). NDV and CTLA-4 block synergy to reject local and distant tumors
[276] Despite the prominent inflammatory response and growth delay seen in distant tumors, complete contralateral tumor rejection with long-term survival was only observed in about 10% of animals (Figure 22M), suggestive of active immunosuppressive mechanisms in the microenvironment tumor. The characterization of tumors injected with NDV and distant ones revealed the positive regulation of CTLA-4 in T cells that infiltrate the tumor (Figure 26), suggesting that the tumor inflammation induced by NDV would make the tumors sensitive to systemic therapy with CTLA-4 blockade. . Notably, combination therapy of NDV with anti-CTLA-4 (Figure 27A) resulted in the rejection of bilateral tumors and long-term survival in most animals, an effect that was not seen with treatment alone (Figure 27B-D). To determine the durability of the observed protection, B16-F10 cells were injected into the right side of the surviving animals on day 90 without any additional treatment. Animals treated with NDV and anti-CTLA-4 combination therapy demonstrated more than 80% protection against the new tumor challenge, compared to 40% protection in animals treated with a single anti-CTLA-4 agent (Figure 27E ). Combination therapy with NDV and CTLA-4 blockade is effective against permissive non-viral tumors
[277] To determine whether this treatment strategy can be extended to other types of tumors, the strategy was evaluated in the immunogenicly weak TRAMP-C2 prostate adenocarcinoma model. Similar to the B16-F10 model, combination therapy caused the regression of the injected tumors to regress (Figure 27F), and delayed both the growth of distant tumors and led to complete regression of the distant tumor with prolonged long-term survival (Figure 2 7F, G). Interestingly, while B16-F10 cells were susceptible to NDV-mediated lysis in vitro, TRAMP C2 cells were strongly resistant, with low cytotoxicity observed at a multiplicity of infection (MOI) of up to 10 (Figure 27H). In both cell lines, NDV infection in vitroresulted in the positive regulation of the MHC surface and co-stimulatory molecules (Figure 27i-K). Class I MHC was uniformly regulated in all cells, although not all cells were infected with NDV at the MOI of 1. Previous studies have shown that NDV induces IFN type I expression in B16-F10 cells (Zamarin et al ., 2009, Mol Ther 17: 697). Both type I IFN (Dezfouli et al., 2003, Immunol. Cell. Biol., 81: 459, Seliger et al., 2001, Cancer Res., 61: 1095) that are known to positively regulate class I MHC in B16-F10 cells, which suggests that, in the context of infected tumors, these mechanisms may play an additional role in increasing tumor immunogenicity. These results therefore suggest that in vitro sensitivity to virus-mediated lysis is not necessary for sensitivity to in vivo NDV therapy and further emphasize the importance of an inflammatory response generated by the virus, rather than direct oncolysis, in antitumor efficacy. observed. Antigen-systemic antitumor effect is restricted to the type of tumor injected
[278] To determine whether the antitumor effect observed in the distant tumor was specific to the type of tumor injected, the combination therapy in animals carrying a unilateral distant B16-F10 tumor and in animals with the tumor types (heterologous colon carcinoma MC38 and melanoma B16-F10) implanted on opposite sides was evaluated (Figure 28A). Although administration of the virus intradermally on the right side without a tumor resulted in a delay in the development of a tumor on the left side; it failed to achieve long-term protection and tumor rejection in animals with bilateral B16-F10 tumors (Figure 28B, C). Likewise, the injection of NDV into the MC38 tumor on the right side of animals carrying tumors on the left side B16-F10 failed to induce rejection of the B16-F10 tumor (Figure 28D, E), suggesting that the immune response induced by antitumor NDV it is similar to the antigen restricted to the injected tumor. Combination therapy with anti-CTLA-4 NDVe induces tumor infiltration with activated lymphocytes
[2 79] To examine the B16-F10 tumor microenvironment in the treated animals, bilateral tumors were collected and processed for infiltrating cell analysis. Analysis of distant injected tumors and treated animals revealed significant inflammatory infiltrates and large areas of tumor necrosis in animals treated with combination therapy (Figure 30A, Figure 29). This correlated with the increase in the number of CD45 + cells and T cells in the combination therapy group (Figure 30A-C, Figure 29A-C). As previously, the observed increase in TILs was mainly due to the infiltration of CD8 + and Tconv, but not in Treg cells, inducing the effector increase for Treg rates (Figure 30D-F, Figure 29C-E). The phenotypic characterization of CD4 + and CD8 + TILs in animals that received the combination treatment demonstrated positive regulation of ICOS, Granzima B, and Ki-67 in animals treated with anti-CTLA-4 and untreated animals (Figure 30G-I) and a higher percentage of CD8 + cells that express IFN gamma in response to re-stimulation with dendritic cells (DC's) pulsed with B16-F10 tumor lysates (Figure 30J). Antitumor activity of NDV combination therapy depends on CD8 + cells, NK cells and type I and II interferons
[280] To determine which components of cellular immunity were responsible for the observed therapeutic effect, treatment was repeated in the presence of antibodies to deplete CD4 +, CD8 +, or NK cells. Adequate cell depletion of each subset of cells was confirmed by peripheral blood flow cytometry (Figure 31). Depletion of both CD8 + and NK cells resulted in the revocation of the therapeutic effect in both tumors with virus injection and distant ones (Figure 32A, B), with a significant reduction in long-term survival (P <0.0001 for CD8 and p = 0 .0011 for NK depletion) (Figure 32C). In line with these results, treatment of animals with a neutralizing anti-IFNy antibody also decreased therapeutic efficacy. In contrast, the depletion of CD4 + T cells did not result in an appreciable change in the antitumor effect, although these results should be interpreted with caution, since anti-CD4 + depletion also results in simultaneous depletion of Tregs.
[281] IFN type I has previously been shown to play an important role in the initiation of CD8 + cells for anti-tumor immune response ((Fuertes et al., 2011, J Exp Med, 2 08: 2 005; Diamond et al, 2011, J Exp Med , 208: 1989) .In order to investigate the role of IFN type I in tumor rejection by NDV, experiments were repeated in mice (IFNAR - / -) IFN type I receptor knockout demonstrated rapid progression of both injected and contralateral tumors and were completely resistant to combination therapy (Figure 32D-F). Overall, these results highlight the importance of the role of both adaptive and innate immune responses to the systemic therapeutic efficacy of the virus observed in this study. NDV therapy leads to positive regulation of PD-L1 in tumor cells and leukocytes that infiltrate tumors
[282] To determine whether other immune targeting signaling pathways in combination with NDV therapy may be beneficial, the effect on the DP-1 - PD-L1 pathway following NDV infection was assessed. As shown in Figure 33, tumor cells infected with NDV both in vitro and in vivo positively regulated the expression of the inhibitory PD-L1 ligand on the cell surface (Figure 33A), which was also seen in distant uninfected tumors. Positive regulation of PD-L1 was not restricted only to tumor cells, but infiltrating leukocytes from both adaptive and innate immune strains were also seen in the tumor (Figure 33B). Combination therapy of NDV with blocking antibodies PD-L1 and DP-1 leads to improved antitumor immunity and long-term survival of animals
[283] The combination of NDV with antibodies that block DP-1 and the combination of NDV with antibodies that block PD-L1 were evaluated in the bilateral melanoma model described above. Notably, similar to CTLA-4 blockade, NDV therapy in combination with anti-DP-1 antibodies or anti-PD-Ll antibody led to better animal survival (figures 34 and 35). Distant tumors from animals treated with the combination of NDV and anti-DP-ls antibody were characterized. As can be seen from Figure 36, the combination of intratumoral NDV with systemic PD-1 blockade led to distant tumor infiltration marked with immune cells, with the increase of CD8 cells that infiltrate the tumor being seen more. Positive proliferation regulated the infiltrating cells, lytic markers Ki67 and Granzima B, respectively (Figure 37). NDV induces positive regulation of immune tumor infiltration of CD4 and CD8 cells in ICOs in distant tumors and injected by viruses and distant tumors and tumor-draining lymph nodes (TDLN)
