Cancer treatment
The present invention provides phagemid vectors and associated phagemid particles for cancer treatment, 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 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 invention also extends to the use of phagemid particles and expression systems for the delivery of transgenes, and for the combination of such treatment with the use of adoptively transferred T cells, for the treatment, prevention, amelioration, or management of cancer.
1. A method of expressing one or more cytokines in a cell, the method comprising transducing the cell with a recombinant phagemid particle comprising M13 phage coat proteins and a phagemid particle genome that expresses a transgene expression cassette in the cell,
wherein the transgene expression cassette comprises a promoter and a nucleic acid sequence encoding the one or more cytokines, and wherein the genome comprises no more than 50% of a bacteriophage genome and encodes neither a pIII capsid minor coat protein nor a p VIII capsid major coat protein, and the one or more cytokines are expressed in the cell.
2. The method according to claim 1 , wherein the one or more cytokines are selected from IL-4, IL-12, IL-15, TNFα, TRAIL, IFN-γ.
3. The method according to claim 1 , wherein the one or more cytokines are one or more hybrid cytokines comprising a signal peptide that is not endogenous to the cytokine.
4. The method according to claim 3 , wherein the signal peptide is an IL-2 signal peptide.
5. The method according to claim 4 , wherein the one or more hybrid cytokines comprise a hybrid TNFα comprising a IL-2 signal peptide configured to precede the TNFα sequence and thereby increase expression and/or secretion of TNFα.
6. The method according to claim 5 , wherein the hybrid TNFα comprises an amino acid sequence substantially as set out in SEQ ID No: 22 or a fragment or variant thereof.
7. The method according to claim 5 , wherein the hybrid TNFα is encoded by a nucleic acid sequence comprising SEQ ID No: 23 or a fragment or variant thereof.
8. The method according to claim 1 , 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 subsequently be packaged into the phagemid particle inside a prokaryotic host.
9. The method according to claim 1 , wherein the genome of the recombinant phagemid particle comprises an origin of replication for enabling replication of double-stranded vector inside a prokaryotic host.
10. The method according to claim 1 , wherein the genome of the recombinant phagemid particle comprises one or more DNA sequences, which favours targeted integration into a host genome.
11. The method according to claim 1 , wherein the at least one transgene expression cassette comprises a viral transgene expression cassette, or wherein the particle comprises multiple transgene expression cassettes.
12. The method according to claim 1 , wherein the at least one transgene expression cassette comprises a mammalian viral transgene expression cassette.
13. The method according to claim 1 , wherein the transgene expression cassette comprises one or more functional elements required that expresses the nucleic acid in the target cell selected from the groups consisting of: a nucleic acid encoding a polyA tail operable linked to the one or more cytokine coding sequences, and either left and/or right Inverted Terminal Repeat sequences (ITRs) or left and/or right Long Terminal repeat sequences (LTRs).
14. The method according to claim 1 wherein the at least one transgene expression cassette comprises a lentivirus transgene expression cassette or an adeno-associated virus (AAV) transgene expression cassette.