KR101227419B1 - Recombinant nucleic acid molecules, expression cassettes, and bacteria, and methods of use thereof - Google Patents

Abstract

The present invention provides recombinant nucleic acid molecules, expression cassettes, and vectors useful for the expression of polypeptides comprising heterologous polypeptides, such as bacteria. Certain recombinant nucleic acid molecules, expression cassettes and vectors include codon-optimized sequences that encode polypeptides and / or signal peptides. Certain recombinant nucleic acid molecules, expression cassettes, and expression vectors include sequences encoding non-Listeria and / or non-secA1 bacterial signal peptides for secretion of a polypeptide. The invention also provides compositions, such as bacteria comprising nucleic acid molecules, expression cassettes, and expression vectors, and vaccines comprising bacteria. Also provided are methods of making and using bacteria, recombinant nucleic acid molecules, and expression cassettes.

Recombinant nucleic acid molecules, expression cassettes, bacteria.

Description

Recombinant Nucleic Acid Molecules, Expression Cassettes, Bacteria, and Methods of Their Use {RECOMBINANT NUCLEIC ACID MOLECULES, EXPRESSION CASSETTES, AND BACTERIA, AND METHODS OF USE THEREOF}

Cross-Reference with the Related Application

This application requires 35 U.S.C. Under §119 (e), each disclosure claims the priority benefit of each of the following United States provisional applications, which are hereby incorporated by reference in their entirety: Thomas W. Dubensky, filed October 6, 2004 (document number 282173003923). US Provisional Serial No. 60 / 616,750 entitled "Bacterial Expression Cassettes, Bacterial Vaccine Compositions, and Methods of Use Thereof," by Jr., et al .; Thomas W. Dubensky, Jr., filed Oct. 1, 2004 (Document No. 282173003922). US Provisional Serial No. 60 / 615,287 entitled "Bacterial Expression Cassettes, Bacterial Vaccine Compositions, and Methods of Use Thereof," et al .; US Provisional Serial No. 60 / 599,377, filed August 5, 2004; US Provisional Application No. 60 / 541,515, filed March 26, 2004; US Provisional Serial No. 60 / 541,515, filed February 2, 2004; And US Provisional Application No. 60 / 532,598, filed December 24, 2003. In addition, there are some continuing applications for each of the following priority applications, each disclosure of which is incorporated herein by reference: International Application No. PCT / US2004 / 23881, filed July 23, 2004; US Patent Application Serial No. 10 / 883,599, filed June 30, 2004; US Patent Application Serial No. 10 / 773,618, filed February 6, 2004; And US Provisional Application No. 10 / 773,792, filed February 6, 2004.

A statement of federally sponsored research or development

The present invention was made, in part, with federal support under SBIR Accession No. 1 R43 CA101421-01 awarded by the US National Institutes of Health. The federal government may have certain rights in the invention.

The field of the invention generally relates to novel polynucleotides and expression cassettes useful for the expression of polypeptides, including heterologous polypeptides, in recombinant bacteria. In particular, the present invention relates to recombinant bacteria comprising novel expression cassettes and / or nucleic acid molecules useful in vaccine compositions.

Microbes have begun to be developed for use as vaccines to deliver heterologous antigens. Heterologous antigen delivery is provided by microorganisms modified to contain nucleic acid sequences encoding proteins or antigens originating from different species. Heterologous antigen delivery is particularly beneficial for the treatment or prevention of diseases or conditions resulting from particularly virulent or deadly causes, such as cancer and pathogens (eg HIV or Hepatitis B), where the infusion of the original infectious agent or cancer cell is a beneficiary. Administration of infectious agents or cells that are potentially harmful to living organisms has proved to be unsuccessful in eliciting an effective immune response, or in this case certainty is acceptable if the infectious agents or cancer cells are sufficiently supervised. Cannot be guaranteed. Recently, certain bacterial strains have been developed as recombinant vaccines. For example, an oral vaccine of directed Salmonella that has been modified to express the antigen of Plasmodium berghei spores has been shown to protect mice from malaria (Aggarwal et al. 1990. J. Exp. Med. 172: 1083).

Listeria monocytogenes (Listeria) are Gram-positive facultative intracellular bacteria that are antigen-specific due to their ability to first stimulate potent CD4 + / CD8 + T-cell mediated responses through MHC class I and class II antigen presenting pathways. It was developed for use in vaccines. See, for example, US Pat. Nos. 6,051,237, 6,565,852, and 5,830,702.

Listeria has been studied for years as a model for stimulating both innate and posterior T-cell-dependent antibacterial immunity. Listeria's ability to effectively stimulate cellular immunity is based on its intracellular life cycle. When infecting a host, the bacteria are rapidly absorbed into the phagolysosomal compartment by macrophages, including macrophages and dendritic cells (DCs). Then most of the bacteria are denatured. Peptides generated by pathogenic proteolytic degeneration in infected APCs accumulate directly on MHC class II molecules, and processed antibodies are expressed on the surface of antigen-presenting cells via class II endosomal pathways, and these MHC II- The peptide complex activates CD4 + “helper” T cells that stimulate antibody production. Within the acidic compartment, certain bacterial genes, including LLO, cholesterol-dependent cytolysis, are activated, which denatures pagolysosomes, releases bacteria into the cytosol compartment of the host cell, and the surviving Listeria propagates. Effective presentation of heterologous antigens via the MHC class I pathway requires new endogenous protein expression by Listeria. Proteins synthesized and secreted by Listeria in the cytoplasm of antigen presenting cells (APC) are sampled and then denatured with proteosomes. The resulting peptide is shuttled to the endoplasmic reticulum by the TAP protein and accumulated in MHC class I molecules. When the MHC I-peptide complex is delivered to the cell surface, it is combined with sufficient co-stimulation (signal 2) to activate and stimulate cytotoxic T lymphocytes (CTLs) with cognate T cell receptors, for example on tumor cells. The MHC I-peptide complexes shown are expanded and then recognized. Activated T cells in the appropriate microenvironment target and kill cancer cells.

Assuming a mechanism by which Listeria programs the presentation of heterologous antigens through the MHC class I pathway, the expression of heterologous genes and the efficacy of the secretion of newly synthesized proteins into the cytoplasm of infected (antigenic) cells from bacteria are both CD8 + T cells. It is directly related to the efficacy of initial stimulation and / or activation. Since the level of Ag-specific T cell superstimulation is directly related to vaccine efficacy, the efficacy of heterologous protein expression and secretion is directly related to vaccine efficacy.

Therefore, there is a need in the art for novel methods to optimize the efficacy of heterologous protein expression and secretion to maximize the efficacy of Listeria-based vaccines and other bacterial-based vaccines. It is also beneficial to optimize the efficacy of heterologous protein expression and secretion in bacterial host expression systems, with the expression and secretion of multiple heterologous proteins preferred.

A brief overview of the invention

The present invention generally provides novel polynucleotides comprising novel recombinant nucleic acid molecules, expression cassettes and vectors for use in expressing and / or secreting polypeptides (eg, heterologous polypeptides) in bacteria, in particular Listeria. . In some embodiments, these polynucleotides provide for increased expression and / or secretion of polypeptides in bacteria. The present invention also generally provides bacteria comprising recombinant nucleic acid molecules, expression cassettes or vectors, and pharmaceutical, immunogenic, and vaccine compositions comprising the bacteria. These bacteria and compositions are useful for the induction of immune responses and for the treatment and / or prevention of a wide range of diseases or other conditions, including cancer, infections and autoimmunity.

In one aspect, the invention encodes a polypeptide (eg, an antigen) that is codon-optimized for expression in bacteria, a first polynucleotide encoding a signal peptide, and in the same translation reading frame as the first polynucleotide. To provide a recombinant nucleic acid molecule comprising a second polynucleotide, wherein the recombinant nucleic acid molecule encodes a fusion protein comprising a signal peptide and a polypeptide. In some embodiments, the second polynucleotide is also codon-optimized for expression in bacteria such as Listeria. The present invention also provides an expression cassette comprising the recombinant nucleic acid molecule, further comprising a promoter operably linked to the recombinant nucleic acid molecule. Also provided are vectors and bacteria comprising recombinant nucleic acid molecules and / or expression cassettes, likewise pharmaceutical compositions, immunogenic compositions and vaccines comprising the bacteria. Also provided are methods for inducing an immune response in a host using a bacterium or a composition comprising a bacterium and / or preventing or treating a condition such as a disease.

In another aspect, the present invention provides a kit comprising: (a) a first polynucleotide encoding a bacterial natural signal peptide, a codon-optimized first polynucleotide for expression in bacteria, and (b) a second polynucleotide encoding a polypeptide. A recombinant nucleic acid molecule comprising a second polynucleotide in the same translation reading frame as the first polynucleotide, wherein the recombinant nucleic acid molecule encodes a fusion protein comprising a signal peptide and a polypeptide. In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous to the signal peptide. In some embodiments, the polypeptide encoded by the second polynucleotide is exogenous to bacteria. The present invention also provides an expression cassette comprising the recombinant nucleic acid molecule, further comprising a promoter operably linked to the recombinant nucleic acid molecule. Also provided are vectors and bacteria comprising recombinant nucleic acid molecules and / or expression cassettes, as well as pharmaceutical compositions, immunogenic compositions and vaccines comprising the bacteria. Also provided are methods of inducing an immune response in a host and / or preventing or treating a condition (eg, a disease) using a bacterium or a composition comprising bacteria.

In another aspect, the invention provides a recombinant Listeria bacterium comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule is (a) a first polynucleotide encoding a signal peptide, wherein the first polydon is codon-optimized for expression in Listeria. Nucleotide, (b) a second polynucleotide encoding a polypeptide, comprising a second polynucleotide in the same translation reading frame as the first polynucleotide, wherein the recombinant nucleic acid molecule encodes a fusion protein comprising a signal peptide and a polypeptide . In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous to the signal peptide. In some embodiments, the polypeptide is exogenous to Listeria bacteria. In some embodiments, the signal peptide is a natural signal peptide of Listeria. Also provided are pharmaceutical compositions, immunogenic compositions and vaccines comprising this Listeria. Also provided are methods for using this Listeria (or compositions comprising Listeria) to induce an immune response in a host and / or to prevent or treat a condition (eg, a disease).

In another aspect, the invention provides a translational reading of a first polynucleotide encoding a non-secA1 bacterial signal peptide, and a second polynucleotide encoding a polypeptide (eg, an antigen), wherein the second polynucleotide is identical to the first polypeptide. In a frame), wherein the recombinant nucleic acid molecule encodes a fusion protein comprising a signal peptide and a polypeptide. In some embodiments, the polypeptide is heterologous to the signal peptide. In some embodiments, the first and / or second polynucleotides are codon-optimized for expression in bacteria such as Listeria. The present invention also provides an expression cassette comprising the recombinant nucleic acid molecule, further comprising a promoter operably linked to the recombinant nucleic acid molecule. Also provided are vectors and bacteria comprising recombinant nucleic acid molecules and / or expression cassettes, as well as pharmaceutical compositions, immunogenic compositions and vaccines comprising the bacteria. Also provided are methods of inducing an immune response in a host using a bacterium or a composition comprising a bacterium and / or treating a condition such as a disease.

In another aspect, the invention provides a recombinant Listeria bacterium comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule is heterologous to (a) a first polynucleotide encoding a non-secA1 bacterial signal peptide, and (b) a signal peptide. A second polynucleotide encoding a polypeptide that is or is exogenous to bacteria, wherein the second polynucleotide is in the same translation reading frame as the first polynucleotide, and the recombinant nucleic acid molecule is a fusion protein comprising a signal peptide and a polypeptide. Coding In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous to the signal peptide, exogenous to the bacterium (ie, heterologous to bacteria), or both. Also provided are pharmaceutical compositions, immunogenic compositions and vaccines comprising this Listeria. Also provided are methods for using this Listeria (or compositions comprising Listeria) to induce an immune response in a host and / or to prevent or treat a condition (eg, a disease).

In another aspect, the invention provides a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising a polynucleotide encoding a polypeptide that is exogenous to Listeria (eg, a cancer or a non-Listeria infectious disease antigen), and comprises an exogenous polypeptide. The polynucleotide encoding is codon-optimized for expression in Listeria. In some embodiments, the recombinant nucleic acid molecule further comprises a polynucleotide encoding a signal peptide that is within the same translation reading frame as the polynucleotide encoding the exogenous polypeptide to Listeria. In some embodiments, the signal peptide is a natural signal peptide of Listeria bacteria. In another embodiment, the signal peptide is a signal peptide exogenous to Listeria bacteria. In some embodiments, polynucleotides encoding signal peptides are also codon-optimized for expression in Listeria. Also provided is a Listeria comprising this recombinant nucleic acid molecule. Also provided are pharmaceutical compositions, immunogenic compositions and vaccines comprising this Listeria. The present invention also provides a method of using the recombinant Listeria bacteria to induce an immune response in a host and / or to prevent or treat a condition (eg, but not limited to a disease).

In another aspect, the invention provides a recombinant Listeria bacterium comprising an expression cassette, wherein the expression cassette is a polynucleotide encoding a polypeptide that is exogenous to Listeria (eg, a cancer or a non-Listeria infectious disease antigen), and an exogenous polypeptide. And a promoter operably linked to a polynucleotide encoding A, wherein the polynucleotide encoding the exogenous polypeptide is codon-optimized for expression in Listeria. In some embodiments, the expression cassette encodes a signal peptide (natural signal peptide or exogenous signal peptide of Listeria bacteria) within the same translational reading frame as a polynucleotide encoding a polypeptide that is exogenous to Listeria and is operably linked to a promoter. It further comprises a nucleotide, whereby the expression cassette expresses a fusion protein comprising a signal peptide and an exogenous polypeptide. In some embodiments, polynucleotides encoding signal peptides are also codon-optimized for expression in Listeria. Also provided are pharmaceutical compositions, immunogenic compositions and vaccines comprising this Listeria. The present invention also provides a method of using the recombinant Listeria to induce an immune response in a host and / or to prevent or treat a condition (eg a disease).

In another aspect, the invention provides a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule is (a) a first polynucleotide encoding a non-Listeria signal peptide, and (b) a polypeptide in the same translation reading frame as the first polynucleotide. Wherein the recombinant nucleic acid molecule encodes a fusion protein comprising both a non-Listeria signal peptide and a polypeptide. The present invention also provides an expression cassette comprising the recombinant nucleic acid molecule, wherein the expression cassette further comprises a promoter operably linked to the first and second polynucleotides of the recombinant nucleic acid molecule. Also provided are vectors comprising recombinant nucleic acid molecules and / or expression cassettes. Also provided are Listeria bacteria comprising recombinant nucleic acid molecules and / or expression cassettes. Also provided are pharmaceutical compositions, immunogenic compositions and vaccines comprising the Listeria bacteria. Also provided are methods for using the Listeria bacteria (or compositions comprising Listeria bacteria) to induce an immune response in a host and / or to prevent or treat a condition (eg, a disease).

In a further aspect, the invention provides a recombinant Listeria bacterium comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule is identical to (a) a first polynucleotide encoding a non-Listeria signal peptide, and (b) a first polynucleotide. And a second polynucleotide encoding a polypeptide within the translation reading frame, wherein the recombinant nucleic acid molecule encodes a fusion protein comprising both the non-Listeria signal peptide and the polypeptide. Also provided are pharmaceutical compositions, immunogenic compositions and vaccines comprising this Listeria. Also provided are methods for using this Listeria to induce an immune response in a host and / or to prevent or treat a condition (eg, a disease).

In another aspect, the invention provides a polynucleotide encoding a non-Listeria signal peptide, a second polynucleotide encoding a polypeptide (eg, an antigen) that is within the same translational reading frame as the first polynucleotide, and a first polynucleotide. And an expression cassette comprising a promoter operably linked to both second polynucleotides (eg, from Listeria Monocytogenes species). The expression cassette encodes a fusion protein comprising both non-Listeria signal peptides and polypeptides. In some embodiments, the first and / or second polynucleotides are codon-optimized for expression in Listeria. Also provided are pharmaceutical compositions, immunogenic compositions and vaccines comprising this Listeria. The invention also provides a method of using the recombinant Listeria bacterium to induce an immune response in a host and / or to prevent or treat a condition (eg a disease).

In addition, the present invention provides a kit comprising: (a) a first polynucleotide encoding a bacterial autolysin, or a catalytically active fragment or catalytically active variant thereof, and (b) a second poly encoding a polypeptide. Providing a recombinant nucleic acid molecule comprising a nucleotide (the second polynucleotide is in the same translation reading frame as the first polynucleotide), wherein the recombinant nucleic acid molecule autolyzes, or catalyzes, the polypeptide encoded by the second polynucleotide. Encodes a protein chimera comprising an actively active fragment or a catalytically active variant, wherein the polypeptide in the protein chimera is fused to, or fused with, a catalytically active variant or autocatalytic fragment thereof It is located inside. Also provided are vectors and bacteria comprising recombinant nucleic acid molecules and / or expression cassettes, as well as pharmaceutical compositions, immunogenic compositions and vaccines comprising the bacteria. Also provided are methods of inducing an immune response in a host using a bacterium or a composition comprising a bacterium and / or treating a condition such as a disease.

In another aspect, the invention provides a recombinant nucleic acid molecule, which encodes at least two individual non-Listeria polypeptides. The present invention also provides an expression cassette comprising the recombinant nucleic acid molecule, further comprising a promoter operably linked to the recombinant nucleic acid molecule. Also provided are vectors comprising recombinant nucleic acid molecules and / or expression cassettes. Also provided are recombinant Listeria bacteria comprising recombinant nucleic acid molecules (and / or expression cassettes). Also provided are pharmaceutical compositions, immunogenic compositions and vaccines comprising this Listeria. Also provided are methods for using this Listeria (or compositions comprising Listeria) to induce an immune response in a host and / or to prevent or treat a condition (eg, a disease).

