Methods of treating cancer

Abstract

To provide methods of treating cancer.SOLUTION: Disclosed herein is a method for treating cancer by administering a heterologous prime-boost regimen of an oncolytic microorganism that enhances or elicits an immune response to a tumor protein not encoded by the oncolytic microorganism. The method provided herein includes a method for eliciting or enhancing immune response in vivo in a subject. The methods described herein feature, inter alia, methods of treating cancer by administering a heterologous prime-boost regimen of oncolytic microorganisms that enhances or elicits an immune response to a tumor protein that is not coded for by the oncolytic microorganisms.SELECTED DRAWING: None

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 789,164, filed Jan. 7, 2019, which is incorporated by reference in its entirety. [Background technology]

[0002] Various immunotherapeutic approaches are being evaluated for the treatment of several cancers. However, there remains a need for immunotherapies and regimens capable of generating robust immune responses that are specifically directed against cancer cells. Summary of the Invention [Means for solving the problem]

[0003] The methods described herein feature, inter alia, methods of treating cancer by administering a heterologous prime-boost regimen of an oncolytic microorganism that enhances or elicits an immune response to an oncoprotein not encoded by the oncolytic microorganism.

[0004] One embodiment provides a method of treating a tumor in a subject comprising: administering to the subject a first recombinant microorganism, where the first recombinant microorganism replicates in tumor cells but does not replicate in non-tumor cells or exhibits attenuated replication in non-tumor cells; and administering to the subject a second recombinant microorganism, where the second recombinant microorganism replicates in tumor cells but does not replicate in non-tumor cells or exhibits attenuated replication in non-tumor cells, wherein the first recombinant microorganism and the second recombinant microorganism are different and at least one of: a) enhancing or eliciting an immune response to tumor proteins that are not encoded or expressed by the first and second recombinant microorganisms; b) enhancing or eliciting an immune response to proteins expressed by tumor-associated cells that are not encoded or expressed by the first and second recombinant microorganisms. In some embodiments, the immune response is demonstrated by one or more of: a decrease in tumor volume in the subject, a decrease in expression levels of one or more tumor proteins in the subject or a sample from the subject, a decrease in the number of tumor sites in the subject, a change in viral load in the subject or a sample from the subject, a change in immune cell population in the subject or a sample from the subject, a change in expression levels of immune cell markers in the subject or a sample from the subject, enhanced B cell proliferation in the subject or a sample from the subject, enhanced CD4+ T cell proliferation in the subject or a sample from the subject, enhanced CD8+ T cell proliferation in the subject or a sample from the subject, enhanced cytokine production in the subject or a sample from the subject, enhanced antigen presenting cell proliferation in the subject or a sample from the subject, or any combination thereof. In some embodiments, the immune response is demonstrated by a decreased expression level of one or more tumor proteins, a change in immune cell population, a change in expression level of immune cell markers, enhanced B cell proliferation, enhanced CD4+ T cell proliferation, enhanced CD8+ T cell proliferation, enhanced cytokine production, enhanced antigen presenting cell proliferation, or any combination thereof in the subject or a sample from the subject, where the sample from the subject is a blood, tissue, urine or saliva sample.

[0005] In some embodiments, the immune response can be detected at the time of administration of the first or second recombinant microorganism or at a time point thereafter. In some embodiments, the first recombinant microorganism does not replicate in non-tumor cells. In some embodiments, the first recombinant microorganism exhibits attenuated replication in non-tumor cells. In some embodiments, the infectivity of the first recombinant microorganism is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% lower in non-tumor cells compared to tumor cells. In some embodiments, the replication efficiency of the first recombinant microorganism is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% lower in non-tumor cells compared to tumor cells. In some embodiments, the second recombinant microorganism does not replicate in non-tumor cells. In some embodiments, the second recombinant microorganism exhibits attenuated replication in non-tumor cells. In some embodiments, the infectivity of the second recombinant microorganism is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% lower in non-tumor cells compared to tumor cells. In some embodiments, the replication efficiency of the second recombinant microorganism is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% lower in non-tumor cells compared to tumor cells. In some embodiments, the first recombinant microorganism is a bacterium, a virus or a parasite. In some embodiments, the second recombinant microorganism is a bacterium, a virus or a parasite. In some embodiments, the first recombinant microorganism is a bacterium. In some embodiments, the first recombinant microorganism is an Enterobacteriaceae bacterium or a Streptococcus bacterium. In some embodiments, the bacterium is an Enterobacteriaceae bacterium. In some embodiments, the Enterobacteriaceae bacterium is a Salmonella bacterium. In some embodiments, the Salmonella bacterium is S. bongori or S. enterica. In some embodiments, the bacterium is a bacterium of the Listeriaceae family. In some embodiments, the Listeriaceae bacterium is Listeria monocytogenes. In some embodiments, the Listeria monocytogenes is L. monocytogenes. In some embodiments, the bacterium is a bacterium of the Streptococcus family. In some embodiments, the Streptococcus family bacterium is a bacterium of the lactococcus genus.In some embodiments, the Lactococcus bacterium is L. chungangensis, L. formosensis, L. fujiensis, L. garvieae, L. hircilactis, L. lactis, L. laudensis, L. nasutitermitis, L. piscium, L. plantarum, L. raffinolactis or L. taiwanensis. In some embodiments, the first recombinant microorganism is a parasite. In some embodiments, the parasite is a sarcocystidae parasite or a trypanosomatida parasite. In some embodiments, the parasite is a sarcocystidae parasite. In some embodiments, the sarcocystidae parasite is a Toxoplasma parasite. In some embodiments, the Toxoplasma parasite is Toxoplasma gondii. In some embodiments, the parasite is a trypanosomatidae parasite. In some embodiments, the Trypanosomatidae parasite is a Leishmania parasite. In some embodiments, the Leishmania parasite is L. aethiopica, L. amazonensis, L. Arabica, L. aristedesi, L. braziliensis, L. chagasi, L. colombiensis, L. deanei, L. donovani, L. enriettii, L. forattinii, L. garnhami, L. guyanensis, L. herreri, L. hertigi, L. infantum, L. killicki, L. lainsoni, L. major, L. mexicana, L. naiffi, L. panamensis, L. peruviana, L. pifanoi, L. shawi, L. tarentolae, L. tropica, L. turanica, or L. venezuelensis. In some embodiments, the first recombinant microorganism is a virus. In some embodiments, the first recombinant microorganism is of the Viral family. In some embodiments, the first recombinant microorganism is a DNA virus. In some embodiments, the first recombinant microorganism is an RNA virus.In some embodiments, the first recombinant microorganism comprises a poxvirus, a picornavirus, an adenovirus, a parvovirus, a herpesvirus, a reovirus, a paramyxovirus, a rhabdovirus, an orthomyxovirus, or a coxsackievirus. In some embodiments, the first recombinant microorganism is a poxvirus. In some embodiments, the poxvirus is an orthopoxvirus, a parapoxvirus, a yatapoxvirus, a leporipoxvirus, a mulluscipoxvirus, a betaentomopoxvirus, a cervidpoxvirus, a gammaentomopoxvirus, a suipoxvirus, a crocodylidpoxvirus, an alphaentomopoxvirus, a capripoxvirus, or an avipoxvirus. In some embodiments, the poxvirus is an orthopoxvirus. In some embodiments, the orthopoxvirus is a vaccinia virus. In some embodiments, the poxvirus is a leporipoxvirus. In some embodiments, the leporipoxvirus is a myxoma virus, hi some embodiments, the poxvirus is a picornavirus.In some embodiments, the picornavirus is an aphthovirus, aquamavirus, avihepatovirus, abyssivirus, cardiovirus, cosavirus, dicipivirus, enterovirus, erbovirus, gallivirus, hepatovirus, hunnivirus, kobuvirus, kunsagivirus, megrivirus, missvirus, or the like. In some embodiments, the picornavirus is an enterovirus. In some embodiments, the enterovirus is a poliovirus or a coxsackievirus. In some embodiments, the picornavirus is a cardiovirus. In some embodiments, the cardiovirus is a mengovirus. In some embodiments, the first recombinant microorganism is an adenovirus. In some embodiments, the adenovirus is an atadenovirus, an avian adenovirus, an ichtadenovirus, a mastadenovirus, or a siadenovirus. In some embodiments, the first recombinant microorganism is a parvovirus.In some embodiments, the parvovirus is an amdoparvovirus, an aveparvovirus, a bocaparvovirus, a chapparvovirus, a copiparvovirus, a dependoparvovirus, an erythroparvovirus, a protoparvovirus, or a tetraparvovirus. In some embodiments, the parvovirus is a dependoparvovirus. In some embodiments, the dependoparvovirus is an adeno-associated virus. In some embodiments, the first recombinant microorganism is a herpesvirus. In some embodiments, the herpesvirus is an alphaherpesvirus, a betaherpesvirus, or a gammaherpesvirus. In some embodiments, the herpesvirus is an alphaherpesvirus. In some embodiments, the alphaherpesvirus is an irtovirus, a mardivirus, a simplexvirus, or a varicellovirus. In some embodiments, the herpesvirus is a beta-herpesvirus. In some embodiments, the beta-herpesvirus is a cytomegalovirus, a muromegalovirus, a proboscivirus or a poseolovirus. In some embodiments, the herpesvirus is a gamma-herpesvirus. In some embodiments, the gamma-herpesvirus is a lymphocryptovirus, a macavirus, a percavirus or a rhadinovirus. In some embodiments, the first recombinant microorganism is a rhabdovirus.In some embodiments, the rhabdovirus is a curiovirus, a cytorhabdovirus, a dichorhavirus, an ephemerovirus, a hapavirus, a ledantevirus, a lyssavirus, a novirhabdovirus, a nucleorhabdovirus, a perhabdovirus, a sigmavirus, a sprivivirus, a sripuvirus, a tibrovirus, a tupavirus, a varicosavirus, or a vesiculovirus. In some embodiments, the rhabdovirus is a vesiculovirus. In some embodiments, the vesiculovirus is a vesicular stomatitis virus or a marba virus. In some embodiments, the first recombinant microorganism is a reovirus. In some embodiments, the reovirus is a sedoreovirus or a spinareovirus. In some embodiments, the reovirus is a sedoreovirus.

[0006] In some embodiments, the cedreovirus is a cardoreovirus, a mimoreovirus, an orbivirus, a phytoreovirus, a rotavirus, or a seaadornavirus. In some embodiments, the reovirus is a spinaleovirus. In some embodiments, the spinaleovirus is an aquareovirus, a coltivirus, a cypovirus, a fijivirus, an orthoreovirus, an idnoreovirus, a dinovernavirus, an oryzavirus, or a mycoreovirus. In some embodiments, the first recombinant microorganism is a paramyxovirus. In some embodiments, the paramyxovirus is an aquaparamyxovirus, an abulavirus, a ferlavirus, a henipavirus, a morbillivirus, a respirovirus, or a rubulavirus. In some embodiments, the paramyxovirus is a morbillivirus. In some embodiments, the morbillivirus is a measles virus. In some embodiments, the first recombinant microorganism is an orthomyxovirus. In some embodiments, the orthomyxovirus is an influenza virus. In some embodiments, the second recombinant microorganism is a bacterium. In some embodiments, the second recombinant microorganism is an Enterobacteriaceae bacterium, a Listeriaceae bacterium, or a Streptococcusceae bacterium. In some embodiments, the bacterium is an Enterobacteriaceae bacterium. In some embodiments, the Enterobacteriaceae bacterium is Salmonella. In some embodiments, the Salmonellaceae bacterium is S. bongori or S. enterica. In some embodiments, the bacterium is a Listeriaceae bacterium. In some embodiments, the Listeriaceae bacterium is Listeria monocytogenes. In some embodiments, the bacterium is a Streptococcusceae bacterium. In some embodiments, the Streptococcusceae bacterium is a Lactococcus genus bacterium.In some embodiments, the Lactococcus bacterium is L. chungangensis, L. formosensis, L. fujiensis, L. garvieae, L. hircilactis, L. lactis, L. laudensis, L. nasutitermitis, L. piscium, L. plantarum, L. raffinolactis, or L. taiwanensis. In some embodiments, the second recombinant microorganism is a parasite. In some embodiments, the parasite is a Sarcocystozoa parasite or a Trypanosomatidae parasite. In some embodiments, the parasite is a Sarcocystozoa parasite. In some embodiments, the Sarcocystozoa parasite is a Toxoplasma parasite. In some embodiments, the Toxoplasma parasite is Toxoplasma gondii. In some embodiments, the parasite is a Trypanosomatidae parasite. In some embodiments, the Trypanosomatidae parasite is a Leishmania parasite. In some embodiments, the Leishmania parasite is L. aethiopica, L. amazonensis, L. Arabica, L. aristedesi, L. braziliensis, L. chagasi, L. colombiensis, L. deanei, L. donovani, L. enriettii, L. forattinii, L. garnhami, L. guyanensis, L. herreri, L. hertigi, L. infantum, L. killicki, L. lainsoni, L. major, L. Mexicana, L. naiffi, L. panamensis, L. peruviana, L. pifanoi, L. shawi, L. tarentolae, L. tropica, L. turanica, or L. venezuelensis. In some embodiments, the second recombinant microorganism is a virus. In some embodiments, the second recombinant microorganism is of the Viridae family. In some embodiments, the second recombinant microorganism is a DNA virus. In some embodiments, the second recombinant microorganism is an RNA virus.In some embodiments, the second recombinant microorganism is a poxvirus, picornavirus, adenovirus, parvovirus, herpesvirus, reovirus, paramyxovirus, rhabdovirus, orthomyxovirus, or coxsackievirus. In some embodiments, the second recombinant microorganism is a poxvirus. In some embodiments, the poxvirus is an orthopoxvirus, a parapoxvirus, a yatapoxvirus, a leporipoxvirus, a mursipoxvirus, a betaentomopoxvirus, a cervidopoxvirus, a gammaentomopoxvirus, a suipoxvirus, a crocodilidopoxvirus, an alphaentomopoxvirus, a capripoxvirus, or an avipoxvirus. In some embodiments, the poxvirus is an orthopoxvirus. In some embodiments, the orthopoxvirus is a vaccinia virus. In some embodiments, the poxvirus is a leporipoxvirus. In some embodiments, the leporipoxvirus is a myxoma virus. In some embodiments, the poxvirus is a picornavirus. In some embodiments, the picornavirus is an aphthovirus, an aquamavirus, an avihepatovirus, an abyssivirus, a cardiovirus, a cosavirus, a dicipivirus, an enterovirus, an erbovirus, a gallivirus, a hepatovirus, a hunnivirus, a kobuvirus, a kunsagivirus, a meghrivirus, a miskivirus, a mosavirus, an osivirus, a parechovirus, a pasivirus, a passerivirus, a rosavirus, a sakobuvirus, a sarivirus, a sapelovirus, a senecavirus, a sissinivirus, a teschovirus, or a tremovirus. In some embodiments, the picornavirus is an enterovirus. In some embodiments, the enterovirus is a poliovirus or a coxsackievirus. In some embodiments, the picornavirus is a cardiovirus.

[0007] In some embodiments, the cardiovirus is a mengovirus. In some embodiments, the second recombinant microorganism is an adenovirus. In some embodiments, the adenovirus is an atadenovirus, an avian adenovirus, an ictoadenovirus, a mastadenovirus, or a siadenovirus. In some embodiments, the second recombinant microorganism is a parvovirus. In some embodiments, the parvovirus is an amdoparvovirus, an abeparvovirus, a bocaparvovirus, a chapparvovirus, a copiparvovirus, a dependoparvovirus, an erythroparvovirus, a protoparvovirus, or a tetraparvovirus. In some embodiments, the parvovirus is a dependoparvovirus. In some embodiments, the dependoparvovirus is an adeno-associated virus. In some embodiments, the second recombinant microorganism is a herpesvirus. In some embodiments, the herpesvirus is an alphaherpesvirus, a betaherpesvirus, or a gammaherpesvirus. In some embodiments, the herpesvirus is an alphaherpesvirus. In some embodiments, the alphaherpesvirus is an irtovirus, a mardivirus, a simplexvirus, or a varicellovirus. In some embodiments, the herpesvirus is a betaherpesvirus. In some embodiments, the betaherpesvirus is a cytomegalovirus, a muromegalovirus, a proboscivirus, or a poseolovirus. In some embodiments, the herpesvirus is a gammaherpesvirus. In some embodiments, the gammaherpesvirus is a lymphocryptovirus, a macavirus, a percavirus, or a rhadinovirus. In some embodiments, the second recombinant microorganism is a rhabdovirus. In some embodiments, the rhabdovirus is a cryovirus, a cytorhabdovirus, a zykolavirus, an ephemerovirus, a hapavirus, a redantevirus, a lyssavirus, a novirhabdovirus, a nucleorhabdovirus, a perhabdovirus, a sigmavirus, a sprivivirus, a thripvirus, a tibrovirus, a tupavirus, a varicosavirus, or a vesiculovirus. In some embodiments, the rhabdovirus is a vesiculovirus.In some embodiments, the vesiculovirus is a vesicular stomatitis virus or a malvavirus. In some embodiments, the second recombinant microorganism is a reovirus. In some embodiments, the reovirus is a cedreovirus or a spinaleovirus. In some embodiments, the reovirus is a cedreovirus. In some embodiments, the cedreovirus is a caldoleovirus, a mimoreovirus, an orbivirus, a phytreovirus, a rotavirus, or a ceradornavirus.

[0008] In some embodiments, the reovirus is a spinaleovirus. In some embodiments, the spinaleovirus is an aquareovirus, a coltivirus, a cypovirus, a fijivirus, an orthoreovirus, an idnoreovirus, a dinovernavirus, an oryzavirus, or a mycoreovirus. In some embodiments, the second recombinant microorganism is a paramyxovirus. In some embodiments, the paramyxovirus is an aquaparamyxovirus, an abulavirus, a ferulavirus, a henipavirus, a morbillivirus, a respirovirus, or a rubulavirus. In some embodiments, the paramyxovirus is a morbillivirus. In some embodiments, the morbillivirus is a measles virus. In some embodiments, the second recombinant microorganism is an orthomyxovirus. In some embodiments, the orthomyxovirus is an influenza virus. In some embodiments, the first recombinant microorganism comprises an exogenous nucleic acid encoding one or more human proteins, and the second recombinant microorganism does not comprise an exogenous nucleic acid encoding the one or more human proteins. In some embodiments, the human protein is a chemokine or a cytokine. In some embodiments, the second recombinant microorganism comprises an exogenous nucleic acid encoding one or more human proteins, and the second recombinant microorganism does not comprise an exogenous nucleic acid encoding the one or more human proteins. In some embodiments, the human protein is a chemokine or a cytokine. In some embodiments, the subject is a human. In some embodiments, the tumor is a solid tumor or a hematological tumor. In some embodiments, the tumor comprises a prostate tumor, a lung tumor, a kidney tumor, a stomach tumor, a colon tumor, an ovarian tumor, a bladder tumor, a breast tumor, a cervical tumor, a esophageal tumor, a testicular tumor, a liver tumor, a pancreatic tumor, a rectal tumor, a thyroid tumor, a uterine tumor, a skin tumor, a muscle tumor, a cartilage tumor, a bone tumor, an endothelial tumor, an epithelial tumor, a leukemia, a lymphoma, a myeloma, a dermal tumor, a basal tumor, a retinal tumor, a skin tumor, or a brain tumor. In some embodiments, the first recombinant microorganism and the second recombinant microorganism are administered to the subject simultaneously.In some embodiments, the first recombinant microorganism is formulated in a delayed release composition, a sustained release composition, an immediate release composition, a stealth release composition, or any combination thereof. In some embodiments, the second recombinant microorganism is formulated in a delayed release composition. In some embodiments, the second recombinant microorganism is administered to the subject after the first recombinant microorganism is administered to the subject. In some embodiments, the second recombinant microorganism is administered about 1-60 days, 1-45 days, 1-30 days, 1-15 days, 1-10 days, or 1-7 days after administration of the first recombinant microorganism.

[0009] In some embodiments, the second recombinant microorganism is administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 21, 28, 35, 56, or 60 days after administration of the first recombinant microorganism. In some embodiments, the second recombinant microorganism is administered to the subject 1, 2, 3, 4, 5, or more times. In some embodiments, the second recombinant microorganism is administered to the subject 2, 3, 4, 5, or more times, with about 1-60 days, 1-45 days, 1-30 days, 1-15 days, 1-10 days, 1-7, 1-5 days, or 1-3 days between each administration. In some embodiments, the first recombinant microorganism is administered to the subject after the second recombinant microorganism is administered to the subject.