[284] The above results demonstrated that the combination of intratumoral NDV with the blocking of the systemic immune signaling pathway results in significant synergy between the two therapeutic approaches. To reinforce these results, the increase in the effector function of T cells within the tumor microenvironment through a relevant co-stimulation pathway could lead to a better immune anti-tumor response was investigated. Previous studies have identified sustained positive regulation of the inducible co-stimulator (ICOS) in T cells as a strong indicator of response to CTLA-4 blockade in patients (Carthon et al., 2010, Clin. Canc. Res., 16: 2861). ICOS is a positively regulated CD28 homologue on the surface of activated T cells that has been shown to be essential for T cell-dependent B lymphocyte responses and the development of all T helper subsets (Simpson et al., 2010 Curr Opin Immunol. 22: 326 ). The role of tumor antitumor ICOS in the effectiveness of CTLA-4 blockade has recently been confirmed in studies in rats, where ICOS deficient rats have been severely compromised in the development of an antitumor response with CTLA-4 blockade (Fu et al., 2011, Cancer Res., 71: 5445).
[285] ICOS expression in bilateral NDV-treated tumor models was characterized to determine whether the receptor could serve as a target in this therapeutic approach. To characterize the local and abscopal effects of NDV intratumoral therapy, bilateral B16-F10 melanoma models were used, with the virus administered to a unilateral tumor (Figure 3 8A). Activation markers that could predict a better response and could be targeted to further improve therapeutic efficacy were evaluated. The example focused on ICOS, as sustained ICOS positive regulation has previously been shown to be associated with more durable therapeutic responses and increased survival in patients treated with anti-CTLA-4 therapy for malignant melanomas. Analysis of lymphocytes isolated from tumors and the identified positive regulation of lymph nodes that drain tumors of the co-stimulatory molecule ICOS as one of the activation markers in the treated animals (Figure 38B, C). Generation and in vitro evaluation of NDV-ICOSL virus
[286] Using the NDV reverse genetics system, mice expressing NDV ICOSL (NDV-ICOSL) were generated (Figure 39A). ICOSL expression on the surface of infected B16-F10 cells was confirmed by flow cytometry 24 hours after infection (Figure 39B). In vitro characterization of the virus revealed that it had lytic properties (Figure 39C) and similar replicates (Figure 39D) for the parental NDV strain. Delayed growth of NDV-ICOSL from distant tumors and induces increased lymphocyte infiltration
[287] To evaluate NDV-ICOSL for therapeutic efficacy in virus-injected and distant tumors, animals carrying bilateral B16-F10 tumors were treated with 4 intratumoral injections of a given virus into a unilateral tumor. Both NDV-ICOSL and wild-type NDV were comparable in their ability to cause tumor regressions in tumors directly injected with the virus (Figure 4 0A). However, when compared to wild-type NDV, NDV-ICOSL resulted in a significant delay in tumor growth from distant tumors with several animals remaining tumor-free for the long term (Figure 40B-C). Analysis of tumors injected with viruses revealed an increase in tumor infiltration with CD4 and CD8 effector cells in animals treated with wild type NDV and NDV-ICOSL, although the differences between the two viruses were not statistically significant, reflecting the similar activity of the two virus against the right side tumors (Figure 40A and 40D). In contrast, analysis of tumors on the left revealed a more prominent increase in CD8 and Tconv cells that infiltrate the tumor in the group treated with NDV-ICOSL (Figure 40E). Interestingly, there was also an absolute increase in the number of regulatory T cells, with the largest increase seen in the NDV-ICOSL group (Figure 40E), although the relative percentage of regulatory T cells was significantly lower in animals treated with NDV (Fig 40F). Combination therapy of NDV-ICOSL and CTLA-4 blocks results in rejection of injected and distant tumors
[288] Overall, the above results demonstrate that, despite the significant inflammatory response seen in distant tumors with intratumoral administration of NDV-ICOSL, most animals have still succumbed to the tumors, suggesting that the active inhibitory mechanisms within the microenvironment prevent tumor rejection by infiltrating immune cells. Thus, the effectiveness of localized NDV-ICOSL combination therapy with systemic CTLA-4 blockade was assessed. For these experiments, tumor challenge doses were increased to levels where no significant therapeutic effect was observed with NDV or anti-CTLA-4 as individual agents. As before, the animals were treated with 4 doses of NDV administered to a unilateral tumor, concomitantly given with systemic anti-CTLA-4 antibody (Figure 41A). In the B16-F10 model, combination therapy with NDV-ICOSL and anti-CTLA-4 led to the regression of most of the injected and distant tumors with long-term survival of the animals, which was significantly superior to the combination of NDV-WT with anti-CTLA-4 (Figure 41B-D). To determine whether these findings can be extended to other tumor models, the same experiment was performed on the bilateral CT26 colon carcinoma model. Despite the low sensitivity of CT26 cells to NDV-mediated lysis in vitro, significant therapeutic efficacy of the combination therapy of NDV and anti-CTLA-4 was observed against both tumor injected and distant tumors, with a higher efficacy again observed in the group using the combination of NDV-ICOSL with anti-CTLA-4 (Figure 42A-D). In both tumor models, the animals that completely extinguished the tumors were again challenged with a lethal dose of tumor cells on day 90 without any further therapy and most animals demonstrated protection against the new challenge (Figure 41E and Figure 42E). Interestingly, while in the CT26 model all cured animals were protected against the new challenge, in the B16-F10 model the animals treated with the combination therapy demonstrated superior protection when compared to animals that were cured only by anti- CTLA-4 (Figure 41E), indicating that the combination leads to a more effective protective memory response approach. Combination therapy leads to better tumor infiltration with innate and adaptive immune cells
[289] Analysis of B16 tumors distant from animals treated with the combination of NDV and anti-CTLA-4 therapy demonstrated significant tumor infiltration with different immune cell subtypes (Figure 43A, B). The increase in infiltration was evident both in the innate immune compartments (Figure 43C, D) and in the adaptive ones (Figure 43E), with the greatest increase observed in the group treated with the combination of NDV-ICOSL and anti-CTLA-4. Interestingly, while this group demonstrated the highest number of CD8 + lymphocyte infiltration, there was also a statistically significant increase in regulatory T cells seen in this group (Figure 43E), although the overall percentage of Tregs was significantly decreased when compared to treated animals. or animals not treated with a single anti-CTLA-4 agent (Figure 43F), with the consequent increase in effectors for Treg rates (Figure 43G). A detailed analysis of TILs showed that TILs isolated from animals treated with the combination NDV-ICOSL and anti-CTLA-4 expressed the highest levels of activation, lytic, and proliferation markers ICOS, Granzima B, and Ki67, respectively (Figure 43H- J). Generation of recombinant NDV that expresses other costimulatory molecules.