In a further aspect, the invention provides a recombinant Listeria bacterium comprising a polycistronic expression cassette, wherein the polycistronic expression cassette encodes at least two individual non-Listeria polypeptides. Also provided are pharmaceutical compositions, immunogenic compositions and vaccines comprising this Listeria. Also provided are methods for using this Listeria (or compositions comprising Listeria) to induce an immune response in a host and / or to prevent or treat a condition (eg, a disease).

In another aspect, the invention provides a second polynucleotide encoding (a) a first polynucleotide encoding a signal peptide, (b) a secreted protein, or a fragment thereof, wherein the second polynucleotide is identical to the first polynucleotide. (C) a third polynucleotide encoding a polypeptide that is heterologous to the secreted protein, or fragment thereof, wherein the third polynucleotide is in the same translation reading frame as the first and second polynucleotides. Wherein the recombinant nucleic acid molecule encodes a protein chimera comprising a signal peptide, a polypeptide encoded by a third polynucleotide, and a secreted protein or fragment thereof, and Polypeptides encoded by the fusion to proteins or fragments thereof secreted from the protein chimera It is located within the combined or secreted protein or fragment thereof. Also provided is an expression cassette comprising this recombinant nucleic acid molecule and further comprising a promoter operably linked to the first, second, and third polynucleotides of the recombinant nucleic acid molecule. Also provided are vectors and bacteria comprising recombinant nucleic acid molecules and / or expression cassettes, as well as pharmaceutical compositions, immunogenic compositions and vaccines comprising the bacteria. Also provided are methods of inducing an immune response in a host and / or preventing or treating a condition using a bacterium or a composition comprising the bacterium.

In some embodiments, a method of inducing an immune response to an antigen in a host comprises a recombinant bacterium described herein (eg, in any of the above aspects, or in the detailed description or examples of the invention below). Administering an effective amount of the composition to a host, wherein the polypeptide encoded by the recombinant nucleic acid molecule, expression cassette and / or vector in the bacteria comprises an antigen. In some embodiments, a method of preventing or treating a condition, such as a disease, in a host comprises administering to the host an effective amount of a composition comprising a recombinant bacterium described herein.

In addition, the present invention provides a recombinant bacterium described herein (eg, in any of the above embodiments, or in the following detailed description or examples) in the manufacture of a medicament for inducing an immune response against an antigen in a host. Polypeptides encoded by recombinant nucleic acid molecules, expression cassettes and / or vectors in bacteria include antigens. In some embodiments, the antigen is a heterologous antigen. The present invention also provides the use of the recombinant bacteria described herein in the manufacture of a medicament for preventing or treating a condition (eg, a disease such as cancer or an infectious disease) in a host. In addition, the present invention provides a recombinant bacterium described herein for use in inducing an immune response to an antigen in a host, wherein the polypeptide encoded by the recombinant nucleic acid molecule, expression cassette and / or vector in the bacterium comprises an antigen. do. In addition, the present invention provides a recombinant bacterium described herein for use in the prevention or treatment of a condition (eg, a disease) in a host.

In a further aspect, the present invention provides an improved method of expressing and secreting heterologous proteins in host bacteria.

Also provided are methods of producing bacteria comprising each recombinant nucleic acid molecule and expression cassette described above. Also provided is a method of producing a vaccine using this bacterium.

In addition, the present invention provides various polynucleotides that encode signal peptides and / or antigens, including codon-optimized polynucleotides for expression in Listeria monocytogenes .

1 shows hly promoter alignment for Listeria monocytogenes DP-L4056 (SEQ ID NO: 1) (bottom sequence) and EDG strain (SEQ ID NO: 2) (top sequence).

Figure 2 shows the sequence of a polynucleotide (SEQ ID NO: 3) encoding a fusion protein comprising an LLO signal peptide, an LLO PEST sequence, and a full length human EphA2 antigen.

Figure 3 shows the sequence of the fusion protein encoded by the polynucleotide shown in Figure 2 (SEQ ID NO: 4).

4 shows the native nucleotide sequence (SEQ ID NO: 5) encoding the human EphA2 extracellular domain (EX2).

5 shows the nucleotide sequence (SEQ ID NO: 6) encoding the codon-optimized human EphA2 extracellular domain for expression in Listeria monocytogenes .

6 shows the amino acid sequence of human EphA2 extracellular domain (EX2) (SEQ ID NO: 7).

7 shows a non-codon-optimized polynucleotide sequence (SEQ ID NO: 8) encoding a fusion protein comprising an LLO signal peptide, an LLO PEST sequence and an extracellular domain of human EphA2.

FIG. 8 shows the sequence of a fusion protein (SEQ ID NO: 9) encoded by the coding sequence shown in FIG. 7.

9 shows an expression cassette (SEQ ID NO: 10) encoding a fusion protein comprising a hly promoter and comprising an LLO signal peptide, an LLO PEST sequence and an extracellular domain of human EphA2. In this sequence, the sequence encoding the human EphA2 extracellular domain is codon-optimized for expression in Listeria monocytogenes .

FIG. 10 shows the amino acid sequence encoded by the expression cassette of FIG. 9 (SEQ ID NO: 11).

FIG. 11 shows an expression cassette (SEQ ID NO: 12) encoding a fusion protein comprising a hly promoter and comprising an LLO signal peptide, an LLO PEST sequence and an extracellular domain of human EphA2. In this sequence, the sequences encoding the LLO signal peptide, LLO PEST and the human EphA2 extracellular domain are all codon-optimized for expression in Listeria monocytogenes .

FIG. 12 shows the amino acid sequence encoded by the expression cassette of FIG. 11 (SEQ ID NO: 13).

FIG. 13 shows an expression cassette (SEQ ID NO: 14) encoding a fusion protein comprising a hly promoter and comprising a phoD Tat signal peptide and a human EphA2 extracellular domain. In this sequence, the sequences encoding the phoD Tat signal peptide and the human EphA2 extracellular domain are codon-optimized for expression in Listeria monocytogenes .

FIG. 14 shows amino acid sequence encoded by the expression cassette of FIG. 13 (SEQ ID NO: 15).

15 shows the natural nucleotide sequence (SEQ ID NO: 16) encoding the human EphA2 intracellular domain (CO).

FIG. 16 shows the nucleotide sequence (SEQ ID NO: 17) encoding the codon-optimized human EphA2 intracellular domain for expression in Listeria monocytogenes .

17 shows the amino acid sequence of the intracellular domain (EX2) of human EphA2 (SEQ ID NO: 18).

FIG. 18 shows a non-codon-optimized polynucleotide sequence (SEQ ID NO: 19) encoding a fusion protein comprising an LLO signal peptide, an LLO PEST sequence and a human EphA2 extracellular domain.

FIG. 19 shows the sequence of a fusion protein (SEQ ID NO: 20) encoded by the coding sequence shown in FIG. 18.

FIG. 20 shows an expression cassette (SEQ ID NO: 21) comprising a hly promoter and encoding a fusion protein comprising an LLO signal peptide, an LLO PEST sequence and a human EphA2 intracellular domain.

FIG. 21 shows amino acid sequence encoded by the expression cassette of FIG. 20 (SEQ ID NO: 22).

FIG. 22 shows an expression cassette (SEQ ID NO: 22) comprising a hly promoter and encoding a fusion protein comprising an LLO signal peptide, an LLO PEST sequence and a human EphA2 intracellular domain. In this sequence, the sequences encoding the LLO signal peptide, LLO PEST and human EphA2 intracellular domain are all codon-optimized for expression in Listeria monocytogenes .

FIG. 23 shows the amino acid sequence encoded by the expression cassette of FIG. 22 (SEQ ID NO: 24).

FIG. 24 shows an expression cassette (SEQ ID NO: 25) encoding a fusion protein comprising a hly promoter and comprising a phoD Tat signal peptide and a human EphA2 intracellular domain. In this sequence, a sequence encoding both the phoD Tat signal peptide and the human EphA2 intracellular domain is codon-optimized for expression in Listeria monocytogenes .

FIG. 25 shows amino acid sequence encoded by the expression cassette of FIG. 24 (SEQ ID NO: 27).

FIG. 26 shows a codon-optimized expression cassette (SEQ ID NO: 27) comprising a hly promoter and encoding a fusion protein comprising an LLO signal peptide and a NY-ESO-1 antigen. Both the signal peptide and the sequence encoding the antigen are codon-optimized for expression in Listeria monocytogenes .

FIG. 27 shows amino acid sequence encoded by the expression cassette of FIG. 26 (SEQ ID NO: 28).

FIG. 28 shows a polynucleotide (SEQ ID NO: 29) comprising a hly promoter operably linked to a codon-optimized sequence encoding a Usp45 signal peptide.

29 shows a polynucleotide (SEQ ID NO: 30) comprising a hly promoter operably linked to a native sequence encoding a p60 signal peptide.

30 shows a polynucleotide (SEQ ID NO: 31) comprising a hly promoter operably linked to a codon-optimized sequence encoding a p60 signal peptide.

Figure 31 shows the sequence of the hlyP-p60 gene fragment (SEQ ID NO: 32).

32 (including FIGS. 32A, 32B and 32C) shows the sequence of pAM401-MCS (SEQ ID NO: 33), which is a pAM401 plasmid containing multiple cloning sites (MCS) from the pPL2 vector.

33 shows the coding sequence (SEQ ID NO: 34) for codon-optimized human mesothelin for expression in Listeria monocytogenes .

34 shows the amino acid sequence of human mesothelin (SEQ ID NO: 35).

35 shows the coding sequence (SEQ ID NO: 36) for codon-optimized murine mesothelin for expression in Listeria monocytogenes .

36 shows the amino acid sequence of murine mesothelin (SEQ ID NO: 37).

37 shows Western blot analysis of proteins secreted from recombinant Listeria encoding native EphA2 CO domain sequences.

38 shows Western blot analysis of proteins secreted from recombinant Listeria encoding natural or codon-optimized LLO secA1 signal peptide fused with a codon-optimized EphA2 EX2 domain sequence.

FIG. 39 shows Western blot analysis of proteins secreted from recombinant Listeria encoding a natural or codon-optimized LLO secA1 signal peptide or a codon-optimized Tat signal peptide fused with a codon-optimized EphA2 CO domain sequence.

FIG. 40 shows Western blot analysis of 293 cell lysates 48 hours after transfection with pCDNA4 plasmid DNA encoding full length native EphA2 sequence.

Figure 41 is a graph showing that long-term survival is provided when immunizing Balb / C mice with CT26.24 (huEphA2 +) lung tumors with recombinant Listeria encoding OVA.AH1 or OVA.AH1-A5.

42 shows increased survival of Balb / C mice with CT26.24 (huEphA2 +) lung tumors when immunized with recombinant Listeria encoding a codon-optimized secA1 signal peptide fused with a codon-optimized EphA2 EX2 domain sequence It is a graph indicating that.

Figure 43 is a graph showing that long-term survival is provided by immunizing Balb / C mice with CT26.24 (huEphA2 +) lung tumors with recombinant Listeria encoding the EphA2 CO domain.

Figure 44 is a graph showing that long-term survival is provided when immunizing Balb / C mice carrying CT26.24 (huEphA2 +) lung tumors with recombinant Listeria encoding the EphA2 CO domain, but not the plasmid DNA encoding full length EphA2. .

45 is a graph showing that Listeria expressing hEphA2 induces EphA2-specific CD8 + T cell responses.

46 is a graph showing that both CD4 + and CD8 + T cell responses are beneficial for hEphA2-designated antitumor efficacy of hEphA2-expressing Listeria.

47 is a codon for secretion in Listeria monocytogenes - sequence of the Listeria monocytogenes strain 10403S hly promoter in the protective antigen signal peptide from B. anthracis optimized operably linked to: show (SEQ ID NO 38). Six additional nucleotides (5'-GGATCC-3 ') corresponding to the BamHI restriction enzyme recognition site are included at the carboxy terminus of the signal peptide sequence, facilitating in-frame binding with any selected coding sequence. The 5 'end of the hly promoter contains a unique KpnI restriction enzyme recognition site.

48 shows effective expression / secretion of full length human tumor antigens from recombinant Listeria. 48A shows mesothelin expression / secretion by constructs consisting of LLO signal peptide fused with human mesothelin using natural codons. 48B shows mesothelin expression / secretion by constructs comprising various signal peptides fused with codon-optimized human mesothelin for expression in Listeria. 48C shows NY-EOS-1 expression / secretion by constructs comprising a codon-optimized LLO signal peptide fused with human mesothelin codon-optimized NY-ESO-1.

49 shows the coding sequence of phEphA2KD (SEQ ID NO: 39).

50 shows the Mlu I subfragment (SEQ ID NO: 40) of codon-optimized human EphA2 containing the actA-plcB intergenic region.

51 shows the sequence of a hly promoter-70 N-terminal p60 amino acid (SEQ ID NO: 41).

52 shows the KpnI-BamHI subfragment (SEQ ID NO: 42) of the plasmid pPL2-hlyP-Np60 CodOp (1-77).

FIG. 53 shows KpnI-BamHI subfragment (SEQ ID NO: 43) of plasmid pPL2-hlyP-Np60 CodOp (1-77) -mesothelin.

FIG. 54 shows Kpn I-BamHI subfragment (SEQ ID NO: 44) of plasmid pPL2-hlyP-Np60 CodOp (1-77) -mesothelin ΔSP / ΔGPI.

FIG. 55 shows Western blot analysis for expression and secretion of antigens from recombinant Listeria including antigen-bacterial protein chimeras.

56 shows Western blot analysis for expression of the intracellular domain (ICD) of EphA2 from bicistron messages.

FIG. 57 is a function of N-terminal fusion with various codon-optimized signal peptides, as demonstrated in different bacterial fractions, secreted protein (FIG. 57A), cell wall (FIG. 57B), and cell lysate (FIG. 57C). Western blot analysis of plasmid-based expression and secretion of murine mesothelin as shown.

58 shows Western blot analysis of chromosome-based expression and secretion of human mesothelin in Listeria monocytogenes . Western blot analysis of mesothelin expression for various bacterial cell fractions is shown along with results from control Listeria and Listeria encoding mesothelin expressed from the indicated signal sequence.

59A and B are graphs showing delivery of heterologous antigen (AH1-A5) to the MHC class I pathway by Listeria vaccine. The Listeria vaccine consists of Listeria or LLO signal peptide expressing the p60-AH1-A5 protein chimera (AH1-A5 embedded in p60) (FIG. 59A) and Listeria expressing a fusion protein comprising AH1-A5 (FIG. 59B). lost.

60A and B are graphs showing Listeria vaccine-mediated delivery of bacteria-specific antigens to the MHC class I pathway, where the vaccine expresses Listeria (AH1-A5 embedded in p60) expressing the p60-AH1-A5 protein chimera. 60A) or Listeria expressing a fusion protein comprising LLO signal peptide and AH1-A5 (FIG. 60B), wherein the test peptide added to the cell-based assay was a non-test peptide (non-irritating) (FIG. 60A), LLO _91-99 (FIG. 60A), non test peptide (FIG. 60B), or p60 _217-225 (FIG. 60B).

FIG. 61 is a graph showing the therapeutic efficacy of Listeria expressing human mesothelin in vaccinated tumor-bearing animals, where tumor cells were engineered to express human mesothelin.

FIG. 62 is a graph showing reduction of lung tumor nodule levels in tumor-bearing mice, where tumor cells were engineered to express human mesothelin.

FIG. 63 is a graph depicting a control study using CT.26 parent target cells, ie cells not engineered to express human mesothelin, demonstrating that the antitumor efficacy of Lm-Meso vaccination is mesothelin specific.

64 is a graph showing that vaccination with Listeria expressing codon-optimized human mesothelin reduces tumor volume.

65 shows the results of an ELISPOT experiment showing the immunogenicity of Listeria ΔactA / ΔinlB- human mesothelin strains in which nucleic acid encoding human mesothelin was integrated into the Listeria genome.

1. Introduction

The present invention provides a variety of recombinant nucleic acid molecules, expression cassettes, and expression vectors useful for the expression and / or secretion of polypeptides, including heterologous polypeptides (eg, antigens and / or mammalian proteins) in bacteria such as Listeria. Provided are polynucleotides. In some embodiments, these polynucleotides may be used for increased expression and / or secretion of polypeptides in bacteria. Some expression cassettes contain codon-optimized coding sequences for polypeptides and / or signal peptides. In addition, some expression cassettes for use in bacteria contain signal peptide sequences derived from other bacterial sources and / or various different secretion pathways. In addition, a bacterium comprising an expression cassette is provided as a composition, such as a vaccine containing the bacterium. Also provided are methods for using the polynucleotides, bacteria and compositions to induce an immune response and / or to prevent or treat a condition such as a disease in a host (eg cancer).

Codon-optimization of signal peptide sequences in expression cassettes enhances expression and / or secretion of heterologous polypeptides (such as antigens) from recombinant bacteria (especially in combination with codon-optimization of heterologous polypeptides), and signal peptide sequences It is based in part on the discovery that even when it is the natural signal peptide of this bacterium (see eg Examples 19 and 27 below). In addition, signal peptide sequences from non-secA1 secretory pathways and / or signal peptide sequences from non-Listeria bacterial sources can also be used to influence the effective expression and / or secretion of heterologous polypeptides from Listeria (eg, See, Examples 19, 27, and 30, below). In addition, the present invention is based in part on the further finding that codon-optimization of coding sequences of heterologous polypeptides enhances expression and / or secretion of heterologous polypeptides in Listeria (see, eg, Example 19 below). In addition, improved expression and / or secretion of heterologous proteins obtained through optimization of expression cassettes has been shown to lead to improved immunogenicity of bacteria including optimized expression cassettes (see, eg, Example 20 below). In addition, expression cassettes encoding protein chimeras comprising heterologous antigens embedded in autolysis have been shown to be useful for influencing the effective expression and secretion of heterologous antigens in Listeria (see, eg, Example 29 below). In addition, self-dissolving protein chimeras have been shown to be immunogenic (see, eg, Example 31A below). In addition, expression cassettes comprising Listeria and / or non-Listeria signal peptides, including codon-optimized expression cassettes, have been shown to be immunogenic, reduce tumor volume, and increase survival in mouse models (eg, , See Examples 31B-E below).