[0010] In some embodiments, the first recombinant microorganism is administered about 1-60 days, 1-45 days, 1-30 days, 1-15 days, 1-10 days, or 1-7 days after administration of the second recombinant microorganism.

[0011] In some embodiments, the first recombinant microorganism is administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 21, 28, 35, 56, or 60 days after administration of the second recombinant microorganism. In some embodiments, the first recombinant microorganism is administered to the subject 1, 2, 3, 4, 5, or more times. In some embodiments, the first recombinant microorganism is administered to the subject 2, 3, 4, 5, or more times with about 1-60 days, 1-45 days, 1-30 days, 1-15 days, 1-10 days, 1-7, 1-5 days, or 1-3 days between each administration. In some embodiments, the first recombinant microorganism is administered intratumorally, intradermally, subcutaneously, intraperitoneally, intramuscularly, or intravenously; and the second composition is administered intratumorally, intradermally, subcutaneously, intraperitoneally, intrathecally, intramuscularly, or intravenously. In some embodiments, the second recombinant microorganism is administered intratumorally, intradermally, subcutaneously, intraperitoneally, intramuscularly; and the second composition is administered intratumorally, intradermally, subcutaneously, intraperitoneally, intrathecally, intramuscularly, or intravenously.

[0012] In some embodiments, the method further comprises administering an anti-cancer therapy. In some embodiments, the anti-cancer therapy comprises chemotherapy, radiation, an immunomodulatory agent, or a cell therapy. In some embodiments, the immunomodulatory agent comprises an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor comprises a protein that binds to PD-1, PD-L1, CTLA-4, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, TIM-3, VISTA, CD160, TIGIT, PSGL-1, or any combination thereof. In some embodiments, the immune checkpoint inhibitor comprises an antibody that specifically binds to PD-1, PD-L1, CTLA-4, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, TIM-3, VISTA, CD160, TIGIT, PSGL-1, or any combination thereof.

[0013] In some embodiments, the immune response is demonstrated by a change in immune cell population, the immune cells comprising myeloid derived suppressor cells, regulatory T cells, natural killer cells, dendritic cells, or any combination thereof. In some embodiments, the immune response is demonstrated by enhanced cytokine production, the cytokines comprising IFN-γ, IL-12, TNFα, or any combination thereof. In some embodiments, the method further comprises administering to the subject an additional recombinant microorganism. In some embodiments, the method further comprises administering to the subject a third recombinant microorganism. In some embodiments, the method further comprises administering to the subject a fourth recombinant microorganism.

[0014] One embodiment provides a kit comprising at least a first and a second container and instructions for use, the first container comprising a first recombinant microorganism and the second container comprising a second recombinant microorganism. In some embodiments, the first recombinant microorganism replicates in tumor cells but does not replicate or exhibits attenuated replication in non-tumor cells, and the second recombinant microorganism replicates in tumor cells but does not replicate or exhibits attenuated replication in non-tumor cells. In some embodiments, the kit further comprises a third container comprising a third recombinant microorganism. In some embodiments, the kit further comprises a fourth container comprising a fourth recombinant microorganism.

[0015] In some embodiments, the third recombinant microorganism replicates in tumor cells but does not replicate or exhibits attenuated replication in non-tumor cells, and the fourth recombinant microorganism replicates in tumor cells but does not replicate or exhibits attenuated replication in non-tumor cells.

[0016] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over such conflicting material. The present invention provides, for example, the following items. (Item 1) 1. A method of treating a tumor in a subject, comprising: administering to the subject a first recombinant microorganism, wherein the first recombinant microorganism replicates in tumor cells but does not replicate or is attenuated in non-tumor cells. a step of indicating duplication; administering to the subject a second recombinant microorganism, wherein the second recombinant microorganism replicates in tumor cells but does not replicate or exhibits attenuated replication in non-tumor cells; the first recombinant microorganism and the second recombinant microorganism are different; and a) enhancing or eliciting an immune response to a tumor protein that is not encoded or expressed by said first microorganism and said second microorganism; b) enhancing or eliciting an immune response to proteins expressed by tumor-associated cells that are not encoded or expressed by said first recombinant microorganism and said second recombinant microorganism. The method is at least one of: (Item 2) The method of claim 1, wherein the immune response is evidenced by one or more of the following: a decrease in tumor volume in the subject, a decrease in expression levels of one or more tumor proteins in the subject or the sample from the subject, a decrease in the number of tumor sites in the subject, a change in viral load in the subject or the sample from the subject, a change in immune cell population in the subject or the sample from the subject, a change in expression levels of immune cell markers in the subject or the sample from the subject, enhanced B cell proliferation in the subject or the sample from the subject, enhanced CD4+ T cell proliferation in the subject or the sample from the subject, enhanced CD8+ T cell proliferation in the subject or the sample from the subject, enhanced cytokine production in the subject or the sample from the subject, enhanced antigen presenting cell proliferation in the subject or the sample from the subject, or any combination thereof. (Item 3) 3. The method of claim 2, wherein the immune response is demonstrated by a decrease in expression level of one or more tumor proteins, a change in immune cell population, a change in expression level of an immune cell marker, an increase in B cell proliferation, an increase in CD4+ T cell proliferation, an increase in CD8+ T cell proliferation, an increase in cytokine production, an increase in antigen presenting cell proliferation, or any combination thereof in the subject or the sample from the subject, and wherein the sample from the subject is a blood, tissue, urine, or saliva sample. (Item 4) The method of any preceding item, wherein the immune response can be detected at the time of administration of the first recombinant microorganism or the second recombinant microorganism or at a subsequent time. (Item 5) The method of any preceding item, wherein the first recombinant microorganism does not replicate in the non-tumor cell. (Item 6) 5. The method according to any one of items 1 to 4, wherein the first recombinant microorganism exhibits attenuated replication in the non-tumor cell. (Item 7) 7. The method of claim 6, wherein the infectivity of the first recombinant microorganism is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% lower in the non-tumor cells compared to the tumor cells. (Item 8) 8. The method of claim 6 or 7, wherein the replication efficiency of the first recombinant microorganism is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% lower in the non-tumor cells compared to the tumor cells. (Item 9) The method of any preceding item, wherein the second recombinant microorganism does not replicate in the non-tumor cell. (Item 10) 9. The method of any one of items 1 to 8, wherein the second recombinant microorganism exhibits attenuated replication in the non-tumor cell. (Item 11) 11. The method of claim 10, wherein the infectivity of the second recombinant microorganism is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% lower in the non-tumor cells compared to the tumor cells. (Item 12) 12. The method of claim 10 or 11, wherein the replication efficiency of the second recombinant microorganism is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% less in the non-tumor cells compared to the tumor cells. (Item 13) The method of any preceding item, wherein the first recombinant microorganism is a bacterium, a virus, or a parasite. (Item 14) The method of any preceding item, wherein the second recombinant microorganism is a bacterium, a virus, or a parasite. (Item 15) 2. The method of any preceding item, wherein the first recombinant microorganism is a bacterium. (Item 16) 16. The method of claim 15, wherein the first recombinant microorganism is an Enterobacteriaceae bacterium, a Listeriaceae bacterium, or a Streptococcusceae bacterium. (Item 17) 17. The method of claim 16, wherein the bacterium is an enterobacterium. (Item 18) 18. The method according to item 17, wherein the enterobacteria is Salmonella. (Item 19) 19. The method of claim 18, wherein the Salmonella bacterium is S. bongori or S. enterica. (Item 20) 17. The method according to item 16, wherein the bacterium is a Listeriaceae bacterium. (Item 21) 21. The method according to item 20, wherein the bacterium of the Listeria family is Listeria monocytogenes. (Item 22) 22. The method of claim 21, wherein the Listeria monocytogenes is L. monocytogenes. (Item 23) 17. The method according to item 16, wherein the bacterium is a bacterium of the Streptococcus family. (Item 24) 24. The method according to item 23, wherein the bacterium of the Streptococcus family is a bacterium of the Lactococcus genus. (Item 25) 25. The method according to item 24, wherein the Lactococcus bacterium is L. chungangensis, L. formosensis, L. fujiensis, L. garvieae, L. hircilactis, L. lactis, L. laudensis, L. nasutitermitis, L. piscium, L. plantarum, L. raffinolactis or L. taiwanensis. (Item 26) 15. The method according to any one of items 1 to 14, wherein the first recombinant microorganism is a parasite. (Item 27) The parasite is a Sarcocystiidae parasite or a Trypanosomatidae parasite. 26. The method according to claim 26. (Item 28) 28. The method according to item 27, wherein the parasite is a Sarcocystodiaceae parasite. (Item 29) 28. The method of claim 27, wherein the Sarcocystodiaceae parasite is a Toxoplasma parasite. (Item 30) 30. The method of claim 29, wherein the toxoplasma parasite is Toxoplasma gondii. (Item 31) 28. The method according to item 27, wherein the parasite is a Trypanosomatidae parasite. (Item 32) 32. The method of claim 31, wherein the Trypanosomatidae parasite is a Leishmania parasite. (Item 33) The Leishmania parasites include L.aethiopica, L.amazonensis, L.Arabica, L.aristedesi, L.braziliensis, L.chagasi, L.colombiensis, L.deanei, L.donovani, L.enriettii, L.forattinii, L.garnhami, L.guyanensis, and L.herrer. i, L. hertigi, L. infantum, L. killicki, L. lainsoni, L. major, L. Mexicana, L. naiffi, L. panamensis, L. peruviana, L. pifanoi, L. shawi, L. tarentolae, L. tropica, L. turanica or L. venezuelensis. (Item 34) 15. The method according to any one of items 1 to 14, wherein the first recombinant microorganism is a virus. (Item 35) 16. The method of claim 15, wherein the first recombinant microorganism is of the Viridae family. (Item 36) 16. The method of claim 15, wherein the first recombinant microorganism is a DNA virus. (Item 37) 16. The method of claim 15, wherein the first recombinant microorganism is an RNA virus. (Item 38) 15. The method of any one of items 1 to 14, wherein the first recombinant microorganism comprises a poxvirus, picornavirus, adenovirus, parvovirus, herpesvirus, reovirus, paramyxovirus, rhabdovirus, orthomyxovirus or coxsackievirus. (Item 39) 16. The method of claim 15, wherein the first recombinant microorganism is a poxvirus. (Item 40) 40. The method of claim 39, wherein the poxvirus is an orthopoxvirus, a parapoxvirus, a yatapoxvirus, a leporipoxvirus, a mursipoxvirus, a betaentomopoxvirus, a cervidopoxvirus, a gammaentomopoxvirus, a suipoxvirus, a crocodilidopoxvirus, an alphaentomopoxvirus, a capripoxvirus, or an avipoxvirus. (Item 41) 41. The method of claim 40, wherein the poxvirus is an orthopoxvirus. (Item 42) 42. The method of claim 41, wherein the orthopoxvirus is a vaccinia virus. (Item 43) 41. The method of claim 40, wherein the poxvirus is a leporipoxvirus. (Item 44) 44. The method of claim 43, wherein the leporipoxvirus is a myxoma virus. (Item 45) 40. The method of claim 39, wherein the poxvirus is a picornavirus. (Item 46) 46. ​​The method of claim 45, wherein the picornavirus is an aphthovirus, aquamavirus, avihepatovirus, abyssivirus, cardiovirus, cosavirus, dicipivirus, enterovirus, erbovirus, gallivirus, hepatovirus, hunnivirus, kobuvirus, kunsagivirus, megrivirus, miskivirus, mosavirus, osivirus, parechovirus, pasivirus, passerivirus, rosavirus, sakobuvirus, sarivirus, sapelovirus, senecavirus, sissinivirus, teschovirus, or tremovirus. (Item 47) 47. The method of claim 46, wherein the picornavirus is an enterovirus. (Item 48) 48. The method of claim 47, wherein the enterovirus is a poliovirus or a coxsackievirus. (Item 49) 47. The method of claim 46, wherein the picornavirus is a cardiovirus. (Item 50) 50. The method of claim 49, wherein the cardiovirus is a mengovirus. (Item 51) 39. The method of claim 38, wherein the first recombinant microorganism is an adenovirus. (Item 52) 52. The method of claim 51, wherein the adenovirus is an adenovirus, an avian adenovirus, an ictoadenovirus, a mastadenovirus or a siadenovirus. (Item 53) 39. The method of claim 38, wherein the first recombinant microorganism is a parvovirus. (Item 54) 54. The method of claim 53, wherein the parvovirus is amdoparvovirus, abeparvovirus, bocaparvovirus, chapparvovirus, copiparvovirus, dependoparvovirus, erythroparvovirus, protoparvovirus or tetraparvovirus. (Item 55) 55. The method of claim 54, wherein the parvovirus is a dependoparvovirus. (Item 56) 56. The method of claim 55, wherein the dependoparvovirus is an adeno-associated virus. (Item 57) 39. The method of claim 38, wherein the first recombinant microorganism is a herpes virus. (Item 58) 58. The method of item 57, wherein the herpesvirus is an alphaherpesvirus, a betaherpesvirus or a gammaherpesvirus. (Item 59) 59. The method of claim 58, wherein the herpesvirus is an alphaherpesvirus. (Item 60) The method of claim 59, wherein the alphaherpesvirus is an Irtovirus, a Mardivirus, a Simplexvirus or a Varicellovirus. (Item 61) 59. The method of item 58, wherein the herpesvirus is a beta-herpesvirus. (Item 62) Item 62. The method of item 61, wherein the beta-herpesvirus is a cytomegalovirus, a muromegalovirus, a proboscivirus or a poseolovirus. (Item 63) 60. The method of claim 59, wherein the herpesvirus is a gamma herpesvirus. (Item 64) 64. The method of claim 63, wherein the gammaherpesvirus is a lymphocryptovirus, a macavirus, a percavirus or a rhadinovirus. (Item 65) 39. The method of claim 38, wherein the first recombinant microorganism is a rhabdovirus. (Item 66) 66. The method of claim 65, wherein the rhabdovirus is a cryovirus, cytorhabdovirus, dicolavirus, ephemerovirus, hapavirus, redantevirus, lyssavirus, novirhabdovirus, nucleorhabdovirus, perahabdovirus, sigmavirus, sprivivirus, thripvirus, tibrovirus, tupavirus, varicosavirus, or vesiculovirus. (Item 67) Item 67. The method of item 66, wherein the rhabdovirus is a vesiculovirus. (Item 68) 68. The method of claim 67, wherein the vesiculovirus is a vesicular stomatitis virus or a marva virus. (Item 69) 39. The method of claim 38, wherein the first recombinant microorganism is a reovirus. (Item 70) 70. The method of claim 69, wherein the reovirus is a cedreovirus or a spinaleovirus. (Item 71) 71. The method of claim 70, wherein the reovirus is a cedreovirus. (Item 72) 72. The method of claim 71, wherein the cedreovirus is a caldreovirus, a mimoreovirus, an orbivirus, a phytoreovirus, a rotavirus, or a ceradornavirus. (Item 73) 71. The method of claim 70, wherein the reovirus is a spinal reovirus. (Item 74) 74. The method of claim 73, wherein the spinaleovirus is an aquareovirus, a coltivirus, a cypovirus, a fijivirus, an orthoreovirus, an idnoreovirus, a dinovernavirus, an oryzavirus, or a mycoreovirus. (Item 75) 39. The method of claim 38, wherein the first recombinant microorganism is a paramyxovirus. (Item 76) 76. The method of claim 75, wherein the paramyxovirus is an aquaparamyxovirus, an abulavirus, a ferulavirus, a henipavirus, a morbillivirus, a respirovirus, or a rubulavirus. (Item 77) 77. The method of claim 76, wherein the paramyxovirus is a morbillivirus. (Item 78) 78. The method of claim 77, wherein the morbillivirus is a measles virus. (Item 79) 39. The method of claim 38, wherein the first recombinant microorganism is an orthomyxovirus. (Item 80) 80. The method of claim 79, wherein the orthomyxovirus is an influenza virus. (Item 81) 2. The method of any preceding claim, wherein the second recombinant microorganism is a bacterium. (Item 82) 82. The method of item 81, wherein the second recombinant microorganism is an Enterobacteriaceae bacterium, a Listeriaceae bacterium, or a Streptococcusceae bacterium. (Item 83) 83. The method of item 82, wherein the bacterium is an enterobacterium. (Item 84) 84. The method of claim 83, wherein the enterobacteria is Salmonella. (Item 85) 85. The method of claim 84, wherein the Salmonella bacterium is S. bongori or S. enterica. (Item 86) 83. The method of claim 82, wherein the bacterium is a Listeriaceae bacterium. (Item 87) 87. The method of claim 86, wherein the Listeriaceae bacterium is Listeria monocytogenes. (Item 88) 88. The method of claim 87, wherein the Listeria monocytogenes is L. monocytogenes. (Item 89) 83. The method of claim 82, wherein the bacterium is a Streptococcus bacterium. (Item 90) 90. The method according to item 89, wherein the bacterium of the Streptococcus family is a bacterium of the Lactococcus genus. (Item 91) Item 91. The method according to item 90, wherein the Lactococcus bacterium is L. chungangensis, L. formosensis, L. fujiensis, L. garvieae, L. hircilactis, L. lactis, L. laudensis, L. nasutitermitis, L. piscium, L. plantarum, L. raffinolactis or L. taiwanensis. (Item 92) 81. The method of any one of items 1 to 80, wherein the second recombinant microorganism is a parasite. (Item 93) 93. The method of claim 92, wherein the parasite is a Sarcocystiidae parasite or a Trypanosomatidae parasite. (Item 94) 94. The method of claim 93, wherein the parasite is a Sarcocystozoa parasite. (Item 95) 95. The method of claim 94, wherein the Sarcocystodiaceae parasite is a Toxoplasma parasite. (Item 96) 96. The method of claim 95, wherein the toxoplasma parasite is Toxoplasma gondii. (Item 97) 94. The method of item 93, wherein the parasite is a Trypanosomatidae parasite. (Item 98) 98. The method of item 97, wherein the Trypanosomatidae parasite is a Leishmania parasite. (Item 99) The Leishmania parasites include L.aethiopica, L.amazonensis, L.Arabica, L.aristedesi, L.braziliensis, L.chagasi, L.colombiensis, L.deanei, L.donovani, L.enriettii, L.forattinii, L.garnhami, L.guyanensis, and L.herrer. i, L. hertigi, L. infantum, L. killicki, L. lainsoni, L. major, L. Mexicana, L. naiffi, L. panamensis, L. peruviana, L. pifanoi, L. shawi, L. tarentolae, L. tropica, L. turanica or L. venezuelensis. (Item 100) 81. The method of any one of items 1 to 80, wherein the second recombinant microorganism is a virus. (Item 101) 101. The method of claim 100, wherein the second recombinant microorganism is of the Viridae family. (Item 102) 101. The method of claim 100, wherein the second recombinant microorganism is a DNA virus. (Item 103) 101. The method of claim 100, wherein the second recombinant microorganism is an RNA virus. (Item 104) 101. The method of claim 100, wherein the second recombinant microorganism is a poxvirus, picornavirus, adenovirus, parvovirus, herpesvirus, reovirus, paramyxovirus, rhabdovirus, orthomyxovirus, or coxsackievirus. (Item 105) 105. The method of claim 104, wherein the second recombinant microorganism is a poxvirus. (Item 106) 106. The method of item 105, wherein the poxvirus is an orthopoxvirus, a parapoxvirus, a yatapoxvirus, a leporipoxvirus, a mursipoxvirus, a betaentomopoxvirus, a cervidopoxvirus, a gammaentomopoxvirus, a suipoxvirus, a crocodilidopoxvirus, an alphaentomopoxvirus, a capripoxvirus, or an avipoxvirus. (Item 107) 107. The method of claim 106, wherein the poxvirus is an orthopoxvirus. (Item 108) 108. The method of claim 107, wherein the orthopoxvirus is a vaccinia virus. (Item 109) 107. The method of claim 106, wherein the poxvirus is a leporipoxvirus. (Item 110) 110. The method of claim 109, wherein the leporipoxvirus is a myxoma virus. (Item 111) 106. The method of claim 105, wherein the poxvirus is a picornavirus. (Item 112) 112. The method of item 111, wherein the picornavirus is an aphthovirus, aquamavirus, avihepatovirus, abyssivirus, cardiovirus, cosavirus, dicipivirus, enterovirus, erbovirus, gallivirus, hepatovirus, hunnivirus, kobuvirus, kunsagivirus, megrivirus, miskivirus, mosavirus, osivirus, parechovirus, pasivirus, passerivirus, rosavirus, sakobuvirus, sarivirus, sapelovirus, senecavirus, sissinivirus, teschovirus, or tremovirus. (Item 113) 13. The method of claim 112, wherein the picornavirus is an enterovirus. (Item 114) 114. The method of claim 113, wherein the enterovirus is a poliovirus or a coxsackievirus. (Item 115) 113. The method of claim 112, wherein the picornavirus is a cardiovirus. (Item 116) 116. The method of claim 115, wherein the cardiovirus is a mengovirus. (Item 117) 105. The method of claim 104, wherein the second recombinant microorganism is an adenovirus. (Item 118) 118. The method of claim 117, wherein the adenovirus is an adenovirus, an avian adenovirus, an ictoadenovirus, a mastadenovirus or a siadenovirus. (Item 119) 105. The method of claim 104, wherein the second recombinant microorganism is a parvovirus. (Item 120) 120. The method of claim 119, wherein the parvovirus is amdoparvovirus, abeparvovirus, bocaparvovirus, chapparvovirus, copiparvovirus, dependoparvovirus, erythroparvovirus, protoparvovirus or tetraparvovirus. (Item 121) 121. The method of item 120, wherein the parvovirus is a dependoparvovirus. (Item 122) 122. The method of claim 121, wherein the dependoparvovirus is an adeno-associated virus. (Item 123) 105. The method of claim 104, wherein the second recombinant microorganism is a herpes virus. (Item 124) 124. The method of item 123, wherein the herpesvirus is an alphaherpesvirus, a betaherpesvirus or a gammaherpesvirus. (Item 125) 125. The method of item 124, wherein the herpesvirus is an alphaherpesvirus. (Item 126) The method of claim 125, wherein the alphaherpesvirus is an Irtovirus, a Mardivirus, a Simplexvirus or a Varicellovirus. (Item 127) Item 125. The method of item 124, wherein the herpesvirus is a beta-herpesvirus. (Item 128) Item 128. The method of item 127, wherein the beta-herpesvirus is a cytomegalovirus, a muromegalovirus, a proboscivirus or a poseolovirus. (Item 129) 126. The method of claim 125, wherein the herpesvirus is a gamma herpesvirus. (Item 130) 130. The method of claim 129, wherein the gammaherpesvirus is a lymphocryptovirus, a macavirus, a percavirus or a rhadinovirus. (Item 131) Item 105. The method of item 104, wherein the second recombinant microorganism is a rhabdovirus. (Item 132) The rhabdovirus may be a cryovirus, a cytorhabdovirus, a dichorhavirus, an ephemerovirus, a hapavirus, a redantevirus, a lyssavirus, a novirhabdovirus, a nucleorhabdovirus, a perahabdovirus, a sigmavirus, a sprivivirus, 132. The method of claim 131, wherein the virus is a Thripvirus, a Tibrovirus, a Tupavirus, a Varicosavirus or a Vesiculovirus. (Item 133) Item 133. The method of item 132, wherein the rhabdovirus is a vesiculovirus. (Item 134) 134. The method of claim 133, wherein the vesiculovirus is a vesicular stomatitis virus or a marvavirus. (Item 135) 105. The method of claim 104, wherein the second recombinant microorganism is a reovirus. (Item 136) 136. The method of claim 135, wherein the reovirus is a cedreovirus or a spinaleovirus. (Item 137) 137. The method of claim 136, wherein the reovirus is a cedreovirus. (Item 138) 138. The method of claim 137, wherein the cedreovirus is a caldreovirus, a mimoreovirus, an orbivirus, a phytoreovirus, a rotavirus, or a ceradornavirus. (Item 139) 137. The method of claim 136, wherein the reovirus is a spinal reovirus. (Item 140) 140. The method of claim 139, wherein the spinaleovirus is an aquareovirus, a coltivirus, a cypovirus, a fijivirus, an orthoreovirus, an idnoreovirus, a dinovernavirus, an oryzavirus, or a mycoreovirus. (Item 141) 105. The method of claim 104, wherein the second recombinant microorganism is a paramyxovirus. (Item 142) 142. The method of claim 141, wherein the paramyxovirus is an aquaparamyxovirus, an abulavirus, a ferulavirus, a henipavirus, a morbillivirus, a respirovirus, or a rubulavirus. (Item 143) 143. The method of claim 142, wherein the paramyxovirus is a morbillivirus. (Item 144) 144. The method of claim 143, wherein the morbillivirus is a measles virus. (Item 145) 105. The method of claim 104, wherein the second recombinant microorganism is an orthomyxovirus. (Item 146) 146. The method of claim 145, wherein the orthomyxovirus is an influenza virus. (Item 147) 2. The method of any preceding claim, wherein the first recombinant microorganism comprises an exogenous nucleic acid encoding one or more human proteins, and the second recombinant microorganism does not comprise an exogenous nucleic acid encoding the one or more human proteins. (Item 148) 148. The method of claim 147, wherein the human protein is a chemokine or cytokine. (Item 149) The method of any preceding item, wherein the second recombinant microorganism comprises an exogenous nucleic acid encoding one or more human proteins, and wherein the second recombinant microorganism does not comprise an exogenous nucleic acid encoding one or more human proteins. (Item 150) 150. The method of claim 149, wherein the human protein is a chemokine or cytokine. (Item 151) The method of any preceding claim, wherein the subject is a human. (Item 152) The method of any preceding claim, wherein the tumor is a solid tumor or a hematological tumor. (Item 153) The method of any preceding item, wherein the tumor comprises a prostate tumor, a lung tumor, a kidney tumor, a stomach tumor, a colon tumor, an ovarian tumor, a bladder tumor, a breast tumor, a cervical tumor, an esophageal tumor, a testicular tumor, a liver tumor, a pancreatic tumor, a rectal tumor, a thyroid tumor, a uterine tumor, a skin tumor, a muscle tumor, a cartilage tumor, a bone tumor, an endothelial tumor, an epithelial tumor, a leukemia, a lymphoma, a myeloma, a dermal tumor, a basal tumor, a retinal tumor, a skin tumor, or a brain tumor. (Item 154) The method of any preceding claim, wherein the first recombinant microorganism and the second recombinant microorganism are administered to the subject simultaneously. (Item 155) 155. The method of claim 154, wherein the first recombinant microorganism is formulated in a delayed release composition, a sustained release composition, an immediate release composition, a stealth release composition, or any combination thereof. (Item 156) 156. The method of claim 154 or 155, wherein the second recombinant microorganism is formulated in a delayed release composition. (Item 157) 154. The method of any one of items 1 to 153, wherein the second recombinant microorganism is administered to the subject after the first recombinant microorganism is administered to the subject. (Item 158) 158. The method of item 157, wherein the second recombinant microorganism is administered about 1 to 60 days, 1 to 45 days, 1 to 30 days, 1 to 15 days, 1 to 10 days, or 1 to 7 days after administration of the first recombinant microorganism. (Item 159) 158. The method of item 157, wherein the second recombinant microorganism is administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 21, 28, 35, 56, or 60 days after administration of the first recombinant microorganism. (Item 160) 158. The method of claim 157, wherein the second recombinant microorganism is administered to the subject 1, 2, 3, 4, 5 or more times. (Item 161) 158. The method of item 157, wherein the second recombinant microorganism is administered to the subject two, three, four, five or more times with about 1-60 days, 1-45 days, 1-30 days, 1-15 days, 1-10 days, 1-7, 1-5 days, or 1-3 days between each administration. (Item 162) 154. The method of any one of items 1 to 153, wherein the first recombinant microorganism is administered to the subject after the second recombinant microorganism is administered to the subject. (Item 163) 163. The method of item 162, wherein the first recombinant microorganism is administered about 1-60 days, 1-45 days, 1-30 days, 1-15 days, 1-10 days, or 1-7 days after administration of the second recombinant microorganism. (Item 164) The first recombinant microorganism is administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 21, 28, 35, 56, or 60 days after administration of the second recombinant microorganism. Item 163. The method according to item 162, (Item 165) 163. The method of claim 162, wherein the first recombinant microorganism is administered to the subject 1, 2, 3, 4, 5 or more times. (Item 166) 163. The method of item 162, wherein the first recombinant microorganism is administered to the subject two, three, four, five or more times with about 1-60 days, 1-45 days, 1-30 days, 1-15 days, 1-10 days, 1-7, 1-5 days, or 1-3 days between each administration. (Item 167) The method of any preceding item, wherein the first recombinant microorganism is administered intratumorally, intradermally, subcutaneously, intraperitoneally, intramuscularly, or intravenously, and the second composition is administered intratumorally, intradermally, subcutaneously, intraperitoneally, intrathecally, intramuscularly, or intravenously. (Item 168) The method of any preceding item, wherein the second recombinant microorganism is administered intratumorally, intradermally, subcutaneously, intraperitoneally, intramuscularly, and the second composition is administered intratumorally, intradermally, subcutaneously, intraperitoneally, intrathecally, intramuscularly, or intravenously. (Item 169) The method of any preceding claim, further comprising administering an anti-cancer therapy. (Item 170) 170. The method of item 169, wherein the anticancer therapy comprises chemotherapy, radiation, an immunomodulator or a cell therapy. (Item 171) 171. The method of claim 170, wherein the immunomodulatory agent comprises an immune checkpoint inhibitor. (Item 172) 172. The method of claim 171, wherein the immune checkpoint inhibitor comprises a protein that binds to PD-1, PD-L1, CTLA-4, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, TIM-3, VISTA, CD160, TIGIT, PSGL-1, or any combination thereof. (Item 173) 172. The method of claim 171, wherein the immune checkpoint inhibitor comprises an antibody that specifically binds to PD-1, PD-L1, CTLA-4, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, TIM-3, VISTA, CD160, TIGIT, PSGL-1, or any combination thereof. (Item 174) 3. The method of claim 2, wherein the immune response is evidenced by the change in an immune cell population, the immune cells comprising myeloid derived suppressor cells, regulatory T cells, natural killer cells, dendritic cells, or any combination thereof. (Item 175) 3. The method of claim 2, wherein the immune response is evidenced by the increased cytokine production, and the cytokines include IFN-γ, IL-12, TNFα, or any combination thereof. (Item 176) 176. The method of any one of items 1 to 175, further comprising administering to the subject an additional recombinant microorganism. (Item 177) 177. The method of claim 176, further comprising administering to the subject a third recombinant microorganism. (Item 178) 178. The method of claim 177, further comprising administering to the subject a fourth recombinant microorganism. (Item 179) A kit comprising at least a first container and a second container and instructions for use, wherein the first container contains a first recombinant microorganism and the second container contains a second recombinant microorganism. (Item 180) 180. The kit of claim 179, wherein the first recombinant microorganism replicates in tumor cells but does not replicate or exhibits attenuated replication in non-tumor cells, and the second recombinant microorganism replicates in tumor cells but does not replicate or exhibits attenuated replication in non-tumor cells. (Item 181) 181. The kit of item 179 or 180, further comprising a third container containing a third recombinant microorganism. (Item 182) 182. The kit of any one of items 179 to 181, further comprising a fourth container containing a fourth recombinant microorganism. (Item 183) The kit of item 182, wherein the third recombinant microorganism replicates in tumor cells but does not replicate or exhibits attenuated replication in non-tumor cells, and the fourth recombinant microorganism replicates in tumor cells but does not replicate or exhibits attenuated replication in non-tumor cells.