[290] Therefore, this example demonstrates that the expression of a co-stimulating ligand by NDV can result in the activation of stronger immune responses, which can lead to more effective antitumor immunity, especially in the context of combination therapies with immune blockade of the pathway. signaling. To evaluate the additional costimulatory molecules, ligands targeting the immunoglobulin receptor superfamily (ICOS and CD28) and the TNF receptor superfamily (GITR, 4-1BB, 0X40, and CD40) were studied. To label CD28, an artificial ligand composed of a chimeric protein with the cytoplasmic and transmembrane domains of the NDV F glycoprotein and the extracellular domain composed of a single chain antibody against CD28 (aCD28-scFv) was manipulated (Figure 44A, B) . To target the ligands targeting the TNF receptor superfamily, the extracellular domain of each ligand was merged with the transmembrane and intracellular domains of the NDV HN glycoprotein to ensure increased expression of the ligands on the surface of infected cells (Figure 44A , B) . In addition, recombinant viruses that express the cytokines of the common gamma chain receptor family (IL-2, IL-15 and IL-21) have been generated. The resulting constructs are illustrated in the diagram in Figure 44C. The recombinant viruses were generated by reverse genetics and the presence of the viruses was confirmed by hemagglutination assays (Figure 45A). To ensure the fidelity of the inserted genes, RNA was isolated from each virus and RT-PCR was produced with annealing primers external to the region of the cloned gene (Figure 45B, C). The sequence of each gene was further confirmed by Sanger sequencing. To confirm the expression of the co-stimulating ligands on the surfaces of the infected cells, the cultured B16-F10 cells were infected in MOI of 2 and analyzed 24 hours later by flow cytometry with specific antibodies for each gene (Figure 46). NDV-4-1BBL induces increased infiltration of tumors with lymphocytes in distant tumors
[291] The ability of the modified viruses to demonstrate any evidence of the enhanced immune response was assessed from NDV-4-1BBL as an example. The rats with B16-F10 melanomas on both sides were treated with intratumor injection of a right tumor of control of NDV or NDV-4-1BBL, as previously described and distant tumors were collected on day 15. As can be seen in Figure 47 , therapy with NDV-4-1BBL shows a better infiltration of both adaptive and innate immune cells in contralateral tumors, consistent with the previous findings that demonstrate similar results with NDV that expresses ICOSL (Figure 40). Overall, these results suggest that the expression of immunostimulatory molecules by NDV in the context of the tumor microenvironment can lead to improved antitumor immunity.
[292] NDV-4-1BBL, NDV-GITRL, NDV-OX40L, NDV-CD40L, NDV-IL-2, NDV-IL-15, NDV-IL-21 viruses are evaluated for their ability to induce infiltration of the immune tumor using similar assays, as described in this Section 7. In addition, for therapeutic evaluation, each virus is evaluated in bilateral tumor models with the virus being administered to a single-sided tumor in combination with systemic antibodies intended for the DP-1, PD-L1, and / or CTLA-4 inhibitory signaling pathways Conclusion
[293] To cause the death of immunogenic tumor cells and an inflammatory response, non-pathogenic NDV was used, which, despite its relatively weak lytic activity, proved to be a potent inducer of IFN type I and DC maturation (Wilden et al. , 2009, Int J Oncol 34: 971; Kato et al., 2005, Immunity 23: 1). A model of bilateral melanoma with staggered implantation of tumors on a schedule that was previously demonstrated is not affected by the concomitant immunity that was used (Turk et al., 2004, J Exp Med 200: 771). This example demonstrates that the intratumoral injection of NDV results in immune distant tumor infiltration in the absence of the spread of the distant virus. Notably, this effect was associated with a relative reduction in the number of Tregs and a marked increase in effector CD4 and CD8 T cells for Treg rates, which has previously been shown to be a marker of a favorable immune response for immunotherapy (Quezada et al ., 2006, J Clin Invest 116: 1935; Curran et al., 2010, Proc Natl Acad Sci USA 107: 4275).
[294] The data in this example demonstrate that NDV increases tumor infiltration with tumor-specific lymphocytes, an effect that was dependent on the identity of the tumor injected with viruses. The increased infiltration of the tumor and the expansion of adoptively transferred lymphocytes also suggest the synergy between oncolytic virus therapy and therapeutic approaches using adoptive T cell transfer. It is plausible that tumor-specific lymphocytes undergoing activation and expansion at the site of the onset of viral infection, followed by their migration to other tumor sites, are likely to be dependent on lymphocyte and chemokine addressing receptors (Franciszkiewicz et al., 2012, Cancer Res 72: 6325). The data in this example also demonstrates that the infiltration of the distant immune tumor was in the non-specific part and can be induced by NDV infection of a heterologous tumor or by transferring serum from treated animals to untreated tumor-bearing rats. The increased vascular permeability induced by inflammatory cytokines, such as IL-6, can contribute strongly to tumor vasculature activation and lymphocyte recruitment in tumors (Fisher et al., 2011, The Journal of clinical investigation 121: 3846).
[295] Despite the pronounced increase in TILs, the therapeutic effect on distant tumors was quite modest with NDV monotherapy, highlighting the immunosuppressive nature of the microenvironment of these tumors (Spranger et al., 2013, Sei Transi Med 5). Notably, the combination of the systemic anti-CTLA-4 antibody with intratumoral NDV led to the rejection of distant B16-F10 tumors with the animals' long-term survival. The animals were also protected against a new tumor challenge, suggestive of establishing long-term memory. Interestingly, therapeutic efficacy was also observed with TRAMP C2 and CT26 tumor models, which exhibit low sensitivity to NDV-mediated cell lysis in vitro. These results highlight the importance of the anti-tumor inflammatory / immune response induced by NDV, instead of direct lysis, while the main mechanism leads to anti-tumor efficacy in this model. In fact, analysis of NDV-injected and distant tumors treated with combination therapy demonstrated prominent infiltration with innate immune cells and activated CD8 + and CD4 + effector cells, while depletion of CD8 + and NK cells nullified therapeutic efficacy. In addition, the combination strategy was completely ineffective in RIFNA - / - mice, which support the role of the type I IFN pathway in inducing antitumor immunity in this system (Fuertes et al., 2 011, J Exp Med 208, 2 005; Diamond et al., 2011, J Exp Med 208: 1989; Swann et al., 2007, J Immunol 178: 7540).
[296] In summary, this example demonstrates localized intratumoral therapy of melanoma B16 with NDV that induces inflammatory responses leading to infiltrated lymphocytes and antitumor effect in distant (uninjected) tumors without spread of the distant virus. The inflammatory effect coincided with the infiltration of the distant tumor with tumor-specific CD4 + and CD8 + T cells that were dependent on the identity of the tumor injected with virus. Combination therapy with localized NDV and systemic CTLA-4 blockade led to rejection of pre-established distant tumors and protection against new tumor challenge in models of poorly immunogenic tumors, regardless of the sensitivity of the tumor cell line to NDV-mediated lysis. The therapeutic effect was associated with infiltration of distant tumor marked with effectors CD8 + and CD4 + and CD8 + activated, but not with regulatory T cells, and was dependent on CD8 + cells, NK cells and type I interferon. This example demonstrates that therapy localized with oncolytic NDV induces inflammatory immune infiltrates in distant tumors, making them susceptible to systemic therapy with immunomodulatory antibodies.
[297] The invention is not to be limited by the scope of the specific modalities described here. In fact, several modifications of the invention in addition to those described, will be evident to those skilled in the art from the detailed description and attached figures. Such modifications are intended to fall within the scope of the appended claims.
[298] All references cited herein are hereby incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or application for a patent or patent was specifically and individually indicated to be incorporated by reference in its entirety for all the ends.

