Cancer Treatment

Abstract

The present invention provides phagemid vectors and related phagemid particles for the treatment of cancer, and in particular provides the use of novel phagemid particles and related expression systems for the treatment, prevention, amelioration, or management of cancer. In particular, the present invention relates to the use of phagemid particles and expression systems for the delivery of transgenes encoding cytokines for the treatment, prevention, amelioration, or management of cancer. The present invention also extends to the use of phagemid particles and expression systems for the delivery of transgenes and in combination with such treatments using adoptively transferred T cells for the treatment, prevention, amelioration, or management of cancer. [Selected figure] Figure 2

Description

[Technical field]

[0001] The present invention relates to the treatment of cancer, and in particular to the use of novel phagemid particles and associated expression systems for the treatment, prevention, amelioration, or management of cancer. In particular, the present invention relates to the use of phagemid particles and expression systems for the delivery of transgenes encoding cytokines for the treatment, prevention, amelioration, or management of cancer. The present invention also extends to the use of phagemid particles and expression systems for the delivery of transgenes and for the combination of such treatments with the use of adoptively transferred T cells for the treatment, prevention, amelioration, or management of cancer. [Background technology]

[0002] Pediatric high-grade gliomas are a heterogeneous group of tumors that account for 15%-20% of all pediatric central nervous system (CNS) tumors in children. The tumors can arise from any site within the CNS. When they arise from the brainstem, especially the pons, they are called DIPGs. Diagnosis is based on imaging studies and histological appearance. Despite many clinical trials, prognosis remains poor, with a 2-year survival rate of less than 10%, making it one of the leading causes of brain tumor-related deaths in childhood cancer. Given the tumor location, the tumors are inoperable, and conventional fractionated radiation is the mainstay of treatment to provide a temporary benefit, with other treatments not showing any benefit over conventional radiation therapy. Thus, due to the poor response of DIPGs to conventional treatments, innovative therapeutic approaches are needed.

[0003] In recent years, efforts have been focused on investigating gene therapy as a technique based on biological mechanisms that specifically target tumor cells and trigger antitumor activity. Gene therapy has traditionally been conceived for the treatment of congenital diseases, but it is increasingly used in the treatment of cancer to improve the efficiency of combination therapies such as chemotherapy and radiotherapy and to directly induce the death of tumor cells. Considering the many side effects resulting from conventional cancer treatments that cannot distinguish between healthy and tumor tissues, the delivery of therapeutic genes via targeted vectors gives more specificity and safety.

[0004] A variety of cytokine genes are suitable for use in cancer immunogene therapy, including interleukins (IL-4, IL-12, and IL-15) and tumor necrosis factor alpha (TNFα) and TNF-related apoptosis-inducing ligand (TRAIL). TRAIL, a member of the TNF family, induces apoptosis in a variety of cancer cell lines, is highly tolerant, and has minimal toxicity to normal tissues. Both local and systemic injections of TRAIL protein exert antitumor effects against human tumor xenografts in mice. However, the rapid clearance of TRAIL after systemic administration and the large doses required to achieve tumor regression limit the efficacy of TRAIL in patients. The currently disclosed particles are ideal and cost-effective for efficient delivery and sustained expression of TRAIL genes in cancer. IL-4 was effective in inducing antitumor effects in mouse models of cancer. Furthermore, a phase I clinical trial has been initiated to determine the safety and tolerability of IL-4 in recurrent human malignant gliomas when injected intratumorally by convection enhanced delivery (CED). No histological evidence of neurotoxicity to normal brain was identified in any patient, and drug-related systemic toxicity was not evident in any treated patient. Six of nine patients demonstrated glioma necrosis, and one patient remained disease-free for more than 18 months after surgery. Yet, again, the large protein dose and expense required to achieve efficacy, as well as the invasiveness of intracranial delivery, pose significant limitations.

[0005] During the past two decades, IL-12 has emerged as one of the most potent cytokines mediating antitumor activity in various preclinical models. Due to its pleiotropic effects on various immune cells that shape the tumor microenvironment, IL-12 establishes a link between innate and adaptive immunity, involving various immune effector cells and cytokines depending on the type of tumor or affected tissue. Although IL-12 has no direct effect on tumor cells, it improves the activation of cytotoxic T and NK effector cells that mediate tumor lysis. In addition, IL-12 improves Th1 cell responses, induces a panel of cytokines including IFN-γ, and exhibits antiangiogenesis. In mice, localized intratumoral adenoviral vector delivery of the IL-12 gene was safely completed, significantly prolonging the survival of the animals and inducing a dramatic regression in tumor size.

[0006] Recently, intracranial injection of recombinant rAAV vectors encoding IL-12 was used for brain tumor gene therapy in a rat model, resulting in antitumor effects associated with an induced increase in activated microglial cells. Finally, TNFα exerts potent antitumor cell effects, either directly through apoptosis or indirectly through its immunomodulatory activity, and also targets and destroys tumor angiogenesis.

[0007] Interleukin-15 participates in the development of important immune antitumor mechanisms. It can activate CD8+ T cells, natural killer (NK) cells, and NK T cells and promote the formation of antitumor antibodies. IL-15 can also protect T effector cells from the action of T regulatory cells and reverse tolerance to tumor-associated antigens.

[0008] The advantages of IL-15 in tumor immunotherapy are due to its special ability to activate key mechanisms of antitumor immunity, including the development and activity of both NK cells and CD8+ T cells, and to promote sustained immune responses through its action on memory T cells. Furthermore, IL-15 is less toxic and less effective at inducing Treg cell activity compared to IL-2, and in certain circumstances can even protect human effector T cells from the action of Treg cells. IL-15 tops the National Cancer Institute's list of agents with the greatest potential applications in tumor immunotherapy, and the first clinical study of recombinant human IL-15 in adults with refractory metastatic melanoma and metastatic renal cell carcinoma is currently recruiting patients (http: / / clinicaltrials.gov / ct2 / show / NCT01021059).

[0009] The survival rate of mice bearing colon cancer was significantly improved by treatment with IL-15 and further improved by blockade of programmed death ligand 1 (PD-L1). Nevertheless, the greatest therapeutic effect was achieved with a combination therapy based on the application of IL-15 and the blockade of both PD-L1 and cytotoxic T-lymphocyte antigen 4 (CTLA-4).

[0010] Unfortunately, despite high expectations, although IL-15 has shown efficacy in treating metastatic malignancies, the lack of biochemical stability in vivo is a significant limitation. Systemic rhIL-15 cytokine in clinical trials has a very short plasma half-life (less than 1 hour) and rapid renal clearance, which easily leads to impedance of efficacy. Furthermore, systemic administration of rhIL15 may cause toxic side effects, including induction of autoimmunity.

[0011] Direct delivery of IL-15 to tumors instead of systemic administration would be ideal to reduce toxicity and increase efficacy. However, direct injection into tumor masses or incorporation of IL-15 as a transgene in adoptive cell transfer is difficult to implement. The presence of multiple metastatic sites or the possibility of excessive proliferation and leukemic transformation of transduced cells may limit the relevance of the treatment in practice.

[0012] Another solution to extend the time window of IL-15 bioavailability was found in gene therapy approaches. The advantages of gene therapy include localized production, the ability to generate fusion constructs, and the versatility of combination strategies. Unfortunately, systemic delivery using eukaryotic viruses has met with limited success due to undesirable uptake by the liver and reticuloendothelial system, insertional mutagenesis, immunogenicity resulting from reactions with the complement system or pre-existing antibodies, and broad tropism of mammalian cells. Viral tropism can be modified by adding tissue-specific ligands to the viral capsid protein to mediate ligand-receptor interactions on target tissues. However, adding these ligands to eukaryotic viruses can alter the structure of the viral capsid, reducing the efficacy of the peptide itself and reducing its targeting properties. Summary of the Invention

[0013] Thus, although the cytokines described above are suitable for use in cancer immunotherapy, the majority of clinical trials involving cytokines have failed to demonstrate sustained anti-tumor responses due to lack of tumor selectivity resulting in systemic toxicity.

[0014] Thus, there is a need for improved methods for the delivery of cytokines in immuno-oncology therapy.

[0015] In a first aspect, there is provided a recombinant phagemid particle for use in a method for treating, preventing or ameliorating cancer, for expressing a transgene in a target tumor cell transduced with the particle, the phagemid particle comprising at least one transgene expression cassette comprising a nucleic acid sequence encoding one or more cytokines and comprising a genome lacking at least 50% of its bacteriophage genome, the method comprising delivering the nucleic acid sequence at least adjacent to the tumor cell such that the one or more cytokines are expressed.

[0016] In preferred embodiments, the transgene expression cassette may encode a cytokine that may have the effect of inducing apoptosis in tumor cells, altering tumor cells to promote an intrinsic anti-tumor response, altering tumor cells to promote other therapies, or altering the tumor microenvironment to promote therapy.

[0017] In certain preferred embodiments, the cytokine may be IL-4, IL-12, IL-15, TNFα, TRAIL, IFN-γ, or any combination thereof. Preferably, the cytokine is IL-15, and preferably, the cytokine is IL-4. Preferably, the cytokine is IL-12. Preferably, the cytokine is TRAIL. Preferably, the cytokine is IFN-γ. In a preferred embodiment, the cytokine is not TNFα.

[0018] However, in another preferred embodiment, the cytokine is TNFα.Preferably, the cytokine is a hybrid TNFα that includes a non-endogenous signal peptide configured to increase the expression and / or secretion of TNFα.Preferably, the signal peptide is a cytokine signal peptide other than that of TNFα.For example, the signal peptide is preferably an IL-2 signal peptide.

[0019] Thus, in one embodiment, the transmembrane domain of TNFα is replaced with a cytokine signal peptide other than that of TNFα, preferably the IL-2 signal peptide. In another embodiment, a signal peptide different from any other cytokine of the present invention, preferably the IL-2 signal peptide, is combined with any other cytokine of the present invention to increase the expression and / or secretion of the resulting hybrid cytokine. In particular, the hybrid cytokine may be any one of hybrid IL-4, IL-12, IL-15, TRAIL, or IFN-γ.

[0020] The hybrid IL-2-TNFα sequence replaces the transmembrane domain of TNFα with the signal peptide of IL-2, leaving behind the sequence encoding the secreted form of TNFα, producing a hybrid TNFα that displays greater expression and / or secretion.

[0021] Those skilled in the art will appreciate that a "signal peptide sequence" can refer to an N-terminal sequence that functions to direct the translocation of a protein to the cell membrane and regulates the secretion of the protein.

[0022] In particular, one of skill in the art will understand that a "non-endogenous signal peptide sequence" can be associated with a cytokine signal peptide, such as an interleukin, that is different from that of the cytokine being expressed.

[0023] One of skill in the art will appreciate that a "hybrid cytokine" may refer to a cytokine that includes a non-endogenous signal peptide sequence.

[0024] In one embodiment, the full length TNFα, including the transmembrane domain, has the amino acid sequence provided herein as SEQ ID NO:12, as follows:

[0025] [ka]

[0026] The nucleic acid sequence encoding the full-length TNFα, including the transmembrane domain, can be represented herein as follows:

[0027] [ka]

[0028] In one embodiment, the secreted form of TNFα has the amino acid sequence provided herein as SEQ ID NO:18, as follows:

[0029] [ka]

[0030] In one embodiment, the nucleic acid sequence encoding a secreted form of TNFα may be represented herein as follows:

[0031] [ka]

[0032] In one embodiment, the IL-2 signal peptide has the amino acid sequence provided herein as SEQ ID NO:20, as follows:

[0033] [ka]

[0034] In one embodiment, the nucleic acid sequence encoding the IL-2 signal peptide can be represented herein as SEQ ID NO:21, as follows:

[0035] [ka]

[0036] Thus, in one embodiment, the hybrid IL-2-TNFα has the amino acid sequence provided herein as SEQ ID NO:22, as follows:

[0037] [ka]

[0038] Thus, in one embodiment, the nucleic acid sequence encoding the hybrid IL-2-TNFα may be represented herein as SEQ ID NO:23, as follows:

[0039] [ka]

[0040] Thus, in one embodiment, the one or more cytokines comprise the amino acid sequence of any one of SEQ ID NOs: 12, 18, 20, and 22, or a fragment or variant thereof.

[0041] Preferably, the one or more cytokines comprise an amino acid sequence substantially as set forth in SEQ ID NO: 22, or a fragment or variant thereof.

[0042] Thus, in one embodiment, the nucleic acid sequence encoding one or more cytokines comprises any one of SEQ ID NOs: 13, 19, 21, and 23, or a fragment or variant thereof.

[0043] Preferably, the nucleic acid sequence encoding the one or more cytokines comprises SEQ ID NO: 23 or a fragment or variant thereof.

[0044] In a second aspect, there is provided a method for treating, preventing or ameliorating cancer in a subject, comprising administering a therapeutically effective amount of a recombinant phagemid particle according to the first aspect to a subject in need of such treatment.

[0045] The method of the second aspect may be used in the management of cancer in a subject.

[0046] In one embodiment, the recombinant phagemid particles are for use in the treatment, prevention, or amelioration of pediatric brain tumors, hi other embodiments, the recombinant phagemid particles are for use in the treatment, prevention, or amelioration of diffuse intrinsic pontine glioma (DIPG) or medulloblastoma.

[0047] In a third aspect, there is provided a system for producing recombinant phagemid particles from a prokaryotic host, comprising: (i) a first vector adapted to persist in a prokaryotic host, the first vector comprising at least one transgene expression cassette comprising a nucleic acid sequence encoding one or more cytokines, and a packaging signal to enable replication of the vector into single-stranded DNA; (ii) a second vector comprising a nucleic acid encoding a structural protein required for packaging of single-stranded DNA, resulting in the formation and extrusion of recombinant phagemid particles from a prokaryotic host.

[0048] In a fourth aspect, there is provided a method for producing recombinant phagemid particles from a prokaryotic host, comprising: (i) introducing into a prokaryotic host cell a first vector adapted to persist in the prokaryotic host, the first vector comprising at least one transgene expression cassette comprising a nucleic acid sequence encoding one or more cytokines and a packaging signal to permit replication of the vector into single-stranded DNA; (ii) introducing into the host a helper phage containing nucleic acid encoding a bacteriophage structural protein; (iii) culturing the host under conditions that result in the single-stranded DNA being packaged by the structural proteins to form and extrude recombinant phagemid particles from the prokaryotic host.

[0049] In a fifth aspect, there is provided a method for producing recombinant phagemid particles from a prokaryotic host, comprising: (i) introducing into a prokaryotic host cell (a) a first vector adapted to persist in a prokaryotic host, the first vector including at least one transgene expression cassette including a nucleic acid sequence encoding one or more cytokines and a packaging signal to enable replication of the vector into single-stranded DNA, and (b) a second vector including a nucleic acid encoding a structural protein required for packaging of the single-stranded DNA; (ii) culturing the host under conditions that result in the single-stranded DNA being packaged by the structural proteins to form and extrude recombinant phagemid particles from the prokaryotic host.

[0050] In a sixth aspect of the present invention, there is provided a pharmaceutical composition comprising a recombinant phagemid viral particle produced by the system according to the third aspect or produced by the method of the fourth or fifth aspects, and a pharma- ceutically acceptable vehicle.

[0051] In a seventh aspect, the present invention also provides a process for making a pharmaceutical composition according to the sixth aspect, comprising contacting a therapeutically effective amount of recombinant phagemid particles produced by a system according to the third aspect or produced by a method of the fourth or fifth aspects, and a pharma- ceutically acceptable vehicle.

[0052] In another aspect of the present invention, there is provided a recombinant phagemid particle for expressing a transgene in a target tumor cell transduced with the particle, the recombinant phagemid particle comprising at least one transgene expression cassette comprising a nucleic acid sequence encoding one or more cytokines and comprising a genome lacking at least 50% of its bacteriophage genome, wherein in use the particle is configured to deliver the nucleic acid sequence at least adjacent to a tumor cell such that the one or more cytokines are expressed, the cytokine being any one of IL-4, IL-12, IL-15, TRAIL, IFN-γ, hybrid TNFα, or any combination thereof.

[0053] Preferably, the cytokine is IL-15, preferably, the cytokine is IL-4. Preferably, the cytokine is IL-12. Preferably, the cytokine is TRAIL. Preferably, the cytokine is IFN-γ.

[0054] Preferably, the cytokine is a hybrid TNFα comprising a non-endogenous signal peptide configured to increase expression and / or secretion of TNFα. Preferably, the signal peptide is a cytokine signal peptide other than that of TNFα. For example, the signal peptide is preferably the IL-2 signal peptide.

[0055] In another embodiment, a signal peptide different from any other cytokine of the present invention, preferably an IL-2 signal peptide, is combined with any other cytokine of the present invention to increase the expression and / or secretion of the resulting hybrid cytokine. In particular, the hybrid cytokine may be any one of hybrid IL-4, IL-12, IL-15, TRAIL, or IFN-γ.

[0056] In a preferred embodiment, the transgene expression cassette may encode a cytokine that may have the effect of inducing apoptosis in tumor cells, altering tumor cells to promote an endogenous anti-tumor response, altering tumor cells to promote other therapies, or altering the tumor microenvironment to promote therapy.

[0057] In a further aspect, there is provided a recombinant phagemid particle for expressing a transgene in a target tumor cell transduced with the particle, the recombinant phagemid particle comprising at least one transgene expression cassette comprising a nucleic acid sequence encoding one or more cytokines and comprising a genome lacking at least 50% of its bacteriophage genome, wherein in use the particle is configured to deliver the nucleic acid sequence at least adjacent to a tumor cell such that one or more cytokines are expressed, the cytokine being any one of IL-4, IL-12, IL-15, TRAIL, IFN-γ, hybrid TNFα, or any combination thereof, for use in therapy or diagnosis.

[0058] Preferably, the cytokine is IL-15, preferably, the cytokine is IL-4. Preferably, the cytokine is IL-12. Preferably, the cytokine is TRAIL. Preferably, the cytokine is IFN-γ.

[0059] Preferably, the cytokine is a hybrid TNFα comprising a non-endogenous signal peptide configured to increase expression and / or secretion of TNFα. Preferably, the signal peptide is a cytokine signal peptide other than that of TNFα. For example, the signal peptide is preferably the IL-2 signal peptide.

[0060] In another embodiment, a signal peptide different from any other cytokine of the present invention, preferably an IL-2 signal peptide, is combined with any other cytokine of the present invention to increase the expression and / or secretion of the resulting hybrid cytokine. In particular, the hybrid cytokine may be any one of hybrid IL-4, IL-12, IL-15, TRAIL, or IFN-γ.

[0061] There is also a need for improved methods of treating cancer for use in adoptive transfer therapies, such as CAR T cell therapy. In the Examples, the inventors have shown that it is possible to use the particles described herein to decorate or label tumor cells with antigens that can be recognized, such as by CAR T cells.

[0062] Thus, according to an eighth aspect of the present invention, there is provided a recombinant phagemid particle for expressing at least one antigen in a target tumor cell transduced with the particle, the recombinant phagemid particle comprising at least one transgene expression cassette comprising a nucleic acid sequence encoding one or more antigens recognized by one or more adoptively transferred T cells and comprising a genome lacking at least 50% of its bacteriophage genome, wherein, in use, the particle is configured to deliver the nucleic acid sequence at least adjacent to the target tumor cell such that the one or more antigens are expressed and recognized by the one or more adoptively transferred T cells.

[0063] In a ninth aspect, there is provided a recombinant phagemid particle according to the eighth aspect for use in therapy or diagnosis.

[0064] In a tenth aspect, there is provided a recombinant phagemid particle according to the eighth aspect for use in the treatment, prevention or amelioration of cancer.

[0065] Advantageously, the particles of the present invention are used to target the delivery and expression of cytokines and antigens to tumor cells, resulting in greater tumor killing and reduced off-target effects associated with conventional therapies. The inventors have developed novel vectors and systems (so-called "hybrid phagemid viral vector systems") that are particularly useful for the delivery of antigens or cytokines to tumor cells. So-called phagemid particles, called phagemid / adeno-associated virions (i.e., PAAV), are provided. Another name the inventors use for the novel vectors they have created is "phasmids." Bacteriophage-induced cytokine therapy is provided herein. Bacteriophage-induced CAR T cell therapy is also provided herein.

[0066] Unlike the prior art AAVP genomes consisting of rAAV cassettes inserted into a filamentous phage genome (Nature protocols 2,523-531 (2007); Cell 125,385-398 (2006)), the PAAV genome of the present invention does not contain any structural bacteriophage (phage) genes, so a prokaryotic helper virus is required to facilitate vector assembly in the host. This is advantageous because AAVPs still have certain inherent limitations of bacteriophages, thus leaving room for significant improvement of AAVP or phage vectors in general. For example, AAVPs are hybrids between two virus species (i.e., bacteriophages and AAVs), and AAVP vectors contain genomes of both eukaryotic and prokaryotic viruses. The prokaryotic genome is functionally or therapeutically irrelevant, despite being essential for AAVP virus replication. Thus, the inclusion of the phage virus genome adversely affects vector efficiency and production methods, resulting in relatively low gene transfer efficiency of AAVPs when compared to mammalian viruses.

[0067] Advantageously, the redesign of hybrid viral vectors (e.g., AAV or lentivirus) into phagemid particles according to the eighth or any of the above aspects substantially lacks the phage genome from which the phagemid particles are derived, dramatically enhancing the functional properties of the resulting vectors (i.e., phagemid particles). The modification of viral expression systems into phagemid-based systems according to the present invention expands the application possibilities of phagemid viral vectors in a broader sense. By eliminating at least 50% of the bacteriophage genome from the genome of the particle, which is more than 50% of the genome size, the particle size of the resulting phagemid particles is dramatically reduced.

[0068] The term "phagemid particle" can refer to a hybrid phagemid genome encapsulated by a phage-derived coat protein. A hybrid phagemid genome is a "phagemid genome" (i.e., a genetic construct that contains two origins of replication, one from a bacteriophage (e.g., F1) and one from a bacterium (e.g., pUC1). In one embodiment, the phagemid genome can contain an integrated "AAV-derived recombinant transgene cassette" (rAAV), and is therefore a hybrid, not a conventional phagemid genome with a normal (i.e., common non-viral) recombinant transgene expression cassette. A phagemid particle can refer to a hybrid phagemid genome (i.e., the present invention) encapsulated by a phage protein derived from a trans-acting agent (e.g., a helper phage).

[0069] While allowing the additional ability to incorporate very large or multiple transgene cassettes, these smaller phagemid particles also show additional advantages in enhancing gene transfer, production yield, biodistribution, and evasion from eukaryotic cell barriers.Another important advantage of using the phagemid particles of the present invention is that they have the capacity to accommodate very large and multiple transgene cassettes or gene inserts, as described below, such as the genes of three plasmids used for recombinant virus (e.g., rAAV or lentivirus) production by transfection.Therefore, by combining the gene components for virus production in single or multiple phagemid vector(s), an efficient commercial scale virus production gene delivery system is designed.Therefore, preferably, the particle contains multiple transgene cassettes.

[0070] In one embodiment, the recombinant phagemid particles are for use in treating a subject who has not been exposed to the delivered antigen or antigens, for example by prior vaccination.

[0071] In one embodiment, the phagemid particles are preferably configured to deliver the transgene expression cassette to cells adjacent to the target tumor cells such that the nucleic acid sequence is expressed, thereby producing the antigen or antigens or cytokines, which can then become associated with or attached to the tumor cell target.

[0072] However, in a preferred embodiment, the phagemid particle is configured to deliver the transgene expression cassette to the target tumor cell.Preferably, the or each antigen is a peptide or protein that is expressed on the cell surface of the target tumor cell.Preferably, the or each antigen is a peptide or protein that is accessible to CAR T cells when expressed by tumor cells.The peptide or protein can be such that when expressed by tumor cells, it is present as a folded peptide protein at or on the cell surface.