[0017] Various features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 shows the therapeutic activity of a heterologous prime-boost regimen of Salmonella and Vaccinia virus in a mouse renal adenocarcinoma (RENCA) tumor model.

[0019] [Diagram 2] 2A and 2B show the therapeutic activity of heterologous prime-boost regimens of vaccinia virus or herpes simplex virus or vesicular stomatitis virus in the murine RENCA tumor model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The following description and examples illustrate the embodiments of the present disclosure in detail.It should be understood that the present disclosure is not limited to the specific embodiments described herein and can be modified as such.Those skilled in the art will recognize that there are numerous embodiments and modifications of the present disclosure, which are encompassed within its scope.

[0021] All terms are intended to be understood as they are understood by one of ordinary skill in the art. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0022] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0023] Although various features of the present disclosure may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may for clarity be described herein in the context of separate embodiments, the present disclosure may also be implemented in a single embodiment.

[0024] I. Specific Definitions The following definitions are directed to this application and should not be attributed to any related or unrelated case, such as any co-owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in carrying out the testing of the present disclosure, the preferred materials and methods are described herein. Therefore, the terms used herein are only intended to describe specific embodiments and are not intended to be limiting.

[0025] The terms used herein are for the purpose of describing particular cases only and are not limiting. As used herein, the singular forms "a," "an," and "the" also include the plural unless the context clearly dictates otherwise. Furthermore, to the extent the terms "contains," "containing," "including," "includes," "having," "has," "with," or variations thereof are used in the detailed description and / or claims, such terms are intended to be as inclusive as the term "comprising."

[0026] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within 1 or more standard deviations per practice for a given value. When a particular value is described in the application and claims, unless otherwise specified, it should be assumed that the term "about" means an acceptable error range of the particular value, e.g., ±10% of the value modified by the term "about."

[0027] The terms "individual," "patient," or "subject" are used interchangeably herein. None of these terms require or are limited to a situation characterized by the supervision (e.g., constant or intermittent) of a health care worker (e.g., a physician, registered nurse, nurse, physician's assistant, janitor, or hospice worker). In some embodiments, a patient, subject, or individual may be under the supervision of a health care worker. The term "subject" can refer to an animal, including, but not limited to, a primate (e.g., a human), cow, sheep, goat, horse, dog, cat, rabbit, rat, or mouse.

[0028] The terms "inhibit," "reduce," or "prevention," or any variation of these terms, referred to herein, include any measurable decrease or complete inhibition that achieves the desired result.

[0029] The terms "treat", "treating" and "treatment" include alleviating or arresting a disorder, disease or condition; or one or more of the symptoms associated with a disorder, disease or condition; or alleviating or eradicating the cause of the disorder, disease or condition itself. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or palliating the disease state, and remission or improved prognosis.

[0030] The terms "systemic delivery" and "systemic administration," as used interchangeably herein, refer to the route of administration of a pharmaceutical agent, recombinant microorganism, or other substance into the circulatory system. Systemic administration can include oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or any combination thereof.

[0031] The term "therapeutically effective amount" refers to an amount of an agent (e.g., a recombinant microorganism described herein) that, when administered, is sufficient to prevent the occurrence or reduce to some extent one or more of the symptoms of the disorder, disease or condition being treated. The term "therapeutically effective amount" can also refer to an amount of an agent (e.g., a recombinant microorganism) that is sufficient to elicit the biological or medical response in a cell, tissue, system, animal or human that is desired by a researcher, veterinarian, physician or clinician.

[0032] The terms "pharmaceutical acceptable carrier", "pharmaceutical acceptable excipient", "physiologically acceptable carrier" or "physiologically acceptable excipient" refer to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. A component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of a pharmaceutical formulation. It is also suitable for use in contact with the tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications, commensurate with a reasonable benefit / risk ratio. Remington: The Science and Practice of Pharmacy, 21st Edition; Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 5th Edition; Rowe et al., Eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd Edition; Ash and Ash Eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Gibson Ed., CRC Press LLC: Boca Raton, FL, See 2004.

[0033] The term "pharmaceutical composition" refers to a mixture of an agent disclosed herein (e.g., a recombinant microorganism described herein) with other chemical components, such as diluents or carriers. A pharmaceutical composition can facilitate administration of a compound to an organism. There are multiple techniques of administering a compound in the art, including but not limited to oral, injection, aerosol, parenteral and topical administration. A pharmaceutical composition can also be obtained by reacting a compound with an inorganic or organic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

[0034] The term "recombinant microorganism," as used interchangeably herein, refers to a microorganism, such as a virus, that has been genetically modified by experimental intervention. For example, a recombinant microorganism contains one or more mutations in its genome, including, but not limited to, a deletion, insertion, inversion, substitution, or combination of a heterologous nucleic acid.

[0035] The term "naturally occurring" as used herein with respect to a microorganism, such as a virus, indicates that the microorganism can be found in nature, i.e., the microorganism can be isolated from its natural source and has not been deliberately altered.

[0036] The term "attenuated" as used herein can mean that the recombinant microorganism has a reduced ability to infect non-cancer cells compared to its ability to infect cancer cells; a reduced ability of the recombinant microorganism to replicate in non-cancer cells compared to its ability to replicate in cancer cells; a reduced replication efficiency in non-cancer cells compared to its replication efficiency in cancer cells; a reduced ability to lyse non-cancer cells compared to its ability to lyse cancer cells. In some cases, the cancer cells and non-cancer cells can be of the same type, e.g., endothelial cells, epithelial cells. In some cases, the cancer cells and non-cancer cells can be of different types, e.g., in some cases, the cancer cells can be endothelial cells and the non-cancer cells can be epithelial cells.

[0037] The term "serologically distinct" as used herein with respect to two or more different microorganisms indicates that there is no or reduced cross-reactivity between antibodies produced by a subject to the two or more different microorganisms.

[0038] As used herein, the term "tumor-associated cells" can refer to non-neoplastic cells present in the microenvironment of a solid tumor in a subject. These cells can include, for example, fibroblasts, endothelial cells, stromal cells, and immune cells.

[0039] The term "tumor protein" as used herein can refer to a protein that is expressed by a tumor cell at an increased level or in a mutated form (or both) compared to the expression level or expressed form by a cell of the same type that is not neoplastic prior to infection with a recombinant microorganism described herein.

[0040] II. Recombinant Microorganisms Recombinant microorganisms as described herein include, but are not limited to, viruses, bacteria, and parasites. In some embodiments, the recombinant microorganism may be a virus. In some embodiments, the recombinant microorganism may be a bacteria. In some embodiments, the recombinant microorganism may be a parasite.

[0041] In some embodiments, the first recombinant microorganism can be a virus and the second recombinant microorganism can be a different virus. In some embodiments, the first recombinant microorganism can be a virus and the second recombinant microorganism can be a bacterium. In some embodiments, the first recombinant microorganism can be a virus and the second recombinant microorganism can be a parasite.

[0042] In some embodiments, the second recombinant microorganism can be a virus and the first recombinant microorganism can be a different virus. In some embodiments, the second recombinant microorganism can be a virus and the first recombinant microorganism can be a bacterium. In some embodiments, the second recombinant microorganism can be a virus and the first recombinant microorganism can be a parasite.

[0043] In some embodiments, the recombinant microorganism may be a bacterium. In some embodiments, the recombinant microorganism is an Enterobacteriaceae bacterium, a Listeriaceae bacterium, or a Streptococcusceae bacterium. In some embodiments, the bacterium may be an Enterobacteriaceae bacterium. In some embodiments, the Enterobacteriaceae bacterium may be a Salmonella bacterium. In some embodiments, the Salmonella bacterium may be an attenuated Salmonella typhimurium, such as an auxotroph (aroA mutant). In some embodiments, the Salmonella bacterium may be S. bongori or S. enterica. In some embodiments, the bacterium may be a Listeriaceae bacterium. In some embodiments, the Listeriaceae bacterium may be Listeria monocytogenes. In some embodiments, the Listeriaceae bacterium may be L. monocytogenes. In some embodiments, the bacterium may be a Streptococcusceae bacterium. In some embodiments, the Streptococcusceae bacterium may be a Lactococcus bacterium. In some embodiments, the bacteria of the Lactococcus genus may be L. chungangensis, L. formosensis, L. fujiensis, L. garvieae, L. hircilactis, L. lactis, L. laudensis, L. nasutitermitis, L. piscium, L. plantarum, L. raffinolactis, or L. taiwanensis.

[0044] In some embodiments, the recombinant microorganism may be a parasite. In some embodiments, the parasite may be a Sarcocystiidae parasite or a Trypanosomatidae parasite. In some embodiments, the parasite may be a Sarcocystiidae parasite. In some embodiments, the Sarcocystiidae parasite may be a Toxoplasma parasite. In some embodiments, the Toxoplasma parasite may be Toxoplasma gondii. In some embodiments, the parasite may be a Trypanosomatidae parasite.