Thus, in one aspect, the present invention provides a polynucleotide comprising a first polynucleotide encoding a signal peptide (the first polynucleotide is codon-optimized for expression in bacteria), and a second poly encoding a polypeptide (eg, an antigen). A recombinant nucleic acid molecule is provided that includes nucleotides (the second polynucleotide is in the same translation reading frame as the first polynucleotide), wherein the recombinant nucleic acid molecule encodes a fusion protein comprising a signal peptide and a polypeptide. In some embodiments, the second polynucleotide is also codon-optimized (typically for expression in bacteria of the same type as the first polynucleotide). In some embodiments, the first polynucleotide or the first and second polynucleotides are codon-optimized for expression in Listeria, Bacillus, Yersinia pestis, Salmonella, Shigella, Brucella, Mycobacteria or E. coli . In some embodiments, the polynucleotides are codon-optimized for expression in Listeria, such as Listeria monocytogenes . In some embodiments, the polypeptide encoded by the second polynucleotide is (or comprises) an antigen, and in some instances may be a non-bacterial antigen. For example, the antigen is in some embodiments a tumor-associated antigen or derived from such as a tumor-associated antigen. For example, in some embodiments, the antigen is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP , Proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, or CEA, or K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY -ESO-1, WT-1, survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, or CEA. For example, in some embodiments, the antigen is mesothelin or an antigen fragment or antigen variant of mesothelin. In some other embodiments, the antigen is NY-ESO-1, or an antigen fragment or antigen variant of NY-ESO-1. In some embodiments, the antigen is an infectious disease antigen or is derived from an infectious disease antigen. In some embodiments, the signal peptide is a bacterium (Listeria or non-Listeria). In some embodiments, the signal peptide encoded by the codon-optimized first polynucleotide is a bacterial natural signal peptide. In another embodiment, the signal peptide encoded by the codon-optimized first polynucleotide is exogenous to bacteria. In some embodiments, the signal peptide is a secA1 signal peptide, such as LLO signal peptide from Listeria monocytogenes , a Usp45 signal peptide from Lactococus lactis , or a protective antigen signal peptide from Bacillus anthracis . In some embodiments, the signal peptide is a secA2 signal peptide. For example, the signal peptide can be a p60 signal peptide from Listeria monocytogenes . In addition, the recombinant nucleic acid molecule optionally comprises a third polynucleotide sequence, or a fragment thereof, that encodes p60 within the same translational reading frame as the first and second polynucleotides, wherein the second polynucleotide is within or Located between the first and third polynucleotides. In another embodiment, the signal peptide is a Tat signal peptide, such as a B. subtilis Tat signal peptide (eg, PhoD). The invention also provides an expression cassette comprising a recombinant nucleic acid molecule and further comprising a promoter operably linked to the recombinant nucleic acid molecule (eg, the first and second polynucleotides (and, if present, the third polynucleotide). In addition, expression vectors comprising an expression cassette and recombinant bacteria (eg Listeria) are provided as pharmaceutical compositions, immunogenic compositions, and vaccines comprising the bacteria. A method of inducing an immune response and / or preventing or treating a condition, such as a disease, is provided, and the use of bacteria in the manufacture of a medicament for inducing an immune response to an antigen in a host is provided. Polypeptides encoded by polynucleotides include antigens.

In a second aspect, the invention provides a kit comprising: (a) a first polynucleotide encoding a bacterial natural signal peptide (the first polynucleotide is codon-optimized for expression in bacteria), and (b) a second encoding a polypeptide. A recombinant nucleic acid molecule is provided that includes a polynucleotide (a second polynucleotide is in the same translation reading frame as the first polynucleotide), wherein the recombinant nucleic acid molecule encodes a fusion protein comprising a signal peptide and a polypeptide. In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous to the signal peptide. In some embodiments, the second polynucleotide is heterologous to the first polynucleotide. In some embodiments, the polypeptide is exogenous to bacteria in which the signal peptide is a natural signal peptide. In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous to the signal peptide, exogenous to the bacteria, or both. In some embodiments, the bacteria from which the signal peptide is derived are intracellular bacteria. In some embodiments, the bacteria are selected from the group consisting of Listeria, Bacillus, Yersinia pestis, Salmonella, Shigella, Brucella, Mycobacteria and E. Coli . In some embodiments, the bacteria are Listeria bacteria (eg, Listeria monocytogenes ). In some embodiments, the second polynucleotide is codon-optimized for expression in bacteria. In some embodiments, codon-optimization of the first and / or second polynucleotides enhances expression and / or secretion in bacteria of the encoded fusion protein (relative to non-codon-optimized sequences). In some embodiments, the polypeptide encoded by the second polynucleotide comprises an antigen. The polypeptide encoded by the second polynucleotide is an antigen. In some embodiments, the antigen is a non-bacterial antigen. In some embodiments, the antigen is a tumor-associated antigen or comprises an antigen derived from a tumor-associated antigen. In some embodiments, the antigen is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protein Third, SPAS-1, SP-17, PAGE-4, TARP, or CEA, or K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, or CEA. For example, in some embodiments, the antigen is mesothelin, or an antigen fragment or variant thereof, or NY-ESO-1, or an antigen fragment or variant thereof. In some other embodiments, the antigen is an infectious disease antigen or is derived from an infectious disease antigen. In some embodiments, the signal peptide is a secA1 signal peptide (eg, LLO signal peptide from Listeria monocytogenes ). Also provided is an expression cassette comprising a promoter comprising a recombinant nucleic acid molecule and further comprising a promoter operably linked to the first and second polynucleotides of the recombinant nucleic acid molecule. Also provided are expression vectors comprising an expression cassette. Also provided is a recombinant bacterium comprising the recombinant nucleic acid molecule, wherein the first polynucleotide is codon-optimized for expression in the recombinant bacterium. In some embodiments, the recombinant bacteria are intracellular bacteria. In some embodiments, the recombinant bacterium is selected from the group consisting of Listeria, Bacillus, Yersinia pestis, Salmonella, Shigella, Brucella, Mycobacteria and E. Coli . In some embodiments, the bacteria are recombinant Listeria bacteria (eg, recombinant Listeria monocytogenes bacteria). In addition, immunogenic compositions are provided comprising recombinant bacteria, and the polypeptide encoded by the second polynucleotide is an antigen. Also provided are methods of inducing an immune response to an antigen in a host comprising administering to the host an effective amount of a composition comprising a recombinant bacterium, wherein the polypeptide encoded by the second polynucleotide is an antigen (or comprises an antigen). do). Also provided is the use of bacteria in the manufacture of a medicament for inducing an immune response to an antigen in a host, wherein the polypeptide encoded by the second polynucleotide comprises an antigen.

In a third aspect, the present invention provides a recombinant Listeria bacterium (eg, Listeria monocytogenes ) comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule comprises (a) a first polynucleotide (a first polynucleotide encoding a signal peptide). Is codon-optimized for expression in Listeria bacteria), and (b) a second polynucleotide encoding the polypeptide (the second polynucleotide is in the same translation reading frame as the first polynucleotide), and the recombinant nucleic acid molecule Encodes a fusion protein comprising a signal peptide and a polypeptide. In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous to the signal peptide. In some embodiments, the recombinant nucleic acid molecule is part of an expression cassette further comprising a promoter operably linked to both the first and second polynucleotides. That is, in some embodiments, the recombinant Listeria bacterium comprises an expression cassette comprising the recombinant nucleic acid molecule, and the expression cassette further comprises a promoter operably linked to both the first and second polynucleotides of the recombinant nucleic acid molecule. In some embodiments, the expression cassette is a polycistronic expression cassette. In some embodiments, the second polynucleotide is codon-optimized for expression in Listeria bacteria. In some embodiments, codon-optimization of the first and / or second polynucleotides enhances expression and / or secretion in Listeria bacteria of the encoded fusion protein (relative to non-codon-optimized sequences). In some embodiments, the polypeptide encoded by the second polynucleotide is exogenous to Listeria bacteria (ie, heterologous to Listeria bacteria). In some embodiments, the polypeptide encoded by the second polynucleotide comprises an antigen (eg, a non-Listeria or non-bacterial antigen). In some embodiments, the polypeptide encoded by the second polynucleotide is an antigen. In some embodiments, the antigen is a tumor-associated antigen or is derived from a tumor-associated antigen. In some embodiments, the antigen is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protein Third, SPAS-1, SP-17, PAGE-4, TARP, and CEA, or K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, and CEA. For example, in some embodiments, the antigen is mesothelin, or an antigen fragment or antigen variant thereof. In some embodiments, the antigen is human mesothelin. In some embodiments, the antigen is human mesothelin with the signal peptide and GPI linker domain removed. In some other embodiments, the antigen is NY-ESO-1, or an antigen fragment or antigen variant thereof. In some other embodiments, the antigen is an infectious disease antigen or an antigen derived from an infectious disease antigen. In some embodiments, the signal peptide is a non-Listeria signal peptide. In some embodiments, the signal peptide is a bacterial signal peptide. In some embodiments, the signal peptide is exogenous to Listeria bacteria. In another embodiment, the signal peptide is a natural signal peptide of Listeria bacteria. In some embodiments, the signal peptide is a secA1 signal peptide (eg, LLO signal peptide from Listeria monocytogenes , Usp45 signal peptide from Lactococcus lactis , and protective antigen signal peptide from Bacillus abthracis ). In some embodiments, the signal peptide is a secA2 signal peptide (eg, a p60 signal peptide from Listeria monocytogenes ). In some embodiments, the signal peptide is a Tat signal peptide (eg, PhoD signal peptide from B. subtilis ). In some embodiments, Listeria bacteria are attenuated. For example, Listeria may be attenuated for intercellular propagation, infiltration or proliferation into non-phagocytic cells. In some embodiments, the recombinant Listeria bacterium has a defect with respect to ActA, internalin B, or both ActA and internalin B (eg, ΔactAΔinlB double deletion mutant). In some embodiments, the recombinant Listeria bacterium is deleted in functional ActA, or internalin B, or both ActA and internalin B. In some embodiments, the nucleic acid of a recombinant bacterium has been modified by reaction with a nucleic acid targeting compound (eg, psoralen compound). The present invention also provides pharmaceutical compositions comprising recombinant Listeria bacteria and pharmaceutically acceptable carriers, and immunogenic compositions comprising recombinant Listeria bacteria, wherein the polypeptide encoded by the second polynucleotide is an antigen. The present invention also provides a vaccine comprising the recombinant Listeria bacteria. Also provided is a method of inducing an immune response in a host comprising administering to the host an effective amount of a composition comprising a recombinant bacterium, wherein the polypeptide encoded by the second polynucleotide is an antigen (or comprises an antigen). Also provided are methods for preventing or treating a condition (a disease such as cancer or an infectious disease) in a host comprising administering to the host an effective amount of a composition comprising recombinant Listeria bacteria. Also provided is the use of bacteria in the manufacture of a medicament for inducing an immune response to an antigen in a host, wherein the polypeptide encoded by the second polynucleotide comprises an antigen.

In a fourth aspect, the invention provides a recombinant nucleic acid molecule comprising a first polynucleotide encoding a non-secA1 bacterial signal peptide, and a second polynucleotide encoding a polypeptide, wherein the second polynucleotide is a first polynucleotide. Within the same translation reading frame as, the recombinant nucleic acid molecule encodes a fusion protein comprising a signal peptide and a polypeptide. In some embodiments, the first polynucleotide and / or the second polynucleotide are codon-optimized for expression in certain types of bacteria. In some embodiments, codon-optimization of the first and / or second polynucleotides enhances expression and / or secretion in bacteria of fusion proteins (relative to non-codon-optimized sequences). In some embodiments, the first polynucleotide and / or second polynucleotide are codon-optimized for expression in Listeria, Bacillus, Yersiniapestis, Salmonella, Shigella, Brucella, Mycobacteria or E. coli . In some embodiments, the polynucleotide (s) are codon-optimized for expression in Listeria, such as Listeria monocytogenes . In some embodiments, the signal peptide encoded by the codon-optimized first polynucleotide is a natural signal peptide of the codon-optimized bacteria. In some embodiments, the first polynucleotide encoding the signal peptide is heterologous to the second polynucleotide. In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous to the signal peptide. In some embodiments, the polypeptide encoded by the second polynucleotide comprises an antigen. In some embodiments, the polypeptide encoded by the second polynucleotide is an antigen and in some cases may be a non-bacterial antigen. In some embodiments, the antigen is a tumor-associated antigen or is derived from a tumor-associated antigen. For example, in some embodiments the antigen is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, Proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, or CEA, or K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY- Derived from ESO-1, WT-1, survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, or CEA. For example, in some embodiments, the antigen is mesothelin or an antigen fragment or antigen variant of mesothelin. In some other embodiments, the antigen is NY-ESO-1, or an antigen fragment or antigen variant of NY-ESO-1. In some embodiments, the antigen is an infectious disease antigen or is derived from an infectious disease antigen. In some embodiments, the signal peptide encoded by the first polynucleotide of the recombinant nucleic acid molecule is a Listeria signal peptide. In other embodiments, the signal peptide is a non-Listeria signal peptide. In some embodiments, the signal peptide is derived from Gram-positive bacteria. In some embodiments, the signal peptide is derived from bacteria belonging to the genus Bacillus, Lactococcus or Straphylococcus . In some embodiments, the signal peptide is a secA2 signal peptide. For example, the signal peptide can be a p60 signal peptide from Listeria mono-cytogenes . In addition, the recombinant nucleic acid molecule optionally comprises a third polynucleotide, or a fragment thereof, that encodes p60 within the same translational reading frame as the first and second polynucleotides, wherein the second polynucleotide is internal to or within the third polynucleotide. Located between the first and third polynucleotides. In another further embodiment, the signal peptide is a Tat signal peptide, such as a B. subtilis Tat signal peptide (eg, a B. subtilis PhoD signal peptide). The present invention also provides an expression cassette comprising a recombinant nucleic acid molecule and further comprising a promoter operably linked to the first and second polynucleotides of the recombinant nucleic acid molecule. In addition, expression vectors and bacteria comprising expression cassettes and / or recombinant nucleic acids are provided as pharmaceutical compositions, immunogenic compositions and vaccines comprising bacteria. In some embodiments, the recombinant bacterium comprising an expression cassette or recombinant nucleic acid molecule is an intracellular bacterium. In some embodiments, the bacterium is a bacterium selected from the group consisting of Listeria, Bacillus, Yersinia pestis, Salmonella, Shigella, Brucella , Mycobacteria or E. Coli . In some embodiments, the bacteria are Listeria bacteria (eg, members of Listeria monocytogenes species). In some embodiments, the polypeptide encoded by the second polynucleotide is exogenous to the bacterium (eg, heterologous to the bacterium). Also provided are methods for using bacteria to induce an immune response in a host and / or to prevent or treat a condition (eg, a disease). In some embodiments, the condition is cancer. In other embodiments, the condition is an infectious disease. Also provided is the use of bacteria in the manufacture of a medicament for inducing an immune response to an antigen in a host, wherein the polypeptide encoded by the second polynucleotide comprises an antigen.