权利要求:
Claims (24)
[0001]
1. Use of a Newcastle disease virus (NDV), CHARACTERIZED because it is in the preparation of a medicine for use in combination with a programmed cell death antagonist 1 (PD1) to treat cancer in an individual in need of it, in that the PD1 antagonist is an antibody that specifically binds to PD1 and blocks binding to its native ligands.
[0002]
2. Use of a Newcastle disease chimeric virus (NDV), CHARACTERIZED for being in the preparation of a drug for use in combination with a PD1 antagonist for the treatment of cancer in a subject in need of it, in which the chimeric NDV comprises a packaged genome comprising a nucleotide sequence encoding a cytokine, where the cytokine is expressed by the virus, and where the PD1 antagonist is an antibody that specifically binds to PD1 and blocks binding to its native ligands.
[0003]
3. Use of a Newcastle disease chimeric virus (NDV), CHARACTERIZED for being in the preparation of a drug for use in combination with a PD1 antagonist for the treatment of cancer in a subject in need of it, in which the chimeric NDV comprises a packaged genome comprising a nucleotide sequence encoding a cytokine, where the cytokine is expressed by the virus, where the cytokine is interleukin-2 (IL-2), and where the PD1 antagonist is an antibody that specifically binds to PD1 and blocks binding to its native ligands.
[0004]
4. Use of a Newcastle disease chimeric virus (NDV), CHARACTERIZED for being in the preparation of a drug for use in combination with a PD1 antagonist for the treatment of cancer in a subject in need of it, in which the chimeric NDV comprises a packaged genome comprising a nucleotide sequence encoding a cytokine, where the cytokine is expressed by the virus, where the cytokine is a stimulating factor for granulocyte and macrophage colonies (GM-CSF), and where the PD1 antagonist is a antibody that specifically binds to PD1 and blocks binding to its native ligands.
[0005]
5. Use of a chimeric Newcastle disease virus (NDV), CHARACTERIZED for being in the preparation of a drug for use in combination with a PD1 antagonist for the treatment of cancer in a subject in need of it, in which the chimeric NDV comprises a packaged genome comprising a nucleotide sequence encoding a cytokine, in which the cytokine is expressed by the virus, and in which the PD1 antagonist is an antibody that specifically binds to PD1 and blocks binding to its native ligands, and in which the packaged genome of the chimeric NDV does not encode an additional heterologous protein.
[0006]
6. Use of a Newcastle disease chimeric virus (NDV), CHARACTERIZED for being in the preparation of a drug for use in combination with a PD1 antagonist for the treatment of cancer in a subject in need of it, in which the chimeric NDV comprises a packaged genome comprising a nucleotide sequence encoding a cytokine, in which the cytokine is expressed by the virus, in which the cytokine is IL-2, and in which the PD1 antagonist is an antibody that specifically binds to PD1 and blocks the binding to its native ligands, and where the packaged genome of the chimeric NDV does not encode an additional heterologous protein.
[0007]
7. Use of a chimeric Newcastle disease virus (NDV), CHARACTERIZED for being in the preparation of a drug for use in combination with a PD1 antagonist for the treatment of cancer in a subject in need of it, in which the chimeric NDV comprises a packaged genome comprising a nucleotide sequence encoding a cytokine, where the cytokine is expressed by the virus, where the cytokine is GM-CSF, and where the PD1 antagonist is an antibody that specifically binds to PD1 and blocks the binding to its native ligands, and where the packaged genome of the chimeric NDV does not encode an additional heterologous protein.
[0008]
8. Use according to claim 2 or 5, CHARACTERIZED by the fact that the cytokine is interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-17 (IL-17), interleukin- 22 (IL-22), interferon-gamma (IFN-gamma), or tumor necrosis factor alpha (TNF-alpha).
[0009]
9. Use according to claim 2 or 5, CHARACTERIZED by the fact that the cytokine is interleukin-21 (IL-21).
[0010]
10. Use according to claim 2 or 5, CHARACTERIZED by the fact that the cytokine is interleukin-15 (IL-15).
[0011]
11. Use, according to any one of claims 2 to 10, CHARACTERIZED by the fact that the NDV is of LaSota strain and the packaged genome comprises a nucleotide sequence that encodes an F protein mutated with the amino acid L289A mutation, in which the mutated F protein is expressed by NDV.
[0012]
12. Use according to claim 1, CHARACTERIZED by the fact that NDV is of La Sota strain and NDV comprises a packaged genome comprising a nucleotide sequence that encodes an F protein mutated with the amino acid L289A mutation, in which the mutated F protein is expressed by NDV.
[0013]
13. Use, according to any one of claims 1 to 10, CHARACTERIZED by the fact that the NDV is of the LaSota strain.
[0014]
14. Use according to any of claims 1 to 13, CHARACTERIZED by the fact that the antibody is a monoclonal antibody.
[0015]
15. NDV, according to any of claims 1 to 14, CHARACTERIZED by the fact that the antibody is MK3475.
[0016]
16. Use according to any one of claims 1 to 15, CHARACTERIZED by the fact that the cancer is melanoma, colon cancer, lung cancer, breast cancer, ovarian cancer, renal cell cancer, cell carcinoma scaly neck or head, pancreatic cancer or liver cancer.
[0017]
17. Use, according to any one of claims 1 to 15, CHARACTERIZED by the fact that the cancer is mesothelioma, colorectal cancer, kidney cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, cancer of esophagus, renal cell carcinoma, cervical cancer, uterine cancer, lung cancer, glioblastoma multiforme, melanoma, sarcoma, brain tumor, thyroid cancer, liver cancer, salivary gland cancer, bladder cancer, Hodgkin's disease, non-lymphoma -Hodgkin, endometrial carcinoma or hepatocellular carcinoma.
[0018]
18. Use, according to any of claims 1 to 17, CHARACTERIZED by the fact that the cancer is metastatic.
[0019]
19. Use, according to any of claims 1 to 18, CHARACTERIZED by the fact that the cancer is refractory or recurrent.
[0020]
20. Use, according to any of claims 1 to 19, CHARACTERIZED by the fact that NDV is formulated for intratumoral administration.
[0021]
21. Use, according to any one of claims 1 to 20, CHARACTERIZED by the fact that the PD1 antagonist is formulated for systemic administration.
[0022]
22. Use according to any one of claims 1 to 20, CHARACTERIZED by the fact that the PD1 antagonist is formulated for intravenous administration.
[0023]
23. Use, according to any one of claims 1 to 20, CHARACTERIZED by the fact that the PD1 antagonist is formulated for subcutaneous administration.
[0024]
24. Use, according to any of claims 1 to 23, CHARACTERIZED by the fact that the subject is a human being.