[0045] In some embodiments, the Trypanosomatidae parasite is a Leishmania parasite. In some embodiments, the Leishmania parasite may be L. aethiopica, L. amazonensis, L. arabica, L. aristedesi, L. braziliensis, L. chagasi, L. colombiensis, L. deanei, L. donovani, L. enriettii, L. forattinii, L. garnhami, L. guyanensis, L. herreri, L. hertigi, L. infantum, L. killicki, L. lainsoni, L. major, L. Mexicana, L. naiffi, L. panamensis, L. peruviana, L. pifanoi, L. shake, L. tarentolae, L. tropica, L. turanica, or L. venezuelensis.

[0046] In some embodiments, the recombinant microorganism may be a virus. In some embodiments, the first recombinant microorganism may be a virus. In some embodiments, the recombinant microorganism may be a DNA virus. In some embodiments, the recombinant microorganism may be an RNA virus.

[0047] In some embodiments, the recombinant microorganism may be a poxvirus, picornavirus, adenovirus, parvovirus, herpesvirus, reovirus, paramyxovirus, rhabdovirus, orthomyxovirus, or coxsackievirus.

[0048] In some embodiments, the recombinant microorganism may be a poxvirus. In some embodiments, the poxvirus may be an orthopoxvirus, a parapoxvirus, a yatapoxvirus, a leporipoxvirus, a mursipoxvirus, a betaentomopoxvirus, a cervidopoxvirus, a gammaentomopoxvirus, a suipoxvirus, a crocodilidopoxvirus, an alphaentomopoxvirus, a capripoxvirus, or an avipoxvirus. In some embodiments, the poxvirus may be an orthopoxvirus. In some embodiments, the orthopoxvirus may be a vaccinia virus. In some embodiments, the poxvirus may be a leporipoxvirus. In some embodiments, the leporipoxvirus may be a myxoma virus.

[0049] In some embodiments, the poxvirus may be a picornavirus. In some embodiments, the picornavirus may be an aphthovirus, an aquamavirus, an avihepatovirus, an abyssivirus, a cardiovirus, a cosavirus, a dicipivirus, an enterovirus, an erbovirus, a gallivirus, a hepatovirus, a hunnivirus, a kobuvirus, a kunsagivirus, a megrivirus, a miskivirus, a mosavirus, an osivirus, a parechovirus, a pasivirus, a passerivirus, a rosavirus, a sakobuvirus, a sarivirus, a sapelovirus, a senecavirus, a sissinivirus, a teschovirus, or a tremovirus. In some embodiments, the picornavirus is an enterovirus. In some embodiments, the enterovirus may be a poliovirus or a coxsackievirus. In some embodiments, the picornavirus may be a cardiovirus. In some embodiments, the cardiovirus may be a mengovirus.

[0050] In some embodiments, the microorganism may be an adenovirus. In some embodiments, the adenovirus may be an atadenovirus, an avian adenovirus, an ictoadenovirus, a mastadenovirus, or a siadenovirus.

[0051] In some embodiments, the recombinant microorganism may be a parvovirus. In some embodiments, the parvovirus may be an amdoparvovirus, an abeparvovirus, a bocaparvovirus, a chapparvovirus, a copiparvovirus, a dependoparvovirus, an erythroparvovirus, a protoparvovirus, or a tetraparvovirus. In some embodiments, the parvovirus may be a dependoparvovirus. In some embodiments, the dependoparvovirus may be an adeno-associated virus.

[0052] In some embodiments, the recombinant microorganism may be a herpesvirus. In some embodiments, the herpesvirus may be an alphaherpesvirus, a betaherpesvirus, or a gammaherpesvirus. In some embodiments, the herpesvirus may be an alphaherpesvirus. In some embodiments, the alphaherpesvirus may be an Irtovirus, a Mardivirus, a Simplexvirus, or a Varicellovirus. In some embodiments, the herpesvirus may be a betaherpesvirus. In some embodiments, the betaherpesvirus may be a cytomegalovirus, a Muromegalovirus, a Proboscivirus, or a Poseolovirus. In some embodiments, the herpesvirus may be a gammaherpesvirus. In some embodiments, the gammaherpesvirus may be a Lymphocryptovirus, a Macavirus, a Percavirus, or a Rhadinovirus.

[0053] In some embodiments, recombinant microorganism can be rhabdovirus.In some embodiments, rhabdovirus can be cryovirus, cytorhabdovirus, dicolavirus, ephemerovirus, hapavirus, redantevirus, lyssavirus, novirhabdovirus, nucleorhabdovirus, perahabdovirus, sigmavirus, sprivivirus, thripvirus, tibrovirus, tupavirus, varicosavirus or vesiculovirus.In some embodiments, rhabdovirus can be vesiculovirus.In some embodiments, vesiculovirus can be vesicular stomatitis virus or malva ...

[0054] In some embodiments, the recombinant microorganism may be a reovirus. In some embodiments, the reovirus may be a cedreovirus or a spinaleovirus. In some embodiments, the reovirus may be a cedreovirus. In some embodiments, the cedreovirus may be a caldreovirus, a mimoreovirus, an orbivirus, a phytoreovirus, a rotavirus, or a seadrnavirus. In some embodiments, the reovirus may be a spinaleovirus. In some embodiments, the spinaleovirus may be an aquareovirus, a coltivirus, a cypovirus, a fijivirus, an orthoreovirus, an idnoreovirus, a dinovernavirus, an oryzavirus, or a mycoreovirus.

[0055] In some embodiments, the recombinant microorganism may be a paramyxovirus. In some embodiments, the paramyxovirus may be an aquaparamyxovirus, an abulavirus, a ferulavirus, a henipavirus, a morbillivirus, a respirovirus, or a rubulavirus. In some embodiments, the paramyxovirus may be a morbillivirus. In some embodiments, the morbillivirus may be a measles virus.

[0056] In some embodiments, the recombinant microorganism may be an orthomyxovirus, hi some embodiments, the orthomyxovirus is an influenza virus.

[0057] In some embodiments, the recombinant microorganism is oncolytic.As used herein, the term "oncolytic microorganism" refers to a microorganism that preferentially replicates in, infects or kills cancer cells compared to non-cancer cells.It is understood that under certain non-limiting circumstances, oncolytic microorganisms (e.g., oncolytic viruses) can promote anti-cancer responses through a dual mechanism that relies on the selective killing of tumor cells as well as the stimulation of host anti-tumor immune responses.

[0058] In some embodiments, the recombinant microbe replicates in cancer cells but is attenuated or does not replicate in non-cancer cells. In some embodiments, the recombinant microbe replicates in cancer cells but does not replicate in non-cancer cells. In some embodiments, the recombinant microbe replicates in cancer cells but is attenuated in non-cancer cells. In some embodiments, the recombinant microbe mediates lysis of a plurality of cancer cells in vivo. In some embodiments, the recombinant microbe mediates lysis of a plurality of cancer cells in vivo and does not replicate in non-cancer cells. In some embodiments, the recombinant microbe mediates lysis of a plurality of cancer cells in vivo and is attenuated in non-cancer cells.

[0059] In some embodiments, oncolytic recombinant microbes can include, but are not limited to, (i) recombinant microbes that naturally replicate preferentially in cancer cells and are non-pathogenic in humans, often due to increased susceptibility to innate antimicrobial signaling or dependency on oncogenic signaling pathways; and (ii) recombinant microbes that are genetically engineered for use.

[0060] The infectivity of the recombinant microorganisms described herein can be determined using standard methods known in the art.For example, viral infectivity can be measured using viral plaque assay, fluorescent focus assay (FFA), end point dilution assay (TCID50), qPCR to determine viral RNA or DNA levels, ELISA to detect specific viral proteins, and transmission electron microscopy to count viral particles.

[0061] Replication of the recombinant microorganisms described herein can be determined using standard methods known in the art. For example, viral replication can be determined using standard plaque assay techniques known in the art. For example, a population of cells can be infected with the virus, and supernatants can be collected at various times after infection and analyzed for viral titer. These assays can be used to establish a semi-quantitative measure of relative viral replication, i.e., replication ratio, in non-cancerous cells (e.g., as described herein) versus cancerous cells (e.g., as described herein). A larger ratio indicates a virus that replicates more efficiently in cancerous cells than in non-cancerous cells.

[0062] In some embodiments, the recombinant microorganism can include an exogenous nucleic acid encoding a proinflammatory protein or a functional domain or fragment thereof. In some embodiments, the proinflammatory protein can be a chemokine or cytokine. In some embodiments, the recombinant microorganism can include an exogenous nucleic acid encoding a receptor for a proinflammatory protein (e.g., a proinflammatory cytokine or chemokine).

[0063] Exemplary proinflammatory cytokines and chemokines can include, but are not limited to, tumor necrosis factor (TNF) (e.g., TNF-α, TNF-β), TNF superfamily molecules (e.g., FasL, CD27L, CD30L, CD40L, Ox40L, 4-1BBL, TRAIL, TWEAK, Apo3L), IL-1 (e.g., IL-1α and IL-1β), IL-2, interferon-γ (IFN-γ), IFN-α / β, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-21, LIF, CCL5, GROα, MCP-1, MIP-1α, MIP-1β, MCSF, GM-CSF, CXCL2, CCL2, and RANTES.

[0064] In some embodiments, the recombinant microorganism can include an exogenous nucleic acid encoding an anti-inflammatory protein or a functional domain or fragment thereof. In some embodiments, the anti-inflammatory protein can be a chemokine or cytokine. In some embodiments, the recombinant microorganism can include an exogenous nucleic acid encoding a receptor for the anti-inflammatory protein (e.g., an anti-inflammatory cytokine or chemokine). Exemplary anti-inflammatory cytokines and chemokines can include, but are not limited to, IL-4, IL-6, IL-10, IL-11, IL-13, and TGFβ.

[0065] In some embodiments, the recombinant microorganism can include an exogenous nucleic acid encoding a cytokine receptor that can cause its cognate cytokine to be expressed in the tumor microenvironment (e.g., IL15-R can have its cognate cytokine IL15 expressed in the tumor microenvironment). In some embodiments, the recombinant microorganism can express a selected chemokine receptor whose cognate chemokine is likely expressed on the tumor (e.g., CXCR4 can have its cognate chemokine CXCL12 expressed on the tumor; CCR2 can have its target CCL2 expressed on the tumor) and can be delivered systemically. Non-limiting examples of chemokine receptors described herein can include, but are not limited to, CXC chemokine receptors, CC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptors, which correspond to the four different subfamilies of chemokines that they bind. Non-limiting embodiments of the present disclosure provide recombinant microorganisms that can include an exogenous nucleic acid encoding a chemokine receptor. In some embodiments, the chemokine receptor is a CXC chemokine receptor, a CC chemokine receptor, a CX3C chemokine receptor, an XC chemokine receptor, or any combination thereof. In some embodiments, the chemokine receptor can be CXCR1, CXCR2, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CX3CR1, XCR1, or any combination thereof.

[0066] In some embodiments, the recombinant microorganism can include an exogenous nucleic acid encoding a chemokine receptor that is a chimeric protein. In some embodiments, at least a portion of its extracellular domain can be derived from a chemokine receptor that promotes tumor-targeted delivery of the recombinant microorganism, and at least a portion of its intracellular domain can be derived from a chemokine receptor that promotes tumor-specific replication, inhibits immunosuppressive activity, or conveys some other beneficial effect, or vice versa. For example, in some embodiments, the recombinant microorganism can include a nucleic acid encoding a protein that has an intracellular GTPase domain of CCR5 and an extracellular chemokine-binding domain of CXCR4 or CCR2. In some embodiments, further improvement in the therapeutic performance of the recombinant microorganism is achieved by combining domains with different functionalities.

[0067] In some embodiments, recombinant microorganisms can contain one or more exogenous nucleic acids encoding at least one chemokine receptor. In some embodiments, recombinant microorganisms can contain exogenous nucleic acids encoding two or more different chemokine receptors that can be simultaneously expressed by recombinant microorganisms. Exemplary chemokine receptors that can be simultaneously expressed from recombinant microorganisms described herein include CXCR4 and CCR2. Recombinant microorganisms expressing multiple chemokine receptors can achieve a combination or synergistic effect on tumor cells in relation to therapeutic application of recombinant microorganisms.

[0068] In some embodiments, the modification of the recombinant microorganism reduces or at least about 1.1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.5, 3.8, 4, 4.2, 4.5, 4.8, 5, 5.2, 5.5, 5.8, 6, 6.2, 6.4 ... .5, 6.8, 7, 7.2, 7.5, 7.8, 8, 8.2, 8.5, 8.8, 9, 9.2, 9.5, 9.8, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 500, 800, 1000, 2500, 5000, 10 4 , 2.5×10 4 , 5×10 4 , 7.5×10 4 , 2.5×10 5 , 5×10 5 , 7.5×10 5 , 10 6 , 2.5×10 6 , 5×10 6 , 7.5×10 6 , 10 7 , 2.5×10 7 , 5×10 7 , 7.5×10 7 , 10 8 , 2.5×10 8 , 5×10 8 , 7.5×10 8 , 10 9 , 2.5×10 9 , 5×10 9 , 7.5×10 9 , 10 10 This can result in a fold increase or more.

[0069] In some embodiments, the recombinant microorganism can contain an exogenous nucleic acid encoding a chemokine receptor, and forced expression of the chemokine receptor by the recombinant microorganism results in an enhanced immune response against the infected tumor. After infecting the tumor, the recombinant microorganism can replicate in the tumor cells, leading to expression of the chemokine receptor on the surface of the tumor cells. These membrane receptors can function as decoy receptors, binding to and sequestering immunosuppressive chemokines within the tumor. As a result, the immunosuppressive microenvironment in the tumor can be altered, leading to enhanced immunotherapeutic activity of the recombinant microorganism compared to an otherwise identical microorganism that contains a nucleic acid encoding a chemokine receptor. In some embodiments, the increase in immunotherapeutic activity is at least about 1.1, 1.1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.5, 3.8, 4, 4.2, 4.5, 4.8, 5, 5.2, 5.5, 5.8, 6, 6.2, 6.5, 6.8, 7, 7.2, 7.5, 7.8, 8, 8.2, 8.5, 8.8, 9, 9.2, 9.5, 9.8, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 500, 800, 1000, 2500, 5000, 10 4 , 2.5×10 4 , 5×10 4 , 7.5×10 4 , 2.5×10 5 , 5×10 5 , 10 6 fold or higher increase.Without being limited, the increased immunotherapy activity can be reflected by increased B cell accumulation in tumor, increased T cell response to tumor-associated immunogen, or both.B cell accumulation can be measured, for example, by quantifying B cells in tumor, and T cell immune activity can be measured, for example, by interferon-γ (interferon-gamma) secretion in ELISPOT assay, ELISA, co-culture assay, flow cytometry, or any combination thereof.

[0070] In some embodiments, the recombinant microorganism comprises an exogenous nucleic acid encoding a chemokine receptor, and forced expression of the chemokine receptor by the recombinant results in increased replication of the microorganism in tumor cells compared to an otherwise identical microorganism that does not comprise a nucleic acid encoding a chemokine receptor. In certain embodiments, the recombinant microorganism comprises an exogenous CCR2-expressing nucleic acid, which increases tumor-specific replication of the recombinant microorganism. In some embodiments, the recombinant microorganism comprises an exogenous CCR5-expressing nucleic acid, which increases tumor-specific replication of the microorganism. In some embodiments, the increase in tumor-specific replication is at least about 1.1, 1.1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.5, 3.8, 4, 4.2, 4.5, 4.8, 5, 5.2, 5.5, 5.8, 6, 6.2, 6.5, 6.8, 7, 7.2, 7.5, 7.8, 8 ,8.2,8.5,8.8,9,9.2,9.5,9.8,10,12,14,15,16,18,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,150,200,250,500,800,1000,2500,5000,10 4 , 2.5×10 4 , 5×10 4 , 7.5×10 4 , 2.5×10 5 , 5×10 5 , 10 6 Exemplary methods for measuring increased viral delivery and spread in tumors include, but are not limited to, fluorescence or bioluminescence based imaging of reporter gene expression, quantitative PCR for detection of tumor concentrations of microbial genomes (e.g., viral genomes), or plaque determination of plaque forming units or immunohistochemistry of microbial proteins (e.g., viral proteins).

[0071] In some embodiments, the recombinant microorganism can include an exogenous nucleic acid encoding a protein that degrades the ECM of the tumor. Exemplary proteins that degrade the ECM can include, but are not limited to, membrane-associated proteins. In some embodiments, the membrane-associated proteins can include a glycosylphosphatidylinisotol (GPI) anchor.

[0072] Hyaluronidases are a family of enzymes that catalyze the degradation of hyaluronan (HA). At least five functional hyaluronidases have been identified in humans: HYAL1, HYAL2, HYAL3, HYAL4 and HYAL5 (also known as PH-20 or SPAM1), of which PH-20 is the only one known to be functional at relatively neutral pH. In some embodiments of the present disclosure, combining hyaluronidase with other tumor-targeting therapeutics (e.g., transgenes, also referred to herein as exogenous nucleic acids) can enhance the therapeutic effect of recombinant microorganisms by at least reducing ECM and enhancing transport of therapeutics into and between tumors.

[0073] In some embodiments, the recombinant microorganism can include an exogenous nucleic acid encoding a membrane-associated protein capable of degrading hyaluronan, such as hyaluronidase. As used herein, the term "hyaluronidase" refers to any enzyme or fragment thereof that catalyzes the degradation of HA in tumors, including, but not limited to, PH-20 and its homologues from other species, as well as other engineered / designed proteins with similar enzymatic functions. Hyaluronidase comprises a class of hyaluronan degrading enzymes. Hyaluronidase includes bacterial hyaluronidase (EC 4.2.2.1 or EC 4.2.99.1), hyaluronidase from leeches, other parasites and crustaceans (EC 3.2.1.36), and mammalian-type hyaluronidase (EC 3.2.1.35). The hyaluronidase can be of any non-human origin, including, but not limited to, murine, canine, feline, lagomorph, avian, bovine, ovine, porcine, equine, fish, frog, bacterial, as well as any from leeches, other parasites and crustaceans. Exemplary non-human hyaluronidases include hyaluronidases from cow, yellow jacket wasp, honeybee, white-face hornet, paper wasp, mouse, pig, rat, rabbit, sheep, chimpanzee, rhesus monkey, orangutan, cynomolgus monkey, guinea pig, Arthrobacter sp. (strain FB24), Bdellovibrio bacteriovorus, Propionibacterium acnes, Streptococcus agalactiae, Staphylococcus aureus; MRSA strain 252, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus suis, Vibrio fischeri, and Streptomyces sp., which is specific for hyaluronic acid and does not cleave chondroitin or chondroitin sulfate. hyaluronolyticus hyaluronidase enzyme may be included.

[0074] In some embodiments, the recombinant microorganism can include an exogenous nucleic acid encoding a secreted hyaluronidase. In some examples, the secreted hyaluronidase can be of microbial origin, such as HysA from Staphylococcus aureus, lin, or sko from Saccoglossus kowalevskii, rv. In some embodiments, the exogenous nucleic acid hysA can include a genomic sequence corresponding to GenBank sequence accession number U21221.1 (SEQ ID NO: 1). In some embodiments, the exogenous nucleic acid hysA encodes a protein HysA that includes an amino acid sequence set forth in SEQ ID NO: 2 (corresponding to Uniprot accession number UniProtKB-Q59801 (HYSA_STAA8)). In some embodiments, expression of a secreted hyaluronidase, such as HysA, enhances the replication, spread, or therapeutic activity (e.g., cancer cell killing ability) of the recombinant microorganism described herein.

[0075] Furthermore, in some embodiments, the recombinant microorganism can include one or more exogenous nucleic acids encoding both hyaluronidase and matrix metalloprotease. Taken together, matrix metalloproteases can degrade all kinds of ECM proteins. Examples can include, but are not limited to, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27 and MMP28.

[0076] In some embodiments, the recombinant microbe comprises an exogenous nucleic acid encoding a protein that degrades the ECM of a tumor and increases microbial spread in and among tumors compared to an otherwise identical microbe that does not comprise a nucleic acid encoding a protein that degrades the ECM of a tumor. In some embodiments, such an increase is at least about 1.1, 1.1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.5, 3.8, 4, 4.2, 4.5, 4.8, 5, 5.2, 5.5, 5.8, 6, 6.2, 6.5, 6.8, 7, 7.2, 7.5, 7.8, 8, 8. .2, 8.5, 8.8, 9, 9.2, 9.5, 9.8, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 500, 800, 1000, 2500, 5000, 10 4 , 2.5×10 4 , 5×10 4 , 7.5×10 4 , 2.5×10 5 , 5×10 5 , 10 6 Exemplary methods for measuring increased microbial spread can include, but are not limited to, fluorescence or bioluminescence based imaging of reporter gene expression, quantitative PCR for detection of tumor concentrations of microbial genomes (e.g., viral genomes), or plaque determination of plaque forming units or immunohistochemistry of microbial proteins (e.g., viral proteins).