[0073] In an eleventh aspect of the present invention, there is provided a recombinant phagemid particle for expressing at least one antigen in a target tumor cell transduced with the particle, the recombinant phagemid particle comprising at least one introduced gene expression cassette comprising a nucleic acid sequence encoding one or more cytokines and comprising a genome lacking at least 50% of its bacteriophage genome, wherein, in use, the particle is configured to deliver the nucleic acid sequence at least adjacent to the target tumor cell such that the one or more cytokines are expressed.

[0074] In a twelfth aspect, there is provided a recombinant phagemid particle according to the eleventh aspect for use in therapy or diagnosis.

[0075] In a thirteenth aspect, there is provided a recombinant phagemid particle according to the eleventh aspect for use in the treatment, prevention or amelioration of cancer.

[0076] In a fourteenth aspect, there is provided a method for treating, preventing or ameliorating cancer in a subject, comprising administering a therapeutically effective amount of a recombinant phagemid particle according to the eleventh aspect to a subject in need of such treatment.

[0077] In a fifteenth aspect, there is provided the use of a helper phage comprising nucleic acid encoding a viral vector structural protein for producing a recombinant phagemid particle according to the eleventh aspect from a prokaryotic host.

[0078] In a sixteenth aspect, there is provided a recombinant phagemid viral particle according to the eleventh aspect, or produced by a system according to the third aspect, or produced by a method of the fourth or fifth aspects, or produced by use of the fifteenth aspect, wherein the recombinant phagemid particle is for the production of a recombinant viral vector comprising or derived from a viral genome within the genome of the phagemid particle, and wherein the recombinant viral vector is used to deliver nucleic acid sequences encoding one or more antigens at least adjacent to a tumor cell such that one or more cytokines are expressed.

[0079] In a seventeenth aspect, there is provided a recombinant vector comprising a rAAV, rep-cap, adeno helper genes, and a nucleic acid sequence encoding one or more antigens or cytokines for use in treating, preventing, or ameliorating cancer.

[0080] In an eighteenth aspect, there is provided a recombinant phagemid particle comprising the vector of the seventeenth aspect for use in a method for the treatment, prevention or amelioration of cancer.

[0081] The or each antigen can be a known target of existing CAR T cells suitable for use in humans.For example, the or each antigen can be selected from the group consisting of MUC1, PSMA, CD19, CD20, estrogen-related receptor beta type 2 (ErRB2), or any combination thereof.The or each antigen can be MUC1 or PSMA.

[0082] In one embodiment, a nucleic acid sequence encoding a suitable antigen (eg, MUC1.CD28.IL4) may be represented herein as follows:

[0083] [ka]

[0084] In another embodiment, a nucleic acid sequence encoding an antigen (eg, MUC1.GPI.IL4) may be represented herein as follows:

[0085] [ka]

[0086] In yet another embodiment, a nucleic acid sequence encoding an antigen (e.g., PSMA) may be represented herein as follows:

[0087] [ka]

[0088] Thus, preferably, the transgene comprises a nucleic acid sequence substantially as set out in any one of SEQ ID NOs: 14 to 16, or a fragment or variant thereof.

[0089] In yet another embodiment, the antigen may comprise or be derived from an amino acid sequence substantially as set forth in SEQ ID NO: 17 (eg, MUC1, Genbank Accession Number: P15941), or a fragment or variant thereof.

[0090] [ka]

[0091] In some embodiments, one antigen type or species, or cytokine is delivered at least adjacent to the target tumor cell. In other embodiments, two or more antigen types or species, or cytokines are delivered at least adjacent to the tumor cell. Two or more antigens or cytokines can be delivered by the same recombinant phagemid particle, or can be delivered separately by two or more recombinant phagemid particles.

[0092] In one embodiment, the or each antigen may be an autoantigen from which the CAR T cells were generated. Autoantigens are considered to be antigens expressed by non-tumor tissues of a subject, and such antigens have been utilized as targets for CAR T cells and are suitable for use in the present invention.

[0093] However, in a preferred embodiment, the or each antigen may be a completely non-self antigen from which the CAR T cells are developed. Non-self antigens may include neo-antigens newly expressed by tumors, which have also been utilized as targets for CAR T cells and are also suitable for use in the present invention. Non-self antigens may include foreign antigens that are not expressed by any tissue of the subject from which the CAR T cells are developed. Such foreign antigens may be advantageous, as treatment may have less "off-target" effects caused by targeting non-tumor tissues that express the target antigen.

[0094] In one embodiment the or each antigen may be derived from a dengue virus or from a yellow fever virus.

[0095] In a preferred embodiment, the recombinant phagemid particle is for use in a method further comprising the use of adoptively transferred T cells.The adoptively transferred T cells can be specific to the antigen or antigens that are introduced by the recombinant phagemid particle into the target tumor cell.Preferably, more than one type of T cells are adoptively transferred.Preferably, more than one type of T cells are specific to the same antigen or different antigens.

[0096] Preferably, the adoptively transferred T cells that recognize one or more antigens expressed at least adjacent to the target tumor cells are selected from the group consisting of chimeric antigen receptor (CAR) T cells, T cell receptor (TCR) transgenic T cells, and tumor infiltrating lymphocytes (TILs). The TCR transgenic T cells can be specific for the introduced antigen or epitopes associated with each antigen. The TILs can be derived from tissues known to contain lymphocytes with specificity for known antigens.

[0097] However, most preferably, the adoptively transferred T cells are chimeric antigen receptor (CAR) T cells. CAR T cells can be specific for the antigen or antigens introduced into the tumor cells by recombinant phagemid particles.

[0098] In a most preferred embodiment, the recombinant phagemid particles are for use in a method comprising specific targeting of tumor cells by the recombinant phagemid particles and subsequent delivery to the tumor cells of one or more sequences encoding one or more antigens or cytokines for expression by the tumor cells, and transfer of CAR T cells associated with the antigen and having specificity for the one or more delivered antigens, and subsequent cytotoxic activity against the tumor cells for the treatment, prevention, or amelioration of cancer.

[0099] In one embodiment, the CAR T cells may be bispecific CAR T cells, which may have specificity for one or more antigens delivered by one or more recombinant phagemid particles. For example, a single type or species of recombinant phagemid particle may deliver two or more antigens for recognition by a single type of bispecific CAR T cell.

[0100] Preferably, the phagemid particle comprises a virion. A preferred embodiment of the genome of a recombinant phagemid particle is shown in Figure 3, with preferred components shown in Figures 4-6.

[0101] Preferably, the genome of the recombinant phagemid particle comprises a packaging signal to allow replication of the phagemid genome into single-stranded DNA, which can then be packaged into the phagemid particle in the prokaryotic host. The packaging signal may preferably comprise an origin of replication. For example, the origin of replication is preferably F1 ori, more preferably F1 bacteriophage. The DNA sequence of one embodiment of F1 ori is represented herein as SEQ ID NO: 1, as follows:

[0102] [ka]

[0103] Preferably, the genome of the recombinant phagemid particle comprises an origin of replication to allow the double-stranded vector to replicate in a prokaryotic host.Preferably, the origin of replication allows the vector to replicate in high copy number in the host.Preferably, the origin of replication comprises a pUC ori.The DNA sequence of one embodiment of the pUC ori is represented herein as SEQ ID NO:2, as follows:

[0104] [ka]

[0105] Alternatively, in another embodiment, phagemid particles can be designed so that they are integrated into the genome of a host cell. In this case, a nucleic acid sequence that favors targeted integration (e.g., by homologous recombination) of the genome of the particle is envisioned. Thus, the genome of recombinant phagemid particles can include one or more DNA sequences that favor targeted integration into the host genome.

[0106] In another embodiment, preferably, the phagemid particles can be used as recombinant vectors for delivery of transgenes to tissue-specific targets (e.g., tumor tissue), regardless of whether the vector is administered systemically or locally to a subject in vivo.

[0107] Preferably, at least one transgene expression cassette comprises a viral transgene expression cassette, more preferably a mammalian viral transgene expression cassette.For example, in a preferred embodiment, at least one transgene expression cassette can comprise a lentiviral transgene expression cassette.At least one transgene expression cassette is preferably an adeno-associated virus (AAV) transgene expression cassette.

[0108] The transgene expression cassette may comprise any nucleic acid that codes for an antigen or antigens or cytokines that are expressed in target tumor cells or tissues.In one embodiment of the present invention, the nucleic acid may be DNA, which may be genomic DNA or cDNA.In some embodiments, non-naturally occurring cDNA may be preferred.In another embodiment, the nucleic acid may be RNA, such as antisense RNA or shRNA.

[0109] As shown in the exemplary example 7, phagemids can be used to deliver genes to tumor cells. In this example, downregulation of mTOR expression in tumor cells (e.g., medulloblastoma cells) can be achieved by treatment with RGD4C-phagemids carrying sequences encoding mTOR / shRNA (RGD4C-mTOR / shRNA). Example 7 provides another illustrative example of the use of phagemids to deliver genes to tumor cells. As shown in the example 7, RGD4C-phagemids can successfully deliver TNFα selectively to DIPGs (diffuse intrinsic pontine gliomas), resulting in apoptosis induction. Thus, RGD4C-phagemids have therapeutic potential for use in targeted therapy against DIPGs using cytokines.

[0110] However, it will be understood that the type of tumor cell targeted by the recombinant phagemid particle will depend on the type of cell targeting ligand that can be expressed on the surface of the particle. Cell targeting ligands are discussed below.

[0111] The transgene expression cassette may comprise one or more functional elements required for the expression of nucleic acid in target tumor cells.For example, preferably, the transgene expression cassette comprises a promoter such as CMV promoter.The DNA sequence of one embodiment of the CMV promoter is represented herein as SEQ ID NO:3, as follows:

[0112] [ka]

[0113] In another preferred embodiment, the transgene expression cassette comprises a promoter that is active only in the target tumor cells. Thus, the promoter can be tumor-activated and / or temozolomide-inducible.

[0114] In one embodiment, the promoter can be the multidrug resistance promoter (MDR), which is highly active in cancer cells.This promoter is activated by cancer drugs, and produces P-gp and ABC transporter to excrete drugs from cells.This promoter can be used for gene therapy in combination with cancer drugs.

[0115] In another embodiment, the promoter can be the human telomerase reverse transcriptase (hTERT) promoter. This promoter is only active in tumor cells, but not in normal proliferating cells. Therefore, this promoter can be used as a tumor-specific promoter.

[0116] In a preferred embodiment, the transgene expression cassette comprises a grp78 promoter. The nucleic acid sequence of one embodiment of the grp78 promoter is represented herein as SEQ ID NO:8, as follows:

[0117] [ka]

[0118] Preferably, the transgene expression cassette comprises a nucleic acid for encoding a polyA tail which can be attached to the or each expressed antigen or cytokine. The DNA sequence of one embodiment of the nucleic acid for encoding the polyA tail is represented herein as SEQ ID NO:4, as follows:

[0119] [ka]

[0120] Preferably, the transgene expression cassette comprises left and / or right inverted terminal repeat sequences (ITR). The ITR can be specific to AAV or long terminal repeat sequences (LTR) specific to the lentivirus serotype and can be any sequence as long as it forms a hairpin loop in its secondary structure. For example, the AAV serotype can be AAV1-9, but is preferably AAV1, AAV2, AAV5, AAV6, or AAV8. The DNA sequence of one embodiment of the ITR (left ITR from a commercially available AAV plasmid) is represented herein as SEQ ID NO:5, as follows:

[0121] [ka]

[0122] The DNA sequence of another embodiment of the ITR (right ITR from a commercially available AAV plasmid) is represented herein as SEQ ID NO:6, as follows:

[0123] [ka]

[0124] Preferably, the genome of the recombinant phagemid particle contains a selectable marker dependent on the host cell harboring the vector, for example to confer ampicillin resistance in the host cell, preferably a bacterium, the marker providing selection pressure during production of the phagemid particle in the host cell.

[0125] Preferably, the recombinant phagemid particle comprises one or more capsid minor coat proteins. The recombinant phagemid particle may comprise a pIII capsid minor coat protein configured to display a cell targeting ligand that enables delivery of the particle to a target tumor cell. Preferably, the recombinant phagemid particle comprises one or more capsid major coat proteins. The recombinant phagemid particle may comprise at least one pVIII capsid major coat protein configured to display a foreign peptide thereon.

[0126] Recombinant phagemid particles can include capsid structure modification, for example, by treatment, or chemical or biochemical binding.Suitable modification examples can include cross-linking of peptide residues onto phagemid particles.In another embodiment, recombinant phagemid particles can include one or functional peptides attached to their capsid.For example, functional peptides can include nuclear localization signals.Thus, phagemid particles can be multifunctional and use the features disclosed in WO2014 / 184528.

[0127] In another embodiment, the recombinant phagemid particles can be combined with a cationic polymer to form a complex with a net positive charge, as described in WO2014 / 184529. The cationic polymer can be selected from the group consisting of chitosan, poly-D-lysine (PDL), diethylaminoethyl (DEAE), diethylaminoethyl-dextran (DEAE.DEX), polyethyleneimine (PEI), polybrene, protamine sulfate, and cationic lipids. Preferably, the cationic lipid is selected from the group consisting of fugene®, lipofectamine®, and DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate). Preferably, the cationic polymer comprises DEAE, more preferably DEAE.DEX.

[0128] Preferably, the phagemid particle comprises a genome that is substantially devoid of the phage genome from which it is derived. Preferably, the genome of the recombinant phagemid particle is devoid of at least 60%, more preferably at least 70%, even more preferably at least 80% of the bacteriophage genome from which it is derived. More preferably, the genome of the recombinant phagemid particle is devoid of at least 90%, more preferably at least 95%, even more preferably at least 99% of the bacteriophage genome from which it is derived. Preferably, the genome of the recombinant phagemid particle is devoid of all of the bacteriophage genome from which it is derived. However, as discussed above, the genome of the phagemid virus particle may, in some embodiments, comprise a bacteriophage origin of replication, i.e., F1 bacteriophage ori, to allow the particle to replicate into single-stranded DNA.

[0129] Preferably, the phagemid particle comprises a genome having a relative genome size of less than 14 Kb. The relative genome size may be less than about 13 Kb, less than about 12 Kb, less than about 11 Kb, less than about 10 Kb, less than about 9 Kb, less than about 8 Kb, or less than about 7 Kb. The relative genome size may be 3 Kb to 9 Kb, 4 Kb to 8 Kb, 5 Kb to 7 Kb, or preferably about 6 Kb.

[0130] Preferably, the phagemid particle lacks bacteriophage structural genes in its genome that are required for the formation, packaging, or extrusion of the particle from a prokaryotic host. Such structural genes encode capsid proteins, etc. Preferably, the phagemid particle comprises a genome that lacks genes encoding the minor or major coat proteins from which the particle is derived. Preferably, the phagemid particle comprises a genome that lacks the pIII capsid minor coat protein or that lacks the pVIII capsid major coat protein. Most preferably, the phagemid particle comprises a genome that lacks both the pIII capsid minor coat protein and the pVIII capsid major coat protein.

[0131] Thus, recombinant phagemid particles include replication-defective virus-like particles or virions that are assembled and displayed from structural components including, but not limited to, proteins and other conjugated compounds derived from a bacteriophage, although the particle's genome does not contain the structural genes of the bacteriophage from which it is derived.

[0132] Thus, given that the genome of the recombinant phagemid particle of the eighth aspect or any of the above aspects lacks a derivative phage genome containing structural genes, an alternative system is needed to package the recombinant phagemid genome into a bacteriophage capsid to provide the necessary structural (i.e., capsid) genes required to produce the particles of the invention. Thus, the inventors have devised a novel system for producing the particles of the eighth aspect or any of the above aspects, which involves the use of a separate so-called "helper virus" vector. Thus, in effect, the particle of the eighth aspect or any of the above aspects is a hybrid phagemid vector containing components of a phagemid and a eukaryotic virus.

[0133] Thus, in a nineteenth aspect, there is provided a system for producing recombinant phagemid particles from a prokaryotic host, comprising: (i) a first vector adapted to persist in a prokaryotic host, the first vector comprising at least one transgene expression cassette comprising a nucleic acid sequence encoding one or more cytokines, or one or more antigens that are one or more adoptively transferred T cells, and a packaging signal to allow replication of the vector into single-stranded DNA; (ii) a second vector comprising a nucleic acid encoding a structural protein required for packaging of single-stranded DNA, resulting in the formation and extrusion of recombinant phagemid particles from a prokaryotic host.

[0134] Advantageously, separating the replication elements of the phagemid particles into a first "therapeutic" vector carrying the transgene encoding the or each antigen recognized by the or each adoptively transferred T cell or cytokine, and a second separate "helper" vector carrying the viral structural genes, substantially reduces the genome / vector size, thereby significantly increasing the transgene capacity. Phagemid particles are used therapeutically (e.g., CAR-T cell therapy), and the increased transgene capacity is a particularly useful advantage for gene therapy applications of the new system. As a result, this improves production yields, gene transfer efficiency, and the flexibility of the vector system for other applications.

[0135] The novelty of the system of the nineteenth aspect is its ability to package a eukaryotic viral (such as AAV or lentivirus) genome provided by a first vector into a prokaryotic viral capsid (i.e., bacteriophage) provided by a second vector. Thus, while the prior art systems (i.e., AAVP) are chimeras of two genomes, the system of the nineteenth aspect (i.e., PAAV) is a chimera between a prokaryotic viral phenotype and a eukaryotic viral genotype.

[0136] The antigen may be any of those disclosed herein. In a preferred embodiment, the or each antigen is a peptide or protein expressed on the cell surface of the target tumor cell. Preferably, the or each antigen is a peptide or protein that is accessible to CAR T cells when expressed by the tumor cell. The peptide or protein may be such that when expressed by the tumor cell, it is present at or as a folded peptide protein on the cell surface. The or each antigen may be a known target of existing CAR T cells suitable for use in humans. For example, the or each antigen may be selected from the group consisting of MUC1, PSMA, CD19, CD20, estrogen-related receptor beta type 2 (ErRB2), or any combination thereof. In one embodiment, the or each antigen may be a dengue virus or yellow fever vaccine antigen.

[0137] Preferably, the system of the 19th aspect is used to produce the recombinant phagemid particle of the 8th aspect. Thus, preferably, the first vector comprises the genome of the recombinant phagemid particle. The packaging signal of the first vector may preferably comprise an origin or replication. Preferably, the replication origin of the first vector is comprised of the F1 ori, more preferably the F1 bacteriophage.

[0138] Preferably, the first vector comprises a second origin of replication to allow the replication of double-stranded vector in prokaryotic host.Preferably, this origin of replication allows the high copy number replication of vector in host.Preferably, the origin of replication comprises pUC ori.Alternatively, the first vector can comprise one or more DNA sequences that promote targeted integration into host genome, thus eliminating the need for any origin of replication.

[0139] The transgene expression cassette comprises a viral transgene expression cassette, more preferably a mammalian viral transgene expression cassette.For example, at least one transgene expression cassette can comprise a lentivirus transgene expression cassette or an AAV transgene expression cassette.AAV transgene expression cassette is preferred.

[0140] A preferred embodiment of the second vector is shown in FIG. 7, with preferred components shown in FIG. 8. The second vector or "helper phage" is preferably a bacteriophage specifically designed to rescue phagemid particles carrying the first vector (i.e., the genome of the phagemid particle) from a prokaryotic host, an embodiment of which is shown in FIG. 3. Thus, the second vector (i.e., the helper phage) is provided to provide its proteins and polypeptides to the first vector (i.e., the genome of the phagemid particle) or other DNA entity that contains a functional packaging signal and / or a single-stranded origin or replication. The second vector is most preferably replication-deficient. Preferably, the second vector contains a disrupted packaging signal that significantly reduces its ability to package itself into a phage particle. Preferably, the second vector contains a disrupted origin of replication. In one embodiment, the disrupted origin of replication is a medium copy number origin such as p15a. In another embodiment, the disrupted origin of replication is a low copy number origin, such as pMB1. Preferably, the first vector (i.e., the genome of the phagemid particle) is configured to outcompete the second vector (i.e., the helper phage) in both replication and packaging.

[0141] The genome of the second vector can be engineered to confer targeting properties (or multifunctional properties as described in WO2014 / 184528) to the resulting recombinant phagemid particle, thus providing structural capsid proteins for phagemid particle assembly. Preferably, the second vector contains nucleic acid encoding one or more capsid minor coat proteins or one or more capsid major coat proteins. All capsid proteins may be wild-type or recombinant, present in single or multiple copies, and modified to display chimeric or synthetic peptides. This includes display of antigens of other viruses for peptide vaccine delivery, or display of adjuvants if a DNA vaccine (delivered by the phagemid particle of the eighth aspect) is desired.

[0142] Thus, in one example, the second vector can include a first nucleic acid sequence encoding a pIII minor coat protein configured to display a cell targeting ligand to enable delivery of the recombinant phagemid particle to a target cell (e.g., tumor). Thus, a 9-amino acid mutation can be induced in the pIII minor coat protein of the recombinant phagemid particle to target tumor cells and α v Beta3 and Alpha v It may be desirable to confer specificity to angiogenic tumor-associated endothelial cells that express β5 integrin. Thus, the genome of the second vector may contain the RGD4C targeting peptide (CDCRGDCFC-SEQ ID NO:7).

[0143] In another example, the second vector can include a second nucleic acid sequence encoding at least one pVIII capsid major coat protein configured to display a foreign peptide thereon. Thus, it may be desirable to mutagenize the wild-type pVIII major coat protein of the recombinant phagemid particle to display a short peptide, e.g., less than 10 amino acids in length. The short peptide may be a targeting moiety and / or have an intrinsic biological / chemical functionality in vivo or in vitro. For example, in in vivo immune stimulation via antigen display, the peptide displayed on the pVIII coat protein is processed via the exogenous pathway and displayed on the MHC II of the target cell, thus displaying another target for the adoptively transferred T cell. Alternatively, at least one pVIII major coat protein can have a binding function to nanoparticles (e.g., gold) in vitro, e.g., by displaying a gold-binding peptide.

[0144] The first vector may be a member of the Retroviridae or Orthoretrovirinae subfamily. The first vector may be a member of the Lentivirus genus. Preferably, the first vector is a member of the Parvoviridae or subfamily. Preferably, the first vector is a member of the Dependoparvovirus or Adeno-associated virus species.

[0145] Once the first vector (i.e., the genome of the phagemid particle) and the second vector (i.e., the helper phage) are constructed, they are used together to produce the recombinant phagemid particles of the eighth embodiment or any of the above embodiments in a prokaryotic host. It will be understood that the packaging signal (e.g., origin of replication) of the first vector, which allows replication of the phagemid genome into single-stranded DNA, functions to signal the second vector (i.e., the helper phage) structural proteins to package the phagemid genome (i.e., they act together in trans within the host) to create the particles used in the eighth embodiment or any of the above embodiments.

[0146] In one preferred embodiment, the first vector (phagemid particle genome) comprises a nucleic acid sequence substantially as set forth in SEQ ID NO:9, or a fragment or variant thereof, where SEQ ID NO:9 is represented as follows, and "N" is a transgene:

[0147] [ka]

[0148] [ka]

[0149] In one preferred embodiment, the second vector (helper phage carrying the RGD sequence) comprises a nucleic acid sequence substantially as set forth in SEQ ID NO: 10, or a fragment or variant thereof, where SEQ ID NO: 10 is represented as follows:

[0150] [ka]

[0151] [ka]

[0152] [ka]

[0153] [ka]

[0154] [ka]

[0155] In a preferred embodiment, the second vector (helper phage without the RGD sequence) comprises a nucleic acid sequence substantially as set forth in SEQ ID NO:11, or a fragment or variant thereof, where SEQ ID NO:11 is represented as follows:

[0156] [ka]

[0157] [ka]

[0158] [ka]

[0159] [ka]

[0160] [ka]

[0161] The system according to the nineteenth aspect can be used to produce particles for use according to any of the uses disclosed herein. The particles can be for use according to the second, ninth, or tenth aspects. In one embodiment, the produced recombinant phagemid particles are for use in treating a subject that has not been exposed to the delivered antigen or antigens, for example by prior vaccination.