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

DE3806565C2|1988-03-01|1991-04-25|Deutsches Krebsforschungszentrum Stiftung Des Oeffentlichen Rechts, 6900 Heidelberg, De|

---

DE3922444A1|1988-03-01|1991-01-10|Deutsches Krebsforsch|Virus modified tumour specific vaccine - contains immunostimulating substances, and is used in immuno-therapy of tumours|

---

US5223409A|1988-09-02|1993-06-29|Protein Engineering Corp.|Directed evolution of novel binding proteins|

---

US5166057A|1989-08-28|1992-11-24|The Mount Sinai School Of Medicine Of The City University Of New York|Recombinant negative strand rna virus expression-systems|

---

US5854037A|1989-08-28|1998-12-29|The Mount Sinai School Of Medicine Of The City University Of New York|Recombinant negative strand RNA virus expression systems and vaccines|

---

US5786199A|1989-08-28|1998-07-28|The Mount Sinai School Of Medicine Of The City University Of New York|Recombinant negative strand RNA virus expression systems and vaccines|

---

ES2196026T3|1993-04-30|2003-12-16|Wellstat Biologics Corp|USE OF NDV IN THE MANUFACTURE OF A MEDICINAL PRODUCT TO TREAT CANCER.|

---

DE69532369T3|1994-07-18|2010-11-04|Conzelmann, Karl-Klaus, Prof. Dr.|Recombinant infectious non-segment shared negative-stranded RNA virus|

---

US5891680A|1995-02-08|1999-04-06|Whitehead Institute For Biomedical Research|Bioactive fusion proteins comprising the p35 and p40 subunits of IL-12|

---

US7153510B1|1995-05-04|2006-12-26|Yale University|Recombinant vesiculoviruses and their uses|

---

AT181112T|1995-08-09|1999-06-15|Schweiz Serum & Impfinst|CDNA CORRESPONDING TO THE GENOM OF MINUS-STRING RNA VIRUS AND METHOD FOR PRODUCING INFECTIOUS MINUS-STRING RNA VIRUS|

---

AU727923B2|1995-09-27|2001-01-04|Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The|Production of infectious respiratory syncytial virus from cloned nucleotide sequences|

---

CA2231603C|1995-10-17|2009-04-21|Wayne State University|Chicken interleukin-15 and uses thereof|

---

AU3799797A|1996-07-15|1998-02-09|Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The|Production of attenuated respiratory syncytial virus vaccines from cloned nucleotide sequences|

---

KR20000048628A|1996-09-27|2000-07-25|윌리암 에이취 캘넌, 에곤 이 버그|3' genomic promoter region and polymerase gene mutations responsible for attenuation in viruses of the order designated mononegavirales|

---

CN1250725C|1997-05-23|2006-04-12|美国政府健康及人类服务部|Prodn of attenuated parainfluenza virus vaccines from cloned nucleotide sequences|

---

DK1012244T3|1997-07-11|2007-09-10|Univ Yale|Rhabdovira with genetically engineered jackets|

---

BR9812232A|1997-09-19|2000-07-18|American Cyanamid Co|Human respiratory syncytial virus human subgroup b, isolated, recombinantly generated, attenuated, vaccine, process to immunize an individual to induce protection against rsv subgroup b, composition, process to produce attenuated infectious rsv subgroup b, and isolated acid molecule nucleic|

---

HU0003911A3|1997-10-09|2007-11-28|Pro Virus|Treatment of neoplasms with viruses|

---

US7780962B2|1997-10-09|2010-08-24|Wellstat Biologics Corporation|Treatment of neoplasms with RNA viruses|

---

US7470426B1|1997-10-09|2008-12-30|Wellstat Biologics Corporation|Treatment of neoplasms with viruses|

---

US20030044384A1|1997-10-09|2003-03-06|Pro-Virus, Inc.|Treatment of neoplasms with viruses|

---

BR9911163A|1998-06-12|2002-01-15|Sinai School Medicine|Vaccine formulation, pharmaceutical formulation, attenuated influenza virus, processes to vaccinate an individual, to prevent an infectious disease in an individual, and, to treat or prevent tumors in an individual|

---

EP0974660A1|1998-06-19|2000-01-26|Stichting Instituut voor Dierhouderij en Diergezondheid |Newcastle disease virus infectious clones, vaccines and diagnostic assays|

---

US6544785B1|1998-09-14|2003-04-08|Mount Sinai School Of Medicine Of New York University|Helper-free rescue of recombinant negative strand RNA viruses|

---

US6146642A|1998-09-14|2000-11-14|Mount Sinai School Of Medicine, Of The City University Of New York|Recombinant new castle disease virus RNA expression systems and vaccines|

---

US7052685B1|1998-10-15|2006-05-30|Trustees Of The University Of Pennsylvania|Methods for treatment of cutaneous T-cell lymphoma|

---

EP1390046A4|1999-04-15|2005-04-20|Wellstat Biologics Corp|Treatment of neoplasms with viruses|