[0077] In some embodiments, the recombinant microorganism does not comprise an exogenous nucleic acid encoding a tumor protein described herein.

[0078] One of the most common types of genes includes BCMA, HER2, GD2, CD19, CD20, and CD22. CD30, CD33, CD123, CD38, CD44, CD70, CD274, CD45, CD123, CD138C D171, ROR1, EGFR, EphA2, FBP, FAP, CEA, EGP2, EGP40, TAG72, PSMA. PSA, PAP, hsp70-2, M-CSF, LAGE-la, p53, NKG2D, B7-H6, IL-13 Rα2, IL-11, Rα, MUC1, MUC16, CA9, GD3, HMW-MAA, CD171, イスY, G250 / CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSC1, PCTA-1, MAGE, ELF2M, IGF-I, IGF-II, IGF-I inducers, hTERT, WT1, MUC1, LMP2, HPV16, HPV18, R.S GL4 MelanA MART ML-IAP AFP BCR ABL CYP1B1 PLAC1 BORIS NY-BR-1 RGS5 SART3 EphA2 5T4, 8H9, ανβ6, B7-H3, B7-H6, CAIX, CA9, CSP G4, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3 / 4, FAP. FAR, FBP, KDR, MCSP, Mucl, Mucl6, NCAM, PRAME, ROR1, CD44v7 / 8, 8H9, NCAM, VEGF-R, TAG72, RAGE-1, MN-CAIX, RU1, RU2(AS), fetal AchR, TEM1, TEM8, PAX5, OY-TES1, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, tie2, PDGFR-β, kallikrein 4, PBF, PRAME, HSDL1, CA125, TADG-1 2, MUC16, Mannan-MIC-1, HERV-K-MEL, KK-LC-1, KM-HN-1, LAGE-1, MAGE-A4, SP17, SSX4, TAG1, TAG2, ENAH, Mammaglobin-A, NY-BR-1, BAGE-1, HERV-K-MEL, K K-LC-1, KM-KN-1, LAGE1, MAGE1A, MAGEA2, mucin k, TRAG3, c-myc, cyclin B1, p62, DKK1, RU2AS, k-ras, ME1, NFYC, STEAP1, FGF5, RU2AS, hsp70-2, ARTC1, B-RAF, beta-catenin, CDC27, CDK4, CDK12, CDKN2A, CLPP, CSNK1A1, FN1, GAS7, GPNMB, HAUS3, LDLR-fucosyltransferase, MART2, MATN, MUM1, MUM2, MUM3, neo-PAP, Myosin, PPP1R3B, PRDX5, PTPRK, RBAF600, SIRT2, SNRPD1, triosephosphate isomerase, OA1, RAB38, TRP1, TRP2, melan-A, BAGE1, GAGE1, GAGE2, GAGE8, GAGE3, GAGE4, ​​GAGE5, GAGE6, GAGE7, GNTVS, LY6K, TRAG3, CASP8, SAGE, DEK-CAN, EFTUD2, FLT3-ITD, cyclin A1, FNDC3B, MAGEAG, G250, hepsin, intestinal caspases These may include, but are not limited to, carboxylesterase, PBF, CASP5, COA1, OGT, OS9, CALCA, MDM2, alpha actinin 4, elongation factor 2, fos-related antigen 1, legumain, seminal protein 17, carbonic anhydrase IX, folate receptor-α, neutrophil elastase, ephrin B2, glioma-associated antigen, β-human chorionic gonadotropin, alpha fetoprotein thyroglobulin, telomerase reverse transcriptase, intestinal carboxylesterase, prostein, survivin, and any functional portion thereof.

[0079] In some embodiments, the recombinant microorganism does not comprise one or more exogenous nucleic acids encoding one or more of the following human proteins or antigens thereof: mesothelin, BCMA, HER2, GD2, CD19, CD20, CD22, CD30, CD33, CD123, CD38, CD44, CD70, CD274, CD45, CD123, CD138, CD171, ROR1, EGFR, EphA2, FBP, FAP, CEA, EGP2, EGP40, TAG72, PSMA, PSA, PAP, hsp70-2, M-CSF, LAGE-la, p53, NKG2D ligand, B7-H6, IL-13 Rα2, IL-11 Rα, MUC1, MUC16, CA9, GD3, HMW-MAA, CD171, Lewis Y, G250 / CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSC1, PCTA-1, MAGE, ELF2M, IGF-I, IGF-II, IGF-I receptor, hTERT, WT1, MUC1, LMP2, HPV16, HPV18, R GL4, MelanA, MART, ML-IAP, AFP, BCR, ABL, CYP1B1, PLAC1, BORIS, NY-BR-1, RGS5, SART3, EphA2, glypican-3, 5T4, 8H9, ανβ6 integrin, B7-H3, B7-H6, CAIX, CA9, CSPG4, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3 / 4, FAP, FAR, FBP, KDR, MCSP, Mucl, Mucl6, NCAM, PRAME, ROR1, CD44v7 / 8, 8H9, NCAM, VEGF-R, TAG72, RAGE-1, MN-CAIX, RU1, RU2(AS), fetal AchR, TEM1, TEM8, PAX5, OY-TES1, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, tie2, PDGFR-β, kallikrein 4, PBF, PRAME, HSDL1, CA125 , TADG-12, MUC16, Mannan-MIC-1, HERV-K-MEL, KK-LC-1, KM-HN-1, LAGE-1, MAGE-A4, SP17, SSX4, TAG1, TAG2, ENAH, Mammaglobin-A, NY-BR-1, BAGE- HERV-1, HERV-2, HERV-3, HERV-4, HERV-5, LAGE1, MAGE1A, MAGEA2, mucin k, TRAG3, c-myc, cyclin B1, p62, DKK1, RU2AS, k-ras, ME1, NFYC, STEAP1, FGF5, RU2AS, hsp70-2, ARTC1, B-RAF, beta-catenin, CDC27, CDK4, CDK12, CDKN2A, CLPP, CSNK1A1, FN1, GAS7, GPNMB, HAUS3, LDLR-fucosyltransferase, MART2, HERV-2, HERV-3, HERV-4, HERV-5, HERV-6, HERV-7, HERV-8, HERV-9, HERV-10, HERV-11, HERV-12, HERV-13, HERV-14, HERV-15, HERV-16, HERV-17, HERV-18, HERV-19, HERV-20, HERV-21, HERV-22, HERV-23, HERV-24, HERV-25, HERV-26, HERV-27, HERV-28, HERV-29, HERV-30, HERV-31, HERV-32, HERV-33, HERV-34, HERV-35, HERV-36, HERV-37, HERV-38, HERV-39, HERV-40, HERV-41, HERV-42, HERV-43, HERV-44, HERV-45, HERV-46, HERV-47, HERV-48, HER , MATN, MUM1, MUM2, MUM3, neo-PAP, myosin, PPP1R3B, PRDX5, PTPRK, RBAF600, SIRT2, SNRPD1, triosephosphate isomerase, OA1, RAB38, TRP1, TRP2, melan-A, BAGE1, GAGE1, GAGE2, GAGE8, GAGE3, GAGE4, ​​GAGE5, GAGE6, GAGE7, GNTVS, LY6K, TRAG3, CASP8, SAGE, DEK-CAN, EFTUD2, FLT3-ITD, cyclin A1, FN DC3B, MAGEAG, G250, hepsin, intestinal carboxylesterase, PBF, CASP5, COA1, OGT, OS9, CALCA, MDM2, alpha actinin 4, elongation factor 2, fos-related antigen 1, legumain, seminal protein 17, carbonic anhydrase IX, folate receptor-α, neutrophil elastase, ephrin B2, glioma-associated antigen, β-human chorionic gonadotropin, alpha fetoprotein thyroglobulin, telomerase reverse transcriptase, intestinal carboxylesterase, prostein and survivin.

[0080] The recombinant microorganisms described herein can be produced by standard molecular biology and microbiology techniques known to those skilled in the art. For example, the recombinant viruses described herein can be grown in suitable host cells, e.g., HeLa, 293 or Vero cells, isolated from the host cells, and stored under conditions that promote the stability and integrity of the virus such that loss of infectivity over time is minimal. In some embodiments, the recombinant viruses described herein can be grown in host cells using cell stacks, roller bottles or perfusion bioreactors. In some examples, downstream methods for purification of the recombinant oncolytic virus can include filtration (e.g., depth filtration, tangential flow filtration or a combination thereof), ultracentrifugation or chromatographic capture. In some embodiments, the recombinant virus can be stored, e.g., by freezing or drying, e.g., by lyophilization. In some embodiments, the stored recombinant virus can be reconstituted (if dried for storage) and diluted in a pharma- ceutically acceptable carrier for administration prior to administration.

[0081] III. Methods of Treating Cancer The methods provided herein include methods of treating cancer in a subject. The terms "cancer" and "tumor" are used interchangeably herein, and the term "cancer" as used herein refers to a hyperproliferative condition. The cancers treated by the compositions and methods included herein can include solid or liquid cancers. The cancers treated by the methods described herein include, but are not limited to, melanoma, hepatocellular carcinoma, breast cancer, lung cancer, peritoneal cancer, prostate cancer, bladder cancer, ovarian cancer, leukemia, lymphoma, kidney cancer, renal adenocarcinoma, pancreatic cancer, epithelial cancer, gastric cancer, colon cancer, duodenal cancer, pancreatic adenocarcinoma, mesothelioma, glioblastoma multiforme, astrocytoma, multiple myeloma, prostate cancer, hepatocellular carcinoma, cholangiosarcoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, colorectal cancer, intestinal type gastric adenocarcinoma, cervical squamous cell carcinoma, osteosarcoma, epithelial ovarian cancer, acute lymphoblastic lymphoma, myeloproliferative neoplasms and sarcomas.

[0082] Cancer cells treated with the methods of this disclosure include cells of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, prostate, skin, stomach, testes, tongue, or uterus. Furthermore, the cancer may be specifically of the following histological types, but is not limited to: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyps; adenocarcinoma, familial polyposis coli; solid tumors; carcinoid tumor, malignant; bronchiolo-alveolar adenocarcinoma. adenocarcinoma); papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; acidophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular Adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; cutaneous adnexal carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucinous epidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous carcinoma Cystadenocarcinoma;Mucinous cystadenocarcinoma;Mucinous adenocarcinoma;Signet ring cell carcinoma;Infiltrating ductal carcinoma;Medullary carcinoma;Lobular carcinoma;Inflammatory carcinoma;Paget's disease, breast;Acinic cell carcinoma;Adenosquamous carcinoma;Adenocarcinoma with squamous metaplasia;Thymoma, malignant;Ovarian stromal tumor, malignant;Theca tumor, malignant;Granulosa cell tumor, malignant;Androblastoma, malignant;Sertoli cell carcinoma;Leydig cell tumor, malignant;Lipid cell mammary tumor, malignant;paraganglioma, malignant;extramammary paraganglioma, malignant;pheochromocytoma;glomangiosarcoma;malignant melanoma;amelanotic melanoma;superficial spreading melanoma;malignant melanoma in giant pigmented nevus;epithelioid cell melanoma;blue nevus, malignant;sarcoma;fibrosarcoma;fibrohistiocytoma, malignant;myxosarcoma;liposarcoma;leiomyosarcoma;rhabdomyosarcoma;embryonal rhabdomyosarcoma;alveolar rhabdomyosarcoma;stromal sarcoma;mixed tumor, malignant;Müllerian mixed tumor;nephroblastoma;hepatoblastoma;carcinosarcoma;mesenchymoma, malignant;Brenner tumor, malignant;phyllodes tumor, malignant;synovial sarcoma;mesothelioma, malignant;dysgerminoma;embryonal carcinoma;teratoma, malignant;ovarian goiter, malignant;choriocarcinoma;mesonephroma, malignant;angiosarcoma;Hemangioendothelioma, malignant;Kaposi's sarcoma;Hemangiopericytoma, malignant;Lymphangiosarcoma;Osteosarcoma;Juxtamoreticular osteosarcoma;Chondrosarcoma;Chondrosarcoma, malignant;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Odontogenic tumor, malignant;Ameloblastic odontosarcoma;Ameloblastic epithelioma, malignant;Ameloblastic fibrosarcoma;Pinealoma, malignant;Chordoma;Glioma, malignant;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrous astrocytoma;Astroblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Early neuroectodermal;Cerebellar sarcoma;Ganglioblastoma;Neuroblastoma;Retinoblastoma; Olfactory neuroblastoma;Meningioma, malignant;Neurofibrosarcoma;Neurilemoma, malignant;Granular cell tumor, malignant;Malignant lymphoma;Hodgkin's disease;Hodgkin's disease;Side granuloma;Malignant lymphoma, small lymphocytic;Malignant lymphoma, large cell, diffuse;Malignant lymphoma, follicular;Mycosis fungoides;Other specific non-Hodgkin's lymphoma;Malignant histiocytosis;Multiple myeloma;Mast cell sarcoma;Immunoproliferative small intestinal disease;Leukemia;Lymphocytic leukemia;Plasma cell leukemia;Erythroleukemia;Lympho-sarcoma cell leukemia;Myeloid leukemia;Basophilic leukemia;Esinophilic leukemia;Monocytic leukemia;Mast cell leukemia;Megakaryoblastic leukemia;Myeloid sarcoma;And hairy cell leukemia.In some cases, solid cancers that are metastatic are treated using the methods provided herein. In some cases, solid cancers that are inaccessible or difficult to access, for example for intratumoral delivery of therapeutic agents, are treated using the methods described herein. Cancers associated with increased expression of free fatty acids can in some cases be treated using the methods described herein;

[0083] The methods provided herein include methods for preventing or preventing the local invasiveness or metastasis or both of any type of primary cancer.For example, the primary cancer can be melanoma, non-small cell lung, small cell lung, lung, liver cancer, retinoblastoma, astrocytoma, glioblastoma, gum, tongue, leukemia, neuroblastoma, head, neck, breast, pancreas, prostate, kidney, bone, testicular, ovarian, mesothelioma, cervical, gastrointestinal, lymphoma, brain, colon or bladder.In some embodiments, the primary cancer is lung cancer, for example non-small cell lung cancer.

[0084] The methods provided herein include methods of preventing cancer, and methods of treating pre-cancerous or pre-malignant cells, including metaplasia, dysplasia and hyperplasia.The methods provided herein further include methods of inhibiting unwanted but benign cells, such as squamous cell metaplasia, dysplasia, benign prostatic hyperplasia cells, hyperplastic lesions, etc.In some embodiments, the progression of cancer or to more severe forms of cancer is stopped, destroyed or delayed by the methods provided herein.

[0085] In some embodiments, the treatment of cancer can be detected by one or more of the following: a decrease in tumor volume in a subject, a decrease in the expression level of one or more tumor markers in a subject or a sample from a subject, or a decrease in the number of tumor sites in a subject. Methods for evaluating the volume and metastasis of solid tumors in humans are known in the art. These can include, for example, positron emission tomography (PET) scanning, magnetic resonance imaging (MRI), ultrasound, CT angiography, and X-ray. Tumor proteins can be detected in vivo or in ex vivo samples from a subject. In some embodiments, the sample from a subject can be blood, tissue, urine, bone marrow, tumor biopsy, or saliva samples. Tumor proteins can be detected through standard assays known to those skilled in the art, including, for example, but not limited to, standard immunoassays, such as ELISA, flow cytometry, immunohistochemistry, and RIA.

[0086] IV. Methods of Enhancing Immune Responses Methods provided herein include methods of inducing or enhancing an immune response in a subject in vivo. In some embodiments, the induced or enhanced immune response is directed to one or more cancer-associated antigens released from one or more cancer cells lysed in vivo by a recombinant microorganism described herein.

[0087] In some embodiments, the immune response may be a cell-mediated or humoral response. In some embodiments, the immune response may be detected by assaying for enhanced B cell proliferation, CD3+ T cell proliferation, CD4+ T cell proliferation, CD8+ T cell proliferation, or any combination thereof. In some embodiments, the immune response may be detected by assaying for enhanced production of IL-2, IFN-γ, IL-1, IL-4, IL-5, IL-6, IL-13, IL-17, IL-21, IL-22, TNFα, CSF, TGFβ, granzymes, and the like. In some embodiments, cytokine release may be quantified using ELISA, flow cytometry, Western blot, or any combination thereof. In some embodiments, the immune response may be detected by enhanced proliferation, function, or any combination thereof of antigen-presenting cells. In some embodiments, the immune response may be detected by one or more of a reduction in tumor volume in the subject, a stabilization of the tumor, a reduction in the expression level of one or more tumor markers in the subject or a sample from the subject, or a reduction in the number of tumor sites in the subject. Methods for assessing the volume and metastasis of solid tumors in humans are known in the art and can include, for example, positron emission tomography (PET) scanning, magnetic resonance imaging (MRI), ultrasound, CT angiography, and X-ray.

[0088] Tumor protein can be detected in vivo or in ex vivo samples from a subject.In some embodiments, the sample from a subject can be blood, tissue, urine, bone marrow or saliva samples.Tumor protein can be detected through standard assays known to those skilled in the art, including, but not limited to, standard immunoassays, such as ELISA, immunohistochemistry, flow cytometry and RIA.

[0089] There are additional types of in vitro and in vivo assays known in the art for measuring immune responses, including measuring humoral and cellular immune responses, including, but not limited to, standard immunoassays, such as RIAs, ELISA assays, immunohistochemistry, intracellular staining (e.g., intracellular cytokine staining, see, e.g., Smith et al., PLoS One (2015) 10(9): e0138042); T cell assays, including, e.g., lymphocyte proliferation (lymphocyte activation) assays, CTL cytotoxicity cell assays, or by assaying for antigen-specific T lymphocytes in sensitized subjects. Such assays are well known in the art. See, e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al., Eur. J. Immunol. (1994) 24:2369-2376. Recent methods of measuring cell-mediated immune responses include measuring intracellular cytokines or cytokine secretion by T cell populations, or measuring epitope-specific T cells (e.g., by tetramer technology) (McMichael, AJ, and O'Callaghan, CA, J. Exp. Med. 187(9)1367-1371, 1998;Mcheyzer-Williams, MG, et al., Immunol. Rev. 150:5-21, 1996;reviewed by Lalvani, A., et al., J. Exp. Med. 186:859-865, 1997). In an exemplary embodiment disclosed herein, an enzyme-linked immunospot (ELISPOT) assay is used to detect and analyze individual cells secreting interferon-gamma (IFN-gamma). ELISPOT IFN-gamma assays and reagents are available from BD Biosciences 2350 Qume Drive San Jose, Calif., 95131. The ELISPOT assay is capable of detecting cytokine-producing cells from activated naive and memory T cell populations, and derives its specificity and sensitivity by using high affinity capture and detection antibodies and enzyme amplification. Additional information regarding the use of the ELISPOT assay is provided in J. Immunol. Methods. 2001, 254(1-2):59. Animal models, such as non-human primates, are known in the art. For example, mice are an accepted model for human immune responses. Mouse NK cell responses to tumors are an accepted model of human NK cell responses to tumors. Furthermore, mouse T cells are a model of human T cells, mouse dendritic cells (DCs) are a model of human DCs, mouse NKT cells are a model of human NKT cells, mouse innate responses are an accepted model of human innate responses, and so on. For example, CD8 +T cells, central memory T cells and effector memory T cells (see, e.g., Walzer, et al. (2002) J. Immunol. 168:2704-2711); two subsets of NK cells (see, e.g., Chakir, et al. (2000) J. Immunol. 165:4985-4993; Smith, et al. (2000) J. Exp. Med. 191:1341-1354; Ehrlich, et al. (2005) J. Immunol. 174:1922-1931; Peritt, et al. (1998) J. Immunol. 161:5821-5824); NKT cells (see, e.g., Couedel, et al. (1998) Eur. J. Immunol. 28:4391-4397;Sakamoto, et al. (1999) J. Allergy Clin. Immunol. 103:S445-S451;Saikh, et al. (2003) J. Infect. Dis. 188:1562-1570;Emoto, et al. (1997) Infection Immunity 65:5003-5009;Taniguchi, et al. (2003) Annu. Rev. Immunol. 21:483-513;see Sidobre, et al. (2004) Proc. Natl. Acad. Sci. 101:12254-12259); monocytes / macrophages (Sunderkotter, et al. (2004) J. Immunol. 172:4410-4417); two lineages of DC (Boonstra, et al. (2003) J. Exp. Med. 197:101-109;Donnenberg, Model studies have been disclosed for: Janssens and Beyaert (2003) Clinical Microb. Revs. 16:637-646. As disclosed, the innate response of mice containing Toll-like receptors (TLRs) is a model for the innate immune response of humans (see, e.g., Janssens and Beyaert (2003) Clinical Microb. Revs. 16:637-646). Mouse neutrophils are an accepted model for human neutrophils (see, e.g., Kobayashi, et al. (2003) Proc. Natl. Acad. Sci. USA 100:10948-10953; Torres, et al. (2004) 72:2131-2139; Sibelius, et al. (1999) Infection Immunity 67:1125-1130; Tvinnereim, et al. (2004) J. Immunol. 173:1994-2002). The mouse immune response to Listeria is an accepted model of the human response to Listeria (see, e.g., Kolb-Maurer, et al. (2000) Infection Immunity 68:3680-3688; Brzoza, et al. (2004) J. Immunol. 173:2641-2651).

