阅读说明：本技术 癌症治疗 (cancer treatment ) 是由 阿明·哈吉图 基蒂萨克·苏万 玛利亚姆·阿尔巴拉尼 萨吉·瓦拉米特 于 2018-04-24 设计创作，主要内容包括：本发明提供了用于癌症治疗的噬菌粒载体和相关的噬菌粒颗粒,并且具体地说,涉及新颖噬菌粒颗粒和相关表达系统的用途,其用于治疗、预防、改善或管理癌症。具体地说,本发明涉及噬菌粒颗粒和表达系统的用途,其用于递送对细胞因子进行编码的转基因以治疗、预防、改善或管理癌症。本发明还扩展到噬菌粒颗粒和表达系统的用途,其用于递送转基因并用于将这种治疗与过继转移的T细胞的使用进行组合以治疗、预防、改善或管理癌症。(The present invention provides phagemid vectors and related phagemid particles for use in cancer therapy and in particular to the use of novel phagemid particles and related expression systems for the treatment, prevention, amelioration or management of cancer. In particular, the invention relates to the use of phagemid particles and expression systems for delivering transgenes encoding cytokines for the treatment, prevention, amelioration or management of cancer. The invention also extends to the use of phagemid particles and expression systems for delivering transgenes and for combining such treatment with the use of adoptively transferred T cells to treat, prevent, ameliorate or manage cancer.)

1. 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, wherein the phagemid particle comprises at least one transgene expression cassette comprising a nucleic acid sequence encoding one or more cytokines and comprises a genome that lacks at least 50% of its phage genome, and wherein the method comprises delivering the nucleic acid sequence at least adjacent to the tumor cell such that one or more cytokines are expressed.

2. The recombinant phagemid particle for use according to claim 1, wherein the transgenic expression cassette encodes a cytokine having the following effects: induction of apoptosis in the tumor cells, alteration of the tumor cells to promote endogenous anti-tumor responses, alteration of the tumor cells to promote other therapies, or alteration of the tumor microenvironment to promote therapies.

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

4. The recombinant phagemid particle for use according to any of the preceding claims, wherein the cytokine is a hybrid cytokine comprising a non-endogenous signal peptide configured to increase expression and/or secretion of the cytokine.

5. The recombinant phagemid particle for use according to claim 4, wherein the non-endogenous signal peptide is an IL-2 signal peptide.

6. The recombinant phagemid particle for use according to claim 5, wherein the hybrid cytokine is a hybrid TNF α comprising an IL-2 signal peptide, the IL-2 signal peptide being configured to increase expression and/or secretion of TNF α.

7. The recombinant phagemid particle for use according to claim 6, wherein the hybrid TNF α comprises an amino acid sequence substantially as depicted in SEQ ID No 22 or a fragment or variant thereof.

8. The recombinant phagemid particle for use according to claim 6 or 7, wherein the hybrid TNF α is encoded by a nucleic acid sequence comprising SEQ ID No 23 or a fragment or variant thereof.

9. Recombinant phagemid particles for use according to any of the preceding claims for use in the treatment, prevention or amelioration of a pediatric brain tumor, optionally a diffuse intracerebroventricular glioma (DIPG) or medulloblastoma.

10. A system for producing recombinant phagemid particles from a prokaryotic host, the system comprising: -

(i) a first vector configured to persist within a prokaryotic host and comprising at least one transgenic expression cassette comprising a nucleic acid sequence encoding one or more cytokines and a packaging signal for enabling the vector to replicate into single-stranded DNA; and

(ii) A second vector comprising a nucleic acid encoding a structural protein required for packaging said single stranded DNA, resulting in the formation and extrusion of a recombinant phagemid particle from said prokaryotic host.