---

US7244558B1|1999-05-05|2007-07-17|University Of Maryland|Production of novel Newcastle disease virus strains from cDNAs and improved live attenuated Newcastle disease vaccines|

---

DE122008000056I1|1999-07-14|2009-04-09|Sinai School Medicine|IN VITRO RECONSTITUTION OF SEGMENTED NEGATIVE RADIUS RNA VIRUSES|

---

WO2001020989A1|1999-09-22|2001-03-29|Mayo Foundation For Medical Education And Research|Therapeutic methods and compositions using viruses of the recombinant paramyxoviridae family|

---

US6896881B1|1999-09-24|2005-05-24|Mayo Foundation For Medical Education And Research|Therapeutic methods and compositions using viruses of the recombinant paramyxoviridae family|

---

ES2246314T3|2000-01-20|2006-02-16|Universitat Zurich Institut Fur Medizinische Virologie|INTRATUMORAL ADMINISTRATION OF NUCLEIC NUDE ACID MOLECULES CODING IL-12.|

---

AU5700101A|2000-04-10|2001-10-23|Sinai School Medicine|Screening methods for identifying viral proteins with interferon antagonizing functions and potential antiviral agents|

---

US6818444B2|2000-08-04|2004-11-16|Heska Corporation|Canine and feline proteins, nucleic acid molecules and uses thereof|

---

FR2823222B1|2001-04-06|2004-02-06|Merial Sas|VACCINE AGAINST NILE FEVER VIRUS|

---

WO2002102404A1|2001-06-18|2002-12-27|Institut National De La Recherche Agronomique|Uses of cytokines|

---

US20030224017A1|2002-03-06|2003-12-04|Samal Siba K.|Recombinant Newcastle disease viruses useful as vaccines or vaccine vectors|

---

WO2003092579A2|2002-04-29|2003-11-13|Hadasit Medical Research Services And Development Company Ltd.|Compositions and methods for treating cancer with an oncolytic viral agent|

---

AU2003241953A1|2002-06-03|2003-12-19|Dnavec Research Inc.|Pramyxovirus vectors encoding antibody and utilization thereof|

---

EP1543418B1|2002-08-07|2016-03-16|MMagix Technology Limited|Apparatus, method and system for a synchronicity independent, resource delegating, power and instruction optimizing processor|

---

SE0203159D0|2002-10-25|2002-10-25|Electrolux Ab|Handle for a motor driven handheld tool|

---

US9068234B2|2003-01-21|2015-06-30|Ptc Therapeutics, Inc.|Methods and agents for screening for compounds capable of modulating gene expression|

---

US20040197312A1|2003-04-02|2004-10-07|Marina Moskalenko|Cytokine-expressing cellular vaccine combinations|

---

AT516305T|2004-02-27|2011-07-15|Inst Nat Sante Rech Med|IL-15 Binding Site for IL-15 RALPHA and Specific IL-15 Mutants Having Activity as Agonists / Antagonists|

---

NZ591733A|2004-11-12|2012-10-26|Bayer Schering Pharma Ag|Recombinant newcastle disease virus comprising a transgene encoding a fusion protein comprising a binding domain and a heterologous domain comprising an enzyme|

---

JP5236461B2|2005-05-17|2013-07-17|ユニバーシティオブコネチカット|Compositions and methods for immunomodulation in organisms|

---

WO2007008918A2|2005-07-08|2007-01-18|Wayne State University|Virus vaccines comprising envelope-bound immunomodulatory proteins and methods of use thereof|

---

EP1777294A1|2005-10-20|2007-04-25|Institut National De La Sante Et De La Recherche Medicale |IL-15Ralpha sushi domain as a selective and potent enhancer of IL-15 action through IL-15Rbeta/gamma, and hyperagonist fusion proteins|

---

EP2529747B1|2005-12-02|2018-02-14|Icahn School of Medicine at Mount Sinai|Chimeric Newcastle Disease virusespresenting non-native surface proteins and uses thereof|

---

CN102796743B|2006-01-13|2015-11-18|美国政府健康及人类服务部国立卫生研究院|For codon optimized IL-15 and the IL-15R α gene of expressing in mammalian cell|

---

WO2007113648A2|2006-04-05|2007-10-11|Pfizer Products Inc.|Ctla4 antibody combination therapy|

---

US20090175826A1|2006-06-05|2009-07-09|Elankumaran Subbiah|Genetically-engineered newcastle disease virus as an oncolytic agent, and methods of using same|

---

WO2008011726A1|2006-07-27|2008-01-31|Ottawa Health Research Institute|Staged immune-response modulation in oncolytic therapy|

---

US8765462B2|2007-05-04|2014-07-01|University Health Network|IL-12 immunotherapy for cancer|

---

AU2008253720B2|2007-05-11|2014-01-16|Altor Bioscience Corporation|Fusion molecules and IL-15 variants|

---

WO2008156712A1|2007-06-18|2008-12-24|N. V. Organon|Antibodies to human programmed death receptor pd-1|

---

WO2009002562A2|2007-06-27|2008-12-31|Marine Polymer Technologies, Inc.|Complexes of il-15 and il-15ralpha and uses thereof|

---

EP2085092A1|2008-01-29|2009-08-05|Bayer Schering Pharma Aktiengesellschaft|Attenuated oncolytic paramyxoviruses encoding avian cytokines|

---

DK2350129T3|2008-08-25|2015-08-31|Amplimmune Inc|PREPARATIONS WITH PD-1 ANTAGONISTS AND PROCEDURES FOR USE THEREOF|

---

US8475790B2|2008-10-06|2013-07-02|Bristol-Myers Squibb Company|Combination of CD137 antibody and CTLA-4 antibody for the treatment of proliferative diseases|

---

CN101787373B|2009-01-23|2013-06-19|中国人民解放军第二军医大学东方肝胆外科医院|Foreign gene-carrying recombinant virus vector efficiently produced in packaging cell and construction method and application thereof|

---

US8591881B2|2009-02-05|2013-11-26|Mount Sinai School Of Medicine|Chimeric Newcastle disease viruses and uses thereof|

---

CA2760446C|2009-04-30|2018-01-02|The United States Of America, As Represented By The Secretary, Department Of Health And Human Services|Inducible interleukin-12|

---

US20100297072A1|2009-05-19|2010-11-25|Depinho Ronald A|Combinations of Immunostimulatory Agents, Oncolytic Virus, and Additional Anticancer Therapy|

---

AU2010282280B2|2009-08-14|2016-06-09|The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services|Use of IL-15 to increase thymic output and to treat lymphopenia|

---

ES2622087T3|2009-08-21|2017-07-05|Merial, Inc.|Recombinant vaccine against avian paramyxovirus and its manufacturing and use procedure|

---

CA2775761C|2009-09-30|2018-08-28|Memorial Sloan-Kettering Cancer Center|Combination immunotherapy for the treatment of cancer|