[0090] In some embodiments, the immune response can be measured by one or more of intracellular cytokine staining (ICS), ELISpot, proliferation assays, cytotoxic T cell assays including chromium release or equivalent assays, and gene expression analysis using any number of polymerase chain reaction (PCR) or RT-PCR based assays described herein, as well as any other suitable assays for measuring the immune response. In some cases, samples of the cell product or apheresis product can be cryopreserved for retrospective analysis of cell phenotype and function.

[0091] V. Administration The method described herein comprises administering at least two recombinant microorganisms (e.g., recombinant viruses) described herein in a prime-boost regimen. Prime-boost regimens are known to those skilled in the art and are known in the art. Prime-boost regimens are generally characterized by administering a first priming dose and one or more subsequent boosting doses.

[0092] In some embodiments, the prime-boost regimen can be heterologous. The heterologous prime-boost regimen described herein can include the use of different means for priming and boosting immune response. The heterologous prime-boost regimen described herein can include administering a first recombinant microorganism (prime) and then administering a second recombinant microorganism (boost), where the first and second recombinant microorganisms are different.

[0093] In some embodiments, the first (prime) recombinant microorganism may be a virus and the second (boost) recombinant microorganism may be a virus, where the first (prime) and second (boost) viruses are different. In some embodiments, the first (prime) and second (boost) viruses may be serologically different. In some embodiments, the first (prime) and second (boost) viruses may be classified into different taxonomic families. In some embodiments, the first (prime) and second (boost) viruses may be classified into different taxonomic species. In some embodiments, the first (prime) recombinant virus may be a virus described herein and the second (boost) recombinant virus may be a different virus described herein.

[0094] In some embodiments, the first (prime) recombinant microorganism may be a bacterium and the second (boost) recombinant microorganism may be a bacterium, where the first (prime) and second (boost) bacteria are different. In some embodiments, the first (prime) and second (boost) bacteria may be serologically different. In some embodiments, the first (prime) and second (boost) bacteria may be classified into different taxonomic families. In some embodiments, the first (prime) and second (boost) bacteria may be classified into different taxonomic species. In some embodiments, the first (prime) recombinant bacterium may be a bacterium as described herein and the second (boost) recombinant bacterium may be a different virus as described herein.

[0095] In some embodiments, the first (prime) recombinant microorganism may be a parasite and the second (boost) recombinant microorganism may be a parasite, where the first (prime) and second (boost) parasites are different. In some embodiments, the first (prime) and second (boost) parasites may be serologically different. In some embodiments, the first (prime) and second (boost) parasites may be classified into different taxonomic families. In some embodiments, the first (prime) and second (boost) parasites may be classified into different taxonomic species. In some embodiments, the first (prime) recombinant parasite may be a virus as described herein and the second (boost) recombinant virus may be a different parasite as described herein.

[0096] In some embodiments, the first (prime) recombinant microorganism may be a virus and the second (boost) recombinant microorganism may be a bacterium. In some embodiments, the first (prime) recombinant virus may be a virus as described herein and the second (boost) recombinant bacterium may be a bacterium as described herein. In some embodiments, the first (prime) recombinant microorganism may be a bacterium and the second (boost) recombinant microorganism may be a virus. In some embodiments, the first (prime) recombinant bacterium may be a bacterium as described herein and the second (boost) recombinant virus may be a virus as described herein.

[0097] In some embodiments, the first (prime) recombinant microorganism may be a virus and the second (boost) recombinant microorganism may be a parasite. In some embodiments, the first (prime) recombinant virus may be a virus described herein and the second (boost) recombinant parasite may be a parasite described herein. In some embodiments, the first (prime) recombinant microorganism may be a parasite and the second (boost) recombinant microorganism may be a virus. In some embodiments, the first (prime) recombinant parasite may be a parasite described herein and the second (boost) recombinant virus may be a virus described herein.

[0098] In some embodiments, the first (prime) recombinant microorganism may be a bacterium and the second (boost) recombinant microorganism may be a parasite. In some embodiments, the first (prime) recombinant bacterium may be a bacterium described herein and the second (boost) recombinant parasite may be a parasite described herein. In some embodiments, the first (prime) recombinant microorganism may be a parasite and the second (boost) recombinant microorganism may be a bacterium. In some embodiments, the first (prime) recombinant parasite may be a parasite described herein and the second (boost) recombinant bacterium may be a bacterium described herein.

[0099] In some embodiments, a prime-boost regimen can include administering a single prime recombinant microorganism and one or more (e.g., 2, 3, 4, 5 or more) subsequent boosting recombinant microorganisms. In some embodiments, a heterologous prime-boost regimen can include administering a single prime recombinant microorganism and two or more (e.g., 3, 4, 5 or more) subsequent boosting recombinant microorganisms, where the two or more subsequent boosting recombinant microorganisms are different from each other. In some embodiments, a heterologous prime-boost regimen can include administering a single prime recombinant microorganism and two or more (e.g., 3, 4, 5 or more) subsequent boosting recombinant microorganisms, where the two subsequent boosting recombinant microorganisms are the same as each other.

[0100] In some embodiments, one or more boosting doses can be administered at intervals including days, weeks or months after administration of the first priming dose. In some embodiments, one or more boosting doses are administered at intervals of 1, 2, 3, 4, 5, 6, 7 or more days after administration of the first priming dose. In some embodiments, one or more boosting doses can be administered at intervals of 1, 2, 3, 4, 5, 6, 7, 8 or more weeks after administration of the first priming dose. In some embodiments, one or more boosting doses can be administered at intervals of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months after administration of the first priming dose. In some embodiments, one or more boosting administrations can be administered at any combination of intervals following administration of the initial priming administration (e.g., 1, 2, 3, 4, 5, 6, 7 or more days, 1, 2, 3, 4, 5, 6, 7, 8 or more weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months).

[0101] In some embodiments, the first boost can be administered within 1, 2, 3, 4, 5, 6, 7 days or weeks after the prime administration. In some embodiments, the second boost can be administered within 1, 2, 3, 4, 5, 6, 7 days or weeks after the first boost administration.

[0102] In embodiments, one or more boost administrations can be administered over a period of about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 12 weeks to about 24 weeks, about 24 weeks to about 48 weeks, about 48 weeks or about 52 weeks or longer.

[0103] The timing of the prime and boost administration can be determined by those skilled in the art. The administration of one or more boost administrations can be determined by those skilled in the art. In some embodiments, the prime dose administered can be lower than the boost dose administered. In some embodiments, the prime dose administered can be higher than the boost dose administered. The prime and boost dose administered can be determined by those skilled in the art.

[0104] In some embodiments, a prime dose can be administered in an amount sufficient to induce oncolysis in at least about 20% of the cells in the tumor, at least about 30% of the cells in the tumor, at least about 40% of the cells in the tumor, at least about 50% of the cells in the tumor, at least about 60% of the cells in the tumor, at least about 70% of the cells in the tumor, at least about 80% of the cells in the tumor, or at least about 90% of the cells in the tumor. In some embodiments, a prime dose can refer to an amount administered to a subject or tumor over a 1, 2, 5, 10, 15, 20, or 24 hour period.

[0105] In some embodiments, a boost dose can be administered in an amount sufficient to induce oncolysis in at least about 20% of the cells in the tumor, at least about 30% of the cells in the tumor, at least about 40% of the cells in the tumor, at least about 50% of the cells in the tumor, at least about 60% of the cells in the tumor, at least about 70% of the cells in the tumor, at least about 80% of the cells in the tumor, or at least about 90% of the cells in the tumor. In some embodiments, a boost dose can refer to an amount administered to a subject or tumor over a 1, 2, 5, 10, 15, 20, or 24 hour period.

[0106] In some embodiments, a subject can be administered a first (prime) dose of a recombinant microorganism described herein and a second (boost) dose of a recombinant microorganism described herein. Prime and boost dosages of the recombinant microorganism can be determined by one of skill in the art.

[0107] In some embodiments, the first (prime) recombinant microorganism dose is an auxotrophic (aroA mutant) Salmonella Typhimurium strain and the second (boost) recombinant microorganism dose is Vaccinia virus (WR.TK-). In some embodiments, the first (prime) recombinant microorganism dose is derived from Vaccinia virus and the second (boost) recombinant microorganism dose is derived from at least one of Herpes Simplex Virus strain or Vesicular Stomatitis Virus (VSV). In one aspect, the Herpes Simplex Virus may be modified. Modifications may include additions, insertions, truncations, mutations and any combination thereof. In one aspect, genes involved in neurovirulence, pathogenesis or replication may be modified. In some embodiments, the HSV modification may be any one of gamma 34.5 or ICP6 deletion.

[0108] In some embodiments, the prime recombinant microorganism can be a recombinant virus and the boost recombinant microorganism can be a different recombinant virus, and the amount of prime and / or boost administered is about 10 3 From 10 12of infectious viral particles or plaque-forming units (PFU), or approximately 10 5 From 10 10 PFU, or about 10 5 From 10 8 PFU, or about 10 8 From 10 10 In some embodiments, the amount of recombinant virus of the disclosure administered to a subject is about 10 3 From 10 12 of virus particles or plaque-forming units (PFU), or approximately 10 5 From 10 10 PFU, or about 10 5 From 10 8 PFU, or about 10 8 From 10 10 In some embodiments, the recombinant virus of the disclosure may be about 10 PFU. 3 Approximately 10 PFU / dose 4 PFU / dose, approximately 10 4 Approximately 10 PFU / dose 5 PFU / dose, approximately 10 5 Approximately 10 PFU / dose 6 PFU / dose, approximately 10 7 Approximately 10 PFU / dose 8 PFU / dose, approximately 10 9 Approximately 10 PFU / dose 10 PFU / dose, approximately 10 10 Approximately 10 PFU / dose 11 PFU / dose, approximately 10 11 Approximately 10 PFU / dose 12 PFU / dose, approximately 10 12 Approximately 10 PFU / dose 13 PFU / dose, approximately 10 13 Approximately 10 PFU / dose 14 PFU / dose, or approximately 10 14 Approximately 10 PFU / dose 15 In some embodiments, the recombinant virus of this disclosure can be administered in a dose comprising about 2×10 PFU / dose. 3 PFU / dose, 3×10 3 PFU / dose, 4×10 3 PFU / dose, 5×10 3PFU / dosage, 6×10 3 PFU / dosage, 7×10 3 PFU / dosage, 8×10 3 PFU / dosage, 9×10 3 PFU / dosage, about 10 4 PFU / dosage, about 2×10 4 PFU / dosage, about 3×10 4 PFU / dosage, about 4×10 4 PFU / dosage, about 5×10 4 PFU / dosage, about 6×10 4 PFU / dosage, about 7×10 4 PFU / dosage, about 8×10 4 PFU / dosage, about 9×10 4 PFU / dosage, about 10 5 PFU / dosage, 2×10 5 PFU / dosage, 3×10 5 PFU / dosage, 4×10 5 PFU / dosage, 5×10 5 PFU / dosage, 6×10 5 PFU / dosage, 7×10 5 PFU / dosage, 8×10 5 PFU / dosage, 9×10 5 PFU / dosage, about 10 6 PFU / dosage, about 2×10 6 PFU / dosage, about 3×10 6 PFU / dosage, about 4×10 6 PFU / dosage, about 5×10 6 PFU / dosage, about 6×10 6 PFU / dosage, about 7×10 6 PFU / dosage, about 8×10 6 PFU / dosage, about 9×10 6 PFU / dosage, about 10 7 PFU / dosage, about 2×10 7 PFU / dosage, about 3×10 7 PFU / dosage, about 4×10 7 PFU / dosage, about 5×10 7 PFU / dosage, about 6×10 7 PFU / dosage, about 7×10 7 PFU / dosage, about 8×10 7 PFU / dosage, about 9×10 7 PFU / dosage, about 10 8PFU / dosage, about 2×10 8 PFU / dosage, about 3×10 8 PFU / dosage, about 4×10 8 PFU / dosage, about 5×10 8 PFU / dosage, about 6×10 8 PFU / dosage, about 7×10 8 PFU / dosage, about 8×10 8 PFU / dosage, about 9×10 8 PFU / dosage, about 10 9 PFU / dosage, about 2×10 9 PFU / dosage, about 3×10 9 PFU / dosage, about 4×10 9 PFU / dosage, about 5×10 9 PFU / dosage, about 6×10 9 PFU / dosage, about 7×10 9 PFU / dosage, about 8×10 9 PFU / dosage, about 9×10 9 PFU / dosage, about 10 10 PFU / dosage, about 2×10 10 PFU / dosage, about 3×10 10 PFU / dosage, about 4×10 10 PFU / dosage, about 5×10 10 PFU / dosage, about 6×10 10 PFU / dosage, about 7×10 10 PFU / dosage, about 8×10 10 PFU / dosage, about 9×10 10 PFU / dosage, about 10 10 PFU / dosage, about 2×10 10 PFU / dosage, about 3×10 10 PFU / dosage, about 4×10 10 PFU / dosage, about 5×10 10 PFU / dosage, about 6×10 10 PFU / dosage, about 7×10 10 PFU / dosage, about 8×10 10 PFU / dosage, about 9×10 10 PFU / dosage, about 10 11 PFU / dosage, about 2×10 11 PFU / dosage, about 3×10 11 PFU / dosage, about 4×10 11 PFU / dosage, about 5×10 11PFU / dose, approximately 6 x 10 11 PFU / dose, approximately 7 x 10 11 PFU / dose, approximately 8 x 10 11 PFU / dose, approximately 9 x 10 11 PFU / dose or approximately 10 12 PFU / dose, approximately 10 12 Approximately 10 PFU / dose 13 PFU / dose, approximately 10 13 Approximately 10 PFU / dose 14 PFU / dose, or approximately 10 14 Approximately 10 PFU / dose 15 Doses including PFU / dose may be administered.

[0109] In some embodiments, the recombinant virus of the disclosure comprises at least about 10 3 Approximately 10 viral particles / dose 4 Viral particles / dose, approximately 10 4 Approximately 10 viral particles / dose 5 Viral particles / dose, approximately 10 5 Approximately 10 viral particles / dose 6 Viral particles / dose, approximately 10 7 Approximately 10 viral particles / dose 8 Viral particles / dose, approximately 10 9 Approximately 10 viral particles / dose 10 Viral particles / dose, approximately 10 10 Approximately 10 viral particles / dose 11 Viral particles / dose, approximately 10 11 Approximately 10 viral particles / dose 12 Viral particles / dose, approximately 10 12 Approximately 10 viral particles / dose 13 Viral particles / dose, approximately 10 13 Approximately 10 viral particles / dose 14 viral particles / dose, or approximately 10 14 Approximately 10 viral particles / dose 15 It may be administered in doses containing viral particles / dose.

[0110] In some embodiments, the recombinant virus of the disclosure comprises at least about 10 3 PFU / kg to about 104 PFU / kg, approximately 10 4 PFU / kg to about 10 5 PFU / kg, approximately 10 5 PFU / kg to about 10 6 PFU / kg, approximately 10 7 PFU / kg to about 10 8 PFU / kg, approximately 10 9 PFU / kg to about 10 10 PFU / kg, approximately 10 10 PFU / kg to about 10 11 PFU / kg, approximately 10 11 PFU / kg to about 10 12 PFU / kg, approximately 10 12 PFU / kg to about 10 13 PFU / kg, approximately 10 13 PFU / kg to about 10 14 PFU / kg, or approximately 10 14 PFU / kg to about 10 15 In some embodiments, the modified oncolytic virus of this disclosure, such as an oncolytic vaccinia virus, can be administered at a dose comprising about 2×10 PFU / kg. 3 PFU / kg, 3×10 3 PFU / kg, 4×10 3 PFU / kg, 5×10 3 PFU / kg, 6×10 3 PFU / kg, 7×10 3 PFU / kg, 8×10 3 PFU / kg, 9×10 3 PFU / kg, approximately 10 4 PFU / kg, approximately 2×10 4 PFU / kg, approximately 3×10 4 PFU / kg, approximately 4×10 4 PFU / kg, approximately 5×10 4 PFU / kg, approximately 6×10 4 PFU / kg, approximately 7×10 4 PFU / kg, approximately 8×10 4 PFU / kg, approximately 9×10 4 PFU / kg, approximately 10 5 PFU / kg, 2×10 5 PFU / kg, 3×10 5 PFU / kg, 4×105 PFU / kg, 5×10 5 PFU / kg, 6×10 5 PFU / kg, 7×10 5 PFU / kg, 8×10 5 PFU / kg, 9×10 5 PFU / kg, about 10 6 PFU / kg, about 2×10 6 PFU / kg, about 3×10 6 PFU / kg, about 4×10 6 PFU / kg, about 5×10 6 PFU / kg, about 6×10 6 PFU / kg, about 7×10 6 PFU / kg, about 8×10 6 PFU / kg, about 9×10 6 PFU / kg, about 10 7 PFU / kg, about 2×10 7 PFU / kg, about 3×10 7 PFU / kg, about 4×10 7 PFU / kg, about 5×10 7 PFU / kg, about 6×10 7 PFU / kg, about 7×10 7 PFU / kg, about 8×10 7 PFU / kg, about 9×10 7 PFU / kg, about 10 8 PFU / kg, about 2×10 8 PFU / kg, about 3×10 8 PFU / kg, about 4×10 8 PFU / kg, about 5×10 8 PFU / kg, about 6×10 8 PFU / kg, about 7×10 8 PFU / kg, about 8×10 8 PFU / kg, about 9×10 8 PFU / kg, about 10 9 PFU / kg, about 2×10 9 PFU / kg, about 3×10 9 PFU / kg, about 4×10 9 PFU / kg, about 5×10 9 PFU / kg, about 6×10 9 PFU / kg, about 7×10 9 PFU / kg, about 8×10 9PFU / kg, approximately 9×10 9 PFU / kg, approximately 10 10 PFU / kg, approximately 2×10 10 PFU / kg, approximately 3×10 10 PFU / kg, approximately 4×10 10 PFU / kg, approximately 5×10 10 PFU / kg, approximately 6×10 10 PFU / kg, approximately 7×10 10 PFU / kg, approximately 8×10 10 PFU / kg, approximately 9×10 10 PFU / kg, approximately 10 10 PFU / kg, approximately 2×10 10 PFU / kg, approximately 3×10 10 PFU / kg, approximately 4×10 10 PFU / kg, approximately 5×10 10 PFU / kg, approximately 6×10 10 PFU / kg, approximately 7×10 10 PFU / kg, approximately 8×10 10 PFU / kg, approximately 9×10 10 PFU / kg, approximately 10 11 PFU / kg, approximately 2×10 11 PFU / kg, approximately 3×10 11 PFU / kg, approximately 4×10 11 PFU / kg, approximately 5×10 11 PFU / kg, approximately 6×10 11 PFU / kg, approximately 7×10 11 PFU / kg, approximately 8×10 11 PFU / kg, approximately 9×10 11 PFU / kg, or approximately 10 12 PFU / kg, approximately 10 12 PFU / kg to about 10 13 PFU / kg, approximately 10 13 PFU / kg to about 10 14 PFU / kg, or approximately 10 14 PFU / kg to about 10 15 In some embodiments, the modified oncolytic virus of this disclosure, such as an oncolytic vaccinia virus, is administered at a dose comprising 5×10 PFU / kg. 9In some embodiments, the modified oncolytic virus of this disclosure, such as an oncolytic vaccinia virus, is administered in a dose comprising up to 5×10 PFU / kg. 9 The dose is administered in the amount of PFU / kg.