11. A method for producing recombinant phagemid particles from a prokaryotic host, the method comprising: -

i) Introducing a first vector into a prokaryotic host cell, the first vector configured to persist within the prokaryotic host and comprising at least one transgenic expression cassette comprising a nucleic acid sequence encoding one or more cytokines and a packaging signal for enabling the vector to replicate into single-stranded DNA;

ii) introducing a helper phage into the host, the helper phage comprising a nucleic acid encoding a phage structural protein; and

iii) culturing said host under conditions that result in packaging of said single stranded DNA by said structural protein to form and extrude recombinant phagemid particles from said prokaryotic host.

12. A method for producing recombinant phagemid particles from a prokaryotic host, the method comprising: -

i) The following were introduced into prokaryotic host cells: (a) a first vector configured to persist within the prokaryotic host and comprising at least one transgenic expression cassette comprising a nucleic acid sequence encoding one or more cytokines and a packaging signal for enabling the vector to replicate into single-stranded DNA; and (b) a second vector comprising a nucleic acid encoding a structural protein required for packaging the single-stranded DNA; and

ii) culturing said host under conditions that result in packaging of said single stranded DNA by said structural protein to form and extrude recombinant phagemid particles from said prokaryotic host.

13. A pharmaceutical composition comprising recombinant phagemid virus particles produced by the system of claim 10 or by the method of claim 11 or 12 and a pharmaceutically acceptable vehicle.

14. A process for preparing a pharmaceutical composition according to claim 13, the process comprising contacting a therapeutically effective amount of recombinant phagemid particles produced by the system according to claim 10 or by the process according to claim 11 or 12 with a pharmaceutically acceptable vehicle.

15. A recombinant phagemid particle for expressing a transgene in a target tumor cell transduced with the particle, wherein the phagemid particle comprises at least one transgene expression cassette comprising a nucleic acid sequence encoding one or more cytokines and comprises a genome that lacks at least 50% of its phage genome, and wherein the particle is configured to deliver the nucleic acid sequence at least adjacent to the tumor cell such that one or more cytokines are expressed when in use, and wherein the cytokine is any one of IL-4, IL-12, IL-15, TRAIL, IFN- γ, hybrid TNF α, or any combination thereof, optionally wherein the cytokine is IL-15.

16. The recombinant phagemid particle according to claim 15 for use in therapy or diagnosis.

17. a recombinant phagemid particle for expressing at least one antigen 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 that lacks at least 50% of its phage genome, and wherein the particle is configured, in use, to deliver nucleic acid sequences at least proximal to the target tumor cell such that the one or more cytokines are expressed.

18. The recombinant phagemid particle according to claim 17 for use in therapy or diagnosis.

19. The recombinant phagemid particle according to claim 17 for use in the treatment, prevention or amelioration of cancer.

20. Use of a helper phage comprising a nucleic acid encoding a viral vector structural protein for producing the recombinant phagemid particle of claim 17 from a prokaryotic host.

21. The recombinant phagemid virus particle according to claim 17, produced by the system of claim 10, produced by the method of claim 11 or 12 or produced for use of claim 20, wherein the recombinant phagemid particle is used to produce a recombinant viral vector comprising or derived from a viral genome within the genome of the phagemid particle, wherein the recombinant viral vector is used to deliver a nucleic acid sequence encoding one or more antigens to at least the vicinity of the tumor cell such that one or more cytokines are expressed.

22. A recombinant vector comprising a rAAV, rep-cap, adenohelper gene, and a nucleic acid sequence encoding one or more antigens or cytokines for use in treating, preventing, or ameliorating cancer.

23. A recombinant phagemid particle comprising the vector of claim 22 for use in a method for treating, preventing or ameliorating cancer.

24. Recombinant phagemid particle according to claim 15 or 17, or produced by the system according to claim 10, or produced by the method according to claim 11 or 12, or produced by the use according to claim 20 or 21, or for use according to any one of claims 1 to 9 and 22 to 23, or the pharmaceutical composition according to claim 13, or the method for preparing a pharmaceutical composition according to claim 14, wherein the phagemid particle is configured to deliver the transgenic expression cassette to the target tumor cell.