[0162] In one embodiment, the recombinant phagemid particles produced by the system of the nineteenth aspect are for use in a method further comprising the use of one or more adoptively transferred T cells.Preferably, the adoptively transferred T cells are selected from the group consisting of chimeric antigen receptor (CAR) T cells, T cell receptor (TCR) transgenic T cells, and tumor infiltrating lymphocytes (TILs).The adoptively transferred T cells can be specific for one or more antigens introduced into the tumor cells by the recombinant phagemid particles.In a preferred embodiment, the adoptively transferred T cells are CAR T cells.

[0163] As described in Example 1, the inventors have devised two alternative approaches (see Figures 9 and 10) for producing recombinant phagemid particles of the invention in a prokaryotic host.

[0164] Thus, in a twentieth aspect, there is provided a method for producing recombinant phagemid particles from a prokaryotic host, comprising: (i) introducing into a prokaryotic host cell a first vector adapted to persist in the prokaryotic host, the first vector comprising at least one transgene expression cassette comprising a nucleic acid sequence encoding one or more cytokines, or one or more antigens recognized by one or more adoptively transferred T cells, and a packaging signal to permit replication of the vector into single-stranded DNA; (ii) introducing into the host a helper phage containing nucleic acid encoding a bacteriophage structural protein; (iii) culturing the host under conditions that result in the single-stranded DNA being packaged by the structural proteins to form and extrude recombinant phagemid particles from the prokaryotic host.

[0165] Advantageously, this method (shown in FIG. 9) results in a very high yield of particles. The first vector (i.e., the genome of the phagemid particle) can be introduced into a host cell, for example by infection. The host cell can then be transformed with a helper phage, thereby producing recombinant phagemid particles. Preferably, this method includes a purification step following the culturing step. Purification can include centrifugation and / or filtration.

[0166] The antigen may be any of those disclosed herein. In a preferred embodiment, the or each antigen is a peptide or protein expressed on the cell surface of the target tumor cell. Preferably, the or each antigen is a peptide or protein that is accessible to CAR T cells when expressed by the tumor cell. The peptide or protein may be such that when expressed by the tumor cell, it is present at or as a folded peptide protein on the cell surface. The or each antigen may be a known target of existing CAR T cells suitable for use in humans. For example, the or each antigen may be selected from the group consisting of MUC1, PSMA, CD19, CD20, estrogen-related receptor beta type 2 (ErRB2), or any combination thereof. In one embodiment, the or each antigen may be a dengue virus or yellow fever vaccine antigen.

[0167] The cytokine may be any of those disclosed herein. In particular, the cytokine may be IL-4, IL-12, IL-15, TNFα, TRAIL, IFN-γ, or any combination thereof. Preferably, the cytokine is IL-15, and preferably, the cytokine is IL-4. Preferably, the cytokine is IL-12. Preferably, the cytokine is TRAIL. Preferably, the cytokine is IFN-γ.

[0168] Preferably, the cytokine is not TNFα. Preferably, the cytokine is TNFα. Preferably, the cytokine is a hybrid TNFα that includes a non-endogenous signal peptide configured to increase the expression and / or secretion of TNFα. Preferably, the signal peptide is a cytokine signal peptide other than that of TNFα.

[0169] Preferably, the method of the twentieth aspect is used to produce the recombinant phagemid particle of the eighth aspect. Thus, preferably, the first vector comprises the genome of the recombinant phagemid particle. The packaging signal of the first vector may preferably comprise an origin or replication. Preferably, the replication origin of the first vector is comprised of the F1 ori, more preferably the F1 bacteriophage.

[0170] The method according to the twentieth aspect can be used to produce particles for use according to any use disclosed herein. The particles can be for use according to the second, ninth, or tenth aspects. In one embodiment, the produced recombinant phagemid particles are for use in treating a subject that has not been exposed to the delivered antigen or antigens, for example by prior vaccination.

[0171] In one embodiment, the recombinant phagemid particles produced by the method of the twentieth aspect are for use in a method further comprising the use of one or more adoptively transferred T cells.Preferably, the adoptively transferred T cells are selected from the group consisting of chimeric antigen receptor (CAR) T cells, T cell receptor (TCR) transgenic T cells, and tumor infiltrating lymphocytes (TILs).The adoptively transferred T cells can be specific for one or more antigens introduced into the tumor cells by the recombinant phagemid particles.In a preferred embodiment, the adoptively transferred T cells are CAR T cells.

[0172] In a twenty-first aspect, there is provided a method for producing recombinant phagemid particles from a prokaryotic host, comprising: (i) introducing into a prokaryotic host cell a first vector adapted to persist in the prokaryotic host, the first vector comprising at least one transgene expression cassette comprising a nucleic acid sequence encoding one or more cytokines, or one or more antigens recognized by one or more adoptively transferred T cells, and a packaging signal to enable replication of the vector into single-stranded DNA, and (b) a second vector comprising a nucleic acid encoding a structural protein necessary for packaging the single-stranded DNA; (ii) culturing the host under conditions that result in the single-stranded DNA being packaged by the structural proteins to form and extrude recombinant phagemid particles from the prokaryotic host.

[0173] Advantageously, this method (as shown in FIG. 10) provides improved safety. The second vector (i.e., helper phage) can be introduced into the host cell, for example by infection. The host cell can then be transformed with the first vector (i.e., the genome of the phagemid particle), thereby resulting in the production of recombinant phagemid particles. Preferably, this method includes a purification step following the culturing step. Purification can include centrifugation and / or filtration.

[0174] The antigen may be any of those disclosed herein. In a preferred embodiment, the or each antigen is a peptide or protein expressed on the cell surface of the target tumor cell. Preferably, the or each antigen is a peptide or protein that is accessible to CAR T cells when expressed by the tumor cell. The peptide or protein may be such that when expressed by the tumor cell, it is present at or as a folded peptide protein on the cell surface. The or each antigen may be a known target of existing CAR T cells suitable for use in humans. For example, the or each antigen may be selected from the group consisting of MUC1, PSMA, CD19, CD20, estrogen-related receptor beta type 2 (ErRB2), or any combination thereof. In one embodiment, the or each antigen may be a dengue virus or yellow fever vaccine antigen.

[0175] The cytokine may be any of those disclosed herein. In particular, the cytokine may be IL-4, IL-12, IL-15, TNFα, TRAIL, IFN-γ, or any combination thereof. Preferably, the cytokine is IL-15. Preferably, the cytokine is IL-4. Preferably, the cytokine is IL-12. Preferably, the cytokine is TRAIL. Preferably, the cytokine is IFN-γ.

[0176] Preferably, the cytokine is not TNFα. Preferably, the cytokine is TNFα. Preferably, the cytokine is a hybrid TNFα that includes a non-endogenous signal peptide configured to increase the expression and / or secretion of TNFα. Preferably, the signal peptide is a cytokine signal peptide other than that of TNFα.

[0177] Preferably, the method of the twenty-first aspect is used to produce the recombinant phagemid particle of the eighth aspect. Thus, preferably, the first vector comprises the genome of the recombinant phagemid particle. It may be preferred that the packaging signal of the first vector comprises an origin of replication. Preferably, the origin of replication of the first vector comprises the F1 ori, more preferably the F1 bacteriophage.

[0178] The method of the twenty-first aspect can be used to produce recombinant phagemid particles for any use disclosed herein. The particles can be for use according to the second, ninth, or tenth aspects. In one embodiment, the recombinant phagemid particles are for use in treating a subject that has not been exposed to the delivered antigen(s), for example by prior vaccination.

[0179] In one embodiment, the recombinant phagemid particles produced by the method of the twenty-first aspect are for use in a method further comprising the use of one or more adoptively transferred T cells.Preferably, the adoptively transferred T cells are selected from the group consisting of chimeric antigen receptor (CAR) T cells, T cell receptor (TCR) transgenic T cells, and tumor infiltrating lymphocytes (TILs).The adoptively transferred T cells can be specific for one or more antigens introduced into the tumor cells by the recombinant phagemid particles.In a preferred embodiment, the adoptively transferred T cells are CAR T cells.

[0180] In a twenty-second aspect, there is provided the use of a helper phage comprising nucleic acid encoding a viral vector structural protein, for producing recombinant phagemid particles according to the eighth aspect from a prokaryotic host.

[0181] There is provided a host cell comprising the first and / or second vector as defined in the twenty-first aspect.

[0182] The host cell is preferably a prokaryotic cell, more preferably a bacterial cell. Examples of suitable host cells include (i) TG1 (genotype: K-12 supE thi-1Δ(lac-proAB)Δ(mcrB-hsdSM)5, (r K - m K - ), plasmid: F'[traD36 proAB + lacI q lacZΔM15]), (ii) DH5α F'IQ™ (genotype: F-φ80lacZΔM15Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rk−, mk+)phoA supE44λ-thi-1 gyrA96 relA1, plasmid: F'proAB+lacIqZΔM15zzf Tn5[KmR], and (iii) XL1-Blue MRF' (genotype: Δ(mcrA)183Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac, plasmid: F'proAB lacIqZΔM15 Tn10(Tetr).

[0183] The particles are used in therapeutic or diagnostic methods, preferably in vivo.

[0184] The present invention can be used to treat a wide variety of cancers due to the target-specific nature and improved transduction efficiency of the recombinant phagemid particles of the present invention.As a result, the therapeutic opportunities of recombinant bacteriophages used in gene therapy can be significantly increased by the present invention due to their ability to carry one or more transgene expression cassettes.The present invention can be used prophylactically to prevent cancer, or to improve, manage, and / or treat cancer after its onset.

[0185] It will be appreciated that the present invention can be used to generate a variety of different recombinant phagemid particles that can be used to treat and / or diagnose various cancers depending on the nature of the particle and the foreign protein (in the case of an antigen) displayed. It will be appreciated that cytokines may not be displayed on tumor cells. The target cells in gene therapy techniques are preferably eukaryotic, preferably mammalian.

[0186] Gene therapy techniques are used to treat, prevent, ameliorate, or manage cancer. The tumor may be a liquid tumor, such as a hematologic malignancy or hematopoietic tissue tumor, or a hematologic malignancy. The tumor may be a solid tumor. The tumor may be in the brain, such as a medulloblastoma, glioblastoma, or diffuse intrinsic pontine glioma (DIPG). The recombinant phagemid particles may be used in combination with conventional therapies such as chemotherapeutic drugs (i.e., doxorubicin, temozolomide, lomustine, cisplatin, vincristine), radiation therapy, or other drugs / xenobiotic compounds, including but not limited to inhibitors of histone deacetylase (HDAC inhibitors), proteasome inhibitors (e.g., MG132, borzotemib, carfilzomib), and anticancer agents derived from natural and dietary sources (i.e., genistein, epigallocatechin gallate (EGCG), resveratrol).

[0187] Recombinant phagemid particles can also be used to display and express antigens of interest directly on the major pVIII coat protein, thus providing an efficient platform for simultaneous delivery of multiple antigens, or proteins, or proteins with related biological activity, by a single phage particle. The subject may be a mammal, preferably a human.

[0188] It will be understood that the recombinant phagemid particles and systems of the present invention (i.e., hereinafter referred to as "agents") can be used in medicines that can be used in monotherapy or as an adjunct to or in combination with known therapies for the treatment, amelioration, management, or prevention of cancer.For example, a combination treatment approach is preferred in which the phagemid particles and systems of the present invention are used in combination with existing chemotherapeutic drugs such as temozolamide, cisplatin, doxorubicin, vincristine, or genistein.

[0189] In another preferred embodiment, the therapeutic method may include a combination of the recombinant phagemid particles and systems of the present invention with an extracellular matrix degrading agent, such as an enzyme or losartan, which the inventors believe should enhance the diffusion of the phagemid within the subject being treated, particularly in solid tumors.

[0190] The agent according to the invention (i.e. the recombinant phagemid particle used in the eighth or any of the above aspects or produced by the system according to the nineteenth aspect) can be combined into a composition having many different forms, depending in particular on the manner in which the composition is to be used. Thus, for example, the composition can be in the form of a powder, tablet, capsule, liquid, etc., or any other suitable form that can be administered to a human or animal in need of treatment. It will be understood that the vehicle of the pharmaceutical according to the invention must be well tolerated by the subject to which it is given.

[0191] The medicament containing the agent according to the present invention can be used in several ways. For example, oral administration may be required, in which case the agent can be contained in a composition that can be taken orally, for example, in the form of a tablet, capsule, or liquid. The composition containing the agent of the present invention can be administered by inhalation (e.g., intranasally). The composition can also be formulated for topical use. For example, a cream or ointment can be applied to the skin.

[0192] The agent according to the present invention can also be incorporated into a sustained or delayed release device. Such a device can be inserted, for example, on or under the skin, and the medicament can be released over a period of weeks or months. The device can be placed at least adjacent to the treatment site. Such a device can be particularly advantageous when long-term treatment with the agent used according to the present invention is required, which usually requires frequent administration (e.g., at least daily injections).

[0193] In a preferred embodiment, the agents and compositions according to the invention may be administered to a subject by injection into the bloodstream or directly to the site in need of treatment. Injection may be intravenous (bolus or infusion), subcutaneous (bolus or infusion), intradermal (bolus or infusion), or may be enhanced by traditional methods (associated with local injection at the disease site - convection enhanced delivery).

[0194] It will be understood that the amount of agent required will in turn be determined by its biological activity and bioavailability, which depends on the mode of administration, the physicochemical properties of the agent (i.e., recombinant phagemid virus particle or system), and whether it is used as a monotherapy or in a combination therapy. The frequency of administration will also be affected by the half-life of the agent in the subject being treated. The optimal dosage to be administered can be determined by those skilled in the art and will vary with the specific agent used, the strength of the pharmaceutical composition, the mode of administration, and the progression of the disease. Additional factors depending on the specific subject being treated, including the subject's age, weight, sex, diet, and administration time, will result in the need to adjust the dosage.

[0195] In general, the daily dose of the agent according to the present invention is 0.01 μg / kg body weight to 500 mg / kg body weight. More preferably, the daily dose is 0.01 mg / kg body weight to 400 mg / kg body weight, more preferably 0.1 mg / kg to 200 mg / kg body weight.

[0196] As discussed in the examples, the agent can be administered before, during, or after the onset of the disease. For example, the agent may be administered shortly after the subject develops the disease. The daily dose may be given systemically as a single dose (e.g., one injection per day). Alternatively, the agent may need to be administered more than once per day. By way of example, the agent can be administered as a daily dose (i.e., assuming a body weight of 70 kg) of between 25 mg and 7000 mg twice (or more depending on the severity of the disease being treated). The patient undergoing treatment can take a first dose upon awakening and a second dose in the evening (for a two-dose regimen) or three or four hours apart thereafter. Alternatively, a sustained release device can be used to provide the patient with an optimal dose of the agent according to the invention without the need to administer repeated doses.

[0197] Known procedures, such as those conventionally used in the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.), can be used to formulate specific formulations containing the particles or systems according to the invention and precise treatment regimens (such as daily dose and frequency of administration of the agent).

[0198] Thus, in a 23rd aspect of the present invention there is provided a pharmaceutical composition comprising a recombinant phagemid virus particle according to the 8th aspect, produced by the system according to the 19th aspect, produced by the method of the 20th or 21st aspects, or produced by the use of the 22nd aspect, and a pharma- ceutically acceptable vehicle.

[0199] The present invention also provides in a 24th aspect a process for making a pharmaceutical composition according to the 23rd aspect, comprising contacting a therapeutically effective amount of recombinant phagemid virus particles produced by the system according to the 8th aspect, or by the system according to the method of the 19th aspect, or by the method of the 20th or 21st aspects, or by the use of the 22nd aspect, with a pharma- ceutically acceptable vehicle.

[0200] A "subject" may be a vertebrate, a mammal, or a livestock animal. Thus, the agents, compositions, and medicaments according to the invention may be used to treat any mammal, such as livestock (e.g., horses), pets, or for other veterinary applications. Most preferably, however, the subject is a human.

[0201] A "therapeutically effective amount" of an agent (i.e., a recombinant phagemid viral particle) is any amount that, when administered to a subject, is the amount of agent necessary to treat a targeted disease or produce a desired effect, e.g., to effect effective delivery of a transgene to a targeted cell or tissue, such as causing tumor death.

[0202] For example, the therapeutically effective amount of the agent used may be from about 0.01 mg to about 800 mg, preferably from about 0.01 mg to about 500 mg.

[0203] A "pharmaceutically acceptable vehicle" as referred to herein is any known compound or combination of known compounds known to those skilled in the art to be useful in formulating a pharmaceutical composition.

[0204] In one embodiment, the pharma- ceutically acceptable vehicle may be solid, and the composition may be in the form of a powder or tablet. The solid pharma- ceutically acceptable vehicle may contain one or more substances that may also act as flavoring agents, lubricants, solubilizers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coating agents, or tablet disintegrants. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid that is mixed with the finely divided active agent according to the present invention. In tablets, the active agent (e.g., the particles or system of the present invention) can be mixed with a vehicle having the necessary compression properties in suitable proportions and compressed into the desired shape and size. Powders and tablets preferably contain up to 99% of the active agent. Suitable solid vehicles include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes, and ion exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel, and the composition may be in the form of a cream or the like.

[0205] However, the pharmaceutical vehicle may be liquid and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used to prepare solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. The particles or systems according to the invention may be dissolved or suspended in a pharma- ceutically acceptable liquid vehicle, such as water, an organic solvent, a mixture of both, or a pharma- ceutically acceptable oil or fat. The liquid vehicle may contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, colorants, viscosity adjusters, stabilizers, or osmolality adjusters. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing the above-mentioned additives, e.g., cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (including monohydric and polyhydric alcohols, e.g., glycols), and their derivatives, and oils (e.g., fractionated coconut oil and peanut oil). For parenteral administration, the vehicle may also be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions may be halogenated hydrocarbons or other pharma- ceutically acceptable propellants.

[0206] Liquid pharmaceutical compositions that are sterile solutions or suspensions can be utilized, for example, by intramuscular, intrathecal, epidural, intraperitoneal, intravenous, and especially subcutaneous injection. The particles or systems (i.e., hybrid vectors) can be prepared as sterile solid compositions that can be dissolved or suspended at the time of administration using sterile water, saline, or other suitable sterile injectable medium.

[0207] The recombinant phagemid particles, systems, and pharmaceutical compositions of the present invention can be orally administered in the form of a sterile solution or suspension containing other solutes or suspending agents (e.g., sufficient saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monooleate, polysorbate 80 (oleic acid ester of sorbitol and its anhydrides copolymerized with ethylene oxide), and the like. The particles and systems according to the present invention can also be orally administered in the form of either liquid or solid compositions. Compositions suitable for oral administration include solid forms such as tablets, capsules, granules, tablets, and powders, and liquid forms such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

[0208] It will be appreciated that adeno-associated viruses (AAV) are often the vectors of choice for gene therapy. As gene delivery vectors, lentiviral vectors also have several important advantages over other systems. They have a large packaging capacity of at least 8 Kb of DNA, which is a key feature in packaging tissue-specific promoters and substantial expression cassettes of transgenes. In addition, lentivectors reduce immunogenicity compared to adenoviral vectors, allowing systemic delivery routes to be explored. However, barriers to using AAV or lentiviruses in laboratory and clinical studies include very high production costs and low yields.

[0209] In addition to showing that the present inventors have useful applications in gene therapy, the recombinant phagemid particles of the present invention can also be used to produce recombinant viral vectors such as AAV or lentivirus in vitro (including in situ) or in vivo. Phage-induced AAV production utilizes the ability of phagemid particles to package large amounts of single-stranded ssDNA. A typical AAV production system consists of three main elements: rAAV, rep-cap, and adeno-helper genes that function together to produce rAAV particles.

[0210] Thus, in a twenty-fifth aspect, there is provided a recombinant phagemid particle according to the eighth aspect, or produced by the solar system of the nineteenth aspect, or produced by the method of the twentieth or twenty-first aspect, or produced by the use of the twenty-second aspect, wherein the recombinant phagemid particle is for the production of a recombinant viral vector comprising or derived from a viral genome within the genome of the phagemid particle, and wherein the recombinant viral vector is used to deliver a nucleic acid sequence encoding one or more antigens or cytokines at least adjacent to a tumor cell, and when the sequence encodes one or more antigens, the one or more antigens are expressed and recognizable by one or more adoptively transferred T cells.

[0211] Recombinant phagemid particles may then be advantageous that can produce recombinant viral vectors that code for antigens for recognition by adoptively transferred T cells (such as CAR T cells). Figure 16 shows the delivery of nucleic acid sequences to malignant tumors, where the production of viral vectors allows for autoinfection of the tumor, resulting in delivery of nucleic acid sequences throughout the tumor. This may be advantageous, as it allows for antigens to be delivered throughout the tumor for recognition by adoptively transferred T cells. This is synergistic with any subsequent adoptive transfer therapy, such as CAR T cell therapy, as it provides a suitable target for CAR T cells. Additionally, it may allow for lower concentrations of the introduced vector (i.e., recombinant phagemid particles) to be used.

[0212] In a preferred embodiment, the or each antigen is a peptide or protein expressed on the cell surface of the target tumor cell. Preferably, the or each antigen is a peptide or protein that is accessible to CAR T cells when expressed by tumor cells. The peptide or protein can be such that when expressed by tumor cells, it is present at or as a folded peptide protein on the cell surface. The or each antigen can be a known target of existing CAR T cells suitable for use in humans. For example, the or each antigen can be selected from the group consisting of MUC1, PSMA, CD19, CD20, estrogen-related receptor beta type 2 (ErRB2), or any combination thereof. In one embodiment, the or each antigen can be a dengue virus or yellow fever vaccine antigen.

[0213] The recombinant phagemid particle according to the twenty-fifth aspect may be for any use disclosed herein. The particle may be for use according to the second, ninth, or tenth aspects. In one embodiment, the recombinant phagemid particle is for use in treating a subject that has not been exposed to the delivered antigen or antigens, for example by prior vaccination.

[0214] In one embodiment, the recombinant phagemid particle of the twenty-fifth aspect is for use in a method further comprising the use of one or more adoptively transferred T cells.Preferably, the adoptively transferred T cells are selected from the group consisting of chimeric antigen receptor (CAR) T cells, T cell receptor (TCR) transgenic T cells, and tumor infiltrating lymphocytes (TILs).The adoptively transferred T cells can be specific for one or more antigens introduced into tumor cells by the recombinant phagemid particle.In a preferred embodiment, the adoptively transferred T cells are CAR T cells.

[0215] Also provided is a method for producing a recombinant viral vector, the method comprising introducing a recombinant phagemid particle according to the eighth aspect or a system according to the nineteenth aspect into a eukaryotic host cell and enabling the host cell to produce the recombinant viral vector.

[0216] Preferably, the recombinant virus product is a recombinant mammalian virus, such as AAV or lentivirus. Preferably, the viral vector product is rAAV. Preferably, the phagemid virus particle according to the eighth aspect, or the system according to the nineteenth aspect, is used in cis and / or trans in eukaryotic host cells, with the delivery and / or presence of other genetic elements required for the production of mammalian viruses, as determined by the genome of the phagemid particle. Methods used to aid or enhance gene transfer into host cells by phagemid particles include those described in WO2014 / 184528 (i.e., multifunctional) and WO2014 / 184529 (i.e., in combination with cationic polymers to form complexes with a net positive charge).

[0217] The eukaryotic host cell may be mammalian. The host cell may comprise or be derived from human embryonic kidney cells (HEK293), Spodoptera frugiperda pupal ovarian tissue (Sf9), or Chinese hamster ovary (CHO). Insect cells are also contemplated.

[0218] In one example, a host cell can be transformed with one or more phagemid particle genomes carrying genes selected from the group consisting of rAAV, lentivirus, capsid, replication, helper protein-encoding genes, and any other genes necessary for mammalian virus expression and packaging.