[0111] In some embodiments, the recombinant virus of the disclosure comprises at least about 10 3 Virus particles / kg to about 10 4 Virus particles / kg, approximately 10 4 Virus particles / kg to about 10 5 Virus particles / kg, approximately 10 5 Virus particles / kg to about 10 6 Virus particles / kg, approximately 10 7 Virus particles / kg to about 10 8 Virus particles / kg, approximately 10 9 Virus particles / kg to about 10 10 Virus particles / kg, approximately 10 10 Virus particles / kg to about 10 11 Virus particles / kg, approximately 10 11 Virus particles / kg to about 10 12 Virus particles / kg, approximately 10 12 Virus particles / kg to about 10 13 Virus particles / kg, approximately 10 13 Virus particles / kg to about 10 14 Virus particles / kg, or approximately 10 14 Virus particles / kg to about 10 15 The dose may be administered in the amount of viral particles / kg.

[0112] Liquid dosage forms of the recombinant viruses described herein, in some embodiments, contain about 10 3 PFU / mL to about 10 4 PFU / mL, approximately 10 4 PFU / mL to about 10 5 PFU / mL, approximately 10 5 PFU / mL to about 10 6 PFU / mL, approximately 10 7 PFU / mL to about 10 8 PFU / mL, approximately 10 9PFU / mL to about 10 10 PFU / mL, approximately 10 10 PFU / mL to about 10 11 PFU / mL, approximately 10 11 PFU / mL to about 10 12 PFU / mL, approximately 10 12 PFU / mL to about 10 13 PFU / mL, approximately 10 13 PFU / mL to about 10 14 PFU / mL, or approximately 10 14 PFU / mL to about 10 15 The viral dose in PFU / mL can be included.

[0113] In some embodiments, the recombinant virus of this disclosure comprises about 2×10 3 PFU / mL, 3×10 3 PFU / mL, 4×10 3 PFU / mL, 5×10 3 PFU / mL, 6×10 3 PFU / mL, 7×10 3 PFU / mL, 8×10 3 PFU / mL, 9×10 3 PFU / mL, approximately 10 4 PFU / mL, approximately 2×10 4 PFU / mL, approximately 3×10 4 PFU / mL, approximately 4×10 4 PFU / mL, approximately 5×10 4 PFU / mL, approximately 6×10 4 PFU / mL, approximately 7×10 4 PFU / mL, approximately 8×10 4 PFU / mL, approximately 9×10 4 PFU / mL, approximately 10 5 PFU / mL, 2×10 5 PFU / mL, 3×10 5 PFU / mL, 4×10 5 PFU / mL, 5×10 5 PFU / mL, 6×10 5 PFU / mL, 7×10 5 PFU / mL, 8×10 5 PFU / mL, 9×10 5 PFU / mL, approximately 10 6PFU / mL, about 2×10 6 PFU / mL, about 3×10 6 PFU / mL, about 4×10 6 PFU / mL, about 5×10 6 PFU / mL, about 6×10 6 PFU / mL, about 7×10 6 PFU / mL, about 8×10 6 PFU / mL, about 9×10 6 PFU / mL, about 10 7 PFU / mL, about 2×10 7 PFU / mL, about 3×10 7 PFU / mL, about 4×10 7 PFU / mL, about 5×10 7 PFU / mL, about 6×10 7 PFU / mL, about 7×10 7 PFU / mL, about 8×10 7 PFU / mL, about 9×10 7 PFU / mL, about 10 8 PFU / mL, about 2×10 8 PFU / mL, about 3×10 8 PFU / mL, about 4×10 8 PFU / mL, about 5×10 8 PFU / mL, about 6×10 8 PFU / mL, about 7×10 8 PFU / mL, about 8×10 8 PFU / mL, about 9×10 8 PFU / mL, about 10 9 PFU / mL, about 2×10 9 PFU / mL, about 3×10 9 PFU / mL, about 4×10 9 PFU / mL, about 5×10 9 PFU / mL, about 6×10 9 PFU / mL, about 7×10 9 PFU / mL, about 8×10 9 PFU / mL, about 9×10 9 PFU / mL, about 10 10 PFU / mL, about 2×10 10 PFU / mL, about 3×10 10 PFU / mL, about 4×10 10 PFU / mL, about 5×10 10PFU / mL, approximately 6×10 10 PFU / mL, approximately 7×10 10 PFU / mL, approximately 8×10 10 PFU / mL, approximately 9×10 10 PFU / mL, approximately 10 10 PFU / mL, approximately 2×10 10 PFU / mL, approximately 3×10 10 PFU / mL, approximately 4×10 10 PFU / mL, approximately 5×10 10 PFU / mL, approximately 6×10 10 PFU / mL, approximately 7×10 10 PFU / mL, approximately 8×10 10 PFU / mL, approximately 9×10 10 PFU / mL, approximately 10 11 PFU / mL, approximately 2×10 11 PFU / mL, approximately 3×10 11 PFU / mL, approximately 4×10 11 PFU / mL, approximately 5×10 11 PFU / mL, approximately 6×10 11 PFU / mL, approximately 7×10 11 PFU / mL, approximately 8×10 11 PFU / mL, approximately 9×10 11 PFU / mL, or approximately 10 12 PFU / mL, approximately 10 12 PFU / mL to about 10 13 PFU / mL, approximately 10 13 PFU / mL to about 10 14 PFU / mL or approximately 10 14 PFU / mL to about 10 15 Doses containing PFU / mL can be administered.

[0114] In some embodiments, the recombinant virus is administered by injection. In some embodiments, the dosage is about 10 3 Viral particles, 10 per injection 4 Viral particles, 10 per injection 5 Viral particles, 10 per injection 6 Viral particles, 10 per injection 7 Viral particles, 10 per injection 8Viral particles, 10 per injection 9 Viral particles, 10 per injection 10 Viral particles, 10 per injection 11 Viral particles, 10 per injection 12 Viral particles, 2 × 10 per injection 12 Viral particles, 10 per injection 13 Viral particles, 10 per injection 14 viral particles, or 10 per injection 15 In some embodiments, the dosage is about 10 per injection. 3 of infectious viral particles per injection, 10 4 of infectious viral particles per injection, 10 5 of infectious viral particles per injection, 10 6 of infectious viral particles per injection, 10 7 of infectious viral particles per injection, 10 8 of infectious viral particles per injection, 10 9 of infectious viral particles per injection, 10 10 of infectious viral particles per injection, 10 11 of infectious viral particles per injection, 10 12 of infectious viral particles, 2 × 10 per injection 12 of infectious viral particles per injection, 10 13 of infectious viral particles per injection, 10 14 of infectious viral particles, or 10 per injection. 15 In an additional embodiment, the recombinant virus of this disclosure can comprise about 10 infectious viral particles. 3 Tissue culture inhibitor dose 50% (TCID 50 ) / kg, 10 4 TCID 50 / kg, 10 4 TCID 50 / kg, 10 4 TCID 50 / kg, 10 4 TCID 50 / kg, 10 4 TCID 50 / kg, 10 4 TCID 50 / kg, 10 4 TCID 50 / kg, 10 4 TCID 50 / kg, 10 4 TCID 50 / kg, 10 4 TCID 50 / kg, 10 4 TCID 50 / kg, 3×10 8 TCID 50 / kg, 4×10 8 TCID 50 / kg, 5×10 8 TCID 50 / kg, 3×10 9 TCID 50 / kg, 4×10 9 TCID 50 / kg, 5×10 9 TCID 50 / kg, 3×10 10 TCID 50 / kg, 4×10 10 TCID 50 / kg, or 4 × 10 10 TCID 50 The dose can be administered at 10 mg / kg.

[0115] The route of administration can vary depending on the location and nature of the tumor. In certain embodiments, the route of administration is percutaneous, parenteral, intramuscular, subcutaneous, regional (e.g., near the tumor, particularly with the tumor's vasculature or adjacent vasculature), transdermal, intrathecal, intranasal, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage, or oral.

[0116] In some embodiments, the injection dose of the pharmaceutical composition can be administered as a bolus injection or slow infusion. In some embodiments, the pharmaceutical composition can be administered to the subject from a source implanted in the subject. In certain embodiments, the administration of the pharmaceutical composition can be by continuous infusion over a selected period of time. In some embodiments, the pharmaceutical composition can be administered at a therapeutically effective dose by infusion over a period of about 15 minutes, about 30 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 100 minutes, or about 120 minutes or longer. In some embodiments, the pharmaceutical composition can be administered as a liquid dosage, with the total volume of administration being about 1 mL to about 5 mL, about 5 mL to 10 mL, about 15 mL to about 20 mL, about 25 mL to about 30 mL, about 30 mL to about 50 mL, about 50 mL to about 100 mL, about 100 mL to 150 mL, about 150 mL to about 200 mL, about 200 mL to about 250 mL, about 250 mL to about 300 mL, about 300 mL to about 350 mL, about 350 mL to about 400 mL, about 400 mL to about 450 mL, about 450 mL to 500 mL, about 500 mL to 750 mL, or about 750 mL to 1000 mL.

[0117] In some embodiments, the method can include administering a prime-boost regimen disclosed herein, followed by administration of an additional recombinant microorganism, such as a third recombinant microorganism, a fourth recombinant microorganism, or any combination thereof. In some embodiments, the method can include administering a prime-boost regimen disclosed herein, followed by one or more prior or concomitant additional therapies. Examples of additional therapies can include, but are not limited to, chemotherapy, radiation, oncolytic virotherapy with additional viruses, treatment with immunomodulatory proteins, anti-cancer agents, or any combination thereof. The additional therapies can be administered concurrently or sequentially with respect to the administration of the prime, boost, or prime-boost administration. In some embodiments, the method can include administering a prime, boost, or prime-boost regimen disclosed herein, followed by one or more prior or concomitant anti-cancer agents or cancer therapies. Anti-cancer agents can include, but are not limited to, chemotherapeutic agents, radiation therapy agents, cytokines, immune checkpoint inhibitors, anti-angiogenic agents, apoptosis inducers, anti-cancer antibodies, and anti-cyclin-dependent kinase agents. In some embodiments, the cancer therapy includes chemotherapy, biological therapy, radiation therapy, immunotherapy, hormone therapy, antivascular therapy, cryotherapy, toxin therapy, surgery, or a combination thereof.

[0118] In certain embodiments, the method can include administering the prime-boost regimen disclosed herein in combination with one or more immunomodulatory agents. The immunomodulatory agent can include any compound, molecule, or substance capable of suppressing anti-recombinant microbial immunity (e.g., anti-viral immunity) associated with a tumor or cancer. In some embodiments, the immunomodulatory agent can suppress innate or adaptive immunity to a recombinant microorganism (e.g., a recombinant virus). Non-limiting examples of immunomodulatory agents include anti-CD33 antibodies or variable regions thereof, anti-CD11b antibodies or variable regions thereof, COX2 inhibitors, such as celecoxib, cytokines, such as IL-12, GM-CSF, IL-2, IFN3, and IFNγ, and chemokines, such as MIP-1, MCP-1, and IL-8. In some embodiments, the immunomodulatory agent can include immune checkpoint modulators, such as, but not limited to, anti-CTLA4, anti-PD-1, and anti-PD-L1, and TLR agonists (e.g., poly I:C). In some examples, the immunomodulatory agent is an immune checkpoint inhibitor, e.g., an antagonist of PD-1 (e.g., an antagonist antibody that binds to PD-1), an antagonist of PD-L1 (e.g., an antagonist antibody that binds to PD-L1), an antagonist of CTLA-4 (e.g., an antagonist antibody that binds to CTLA-4), an antagonist of A2AR (e.g., an antagonist antibody that binds to A2AR), an antagonist of B7-H3 (e.g., an antagonist antibody that binds to B7-H3 ... These can include antagonists of H4 (e.g., an antagonist antibody that binds to B7-H4), antagonists of BTLA (e.g., an antagonist antibody that binds to BTLA), antagonists of IDO (e.g., an antagonist antibody that binds to IDO), antagonists of KIR (e.g., an antagonist antibody that binds to KIR), antagonists of LAG3 (e.g., an antagonist antibody that binds to LAG3), and antagonists of TIM-3 (e.g., an antagonist antibody that binds to TIM3).In some embodiments, the immune checkpoint inhibitor can include a protein that binds to PD-1, PD-L1, CTLA-4, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, TIM-3, VISTA, CD160, TIGIT, PSGL-1, or any combination thereof.

[0119] In some embodiments, the additional therapy may include administering an immune checkpoint modulator. In some embodiments, the immune checkpoint modulator may be TGN1412. In some embodiments, the immune checkpoint modulator may be NKTR-214. In some embodiments, the immune checkpoint modulator may be MEDI0562. In some embodiments, the immune checkpoint modulator may be MEDI6469. In some embodiments, the immune checkpoint modulator may be MEDI6383. In some embodiments, the immune checkpoint modulator may be JTX-2011. In some embodiments, the immune checkpoint modulator may be Keytruda (pembrolizumab). In some embodiments, the immune checkpoint modulator may be Opdivo (nivolumab). In some embodiments, the immune checkpoint modulator may be Yervoy (ipilimumab). In some embodiments, the immune checkpoint modulator may be tremelimumab. In some embodiments, the immune checkpoint modulator may be Tecentriq (atezolizumab). In some embodiments, the immune checkpoint modulator may be MGA271. In some embodiments, the immune checkpoint modulator may be indoximod. In some embodiments, the immune checkpoint modulator may be epacadostat. In some embodiments, the immune checkpoint modulator may be lirilumab. In some embodiments, the immune checkpoint modulator may be BMS-986016. In some embodiments, the immune checkpoint modulator may be MPDL3280A. In some embodiments, the immune checkpoint modulator may be avelumab. In some embodiments, the immune checkpoint modulator may be durvalumab. In some embodiments, the immune checkpoint modulator may be MEDI4736. In some embodiments, the immune checkpoint modulator may be MEDI4737. In some embodiments, the immune checkpoint modulator may be TRX518. In some embodiments, the immune checkpoint modulator may be MK-4166.In some embodiments, the immune checkpoint modulator can be urelumab (BMS-663513). In some embodiments, the immune checkpoint modulator can be PF-05082566 (PF-2566).

[0120] In some embodiments, the additional therapy may be radiation. Exemplary doses include, but are not limited to, 5,000 rads (50 Gy) to 100,000 rads (1000 Gy), or 50,000 rads (500 Gy), or other suitable doses within the recited ranges. In some embodiments, the radiation dose may be about 30 to 60 Gy, about 40 to about 50 Gy, about 40 to 48 Gy, or about 44 Gy, or other suitable doses within the recited ranges, the dose being determined, for example, by means of the dosimetry tests described above. As used herein, "Gy" may refer to a unit of specific absorbed dose of radiation equivalent to 100 rads. Gy is an abbreviation for "gray."

[0121] In some embodiments, the additional therapy may be chemotherapy.Exemplary chemotherapeutic agents may include, but are not limited to, alkylating agents (e.g., nitrogen mustard derivatives, ethyleneimines, alkylsulfonates, hydrazines and triazines, nitrosurea and metal salts), plant alkaloids (e.g., vinca alkaloids, taxanes, podophyllotoxins and camptothecan analogs), antitumor antibiotics (e.g., anthracyclines, chromomycins, etc.), antimetabolites (e.g., folate antagonists, pyrimidine antagonists, purine antagonists and adenosine deaminase inhibitors), topoisomerase I inhibitors, topoisomerase II inhibitors and a wide variety of antineoplastic agents (e.g., ribonucleotide reductase inhibitors, corticosteroid inhibitors, enzymes, microtubule inhibitors and retinoids). Exemplary chemotherapeutic agents include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cyclophosphamide ... Famide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®),Fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), These agents may include, but are not limited to, rifabutin, mylotarg, paclitaxel (Taxol®), phoenix (yttrium 90 / MX-DTPA), pentostatin, polipheprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®) and vinorelbine (Navelbine®), ibrutinib, idelalisib and brentuximab vedotin.

[0122] Exemplary alkylating agents include nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes: uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard (Aminouracil Mustard®), Mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™), ifosfamide (Mitoxana®), melphalan (Alkeran®), chlorambucil (Leukeran®), pipobroman (A medel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and dacarbazine (DTIC-Dome®).Additional exemplary alkylating agents include oxaliplatin (Eloxatin®); temozolomide (Temodar® and Temodal®); dactinomycin (Actinomycin D, also known as Cosmegen®); melphalan (L-PAM, also known as L-sarcolysin and phenylalanine mustard, Alkeran®); altretamine (hexamethylmelamine (HMM), also known as Hexalen®); carmustine. (BiCNU®); bendamustine (Treanda®); busulfan (Busulfex® and Myleran®); carboplatin (Paraplatin®); lomustine (CCNU, also known as CeeNU®); cisplatin (CDDP, also known as Platinol® and Platinol®-AQ); chlorambucil (Leukeran®); cyclophosphamide (Cytoxan®); trademark) and Neosar®; dacarbazine (also known as DTIC, DIC and imidazole carboxamide, DTIC-Dome®); altretamine (hexamethylmelamine (HMM), also known as Hexalen®); ifosfamide (Ifex®); prednumustine; procarbazine (Matulane®); mechlorethamine (nitrogen mustard, mustine and mechlorethamine hydrochloride (mech loroethamine), also known as Mustargen®; streptozocin (Zanosar®); thiotepa (thiophosphoamide, also known as TESPA and TSPA, Thioplex®); cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®); and bendamustine HCl (Treanda®).

[0123] Exemplary anthracyclines include, but are not limited to, e.g., doxorubicin (Adriamycin® and Rubex®); bleomycin (Lenoxane®); daunorubicin (daunorubicin hydrochloride, daunomycin and rubidomycin hydrochloride, Cerubidine®); daunorubicin liposome (daunorubicin citrate liposome, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®, Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; rabidomycin; and desacetylrabidomycin.

[0124] Exemplary vinca alkaloids can include, but are not limited to, vinorelbine tartrate (Navelbine®), vincristine (Oncovin®), and vindesine (Eldisine®); vinblastine (also known as vinblastine sulfate, vincaleukoblastine, and VLB, Alkaban-AQ®, and Velban®); and vinorelbine (Navelbine®).

[0125] Exemplary proteasome inhibitors are bortezomib (Velcade®); carfilzomib (PX-171-007, (S)-4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)-pentaerythritol; ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O-methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(1S)-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (ONX-0912).

[0126] In various embodiments, the additional therapy can be administered in a liquid dosage form, a solid dosage form, a suppository, an inhalable dosage form, an intranasal dosage form, a liposomal formulation, a dosage form that includes nanoparticles, a dosage form that includes microparticles, a polymeric dosage form, or any combination thereof. In certain embodiments, the additional therapy can be administered for about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 12 weeks to about 24 weeks, about 24 weeks to about 48 weeks, about 48 weeks or about 52 weeks, or longer. In certain cases, the frequency of administration of the additional therapy may be once a day, twice a day, once a week, once every 3 weeks, once every 4 weeks (or once a month), once every 8 weeks (or once every 2 months), once every 12 weeks (or once every 3 months), or once every 24 weeks (or once every 6 months).

[0127] In some embodiments, the additional therapy may include a vaccine, a colony stimulating agent, an interferon, an interleukin, a virus, an anti-angiogenic agent, an antigen, a co-stimulatory agent, an immunogenic agent, an immunomodulatory agent, or an immunotherapeutic agent.

[0128] VI. Pharmaceutical Compositions Compositions described herein include pharmaceutical compositions containing one or more recombinant microorganisms described herein. In some embodiments, the pharmaceutical compositions herein may be in unit dosage form. The pharmaceutical compositions may be prepared as solutions, dispersions in glycerol, liquid polyethylene glycols, and any combination thereof in oils, as solid dosage forms, as inhalable dosage forms, as intranasal dosage forms, as liposomal formulations, as nanoparticle-containing dosage forms, as microparticle-containing dosage forms, as polymeric dosage forms, or any combination thereof. In some embodiments, the pharmaceutical compositions include a solubilizing agent, e.g., sterile water, Tris buffer. In some embodiments, the pharmaceutical compositions include an excipient. Exemplary excipients are known in the art, e.g., as described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986). Examples of excipients include, but are not limited to, buffering agents, preservatives, stabilizers, binders, compaction agents, lubricants, chelators, dispersion enhancers, disintegrants, flavoring agents, sweeteners, and coloring agents.