In another aspect, the invention provides a recombinant Listeria bacterium comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule comprises (a) a first polynucleotide encoding a non-secA1 bacterial signal peptide, and (b) a polypeptide encoding a polypeptide. 2 polynucleotides (the second polynucleotide is in the same translation reading frame as the first polynucleotide) and the recombinant nucleic acid molecule encodes a fusion protein comprising a signal peptide and a polypeptide. In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous to the signal peptide, exogenous to the bacteria, or both. In some embodiments, the Listeria bacteria belong to the Listeria monocytogenes species. In some embodiments, the recombinant nucleic acid molecule is part of an expression cassette further comprising a promoter operably linked to both the first and second polynucleotides. That is, in some embodiments, the recombinant Listeria bacterium comprises an expression cassette comprising the recombinant nucleic acid molecule, and the expression cassette further comprises a promoter operably linked to both the first and second polynucleotides of the recombinant nucleic acid molecule. In some embodiments, the expression cassette is a polycistronic expression cassette. In some embodiments, the first polynucleotide, second polynucleotide, or first and second polynucleotides are codon-optimized for expression in Listeria bacteria (eg, Listeria monocytogenes ). In some embodiments, codon-optimization of the first and / or second polynucleotides enhances expression and / or secretion in bacteria of fusion proteins (relative to non-codon-optimized sequences). In some embodiments, the first and second polynucleotides are heterologous to one another. In some embodiments, the polypeptide and signal peptide encoded by the second polynucleotide are heterologous to one another. In some embodiments, the polypeptide encoded by the second polynucleotide is exogenous to Listeria bacteria (eg, heterologous to Listeria bacteria). In some embodiments, the polypeptide encoded by the second polynucleotide comprises an antigen. In some embodiments, the polypeptide encoded by the second polynucleotide is an antigen (eg, a non-Listeria or non-bacterial antigen). In some embodiments, the antigen is a tumor-associated antigen or is derived from a tumor-associated antigen. In some embodiments, the antigen is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protein Third, SPAS-1, SP-17, PAGE-4, TARP, and CEA, or K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, and CEA. For example, in some embodiments, the antigen is mesothelin or an antigen fragment or antigen variant thereof. In some embodiments, the antigen is human mesothelin. In some embodiments, the antigen is human mesothelin lacking a signal peptide and a GPI linker domain. In some other embodiments, the antigen is an infectious disease antigen or is derived from an infectious disease antigen. In some embodiments, the signal peptide is a non- Listeria signal peptide. In some embodiments, the non-secA1 bacterial signal peptide is a Listeria signal peptide. In another embodiment, the non-secA1 bacterial signal peptide is a non- Listeri a signal peptide. In some embodiments, the signal peptide is a secA2 signal peptide (eg, a p60 signal peptide from Listeria monocytogenes ). In some embodiments, a recombinant nucleic acid molecule comprising a secA2 signal peptide is a secA2 autosolubilizer (eg, p60 or N-acetylmuramidase), or fragments thereof, within the same translational reading frame as the first and second polynucleotides Further comprising a third polynucleotide encoding (eg, a catalytically active fragment), wherein the second polynucleotide is located within the third polynucleotide of the recombinant nucleic acid molecule or between the first and third polynucleotides. In some embodiments, the second polynucleotide is located inside the third polynucleotide. In some embodiments, the signal peptide is a Tat signal peptide. In some embodiments, the signal peptide is a B. subtilus signal peptide derived from a Tat signal peptide (eg, a PhoD signal peptide from B. subtilus ). In some embodiments, Listeria bacteria are attenuated. For example, Listeria is attenuated for intercellular propagation, penetration into non-phagocytic cells, or proliferation. In some embodiments, the recombinant Listeria bacterium has a defect with respect to functional ActA, or internalin B, or both ActA and internalin B. In some embodiments, the recombinant Listeria bacterium is deleted in functional ActA, or internalin B, or both ActA and internalin B (eg, an ΔactAΔinlB double deletion mutation). In some embodiments, the nucleic acid of a recombinant bacterium has been modified by reaction with a nucleic acid targeting compound (eg, a soraren compound). The present invention also provides a pharmaceutical composition comprising the recombinant Listeria bacterium and a pharmaceutically acceptable carrier. The present invention also provides an immunogenic composition comprising recombinant bacteria, wherein the polypeptide encoded by the second polynucleotide is an antigen. The present invention also provides a vaccine comprising the recombinant Listeria bacteria. Also provided are methods of inducing an immune response to an antigen in a host comprising administering to the host an effective amount of a composition comprising a recombinant bacterium, wherein the polypeptide encoded by the second polynucleotide is an antigen (or comprises an antigen). do). Also provided are methods for preventing or treating a condition (a disease such as cancer or an infectious disease) in a host comprising administering to the host an effective amount of a composition comprising recombinant Listeria bacteria. Also provided is the use of bacteria in the manufacture of a medicament for inducing an immune response to an antigen in a host, wherein the polypeptide encoded by the second polynucleotide comprises an antigen.

In another aspect, the present invention provides a recombinant nucleic acid molecule comprising a polynucleotide encoding a polypeptide that is exogenous to Listeria bacteria (an antigen such as a cancer antigen or a non-Listeria antigen), wherein the polynucleotide is codon for expression in Listeria . Optimized. In some embodiments, the codon-optimization of the polynucleotides enhances expression and / or secretion of the polypeptide in Listeria bacteria (relative to non-codon-optimized sequences). In some embodiments, the exogenous polypeptide comprises an antigen. In some embodiments, the exogenous polypeptide is an antigen. In some embodiments, the antigen is a non-bacterial antigen. For example, in some embodiments, the antigen is a tumor-associated antigen or is derived from a tumor-associated antigen. For example, in some embodiments, the polypeptide is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP , Proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, or CEA, or K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY -ESO-1, WT-1, survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, or CEA. In some embodiments, the antigen is mesothelin or an antigen fragment or antigen variant of mesothelin. In some embodiments, the antigen is NY-ESO-1, or an antigen fragment or variant of NY-ESO-1. In some other embodiments, the antigen is an infectious disease antigen or is derived from an infectious disease antigen. In some embodiments, the recombinant nucleic acid molecule further comprises a polynucleotide encoding a signal peptide within the same translation frame as the exogenous polypeptide such that the recombinant nucleic acid molecule encodes a fusion protein comprising the signal peptide and the exogenous polypeptide. (This is may be of Listeria original or may not) In some embodiments, the polynucleotide encoding the signal peptide codons for expression in Listeria monocytogenes - are optimized. The invention further provides an expression cassette comprising a recombinant nucleic acid molecule and further comprising a promoter operably linked to the first and second polynucleotides of the recombinant nucleic acid molecule. Also provided are vectors (eg, expression vectors) comprising recombinant nucleic acid molecules and / or expression cassettes. The present invention also provides recombinant Listeria bacteria comprising recombinant nucleic acid molecules and / or expression cassettes. In some embodiments, the Listeria bacteria belong to the Listeria monocytogenes species. Also provided are pharmaceutical compositions, immunogenic compositions and vaccines comprising recombinant Listeria bacteria. In addition, the present invention provides a method of inducing an immune response to an antigen in a host comprising administering to the host an effective amount of a composition comprising recombinant Listeria bacteria, wherein the polypeptide is an antigen (or comprises an antigen). The present invention also provides methods of inducing an immune response and / or preventing or treating a condition (eg a disease) using recombinant Listeria bacteria. Also provided is the use of bacteria in the manufacture of a medicament for inducing an immune response to an antigen in a host, wherein the exogenous polypeptide comprises an antigen.

In a further aspect, the present invention provides a recombinant Listeria bacterium comprising an expression cassette, the expression cassette is an exogenous polypeptide to Listeria bacteria (cancer antigen or a non - Listeria antigen, such as a bacterial antigen), and a polynucleotide encoding an exogenous polypeptide A promoter operably linked to the polynucleotide is codon-optimized for expression in Listeria . In some embodiments, the Listeria bacteria belong to the Lsteria monocytogenes species. In some embodiments, codon-optimization of the polynucleotides increases expression and / or secretion in Listeria of the polypeptide (relative to non-codon-optimized sequences). In some embodiments, the exogenous polypeptide comprises an antigen. In some embodiments, the exogenous polypeptide is an antigen and in some embodiments can be a non-bacterial antigen. For example, in some embodiments, it is a tumor-associated antigen or is derived from a tumor-associated antigen. For example, in some embodiments the polypeptide is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, Proteinase 3, SPAS-1, SP-17, PAGE-4, TARP or CEA, or K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO -1, WT-1, survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP or CEA. In some embodiments, the antigen is mesothelin, or an antigen fragment or antigen variant of mesothelin. In some other embodiments, the antigen is NY-ESO-1, or an antigen fragment or antigen variant of NY-ESO-1. In some other embodiments, the antigen is an infectious disease antigen or is derived from an infectious disease antigen. In some embodiments, the expression cassette further comprises a polynucleotide encoding a signal peptide within the same transcription frame as the exogenous polypeptide such that the expression cassette is operably linked to the promoter and the expression cassette encodes a fusion protein comprising the signal peptide and the exogenous polypeptide. . In some embodiments, the polynucleotide encoding a signal peptide, which may or may not be native to Listeria , is codon-optimized for expression in Listeria monocytogenes . Also provided are pharmaceutical compositions, immunogenic compositions and vaccines comprising recombinant Listeria bacteria. The invention further provides a method of inducing an immune response against an antigen in a host comprising administering to the host an effective amount of a composition comprising a recombinant Listeria bacterium. The present invention also provides a method of using recombinant Listeria bacteria to induce an immune response and / or to prevent or treat a condition (eg a disease). Also provided is the use of bacteria in the manufacture of a medicament for inducing an immune response to an antigen in a host, wherein the exogenous polypeptide comprises an antigen.

In a further aspect, the invention provides a recombinant Listeria bacterium (eg, Listeria monocytogenes ) comprising a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising (a) a first polynucleotide encoding a Listeria signal peptide; And (b) a second polynucleotide encoding a polypeptide that is within the same translation reading frame as the first polynucleotide, wherein the recombinant nucleic acid molecule encodes a fusion protein comprising both the non-Listeria signal peptide and the polypeptide. In some embodiments, the recombinant nucleic acid molecule is located in an expression cassette, and the expression cassette further comprises a promoter operably linked to both the first and second polynucleotides. Thus, in some embodiments, the recombinant Lsiteria bacteria comprises an expression cassette comprising a recombinant nucleic acid molecule, wherein the expression cassette further comprises a promoter operably linked to the first and second polynucleotides of the recombinant nucleic acid molecule. In some embodiments, the expression cassette is a polycistron expression cassette (eg, bicistron expression cassette). In some embodiments, the first polynucleotide, the second polynucleotide, or both the first and second polynucleotides are codon-optimized for expression in Listeria (eg, Listeria monocytogenes ). In some embodiments, codon-optimization of the first and / or second polynucleotides enhances expression and / or secretion of fusion proteins in bacteria (relative to non-codon-optimized sequences). In some embodiments, the first and second polynucleotides are heterologous to one another. In some embodiments, the polypeptide and signal peptide encoded by the second polynucleotide are heterologous to one another. In some embodiments, the polypeptide encoded by the second polynucleotide is exogenous to Listeria bacteria (ie, heterologous to Listeria bacteria). In some embodiments, the polypeptide encoded by the second polynucleotide comprises an antigen (eg, a non-Listeria antigen). In some embodiments, the polypeptide encoded by the second polynucleotide is an antigen. In some embodiments, the antigen is a tumor-associated antigen or is derived from a tumor-associated antigen. In some embodiments, the antigen is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protein Third, SPAS-1, SP-17, PAGE-1OO, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, and CEA, or K-Ras , H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17 , PAGE-4, TARP, and CEA. For example, in some embodiments, the antigen is mesothelin or an antigen fragment or antigen variant thereof. In some embodiments, the antigen is human mesothelin. In some embodiments, the antigen is human mesothelin lacking a signal peptide and a GPI linker domain. In some other embodiments, the antigen is an infectious disease antigen or is derived from an infectious disease antigen. In some embodiments, the signal peptide is a bacterial signal peptide. In some embodiments, the signal peptide is derived from intracellular bacteria. In some embodiments, the signal peptide is derived from Gram-positive bacteria. In some embodiments, the signal peptide is derived from a bacterium belonging to the genus Bacillus, Staphylococcus, or Lacotococcus (eg, Bacillusanthracis, Bacillus subtilis, Staphylococcus aureus, or Lactococcus lactis ). In some embodiments, the signal peptide is a secA1 signal peptide (eg, a Usp45 signal peptide of Lactococcus lactis or a protective antigen signal peptide of Bacillus anthracis ). In some embodiments, the signal peptide is a secA2 signal peptide. In some embodiments, the signal peptide is a Tat signal peptide (eg, PhoD signal peptide of B. subtilus ). In some embodiments, Listeria bacteria are attenuated. For example, in some embodiments, Listeria is attenuated for intercellular propagation, penetration into non-phagocytic cells, or proliferation. In some embodiments, the recombinant Listeria bacterium has a defect with respect to ActA, or internalin B, or both ActA and internalin B (eg, ΔactAΔinlB double deletion mutant). In some embodiments, the recombinant Listeria bacterium is deleted in functional ActA, or internalin B, or both Act A and internalin B. In some embodiments, the nucleic acid of a recombinant bacterium has been modified by reaction with a nucleic acid targeting compound (eg, a soraren compound). The present invention also provides a pharmaceutical composition comprising the recombinant Listeria bacterium and a pharmaceutically acceptable carrier. The invention further provides an immunogenic composition comprising recombinant bacteria, wherein the polypeptide encoded by the second polynucleotide is an antigen. The present invention also provides a vaccine comprising the recombinant Listeria bacteria. Also provided are methods of inducing an immune response to an antigen in a host comprising administering to the host an effective amount of a composition comprising a recombinant Listeria bacterium, wherein the polypeptide encoded by the second polynucleotide is an antigen (or an antigen). Includes). Also provided are methods for preventing or treating a condition (a disease such as cancer or an infectious disease) in a host comprising administering to the host an effective amount of a composition comprising recombinant Listeria bacteria. Also provided is the use of bacteria in the manufacture of a medicament for inducing an immune response to an antigen in a host, wherein the polypeptide encoded by the second polynucleotide comprises an antigen.

In another embodiment, the present invention provides a method for a non- Listeria signal peptide comprising a first polynucleotide encoding a non- Listeria signal peptide, a second polynucleotide encoding a polypeptide within the same translational reading frame as the first polynucleotide, and both the first and second polynucleotides. Provided are recombinant Listeria bacteria (eg, from Listeria monocytogenes species) comprising an expression cassette comprising a promoter operably linked thereto. The expression cassette encodes a fusion protein comprising both non-Listeria signal peptides and polypeptides. In some embodiments, Listeria bacteria are directed for intercellular propagation, penetration into non-phagocytic cells, or proliferation. In some embodiments, the first polynucleotide, second polynucleotide, or first and second polynucleotides are codon-optimized for expression in Listeria. In some embodiments, codon-optimization of the first and / or second polynucleotides enhances expression and / or secretion in bacteria of the encoded fusion protein (relative to non-codon-optimized sequences). In some embodiments, the first polynucleotide and / or the second polynucleotide are codon-optimized for expression in Listeria monocytogenes . In some embodiments, the polypeptide encoded by the second polynucleotide comprises an antigen. In some embodiments, the polypeptide encoded by the second polynucleotide is an antigen and in some embodiments may be a non-bacterial antigen. For example, the antigen is, in some embodiments, a tumor-associated antigen or derived from a tumor-associated antigen. For example, in some embodiments, the antigen is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP , Proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, or CEA, or K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY -ESO-1, WT-1, survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, or CEA. For example, in some embodiments, the antigen is mesothelin or an antigen fragment or antigen variant of mesothelin. In some other embodiments, the antigen is NY-ESO-1, or an antigen fragment or antigen variant of NY-ESO-1. In some embodiments, the antigen is an infectious disease antigen or is derived from an infectious disease antigen. In a preferred embodiment, the signal peptide is a bacterial signal peptide. In some embodiments, the signal peptide is derived from a bacterium belonging to the genus Bacillus, Staphylococcus, or Lactococcus . For example, in some embodiments, the signal peptide is derived from Bacillusanthracis, Bacillus subtilis, Staphylococcus aureus, or Lactococcus lactis . In some embodiments, the signal peptide is a secA1 signal peptide, such as a Usp45 signal peptide from Lactococcus lactis or a protective antigen signal peptide from Bacillus anthracis . In some embodiments, the signal peptide is a secA2 signal peptide. In other embodiments, the signal peptide is a Tat signal peptide such as B. subtilis Tat signal peptide (eg, PhoD). Also provided are pharmaceutical compositions, immunogenic compositions and vaccines comprising the recombinant Listeria bacteria described herein. The present invention also provides a method of using recombinant Listeria bacteria to induce an immune response and / or to prevent or treat a condition such as a disease. Also provided is the use of bacteria in the manufacture of a medicament for inducing an immune response to an antigen in a host, wherein the polypeptide encoded by the second polynucleotide comprises an antigen.

The present invention also provides a kit comprising: (a) a first polynucleotide encoding a bacterial autolysis, or a catalytically active fragment or catalytically active variant thereof; And (b) a second polynucleotide encoding the polypeptide, wherein the second polynucleotide is in the same translation reading frame as the first polynucleotide, wherein the recombinant nucleic acid molecule is defined by the second polynucleotide. Encodes a protein chimera comprising a polypeptide to be encoded and self-dissolving, or a catalytically active fragment or catalytically active variant thereof, wherein in the protein chimera the polypeptide is auto-dissolving, or a catalytically active fragment thereof or It is fused with a catalytically active variant, or is located within a self-dissolved, or catalytically active fragment or catalytically active variant thereof. In some embodiments, the first polynucleotide encodes bacterial autolysis. In some embodiments, the protein chimera is catalytically active as autodissolver. In some embodiments, bacterial autolysis is derived from intracellular bacteria (eg, Listeri a). In some embodiments, the bacterial autolysis is Listeria autolysis. In some embodiments, the second polynucleotide encoding the polypeptide is located within the first polynucleotide encoding the autolytic, or catalytically active fragment or catalytically active variant thereof, and the recombinant nucleic acid molecule is a protein The chimera is encoded, and the polypeptide is located within the autolytic, or catalytically active fragment, or catalytically active variant thereof (ie, the polypeptide is within the autolytic, or catalytically active fragment or variant thereof) Is embedded). In some other embodiments, the second polynucleotide is located outside of the first polynucleotide that encodes a self-dissolution, or a catalytically active fragment or catalytically active variant thereof, wherein the recombinant nucleic acid molecule comprises a protein chimera. And encode, the polypeptide is fused with autolytic soluble or catalytically active fragment or catalytically active variant thereof. In some embodiments, the recombinant nucleic acid molecule further comprises (c) a third polynucleotide encoding a signal peptide in the same translation reading frame as the first and second polynucleotides, wherein the recombinant nucleic acid molecule is a signal peptide, second polynucleotide And a protein chimera comprising a polypeptide encoded by and autolytic soluble or a catalytically active fragment or catalytically active variant thereof. In some embodiments, the signal peptide is a secA2 signal peptide (eg, p60). In some embodiments, the signal peptide is a signal peptide combined with natural autolysis (eg, the signal peptide is p60 and autolysis is p60). In some embodiments, the autolysis is secA2-dependent autolysis. In some embodiments, the autolysis is a peptidoglycan hydrolase (eg, N-acetylmuramidase or p60). In some embodiments, the polypeptide encoded by the second polynucleotide comprises an antigen. In some embodiments, the polypeptide is an antigen (eg, a tumor-associated antigen, an antigen derived from a tumor-related antigen, an infectious disease antigen, or an antigen derived from an infectious disease antigen). In some embodiments, the antigen is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, protein Third, SPAS-1, SP-17, PAGE-4, TARP, or CEA, or K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, or CEA. For example, in some embodiments, the antigen is mesothelin, or an antigen fragment or antigen variant thereof. In some embodiments, the antigen is human mesothelin. In some embodiments, the antigen is human mesothelin lacking a signal peptide and a GPI anchor. The present invention also provides an expression cassette comprising a recombinant nucleic acid molecule and further comprising a promoter operably linked to the first and second polynucleotides of the recombinant nucleic acid molecule, and an expression vector comprising the expression cassette. In addition, the present invention provides a recombinant bacterium comprising the recombinant nucleic acid molecule. In some embodiments, the recombinant bacteria are intracellular bacteria, such as Listeria bacteria (eg, Listeria monocytogenes ). In some embodiments, the polypeptide encoded by the second polynucleotide is exogenous to recombinant bacteria. Also provided is a pharmaceutical composition comprising (a) a recombinant bacterium and (b) a pharmaceutically acceptable carrier. Also provided are immunogenic compositions comprising recombinant bacteria, wherein the polypeptide encoded by the second polynucleotide is an antigen. Also provided are vaccines comprising recombinant bacteria, wherein the polypeptide encoded by the second polynucleotide is an antigen. The present invention also provides a method of inducing an immune response to an antigen in a host comprising administering to the host an effective amount of a composition comprising a recombinant bacterium, wherein the polypeptide encoded by the second polynucleotide is an antigen (or Antigen). Also provided are methods for preventing or treating a condition in a host comprising administering to the host an effective amount of a composition comprising a recombinant bacterium. Also provided is the use of bacteria in the manufacture of a medicament for inducing an immune response to an antigen in a host, wherein the polypeptide encoded by the second polynucleotide comprises an antigen.