[0219] For example, in hybrid phagemid particle-induced rAAV production, the rAAV genes can be carried by the recombinant phagemid virus particle according to the eighth embodiment, as shown in Figure 3, or the adeno-helper and rep-cap genes can be carried on separate vectors or integrated into the eukaryotic host genome. For example, Figure 12 shows the adeno-helper genes on one vector, and Figure 13 shows the rep-cap on a separate vector. Any combination of rAAV, rep-cap, and adeno-helper genes can be carried on one or more vectors, i.e., in cis or trans configuration. Alternatively, the rep-cap or adeno-helper proteins can be integrated or introduced into the eukaryotic host as stably expressed accessory DNA (e.g., a plasmid) in the context of rAAV production, whereby the hybrid phagemid particle provides the recombinant viral genome for packaging into recombinant virus, as determined by the transgene cassette in the genome of the phagemid particle.

[0220] In one preferred embodiment, the rAAV, rep-cap, and adeno helper genes are carried on a single vector, as shown in Figures 14 and 15. We believe this is the first time that all three sets of genes have been contained on the same vector.

[0221] Thus, in a twenty-sixth aspect, there is provided a recombinant vector comprising an rAAV, rep-cap, an adeno helper gene, and a nucleic acid sequence encoding one or more cytokines or one or more antigens for recognition by one or more adoptively transferred T cells, for use in treating, preventing, or ameliorating cancer.

[0222] The recombinant vector may be for any use or method disclosed herein.

[0223] In a twenty-seventh aspect, there is provided a recombinant phagemid particle comprising the vector of the twelfth aspect for use in a method for the treatment, prevention or amelioration of cancer.

[0224] The recombinant phagemid particles can be for any use or method disclosed herein.

[0225] There is provided use of a vector according to the 26th aspect or a particle of the 27th aspect for producing a recombinant AAV viral vector comprising or derived from the viral genome of the phagemid particle.

[0226] A method for producing a recombinant AAV viral vector is provided, the method comprising introducing into a eukaryotic host cell a vector according to the 26th aspect or a particle of the 27th aspect and enabling the host cell to produce the recombinant viral vector.

[0227] When introduced into the same eukaryotic host cell (see Figures 11 and 14), the rep-cap and adeno helper genes on the vector behave in the context of rAAV production as trans-acting or cis-acting, or as a combination of both elements that promote packaging of the rAAV genome in the AAV viral capsid. This production process is comparable to the transient co-transfection of multiple plasmids, usually including three plasmids. However, in this embodiment, the plasmid is replaced with the recombinant phagemid particles of the present invention that also carry the same elements and are targeted to eukaryotic cells (preferably mammalian cells).

[0228] The method can be carried out in vivo, in vitro, ex vivo, or in situ. For in situ production, the recombinant phagemid particle preferably contains a targeting moiety for the target eukaryotic cell, which is a designated eukaryotic host. Preferably, in the context of in situ, ex vivo, and in vivo virus production, the designated eukaryotic host cell type is a diseased cell. Preferably, the diseased cell is a malignant or benign tumor. In the context of in vitro virus production, the eukaryotic host is preferably a derivative of any of the eukaryotic hosts listed above. The application of the recombinant phagemid particle and the genetic elements required for the production of recombinant virus (determined by the transgene cassette in the hybrid phagemid particle) can be in any manner as indicated above, either in cis-acting or trans-acting combination, inside the eukaryotic host cell.

[0229] In a twenty-eighth aspect, there is provided a recombinant phagemid particle for expressing a transgene in a target tumor cell transduced with the particle, for use in a method for treating, preventing or ameliorating cancer, the recombinant phagemid particle comprising at least one transgene expression cassette comprising a nucleic acid sequence encoding one or more cytokines, or an antigen recognizable by one or more adoptively transferred T cells, and a genome lacking at least 50% of its bacteriophage genome, the method comprising delivering the nucleic acid sequence at least adjacent to a tumor cell, and wherein when the nucleic acid encodes one or more antigens, the one or more cytokines are expressed and recognizable by the one or more adoptively transferred T cells.

[0230] It will be understood that the present invention includes any nucleic acid or peptide that substantially comprises the amino acid or nucleic acid sequence of any of the sequences mentioned herein, or a variant, derivative, or analogue thereof, as well as a functional variant or functional fragment thereof. The terms "substantially amino acid / polynucleotide / polypeptide sequence", "functional variant" and "functional fragment" may be a sequence that has at least 40% sequence identity with the amino acid / polynucleotide / polypeptide sequence of any one of the sequences mentioned herein, for example a sequence that has 40% identity with the nucleic acids identified herein.

[0231] Amino acid / polynucleotide / polypeptide sequences having greater than 65% sequence identity with any of the sequences mentioned, more preferably greater than 70%, even more preferably greater than 75%, even more preferably greater than 80% are also envisaged. Preferably, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity with any of the sequences mentioned herein, more preferably at least 90% identity with any of the sequences mentioned herein, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, and most preferably at least 99% identity.

[0232] Those skilled in the art will understand how to calculate the percentage of identity between two amino acid / polynucleotide / polypeptide sequences.To calculate the percentage of identity between two amino acid / polynucleotide / polypeptide sequences, the alignment of the two sequences must first be prepared, and then the sequence identity value must be calculated.The percentage of identity between two sequences can take different values ​​depending on (i) the method used to align sequences, such as ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison, and (ii) the parameters used by the alignment method, such as local vs. global alignment, the pair score matrix used (such as BLOSUM62, PAM250, Gonnet, etc.), and gap penalties, such as functional form and constants.

[0233] Once aligned, there are many different ways to calculate the percentage identity between two sequences. For example, the number of identities can be divided by: (i) the length of the shortest sequence, (ii) the length of the alignment, (iii) the average length of the sequences, (iv) the number of non-gap positions, or (v) the number of equivalent positions excluding overhangs. Furthermore, it will be understood that the percentage identity is also strongly dependent on length. Thus, the shorter the pair of sequences, the higher the sequence identity that can be expected to occur by chance.

[0234] It will therefore be appreciated that accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is the preferred method for generating protein or DNA multiple alignments according to the present invention. The preferred parameters for ClustalW may be as follows: for DNA alignment: gap opening penalty=15.0, gap extension penalty=6.66, and matrix=identity; for protein alignment: gap opening penalty=10.0, gap extension penalty=0.2, and matrix=Gonnet; for DNA and protein alignment: ENDGAP=-1, and GAPDIST=4. Those skilled in the art will recognize that these and other parameters may need to be varied for optimal sequence alignment.

[0235] Preferably, then, calculation of the percentage identity between two amino acid / polynucleotide / polypeptide sequences is calculated from the alignment as (N / T)*100, where N is the number of positions where the sequences share identical residues and T is the total number of compared positions including gaps but excluding overhangs. Thus, the most preferred method for calculating the relative percentage identity between two sequences includes (i) preparing a sequence alignment using, for example, the ClustalW program with a suitable set of parameters as described above, and (ii) inserting the values ​​of N and T into the following formula: sequence identity=(N / T)*100.

[0236] Alternative methods for identifying similar sequences will be known to those of skill in the art. For example, a substantially similar nucleotide sequence is encoded by a sequence that hybridizes under stringent conditions to the nucleic acid sequences described herein or their complements. Stringent conditions mean that the nucleotides hybridize to filter-bound DNA or RNA in 3x sodium chloride / sodium citrate (SSC) at about 45°C, followed by at least one wash in 0.2x SSC / 0.1% SDS at about 20-65°C. Alternatively, a substantially similar polypeptide may differ from the sequences shown herein by at least 1, 5, 10, 20, 50, or less than 100 amino acids.

[0237] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence can be altered to provide a functional variant thereof without substantially affecting the sequence of the protein encoded thereby. Suitable nucleotide variants are those that have a sequence altered by substitution of different codons that code for the same amino acid in the sequence, thus resulting in a silent change. Other suitable variants are those that have a homologous nucleotide sequence but contain all or part of a sequence that is modified by substitution of different codons that code for amino acids with side chains that have similar biophysical properties to the amino acid that it replaces, producing a conservative change. For example, small non-polar hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. Positively charged (basic) amino acids include lysine, arginine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Thus, it will be understood which amino acids can be replaced with amino acids having similar biophysical properties, and one of skill in the art will know the nucleotide sequences that code for these amino acids.

[0238] All of the features described herein (including any accompanying claims, abstracts, and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. For the avoidance of doubt, reference to a cytokine may preferably relate to IL-4, IL-12, IL-15, TNFα, TRAIL, IFN-γ, or any combination thereof. Preferably, the cytokine is IL-15, and preferably, the cytokine is IL-4. Preferably, the cytokine is IL-12. Preferably, the cytokine is TRAIL. Preferably, the cytokine is IFN-γ.

[0239] However, in another preferred embodiment, the cytokine is TNFα.Preferably, the cytokine is a hybrid TNFα that includes a non-endogenous signal peptide configured to increase the expression and / or secretion of TNFα.Preferably, the signal peptide is a cytokine signal peptide other than that of TNFα.For example, the signal peptide is preferably an IL-2 signal peptide.

[0240] For a better understanding of the present invention, and to show how embodiments thereof may be carried into effect, reference will now be made, by way of example, to the accompanying drawings in which: [Brief description of the drawings]