25. A particle according to claim 15, 17 or 24, or produced by a system according to claim 10, produced by a method according to claim 11 or 12, or produced according to the use of claim 20 or 21, or for use according to any one of claims 1 to 9 and 22 to 23, or a pharmaceutical composition according to claim 13, or a method for preparing a pharmaceutical composition according to claim 14, wherein the genome of the recombinant phagemid particle comprises a packaging signal for enabling replication of the phagemid genome into single-stranded DNA which can then be packaged into the phagemid particle in a prokaryotic host, optionally wherein the packaging signal comprises an origin of replication, optionally F1 ori.

26. A particle according to claim 15, 17, 24 or 25, or produced by a system according to claim 10, or produced by a method according to claim 11 or 12, or produced by a use according to claim 20 or 21, or for use according to any one of claims 1 to 9 and 22 to 23, or a pharmaceutical composition according to claim 13, or a method for the preparation of a pharmaceutical composition according to claim 14, wherein the genome of the recombinant phagemid particle comprises an origin of replication for enabling a double stranded vector to replicate in a prokaryotic host, optionally a pUC ori.

27. A particle according to any one of claims 15, 17, 24 to 26, or produced by the system of claim 10, or produced by the method of claim 11 or 12, or produced by the use of claim 20 or 21, or for use according to any one of claims 1 to 9 and 22 to 23, or the pharmaceutical composition of claim 13, or the method of preparing a pharmaceutical composition according to claim 14, wherein the genome of the recombinant phagemid particle comprises one or more DNA sequences that facilitate targeted integration into a host genome.

28. A particle according to any one of claims 15, 17, 24 to 27, or produced by a system according to claim 10, or produced by a method according to claim 11 or 12, or produced according to the use of claim 20 or 21, or for use according to any one of claims 1 to 9 and 22 to 23, or a pharmaceutical composition according to claim 13, or a method for preparing a pharmaceutical composition according to claim 14, wherein the at least one transgene expression cassette comprises a viral transgene expression cassette.

29. A particle according to any one of claims 15, 17, 24 to 28, or produced by the system of claim 10, or produced by the method of claim 11 or 12, or produced by the use of claim 20, or for use in the method of any one of claims 1 to 9 and 22 to 23, or the pharmaceutical composition of claim 13, or the method of claim 14 for preparing a pharmaceutical composition, comprising a plurality of transgene expression cassettes.

30. A particle according to any one of claims 15, 17, 24 to 29, or produced by a system according to claim 10, produced by a method according to claim 11 or 12, or produced according to the use of claim 20 or 21, or for use according to any one of claims 1 to 9 and 22 to 23, or a pharmaceutical composition according to claim 13, or a method for preparing a pharmaceutical composition according to claim 14, wherein the at least one transgene expression cassette comprises a mammalian viral transgene expression cassette.

31. A particle according to any one of claims 15, 17, 24 to 30, or produced by a system according to claim 10, or produced by a method according to claim 11 or 12, or produced according to the use of claim 20 or 21, or for use according to any one of claims 1 to 9 and 22 to 23, or a pharmaceutical composition according to claim 13, or a method for preparing a pharmaceutical composition according to claim 14, wherein the transgenic expression cassette comprises one or more functional elements required for expression of the nucleic acid in a target cell, the one or more functional elements being selected from the group consisting of: a promoter, a nucleic acid encoding a polyA tail capable of attaching to an expressed agent, and a left and/or right Inverted Terminal Repeat (ITR) or a left and/or right Long Terminal Repeat (LTR).

32. a particle according to any one of claims 15, 17, 24 to 31, or produced by a system according to claim 10, or produced by a method according to claim 11 or 12, or produced according to the use of claim 20 or 21, or for use according to any one of claims 1 to 9 and 22 to 23, or a pharmaceutical composition according to claim 13, or a method for preparing a pharmaceutical composition according to claim 14, wherein the at least one transgene expression cassette comprises a lentiviral transgene expression cassette.