[0129] In some embodiments, the excipient may be a buffering agent.Non-limiting examples of buffering agents may include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate and calcium bicarbonate.As buffering agents, sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium gluconate, aluminum hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, calcium hydroxide and other calcium salts or combinations thereof may be used in pharmaceutical formulations.

[0130] In some embodiments, the excipient can include a preservative.Non-limiting examples of the preservative can include antioxidants, such as alpha tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol and phenol.Antioxidants can further include, but are not limited to, EDTA, citric acid, ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), sodium sulfite, p-aminobenzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol and N-acetylcysteine. In some cases, the preservatives include validamycin A, TL-3, sodium orthovanadate, sodium fluoride, Na-tosyl-Phe-chloromethylketone, Na-tosyl-Lys-chloromethylketone, aprotinin, phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, kinase inhibitors, phosphatase inhibitors, caspase inhibitors, granzyme inhibitors, cell adhesion inhibitors, cell division inhibitors, cell cycle inhibitors, lipid signaling inhibitors, protease inhibitors, reducing agents, alkylating agents, antimicrobial agents, oxidase inhibitors or other inhibitors.

[0131] In some embodiments, the pharmaceutical composition can include a binder as an excipient. Non-limiting examples of binders include starch, pregelatinized starch, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamide, polyvinyloxoazolidone, polyvinyl alcohol, C 12 ~C 18It can include fatty acid alcohols, polyethylene glycols, polyols, saccharides, oligosaccharides and combinations thereof.The binder used in the pharmaceutical formulation is selected from starches, such as potato starch, corn starch, wheat starch; sugars, such as sucrose, glucose, dextrose, lactose, maltodextrin; natural and synthetic gums; gelatin; cellulose derivatives, such as microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, methyl cellulose, ethyl cellulose; polyvinylpyrrolidone (povidone); polyethylene glycol (PEG); wax; calcium carbonate; calcium phosphate; alcohols, such as sorbitol, xylitol, mannitol and water or combinations thereof.

[0132] In some embodiments, the pharmaceutical composition may include a lubricant as an excipient. Non-limiting examples of lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oil, sterotex, polyoxyethylene monostearate, talc, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil. The lubricant used in the pharmaceutical formulation is selected from metal stearates (e.g., magnesium stearate, calcium stearate, aluminum stearate), fatty acid esters (e.g., sodium stearyl fumarate), fatty acids (e.g., stearic acid), fatty alcohols, glyceryl behenate, mineral oil, paraffin, hydrogenated vegetable oil, leucine, polyethylene glycol (PEG), metal lauryl sulfates (e.g., sodium lauryl sulfate, magnesium lauryl sulfate), sodium chloride, sodium benzoate, sodium acetate, and talc, or combinations thereof.

[0133] In some embodiments, the pharmaceutical formulation can include dispersion enhancers as excipients.Non-limiting examples of dispersion agents include starch, alginic acid, polyvinylpyrrolidone, guar gum, kaolin, bentonite, refined wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose as high HLB emulsifying surfactants.

[0134] In some embodiments, the pharmaceutical composition may include a disintegrant as an excipient. In some embodiments, the disintegrant may be a non-effervescent disintegrant. Non-limiting examples of non-effervescent disintegrants include starches, such as corn starch, potato starch, pregelatinized and modified starches, sweeteners, clays, such as bentonite, microcrystalline cellulose, alginates, sodium starch glycolate, gums, such as agar, guar, carob, karaya gum, pectin and tragacanth gum. In some embodiments, the disintegrant may be an effervescent disintegrant. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.

[0135] In some embodiments, the pharmaceutical composition may include a chelator. In some embodiments, the chelator may be a fungicidal chelator. Examples include, but are not limited to, ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA); disodium, trisodium, tetrasodium, dipotassium, tripotassium, dilithium and diammonium salts of EDTA; barium, calcium, cobalt, copper, dysprosium, europium, iron, indium, lanthanum, magnesium, manganese, nickel, samarium, strontium or zinc chelates of EDTA; trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid monohydrate; N,N-bis(2-hydroxyethyl)glycine; 1,3-diamino-2-hydroxypropane-N,N,N',N'-tetraacetic acid; 1,3-diaminopropane-N,N,N',N'-tetraacetic acid; ethylenediamine-N,N'-diacetic acid; ethylenediamine-N,N'-diacetic acid; Amine-N,N'-dipropionic acid dihydrochloride;Ethylenediamine-N,N'-bis(methylenephosphonic acid) hemihydrate;N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid;Ethylenediamine-N,N,N',N'-tetrakis(methylenephosphonic acid);O,O'-bis(2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid;N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid;1,6-hexamethylenediamine-N,N,N',N'-tetraacetic acid;N-(2-hydroxyethyl)iminodiacetic acid;Iminodiacetic acid;1,2-diaminopropane-N,N,N',N'-tetraacetic acid;Nitrilotriacetic acid;Nitrilotripropionic acid;Nitrilotris(methylenephosphoric acid trisodium salt of 7,19,30-trioxa-1,4,10,13,16,22,27,33-octaazabicyclo[11,11,11]pentatriacontane hexahydrobromide; or triethylenetetraamine-N,N,N',N'',N''',N'''-hexaacetic acid.

[0136] Also contemplated herein are combination products that can include one or more recombinant microorganisms disclosed herein and one or more other antimicrobial or antifungal agents, such as polyenes, e.g., amphotericin B, amphotericin B lipid complex (ABCD), liposomal amphotericin B (L-AMB) and liposomal nystatin, azoles and triazoles, such as voriconazole, fluconazole, ketoconazole, itraconazole, posaconazole, and the like; glucan synthase inhibitors, e.g., caspofungin, micafungin (FK463) and V-echinocandin (LY303366); griseofulvin; allylamines such as terbinafine; flucytosine or other antifungal agents, including those described herein. It is further contemplated that the peptides can be combined with topical antifungal agents, such as ciclopirox olamine, haloprogin, tolnaftate, undecylenate, topical nystatin, amorolfine, butenafine, naftifine, terbinafine and other topical agents. In some embodiments, the pharmaceutical composition comprises an additional agent. In some embodiments, the additional agent can be present in the pharmaceutical composition in a therapeutically effective amount.

[0137] In some embodiments, the pharmaceutical compositions described herein may contain a preservative, for example to prevent the growth of microorganisms. In some embodiments, the pharmaceutical compositions may contain a preservative. In some embodiments, the pharmaceutical compositions are free of preservatives. Suitable pharmaceutical forms for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The pharmaceutical compositions may contain a carrier, which is a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and / or vegetable oils, or any combination thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial activity can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable composition can be brought about by using agents that delay absorption, for example, aluminum monostearate and gelatin, in the composition.

[0138] For parenteral administration in aqueous solution, for example, the liquid dosage form can be suitably buffered as necessary, and the liquid diluent can be made isotonic with sufficient salts or glucose. The liquid dosage form is particularly suitable for intravenous, intramuscular, subcutaneous, intratumoral and intraperitoneal administration. In this regard, the sterile aqueous medium that can be used will be known to those skilled in the art through this disclosure. For example, a single dosage can be dissolved in 1 mL to 20 mL of isotonic NaCl solution and added to 100 mL to 1000 mL of fluid, such as sodium bicarbonate buffered saline, or injected into the proposed injection site.

[0139] In certain embodiments, a sterile injectable solution can be prepared by incorporating the modified oncolytic virus according to the present disclosure, such as the oncolytic vaccinia virus described herein, or a pharmaceutical composition containing it, in the required amount in an appropriate solvent, together with various other ingredients as listed above as necessary, followed by sterilization by filtration. In general, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and other required ingredients from those listed above. The compositions disclosed herein can be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and are also formed with inorganic acids, such as hydrochloric or phosphoric acid, or organic acids, such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron (III) hydroxide, and organic bases, such as isopropylamine, trimethylamine, histidine, procaine, and the like. Once formulated, pharmaceutical compositions can be administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically effective.

[0140] In certain embodiments, the pharmaceutical composition of this disclosure can include an effective amount of the modified virus disclosed herein in combination with a pharma- ceutically acceptable carrier. As used herein, "pharmaceutically acceptable" includes any carrier that does not interfere with the effectiveness of the biological activity of the active ingredient and / or is not toxic to the patient to whom it is administered. Non-limiting examples of suitable pharmaceutical carriers can include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, and sterile solutions. Additional non-limiting examples of pharma- ceutically compatible carriers include gels, bioabsorbable matrix materials, implanted elements containing the modified oncolytic virus, or any other suitable vehicle, delivery or administration means or material. Such carriers can be formulated by conventional methods and administered to a subject in an effective amount.

[0141] VII. Kit Provided herein is a kit comprising the prime composition, one or more boost compositions, or a prime composition and one or more boost compositions described herein. In some embodiments, the kit can comprise a recombinant microbial composition for prime administration. In some embodiments, the kit can comprise one or more recombinant microbial compositions for one or more boost administrations. In some embodiments, the kit can comprise a recombinant microbial composition for prime administration and one or more recombinant microbial compositions for one or more boost administrations.

[0142] In certain embodiments, the kit can include one or more containers containing a recombinant microbial composition for a prime administration. In certain embodiments, the kit can include one or more containers containing a recombinant microbial composition for a boost administration. In some embodiments, the kit can include one or more containers containing a recombinant microbial composition for a prime administration and one or more containers containing a recombinant microbial composition for one or more boost administrations.

[0143] In some embodiments, the kit includes instructions for use, a device for administering the recombinant microbial composition to a subject, or a device for administering an additional agent or compound to a subject. In some embodiments, the instructions can include a description of the modified recombinant microorganism and other components optionally included in the kit, as well as a method for administration, including a method for determining the appropriate condition of the subject for administering the recombinant microorganism, the appropriate dosage, and the appropriate method of administration. The instructions can also include guidelines for monitoring the subject during the treatment period. In some embodiments, the kit can include one or more agents to be administered in combination with the recombinant microbial composition described herein, such as at least one anti-cancer agent, an immunomodulatory agent, or any combination thereof.

[0144] In some embodiments, the kit includes a device for administering the recombinant microbial composition to a subject. Any of a variety of devices known in the art for administering pharmaceuticals and pharmaceutical compositions may be included in the kits provided herein. For example, and not by way of limitation, such devices include hypodermic needles, intravenous needles, catheters, needle-free injection devices, inhalers, and liquid dispensers, such as eye droppers. In certain embodiments, recombinant microorganisms delivered systemically, for example by intravenous injection, intratumoral injection, intraperitoneal injection, are included in the kit along with hypodermic needles and syringes. EXAMPLES

[0145] The examples below further illustrate the described embodiments without limiting the scope of this disclosure.

[0146] Example 1: Heterologous prime-boost regimens of Salmonella or Vaccinia virus in a mouse renal adenocarcinoma tumor model A heterologous prime-boost regimen was tested using RENCA tumor-bearing BALB / c mice as follows. The regimen used a tumor-selective strain of vaccinia virus with a TK deletion and an attenuated, auxotrophic (aroA mutant) Salmonella typhimurium strain, which were given to the mice by intratumoral injection at suboptimal doses. Mice were administered 1 × 10 5 RENCA cells were injected subcutaneously, and tumors were 50–100 mm 3 Treatment was initiated when the tumor size reached 1. Mice were assigned to one of four groups as shown in Table 1. Table 1: Treatment groups for RENCA tumor mice dosed with Salmonella or Vaccinia virus. [Table 1]

[0147] The therapeutic activity of the two prime-boost regimens was evaluated in comparison to a control and a single administration of a single agent, as shown in FIG. 1. Group 1 Balb / c mice was the control group. Group 2 of Balb / c mice was injected with single agent vaccinia virus (WR.TK-) on day 1. Group 3 of mice was injected with an auxotrophic (aroA mutant) Salmonella typhimurium strain on day 1 (prime) and received vaccinia virus (WR.TK-) on day 7 (boost). Group 4 of mice was injected with vaccinia virus (WR.TK-) on day 1 (prime) and received an auxotrophic (aroA mutant) Salmonella typhimurium strain on day 7 (boost). Tumor volumes were then evaluated for each group on day 14 of treatment, and the results are shown in FIG. 1.

[0148] The results shown in Figure 1 show enhanced therapeutic activity in group 3, where Salmonella was given as a prime and Vaccinia was given as a boost. However, when the same recombinant microorganisms were given in the reverse order, the therapeutic activity was reduced (as shown for group 4). Thus, these results indicate that the enhanced therapeutic activity was not due to a purely additive effect.

[0149] Example 2: Heterologous prime-boost regimens of vaccinia virus or herpes simplex virus (HSV) or vesicular stomatitis virus (VSV) in the murine RENCA tumor model The therapeutic efficacy of heterologous prime-boost regimens compared with single-agent administration was examined. In each group, RENCA tumors (50-100 mm 3 ) were used.

[0150] Mice were dosed intratumorally with a tumor-selective strain of vaccinia virus with a TK deletion (designated WO00001), a herpes simplex virus strain with deletions of the gamma34.5 and ICP6 genes, vesicular stomatitis virus (VSV) MΔ51 strain, or a control (VFB). Mice were assigned to one of six groups, as shown in Table 2. Table 2: Treatment groups of RENCA tumor-bearing mice dosed with vaccinia virus, herpes simplex virus (HSV) or vesicular stomatitis virus (VSV). [Table 2-1] [Table 2-2]

[0151] As shown in Figures 2A and 2B, the therapeutic activity of the two heterologous prime-boost combinations was evaluated in comparison to a control and three doses of single agents. The first group of Balb / c mice served as a control. The second group of Balb / c mice was injected with approximately 1 x 10 single agent vaccinia virus (WR.TK-) on day 1. 7 The third group of Balb / c mice was injected with vaccinia virus (WR.TK-) strain at a dose of approximately 1 × 10 7 PFU / mouse, and on day 7, approximately 3 × 10 6 The fourth group of Balb / c mice was injected with vaccinia virus (WR.TK-) strain at a dose of approximately 1 × 10 7 PFU / mouse, and on day 7, vesicular stomatitis virus (MΔ51) strain was injected at a dose of approximately 1 × 10 7 Group 5 was injected with a single-agent herpes simplex virus (gamma 34.5 and ICP6 deleted) strain on day 1 at a dose of 3 × 10 6 Group 6 of Balb / c mice was injected with a single-agent vesicular stomatitis virus (MΔ51) strain at a dose of approximately 1 × 10 PFU / mouse on day 1. 7 Tumor volumes were then assessed for each group on day 18 of treatment, with the results shown in Figure 2A and on day 21 of treatment in Figure 2B (Day 21 NB, tumor burden >1400 mm 3 or tumor burden >1400mm 3 Mice were killed before for 1400 mm 3 The tumor burden was recorded as 100 mg / kg / day.

[0152] The results shown in Figures 2A and 2B demonstrate that synergistic therapeutic activity was only seen in the prime-boost regimens of Groups 3 and 4, in which vaccinia was given as the prime and either the HSV or VSV strain was given as the boost. Table 3: Sequences [Table 3-1] [Table 3-2]

Claims

1. 1. A pharmaceutical composition for use in treating a tumor in a subject, comprising: a first oncolytic virus, said first oncolytic virus replicating in tumor cells but not replicating or exhibiting attenuated replication in non-tumor cells; a second oncolytic virus that replicates in tumor cells but does not replicate or exhibits attenuated replication in non-tumor cells; A pharmaceutical composition, wherein the use of said first oncolytic virus and said second oncolytic virus enhances or elicits an immune response to proteins expressed by tumor-associated cells that are not encoded or expressed by said first oncolytic virus and said second oncolytic virus.

2. The pharmaceutical composition of claim 1, wherein the immune response is demonstrated by one or more of the following: a decrease in tumor volume in the subject, a decrease in expression levels of one or more tumor proteins in the subject or the sample from the subject, a decrease in the number of tumor sites in the subject, a change in viral load in the subject or the sample from the subject, a change in immune cell population in the subject or the sample from the subject, a change in expression levels of immune cell markers in the subject or the sample from the subject, enhanced B cell proliferation in the subject or the sample from the subject, enhanced CD4+ T cell proliferation in the subject or the sample from the subject, enhanced CD8+ T cell proliferation in the subject or the sample from the subject, enhanced cytokine production in the subject or the sample from the subject, enhanced antigen presenting cell proliferation in the subject or the sample from the subject, or any combination thereof.

3. The pharmaceutical composition of claim 2, wherein the immune response is demonstrated by a decrease in expression levels of one or more tumor proteins, a change in immune cell population, a change in expression levels of immune cell markers, an increase in B cell proliferation, an increase in CD4+ T cell proliferation, an increase in CD8+ T cell proliferation, an increase in cytokine production, an increase in antigen presenting cell proliferation, or any combination thereof in the subject or in the sample from the subject, and wherein the sample from the subject is a blood, tissue, urine or saliva sample.

4. The pharmaceutical composition of claim 1, wherein the immune response is detectable at the time of administration of the first oncolytic virus or the second oncolytic virus or at a time thereafter.

5. The pharmaceutical composition of claim 1, wherein the first oncolytic virus does not replicate in the non-tumor cells.

6. The pharmaceutical composition of claim 1, wherein the first oncolytic virus exhibits attenuated replication in the non-tumor cells.

7. The pharmaceutical composition of claim 1 , wherein said second oncolytic virus does not replicate in said non-tumor cells.

8. 2. The pharmaceutical composition of claim 1, wherein said second oncolytic virus exhibits attenuated replication in said non-tumor cells.

9. The pharmaceutical composition of claim 1, wherein the first oncolytic virus comprises a poxvirus, picornavirus, adenovirus, parvovirus, herpesvirus, reovirus, paramyxovirus, rhabdovirus, orthomyxovirus or coxsackievirus.

10. The pharmaceutical composition of claim 1, wherein the second oncolytic virus is a poxvirus, picornavirus, adenovirus, parvovirus, herpesvirus, reovirus, paramyxovirus, rhabdovirus, orthomyxovirus or coxsackievirus.

11. The pharmaceutical composition of claim 1, wherein the first oncolytic virus and the second oncolytic virus are administered to the subject simultaneously.

12. 12. The pharmaceutical composition of claim 11, wherein the first oncolytic virus is formulated as a delayed release composition, a sustained release composition, an immediate release composition, a stealth release composition, or any combination thereof.

13. The pharmaceutical composition of claim 1, wherein the second oncolytic virus is administered to the subject after the first oncolytic virus is administered to the subject.

14. 14. The pharmaceutical composition of claim 13, wherein the second oncolytic virus is administered 1 to 60 days, 1 to 45 days, 1 to 30 days, 1 to 15 days, 1 to 10 days or 1 to 7 days after administration of the first oncolytic virus.

15. 14. The pharmaceutical composition of claim 13, wherein the second oncolytic virus is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 21, 28, 35, 56 or 60 days after administration of the first oncolytic virus.

16. The pharmaceutical composition of claim 13, wherein the second oncolytic virus is administered to the subject one or more times.

17. 14. The pharmaceutical composition of claim 13, wherein the second oncolytic virus is administered to the subject 2, 3, 4, 5 or more times with 1-60 days, 1-45 days, 1-30 days, 1-15 days, 1-10 days, 1-7 days, 1-5 days or 1-3 days between each administration.

18. The pharmaceutical composition of claim 1, wherein the first oncolytic virus is administered to the subject after the second oncolytic virus is administered to the subject.

19. 2. The pharmaceutical composition of claim 1, wherein the first oncolytic virus is administered intratumorally, intradermally, subcutaneously, intraperitoneally, intramuscularly or intravenously and the second oncolytic virus is administered intratumorally, intradermally, subcutaneously, intraperitoneally, intrathecally, intramuscularly or intravenously.

20. The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition is administered in combination with an anti-cancer therapy.

21. A pharmaceutical composition according to any one of claims 1 to 20, comprising: the first oncolytic virus comprises an exogenous nucleic acid encoding a cytokine receptor; The pharmaceutical composition, wherein said second oncolytic virus comprises an exogenous nucleic acid encoding a cytokine receptor.

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