[0241] [Figure 1] FIG. 1 is a table showing the characteristics of phagemid-AAV (PAAV) viral particles according to the invention compared to prior art AAVP viral particles. [Diagram 2]Figure 2 shows a schematic diagram of an embodiment of the helper phage and phagemid genome (PAAV) according to the present invention, and the phagemid-AAV (PAAV) particle produced by the helper and phagemid. The structural genes are essential for packaging DNA into the viral particle and are provided by the replicating helper phage. The phagemid genome is highly parasitic on the helper phage. Finally, the PAAV particles are produced at a yield far exceeding that of the prior art systems. [Diagram 3] FIG. 3 is a schematic representation of one embodiment of a phagemid genome (PAAV). [Figure 4] FIG. 4 shows the locations of the f1 ori and the pUC ori on the phagemid genome shown in FIG. [Diagram 5] FIG. 5 shows the location of the selectable marker gene (AmpR) on the recombinant adeno-associated virus (rAAV) transgene cassette on the phagemid genome shown in FIG. [Figure 6] Figure 6 shows an rAAV transgene cassette on the phagemid genome shown in Figure 3, containing a gene of interest (e.g., GFP) whose expression is driven by a CMV promoter and / or enhancer sequence and tailed with a polyA signal. The entire transgene cassette is flanked by inverted terminal repeats (ITRs) from AAV. [Figure 7] FIG. 7 shows one embodiment of a helper phage, which is a bacteriophage engineered to rescue phagemid particles from a prokaryotic host harboring a phagemid genome as shown in FIG. [Figure 8] FIG. 8 shows a portion of the genome of the helper phage shown in FIG. 5, including the RGD4C targeting peptide in the pIII minor coat protein, as shown in SEQ ID NOs:26 and 27. [Figure 9-1] FIG. 9 shows a first embodiment of a method for producing phagemid-AAV (PAAV) particles. [Figure 9-2] FIG. 9 shows a first embodiment of a method for producing phagemid-AAV (PAAV) particles. [Figure 10-1] FIG. 10 shows a second embodiment of a method for producing phagemid-AAV (PAAV) particles. [Figure 10-2] FIG. 10 shows a second embodiment of a method for producing phagemid-AAV (PAAV) particles. [Figure 11] FIG. 11 shows one embodiment of a phage-based approach for in vitro AAV production showing three vectors: (i) phagemid-AAV (PAAV), (ii) Rep-Cap phagemid, and (iii) adeno-helper phagemid. [Figure 12] FIG. 12 shows a genomic map of one embodiment of the adeno-helper phagemid vector shown in FIG. [Figure 13] FIG. 13 shows a genomic map of one embodiment of the Rep-Cap phagemid vector shown in FIG. [Figure 14] FIG. 14 shows an embodiment of an integrated adenohelper / Rep-cap / phagemid-AAV (PAAV) vector. [Figure 15] FIG. 15 shows a genomic map of one embodiment of the integrated adenohelper-Rep-Cap phagemid vector shown in FIG. [Figure 16] FIG. 16 shows one embodiment of in situ AAV production using any of the three phagemid vectors shown in FIGS. 11-13, or the integrated adenohelper-Rep-Cap-AAV phagemid vector shown in FIGS. 14 and 15. [Figure 17] Figure 17 shows transmission electron microscopy (TEM) of known AAVP vectors and PAAV vectors according to the invention. (A) RGD.AAVP.GFP filaments (pink) are typically 1455.02 nm long. (B) RGD.PAAV.GFP filaments (blue) are typically 729.96 nm long, and helper phage present in the virus sample (green) are typically 1186.03 nm long. [Figure 18]Figure 18 shows the internalization of known AAVP vectors and the PAAV vector according to the invention in (A) 293AAV and (B) U87 cells after 2 and 4 hours. Flow cytometry analysis was used with a gate threshold set at 20,000 events in the total cell population. (n=3) *=p<0.05, **=p<0.01. [Figure 19] Figure 19 shows quantification of GFP positive cells 9 days after transduction in (A) 293AAV, (B) 293AAV with the addition of DEAE.DEXTRAN, (C) U87, and (D) U87 with the addition of DEAE.DEXTRAN. Flow cytometry analysis was used with a gate threshold set at 20000 events of the total cell population. (n=3) *=p<0.05, **=p<0.01. [Figure 20] Figure 20 shows quantification of genomic copy numbers of rAAV-GFP from cell lysates following phagemid-directed gene transfer (A) or transfection of rAAV expression elements (B). (Experiment A: n=1, Experiment B: n=3). [Figure 21] Figure 21 shows immunofluorescence staining of UW228 and DAOY human medulloblastoma cells to demonstrate expression of αV, β3, and β5 integrin subunits, which are receptors for RGD4C-phagemid. Tumor cells were stained with primary rabbit anti-αV, β3, or β5 antibodies (diluted 1:50 in PBS-1% BSA), followed by goat anti-rabbit AlexaFluor-488 secondary antibody (shown in green), and counterstained with 0.05 μg / ml DAPI (blue). Images were taken using a confocal microscope. [Figure 22] Figure 22 shows targeted gene delivery to pediatric medulloblastoma cells by RGD4C-phagemid. Medulloblastoma cells (UW228) were grown on 96-well plates and then transduced with RGD4C-phagemid vector carrying the luciferase gene (RGD). Untreated cells or cells treated with a non-targeted vector (M13) were used as negative controls. Luciferase expression was monitored over time from day 2 to day 4 after transduction. [Figure 23]Figure 23 shows a Western blot analysis showing downregulation of mTOR expression in pediatric UW228 and DAOY medulloblastoma cells after treatment with RGD4C-phagemid carrying a sequence encoding mTOR / shRNA (RGD4C-mTOR / shRNA). Cell lysates were harvested 4 days after vector treatment and total protein was measured by BCA assay. Western blots were probed with a monoclonal antibody against human mTOR (cell signaling). Untreated cells (control) and cells treated with RGD4C-phagemid lacking mTOR / shRNA (RGD4C) were used as negative controls. [Figure 24] Figure 24 shows the combined treatment of temozolomide (TMZ) and RGD4C-phagemid carrying a sequence encoding shRNA against mTOR in medulloblastoma. Medulloblastoma cells (UW228 and DAOY) were transduced with RGD4C-phagemid (RGD4C) or RGD4C-phagemid carrying a sequence encoding mTOR / shRNA (RGD4C-mTOR / shRNA). Untreated cells were also used as a control. On the 7th day after vector treatment, temozolomide (TMZ, 100 μM) was added to a small number of wells to evaluate the combined effect of vector and chemotherapy. Images were taken on the 8th day after vector treatment. [Diagram 25] Figure 25 shows treatment of medulloblastoma cells with TNFα phagemid vector. UW228 cells were treated with RGD4C-phagemid-TNFα (RGD4C / TNFα) and non-targeting (ctr). A) Cell viability after expression of TNFα using MTT assay. B) Expression of TNFα in the medium of vector-treated cells measured using human TNFα ELISA Max. Error bars: mean ± SEM. [Figure 26] Figure 26 shows immunofluorescence staining of DIPG cells to demonstrate expression of αV, β3, and β5 integrin subunits, which are receptors for RGD4C-phagemid. Cells were stained using a primary rabbit antibody followed by a goat anti-rabbit AlexaFluor-488 secondary antibody. Control cells received only the secondary antibody. Images were taken using a confocal microscope. [Figure 27A] Figure 27 shows selective and dose-dependent delivery of gene expression by RGD4C-phagemid / AAV to UW228, DAOY, DIPG cells. Cells were treated with increasing vector doses of 1x106 or 2x106 TU / cell of RGD4C-phagemid-Luc (RGD4C) carrying the reporter Luc (luciferase) gene. Expression of Luc was measured daily. A non-targeting vector lacking RGD4C (ctr) was used as a negative control for targeting. Error bars: mean ± SEM. (A) shows treatment of DIPG cells, (B) shows treatment of UW228, and (C) shows treatment of DAOY cells. [Figure 27B] Figure 27 shows selective and dose-dependent delivery of gene expression by RGD4C-phagemid / AAV to UW228, DAOY, DIPG cells. Cells were treated with increasing vector doses of 1x106 or 2x106 TU / cell of RGD4C-phagemid-Luc (RGD4C) carrying the reporter Luc (luciferase) gene. Expression of Luc was measured daily. A non-targeting vector lacking RGD4C (ctr) was used as a negative control for targeting. Error bars: mean ± SEM. (A) shows treatment of DIPG cells, (B) shows treatment of UW228, and (C) shows treatment of DAOY cells. [Figure 27C] Figure 27 shows selective and dose-dependent delivery of gene expression by RGD4C-phagemid / AAV to UW228, DAOY, DIPG cells. Cells were treated with increasing vector doses of 1x106 or 2x106 TU / cell of RGD4C-phagemid-Luc (RGD4C) carrying the reporter Luc (luciferase) gene. Expression of Luc was measured daily. A non-targeting vector lacking RGD4C (ctr) was used as a negative control for targeting. Error bars: mean ± SEM. (A) shows treatment of DIPG cells, (B) shows treatment of UW228, and (C) shows treatment of DAOY cells. [Figure 28-1]Figure 28 shows treatment of UW288, DAOY, or DIPG cells with RGD4C-phagemid-TNFα. DIPG were transduced with 2x106TU / cell RGD4C-phagemid-TNFα (RGD4C) and a non-targeting vector as a negative control (ctr). UW288 or DAOY cells were transduced with 1x106TU / cell with or without DEAE-dextran. Apoptotic activity was measured 9 days after vector treatment by measuring the percentage of viable cells or by using the Caspase-Glo assay (Caspase 3 / 7, Caspase 8, and Caspase 9). Error bars: mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. [Figure 28-2] Figure 28 shows treatment of UW288, DAOY, or DIPG cells with RGD4C-phagemid-TNFα. DIPG were transduced with 2x106TU / cell RGD4C-phagemid-TNFα (RGD4C) and a non-targeting vector as a negative control (ctr). UW288 or DAOY cells were transduced with 1x106TU / cell with or without DEAE-dextran. Apoptotic activity was measured 9 days after vector treatment by measuring the percentage of viable cells or by using the Caspase-Glo assay (Caspase 3 / 7, Caspase 8, and Caspase 9). Error bars: mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. [Figure 29] FIG. 29 shows luciferase expression following transduction with RGD.PAAV at various transducing units of RGD.PAAV. [Diagram 30] Figure 30 shows the percentage of PAAV vectors bound to the cell surface of 293 AAV cells. Compared to the respective controls, the RGD.PAAV vector had a binding efficiency of 58.2%, while the M13.PAAV vector had a binding efficiency of 7.1%. [Figure 31a]Figure 31 shows schematics of embodiments of expression plasmid constructs for bacteriophage-guided CAR T cell therapy, where Figure 31a represents a MUC1-CD28.IL4 expression plasmid driven by the CMV promoter, Figure 31b represents a MUC1-GPI.IL4 expression plasmid driven by the CMV promoter, Figure 31c represents a PSMA expression plasmid driven by the CMV promoter, Figure 31d represents a MUC1-CD28.IL4 expression plasmid driven by the Grp78 promoter, Figure 31e represents a MUC1-GPI.IL4 expression plasmid driven by the Grp78 promoter, and Figure 31f represents a PSMA expression plasmid driven by the Grp78 promoter. [Figure 31b] Figure 31 shows schematics of embodiments of expression plasmid constructs for bacteriophage-guided CAR T cell therapy, where Figure 31a represents a MUC1-CD28.IL4 expression plasmid driven by the CMV promoter, Figure 31b represents a MUC1-GPI.IL4 expression plasmid driven by the CMV promoter, Figure 31c represents a PSMA expression plasmid driven by the CMV promoter, Figure 31d represents a MUC1-CD28.IL4 expression plasmid driven by the Grp78 promoter, Figure 31e represents a MUC1-GPI.IL4 expression plasmid driven by the Grp78 promoter, and Figure 31f represents a PSMA expression plasmid driven by the Grp78 promoter. [Fig. 31c]Figure 31 shows schematics of embodiments of expression plasmid constructs for bacteriophage-guided CAR T cell therapy, where Figure 31a represents a MUC1-CD28.IL4 expression plasmid driven by the CMV promoter, Figure 31b represents a MUC1-GPI.IL4 expression plasmid driven by the CMV promoter, Figure 31c represents a PSMA expression plasmid driven by the CMV promoter, Figure 31d represents a MUC1-CD28.IL4 expression plasmid driven by the Grp78 promoter, Figure 31e represents a MUC1-GPI.IL4 expression plasmid driven by the Grp78 promoter, and Figure 31f represents a PSMA expression plasmid driven by the Grp78 promoter. [Fig. 31d] Figure 31 shows schematics of embodiments of expression plasmid constructs for bacteriophage-guided CAR T cell therapy, where Figure 31a represents a MUC1-CD28.IL4 expression plasmid driven by the CMV promoter, Figure 31b represents a MUC1-GPI.IL4 expression plasmid driven by the CMV promoter, Figure 31c represents a PSMA expression plasmid driven by the CMV promoter, Figure 31d represents a MUC1-CD28.IL4 expression plasmid driven by the Grp78 promoter, Figure 31e represents a MUC1-GPI.IL4 expression plasmid driven by the Grp78 promoter, and Figure 31f represents a PSMA expression plasmid driven by the Grp78 promoter. [Figure 31e]Figure 31 shows schematics of embodiments of expression plasmid constructs for bacteriophage-guided CAR T cell therapy, where Figure 31a represents a MUC1-CD28.IL4 expression plasmid driven by the CMV promoter, Figure 31b represents a MUC1-GPI.IL4 expression plasmid driven by the CMV promoter, Figure 31c represents a PSMA expression plasmid driven by the CMV promoter, Figure 31d represents a MUC1-CD28.IL4 expression plasmid driven by the Grp78 promoter, Figure 31e represents a MUC1-GPI.IL4 expression plasmid driven by the Grp78 promoter, and Figure 31f represents a PSMA expression plasmid driven by the Grp78 promoter. [Fig. 31f] Figure 31 shows schematics of embodiments of expression plasmid constructs for bacteriophage-guided CAR T cell therapy, where Figure 31a represents a MUC1-CD28.IL4 expression plasmid driven by the CMV promoter, Figure 31b represents a MUC1-GPI.IL4 expression plasmid driven by the CMV promoter, Figure 31c represents a PSMA expression plasmid driven by the CMV promoter, Figure 31d represents a MUC1-CD28.IL4 expression plasmid driven by the Grp78 promoter, Figure 31e represents a MUC1-GPI.IL4 expression plasmid driven by the Grp78 promoter, and Figure 31f represents a PSMA expression plasmid driven by the Grp78 promoter. [Diagram 32]Figure 32 shows the expression of MUC-1.CD28, MUC-1.GPI, and PSMA antigens 6 days after transduction. HEK 293 cells were transduced with either RGD-targeted PAAV (RGD) or non-targeted PAAV (NT) at 106TU / cell. Untreated HEK 293 cells (Ctrl) and targeted PAAV-transduced-HEK 293 cells with only 488 secondary antibody staining (Ant.488) were shown as controls. A represents MUC-1 or PSMA expression of HEK 293 cells transduced with CMV promoter-driven PAAV vectors (constructs in Figures 31a, 31b, and 31c). DEAE-dextran was added. B represents MUC-1 or PSMA expression of HEK 293 cells transduced with CMV promoter-driven PAAV vectors (constructs in Figures 31a, 31b, and 31c) without the addition of DEAE-dextran. C represents MUC-1 expression in HEK 293 cells transduced with Grp78 promoter-driven PAAV vectors (constructs of Figures 31d and 31e) without the addition of DEAE dextran. [Figure 33a] Figure 33 shows a schematic diagram of expression plasmid constructs containing a puromycin resistance gene for stable cell line selected bacteriophage guided CAR T cell therapy, where Figure 33a represents a MUC1-CD28.IL4 expression plasmid driven by a CMV promoter, Figure 33b represents a MUC1-GPI.IL4 expression plasmid driven by a CMV promoter, Figure 33c represents a PSMA expression plasmid driven by a CMV promoter, Figure 33d represents a MUC1-CD28.IL4 expression plasmid driven by a Grp78 promoter, Figure 33e represents a MUC1-GPI.IL4 expression plasmid driven by a Grp78 promoter, and Figure 33f represents a PSMA expression plasmid driven by a Grp78 promoter. [Figure 33b]Figure 33 shows a schematic diagram of expression plasmid constructs containing a puromycin resistance gene for stable cell line selected bacteriophage guided CAR T cell therapy, where Figure 33a represents a MUC1-CD28.IL4 expression plasmid driven by a CMV promoter, Figure 33b represents a MUC1-GPI.IL4 expression plasmid driven by a CMV promoter, Figure 33c represents a PSMA expression plasmid driven by a CMV promoter, Figure 33d represents a MUC1-CD28.IL4 expression plasmid driven by a Grp78 promoter, Figure 33e represents a MUC1-GPI.IL4 expression plasmid driven by a Grp78 promoter, and Figure 33f represents a PSMA expression plasmid driven by a Grp78 promoter. [Figure 33c] Figure 33 shows a schematic diagram of expression plasmid constructs containing a puromycin resistance gene for stable cell line selected bacteriophage guided CAR T cell therapy, where Figure 33a represents a MUC1-CD28.IL4 expression plasmid driven by a CMV promoter, Figure 33b represents a MUC1-GPI.IL4 expression plasmid driven by a CMV promoter, Figure 33c represents a PSMA expression plasmid driven by a CMV promoter, Figure 33d represents a MUC1-CD28.IL4 expression plasmid driven by a Grp78 promoter, Figure 33e represents a MUC1-GPI.IL4 expression plasmid driven by a Grp78 promoter, and Figure 33f represents a PSMA expression plasmid driven by a Grp78 promoter. [Fig. 33d]Figure 33 shows a schematic diagram of expression plasmid constructs containing a puromycin resistance gene for stable cell line selected bacteriophage guided CAR T cell therapy, where Figure 33a represents a MUC1-CD28.IL4 expression plasmid driven by a CMV promoter, Figure 33b represents a MUC1-GPI.IL4 expression plasmid driven by a CMV promoter, Figure 33c represents a PSMA expression plasmid driven by a CMV promoter, Figure 33d represents a MUC1-CD28.IL4 expression plasmid driven by a Grp78 promoter, Figure 33e represents a MUC1-GPI.IL4 expression plasmid driven by a Grp78 promoter, and Figure 33f represents a PSMA expression plasmid driven by a Grp78 promoter. [Figure 33e] Figure 33 shows a schematic diagram of expression plasmid constructs containing a puromycin resistance gene for stable cell line selected bacteriophage guided CAR T cell therapy, where Figure 33a represents a MUC1-CD28.IL4 expression plasmid driven by a CMV promoter, Figure 33b represents a MUC1-GPI.IL4 expression plasmid driven by a CMV promoter, Figure 33c represents a PSMA expression plasmid driven by a CMV promoter, Figure 33d represents a MUC1-CD28.IL4 expression plasmid driven by a Grp78 promoter, Figure 33e represents a MUC1-GPI.IL4 expression plasmid driven by a Grp78 promoter, and Figure 33f represents a PSMA expression plasmid driven by a Grp78 promoter. [Fig. 33f]Figure 33 shows a schematic diagram of expression plasmid constructs containing a puromycin resistance gene for stable cell line selected bacteriophage guided CAR T cell therapy, where Figure 33a represents a MUC1-CD28.IL4 expression plasmid driven by a CMV promoter, Figure 33b represents a MUC1-GPI.IL4 expression plasmid driven by a CMV promoter, Figure 33c represents a PSMA expression plasmid driven by a CMV promoter, Figure 33d represents a MUC1-CD28.IL4 expression plasmid driven by a Grp78 promoter, Figure 33e represents a MUC1-GPI.IL4 expression plasmid driven by a Grp78 promoter, and Figure 33f represents a PSMA expression plasmid driven by a Grp78 promoter. [Diagram 34] Figure 34 shows the expression of MUC-1.CD28, MUC-1.GPI, and PSMA antigens in cancer cells 6 days after transduction. Cancer cells were transduced with either RGD-targeted PAAV (RGD) or non-targeted PAAV (M13) at 106 TU / cell. Untreated cells without any antibody staining (CTRL) were shown as a control, and untreated cells were stained with primary and secondary antibodies (staining) to confirm the internal expression of antigens by FACS. A represents MUC-1 or PSMA expression in A549 cells transduced by CMV promoter-driven PAAV vectors. B represents MUC-1 or PSMA expression in Suit2 cells transduced by CMV promoter-driven PAAV vectors. C represents MUC-1 expression in UW 228 cells transduced by CMV promoter-driven PAAV vectors. [Diagram 35]Figure 35 shows the expression of MUC-1.CD28, MUC-1.GPI, and PSMA antigens in stable cancer cells. RGD4C-PAAV stably transduced cancer cells (stable cells) expressing MUC1 or PSMA antigens were selected with puromycin antibiotic and used to perform FACS analysis for MUC-1 or PSMA expression. To confirm the internal expression of antigens, untreated cells without any antibody staining (CTRL) were shown as a control, and untreated cells were stained with primary and secondary antibodies (staining). A represents MUC-1 or PSMA expression in A549 cells. B represents MUC-1 or PSMA expression in Suit2 cells. C represents MUC-1 expression in UW 228 cells. [Diagram 36] Figure 36 shows the treatment and comparison of DIPG cell killing in vitro between PAAV carrying either transmembrane tmTNFα or secreted sTNFα. DIPG cells were treated with either RGD4C-PAAV-tmTNFα or RGD4C-PAAV-sTNFα (RGD4C and non-targeted M13[ctr]) and cell viability was measured 7 days after vector treatment. RGD4C-PAAV-sTNFα particles carrying secreted sTNFα were more potent in inducing DIPG cell killing than RGD4C-PAAV-tmTNFα, even in a transient transduction situation where only a small population of cells was transduced by the vector. [Figure 37-1] FIG. 37 shows schematic diagrams of embodiments of plasmid constructs used in the presently disclosed methods, with 37a representing the PAAV-CMV-IRES GFP plasmid and 37b representing PAAV.Grp78.IRES.GFP. [Figure 37-2] FIG. 37 shows schematic diagrams of embodiments of plasmid constructs used in the presently disclosed methods, with 37a representing the PAAV-CMV-IRES GFP plasmid and 37b representing PAAV.Grp78.IRES.GFP. [Figure 38]Figure 38 shows the expression of TNFα after PAAV-tm.TNFα transduction. (A) UW228 and Daoy were seeded in 96-well plates and transduced with 1x106TU / cell containing DEAE dextran, the supernatants were collected on day 6, and TNFα in the supernatants was measured by ELISA. Data are presented as mean ± SEM. [Figure 39] Figure 39 shows the effect of TNFα and cisplatin combination treatment in UW228 cells. Cell viability was measured at various time points using sulforhodamine B assay. Stably transduced UW228 were treated with 1 μM and 5 μM cisplatin chemotherapy 48 hours after cell seeding. Data are expressed as mean ± SEM. ***P≦0.001. The figure shows that combination with cisplatin (cis) chemotherapy increases tmTNFα cytokine gene therapy for medulloblastoma. [Diagram 40] Figure 40 shows the expression of TNF after PAAV-sTNF transduction. DIPGs were seeded in 96-well plates and transduced with 2x106 TU / cell containing DEAE dextran, the supernatants were collected on day 3, and TNFα in the supernatants was measured by ELISA. [Diagram 41] FIG. 41 shows the generation of PAAV-CMV-tmTNFα. [Diagram 42] FIG. 42 shows the production of PAAV-sTNFα. [Diagram 43] Figure 43 shows a targeted PAAV vector in which the RGD-4C ligand is displayed on the pIII coat protein of M13 filamentous phage. The hybrid genome shows the key gene fragments required for expression of the desired gene. [Diagram 44] FIG. 44 shows a schematic for producing RGD pVIII helper virus. [Diagram 45] FIG. 45 shows a schematic for producing RGD pVIII PAAV-GFP and RGD pVIII PAAV-lucia, which were used in in vitro transduction experiments to assess vector efficiency and gene expression levels. [Figure 46-1]FIG. 46 shows a schematic for producing PAAV-hTRAIL (SnapGene image). [Figure 46-2] FIG. 46 shows a schematic for producing PAAV-hTRAIL (SnapGene image). [Figure 47] Figure 47 shows fluorescent microscopy images of DIPG cells incubated with a primary anti-phage antibody and an Alexa Fluor-488 labeled secondary antibody (green) to assess integrin (αv / β3 / β5) expression. To account for background fluorescence, images were taken of control cells containing no antibody or only the secondary antibody. Nuclei were stained with DAPI (blue). [Figure 48] FIG. 48 shows the theoretical construct of the RGD-4C ligand displayed on the pIII (left) or pVIII (right) coat protein of M13 filamentous phage. [Figure 49] Figure 49 shows fluorescent microscopy images of HEK293T cells incubated with non-targeted RGD pIII PAAV-GFP or RGD pVIII PAAV-GFP vectors at 0.1 m TU, 0.5 m TU, and 1 m TU on day 6 post-transduction. GFP expression is highest with RGD pIII PAAV-GFP at all Tus. [Figure 50] Figure 50 shows the RLU of HEK293T cells incubated with non-targeted RGD pIII PAAV-lucia or RGD pVIII PAAV-lucia vectors at 0.1 m TU, 0.5 m TU, and 1 m TU on day 6 post-transduction. RLU is highest with RGD pIII PAAV-lucia at all TUs. Error bars are + / - 1 standard error. [Figure 51] Figure 51 shows the RLU of HEK293T cells incubated with vectors 4 days after transduction. RLU is highest with H5W RGD pIII PAAV-lucia across all TUs. Error bars are + / - 1 standard error. (Sajee Waramit, unpublished data). [Figure 52]Figure 52 shows the RLU of DIPG cells incubated with non-targeted RGD pIII PAAV-lucia or H5W RGD pIII PAAV-lucia vectors at 1m TU and 2m TU on day 3 post-transduction. RLU is highest with H5W RGD pIII PAAV-lucia at all TUs. Error bars are + / - 1 standard error. [Figure 53] Figure 53 shows microscopy images of DIPG cells transfected with PAAV-hTRAIL and control PAAV-GFP plasmid at 0.2 ng, 0.4 ng, and 0.6 ng of DNA. Images were taken 18 hours after transfection. Cell viability is reduced in cells transfected with PAAV-hTRAIL at all DNA concentrations. [Figure 54] Figure 54 shows a bar graph depicting IL-12 concentrations in media collected 6 days after transduction with PAAV-CMV-IL-12 normalized to 1 μg protein. Control transduction with targeted and non-targeted empty vector using the CMV promoter and mock transduction are shown. The outer selection bar shows analysis of IL-12 production data for all vector titers and controls by 2-way ANOVA. The inner selection bar shows comparison of vector to control at each titer by unpaired t-test. Experiments were performed in triplicate. [Figure 55] Figure 55 shows a line graph depicting mouse IL-12 concentrations in media sampled on various days following transduction with PAAV-CMV-mIL-12. Control transduction with targeted and non-targeted empty vector using the CMV promoter and mock transduction are shown. The outer selection bar shows analysis of mouse IL-12 production data from all sampling days for vector and control by two-way ANOVA. Asterisks on specific data points indicate comparison between vector and control on each specified day by unpaired t-test. [Figure 56]Figure 56 shows a bar graph depicting the average tumor size (mm3) of B16-F10 mouse melanoma tumors in C57BL / 6 mice 7 days after treatment with RGD-4C-targeted PAAV-CMV-mIL-12 (n=4), with RGD-4C-targeted PAAV-CMV, with no transgene (n=4), and with no treatment control (n=4). Three doses of 5x1010TU vector were administered intravenously on days 0, 2, and 5. Data were analyzed by two-way ANOVA with Tukey's multiple comparison test. [Figure 57] FIG. 57 shows the IL2 signal sequence. [Figure 58] FIG. 58 shows the Il-2 / TNFα construct. [Figure 59] Figure 59 shows the cell killing efficiency of RGD4C-sTNFα and RGD4C-tmTNFα in DIPG. DIPG cells were transduced with PAAV targeted (RGD4C) or non-targeted (M13) carrying secreted or transmembrane TNFα (sTNFα) transgenes. Cells were seeded in 96-well plates. After 2 days, cells were transduced with 2×106 TU / cell with 40 ng / μg protein DEAE-dextran. Viability was measured by sulforhodamine B (SRB) assay. Statistical significance was determined by student's t-test. Data are expressed as mean ± SEM. *P≦0.05, **P≦0.01. [Figure 60] Expression of TNFα after transduction with PAAV-sTNFα and PAAV-tmTNFα. DIPG cells were seeded in 6-well plates and transduced with RGD4C and M13 carrying a secreted or transmembrane TNFα transgene. A) RNA was extracted and expression of TNFα was determined by qRT-PCR. B) Supernatants were harvested and TNFα in the supernatants was determined by ELISA. Data are presented as mean ± SEM. **P ≤ 0.01***P ≤ 0.001. Statistical significance was determined by Student's t-test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0242] background The development of gene delivery technologies is beneficial for the successful translation of basic research to society. In the past decade, numerous viral and non-viral vectors have emerged as potential delivery vectors for industrial and therapeutic applications. A key characteristic of a vector is that, in addition to being effective in delivering genes, it must be easily produced and commercially viable. In 2006, Hajitou et al. attempted to meet the need for such vectors by creating a hybrid between a recombinant adeno-associated virus (rAAV) and a filamentous bacteriophage (phage), called adeno-associated virus / phage (AAVP) (Nature protocols 2,523-531(2007);Cell 125,385-398(2006)). The resulting AAVP vectors possess the favorable properties of mammalian and prokaryotic viruses but do not suffer from the drawbacks that their individual vectors usually possess. However, there are certain aspects of AAVP vectors that still leave room for significant improvement. Among others, this includes the genetic design of the vector, which affects its production and therapeutic properties. Ultimately, this results in a relatively low gene transfer efficiency of AAVP when compared to mammalian viruses.

[0243] The work described herein concerns the design of the most advanced version of phage gene delivery vectors by using the so-called phagemid system, which uses a new phagemid vector called phagemid / adeno-associated virion phagemid (i.e., PAAV), and their superiority over the known and existing phage vector, AAVP. Unlike the AAVP genome, which consists of a rAAV cassette that inserts into a filamentous phage genome, the PAAV genome does not contain any structural phage genes - a prokaryotic helper virus is required to facilitate vector assembly (Mol Ther 3,476-484; Pharmaceutical research 27,400-420 (2010)). By separating the reproductive and therapeutic elements of the virus into a therapeutic vector carrying the transgene and a separate helper virus carrying the structural gene, the genome / vector size is substantially reduced, thereby significantly increasing the transgene capacity, a useful advantage for the application of the new system to gene therapy. This results in the encapsidation of the eukaryotic viral genome into a prokaryotic viral capsid, resulting in the vector as a hybrid between the eukaryotic genome and the prokaryotic capsid with enhanced production yields, gene transfer efficiency, and flexibility of the vector system for other applications.

[0244] As described in the Examples below, the inventors 1. Design and construct a hybrid phagemid-AAV vector (PAAV) particle expression system 2. Characterize and determine whether phagemid / AAV vectors (PAAV) are more efficient in gene transfer than known AAVP systems at various stages, including but not limited to: A binding to the cell surface, b. internalization of the vector from the cell surface; c. Expression of recombinant transgenes.

[0245] 3. It was determined whether the hybrid phagemid-PAAV vector system was capable of producing rAAV from mammalian producer cell lines.

[0246] 4. We demonstrated that this system can be used in CAR-T therapy for the treatment of cancer.

[0247] 5. We demonstrated that this system can be used to deliver the cytokines IL-12, TRAIL, and hybrid TNFα to target cells for the treatment of cancer.

[0248] 6. Phagemid particles containing hybrid TNFα constructs were designed and constructed.

[0249] 7. We demonstrated that hybrid TNFα constructs exhibit increased expression, secretion, and cell killing efficiency when compared to full-length TNFα.

[0250] First, referring to FIG. 1, a table is shown comparing the characteristics of the phagemid-AAV (PAAV) particles (i.e., virions) according to the present invention with the AAVP viral particles of the prior art. As can be seen, the PAAV particles of the present invention (6 kb) are much smaller than known AAVP particles (14 kb), i.e., 42% less DNA and 50% shorter viral particles, and the PAAV particles are produced with a yield that far exceeds that of the prior art system, AAVP (100-fold). As a result, the PAAV particles of the present invention can carry a larger payload, which is very useful for delivering multiple transgenes in gene therapy approaches. Thus, the present inventors have demonstrated that the modified bacteriophage expression system (PAAV) can be used as a highly viral vector for gene therapy or large-scale production of viral vectors.

[0251] Example 1 - Phagemid-AAV Vector (PAAV) Construction Referring to Figure 2, there is shown an embodiment of the helper phage genome and phagemid genome (PAAV DNA) of the present invention, which are combined together upon expression in prokaryotes to produce phagemid-AAV (PAAV) particles, as also shown in Figure 1. The structural genes are essential for packaging of DNA into viral particles and are supplied by a replication-deficient helper phage, discussed in detail below. The phagemid genome is highly parasitic to the helper phage, meaning that it outcompetes the replication-deficient helper phage in both replication and packaging.

[0252] A) Phagemid / AAV vectors Referring now to Figure 3, there is shown one embodiment of a phagemid genome, which is a plasmid containing two origins of replication and two other genetic elements. The phagemid genome requires two origins of replication to facilitate both its replication in a prokaryotic (e.g., bacterial) host and its packaging into a phagemid particle when rescued by a helper virus.

[0253] Referring to Figure 4, the first origin of replication (ori) is a high copy number origin of replication (pUC ori) that allows replication of double-stranded phagemids (dsDNA) in large numbers of prokaryotic hosts. The second origin of replication is a phage origin of replication (f1 ori) that allows replication of the plasmid into single-stranded DNA, which can then be packaged into a phagemid vector particle (PAAV).

[0254] Referring to Figure 5, the phagemid genome contains a selection marker gene. In order for the phagemid genome to replicate efficiently in a prokaryotic host, a selection marker (e.g., ampicillin resistance) is used to ensure expression and provide selective pressure to prevent loss of the phagemid genome in the form of an antibiotic resistance gene (with its own promoter). This ensures expression (and replication) of the phagemid genome when the prokaryotic host is cultured in the presence of the antibiotic to which the selection marker confers resistance.

[0255] With reference to FIG. 6, the phagemid genome further comprises a recombinant (adeno-associated virus, AAV) transgene cassette containing the transgene of interest. This may include, but is not limited to, a polypeptide / protein, a short hairpin / small interfering / short inducing RNA, or a combination of both. By way of example only, the transgenes shown in FIG. 6 encode GFP and human β-globin. Expression of the transgene is driven by a viral promoter (e.g., CMV) and / or enhancer sequence and tailed with a polyA signal. The promoter can also be a mammalian and tumor-specific promoter in cancer gene therapy applications (i.e., the promoter of glucose-regulated protein [grp78]). The entire transgene cassette is flanked by AAV-derived inverted terminal repeats (ITRs), which form a protective hairpin structure that allows the transgene cassette to be stably maintained as a concatemeric episomal (extrachromosomal) DNA within the mammalian cell nucleus transduced by the phagemid particle. The ITRs allow for AAV concatamerization and subsequent long-term stable expression of the AAV transgene cassette.

[0256] The phagemid cannot package itself into a particle because it lacks structural phage genes. As a result, it requires "rescue" by a helper virus, as shown in Figure 7, which provides the structural (i.e., capsid) proteins necessary for particle formation and extrusion from the prokaryotic host. In conventional thinking, genetic elements in vectors are common and widely used in genetic engineering.

[0257] B) Helper phage With reference to Figure 7, the helper phage (herein referred to as M13KO7) is a bacteriophage specifically designed to rescue phagemid particles (i.e., PAAV) from prokaryotic hosts harboring and / or containing the phagemid genome depicted in Figure 3. The helper phage contains a disrupted origin of replication (p15a, medium copy number) and a packaging signal that severely reduces its ability to package itself into phage particles. As a result, the phagemid genome will outcompete the helper phage in both replication and packaging.

[0258] To confer phagemid targeting properties (or multifunctional properties as described in the pamphlet in WO2014 / 184528), the genome of the helper phage must be engineered to do so, since it provides the structural capsid proteins for phagemid particle assembly. For example, the helper genome may encode a pIII capsid minor coat protein configured to display a cell targeting ligand that enables delivery of the resulting PAAVP particle to a desired target cell (e.g., tumor). It may also encode at least one pVIII major coat protein configured to display a foreign peptide on the resulting PAAV particle. Thus, in one embodiment, the α V Beta3 and Alpha V It is desirable to introduce nine amino acid mutations into the pIII minor coat protein to confer specificity to tumor cells expressing the β5 integrin receptor and to endothelial cells associated with angiogenic tumors. Thus, referring to FIG. 8, the genome of the helper phage is constructed as follows: v Beta3 and Alpha v It contains the RGD4C peptide (CDCRGDCFC - SEQ ID NO: 7) which targets β5 integrin.

[0259] Once the PAAV phagemid genome and helper phage are constructed, they are used together to produce phagemid-AAV vector (PAAV) particles in a prokaryotic host, as discussed below.

[0260] Example 2 - Production of phagemid-AAV vectors (PAAV) The inventors have devised two different methods (Methods 1 and 2) for producing phagemid-AAV vector (PAAV) particles, which are shown in Figures 9 and 10.

[0261] Notes: -TG1: E. coli strain carrying the reproductive factor (F' fimbriae). -2×YT:TG1 Liquid broth used to cultivate E. coli. - Kanamycin: an antibiotic resistance selection marker present on the helper phage. - Ampicillin: antibiotic resistance selection marker present on the phagemid vector. -TYE Top Agar: A solid medium used to cultivate TG1 E. coli, adapted from 2xTY by the addition of 1.25% bacteriological agar.

[0262] Phagemid / AAV Vector (PAAV) Production Method 1: Infectious Rescue Referring to FIG. Add 4–5 ml of precultured (overnight) TG1 E. coli harboring the PAAV genome to 60 ml of 2xYT (100 µg / mL ampicillin) supplemented with 1.1% glucose. 2. Incubate cultures at 37° in a shaker (250 RPM). 3.OD 600 As soon as the range of 0.5-0.8 (log phase) is reached, the number of 10 A transducing unit of helper phage (M13KO7) is added to the culture. 4. Invert to mix. Incubate at 37° for 30 minutes. 5. Pour the infected starter culture from step 3 into a 2 L flask containing 2xYT (100 µg / mL ampicillin + 25 µg / mL kanamycin) supplemented with 1% glucose to a final volume of 400-450 mL. 6. Incubate overnight at 37 °C and 250 rpm on an orbital shaker for 16-20 h. 7. Purify phagemid (PAAV) particles from culture supernatant.