---

US10238734B2|2010-03-23|2019-03-26|The Regents Of The University Of California|Compositions and methods for self-adjuvanting vaccines against microbes and tumors|

---

WO2012000188A1|2010-06-30|2012-01-05|Tot Shanghai Rd Center Co., Ltd.|Recombinant tumor vaccine and method of producing such|

---

ES2651170T3|2010-09-21|2018-01-24|Altor Bioscience Corporation|Soluble multimeric fusion molecules of IL-15 and methods for making and using them|

---

US11149254B2|2011-04-15|2021-10-19|Genelux Corporation|Clonal strains of attenuated vaccinia viruses and methods of use thereof|

---

EP2537933A1|2011-06-24|2012-12-26|Institut National de la Santé et de la Recherche Médicale |An IL-15 and IL-15Ralpha sushi domain based immunocytokines|

---

EP2766035B1|2011-10-11|2018-03-28|Universität Zürich Prorektorat MNW|Combination medicament comprising il-12 and an agent for blockade of t-cell inhibitory molecules for tumour therapy|

---

WO2013112942A1|2012-01-25|2013-08-01|Dna Trix, Inc.|Biomarkers and combination therapies using oncolytic virus and immunomodulation|

---

EP2669381A1|2012-05-30|2013-12-04|AmVac AG|Method for expression of heterologous proteins using a recombinant negative-strand RNA virus vector comprising a mutated P protein|

---

US20150250837A1|2012-09-20|2015-09-10|Morningside Technology Ventures Ltd.|Oncolytic virus encoding pd-1 binding agents and uses of the same|

---

EP2911684B1|2012-10-24|2019-06-19|Novartis Ag|Il-15r alpha forms, cells expressing il-15r alpha forms, and therapeutic uses of il-15r alpha and il-15/il-15r alpha complexes|

---

US20160015760A1|2013-03-14|2016-01-21|Icahn School Of Medicine At Mount Sinai|Newcastle disease viruses and uses thereof|

---

WO2014170032A1|2013-04-19|2014-10-23|Cytune Pharma|Cytokine derived treatment with reduced vascular leak syndrome|

---

DK3030575T3|2013-08-08|2018-10-22|Cytune Pharma|IL-15 AND IL-15R-ALFA-SUSHI DOMAIN-BASED MODULOKINES|

---

ES2760249T3|2013-08-08|2020-05-13|Cytune Pharma|Combined pharmaceutical composition|

---

ME03345B|2013-09-03|2019-10-20|Medimmune Ltd|Compositions featuring an attenuated newcastle disease virus and methods of use for treating neoplasia|

---

AU2015222685A1|2014-02-27|2016-09-08|Viralytics Limited|Combination method for treatment of cancer|

---

EP2915569A1|2014-03-03|2015-09-09|Cytune Pharma|IL-15/IL-15Ralpha based conjugates purification method|

---

JP6655061B2|2014-07-29|2020-02-26|ノバルティス アーゲー|IL-15 and IL-15Ralpha heterodimer dose escalation regimens for treating conditions|

---

WO2016048903A1|2014-09-22|2016-03-31|Intrexon Corporation|Improved therapeutic control of heterodimeric and single chain forms of interleukin-12|

---

RU2017123117A3|2014-12-09|2019-07-17|

---

CN106166294A|2015-05-18|2016-11-30|国科丹蓝生物科技（北京）有限公司|A kind of compound for preoperative intervention radiotherapy in the treatment tumor|

---

EP3316902A1|2015-07-29|2018-05-09|Novartis AG|Combination therapies comprising antibody molecules to tim-3|

---

EP3328418A1|2015-07-29|2018-06-06|Novartis AG|Combination therapies comprising antibody molecules to pd-1|

---

EP3317301B1|2015-07-29|2021-04-07|Novartis AG|Combination therapies comprising antibody molecules to lag-3|

---

WO2017062953A1|2015-10-10|2017-04-13|Intrexon Corporation|Improved therapeutic control of proteolytically sensitive, destabilized forms of interleukin-12|

---

CN108350037A|2015-11-09|2018-07-31|免疫设计股份有限公司|Include the composition and its application method of the slow virus carrier of expression IL-12|

---

EP3805376A1|2016-01-08|2021-04-14|Replimune Limited|Engineered virus|

---

JP2019503398A|2016-01-15|2019-02-07|アールエフイーエムビー ホールディングス リミテッド ライアビリティ カンパニー|Immunological treatment of cancer|

---

US10344067B2|2016-02-25|2019-07-09|Deutsches Krebsforschungszentrum|RNA viruses expressing IL-12 for immunovirotherapy|

---

CN105734023B|2016-03-28|2019-04-26|江苏康缘瑞翱生物医药科技有限公司|A kind of recombinant Newcastle disease virus is preparing the application in medicines resistant to liver cancer|

---

EP3448401B1|2016-04-29|2021-10-27|Virogin Biotech Canada Ltd|Hsv vectors with enhanced replication in cancer cells|EP2529747B1|2005-12-02|2018-02-14|Icahn School of Medicine at Mount Sinai|Chimeric Newcastle Disease virusespresenting non-native surface proteins and uses thereof|

---

WO2008156712A1|2007-06-18|2008-12-24|N. V. Organon|Antibodies to human programmed death receptor pd-1|

---

US8591881B2|2009-02-05|2013-11-26|Mount Sinai School Of Medicine|Chimeric Newcastle disease viruses and uses thereof|

---

US20160015760A1|2013-03-14|2016-01-21|Icahn School Of Medicine At Mount Sinai|Newcastle disease viruses and uses thereof|

---

ME03345B|2013-09-03|2019-10-20|Medimmune Ltd|Compositions featuring an attenuated newcastle disease virus and methods of use for treating neoplasia|

---

RS59480B1|2013-12-12|2019-12-31|Shanghai hengrui pharmaceutical co ltd|Pd-1 antibody, antigen-binding fragment thereof, and medical application thereof|

---

US10618963B2|2014-03-12|2020-04-14|Yeda Research And Development Co. Ltd|Reducing systemic regulatory T cell levels or activity for treatment of disease and injury of the CNS|

---

CA2942245C|2014-03-12|2021-11-02|Yeda Research And Development Co. Ltd.|Reducing systemic regulatory t cell levels or activity for treatment of disease and injury of the cns|

---

US9394365B1|2014-03-12|2016-07-19|Yeda Research And Development Co., Ltd|Reducing systemic regulatory T cell levels or activity for treatment of alzheimer's disease|

---

US10519237B2|2014-03-12|2019-12-31|Yeda Research And Development Co. Ltd|Reducing systemic regulatory T cell levels or activity for treatment of disease and injury of the CNS|

---

SG11201700476VA|2014-07-21|2017-02-27|Novartis Ag|Treatment of cancer using humanized anti-bcma chimeric antigen receptor|