In another aspect, the invention provides a recombinant Listeria bacterium comprising a polycistronic expression cassette, wherein the polycistronic expression cassette encodes at least two individual non- Listeria polypeptides. For example, in some embodiments, the expression cassette operates on a first polynucleotide encoding a first non- Listeria polypeptide, a second polynucleotide encoding a second non- Listeria polypeptide, and a first and second polynucleotide. Possibly linked promoters. In some embodiments, the expression cassette further comprises an intergenic sequence between the first and second polynucleotides. In some embodiments, the polycistronic expression cassette is a bicistron expression cassette encoding two separate non- Listeria polypeptides. In some embodiments, the recombinant Listeria bacteria belongs to Listeria monocytogenens species. In some embodiments, at least one of the non- Listeria polypeptides encoded by the polycistron expression cassette comprises an antigen. In some embodiments, at least two non-Listeria polypeptides each comprise a fragment of the same antigen. In some embodiments, the antigen is a tumor-associated antigen or is derived from a tumor-associated antigen. For example, in some embodiments the antigen is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, Antigen selected from the group consisting of proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, and CEA, or K-Ras, H-Ras, N-Ras, 12-K-Ras, Antigen selected from the group consisting of mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, and CEA Derived from. In some embodiments, the antigen is mesothelin, or an antigen fragment or antigen variant thereof. In some embodiments, the antigen is human mesothelin. In some embodiments, the antigen is human mesothelin lacking a signal peptide and a GPI anchor. In some embodiments, the antigen is an infectious disease antigen or is derived from an infectious disease antigen. In some embodiments, at least one non- Listeria polypeptide encoded by the polycistron expression cassette comprises a signal peptide ( Listeria signal peptide or non- Listeria signal peptide). In some embodiments, the signal peptide is a secA1 signal peptide. In some embodiments, the signal peptide is a secA2 signal peptide. In other embodiments, the signal peptide is a Tat signal peptide. In some embodiments, the expression cassette comprises a polynucleotide encoding a signal peptide and the polynucleotide encoding the signal peptide is codon-optimized for expression in Listeria . The present invention also provides a pharmaceutical composition comprising (a) a recombinant Listeria Valkeria and (b) a pharmaceutically acceptable carrier. Also provided are immunogenic compositions comprising recombinant Listeria bacteria. Also provided are vaccines comprising recombinant Listeria bacteria. Also provided are methods for inducing an immune response to an antigen in a host comprising administering to the host an effective amount of a composition comprising a recombinant Listeria bacterium, wherein the at least one non- Listeria polypeptide comprises an antigen. Also provided are methods for preventing or treating a condition in a host comprising administering to the host an effective amount of a composition comprising recombinant Listeria bacteria. Also provided is the use of bacteria in the manufacture of a medicament for inducing an immune response to an antigen in a host, wherein at least one non- Listeria polypeptide encoded by a polycistronic expression cassette comprises an antigen.

In another aspect, the invention provides a second polynucleotide encoding (a) a first polynucleotide encoding a signal peptide, (b) a secreted protein, or a fragment thereof, wherein the second polynucleotide is identical to the first polynucleotide. (C) a third polynucleotide encoding a polypeptide that is heterologous to the secreted protein, or fragment thereof, wherein the third polynucleotide is in the same translation reading frame as the first and second polynucleotides. Wherein the recombinant nucleic acid molecule encodes a protein chimera comprising a signal peptide, a polypeptide encoded by a third polynucleotide and a secreted protein or fragment thereof, and is encoded by the third polynucleotide The encoded polypeptide is fused with a protein or fragment thereof secreted from the protein chimera. Or, or, or secreted proteins are located within its segment. In some embodiments, the secreted protein is a naturally secreted protein (ie, a protein secreted from natural cells). In some embodiments, the third polynucleotide is located within the second polynucleotide in the recombinant nucleic acid molecule, and the polypeptide encoded by the third polynucleotide is combined with a protein or fragment thereof secreted from the protein chimera encoded by the recombinant nucleic acid molecule. Are located together. In some embodiments, the third polynucleotide is located outside of the second polynucleotide in the recombinant nucleic acid molecule, and the polypeptide encoded by the third polynucleotide is fused with a protein or fragment thereof secreted from the protein chimera. Also provided are expression cassettes comprising a promoter comprising a recombinant nucleic acid molecule and further comprising a promoter operably linked to the first, second, and third polynucleotides of the recombinant nucleic acid molecule. In some embodiments, the polypeptide encoded by the second polynucleotide comprises an antigen. In some embodiments, the polypeptide encoded by the second polynucleotide is an antigen. For example, in some embodiments, the antigen is a tumor-associated antigen or is derived from a tumor-associated antigen (eg, the antigen is K-Ras, H-Ras, N-Ras, 12-K-Ras, Selected from the group consisting of mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, and CEA Or K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, proteinase 3, SPAS -1, SP-17, PAGE-4, TARP, and CEA). In some embodiments, the antigen is mesothelin, or an antigen fragment or antigen variant thereof. For example, in some embodiments, the antigen is human mesothelin or human mesothelin lacking a signal peptide and a GPI anchor. In other embodiments, the antigen is an infectious disease antigen or is derived from an infectious disease antigen. Also provided are expression vectors comprising an expression cassette. In addition, recombinant bacteria comprising recombinant nucleic acid molecules are provided. In addition, recombinant Listeria bacteria (eg, Listeria monocytogenes ) are provided, and in some embodiments the polypeptide encoded by the third nucleotide is exogenous to Listeria bacteria. The present invention also provides an immunogenic composition comprising recombinant bacteria, wherein the polypeptide encoded by the third polypeptide is an antigen. Also provided are methods of inducing an immune response to an antigen in a host comprising administering to the host an effective amount of a composition comprising a recombinant bacterium, wherein the polypeptide encoded by the third polynucleotide is an antigen. Also provided is a method of inducing an immune response to an antigen in a host comprising administering to the host an effective amount of a composition comprising a recombinant bacterium, wherein the polypeptide encoded by the third polynucleotide is an antigen (or comprises an antigen). do). Also provided are methods of using recombinant bacteria or compositions comprising bacteria to prevent or treat conditions in a host, and pharmaceutical compositions and vaccines comprising bacteria are provided. Also provided is the use of bacteria in the manufacture of a medicament for inducing an immune response to an antigen in a host, wherein the polypeptide encoded by the third polynucleotide comprises an antigen.

In a further aspect, the present invention provides an improved method of expressing and secreting heterologous proteins in host bacteria. The present invention also provides a method for improving the expression and secretion of heterologous proteins in bacteria. The invention further provides methods for making the recombinant nucleic acid molecules, expression cassettes, expression vectors, and recombinant bacteria described herein.

The present invention also provides a variety of polynucleotides useful for optimizing the expression of heterologous polynucleotides in bacteria such as Listeria .

Unless otherwise indicated by description or context, embodiments presented in the Markersi group, Markersi claim, or by the method of “or language” are each individual embodiment, any combination of each individual embodiment, and each It will be understood that the invention comprises the invention, which may consist of or include both individual embodiments.

In addition, further embodiments and aspects of the invention are provided, as well as descriptions of the aspects and embodiments described above.

II. Recombinant nucleic acid molecule

The present invention provides a variety of polynucleotides useful for the expression of polynucleotides such as heterologous polynucleotides in bacteria such as Listeria . For example, there is also provided a recombinant nucleic acid molecule comprising a novel combination of a coding sequence of a polypeptide, such as a heterologous antigen, with a sequence encoding a signal peptide (or polypeptide comprising a signal peptide). Recombinant nucleic acid molecules are provided that include codon-optimized polynucleotide sequences. In some embodiments, these recombinant nucleic acid molecules are heterologous in that they are polynucleotides (ie, polynucleotide sequences) that are not found in nature in combination with each other as part of the same nucleic acid molecule. In some embodiments, recombinant nucleic acid molecules are isolated. In some embodiments, the recombinant nucleic acid molecule is located in the sequence of an expression cassette, expression vector, plasmid DNA, and / or even bacterial genomic DNA (after insertion) in a bacterium. In some embodiments, recombinant nucleic acid molecules provide enhanced expression and / or secretion of polypeptides (eg, heterologous polypeptides) in bacteria.

In some embodiments, the recombinant nucleic acid molecule is DNA. In some embodiments, the recombinant nucleic acid molecule is RNA. In some embodiments, the recombinant nucleic acid is single-stranded. In other embodiments, the recombinant nucleic acid is double-stranded.

In some embodiments, the recombinant nucleic acid molecules described herein encode a fusion protein, such as a fusion protein comprising a signal peptide and other polypeptides such as polypeptides heterologous to the signal peptide. In some embodiments, the signal peptide is a bacterial signal peptide. The listed polypeptide components of the fusion protein are not essential, but may also be fused directly to each other. The polypeptide component of the fusion protein may, in some embodiments, be separated on the polypeptide sequence by one or more involved amino acid sequences. In some embodiments, the other polypeptide is a non-bacterial polypeptide, eg, a mammalian or viral polypeptide.

For example, in one aspect, the present invention provides a recombinant nucleic acid molecule comprising: (a) a first polynucleotide encoding a signal peptide (the first polynucleotide is codon-optimized for expression in bacteria) ; And (b) a second polynucleotide encoding a polypeptide (eg, an antigen), wherein the second polynucleotide is in the same translation reading frame as the first polynucleotide, and the recombinant nucleic acid molecule comprises a fusion comprising a signal peptide and a polypeptide. Code the protein. In further embodiments, the second polynucleotide (polynucleotide encoding a polypeptide such as an antigen) is also codon-optimized for expression in bacteria. The bacterium to which the first and / or second polynucleotides are codon-optimized must be of the type of bacterium to which the recombinant nucleic acid molecule is intended to be located.

In another aspect, the present invention provides a kit comprising: (a) a first polynucleotide encoding a bacterial natural signal peptide (the first polynucleotide is codon-optimized for expression in bacteria), and (b) a second poly encoding a polypeptide. A recombinant nucleic acid molecule is provided that comprises nucleotides (the second polynucleotide is in the same translation reading frame as the first polynucleotide), wherein the recombinant nucleic acid molecule encodes a fusion protein comprising a signal peptide and a polypeptide. In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous to the signal peptide. In some embodiments, the second polynucleotide is heterologous to the first polynucleotide. In some embodiments, the polypeptide is heterologous to a bacterium in which the signal peptide is a natural signal peptide (ie, exogenous to the bacterium). In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous to the signal peptide, exogenous to the bacteria, or both. In some embodiments, the bacteria from which the signal peptide is derived are intracellular bacteria. In some embodiments, the bacteria are selected from the group consisting of Listeria, Bacillus, Yersinia pestis, Salmonella, Shigella, Brucella, Mycobacteria and E. coli . In some embodiments, the signal peptide is naturally occurring in Listeria bacteria Signal peptide. In some embodiments, the signal peptide is a natural signal peptide of Listeria bacteria belonging to the Listeria monocytogenes species. In some embodiments, the second polynucleotide is codon-optimized for expression in bacteria.

In another aspect, the present invention provides a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule is (a) a first polynucleotide encoding a signal peptide (the first polynucleotide is codon-optimized for expression in Listeria bacteria), and ( b) a second polynucleotide encoding the polypeptide (the second polynucleotide is in the same translation reading frame as the first polynucleotide), and the recombinant nucleic acid molecule encodes a fusion protein comprising a signal peptide and a polypeptide. In some embodiments, the signal peptide is a natural signal peptide of Listeria bacteria. In some other embodiments, the signal peptide is exogenous to Listeria bacteria. In some embodiments, the signal peptide is exogenous to the polypeptide encoded by the second polynucleotide. In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous to Listeria bacteria. In some embodiments, the Listeria bacteria belong to the Listeria monocytogenes species.

The invention also provides a recombinant nucleic acid molecule comprising a polynucleotide encoding a polypeptide that is exogenous to a Listeria bacterium (eg, a cancer or a non- Listeria infectious disease antigen), wherein the polynucleotide encoding the exogenous polypeptide is Listeria. Codon-optimized for expression in bacteria.

In another aspect, the invention provides an antibody comprising (a) a first polynucleotide encoding a non-secA1 bacterial signal peptide, and (b) a second polynucleotide encoding a polypeptide, such as an antigen, wherein the second polynucleotide is combined with the first polynucleotide. Within the same translation reading frame), wherein the recombinant nucleic acid molecule encodes a fusion protein comprising a signal peptide and a polypeptide. In some embodiments, the non-secA1 bacterial signal peptide is a secA2 signal peptide or a Tat signal peptide. In some embodiments, the first polynucleotide encoding a non-secA1 bacterial signal peptide is codon-optimized for expression in bacteria (eg, Listeria ) where the recombinant nucleic acid molecule is intended to be located. In some embodiments, the second polynucleotide encoding a polypeptide such as an antigen is codon-optimized for expression in bacteria in which the recombinant nucleic acid molecule is intended to be located. In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous to the signal peptide. In some embodiments, the polypeptide encoded by the second polynucleotide is exogenous to the bacterium to which the recombinant nucleic acid molecule is or will be integrated. In some embodiments, the polypeptide encoded by the second polynucleotide is exogenous to the bacterium to which the recombinant nucleic acid molecule is incorporated or integrated, and the polypeptide encoded by the second polynucleotide is heterologous to the signal peptide.

The present invention provides the same translational reading as a first polynucleotide encoding a non-secA1 bacterial signal peptide, a second polynucleotide encoding a polypeptide (eg, a heterologous protein and / or antigen), and a first and second polynucleotide. Further provided is a recombinant nucleic acid molecule comprising a third polynucleotide encoding SecA2 autolysis, or a fragment thereof, in a frame, wherein the second polynucleotide is within the third polynucleotide or between the first and third polynucleotides. Located. In some embodiments, the recombinant nucleic acid molecule encodes a fusion protein comprising a signal peptide, a polypeptide, and autolysis. In some embodiments, fragments of autodissolution are catalytically active, such as autodissolution. In some embodiments, autolysis is derived from intracellular bacteria. In some embodiments, the autolysis is a peptidoglycan hydrolase. In some embodiments, the bacterial autolysis is Listeria autolysis. In some embodiments, the autolysis is p60. In some embodiments, the autolysis is N-acetylmuramidase.

The present invention also provides a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising (a) a first polynucleotide encoding a non- Listeria signal peptide; And (b) a second polynucleotide encoding a polypeptide that is within the same translation reading frame as the first polynucleotide, wherein the recombinant nucleic acid molecule encodes a fusion protein comprising both a non- Listeria signal peptide and a polypeptide. In some embodiments, the non- Listeria signal peptide is heterologous to the polypeptide encoded by the second polynucleotide. In some embodiments, the first polynucleotide, second polynucleotide, or first and second polynucleotides are codon optimized for expression in Listeria bacteria.