[0263] The advantage of method 1 is its very high yield.

[0264] Phagemid / AAV vector (PAAV) production method 2: Stable producer cell lines Referring to FIG. Part 1: Generation of competent producer cell lines 1. Transform TG1 competent E. coli (Zymo Research, USA) with the ssDNA genome from helper hedge M13KO7 and plate on TYE top agar (50 μg / mL kanamycin). 1. Select an individual colony and inoculate 5 mL 2xYT medium (50 μg / mL kanamycin) supplemented with 1% glucose. 2. Incubate overnight at 37 °C and 250 rpm on an orbital shaker for 16-20 h. 3. Verify true positive transformants by extracting DNA from 5 mL of overnight culture using a commercial extraction kit (QIAGEN, Netherlands) and running it on a 1% agarose gel (100 volts, 2.5 mA) against a DNA ladder. 4. Prepare chemically competent cells from the correct transformants identified in step 4 using published protocols (adapted from those published by Krantz et al., UC Berkeley).

[0265] Part 2: Production of PAAV phagemid particles 1. Transform the competent cell line created in part 1 with the phagemid / AAV genome and plate on TYE top agar (100 μg / mL ampicillin + 50 μg / mL kanamycin). 2. Select a colony and inoculate 5 mL 2xYT (100 μg / mL ampicillin + 50 μg / mL kanamycin) supplemented with 1% glucose. 3. Incubate for 4 hours at 37° and 250 rpm on an orbital shaker. 4. Pour the infected starter culture from step 3 into a 2 L flask containing 2xYT (100 µg / mL ampicillin + 25 µg / mL kanamycin) supplemented with 1% glucose to a final volume of 400-450 mL. 5. Incubate overnight at 37 °C and 250 rpm on an orbital shaker for 16-20 h. 6. Purify phagemid particles from the culture supernatant.

[0266] PAAV phagemid particle purification 1. Transfer the warm overnight culture to a centrifuge bottle and pellet the bacteria by centrifugation at 3300G, 4° for 30 minutes. 2. Discard the pellet and transfer the supernatant to a clean centrifuge bottle. 3. Add 30% volume of supernatant to each bottle with ice cold 20% PEG-8000 / 2.5M NaCl and vortex to mix. 4. Incubate on ice for 4 to 24 hours 5. Precipitate the phagemid particles by centrifugation at 10,000 G for 30 minutes at 4°. Discard the supernatant. 6. Dry the phagemid particle pellet by centrifugation at 10,000G, 4° for 1 minute. 7. Remove remaining supernatant with PEG / NaCl 8. Resuspend the phagemid particle pellet in 0.5–2 mL of PBS 9. Filter the resuspended phagemid particle preparation using a 0.45 micron filter. 10. Keep preparation at 4°. This preparation is stable at 4° for up to 2 years. 25% glycerol stocks can be stored at -80° indefinitely.

[0267] Example 3 - Use of phagemid-AAV vectors (PAAV) for gene therapy techniques Examples 1 and 2 describe the components of the invention required to produce phagemid-AAV vector (PAAV) particles and two production methods (i.e., the phagemid genome shown in Figure 3 and the helper phage shown in Figure 7). Once produced and purified, PAAV particles can have a range of uses, such as gene therapy.

[0268] As an example, the PAAV particles described herein carry a GFP transgene, as successful delivery to target cells can be easily detected by known assays. For therapy, any transgene can be selected and engineered into the phagemid genome shown in FIG. 3 and carried in the resulting PAAV particle. For example, the transgene can be any gene that encodes a protein that may have therapeutic or industrial utility. For example, the transgene may encode one or more antigens for recognition by adoptively transferred T cells, such as CAR T cells. The transgene may also encode short hairpin / small interfering / short guided RNA molecules for use in RNAi therapy. The transgene may encode multiple polypeptides, nucleic acids, or a combination of both, fused together using an internal ribosome entry site (IRES) or a viral fusion peptide (T2A peptide for in-frame fusion).

[0269] Example 4 - Use of phagemid-AAV vectors (PAAV) for in vitro AAV production In addition to gene therapy, the PAAV particles described herein can be used in a novel method for producing adeno-associated viruses (AAV). Phage-induced AAV production takes advantage of the ability of phagemid particles to package large amounts of dsDNA. A typical AAV production system consists of three main elements: rAAV, rep-cap, and adeno helper genes that function together to produce recombinant AAV particles. We have devised two different strategies.

[0270] Referring to Figure 11, the first strategy used is to produce three different phagemid vectors carrying rAAV production elements. These are the phagemid-AAV vector (PAAV) (see Figure 3), the adeno-helper phagemid particle (see Figure 12), and the rep-cap phagemid particle (see Figure 13). The basic structure of these particles is similar, as they contain two origins of replication and a selection marker, as described in the phagemid / AAV construction section. However, the key difference is the transgene cassette. As shown in Figure 3, the phagemid-AAV (PAAV) genome contains the AAV transgene cassette, while the adeno-helper and rep-cap particles contain the adeno-helper transgene or the rep-cap transgene, as shown in Figures 12 and 13, respectively.

[0271] In another embodiment, the inventors engineered so-called "integrated constructs" that contain all of the necessary elements within a single vector genome, as shown in Figures 14 and 15.

[0272] When introduced into the same mammalian producer cell (see Figures 11 and 14), either on separate vectors or on the same integrated vector, the rep-cap and adeno-helper genes behave as trans-acting elements that facilitate packaging of the rAAV genome into a phagemid / AAV vector. This production process is comparable to transient co-transfection of the three plasmids. However, in this case, the plasmid is replaced with a phagemid vector carrying the exact same elements.

[0273] The following describes a protocol for PAAV phagemid-induced production of adeno-associated virus (AAV).

[0274] Notes: DMEM: Dulbecco's modified Eagle's medium. FBS: fetal bovine serum, growth supplement. Complete medium: DMEM+10%FBS. EDTA: Ethyl-diaminetetraacetic acid, an ion chelator used to dissociate cells by sequestering calcium ions required for tight junction formation. GlutaMax: A growth supplement, an analogue of L-glutamine.

[0275] Protocol for phagemid-guided AAV production: HEK293 cells are seeded in 1.15 cm tissue culture plates in complete medium (DMEM supplemented with 10% FBS, 20 mM GlutaMax, penicillin / streptomycin, and non-essential amino acids) and grown for a minimum of 48 hours until they reach 80% confluence. 2. Mix phagemid / AAV, rep-cap phagemid, and adenohelper phagemid to achieve a 1:1:1 transducing unit ratio in less than 5 mL total volume, or aliquot integrated vectors (single vector containing all three elements in a single particle) to achieve one million transducing units per cell. 3. Add an equal volume of serum-free DMEM (supplemented with 20 mM GlutaMax) to the transduction mixture made in step 3. 4. Invert to mix. Incubate at room temperature for 15 minutes. 5. Wash the HEK293 cells plated in step 1 with PBS three times. 6. Add the transfection mixture and vortex gently to evenly distribute the mixture. 7. Incubate for 72 hours in a cell culture incubator at 37 °C, 5% CO2: a. After 6 hours of incubation with the transduction mixture, replenish with an equal volume of complete medium (DMEM supplemented with 10% FBS, 20 mM GlutaMax, penicillin / streptomycin, and non-essential amino acids); b. After 24 hours, replace the medium with complete medium (DMEM supplemented with 10% FBS, 20 mM GlutaMax, penicillin / streptomycin, and non-essential amino acids).

[0276] rAAV purification: 1. Add 0.5 M EDTA solution to the medium in the tissue culture plate to a final concentration of 0.010 M and incubate at room temperature for 5 minutes. 2. Aspirate the cells and media, triturate and transfer to a 50 mL centrifuge tube. 3. Pellet the cells by centrifugation at 1500 RPM for 5 minutes at room temperature: a. Optional: Collect the supernatant for further AAV purification. 4.Resuspend the cell pellet in 2-5 mL of serum-free DMEM. 5. Lyse the cells in suspension by four freeze-thaw cycles in an ethanol-dry ice bath and a water bath set at 37°. 6. Centrifuge the cell lysate at 10000G for 10 minutes at room temperature: a. Aliquot the supernatant for quantification / further purification / concentration.

[0277] b. Discard the pellet (debris).

[0278] Example 5 - Use of phagemid-AAV vectors (PAAV) for in situ AAV production Referring to FIG. 16, the inventors have devised a method for the in situ production of AAV particles using PAAV.

[0279] First, an optimal dose (or multiple doses) of the three phagemid vectors or integrated vectors is introduced in vivo via intravenous / subcutaneous / intraperitoneal or intramuscular / subcutaneous (or any of the aforementioned routes of administration). The diseased tissue is a tumor that displays the relevant integrin, and therefore the targeting moiety on the phagemid PAAV particle is the RGD4C sequence. The tumor should begin to produce rAAV containing the viral transgene encoded in the hybrid phagemid particle, rather than wild-type AAV. These AAV therapeutic particles naturally have a high affinity for mammalian tissues, and therefore should autoinfect nearby sites and eradicate the tumor over a given time period.

[0280] Example 6 - Creation of engineered pseudoviruses for large-scale targeted gene transfer and recombinant adeno-associated virus production Transmission electron microscopy In characterizing the particles, we imaged PAAV particles to show that vector size is substantially reduced when using a phagemid-based vector system. Using transmission electron microscopy, we imaged and measured the length of the PAAV of the present invention and known AAVP particles on a mesh copper TEM grid after negative staining with uranyl acetate (see FIG. 17). We found that the average AAVP particle was 1455.02 nm in length (FIG. 17A), while a typical PAAV particle according to the present invention was only 729.96 nm in length (FIG. 17B), which corresponds to a reduction in particle size of about 50%. Compared to the helper phage used to produce PAAV particles (typically 1186.03 nm, FIG. 17B), the relative vector size is about 38% shorter than the helper virus.

[0281] The difference in vector size forms the basis of the theory that PAAV may be more efficient as a gene delivery vector than AAVP, not only in production yield but also in the subsequent infection process in introducing and expressing genes in mammalian cells. Therefore, we investigated vector efficiency at various stages of infection, including binding, internalization, and gene expression in 293AAV (a derivative of human embryonic kidney 293) and U87 glioblastoma cell lines.

[0282] Vector internalization After binding, the vector undergoes receptor-mediated endocytosis by the target cells. To examine potential differences in vector internalization, we used flow cytometry (see FIG. 18) to assay the number of internalized vectors in target cells at two time points (2 hours, 2H, 4 hours, 4H). When compared to AAVP in both cell lines, PAAV vectors were found to be more effectively internalized at 2 hours (median fluorescence intensity (MFI)=1031.7, 335 higher than AAVP, p<0.05), and to a greater overall extent at 4 hours. The MFI of PAAV at 2 hours was significantly higher than AAVP, 335 for 293AAV and 207 for U87 cells (p<0.05). Four hours after transduction, this difference was significantly greater for 293AAV (829 MFI, p<0.05), but less so for U87 (157 MFI, not significant). Overall, the MFI peaked at 2092 (293AAV, p<0.05, FIG. 18A) and 1137 (U87, FIG. 18B) for PAAV1-treated cells, significantly higher than AAVP, which peaked at 1063 (293AAV) and 980 (U87), respectively. This data demonstrates that PAAV consistently performed better than AAVP in the rate and extent of internalization for both time points in both cell lines.

[0283] Green fluorescent protein expression after AAVP- and PAAV-mediated gene transfer To investigate whether differences in vector internalization lead to increased gene expression, we performed GFP expression assays using RGD and NT PAAV.GFP and AAVP.GFP vectors (see FIG. 19). In this experiment, we also tested whether the addition of cationic polymer DEAE.DEXTRAN (Dex), as described in WO2014 / 184529, could enhance gene transfer by increasing the bioavailability and endosomal escape of PAAV vectors. Nine days after transduction, cells were trypsinized, counted, and analyzed using a flow cytometer. Regardless of whether Dex was used to assist vector transduction, transgene expression was generally higher in 293 AAV cells than in U87. When using vector alone, the targeted RGD.PAAV.GFP vector transduced target cells with higher efficacy (7.7% in GFP+ve cells, p<0.01 and 1.4% in GFP+ve cells, p<0.05 in 293AAV and U87 cells, respectively) compared to AAVP, which represents a 2.44- and 1.56-fold increase in 293AAV and U87 cells, respectively (Figure 19A,C).

[0284] However, the addition of Dex dramatically increases gene expression for RGD.AAVP and RGD.PAAV vectors. In 293AAV cells, GFP expression in RGD.AAVP.GFP treated cells increased to 25% for RGD. PAAV.GFP treated cells substantially increased to 50% (all p<0.01), and the addition of Dex resulted in an increase in gene expression of 7.9-fold for RGD.AAVP and 6.5-fold for RGD. PAAVP (Figure 19B,D). In U87 cells, which are considered to be highly active for transduction, Dex was able to increase gene expression of RGD.PAAV.GFP by more than 3.6-fold (p<0.01) versus 4.8% GFP+ve cells - this was not the case for RGD.PAAV.GFP, as Dex increased gene expression by only 1.5-fold versus 1.3% GFP+ve cells (p<0.05). Interestingly, Dex enabled transduction by NT.PAAV (non-targeted) vectors in 293AAV cells (7.34%) but not in U87.

[0285] Phagemid-derived recombinant adeno-associated virus production To evaluate whether PAAV and phagemid-derived vectors could be used to produce rAAV in commercially available producer cell lines, we transduced 293AAV cells with three target vectors that normally require transfection for gene transfer. They were able to harvest rAAV particles from cell lysates and quantify the rAAV gene copy number (GC) per mL over three time points after phagemid-induced transduction (Figure 20A). Compared to conventional transfection with FuGene6 (transfection reagent, 3.99e11 GC / mL, Figure 20B), phagemid-induced rAAV production results in a more than 1.9-fold increase in rAAV yield at 168 hours (7.69e11 GC / mL, Figure 21A). Phagemid-induced gene transfer requires extensive intracellular processing (unlike transfection) and therefore requires longer times for viral genes to be expressed and packaged into functional particles. However, when comparing yields at the same 72 hour time point, transfection yielded 1.76e11 GC / mL higher than phage-induced rAAV production. rAAV yields per mL of culture supernatant from transfection or phagemid-induced production dishes at all time points were approximately 8-9e10 GC / mL with no observable trend (data not shown).

[0286] Example 7 - Construction and use of RGD4C-phagemid The tripeptide RGD is found in proteins of the extracellular matrix, including fibronectin. Integrins are α V The phagmid particles act as receptors for fibronectin by binding to the RGD motif located on fibronectin at the cell adhesion site to β3 integrin. The specificity was confirmed by sequentially inducing nine amino acid mutations in the pIII minor coat protein of the recombinant phagemid particles. V Beta3 and Alpha V The second vector's genome thus contains the RGD4C targeting peptide (CDCRGDCFC-SEQ ID NO: 7) which is delivered to tumor cells expressing β5 integrin and to angiogenic tumor-associated endothelial cells.

[0287] Referring to FIG. 21, α V Shown is immunofluorescence staining of UW228 and DAOY human medulloblastoma cells, demonstrating expression of the receptors for the β3 and β5 integrin subunits, RGD4C-phagemid. These data demonstrate that phagemid vectors containing the RGD4C targeting peptide can be used for targeted gene delivery and gene therapy in the pediatric brain tumor, medulloblastoma.

[0288] Referring to Figure 22, targeted gene delivery by RGD4C-phagemid to pediatric medulloblastoma cells is shown over a 4 day time course. This data indicates that RGD4C-phagemid mediates efficient and selective gene delivery that increases over time in medulloblastoma.

[0289] Figure 23 shows Western blot analysis showing downregulation of mammalian target of rapamycin (mTOR) expression in pediatric UW228 and DAOY medulloblastoma cells after treatment with RGD4C-phagemid carrying a sequence encoding mTOR / shRNA (RGD4C-mTOR / shRNA). These data demonstrate that RGD4C-phagemid can be successfully used to deliver shRNA in tumor cells to knock down the expression of therapeutic target mTOR in a selective and efficient manner.

[0290] Figure 24 shows the combined treatment of temozolomide (TMZ) and RGD4C-phagemid carrying a sequence encoding shRNA against mTOR in medulloblastoma cells, which are known to be temozolomide resistant. This data demonstrates that targeted RGD4C-mTOR / shRNA can resensitize medulloblastoma cells to TMZ and achieve complete tumor cell eradication. Therefore, targeted knockdown of mTOR expression by RGD4C-phagemid is an efficient strategy to be used in combination with temozolomide against chemotherapy-resistant tumor cells such as medulloblastoma.

[0291] Figure 25 shows treatment of medulloblastoma cells with a TNFα vector. Thus, RGD4C / TNFα has therapeutic potential for use in killing targeted tumors such as medulloblastoma. Figure 26 shows the α receptor for the RGD4C-phagemid. V Figure 1 shows immunofluorescence staining of DIPG cells demonstrating expression of β, β1, and β2 integrin subunits. These data demonstrate that phagemid vectors containing the RGD4C targeting peptide can be used for targeted gene delivery and gene therapy in the pediatric brain tumor, DIPG.

[0292] Figure 27 shows selective and dose-dependent delivery of gene expression to UW288, DAOY, or DIPG cells by RGD4C-phagemid / AAV. These data demonstrate that RGD4C-phagemid can successfully deliver gene expression to DIPG in a dose-dependent and selective manner. These data also show that RGD4C-PAAV shows efficient gene transfer to medulloblastoma in vitro, which increases with time. There was no nonspecific uptake in cells transduced with the control (non-targeted PAAV-Luc). The cationic polymer DEAE-dextran for medulloblastoma cell lines improved transduction efficiency.

[0293] FIG. 28 shows treatment with RGD4C-phagemid-TNFα. These data demonstrate that RGD4C-phagemid selectively delivers TNFα to DIPGs, resulting in apoptosis induction. Thus, RGD4C-phagemid-TNFα has therapeutic potential for use in targeted therapy against DIPGs. FIG. 28 also shows that medulloblastoma is a good candidate for treatment with RGD4C-phagemid-TNFα, as the treatment kills tumor cells when tested on either cell line UW288 or DAOY. For example, UW288 showed approximately 60% cell death at day 6 compared to the control. The cationic polymer DEAE-dextran further promoted tumor cell killing.

[0294] Example 8 - Luciferase expression of RGD4C-phagemid protocol: HEK cells were plated in 48-well plates in complete medium (DMEM, 10% FCS, 1% glutamine, 1% penicillin / streptomycin) and incubated for at least 48 h until they reached 70–80% confluence. Cells were then washed with PBS and transduced for 12 h with hybrid phage / phagemid vectors suspended in serum-free medium (DMEM), after which the medium was supplemented with complete medium. Luciferase expression was measured by adding 10 μL of medium to 50 μL of prepared Quanti-luc (InvivoGen, USA) reagent. Light emission was measured using a plate reader equipped with a luminometer (Promega, USA).

[0295] Figure 29 shows luciferase expression after transduction with RGD.PAAV at various concentrations of transduction units. The graph demonstrates a dose-dependent exponential relationship between time and expression of luciferase after incubation with various concentrations of hybrid phage / phagemid vectors. The figures demonstrate that quantifiable gene expression can be achieved with phagemid vectors via assay of secreted luciferase.

[0296] Example 9 - Binding of RGD.PAAV Vectors to 293 AAV Cells protocol: 293AAV cells were plated in 24-well plates in complete medium (DMEM + 10% FCS, 1% glutamine, 1% penicillin / streptomycin) and allowed to reach 70-90% confluence for a minimum of 48 hours. Cells were washed twice with 500uL PBS and placed on ice before being transduced with 200000 TU / cell (transducing units / cell) of PAAV vector suspended in 200uL of serum-free DMEM. After 1 hour incubation on ice, media was harvested from the wells and the amount of phagemid particles was titrated in TG1 E. coli and quantified by colony counting.

[0297] Referring to Figure 30, the percentage of PAAV vector bound to the cell surface of 293 AAV cells is shown. The RGD.PAAV vector had a binding efficiency of 58.2% and the M13.PAAV vector had a binding efficiency of 7.1% relative to the respective controls.

[0298] Example 10 - Transduction of tumor cells with PAAV expressing either MUC1 or PSMA antigens on the cell surface so that they can be targeted by specific CAR T cells Currently, conventional treatment for cancer consists of one or more of three options: surgery, chemotherapy, and radiation therapy. Although these interventions can sometimes cure the disease, in many cases, cancer cells are not completely eliminated, resulting in a high recurrence rate. To make matters worse, chemotherapy and radiation therapy are associated with unpleasant side effects. As a result, there has been great interest in developing alternative approaches to cancer treatment. One of the most promising of these new therapies is cancer immunotherapy, which aims to harness the power and specificity of the patient's own immune system to eliminate cancer cells. Cancer immunotherapy is a developing form of treatment. The two main strategies include active immunotherapy, which targets tumor-associated antigens (TAA), and passive immunotherapy, which enhances existing antitumor responses.

[0299] Examples of cancer include pediatric brain tumors such as medulloblastoma and diffuse intrinsic pontine glioma (DIPG). Medulloblastoma is the most common brain tumor and occurs in the cerebellum with a 5-year survival rate following current treatment strategies consisting of surgical resection, radiation therapy, and chemotherapy (Rudin et al., 2009). However, survivors often have long-term endocrine and neurocognitive side effects. Therefore, there is an urgent need to develop new therapeutic approaches that are non-invasive, tumor-specific, safer, cost-effective, and efficient to avoid long-term side effects from current treatments. On the other hand, diffuse intrinsic pontine glioma (DIPG) is the most aggressive brain tumor that occurs only in children with a 2-year survival rate of only 6-10%. Due to its diffuse nature, there is no effective treatment strategy for this type of cancer (Jansen et al., 2012, Mueller and Chang, 2009).

[0300] Due to the special properties of the immune system and its central and universal role in the organism, immunotherapy has great potential to treat cancer, potentially offering long-term protection while causing fewer side effects than other treatments. One particular approach, adoptive cell therapy (ACT), involves the transplantation of immune cells with antitumor activity. These cells can be T cells already present in the tumor, known as tumor-infiltrating lymphocytes (TILs), some of which will be specific for TAAs. These cells are isolated from resected tumor tissue, cultured, activated, and expanded ex vivo, and then reinfused back into the patient. Other types of cells useful for ACT include genetically engineered T cells that express either modified T cell receptors (TCRs) or chimeric antigen receptors (CARs). These artificial receptors specifically orient T cells to target antigens expressed by tumor cells (Blankenstein T, et al. The determinants of tumor immunogenicity. Nat Rev Cancer. 2012;12(4):307-13.; Sharpe M and Mount N. Genetically modified T cells in cancer therapy: opportunities and challenges. Dis Model Mech. 2015;8(4):337-50.).

[0301] CAR proteins are expressed on the surface of T cells and contain an extracellular binding domain that tightly binds to a specific tumor antigen, a hinge region connecting the extracellular domain and the transmembrane domain, a transmembrane domain, and an intracellular signaling domain (also called a costimulatory domain) such as CD28 and OX40 (tumor necrosis factor receptor). The costimulatory signals mediated by these domains improve the efficiency of T cells and prolong their antitumor activity (Sharpe M and Mount N. Genetically modified T cells in cancer therapy: opportunities and challenges. Dis Model Mech. 2015;8(4):337-50; Till BG,et al. CD20-specific adoptive immunotherapy for lymphoma using a chimeric antigen receptor with both CD28 and 4-1BB domains: pilot clinical trial results. Blood. 2012;119(17):3940-50; Koehler H,et al. CD28 co-stimulation overcomes transforming growth factor-beta-mediated repression of proliferation of redirected human CD4+ and CD8+ T cells in an antitumor cell attack. Cancer Res. 2007;67(5):2265-73). CAR T cells can be engineered to recognize a wide variety of antigens that may be expressed on the cell surface of tumors. Potential target antigens include proteins, carbohydrates, and glycolipids. CAR T cells, unlike conventional T cells and transgenic TCR T cells, do not require antigens to be processed and presented by MHC.Therefore, the same CAR-based strategy can be applied to all patients expressing the same tumor antigen regardless of the patient's MHC haplotype (Sharpe M and Mount N. Genetically modified T cells in cancer therapy: opportunities and challenges. Dis Model Mech. 2015;8(4):337-50, Haji-Fatahaliha M, et al. CAR-modified T-cell therapy for cancer: an updated review. Artif Cells Nanomed Biotechnol. 2015:1-11).

