Delivery of proteins into eukaryotic cells with recombinant Yersinia

An immunogenic composition, comprising a recombinant Yersinia enterocolitica MRS40 (pABL403), wherein said Yersinia enterocolitica is deficient in the production of any functional effector protein and is transformed with an expression vector which comprises, in the 5′ to 3′ direction:

• a promoter from a Yersinia virulon gene;
• a first DNA sequence encoding a delivery signal from a Yersinia effector protein, operably linked to said promoter;
• and
• a second DNA sequence coding for a heterologous protein, fused in frame to the 3′ end of said first DNA sequence, wherein said heterologous protein comprises at least one epitope of a tumor-associated protein.

A method of inducing a cytotoxic T-lymphocyte (CTL) response specific for a heterologous protein in a subject in need of such response, comprising the steps of:

• (a) obtaining from said subject an antigen-presenting cell expressing an MHC molecule;
• (b) forming a cell mixture by contacting said antigen-presenting cell with a recombinant Yersina, wherein said Yersinia comprises at least one mutation in at least one Yersinia effector-encoding gene;
• and said Yersinia is deficient in the production of at least one functional effector protein and is transformed with an expression vector, wherein said expression vector comprises in 5′ to 3′ direction:
• a promoter from a Yersinia virulon gene;
• a first DNA sequence encoding a delivery signal from a Yersinia effector protein, operably linked to said promoter;
• and
• a second DNA sequence fused in frame to the 3′ end of said first DNA sequence, wherein said second DNA sequence codes for at least one epitope of said heterologous protein which is presented by said MHC molecule of said antigen-presenting cell;
• (c) contacting a sample containing peripheral blood lymphocytes taken from said subject, with the cell mixture formed in step (b), thereby producing CTLs specific for said heterologous protein;
• and
• (d) administering CTLs produced in step (c) to said subject thereby inducing a CTL response specific for said heterologous protein in said subject.

A method for treating a tumor in a subject, comprising administering a recombinant Yersinia to said subject, wherein said Yersinia is deficient in the production of any functional effector protein and is transformed with an expression vector which comprises, in the 5′ to 3′ direction:

• a promoter from a Yersinia virulon gene;
• a first DNA sequence encoding a delivery signal from a Yersinia effector protein, operably linked to said promoter;
• and
• a second DNA sequence coding for a heterologous protein, fused in frame to the 3′ end of said first DNA sequence;
• and wherein said heterologous protein is a tumor-associated protein and elicits an immune response specific to said tumor.

A method for inducing in a subject a cytotoxic T-lymphocyte (CTL) response specific to a tumor-associated antigen, comprising administering a recombinant Yersinia to said subject, wherein said Yersinia comprises at least one mutation in at least one Yersinia effector-encoding gene;

• and said Yersinia is deficient in the production of at least one functional effector protein and is transformed with an expression vector which comprises, in the 5′ to 3′ direction:
• a promoter from a Yersinia virulon gene;
• a first DNA sequence encoding a delivery signal from a Yersinia effector protein, operably linked to said promoter;
• and
• a second DNA sequence coding for said tumor-associated antigen, fused in frame to the 3′ end of said first DNA sequence;
• and wherein said tumor-associated antigen elicits a CTL response specific to said tumor-associated antigen.