In addition, the present invention provides a composition comprising (a) a first polynucleotide encoding bacterial autolysis, or a catalytically active fragment or catalytically active variant thereof, and (b) a second polynucleotide encoding a polypeptide. The second polynucleotide is in the same translation reading frame as the first polynucleotide), wherein the recombinant nucleic acid molecule is a second polynucleotide and autolysates, or a catalytically active fragment or catalytic thereof. And encodes a protein chimera comprising a polypeptide encoded by an active variant, wherein the polypeptide in the protein chimera is fused with, or fused with, a catalytically active variant or a catalytically active fragment thereof. Its catalytically active fragments or catalytically active mutations It is located therein. In some embodiments, the second polynucleotide is located within the first polynucleotide, and the recombinant nucleic acid molecule is characterized in that the polypeptide encoded by the second polynucleotide autolyses, or a catalytically active fragment or catalytically active fragment thereof. It encodes a protein chimera located within a phosphor variant. In some embodiments, the second polynucleotide is located outside the second polynucleotide, and the recombinant nucleic acid molecule is characterized in that the polypeptide encoded by the second polynucleotide self-dissolves, or a catalytically active fragment or catalytically active thereof. Encodes protein chimeras that are fused to phosphorus variants. In some embodiments, the first polynucleotide encodes autolysis. In some embodiments, the recombinant nucleic acid molecule comprises (c) a third polynucleotide encoding a signal peptide within the same translational reading frame as the first and second polynucleotides, wherein the recombinant nucleic acid molecule is a signal peptide, second polynucleotide Encodes a protein chimera comprising a polypeptide encoded by and a self-dissolution, or a catalytically active fragment or catalytically active variant thereof. In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous to autolysis. In some embodiments, the fragment of autolysis is at least about 30, at least about 40, at least about 50, or at least about 100 amino acids in length. In some embodiments, autolysis is derived from intracellular bacteria. In some embodiments, the bacterial autolysis is Listeria autolysis. Catalytically active variants of autodissolution include variants that differ from the original autodissolution in one or more substitutions, deletions, additions, and / or insertions. In some embodiments, the autolysis is a peptidoglycan hydrolase. In some embodiments, the autolysis is p60. In some embodiments, the autolysis is N-acetylmuramidase.

Additional self-dissolution can be identified or characterized by zymography, a technique known to those skilled in the art (see, eg, Lenz, et al. (2003) Proc. Natl. Acad. Sci. USA 100: 12432-12437). ). Zymography can also be used to determine whether a given fragment and / or variant of autolysis is catalytically active, such as autolysis. The technique can also be used to assess whether a particular protein chimera is catalytically active, such as autolysis.

In some embodiments, the catalytically active fragments and / or variants of autodissolution are at least about 10%, at least about 30%, at least about 50%, at least about 75%, at least about 90 as autodissolution, such as natural autodissolution. %, Or at least about 95% catalytically active.

In some embodiments, the protein chimera is catalytically active as autodissolver. In some embodiments, the protein chimera is at least about 10%, at least about 30%, at least about 50%, at least about 75%, at least about 90%, or at least about 95% catalytically active as natural autolysis.

Another option for heterologous protein expression is to use a protein “scaffold” in which the heterologous protein is functionally inserted “in frame”. In this configuration, for example, the entire gene or component of a gene corresponding to an MHC class I or MHC class II epitope is inserted into and through the scaffold protein. The scaffold protein may be a highly expressed bacterial protein (such as a Listeria protein such as LLO or p60), but in other embodiments it is selected for its high expression, stability, secretion, and / or immunogenicity Can be a heterologous protein. Representative examples of scaffold proteins are chicken egg white albumin or other human proteins such as β-globin or albumin.

The invention also provides a second polynucleotide encoding (a) a first polynucleotide encoding a signal peptide, (b) a secreted protein, or a fragment thereof, wherein the second polynucleotide is the same translational reading as the first polynucleotide. (C) a third polynucleotide encoding a polypeptide that is heterologous to the secreted protein, or fragment thereof, wherein the third polynucleotide is in the same translation reading frame as the first and second polynucleotides. Providing a recombinant nucleic acid molecule comprising a recombinant nucleic acid molecule encoding a protein chimera comprising a signal peptide, a polypeptide encoded by a second polynucleotide, and a secreted protein, or fragment thereof, wherein the polypeptide is a protein chimera, Proteins or fragments thereof fused to or secreted proteins, or fragments thereof Is located within. In some embodiments, the second polynucleotide encodes a secreted protein. In some embodiments, the secreted protein is a protein secreted from its natural cells. In some embodiments, the third polynucleotide is located within the second polynucleotide in the recombinant nucleic acid molecule, and the polypeptide encoded by the third polynucleotide is secreted from, or secreted from, the protein chimera encoded by the recombinant nucleic acid molecule. Is located within the fragment. In some embodiments, the third polynucleotide is located outside of the second polynucleotide in the nucleic acid molecule and the polypeptide encoded by the third polynucleotide is fused to the secreted protein or fragment thereof in the protein chimera. In some embodiments, the secreted protein is egg white albumin. In some embodiments, truncated forms of egg white albumin are used. In some embodiments, the secreted protein is p60. In some embodiments, the secreted protein is N-acetylmuramidase. In some embodiments, the signal peptide is a signal peptide normally bound to the secreted protein. In some embodiments, the signal peptide is heterologous to the secreted protein. In some embodiments, the secreted protein is at least about 30, at least about 40, at least about 50, or at least about 100 amino acids in length.

In some embodiments, a recombinant nucleic acid molecule, expression cassette, or expression vector comprises a coding sequence for a polypeptide that is exogenous to a bacterium, embedded within some or the entire coding sequence of a protein that is highly expressed in the bacterium. In some embodiments, the highly expressed sequence is the native sequence of the bacterium to which the sequence is to be expressed. In other embodiments, the highly expressed sequence is the native sequence of the bacterium to which it is to be expressed, but provides sufficient expression.

In another aspect, the invention provides a recombinant nucleic acid molecule wherein the nucleic acid molecule encodes at least two individual non- Listeria polypeptides. In some embodiments, polynucleotides encoding non- Listeria polypeptides are codon-optimized for expression in Listeria bacteria.

Methods of making recombinant nucleic acid molecules comprising those described above are well known to those skilled in the art. For example, recombinant nucleic acid molecules can be prepared by synthesizing long oligonucleotides on a DNA synthesizer, nesting them together, and then performing expansion reactions and / or PCR to produce the desired amount of double-stranded DNA. Double-stranded DNA can be cleaved with restriction enzymes and inserted into the desired expression or cloning vector. Sequencing may be performed to verify that the correct sequence has been obtained. Also, by way of non-limiting example, one or more portions of the recombinant nucleic acid molecule may alternatively be obtained from a plasmid containing the portions. PCR of the relevant portion of the plasmid and / or restriction enzyme cleavage of the relevant portion of the plasmid was followed by ligation and / or PCR to combine the relevant polynucleotides to produce the desired recombinant nucleic acid molecule. Such techniques are customary in the art. Standard cloning techniques can also be used to insert the recombinant nucleic acid sequence into a plasmid and replicate the recombinant nucleic acid in a host such as a bacterium. Recombinant nucleic acid can then be isolated from the host cell.

The present invention also provides methods of using any of the recombinant nucleic acid molecules described herein to produce recombinant bacteria (eg, recombinant Listeria bacteria). In some embodiments, methods of using the recombinant nucleic acid molecules described herein to make recombinant bacteria include introducing the recombinant nucleic acid molecules into bacteria. In some embodiments, the recombinant nucleic acid molecule is integrated into the genome of the bacterium. In some other embodiments, the recombinant nucleic acid molecule is on a plasmid that integrates into the bacterium. In some embodiments, integration of recombinant nucleic acid molecules into bacteria occurs by conjugation. Introduction to bacteria can be accomplished by any standard technique known in the art. For example, integration of recombinant nucleic acid molecules into bacteria can occur by conjugation, transduction (transfection), or transformation.

III. Signal peptide

In some embodiments, recombinant nucleic acid molecules, expression cassettes, and / or vectors of the invention encode a fusion protein or protein chimera that includes signal peptides and is suitable for expression in and secretion from host cells, such as bacteria. Thus, in some embodiments, recombinant nucleic acid molecules, expression cassettes and / or vectors of the invention comprise polynucleotides encoding signal peptides.

The terms "signal peptide" and "signal sequence" are used interchangeably herein. In some embodiments, the signal peptide facilitates transport of the polypeptide fused to the signal peptide through the cell membrane of the cell (eg, bacterial cell) to allow the polypeptide to be secreted from the cell. Thus, in some embodiments, the signal peptide is a "secretory signal peptide" or "secretory sequence". In some embodiments, the signal peptide is located at the N-terminus of the secreted polypeptide.

In some embodiments, the sequence encoding a signal peptide in a recombinant nucleic acid molecule or expression cassette is located in the recombinant nucleic acid molecule or expression cassette such that the encoded signal peptide secretes a polypeptide fused from a desired host cell (eg, a bacteria). Will affect. In some embodiments, in a recombinant nucleic acid molecule or expression cassette, the polynucleotide encoding the signal peptide is in-frame (directly or directly at the 5 'end of the polynucleotide encoding the secreted polypeptide (eg, the polypeptide comprising an antigen). Separated by entrapment between polynucleotides).

In some embodiments, the signal peptide that is part of the fusion protein and / or protein chimera encoded by the recombinant nucleic acid molecule, expression cassette and / or expression vector is heterologous to at least one other polypeptide sequence in the fusion protein and / or protein chimera. In some embodiments, the signal peptide encoded by the recombinant nucleic acid molecule, expression cassette and / or expression vector is heterologous (ie exogenous) to the bacterium to which the recombinant nucleic acid molecule, expression cassette and / or expression vector will be integrated or integrated. In some embodiments, the signal peptide is the natural signal peptide of the bacterium to which the recombinant nucleic acid molecule, expression cassette and / or expression vector will be integrated.

In some embodiments, the polynucleotides encoding the signal peptide are codon-optimized for expression in bacteria (eg, Listeria , such as Listeria monocytogenes ). In some embodiments, the codon-optimized polypeptide for a particular bacterium is exogenous to the bacterium. In another embodiment, the codon-optimized polynucleotide for a particular bacterium is native to that bacterium.

A wide variety of signal peptides are known in the art. In addition, various algorithms and software programs are available in the art, such as, for example, a "SignalP" algorithm that can be used to predict signal peptide sequences. See, eg, Antelmann et al., Genome Res., 11: 1484-502 (2001); Menne et al., Bioinformatics, 16: 741-2 (2000); Nielsen et al., Protein Eng., 10: 1-6 (1997); Zhang et al., Protein Sci., 13: 2819-24 (2004); Bendtsen et al., J. Mol. Biol., 340: 783-95 (2004) (regarding Signal P 3.0); Hiller et al., Nucleic Acids Res., 32: W375-9 (2004); Schneider et al., Proteomics 4: 1571-80 (2004); Chou, Curr. Protein Pept. Sci., 3: 615-22 (2002); Shah et al., Bioinformatics, 19: 1985-96 (2003); And Yuan et al., Biochem. Biophys. Res. Commun. 312: 1278-83 (2003).

In some embodiments, the signal peptide is a prokaryotic cell. In some alternative embodiments, the signal peptide is a eukaryotic cell. The use of eukaryotic cell signal peptides for the expression of proteins in Escherichia coli is described, for example, in Humphreys et al., Protein Expression and Purification, 20: 252-264 (2000).

In some embodiments, the signal peptide is a bacterial signal peptide. In some embodiments, the signal peptide is a non- Listeria signal peptide. In some embodiments, the signal peptide is a Listeria signal peptide. In some embodiments, the signal peptide is derived from Gram-positive bacteria. In some embodiments, the signal peptide is derived from intracellular bacteria.

In some embodiments, the signal peptide (eg, non-secA1 bacterial signal peptide) used in the recombinant nucleic acid molecule, expression cassette, or expression vector is derived from Listeria . In some embodiments, the signal peptide is derived from Listeria monocytogenes . In some embodiments, the signal peptide is a signal peptide from Listeria monocytogenes . In some embodiments, the signal peptide is not derived from Listeri a but instead is derived from bacteria excluding bacteria belonging to the Listeria genus. In some embodiments, the bacterial signal peptide is derived from Bacillus bacteria. In some embodiments, the bacterial signal peptide is derived from Bacillus subtitis . In some embodiments, the bacterial signal peptide is derived from bacteria belonging to the genus Straphylococcus . In some embodiments, the bacterial signal peptide is derived from Lactococcus bacteria. In some embodiments, the bacterial signal peptide is derived from Bacillus, Straphylococcus, or Lactococcus bacteria. In some embodiments, the bacterial signal peptide is a signal peptide from Bacillus, Straphylococcus, or Lactococcus bacteria. In some embodiments, the bacterial signal peptide is a signal peptide derived from Bacillus anthracis, Bacillus subtilis, Staphyloccus aureus, or Lactococcus lactis . In some embodiments, the bacterial signal peptide is a signal peptide from Bacillus anthracis . In some embodiments, the bacterial signal peptide is a signal peptide from Bacillus subtilis . In some embodiments, the bacterial signal peptide is a signal peptide from Lactococcus lactis . In some embodiments, the bacterial signal peptide is a signal peptide from Staphyloccus aureus .

In some embodiments of the polynucleotides described herein, the signal peptide derived from a microorganism such as a bacterium is identical to the naturally occurring signal peptide sequence obtained from the microorganism. In other embodiments, the signal peptide encoded by the recombinant nucleic acid molecule, expression cassette, and / or expression vector may be a naturally occurring signal peptide sequence, ie, a fragment of a naturally occurring signal peptide sequence in which a fragment or variant still acts as a signal peptide and / or Or from variants. Variants include polypeptides that differ from the original sequence by one or more substitutions, deletions, additions, and / or insertions. For example, in some embodiments, the signal peptide encoded by the polynucleotide contains one or more conservative mutations. Possible conservative amino acid changes are well known to those skilled in the art. For example, for further information regarding conservative amino acid changes, see paragraph IV of the detailed family name below.

Signal peptides derived from other signal peptides (ie, fragments and / or variants of other signal peptides) are preferably substantially equivalent to the original signal peptide. For example, the ability of a signal peptide derived from another signal peptide to act as a signal peptide will not be substantially affected by changes (deletions, mutations, etc.) that occur in the original signal peptide sequence. In some embodiments, the induced signal peptide may act at least about 70%, at least about 80%, at least about 90%, or at least about 95% as a signal peptide, such as a natural signal peptide sequence. In some embodiments, the signal peptide has at least about 70%, at least about 80%, at least about 90%, or at least about 95% identity with the original signal peptide in the amino acid sequence. In some embodiments, the only alteration made in the sequence of the signal peptide is a conservative amino acid substitution. The fragment of the signal peptide is preferably at least about 80% or at least about 90% of the length of the original signal peptide.

In some embodiments, the signal peptide encoded by the polynucleotide in a recombinant nucleic acid molecule, expression cassette, or expression vector is a secA1 signal peptide, secA2 signal peptide, or a twin-arginine translocation (Tat) signal peptide. In some embodiments, the signal peptide is a secA1 signal peptide. In some embodiments, the signal peptide is a non-secA1 bacterial signal peptide. In some embodiments, the signal peptide is a secA2 signal peptide. In some embodiments, the signal peptide is a twin-arginine translocation (Tat) signal peptide. In some embodiments, these secA1, secA2, or Tat signal peptides are from Listeria . In some embodiments, these secA1, secA2, or Tat signal peptides are non- Listeria signal peptides. For example, in some embodiments, the secA1, secA2, and Tat signal peptides are derived from bacteria belonging to one of the following genera: Bacillus, Straphylococcus, or Lactococcus.

Bacteria use a variety of pathways for protein secretion, including secA1, secA2, and Tween-Arg translocation (Tat). This pathway is largely determined by the type of signal sequence located at the N-terminus of the pre-protein. Most secreted proteins use the Sec pathway, which is translocated through bacterial membrane-embedded proteolytic Sec pores in a non-folding form. In contrast, proteins that utilize the Tat pathway are secreted in the folded form. Nucleotide sequences encoding signal peptides corresponding to any of these protein secretion pathways can be genetically in-frame fused to the desired heterologous protein coding sequence. Signal peptides optionally contain signal peptidase cleavage sites at their carboxy terminus for release of the truly desired protein into the extracellular environment (Sharkov and Cai. 2002 J. Biol. Chem. 277: 5796-5803; Nielsen et. al. 1997 Protein Engineering 10: 1-6; and, www.cbs.dtu.dk/services/SignalP/).

Signal peptides used in the polynucleotides of the present invention can be derived from various bacterial genera as well as various secretory pathways. Signal peptides generally have a general structural organization, with charged N-terminus (N-domain), hydrophobic key region (H-domain), and more polar C-terminal region (C-domain), but they do not have sequence conservation Don't show In some embodiments, the C-domain of the signal peptide contains a type I signal peptide (SPase I) cleavage site, having the matching sequence AXA at the −1 and −3 positions relative to the cleavage site. Proteins secreted via the sec pathway have signal peptides averaging 28 residues. secA2 protein secretion pathway was first discovered in Listeria monocytogenes ; Mutations in the secA2 paralogue are characterized by coarse colony phenotype on agar media, and directed toxic phenotype in mice (Lenz and Portnoy, 2002 Mol. Microbiol. 45: 1043-1056; and, Lenz et. al 2003 PNAS 100: 12432-12437). Signal peptides related to proteins secreted by the Tat pathway have three organisms similar to Sec signal peptides, but have an RR-motif located at the N-domain / H-domain edge (RRX-#-# where # is a hydrogenated residue). It is characterized by having). The bacterial Tat signal peptide averages 14 amino acids longer than the sec signal peptide. The Bacillus subtilis secretome may contain as many as 69 putative proteins using the Tat secretion pathway, 14 of which contain a SPase I cleavage site (Jongbloed et. Al. 2002 J. BioL Chem. 277: 44068). -44078; Thalsma et.al., 2000 Microbiol.Mol. Biol. Rev. 64: 515-547).

Non-limiting examples of signal peptides that can be used in fusion compositions (including protein chimeric compositions) with selected other polypeptides, such as heterologous polypeptides, can result in secretion of the encoded protein from bacteria.