[0302] Despite its many advantages, CAR T cell therapy has some limitations. For example, tumors may not express a suitable target antigen. This can occur for a variety of reasons, including the neoantigen being unknown, expressed at inappropriate levels, expressed only in a subpopulation of tumor cells, expressed in non-tumor tissues, not expressed in a manner that is suitable for targeting CAR T cells to the tissue, or when expression of the antigen by the tumor is likely to decrease or disappear during treatment.

[0303] For example, some CAR T cell therapies have been reported to have undesirable toxicities in both animal models and clinical trials. This problem may arise when the antigen recognized by CAR T cells is not just expressed on tumor cells, but is also presented on normal cells leading to damage of normal tissues (Palmer DC,et al. Effective tumour treatment targeting a melanoma / melanocyte-associated antigen triggers severe ocular autoimmunity (Proc Natl Acad Sci USA.2008;105(23):8061-6; Morgan RA,et al. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther.2010;18(4):843-51; Lamers CHJ,et al. Treatment of Metastatic Renal Cell Carcinoma With Autologous T-Lymphocytes Genetically Retargeted Against Carbonic Anhydrase IX: First Clinical Experience. Journal of Clinical Oncology.2006;24(13):e20-e2; Grupp SA,et al.Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.N Engl J Med.2013;368(16):1509-18.). Thus, availability and selection of target antigens is an issue. Ideally, antigens are presented only by tumor cells, or alternatively by normal cells that are not essential for survival (Rosenberg SA,Restifo NP.Adoptive cell transfer as personalized immunotherapy for human cancer.Science.2015;348(6230):62-8). Currently, many CAR T cells have been successfully developed against various tumor antigens, such as mucin 1 (MUC1) for the treatment of prostate and breast cancer (Sanchez C, et al. Combining T-cell immunotherapy and anti-androgen therapy for prostate cancer. Prostate Cancer Prostatic Dis. 2013;16(2):123-31,S1; Wilkie S, et al. Retargeting of Human T Cells to Tumor-Associated MUC1:The Evolution of a Chimeric Antigen Receptor. The Journal of Immunology. 2008;180(7):4901-9), prostate-specific membrane antigen (PSMA) for the treatment of prostate cancer (Maher J, et al. Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCR[zeta] / CD28 receptor. Nat Biotech. 2002;20(1):70-5.), CD19 and CD20 for the treatment of B-cell malignancies(Brentjens RJ,et al.Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias.Blood.2011;118(18):4817-28.)and estrogen-related receptor beta type 2(ErRB2)for the treatment of prostate and breast cancer(Pinthus JH,et al.Immuno-Gene Therapy of Established Prostate Tumors Using Chimeric Receptor-redirected Human Lymphocytes.Cancer Research.2003;63(10):2470-6.). Furthermore, in pursuit of higher selectivity for tumor cells, T cells have been developed through several strategies, including dual CAR T cells engineered to express two CARs that specifically target two different antigens expressed on the same tumor cells (Kloss CC,et al.Combinatorial antigen recognition with balanced signalling promotes selective tumor eradication by engineered T cells.Nat Biotechnol.2013;31(1):71-5;Wilkie S,et al.Dual targeting of ErbB2 and MUC1 in breast cancer using chimeric antigen receptors engineered to provide complementary signalling.J Clin Immunol.2012;32(5):1059-70.).

[0304] Molecular Cloning and Genetic Engineering Each PAAV-CMV-CD28-IL4, PAAV-CMV-GPI-IL4, and PAAV-CMV-PSMA (Figure 32f) was constructed by combining the PAAV-CMV-GFP plasmid (Figure 3) with either the pUC57-CD28-IL4 plasmid, pUC57-GPI-IL4 or pUC57-PSMA plasmid, and the PAAV-Grp78-GFP plasmid was combined with either the pUC57-CD28-IL4 plasmid, pUC57-GPI-IL4 or pUC57-PSMA plasmid to construct PAAV-Grp78-CD28-IL4, PAAV-Grp78-GPI-IL4, and PAAV-Grp78-PSMA.

[0305] The new plasmids; PAAV.CMV.MUC1.CD28.IL4, PAAV.CMV.MUC1.GPI.IL4, PAAV.CMV.PSMA, PAAV.Grp78.MUC1.CD28.IL4, PAAV.Grp78.MUC1.GPI.IL4, and PAAV.Grp78.PSMA, were synthesized by restriction enzyme digestion and ligation, transformed into TG1 competent E. coli, and plated on 2xYT top agar containing ampicillin. All constructs were first verified by restriction digestion and gel electrophoresis, and then by DNA sequencing (MRC CSC Genomics Core Laboratory, UK).

[0306] Figure 31 shows a schematic diagram of an embodiment of an expression plasmid construct for bacteriophage-guided CAR T cell therapy, where 32a represents a MUC1-CD28.IL4 expression plasmid driven by a CMV promoter, 32b represents a MUC1-GPI.IL4 expression plasmid driven by a CMV promoter, 32c represents a PSMA expression plasmid driven by a CMV promoter, 32d represents a MUC1-CD28.IL4 expression plasmid driven by a Grp78 promoter, 32e represents a MUC1-GPI.IL4 expression plasmid driven by a Grp78 promoter, and 32f represents a PSMA expression plasmid driven by a Grp78 promoter.

[0307] Expression of CD28, GPI, and PSMA antigens Approximately 60% confluent HEK 293 cells in 12-well plates were incubated for 24 h with each vector (1,000,000TU / cell) in complete medium consisting of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), L-glutamine (2 mM, Sigma), penicillin (100 units / ml, Sigma), and streptomycin (100 mg / ml, Sigma). Untreated cells were used as negative controls. The next day, all vectors were removed from the cultures and cells were maintained as a monolayer in complete medium in a humidified incubator at 37 °C with 5% CO2. Medium was refreshed every 2 days.

[0308] Six days after transduction, cells were harvested with cell dissociation buffer (Invitrogen), washed with wash buffer (PBS containing 2% FBS and 0.1% NaN3) and incubated with Clear Back (human Fc receptor blocking agent, MBL) for 20 min at room temperature. Cells were then incubated with either HMFG2 antibody provided by Dr. John Maher (King's College London, UK) or PSMA antibody (MBL) diluted in wash buffer overnight at 4°C. The next day, cells were washed with wash buffer and incubated with anti-mouse IgG Alexa fluor 488-conjugated secondary antibody (Invitrogen) for 30 min at room temperature. Cells were finally washed with wash buffer and run on a FAC calibur Flow cytometer (BD Biosciences). Mean fluorescence intensity was measured for at least 20,000 gated cells per well in triplicate. Results were analyzed using Flowjo (TreeStar) software.

[0309] MUC-1.CD28, MUC-1.GPI, and PSMA antigen expression 6 days after transduction HEK 293 cells were cultured at 10 6TU / cells were transduced with either RGD-targeted PAAV (RGD) or non-targeted PAAV (NT). Figure 33 shows the data obtained. Untreated HEK 293 cells (Ctrl) and targeted PAAV-transduced-HEK 293 cells with only 488 secondary antibody staining (Ant.488) were shown as controls. (A) MUC-1 or PSMA expression of HEK 293 cells transduced with CMV promoter-driven PAAV vectors. DEAE-dextran was added. (B) MUC-1 or PSMA expression of HEK 293 cells transduced with CMV promoter-driven PAAV vectors without the addition of DEAE-dextran. (C) MUC-1 expression of HEK 293 cells transduced with Grp78 promoter-driven PAAV vectors without the addition of DEAE-dextran.

[0310] Stable cell line selection To select stable cell lines expressing MUC-1.CD28, MUC-1.GPI, or PSMA antigens on the cell surface, a puromycin resistance sequence was inserted into the PAAV-CMV-CD28-IL4, PAAV-CMV-GPI-IL4, PAAV-CMV-PSMA, PAAV-Grp78-CD28-IL4, PAAV-Grp78-GPI-IL4, and PAAV-Grp78-PSMA plasmids.

[0311] Figure 34 shows a schematic diagram of expression plasmid constructs containing puromycin resistance gene for stable cell line selected bacteriophage guided CAR T cell therapy therapy, where 34a represents MUC1-CD28.IL4 expression plasmid driven by CMV promoter, 34b represents MUC1-GPI.IL4 expression plasmid driven by CMV promoter, 34c represents PSMA expression plasmid driven by CMV promoter, 34d represents MUC1-CD28.IL4 expression plasmid driven by Grp78 promoter, 34e represents MUC1-GPI.IL4 expression plasmid driven by Grp78 promoter, and 34f represents PSMA expression plasmid driven by Grp78 promoter.

[0312] New plasmids were made by restriction enzyme digestion and ligation, transformed into TG1 competent E. coli and plated on 2xYT top agar containing ampicillin. All constructs were first verified by restriction digestion and gel electrophoresis, and then by DNA sequencing (MRC CSC Genomics Core Laboratory, UK).

[0313] result PAAV vectors encoding either MUC1 or PSMA transgenes were produced using a helper phage system. These transgene products are displayed on the tumor cell surface after transduction by PAAV. MUC1 and PSMA antigens are specifically recognized by MUC1-CAR T cells and PSMA-CAR T cells.

[0314] Data are presented herein showing that the phagemid particles of the invention can be used to transduce cancer cells which subsequently stably display the delivered antigen in a manner suitable for use as a target for adoptively transferred T cells.

[0315] discussion There is strong evidence to suggest that targeted PAAV vectors are more efficient than AAVP vectors in gene transfer in both commercial and disease cell lines. Both internalization and gene expression data concordantly show that PAAV is more efficient than AAVP. Evidence has also been provided to suggest a strong synergy between Dex and PAAV vectors over AAVP in gene transfer. Although these data suggest that PAAV is superior to AAVP, it must also be considered that PAAV vector samples contain helper phage contamination. Despite efforts to optimize experimental conditions during vector production, helper phage contamination (in this case, about 1 / 10) is inevitable, and it also displays the RGD targeting sequence on its small coat protein, thus competitively inhibiting transduction. Considering this, the internalization and gene expression data may greatly underestimate the "true" efficacy of RGD.PAAV. Furthermore, because internalization assays utilize staining of intracellular phage capsids for signal detection, the small overall size (and available capsid protein per particle) of PAAV means that the proportional number of internalized particles cannot be directly compared to the proportional number of AAVP, which have been shown using TEM to be twice as long as PAAV particles. Therefore, the methods of the invention include a purification step (e.g., FPLC) to remove helper phage.

[0316] In addition to providing mechanistic insights, it is essential that future studies must encompass replicates of all experiments using pure PAAV samples. In particular, phagemid-induced rAAV production can greatly benefit from reduced competitive inhibition due to helper phage contamination and multiple-fold higher yields of rAAV particles compared to traditional transfection protocols.

[0317] summary Hybrid phagemid vectors have been described that are highly efficient in gene transfer into mammalian cells. These phagemid / AAV (PAAV) vectors have a very large cloning capacity and target mammalian cells, meaning that no transfection reagents are required. This platform allows the production of vectors suitable for therapeutic gene therapy. Evidence is provided that this platform can deliver genes to tumor cells containing antigens suitable for targeting by adoptive T cell transfer therapy or CAR T cell therapy.

[0318] Example 11 - A superior phagemid / AAV hybrid vector for inducible delivery of the TRAIL gene into pediatric DIPG cells.

[0319] The use of cytokines in gene therapy has been explored because they perform diverse functions as differentiation, proliferation, activation, or induction of cell death by apoptosis. The tumor necrosis factor (TNF) superfamily is one group of molecules of interest due to their ability to induce tumor cell death. Members of the TNF superfamily, including Fas ligand (FasL), CD95 ligand (CD95L), and TNFα, have been identified as important therapeutic agents for the biological treatment of cancer. Their administration can induce apoptosis in different cancer cells, but causes severe toxicity to the liver, hindering their application in the clinic.

[0320] Given the dilemma of systemic toxicity, another member of the TNF superfamily, TRAIL, has risen as a promising cancer treatment. Preclinical and early clinical trials using recombinant TRAIL and antibodies against the TRAIL receptor have shown that TRAIL exhibits preferential toxicity to tumor cells with little or no toxicity to normal tissues while retaining its antitumor properties. TRAIL is constitutively present at the mRNA level in many tissues, primarily in the spleen, lung, and prostate, and is expressed primarily by cells of the immune system, such as natural killer (NK) cells and macrophages.

[0321] TRAIL is synthesized as a type II transmembrane protein and can also be proteolytically cleaved by cysteine ​​proteases to generate a secreted form. TRAIL is biologically active as a homotrimer, and the membrane-bound conformation appears to be more potent, as this particular conformation presumably facilitates cross-linking of the ligand-receptor complex, thereby enhancing signaling strength. Although the secreted form is less potent, the effect can be enhanced by engineering the extracellular domain fused to motifs such as leucine zippers that help stabilize and form homotrimers.

[0322] Like other TNF superfamily members, TRAIL induces apoptosis through interaction with cross-linking receptor molecules on the surface of target cells5,10. There are five receptors that have been identified: TRAIL R-1 and R-2 are death receptors that contain the cytoplasmic sequence death domain (DD) that triggers apoptosis, and TRAIL R-3, R-4 and osteoprotegerin are decoy receptors that prevent apoptosis5,6,10.

[0323] When homotrimeric TRAIL binds to death receptors, the receptors form trimers and recruit the adaptor protein Fas-associated death domain (FADD). FADD recruits initiator caspases 8 or 10, forming the death-inducing signaling complex (DISC), which is automatically activated by proteolysis. Activated caspases 8 or 10 cleave effector caspase 3, leading to cleavage of death substrates and cell death. If TRAIL instead binds to a decoy receptor, FADD is not recruited and apoptosis is not induced. Even in cells resistant to TRAIL-induced apoptosis, TRAIL can induce necroptosis. The TRAIL receptor system can induce direct killing of tumor-supporting immune cells, and its expression in NK cells is an important mechanism used by the immune system to kill cancer cells.

[0324] Current clinical trials to deliver agonists of the TNF superfamily, including recombinant TRAIL or agonist antibodies to the TRAIL receptor, have unfortunately failed to provide clinical benefit to cancer patients due to insufficient agonist activity and drug half-life. Furthermore, these drugs are often limited in their effectiveness by design due to concerns that systemic delivery of more potent agents may cause lethal side effects. To ensure delivery of TRAIL at optimal concentrations to produce clinical results, a suitable vector is needed to selectively target and transport TRAIL to cancer cells.

[0325] Using phage display-based technology, TRAIL can be delivered using viral vectors that display ligands that target and bind receptors selectively expressed in tumor tissues. Most studies have focused on the use of eukaryotic viruses, such as retroviruses and adenoviruses, as vectors, as they provide excellent transgene delivery. However, their broad tropism for mammalian cell membrane receptors has limited success in systemic gene therapy, leading to undesired uptake by the liver, reticuloendothelial system, and undesired tissues, as well as immunogenicity. In contrast, prokaryotic viruses are advantageous because they do not require removal of their natural tropism for use in mammalian cells, are cost-effective, and are easily produced at high titers. Due to their lack of tropism, they are inherently poor vehicles for transduction of mammalian cells. However, phages can be internalized into cells by modifying their coat proteins to display selective ligand peptide motifs.

[0326] Effective administration of therapy solely to the tumor for long-term effects without systemic toxicity can be achieved using bacterial virus, bacteriophage, or phage-based vectors. These bacteriophage-based vectors can be engineered to display selective ligand peptide motifs on their coat proteins, allowing viral binding to target cells and subsequent internalization for ligand-directed delivery of gene 19. By combining the favorable biological properties of eukaryotic and prokaryotic viruses, a chimeric viral vector was constructed that includes a recombinant adeno-associated virus (AAV) and an M13-derived filamentous phage, named AAV / phage or AAVP13. The pIII coat protein of the phage was engineered to display the bicyclic peptide CDCRGDCFC (RGD-4C, SEQ ID NO: 7), which binds to specific αv integrin receptors (αvβ3 or αvβ5) overexpressed in both the tumor and the supporting vascular system. This makes it an excellent targeting platform for ligand-directed delivery and cellular transduction of therapeutic transgenes, and these functional properties have been confirmed in preclinical models of several cancers, including prostate, breast, and soft tissue sarcoma.

[0327] The inventors have improved on known vector platforms by using the phagemid system to produce a next-generation vector known as phagemid-AAV (PAAV) (Figure 43). The PAAV vector is a chimeric virus, an M13 filamentous phage containing a hybrid genome constructed using AAV serotype 2 DNA sequences. The control gene is regulated by a constitutively active cytomegalovirus, CMV promoter, and is flanked by full-length inverted terminal repeats (ITRs). The phagemid contains the f1 origin of replication, which is used for single-stranded replication and packaging into phage particles, as well as the origin of replication for double-stranded replication after entering the target cell. During cloning and production of the viral vector, selection can be performed using ampicillin.

[0328] In this hybrid vector model, most of the phage genome is removed and it can accommodate longer DNA sequences, but it requires the use of a helper virus to provide the capsid and other phage components. The RGD-4C peptide motif is first displayed on the pIII coat protein of M13 bacteriophage to produce a targeting backbone helper virus. The helper virus is then used to transduce TG1 E. coli bacteria containing the engineered phagemid, and antibiotic selection pressure can be used to select for the phagemid-containing targeting vector. The new model can accommodate longer DNA sequences, has a higher transduction efficiency, and can be produced at higher titers than the original AAVP.

[0329] Without wishing to be bound by any particular theory, two methods could further improve the efficiency of transduction. First, by engineering the RGD-4C peptide to be displayed on the pVIII rather than the pIII coat protein, the major coat protein, pVIII is expressed at up to 2700 copies, whereas pIII is expressed at only up to 5 copies. Thus, it is believed that the more RGD-4C ligands available for integrin targeting and binding, the more efficient the cell binding and transduction.

[0330] Without wishing to be bound by a particular theory, another strategy to increase efficiency is by displaying the histidine-rich H5W ligand, an endosomal escape peptide, on the recombinant pVIII coat protein. Intracellular barriers such as endosomes may limit the rate of gene expression by trapping the vector and preventing it from exerting its therapeutic effect, even when the efficiency of internalization is high. The histidine side chain forms a zwitterion, which allows it to act as a proton sponge and buffer the low pH of the endosome following ligand-directed endocytosis of the phage vector. When protons enter the endosome, water is drawn into the endosome via the vacuolar membrane proton pump, forming a pore in the endosome, thereby releasing the phage vector.

[0331] The aim of this study was to develop a superior PAAV vector with optimal transduction efficiency of DIPG cells by modifying the peptide displayed on the vector's coat protein. We then used the optimal PAAV vector to deliver a TRAIL transgene and evaluate the efficacy of TRAIL as a therapeutic gene in the treatment of DIPG.

[0332] Without wishing to be bound by any particular theory, the inventors hypothesize that: 1. The specificity of targeting and the efficiency of gene transfer can be enhanced by displaying the RGD-4C ligand on the pVIII coat protein or the H5W peptide on the recombinant pVIII coat protein. 2.TRAIL is an effective therapeutic gene that specifically induces DIPG cell death in in vitro experiments and in vivo orthotopic DIPG animal models.

[0333] Research Objective 1. Design and produce viral vectors to specifically target DIPG cells with optimal expression of desired genes.

[0334] 2. Evaluate and explore the killing potential of the therapeutic gene TRAIL against DIPG 3. To evaluate the specificity of targeting TRAIL expression to DIPGs in vivo and to investigate the therapeutic efficacy following intravenous administration of the vector into immunodeficient mice with established orthotopic human DIPGs.

[0335] protocol cell culture HEK293T cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. DIPG tumor cell lines were obtained from Sant Joan de Barcelona Hospital. They were maintained in a tumor stem medium (TSM) base supplemented with 10% FBS. TSM was made according to the provided recipe (Table XXX).

[0336] Integrin staining DIPG cells were seeded on poly-D-lysine-coated coverslips in 24-well plates and grown until 60-70% confluent. Five wells were stained using: control, secondary antibody only, αv, β3, and β5 integrin. Cells were washed and fixed with 4% formaldehyde for 10 min at room temperature (RT). Cells were washed three times with PBS and incubated with 50 mM ammonium chloride for 5 min. Cells were washed again three times with PBS and blocked with 2% bovine serum albumin (BSA-PBS) for 30 min. Cells were then incubated with primary antibodies anti-αv (1:100), anti-β3 (1:50), and anti-β5 integrin (1:100) diluted in 1% BSA-PBS for 1 day and stored in a moist chamber at 4 °C. Afterwards, the cells were washed three times with 1% BSA-PBS and incubated with secondary Alex Fluor 488-conjugated antibody (1:750) and 4',6-diamidino-2-phenylindole (DAPI, 1:2000) for 1 h. The coverslips were washed three times with 1% BSA-PBS, three times with PBS, and once with sterile water. After air drying, the coverslips were mounted in Mowiol mounting medium. The cells were observed and images were taken with a fluorescent microscope.

[0337] Production, purification, and titration of helper phage with RGD-4C ligand displayed on pVIII coat protein (Figure 44) Wild-type M13 phage DNA was modified to express RGD-4C on the pVIII coat protein by polymerase chain reaction (PCR). The primers used in PCR were designed by a colleague (Sajee Waramit) prior to the start of this project. PCR products were ligated using T4 DNA ligase (NEB) and transformed into DH5α bacteria for cloning. Plasmids were extracted by Miniprep (QIAGEN), verified by restriction digestion and gel electrophoresis, and then sent for sequencing to ensure the integrity of the DNA sequence (MRC CSC Genomics Core). Selected clones were then transformed into TG1 competent E. coli (Zymo research) and plated on 2xYT (Sigma) agar containing 50 μg / ml kanamycin in a 37 °C incubator.

[0338] After colonies formed, a single colony was selected and grown in 1 L of 2xYT medium with kanamycin in a shaking incubator (32 °C, 220 rpm) for 18 h. The overnight culture was centrifuged at 6000 g for 30 min at 4 °C. The phage supernatant was collected and 20% v / v PEG / NaCl (20% PEG6000, 2.5 M NaCl) was added and mixed thoroughly. After overnight incubation in the cold room, the supernatant was centrifuged again at 10000 g for 30 min at 4 °C. The phage pellet was resuspended in 16 mL of phosphate-buffered saline (PBS) and 20% v / v PEG / NaCl and kept overnight in the cold room. The final phage suspension was centrifuged at 10,000 g for 30 min at 4 °C and the supernatant was discarded. The purified phage pellet was resuspended in 1–2 mL of PBS (depending on pellet size) and sterile filtered through a 0.45 μm filter cartridge.

[0339] Titrations were performed using serial dilutions with 10-fold increments. 5 μL of each dilution of phage was used to infect 500 μL of naive TG1 E. coli grown to log phase (OD600 = 0.45-0.50) by incubating at 37 °C for 30 min. After incubation, 100 μL of bacteria were plated on 2xYT agar containing kanamycin overnight in a 37 °C incubator. Colonies were counted the next morning after 18 h of incubation, and viral titers were estimated and expressed as bacterial transducing units (TU).

[0340] Production, purification, and titration of RGD pVIII vectors containing reporter genes (green fluorescent protein [GFP] and lucia luciferase [lucia]) (Figure 45) To evaluate the efficiency of transduction of DIPG cells, we constructed a vector containing a phagemid containing the reporter genes green fluorescent protein (GFP) and lucia (InvivoGen). GFP expression can be detected by fluorescence microscopy, while lucia is a secreted luciferase, which can be quantified with its substrate Quanti-Luc by measuring relative luciferase units (RLU). In the following protocol, we use either the PAAV-GFP plasmid or the PAAV-lucia plasmid.