33. The particle according to any one of claims 15, 17, 24 to 32, or produced by the system of claim 10, or produced by the method of claim 11 or 12, or produced by the use of claim 20 or 21, or for use in the pharmaceutical composition of any one of claims 1 to 9 and 22 to 23, or the pharmaceutical composition of claim 13, or the method for preparing a pharmaceutical composition of claim 14, wherein the at least one transgene expression cassette comprises an adeno-associated virus (AAV) transgene expression cassette.

34. The particle of any one of claims 15, 17, 24 to 33, or produced by the system of claim 10, or produced by the method of claim 11 or 12, or produced by the use of claim 20 or 21, for use in the pharmaceutical composition of any one of claims 1 to 9 and 22 to 23, or the pharmaceutical composition of claim 13, or the method for preparing a pharmaceutical composition of claim 14, wherein the transgenic expression cassette further comprises a nucleic acid encoding an agent having therapeutic or industrial utility in the target cell or tissue, optionally wherein the agent encoded by the nucleic acid is a polypeptide or a protein.

35. The particle of any one of claims 15, 17, 24 to 34, or produced by the system of claim 10, or produced by the method of claim 11 or 12, or produced by the use of claim 20 or 21, or for use in the pharmaceutical composition of any one of claims 1 to 9 and 22 to 23, or the pharmaceutical composition of claim 13, or the method for preparing a pharmaceutical composition of claim 14, wherein the recombinant phagemid particle comprises one or more capsid minor coat proteins, optionally wherein the recombinant phagemid particle comprises a pIII capsid minor coat protein configured to display a cellular ligand targeting for enabling the particle to be delivered to the target tumor cell.

36. Particles according to any one of claims 15, 17, 24 to 35, or produced by the system of claim 10, or produced by the method of claim 11 or 12, or produced by the use of claim 20 or 21, or for use according to any one of claims 1 to 9 and 22 to 23, or the pharmaceutical composition of claim 13, or the method for preparing a pharmaceutical composition of claim 14, wherein the recombinant phagemid particles comprise one or more capsid main coat proteins, optionally wherein the recombinant phagemid particles comprise at least one pVIII capsid main coat protein configured to display exogenous peptides thereon.

37. The particle of any one of claims 15, 17, 24 to 36, or produced by the system of claim 10, or produced by the method of claim 11 or 12, or produced by the use of claim 20 or 21, or for use in the pharmaceutical composition of any one of claims 1 to 9 and 22 to 23, or the pharmaceutical composition of claim 13, or the method for preparing a pharmaceutical composition of claim 14, wherein the recombinant phagemid particle is combined with a cationic polymer to form a complex having a net positive charge, optionally wherein the cationic polymer is selected from the group consisting of: chitosan; poly-D-lysine (PDL); diethylaminoethyl (DEAE); diethylaminoethyl-dextran (deae. dex); polyethyleneimine (PEI); coagulating the polyamine; protamine sulfate; 5 and a cationic lipid.

38. Particles according to any one of claims 15, 17, 24 to 37, or produced by a system according to claim 10, or produced by a method according to claim 11 or 12, or produced according to the use of claim 20 or 21, or for use according to any one of claims 1 to 9 and 22 to 23, or a pharmaceutical composition according to claim 13, or a process for preparing a pharmaceutical composition according to claim 14, wherein:

a. The genome of the recombinant phagemid particle lacks at least 60%, 70% or at least 80% of the phage genome from which it is derived;

b. the particle of any one of the preceding claims, wherein the genome of the recombinant phagemid particle lacks at least 90%, 95% or at least 99% of the phage genome from which it is derived; or

c. the particle of any one of the preceding claims, wherein the phagemid particle lacks in its genome a bacteriophage structural gene required to form, package or extrude the particle from a prokaryotic host, preferably a structural gene encoding the capsid protein.