---

KR20170029009A|2014-07-21|2017-03-14|노파르티스 아게|Treatment of cancer using a cd33 chimeric antigen receptor|

---

WO2016014530A1|2014-07-21|2016-01-28|Novartis Ag|Combinations of low, immune enhancing. doses of mtor inhibitors and cars|

---

US20170211055A1|2014-07-21|2017-07-27|Novartis Ag|Sortase synthesized chimeric antigen receptors|

---

EP3660042A1|2014-07-31|2020-06-03|Novartis AG|Subset-optimized chimeric antigen receptor-containing t-cells|

---

CA2958200A1|2014-08-14|2016-02-18|Novartis Ag|Treatment of cancer using a gfr alpha-4 chimeric antigen receptor|

---

PL3183268T3|2014-08-19|2020-09-07|Novartis Ag|Anti-cd123 chimeric antigen receptorfor use in cancer treatment|

---

BR112017005390A2|2014-09-17|2017-12-12|Novartis Ag|target cytotoxic cells with chimeric receptors for adoptive immunotherapy|

---

MA41044A|2014-10-08|2017-08-15|Novartis Ag|COMPOSITIONS AND METHODS OF USE FOR INCREASED IMMUNE RESPONSE AND CANCER TREATMENT|

---

CR20170143A|2014-10-14|2017-06-19|Dana-Farber Cancer Inst Inc|ANTIBODY MOLECULES THAT JOIN PD-L1 AND USES OF THE SAME|

---

WO2016090034A2|2014-12-03|2016-06-09|Novartis Ag|Methods for b cell preconditioning in car therapy|

---

CN105985966A|2015-03-06|2016-10-05|普莱柯生物工程股份有限公司|Gene VII-type newcastle disease virus strain, vaccine composition thereof and preparing method and application of vaccine composition|

---

RU2752918C2|2015-04-08|2021-08-11|Новартис Аг|Cd20 therapy, cd22 therapy and combination therapy with cells expressing chimeric antigen receptork cd19|

---

GB201509338D0|2015-05-29|2015-07-15|Bergenbio As|Combination therapy|

---

EP3322732A2|2015-07-13|2018-05-23|Cytomx Therapeutics Inc.|Anti-pd-1 antibodies, activatable anti-pd-1 antibodies, and methods of use thereof|

---

BR112018000903A2|2015-07-16|2018-09-11|Biokine Therapeutics Ltd.|compositions and methods for cancer treatment|

---

EP3317301B1|2015-07-29|2021-04-07|Novartis AG|Combination therapies comprising antibody molecules to lag-3|

---

EP3316902A1|2015-07-29|2018-05-09|Novartis AG|Combination therapies comprising antibody molecules to tim-3|

---

US20180371093A1|2015-12-17|2018-12-27|Novartis Ag|Antibody molecules to pd-1 and uses thereof|

---

EP3805376A1|2016-01-08|2021-04-14|Replimune Limited|Engineered virus|

---

US20200281973A1|2016-03-04|2020-09-10|Novartis Ag|Cells expressing multiple chimeric antigen receptormolecules and uses therefore|

---

CN110225927A|2016-10-07|2019-09-10|诺华股份有限公司|Chimeric antigen receptor for treating cancer|

---

EP3538546A1|2016-11-14|2019-09-18|Novartis AG|Compositions, methods, and therapeutic uses related to fusogenic protein minion|

---

US20200024351A1|2017-04-03|2020-01-23|Jounce Therapeutics, Inc.|Compositions and Methods for the Treatment of Cancer|

---

EP3615055A1|2017-04-28|2020-03-04|Novartis AG|Cells expressing a bcma-targeting chimeric antigen receptor, and combination therapy with a gamma secretase inhibitor|

---

EP3615068A1|2017-04-28|2020-03-04|Novartis AG|Bcma-targeting agent, and combination therapy with a gamma secretase inhibitor|

---

TW201900191A|2017-05-12|2019-01-01|美國西奈山伊坎醫學院|Xincheng chicken plague virus and its use|

---

EP3746116A1|2018-01-31|2020-12-09|Novartis AG|Combination therapy using a chimeric antigen receptor|

---

WO2019227003A1|2018-05-25|2019-11-28|Novartis Ag|Combination therapy with chimeric antigen receptortherapies|

---

BR112020025048A2|2018-06-13|2021-04-06|Novartis Ag|BCMA CHEMICAL ANTIGEN RECEPTORS AND USES OF THE SAME|

---

EP3856779A1|2018-09-28|2021-08-04|Novartis AG|Cd22 chimeric antigen receptortherapies|

---

EP3856782A1|2018-09-28|2021-08-04|Novartis AG|Cd19 chimeric antigen receptorand cd22 car combination therapies|

---

WO2020075672A1|2018-10-09|2020-04-16|バイオコモ株式会社|Anticancer agent, pharmaceutical composition for cancer treatment, and kit|

---

CN109627336A|2018-12-20|2019-04-16|南京昂科利医药科技创新研究院有限公司|A kind of preparation method and application of newcastle disease oncolytic virus that expressing PD-L1 single-chain antibody|

---

WO2020185739A1|2019-03-11|2020-09-17|Jounce Therapeutics, Inc.|Anti-icos antibodies for the treatment of cancer|

---

CA3134144A1|2019-03-29|2020-10-08|Myst Therapeutics, Llc|Ex vivo methods for producing a t cell therapeutic and related compositions and methods|

---

EP3725370A1|2019-04-19|2020-10-21|ImmunoBrain Checkpoint, Inc.|Modified anti-pd-l1 antibodies and methods and uses for treating a neurodegenerative disease|

---

CN110564766A|2019-09-20|2019-12-13|华农（肇庆）生物产业技术研究院有限公司|Preparation method of whole genome expression vector pBR322-DHN3|

---

WO2021091960A1|2019-11-05|2021-05-14|Jounce Therapeutics, Inc.|Methods of treating cancer with anti-pd-1 antibodies|

---

WO2021108613A1|2019-11-26|2021-06-03|Novartis Ag|Cd19 and cd22 chimeric antigen receptors and uses thereof|

---

WO2021108727A1|2019-11-27|2021-06-03|Myst Therapeutics, Inc.|Method of producing tumor-reactive t cell composition using modulatory agents|

---

WO2021174208A1|2020-02-27|2021-09-02|Myst Therapeutics, Llc|Methods for ex vivo enrichment and expansion of tumor reactive t cells and related compositions thereof|

法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-02-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-10-29| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-06-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-11-10| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 04/03/2014, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201361782994P| true| 2013-03-14|2013-03-14|

---

US61/782,994|2013-03-14|

---

PCT/US2014/020299|WO2014158811A1|2013-03-14|2014-03-04|Newcastle disease viruses and uses thereof|

---