[0341] The plasmid was first transformed into TG1 competent E. coli (Zymo research) and plated on 2xYT (Sigma) agar containing 50 μg / ml ampicillin overnight in a 37 °C incubator. After colonies formed, a single colony was selected and grown in 2xYT medium containing ampicillin in a shaking incubator (32 °C, 220 rpm). 100 mL of bacteria was grown to log phase (OD600 = 0.45-0.50), after which 15-20 μL of RGD pVIII helper phage was added and incubated in a shaking incubator (32 °C, 150 rpm). The bacteria were then grown in 1 L of 2xYT medium containing kanamycin and ampicillin for 18 h in a shaking incubator (32 °C, 150 rpm). Purification was performed according to previous investigations. To estimate the titers of the reporter gene vector and the helper phage, respectively, titrations were performed by growing the bacteria on separate plates containing 2xYT only ampicillin and only kanamycin.

[0342] Production, purification, and titration of H5W peptide RGD pIII vector containing a reporter gene (lucia) H5W RGD pIII helper phage was produced by a colleague (Sajee Waramit) prior to the start of this project. Production was largely similar to the previously described protocol, except that 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was also added to select for the H5W RGD pIII vector. Purification and titration of the vector were performed according to our previous investigation.

[0343] Transduction of cells with vectors The cells used for transduction (HEK293T or DIPG) were seeded in plates until 60-70% confluent. The medium was then removed and the cells were incubated overnight with vector diluted in OptiMEM, the amount of vector being based on the viral titer obtained from titration. The transduction medium was then removed and replaced with complete medium. For cells transduced with GFP, the cells were observed under fluorescence at the designated time points and images were taken to look for cells expressing GFP. For cells transduced with lucia, at the designated time points, 10 μl of cell medium and 25 μl of Quanti-Luc (InvivoGen), a substrate for lucia, were mixed in a white polystyrene 96-well plate and then incubated at room temperature for 5 min. Luciferase expression was quantified using a Promega Glomax microplate reader.

[0344] To evaluate and explore the killing potential of the therapeutic gene TRAIL against DIPG Production of PAAV-human TRAIL (hTRAIL) plasmid for transfection (Figure 46) The GFP sequence of the PAAV-GFP plasmid was replaced with the hTRAIL gene sequence by PCR. The hTRAIL gene was amplified by cloning into pUNO1-hTRAIL (InvivoGen) using specific primers, designed to contain EcoRI and SalI restriction sites at the ends (Table 1). Molecular cloning, plasmid extraction, and DNA sequence verification were performed according to previous investigations. The selected clones were transformed into TG1 bacteria for amplification, and then extracted with Maxiprep (QIAGEN).

[0345] [Table 1]

[0346] Transfection of plasmids into cells DIPG cells were seeded in 48-well plates until 60-70% confluent. Before starting the experiment, cells were incubated in OptiMEM reduced serum medium for 1-2 h. For 48-well plates, the FuGENE HD transfection protocol (Promega) recommends aiming for a medium volume of 0.5-2.0 ml, a FuGENE volume of 0.6-1.8 μl, and a DNA amount of 0.2-0.6 μg. Transfection efficiency depends on the ratio of FuGENE to DNA; in this case, a ratio of 3:1 is used.

[0347] Eppendorf tubes were prepared and labeled for use in the experiment. Depending on the volume of FuGENE used, up to 20 μl of OptiMEM was added to each tube. FuGENE was then added directly to the medium without contacting the walls of the plastic tube and the mixture was incubated at RT for 5 min. Plasmid DNA was added to the tube and the transfection reagent:DNA complex was incubated at RT for 15 min. This complex was then added to the DIPG cells in a dropwise fashion, swirling the plate to ensure uniform distribution. After incubating the cells for 6 h at 37 °C, the transfection medium was replaced with TSM medium supplemented with FBS without antibiotics. Replace the cells in the incubator and observe the cell viability for the next 24-48 h.

[0348] result DIPG cells express integrin receptors for RGD-4C ligand binding To establish the suitability of this vector model in this cell line for targeted gene delivery, the cells were first examined for expression of integrins αvβ3 and αvβ5 by means of fluorescence microscopy. As shown in Figure 47, the DIPG cells tested were positive for expression of αv, β3, and β5 units, and no fluorescence was observed in cells incubated without antibody or secondary antibody alone.

[0349] RGD-4C ligand displayed on pIII coat protein resulted in higher transduction efficiency and gene expression levels than pVIII coat protein Previous data from the inventors' laboratory showed that targeted RGD-4C pIII vector-mediated gene delivery and expression was selective and efficient compared to non-targeted vectors. To optimize transduction, one of the hypotheses was to display RGD-4C on the pVIII coat protein to increase the copy number of the ligand per phage (Figure 48).

[0350] Transduction efficiency was compared using human embryonic kidney cells HEK293T, a cell model that was continuously used to characterize phage vectors using reporter gene vectors (GFP or lucia). Cells were incubated with a non-targeting vector (control lacking RGD-4C), a targeting RGD pIII PAAV-GFP or RGD pVIII PAAV-GFP vector and observed under a fluorescent microscope (Figure 49). The highest GFP expression was seen in cells transduced with RGD pIII PAAV-GFP across all TUs.

[0351] Similarly, in HEK293T cells transduced with non-targeting RGD pIII PAAV-lucia or RGD pVIII PAAV-lucia vectors, the highest RLU was observed with RGD pIII PAAV-lucia (Figure 50), suggesting that pIII-displayed RGD is superior to pVIII in transduction and gene expression.

[0352] H5W peptide displayed on recombinant pVIII coat protein improves transduction efficiency and gene expression Another strategy to optimize the vector was to display the H5W peptide on the recombinant pVIII coat protein. Previous data from the laboratory showed that display of the H5W peptide increased luciferase expression in HEK293T cells compared to RGD pIII alone in transduction experiments using a vector containing the reporter gene lucia (Sajee Waramit, unpublished data) (Figure 51).

[0353] To examine whether this finding could be applied to DIPG cells, DIPG cells were incubated with non-targeting RGD pIII PAAV-lucia or H5W RGD pIII PAAV-lucia vectors. Consistent with the HEK293T data, the highest RLU in DIPG cells is observed with H5W RGD pIII PAAV-lucia (Figure 52). This suggested that the H5W peptide could improve vector efficiency in DIPG cells and should be used in the final vector construct.

[0354] Transfection of PAAV-hTRAIL plasmid into DIPG cells induced cell death After vector optimization and before conducting further experiments, it was essential to determine whether the selected gene of interest, human TRAIL (hTRAIL), was able to induce cell death in DIPG cells via transfection.

[0355] DIPG cells were transfected with either PAAV-hTRAIL or the control PAAV-GFP plasmid. Microscopic images taken 18 hours post-transfection at all DNA concentrations showed significantly more cell death in cells transfected with PAAV-hTRAIL (Figure 53).

[0356] discussion Without wishing to be bound by any particular theory, the data indicate so far that vectors expressing H5W and RGD-4C on the pIII coat protein were the most optimal in targeting and inducing expression of the desired gene in DIPG cells. Data from transfection experiments also suggest that the therapeutic gene TRAIL can induce DIPG cell death, which warrants further investigation.

[0357] Example 12 - Hybrid IL2-TNFa To increase targeted local-regional production of TNFa within tumor sites, we constructed a phagemid encoding a secreted form of TNFa by inserting the signal peptide from IL-2 to precede a TNFa sequence lacking the transmembrane domain. To the inventors' knowledge, this is the first time that a hybrid IL2-TNFa has been engineered, and their data suggest that preceding the TNFa gene with an IL2 signal peptide sequence significantly enhanced the expression and secretion of TNFa in cancer cells. Such modifications represent a significant advancement in the technology available for targeted production and release of TNFa in the tumor microenvironment and should be considered to increase therapeutic levels of TNFa.

[0358] protocol Construction of PAAV.Grp78.IL-2SP.hTNFa The coding therapeutic sequence inserted into the phagemid is a hybrid sequence containing the tumor-specific promoter of glucose-regulated protein (Grp78), the signal peptide (SP) sequence from IL-2 (Figure 57), and the human sequence of TNFα. The Grp78 promoter is stress-inducible, being potently activated by conditions of glucose deprivation, chronic anoxia, and acidic pH, and persists in aggressive, poorly perfused tumors. Furthermore, the Grp78 promoter is induced in a wide variety of tumors, making it an attractive candidate for use in gene therapy. Previous studies have demonstrated several advantages of this promoter over viral promoters. The safety and tumor specificity of this promoter have also been elegantly reported in transgenic mice carrying a LacZ transgene. Tumors established in these transgenic mice showed high LacZ expression, whereas no promoter activity was detected in major normal tissues. Furthermore, unlike viral promoters used in gene therapy vectors, mammalian promoters such as Grp78 are not silenced in eukaryotic cells. In the inventors' previously published work, they reported that dual-targeted RGD4C / phage-Grp78 provided more sustained transgene expression than RGD4C / phage-CMV carrying the cytomegalovirus CMV promoter. Inclusion of both the RGD4C ligand and the Grp78 promoter generates a vector with dual tumor targeting at both the cell entry and transcription levels.

[0359] TNFα is a transmembrane molecule, and to generate a secreted form of TNFα that is more accessible to its receptors on the cell surface of tumor cells, we generated a secreted form of TNFα by removing the transmembrane domain of TNFα and replacing it with the signal peptide of interleukin 2 (IL-2). IL-2 was previously used as an effective signal peptide tested in various studies to promote the secretion of other cytokines or growth factors into the extracellular environment and subsequently increase the availability and availability of their corresponding receptors. The IL-2 signal peptide sequence (Thermoscientific, UK) flanked by BamHI and EcoRI restriction sites was ligated into the PAAV.Grp78 backbone. The hTNFα sequence flanked by EcoRI and SalI restriction sites was provided from the pUNO1-hTNFα plasmid (Invivogen, France) by polymerase chain reaction (PCR) and then ligated into the PAAV.Grp78.IL-2 backbone. Molecular cloning steps were performed using restriction enzyme digestion (NEB, UK) and Quick T4 DNA ligase (NEB, UK) for ligation. The modified plasmid was transformed into TG1 competent E. coli (Zymo research, USA). Bacteria carrying the plasmid were then selected by ampicillin selection on 2xYT agar plates. The construct was verified by restriction digestion and gel electrophoresis, and DNA sequencing (MRC CSC Genomics Core Laboratory, UK) (see Figure 58).

[0360] Example 13 - Application of PAAV-delivered TNFα cytokine gene therapy in DIPG Introduction Diffuse intrinsic pontine gliomas (DIPGs) are the most aggressive brain tumors occurring only in children with a 2-year survival rate of only 6–10%. Due to its diffuse nature and its delicate location within the brainstem, surgical resection is not possible and there are no effective treatment strategies for this type of cancer. The current standard treatment for DIPGs is radiation therapy, which, even in combination with radiosensitizers, has not shown any success as all children subsequently relapse. Many clinical trials have tested the effect of chemotherapy in combination with conventional radiation therapy, but there is no improvement in terms of extending overall survival, even when combined with high doses of chemotherapy drugs. This resistance to chemotherapy is due to the anatomical location of DIPGs in the pons without an intact blood-brain barrier, making it more difficult for any drug to reach the target location. To overcome this problem, a clinical trial applied the use of convection-enhanced delivery (CED) to deliver the chemical agent topotecan for the treatment of DIPGs in two children. In this pilot study, both patients died after treatment, although initially with a reduction in tumor size. CED is a technique that directs the delivery of chemotherapy drugs to brain tumors through continuous flow, and has recently been used in clinical trials to overcome drug delivery challenges by bypassing the blood-brain barrier.

[0361] Targeted therapy against DIPGs seems the best solution due to their diffuse nature and delicate location. In an attempt to target DIPG tumors, an ongoing clinical trial (clinicaltrials.gov ID NCT01502917) applies the concept of targeted radiation by labeling the radioactive substance 124I with the 8H9 antibody, known to selectively bind to cancer cells sparing healthy brain cells [8]. Another candidate to target brain tumors, suggested by our group (Hajitou's laboratory) and which showed high selectivity in glioblastoma, is the integrins αvβ3 and αvβ5 through the binding of the RGD4C peptide, which will be discussed in chapter 3. Indeed, RGD4C targeting DIPGs is currently being used in an ongoing phase I clinical trial (clinicaltrials.gov ID NCT03178032). In this trial, the oncolytic adenovirus DNX2401 is used to target tumor cells and induce cell death. Among the 30 ongoing clinical trials for DIPG, the majority are focused on testing various drugs and radiation therapies, and other treatments, including gene therapy, have not yet been applied. TNFα is a proinflammatory cytokine known for its anticancer properties by mediating apoptosis, necrosis, and activation of immune cells. Depending on the cellular context, it can also induce cell proliferation and angiogenesis. Its antitumor activity has been studied in many solid tumors, including colon cancer, esophageal adenocarcinoma, melanoma, pancreatic cancer, and many others. However, its efficiency as a therapeutic agent has been very limited due to its significant toxicity when introduced systemically. This has limited the clinical use of TNFα in the clinic as a cancer treatment, where it is used for soft tissue sarcoma, melanoma, and other unresectable tumors confined to the limbs in the form of isolated limb perfusion (ILP) to avoid limb amputation. Indeed, TNFα acts synergistically with chemotherapeutic drugs in vivo by targeting the tumor vascular system to disrupt VE-cadherin, thus increasing the penetration of chemotherapeutic drugs into the tumor environment. Therefore, efforts must be made to overcome the limitations of systemic toxicity to enhance the therapeutic efficacy of TNFα.One strategy to reduce the toxicity of TNFα while still retaining its antitumor activity has been created by generating mutant forms of TNFα.This mutant TNFα further targeted the tumor environment through the signaling peptide RGD4C and enhanced the efficiency of chemotherapy drugs in hepatocarcinoma and sarcoma allografts.

[0362] In vitro studies on DIPGs are very limited, with few human tissues available for study, limiting our understanding of this devastating type of brain tumor. Applications for treatments other than chemotherapy drugs and radiation therapy are also hindered by a lack of in vitro application and understanding. Therefore, we investigated the therapeutic effects of two forms of TNFα, transmembrane (tmTNFα), and secreted (sTNFα), on DIPGs. TNFα was used in the form of gene therapy, expressed and delivered to cells using PAAV targeted by the signaling peptide RGD4C. Without wishing to be bound by a particular theory, the use of RGD4C-targeted PAAV as a therapeutic gene delivery vehicle to the tumor site would ensure safer, selective, and efficient gene transfer for the treatment of DIPGs.

[0363] To understand the mechanism of cell death induced by TNFα, we studied the viability and apoptotic activity in response to transduction of PAAV carrying two different forms of TNFα, tmTNFα, and sTNFα. Apoptotic activity was determined by measuring the activity of caspase 3 / 7, caspase 8, and caspase 9.

[0364] Finally, we studied the effect of the chemotherapy drug cisplatin in enhancing gene therapy. Thus, two different promoters upstream of TNFα, GRP78 and CMV, were used to understand the synergistic effect of gene therapy with cisplatin. The lucia reporter gene under the control of the GRP78 and CMV promoters was used for quantitative analysis of gene expression after cisplatin treatment to further understand the role of cisplatin in enhancing gene expression.

[0365] result Transduction of DIPGs with PAAV carrying transmembrane tmTNFα and secreted sTNFα transgenes: The transmembrane TNFα gene and secreted TNFα were cloned into PAAV vectors, PAAV-tmTNFα and PAAV-sTNFα (including the vectors shown in Example 12), followed by production of targeted (RGD4C) and non-targeted (M13 / control) viruses. DIPG cells were transduced and cell viability was measured 7 days after transduction. 40ng / μg of phage protein DEAE-dextran further improved transduction efficiency. DIPG cells responded better to PAAV-sTNFα in inducing cell death compared to PAAV-tmTNFα, with the former showing about 50% cell death at 7 days compared to only 20% cell death induced by the transmembrane form shown in Figure 59. Together, these data suggest that sTNFα is a good candidate for the treatment of DIPG.

[0366] Gene delivery and TNFα expression following transduction with PAAV: The efficiency of PAAV gene delivery was evaluated by measuring mRNA levels after transduction. As shown in Figure 60A, both forms of TNFα were expressed with high specificity in transduced cells, as the non-targeted (M13) form shows negligible mRNA expression of TNFα compared to the targeted PAAV (RGD4C). At the protein level, transduction of PAAV-tmTNFα did not cause secretion of TNFα in the medium, as detected by ELISA (Figure 60B). Although transduction with both forms shows TNFα expression at the mRNA level, only the secreted form was associated with the level of TNFα protein. This further confirms that transgene transduction is efficient and may suggest that the inefficiency of induction of cell death by the transmembrane form is due to the lack of soluble secreted protein. The release of sTNFα from the initial membrane-bound form requires the enzymatic activity or expression of TNFα-converting enzyme (TACE). Therefore, the expression of TACE enzyme was measured by Western blot and compared with other pediatric brain tumor cells (medulloblastoma). DIPG expression of TACE enzyme was 4-fold lower than that of UW228 and 2-fold lower than that of Daoy cells.

Claims

1. A recombinant phagemid / adeno-associated virus (AAV) particle for expressing a transgene in a target tumor cell transduced with said recombinant phagemid / AAV particle for use in a method of treating, preventing, or ameliorating cancer, said recombinant phagemid / AAV particle comprising at least one transgene expression cassette comprising a nucleic acid sequence encoding one or more cytokines, and comprising a genome lacking at least 95% of the M13 bacteriophage genome from which said recombinant phagemid / AAV particle is derived, said recombinant phagemid / AAV particle lacking M13 bacteriophage structural genes necessary for the formation, packaging, or extrusion of said recombinant phagemid / AAV particle from a prokaryotic host. wherein the recombinant phagemid / AAV particle comprises a genome lacking the M13 bacteriophage structural genes encoding capsid proteins, wherein the recombinant phagemid / AAV particle comprises a genome lacking a gene encoding a pIII capsid minor coat protein, wherein the recombinant phagemid / AAV particle comprises a genome lacking a gene encoding a pVIII capsid major coat protein, wherein the genome of the recombinant phagemid / AAV particle comprises a bacteriophage origin of replication, and wherein the method comprises delivering the transgene expression cassette at least adjacent to the tumor cell such that one or more cytokines are expressed.

2. The recombinant phagemid / AAV particle for use according to claim 1, wherein the introduced gene expression cassette encodes a cytokine having the effect of inducing apoptosis in the tumor cells, altering the tumor cells to promote an endogenous anti-tumor response, altering the tumor cells to promote other therapies, or altering the tumor microenvironment to promote therapy.

3. The recombinant phagemid / AAV particle for use according to claim 1 or 2, wherein the cytokine is IL-4, IL-12, IL-15, TNFα, TRAIL, IFN-γ, or any combination thereof, optionally wherein the cytokine is IL-15.

4. The recombinant phagemid / AAV particle for use according to any one of claims 1 to 3, wherein said cytokine is a hybrid cytokine comprising a non-endogenous signal peptide configured to increase expression and / or secretion of said cytokine, optionally wherein said non-endogenous signal peptide is an IL-2 signal peptide.

5. Optionally, the hybrid cytokine is a hybrid TNFα comprising an IL-2 signal peptide configured to increase expression and / or secretion of TNFα; i) the hybrid TNFα comprises an amino acid sequence substantially as set forth in SEQ ID NO: 22 or a fragment or variant thereof; and / or ii) The recombinant phagemid / AAV particle for use according to claim 4, wherein said hybrid TNFα is encoded by a nucleic acid sequence comprising SEQ ID NO: 23 or a fragment or variant thereof.

6. Recombinant phagemid / AAV particles for use according to any one of claims 1 to 5 for use in the treatment, prevention or amelioration of childhood brain tumours, optionally diffuse intrinsic pontine glioma (DIPG) or medulloblastoma.

7. A recombinant phagemid / adeno-associated virus (AAV) particle for expressing a transgene in a target tumor cell transduced with said recombinant phagemid / AAV particle, said recombinant phagemid / AAV particle comprising at least one transgene expression cassette comprising a nucleic acid sequence encoding one or more cytokines, and comprising a genome lacking at least 95% of the M13 bacteriophage genome from which said recombinant phagemid / AAV particle is derived, said recombinant phagemid / AAV particle comprising a genome lacking M13 bacteriophage structural genes necessary for formation, packaging, or extrusion of said recombinant phagemid / AAV particle from a prokaryotic host, said recombinant phagemid / AAV particle comprising a genome lacking said M13 bacteriophage structural genes encoding capsid proteins, a recombinant phagemid / AAV particle comprising a genome lacking a gene encoding a pIII capsid minor coat protein, said recombinant phagemid / AAV particle comprising a genome lacking a gene encoding a pVIII capsid major coat protein, said recombinant phagemid / AAV particle genome comprising a bacteriophage origin of replication, and further configured, in use, for delivering said transgene expression cassette at least adjacent to said tumor cell such that one or more cytokines are expressed, said cytokine being any one of IL-4, IL-12, IL-15, TRAIL, IFN-γ, hybrid TNFα, or any combination thereof, and optionally said cytokine being IL-15.

8. A pharmaceutical composition comprising the recombinant phagemid / AAV particle of claim 7 and a pharma- ceutically acceptable vehicle.

9. A recombinant phagemid / AAV particle according to claim 7 for use in therapy or diagnosis.

10. A recombinant phagemid / AAV particle according to claim 7 or for the use according to any one of claims 1 to 6 and 9, or a pharmaceutical composition according to claim 8, comprising i) the recombinant phagemid / AAV particles are configured to deliver the transgene expression cassette to the target tumor cells; ii) the genome of the recombinant phagemid / AAV particle comprises an origin of replication, optionally a pUC ori, to allow replication of the double-stranded vector into a prokaryotic host; iii) the genome of the recombinant phagemid / AAV particle comprises one or more DNA sequences that are favorable for targeted integration into a host genome; and / or iv) the recombinant phagemid / AAV particles comprise multiple transgene expression cassettes; Recombinant phagemid / AAV particles or pharmaceutical compositions.

11. A recombinant phagemid / AAV particle according to claim 7 or 10, or for the use according to any one of claims 1 to 6 and 9, or a pharmaceutical composition according to claim 8, i) the transgene expression cassette comprises one or more functional elements required for expression of the nucleic acid in the target cell selected from the group consisting of a promoter, a nucleic acid for encoding a polyA tail attachable to the expressed substance, and either a left and / or right inverted terminal repeat (ITR) or a left and / or right long terminal repeat (LTR); ii) the transgene expression cassette further comprises a nucleic acid encoding a substance having therapeutic or industrial utility in the target cell or tissue, optionally wherein the substance encoded by the nucleic acid is a polypeptide or protein; iii) the recombinant phagemid / AAV particle comprises one or more capsid minor coat proteins, optionally wherein the recombinant phagemid / AAV particle comprises a pIII capsid minor coat protein configured to display a cell targeting ligand to enable delivery of the recombinant phagemid / AAV particle to the target tumor cell; iv) said recombinant phagemid / AAV particles comprise one or more capsid major coat proteins, optionally said recombinant phagemid / AAV particles comprise at least one pVIII capsid major coat protein configured to display a foreign peptide thereon; and / or v) the recombinant phagemid / AAV particles are combined with a cationic polymer to form a complex having a net positive charge, optionally the cationic polymer is selected from the group consisting of chitosan; poly-D-lysine (PDL); diethylaminoethyl (DEAE); diethylaminoethyl-dextran (DEAE.DEX); polyethyleneimine (PEI); polybrene; protamine sulfate; and cationic lipids; Recombinant phagemid / AAV particles or pharmaceutical compositions.

12. A recombinant phagemid / AAV particle according to claim 7, 10 or 11, or for the use according to any one of claims 1 to 6 and 9, or a pharmaceutical composition according to claim 8, A recombinant phagemid / AAV particle or pharmaceutical composition, wherein the genome of the recombinant phagemid / AAV particle lacks at least 99% of the M13 bacteriophage genome from which it is derived.

Images