Thus, in some embodiments, the sequence encoding a signal peptide encodes a secA1 signal peptide. An example of a secA1 signal peptide is the Listeriolisin O (LLO) signal peptide from Listeria monocytogenes . In some embodiments, the recombinant nucleic acid molecule or expression cassette comprising a polynucleotide encoding an LLO signal peptide further comprises a polynucleotide sequence encoding an LLO PEST sequence. Other examples of secA1 signal peptides suitable for use in the present invention include the Usp45 gene in Lactococcus lactis (see Table 1 above and Example 12 below) and the signal peptide from the Pag (protective antigen) gene from Bacillus anthracis . Thus, in some embodiments the signal peptide is a protective antigen signal peptide from Bacillus anthracis . In some other embodiments, the signal peptide is a secA1 signal peptide except for a protective antigen signal peptide from Bacillus anthracis . Another example of secA1 signal peptide is SpsB signal peptide from Straphylococcus aureus (Sharkov et al, J. of Biological Chemistry, 277: 5796-5803 (2002)).

In some alternative embodiments, the heterologous coding sequence is genetically fused with a signal peptide recognized by the secA2 pathway protein secretion complex. Secondary SecA paralogs (SecA2) have been identified as nine Gram-positive bacteria that cause serious or fatal infections in humans. SecA2 is required for the secretion of subsets of Listeria, Mycobacteria, and exported protein bodies (secretory proteins) of Streptococci (Braunstein et al., Mol. Microbiol. 48: 453-64 (2003); Bensing et al., Mol Microbiol., 44: 1081-94 (2002); Lenz et al., Mol.Microbiol., 45: 1043-1056 (2002); and Braunstein et al., J. Bacteriology, 183: 6979-6990 (2001). ). Listeria monocytogenes SecA2 was identified through its smooth-rough changes in bacteria and its relationship with mutations in secA2 reduced toxicity of L. monocytogenes and Mycobacterium tuberculosis .

For example, Listeria protein p60 is a peptidoglycan autolyse secreted by the secA2 pathway. By way of example, the signal peptidase cleavage site from secA2 signal peptide and p60 can be genetically linked to the amino terminus of the desired protein (eg, antigen) -coding gene. In one embodiment, the pre-protein comprising secA2 signal peptide and signal peptidase-antigen fusion is transcribed from an intracellular bacterial cassette transported through the Gram-positive cell wall, and the true heterologous protein is released into the extracellular environment do.

Alternatively, the heterologous sequence can be integrated "in-frame" at p60 such that the heterologous protein is secreted in the form of a chimeric p60-heterologous protein. Insertion of the in-frame heterologous protein coding sequence into p60 may occur, for example, at the junction between the signal peptidase cleavage site and the mature p60 protein. In this embodiment, the chimeric protein possesses an appropriate secA2 secretion signal, and also its autolytic activity, meaning that the heterologous protein is secreted as a free passenger of p60. In-frame integration of the heterologous antigen into p60 can be engineered at any point in p60 that retains both secretory and autolytic activity of the p60 protein. Examples of partial expression cassettes suitable for insertion of a desired antigen or other heterologous polypeptide coding sequence are described in Example 13 below.

In some embodiments, the fusion protein encoded by the recombinant nucleic acid molecule is chimeric, including bacterial proteins having certain desired properties (in addition to the desired heterologous protein, such as an antigen). In some embodiments, the chimera comprises a hydrolase. In some embodiments, the recombinant nucleic acid molecule encodes a p60 chimera comprising endopeptidase p60, a peptidoglycan hydrolase that disrupts bacterial cell walls. In some embodiments, the fusion protein encoded by the recombinant nucleic acid molecule is, for example, p60 (eg, Genbank Accession No. NP — 464110) or N-acetylmuramidase (NamA) (Genbank Accession No. NP — 466213), both L. monocytogenes hydrolase, such as secA2 dependent secreted protein that disrupts cell walls. Such protein chimeric compositions utilize the molecular chaperones required for the secretion of bacterial proteins, as well as the activity of bacterial proteins that can promote their secretion. Certain protein chimeras, consisting of the correct placement of heterologous protein coding sequences with L. monocytogenes hydrolase, result in sufficient expression and secretion of heterologous proteins (see, eg, Specific Examples, Example 29). Thus, in some embodiments, the signal peptide encoded by the recombinant nucleic acid molecule as part of the fusion protein is a p60 signal peptide. In some embodiments, the signal peptide encoded by the recombinant nucleic acid molecule as part of a fusion protein is a NamA signal peptide.

In some embodiments, the recombinant nucleic acid molecule encodes a p60 protein in the same translation reading frame as the first polynucleotide encoding the p60 signal peptide and the second polynucleotide encoding another polypeptide (eg, an antigen). Polynucleotide sequences, or fragments thereof. Subsequently, the recombinant nucleic acid molecule encodes a fusion protein comprising a signal peptide, a polypeptide encoded by a second polynucleotide (eg, an antigen), and a p60 protein, or fragment thereof. In this embodiment, the second polynucleotide is preferably located in the third polynucleotide or between the first and third polynucleotides.

In some embodiments, the secA2 signal peptide is a secA2 signal peptide derived from Listeria . For example, in some embodiments, the signal peptide is a secA2 signal peptide such as a p60 signal peptide or an N-acetylmuramidase (NamA) signal peptide from L. monocytogenes . In addition, other L. monocytogenes proteins have not been secreted in the absence of secA2 (Lenz et al., Mol. Microbiology 45: 1043-1056 (2002)) and polynucleotides encoding signal peptides from these proteins are in some embodiments. Can be used. In addition , secA2 signal peptides from bacteria other than Listeria can be used for expression and secretion of heterologous proteins from recombinant Listeria or other bacteria. For example, by way of non-limiting example, secA2 signal peptide from B. anthracis can be used in recombinant nucleic acid molecules and / or expression cassettes. In another embodiment, secA2 signal peptide from S. aureus is used. See Table 1. In addition, secreted proteins have been identified through the secA2 pathway in other bacteria (eg, Braunstein et al., Mol. Microbiol., 48: 453-64 (2003) and Bensing et al., Mol. Microbiol. 44: 1081-94 (2002)).

Additional secreted proteins can be identified via the secA2 pathway. secA2 homologues have been identified in numerous bacterial species (see, eg, Lenz et al., Mol. Microbiology 45: 1043-1056 (2002) and Braunstein et al., J. Bacteriology, 183: 6979-6990 (2001)). . Additional secA2 homologues can be identified by further sequence comparison using techniques known to those skilled in the art. Once homology is identified, the homologues can be deleted from bacterial organisms resulting in ΔsecA2 mutations. Supernatant proteins from wild-type and mutant bacterial cultures can be analyzed by any proteomic technique known to those skilled in the art to TCA-precipitate and determine whether the protein is secreted by wild-type bacteria or by ΔsecA2 mutations. have. For example, secreted proteins can be analyzed via SDS-PAGE and silver staining. The resulting bands can be compared to identify those proteins in which secretion did not occur in the absence of SecA2. (See, eg, Lenz et al., Mol. Microbiology 45: 1043-1056 (2002)). Subsequently, the N-terminal sequence of these proteins can be analyzed to determine the secA2 signal peptide sequence used by that protein (eg, along with an algorithm to predict signal peptide cleavage sites). N-terminal sequencing by automated Edman degradation can be performed to identify the sequence of the signal peptide.

In alternative embodiments, the polynucleotides encode polypeptides (eg, heterologous polypeptide sequences) genetically fused with a signal peptide recognized by the Tat pathway protein secretion complex. The Tat secretion pathway is used to produce Listeria spp. It is used by bacteria containing. For example, the Listeria innocua protein YwbN has a putative Tat motif at its amino terminus and uses the Tat pathway for secretion therein (Genbank Accession No. NP_469731 [gi ｜ 16799463 ｜ ref ｜ NP_46973 ｜ 1.1. B. subtilis Conserved hypothetical protein similar to YwbN protein (Listeria innocua), incorporated herein by reference). Another protein containing the Tat signal peptide is the YwbN protein from Listeria monocytogenes strain EGD (e) (Genbank Accession No. NP_463897 [gi ｜ 16802412 | ref ｜ NP_463897.1 ｜ B. subtilis A hypothetical protein similar to the YwbN protein (Listeria monocytogenes) EGD (e)]). For example, the YwbN signal peptide and the signal peptidase cleavage site from YwbN can be genetically linked to the amino terminus of the desired protein (eg antigen) -coding gene. The pre-protein consisting of Tat signal peptide and signal-peptidase antigen fusion will be transcribed from the expression cassette in bacteria and transported through the Gram-positive cell wall, and this truly heterologous protein is released into the extracellular environment. Another protein predicted to be secreted from Listeria innocua via the pathway is 3- oxoxacyl -acyl carrier protein synthase (Genbank Accession No. NP_4). 71636 [gi | 16801368 | ref ｜ NP_471636.1 ｜ 3 (Osocyl (acyl (similar to Listeria innocua )). From any of these proteins predicted to be secreted from Listeria via the Tat secretion pathway The polynucleotide coding signal sequence of may be used in the polynucleotides, expression cassettes, and / or expression vectors described herein.

In addition, Tat signal sequences from other bacteria can be used as signal peptides including, but not limited to, PhoD from B. Subtilis . Examples of Tat signal peptides from Bacillus subtilis such as phoD are described in Jongbloed et al., J. of Biological Chemistry, 277: 44068-44078 (2002), which are incorporated by reference in their entirety; Jongbloed et al., J: of Biological Chemistry, 275: 41350-41357 (2000), Pop et al., J: of Biological Chemistry, 277: 3268-3273 (2002); van Dijl et al., J of Biotechnology, 98: 243-254 (2002); and Tjalsma et al., Microbiology and Molecular Biology Reviews, 64: 515-547 (2000). Other proteins identified in B. Subtilis predicted to be secreted by the Tat pathway include sequences with the following, all of which are incorporated herein by reference: Genbank / Embl Accession Number: CAB 15017 [gi | 2635523 | emb | CAB 15017.1 | The two are similar (component sensor histidine kinase (YtsA) (Bacillus subtilis)]; CAB12056 [gi | 2632548 | emb | CAB12056.1 | phosphodiesterase / alkali phosphatase D ( Bacillus subtilis)]; CAB12081 [gi | 2632573 | emb | CAB12081.1 | Similar to the protein of hypothesis (Bacillus subtilis)]; CAB13278 [gi | 2633776 | emb | CAB 13278.1 | CAB14172 [gi ｜ 2634674 ｜ emb ｜ CAB14172.1 | Metaquinol: cytochrome c redox enzyme (iron (sulfur subunit) (Bacillus subtilis)]; CAB15089 [gi ｜ 2635595 ｜ emb ｜ CAB15089.1 ｜ yubF (Bacillus subtilis)] and CAB15852 [gi | 2636361 | emb | CAB15852.1 | Alternative gene name: ipa (29d ~ hypothetical) Thus, in some embodiments, the signal peptide encoded by the polynucleotides in the recombinant nucleic acid molecule and / or expression cassette is a Tat signal peptide derived from B. Subtilis from Pseudomonas aeruginosa . Tat signal peptide phase information is provided in Ochsner et al., PNAS, 99: 8312-8317 (2002) In addition, Tat signal peptides from a variety of other bacteria are described by Dilks et. al., J. of Bactoriology, 185: 1478-1483 (2003) and Berks et al., Molecular Microbiology, 35: 260-274 (2000).

Additional Tat signal peptides may be identified and distinguished from Sec-type signal peptides by their "twin-arginine" matching motifs. As pointed out above, the signal peptide associated with the protein secreted by the Tat pathway has three organisms similar to the Sec signal peptide, but has an RR-motif (RRX-#-#, # located at the N-domain / H-domain edge). Is a hydrophobic moiety). In addition, Tat signal peptides are generally longer and less hydrophobic than Sec-type signal peptides. See, eg, Berks et al., Adv. Microb. Physiol., 47: 187-254 (2003) and Berks et al., Mol. Microbiol. 35: 260-74 (2000).

In addition, techniques similar to those described above to identify novel proteins and their corresponding SecA2 signal peptides secreted by the SecA2 pathway can also be used to identify novel proteins and their signal peptides secreted via the Tat pathway. have. Jongbloed et al., J. Biological Chem., 277: 44068-44078 (2002), can be used to identify proteins expressed by types of bacteria, such as proteins secreted via the twin-arginine translocation pathway. Provide an example of the technique.

IV. Polypeptide

The recombinant nucleic acid molecules described herein, and the expression cassettes or expression vectors described herein, can be used to encode any desired polypeptide. In particular, recombinant nucleic acid molecules, expression cassettes, and expression vectors are useful for expressing heterologous polypeptides in bacteria.

In some embodiments, the polypeptide encoded by the recombinant nucleic acid molecule, expression cassette, and / or polynucleotide of the expression vector (depending on the recombinant nucleic acid molecule, expression cassette or expression vector used) is part of a fusion protein along with the signal peptide. Is coded. In other embodiments, the encoded polypeptide is encoded as each polypeptide by a recombinant nucleic acid molecule, expression cassette, or expression vector. In still other embodiments, the polypeptide encoded by the recombinant nucleic acid molecule, expression cassette, or polynucleotide of the expression vector is encoded as part of a fusion protein that does not comprise a signal peptide. In yet another embodiment, a polypeptide encoded by a recombinant nucleic acid molecule, expression cassette, or polynucleotide of an expression vector of the invention is encoded as part of a fusion protein (also referred to herein as a protein chimera), and the polypeptide is another polypeptide. It is embedded in the sequence.

Thus, each of the polypeptides listed herein (hereinafter and elsewhere) encoded by the recombinant nucleic acid molecule, expression cassette, or polynucleotide of the expression vector of the present invention are fused to a fusion protein (signal peptide and / or other polypeptide). Or as a separate polypeptide by a recombinant nucleic acid molecule, expression cassette, or expression vector, depending on the specific recombinant nucleic acid molecule, expression cassette or expression vector used. For example, in some embodiments, a recombinant nucleic acid molecule comprising a polynucleotide encoding an antigen CEA will encode CEA as a fusion protein with a signal peptide.

In some embodiments, the polypeptide is part of a fusion protein encoded by a recombinant nucleic acid molecule, expression cassette, or expression vector and is heterologous to the signal peptide of the fusion protein. In some embodiments, the polypeptide is located in another polypeptide sequence that is heterologous (eg, secreted protein or autolysate, or fragment or variant thereof).

In some embodiments, the polypeptide is a bacterial polypeptide (Listeria or non-Listeria). In some embodiments, the polypeptide is not a bacterial polypeptide. In some embodiments, the polypeptide encoded by the polynucleotide is a mammalian polypeptide. For example, a polypeptide can match a polypeptide sequence found in a human (ie, a human polypeptide). In some embodiments, the polypeptide is a Listeria polypeptide. In some embodiments, the polypeptide is a non-Listeria polypeptide. In some embodiments, the polypeptide is not (i.e. exogenous) a recombinant nucleic acid molecule, expression cassette, and / or bacterial natural polypeptide to which the expression vector is incorporated.

In another embodiment, the polynucleotide encoding the polypeptide is codon-optimized for expression in bacteria. In some embodiments, polynucleotides encoding polypeptides are fully codon-optimized for expression in bacteria. In some embodiments, the polypeptide encoded by the codon-optimized polynucleotide is exogenous to the bacterium (ie, heterologous to the bacterium).

The term "polypeptide" is used herein interchangeably with "peptide" and "protein" and is not intended to be limiting with respect to the length or size of amino acid sequences included herein. Typically, however, the polypeptide will comprise at least about 6 amino acids. In some embodiments, the polypeptide will comprise at least about 9, at least about 12, at least about 20, at least about 30, or at least about 50 amino acids. In some embodiments, the polypeptide comprises at least about 100 amino acids. In some embodiments, a polypeptide is one particular domain of a protein (eg, extracellular domain, intracellular domain, catalytic domain, or binding domain). In some embodiments, the polypeptide comprises a whole (ie full length) protein.

In some embodiments, the polypeptide encoded by the recombinant nucleic acid molecule, expression cassette, and / or polynucleotide of the expression vector comprises an antigen or protein that provides palliative treatment for the disease. In some embodiments, the polypeptide encoded by the recombinant nucleic acid molecule, expression cassette, and / or polynucleotide of the expression vector is an antigen or protein that provides palliative treatment for the disease. In some embodiments, the polypeptide encoded is a therapeutic protein (or includes a therapeutic protein).

In some embodiments, a polypeptide encoded by a recombinant nucleic acid molecule, expression cassette, and / or polynucleotide of a vector comprises an antigen (eg, any of the antigens described herein). In some embodiments, the polypeptide encoded by the recombinant nucleic acid molecule, expression cassette, and / or polynucleotide of the vector is an antigen. In some embodiments, the antigen is a bacterial antigen. In some embodiments, the antigen is a non-Listeria antigen. In other embodiments, the antigen is a non-bacterial antigen. In some embodiments, the antigen is a mammalian antigen. In some embodiments, the antigen is a human antigen. In some embodiments, the polypeptide is (or comprises an antigen) an antigen comprising one or more immunogenic epitopes. In some embodiments, the antigen comprises one or more MHC class I epitopes. In other embodiments, the antigen comprises one or more MHC class II epitopes. In some embodiments, the epitope is a CD4 + T-cell epitope. In another embodiment, the epitope is a CD8 + T-cell epitope.

The polynucleotide encoding the antigen is not limited to any precise nucleic acid sequence (eg, encoding a naturally occurring, full length antigen), but may be any case administered to an individual within a bacterium or composition of the invention. Also, as used herein, the term “antigen” is understood to include fragments of large antigenic proteins, so long as the fragment is an antigen (ie, immunogenic). In addition, in some embodiments, the antigen encoded by the recombinant nucleic acid molecule, expression cassette, or polynucleotide of the expression vector may be various naturally occurring antigen sequences. (Similar to polynucleotides encoding other, non-antigenic proteins, the sequence of the polynucleotides encoding a given protein may be as long as it provides the desired effect (eg, a mitigating effect) when the desired protein to be expressed is administered to the individual. It may vary.

