Process for the production of a DNA vaccine for cancer immunotherapy

1. Method for producing a DNA vaccine for cancer immunotherapy comprising at least the following steps:

a) transforming an attenuated strain of Salmonella with at least one DNA molecule comprising at least one eukaryotic expression cassette encoding at least one antigen or at least one fragment thereof;

b) characterizing at least one transformed cell clone obtained in step (a);

c) selecting at least one of the transformed cell clone(s) characterized in step (b) and further characterizing said at least one selected transformed cell clone;

wherein step (b) comprises at least one of the following substeps:

bi) assessing the cell growth of at least one transformed cell clone obtained in step (a) over time;

bii) assessing the stability of the at least one DNA molecule comprising at least one expression cassette encoding at least one antigen or at least one fragment thereof in the at least one transformed cell clone obtained in step (a);

biii) isolating the at least one DNA molecule comprising at least one expression cassette encoding at least one antigen or at least one fragment thereof from at least one transformed cell clone obtained in step (a) and characterizing the at least one isolated DNA molecule by restriction analysis and/or sequencing;

biv) isolating the at least one DNA molecule comprising at least one expression cassette encoding at least one antigen or at least one fragment thereof from at least one transformed cell clone obtained in step (a), transfecting the at least one isolated DNA molecule into at least one eukaryotic cell and assessing the expression of the at least one antigen or the at least one fragment thereof in said at least one eukaryotic cell;

wherein step (c) comprises at least one of the following substeps:

ci) assessing the number of viable cells per ml cell suspension of the at least one transformed cell clone selected in step (c);

cii) assessing the stability of the at least one DNA molecule comprising at least one expression cassette encoding at least one antigen or at least one fragment thereof in the at least one transformed cell clone selected in step (c);

ciii) isolating the at least one DNA molecule comprising at least one expression cassette encoding at least one antigen or at least one fragment thereof from the at least one transformed cell clone selected in step (c) and characterizing the at least one isolated DNA molecule by restriction analysis and/or sequencing;

civ) isolating the at least one DNA molecule comprising at least one expression cassette encoding at least one antigen or at least one fragment thereof from the at least one transformed cell clone selected in step (c), transfecting the at least one isolated DNA molecule into at least one eukaryotic cell and assessing the expression of the at least one antigen or the at least one fragment thereof in said at least one eukaryotic cell;

cv) testing for the presence of bacterial, fungal and/or viral contaminants in at the least one transformed cell clone selected in step (c);

cvi) verifying the bacterial strain identity of the at least one transformed cell clone selected in step (c); and

wherein the attenuated strain of Salmonella is Salmonella typhi Ty21a.

2. The method of claim 1 , wherein said at least one antigen is selected from the group consisting of a tumor antigen and a tumor stroma antigen.

3. The method of claim 1 , wherein said at least one DNA molecule comprises the kanamycin antibiotic resistance gene, the pMB1 ori, and a eukaryotic expression cassette encoding said antigen under the control of a CMV promoter, wherein said DNA molecule is a DNA plasmid.

4. The method of claim 1 , wherein in step (a) said attenuated strain of Salmonella is transformed by electroporation with said at least one DNA molecule comprising at least one expression cassette encoding at least one antigen or at least one fragment thereof.

5. The method of claim 1 , wherein step (b) comprises one, two, three, or all four of said substeps (bi), (bii), (biii) and (biv).

6. The method of claim 1 , wherein step (c) comprises one, two, three, four, five, or all six of said substeps (ci), (cii), (ciii), (civ), (cv) and (cvi).

7. The method of claim 1 , wherein in step (cv) the presence of bacterial and/or fungal contaminants is tested by growing the at least one transformed cell clone selected in step (c) in or on at least one suitable selective medium.

8. The method of claim 1 , wherein in step (cvi) the bacterial strain identity is verified by growing the at least one transformed cell clone selected in step (c) on bromothymol blue galactose agar and/or on Kligler iron agar and/or by assessing the presence of Salmonella 05 and/or 09-surface antigen(s).

9. The method of claim 1 , wherein the at least one antigen is a tumor antigen and wherein the tumor antigen is a neoantigen.

10. The method of claim 1 , wherein the at least one antigen fragment comprises from 1 to 25 epitopes.

11. The method of claim 1 , wherein the at least one tumor antigen and/or tumor stroma antigen is a patient specific tumor antigen and/or tumor stroma antigen.

12. The method of claim 11 , wherein the at least one tumor antigen and/or stroma antigen was shown to be expressed by tumor cells or the tumor stroma cells of one specific patient.

13. The method of claim 12 , wherein the at least one patient specific tumor antigen and/or tumor stroma antigen was identified by assessing the expression profile of a patient's tumor and/or tumor stroma either on mRNA or on protein level or by assessing pre-existing T cell immune responses to tumor antigens and/or stroma cells of a patient.