Antigens derived from other antigens include antigens that are antigen (ie, immunogenic) fragments of other antigens, antigen variants of other antigens, or antigen variants of fragments of other antigens. Variants of an antigen include antigens that differ from the original antigen in one or more substitutions, deletions, additions, and / or insertions.

The antigen fragments may be of any length, but are typically typically at least 6 amino acids, at least about 9 amino acids, at least about 12 amino acids, at least about 20 amino acids, at least about 30 amino acids, at least about 50 amino acids, or at least about 100 amino acids. Antigen fragments of the antigen include at least one epitope from the antigen. In some embodiments, the epitope is an MHC class I epitope. In other embodiments, the epitope is an MHC class II epitope. In some embodiments, the epitope is a CD4 + T-cell epitope. In another embodiment, the epitope is a CD8 + T-cell epitope.

Various algorithms and software packages useful for predicting antigenic regions (including epitopes) in proteins are available to those skilled in the art. For example, algorithms that can be used to select epitopes that bind to MHC class I and class II molecules are commercially available. For example, commercially available "SYFPEITHI" algorithms can be used to predict MHC-binding peptides (Rammensee et al. (1999) Immunogenetics 50: 213-9). For other examples of commercially available algorithms, see the following references: Parker et al. (1994) J. Immunol 152: 163-75; Singh and Raghava (2001) Bioinformatics 17: 1236-1237; Singh and Raghava (2003) Bioinformatics 19: 1009-1014; Mallios (2001) Bioinformatics 17: 942-8; Nielsen et al. (2004) Bioinformatics 20: 1388-97; Donnes et al. (2002) BMC Bioinformatics 3:25; Bhasin, et al. (2004) Vaccine 22: 3195-204; Guan et al. (2003) Nucleic Acids Res 31: 3621-4; Reche et al. (2002) Hum. Immunol. 63: 701-9; Schirle et al. (2001) J. Immunol Methods 257: 1-16; Nussbaum et al. (2001) Immunogenetics (2001) 53: 87-94; Lu et al. (2000) Cancer Res. 60: 5223-7. Also, for example, Vector NTI? Suite (Informax, Inc., Bethesda, MD), GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.), Welling, et al. (1985) FEBS Lett. 188: 215-218, Parker, et al. (1986) Biochemistry 25: 5425-5432, Van Regenmortel and Pellequer (1994) Pept. Res. 7: 224-228, Hopp and Woods (1981) PNAS 78: 3824-3828, and Hopp (1993) Pept. Res. See 6: 183-190. Any algorithm or software package discussed in the references listed above in this paragraph can be used to make predictions for MHC class I and / or class II binding peptides or epitopes, else for identification of proteosome cleavage sites, and still others This relates to the prediction of antigenicity based on hydrophobicity.

Once a candidate antigen fragment that is believed to contain at least one native epitope of interest is identified, the polynucleotide sequence encoding that sequence can be integrated into an expression cassette and introduced into a Listeria vaccine vector or other bacterial vaccine vector. Subsequently, the immunogenicity of the antigen fragments can be confirmed by evaluating the immune response generated by Listeria or other bacteria expressing the fragments. Standard immunological assays such as ELISPOT assays, intracellular cytokine staining (ICS) assays, cytotoxic T-cell activity assays and the like can be used to confirm that fragments of selected antigens maintain the desired immunogenicity. Examples of these types of assays are provided in the Examples below (see, eg, Example 21). In addition, the anti-tumor effect of Listeria and / or bacterial vaccines can be assessed using the methods described below in the Examples (eg, after transplantation of CT26 murine colon cells expressing antigen fragments in mice, candidates Vaccination of mice with the vaccine and observation of tumor size, metastasis, survival, etc. compared to control and / or full length antigens).

Additionally, large databases containing epitope and / or MHC ligand information used to identify antigen fragments are commercially available. For example, Brusic et al. (1998) Nucleic Acids Res. 26: 368-371; Schonbach et al. (2002) Nucleic Acids Research 30: 226-9; and Bhasin et al. (2003) Bioinformatics 19: 665-666; and Rammensee et al. (1999) Immunogenetics 50: 213-9.

The amino acid sequence of the antigen variant has at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% identity to the original antigen.

In some embodiments, the antigen variant is a conservative variant having at least about 80% identity to the original antigen and the substitution between the antigen variant and the sequence of the original antigen is a conservative amino acid substitution. The following substitutions are considered conservative amino acid substitutions: valine, isoleucine, or leucine are substituted with alanine; Lysine, glutamine, or asparagine is substituted with arginine; Glutamine, histidine, lysine, or arginine is substituted with asparagine; Glutamic acid is substituted with aspartic acid; Serine is substituted with cysteine; Asparagine is substituted with glutamine; Aspartic acid is substituted with glutamic acid; Proline or alanine is substituted with glycine; Asparagine, glutamine, lysine or arginine is substituted with histidine; Leucine, valine, methionine, alanine, phenylalanine, or norleucine are substituted with isoleucine; Norleucine, isoleucine, valine, methionine, alanine, or phenylalanine are substituted with leucine; Arginine, glutamine, or asparagine is substituted with lysine; Leucine, phenylalanine, or isoleucine are substituted with methionine; Leucine, valine, isoleucine, alanine, or tyrosine are substituted with phenylalanine; Alanine is substituted with proline; Threonine is substituted with serine; Serine is substituted with threonine; Tyrosine or phenylalanine is substituted with tryptophan; Tryptophan, phenylalanine, threonine, or serine is substituted with tyrosine; Tryptophan, phenylalanine, threonine, or serine is substituted with tyrosine; Isoleucine, leucine, methionine, phenylalanine, alanine, or norleucine are substituted with valine. In some embodiments, the antigen variant is a conservative variant that has at least about 90% identity to the original antigen.

In some embodiments, antigens derived from other antigens are substantially equivalent to other antigens. Antigens derived from other antigens are substantially equivalent to the original antigens derived if the derived antigens are at least about 70% identical in amino acid sequence to the original antigen and retain at least about 70% immunogenicity to the original antigen. Is equivalent to In some embodiments, the substantially equivalent antigen has at least about 80%, at least about 90%, at least about 95%, or at least about 98% identity with respect to the original antigen in the amino acid sequence. In some embodiments, substantially equivalent antigens comprise only conservative substitutions as compared to the original antigen. In some embodiments, the substantially equivalent antigen maintains at least about 80%, at least about 90%, or at least about 95% immunogenicity with respect to the original antigen. Antigens derived from any of the numerous immunogenicity assays known to those skilled in the art to determine the immunogenicity of a particular derived antigen and to determine whether the derived antigen is substantially equivalent to that of the original antigen. All original antigens can be tested. For example, Listeria expressing the original antigen or derived antigen can be prepared as described herein. Listeria's ability to express different antigens to produce an immune response was achieved by inoculating mice with Listeria and then using standard techniques such as ELISPOT assay, intracellular cytokine staining (ICS) assay, and cytotoxic T-cell activity assay. It can be measured by assessing the immunogenic response. Examples of these types of assays are provided in the Examples below (see, eg, Example 21).

In some embodiments, a polypeptide encoded by a recombinant nucleic acid molecule, expression cassette, and / or polynucleotide of a vector comprises an antigen. In some embodiments, the antigen is selected from the group consisting of a tumor-associated antigen, a polypeptide derived from a tumor-associated antigen, an infectious disease antigen, and a polypeptide derived from an infectious disease antigen.

In some embodiments, a polypeptide encoded by a polynucleotide of a recombinant nucleic acid molecule, expression cassette, and / or vector comprises a tumor-associated antigen or comprises an antigen derived from a tumor-associated antigen. In some embodiments, the polypeptide comprises a tumor-associated antigen. In some embodiments, the encoded polypeptide comprises one or more antigens that are tumor-associated antigens or antigens derived from tumor-associated antigens. For example, in some embodiments, the encoded polypeptide is mesothelin (or antigen fragment or antigen variant thereof) and K-Ras, 12-K-Ras, or PSCA (or K-Ras, we-K-Ras, Or antigenic fragments or antigenic variants of PSCA).

In some embodiments, the antigen encoded by the recombinant nucleic acid molecule, expression cassette and / or polynucleotide of the expression vector is a tumor-associated antigen or is derived from a tumor-associated antigen. In some embodiments, the antigen is a tumor-associated antigen.

In some embodiments, a polynucleotide of a recombinant nucleic acid molecule, expression cassette and / or expression vector encodes an antigen that is not identical to a tumor-associated antigen but is an antigen derived from a tumor-associated antigen (or a polypeptide comprising an antigen). Code). For example, in some embodiments, an antigen encoded by a recombinant nucleic acid molecule, expression cassette, and / or polynucleotide of an expression vector can be a fragment of a tumor-associated antigen, a variant of a tumor-associated antigen, or a tumor-associated antigen. It may also include variants of the fragments. In some cases, an antigen, such as a tumor antigen, can induce a more pronounced immune response in the vaccine if the amino acid sequence is slightly different from the sequence that is endogenous to the host. In other cases, the induced antigen elicits a less pronounced immune response than the original antigen, but is convenient for heterologous expression in Listeria vaccines, for example due to similar size. In some embodiments, the amino acid sequence of a variant of a tumor-associated antigen, or of a fragment of a tumor-associated antigen, differs by one or more amino acids from the sequence of a tumor-associated antigen, or a corresponding fragment thereof. Antigens derived from tumor-associated antigens will comprise at least one epitope sequence capable of eliciting a desired immune response upon expression of a polynucleotide encoding an antigen in a host.

Thus, in some embodiments, a polynucleotide of a recombinant nucleic acid molecule, expression cassette, or vector encodes a polypeptide comprising an antigen derived from a tumor-associated antigen, wherein the antigen comprises at least one antigen fragment of a tumor-associated antigen. do. In some embodiments, the polynucleotide of a recombinant nucleic acid molecule, expression cassette, or vector encodes an antigen derived from a tumor-associated antigen, wherein the antigen comprises at least one antigen fragment of a tumor-associated antigen. Antigen fragments comprise at least one epitope of tumor-associated antigen. In some embodiments, an antigen derived from another antigen is an antigen (ie, immunogenic) fragment or antigen variant of another antigen. In some embodiments, the antigen is an antigen fragment of another antigen. In some embodiments, the antigen is an antigenic variant of another antigen.

Numerous tumor-associated antigens recognized by T-cells have been identified (Renkvist et al., Cancer Immunol Innumother 50: 3-15 (2001)). These tumor-associated antigens may include differentiation antigens (eg PSMA, tyrosinase, gp100), tissue-specific antigens (eg PAP, PSA), generator antigens, tumor-associated viral antigens (eg HPV 16 E7) ), Testicular cancer antigens (eg MAGE, BAGE, NY-ESO-1), embryonic antigens (eg CEA, alpha-fetoprotein), oncogenic protein antigens (eg Ras, p53), overexpressed proteins Antigen (eg ErbB2 (Her2 / Neu), MUC1), or a mutated protein antigen. Tumor-associated antigens that may be encoded by heterologous nucleic acid sequences include 707-AP, Annexin II, AFP, ART-4, BAGE, β-catenin / m, BCL-2, bcr-abl, bcr-abl p190, bcr abl p210, BRCA-1, BRCA-2, CAMEL, CAP-1, CASP-8, CDC27 / m, CDK-4 / m, CEA (Huang et al., Exper Rev. Vaccines (2002) 1: 49- 63), CT9, CT10, Cyp-B, Dek-cain, DAM-6 (MAGE-B2), DAM-10 (MAGE-B1), EphA2 (Zantek et al., Cell Growth Differ. (1999) 10: 629 Carles-Kinch et al., Cancer Res. (2002) 62: 2840-7), ELF2M, EphA2 (Zantek et al., Cell Growth Differ. (1999) 10: 629-38; Carles-Kinch et al. Cancer Res. (2002) 62: 2840-7), ETV6-AML1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE -8, GnT-V, gp100, HAGE, HER2 / neu, HLA-A * 0201-R170I, HPV-E7, H-Ras, HSP70-2M, HST-2, hTERT, hTRT, iCE, apoptosis inhibitors (e.g. For example, survivin), KIAA0205, K-Ras, 12-K-Ras (K-Ras with codon 12 mutations), LAGE, LAGE-1, LDLR / FUT, MAGE-1, MAGE-2, MAGE-3, MAGE -6, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE -A12, MAGE-B5, MAGE-B6, MAGE-C2, MAGE-C3, MAGE-D, MART-1, MART-1 / Melan-A, MC1R, MDM-2, Mesothelin, Myosin / m, MUC1, MUC2, MUM-1, MUM-2, MUM-3, neo-polyA polymerase, NA88-A, N-Ras, NY-ESO-1, NY-ESO-1a (CAG-3), PAGE-4, PAP, proteinase 3 (PR3) (Molldrem et al., Blood (1996) 88: 2450-7; Molldrem et al., Blood (1997) 90: 2529-34), P15, p190, Pm1 / RARα, PRAME, PSA, PSM, PSMA, RAGE, RAS, RCASI, RU1, RU2, SAGE, SART-1, SART- 2, SART-3, SP17, SPAS-1, TEL / AML1, TPI / m, tyrosinase, TARP, TRP-1 (gp75), TRP-2, TRP-2 / INT2, WT-1, and NY-ESO- Alternatively transcribed NY-ESO-ORF2 and CAMEL proteins derived from the 1 and LAGE-1 genes.

In some embodiments, antigens encoded by recombinant nucleic acid molecules, expression cassettes, and / or polynucleotides of a vector include antigens that have not yet been identified, which tumor-associated antigens can induce tumor-specific immune responses. It may also include. In some embodiments, the polynucleotides of a recombinant nucleic acid molecule, expression cassette, and / or vector encode one or more tumor-associated antigens.

In some embodiments, the antigen is mesothelin (Argani et al., Clin Cancer Res. 7 (12): 3862-8 (2001)), Sp17 (Lim et al., Blood 97 (5): 1508-10 (2001) )), gp100 (Kawakami et al., Proc. Natl. Acad. Sci. USA 91: 6458 (1994)), PAGE-4 (Brinkmann et al., Cancer Res. 59 (7): 1445-8 (1999) ), TARP (Wolfgang et al., Proc. Natl. Acad. Sci. USA 97 (17): 9437-42 (2000)), EphA2 (Tatsumi et al., Cancer Res. 63 (15): 4481-9 ( 2003)), PR3 (Muller-Berat et al., Clin. Immunol. Immunopath. 70 (1): 51-9 (1994)), prostate stem cell antigen (PSCA) (Reiter et al., Proc. Natl. Acad Sci., 95: 1735-40 (1998); Kiessling et al., Int. J. Cancer, 102: 390-7 (2002)), or SPAS-1 (US Patent Application Publication No. 2002/0150588). .

In some embodiments of the invention, the antigen encoded by the recombinant nucleic acid molecule or expression cassette is CEA. In other embodiments, the antigen is an antigen fragment and / or antigen variant of CEA. CEA is a 180-kDA membrane intracellular adhesion glycoprotein that is overexpressed in a significant portion of human tumors, including 90% of colon, stomach, and pancreatic cancer, 70% of non-small cell lung cancer, and 50% of breast cancer ( Hammarstrom, Semin. Cancer Biol., 9: 67-81). Numerous immunotherapies such as anti-factor monoclonal antibody mimicking CEA (Foon et al., Clin. Cancer Res., 87: 982-90 (1995)) or inoculation using recombinant vaccinia virus expressing CEA (Tsang et al. , J. Natl. Cancer Inst., 87: 982-90 (1995)), but unfortunately have had limited success, nevertheless, investigators have found HLA * 0201-restricted epitope, CAP-1 (CEA605). -613), which is recognized by human T cell lines generated from inoculated patients Inoculation of DC pulsed patients with this epitope did not induce a clinical response (Morse et al., Clin. Cancer Res., 5: 1331-8 (1999)) Recently, the CEA605-613 peptide agonist was identified with irregular aspartate for asparagine substitution at position 610 (CAP1-6D). While not altering MHC binding affinity, the use of altered peptide ligands (APLs) This resulted in improved production of CEA-specific cytotoxic T lymphocytes (CTLs) in vitro CAP1-6D-specific CTLs retained their ability to recognize and lyse tumor cells expressing native CEA (Zaremba et. al., Cancer Res., 57: 4570-7 (1997); Salazar et al., Int. J. Cancer, 85: 829-38 (2000)) Fong et al. Flt3-ligand expansion incubated with this APL Introduced the introduction of CEA-specific immunity in patients with colon cancer inoculated with pretreated DCs Encouragingly, two out of 12 patients with dramatic tumor regression correlated with the introduction of peptide-MHC tetramer + T cells after inoculation. (Fong et al., Proc. Natl. Acad. Sci. USA, 98: 8809-14 (2001)).

In other embodiments, the antigen is proteinase-3 or is derived from proteinase-3. For example, in one embodiment, the antigen comprises the HLA-A2.1-restricted peptide PR1 (aa 169-177; VLQELNVTV (SEQ ID NO: 63)). Proteinase-3 and / or PR1 epitopes are available in the following references: US Pat. No. 5,180,819, Molldrem, et al., Blood, 90: 2529-2534 (1997); Molldrem et al., Cancer Research, 59: 2675-2681 (1999); Molldrem, et al., Nature Medicine, 6: 1018-1023 (2000); And Molldremet al., Oncogene, 21: 8668-8673 (2002).

In some embodiments, the polypeptide encoded by a polynucleotide in a recombinant nucleic acid molecule, expression cassette, and / or vector is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY- Comprises an antigen selected from the group consisting of ESO-1, WT-1, survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, and CEA, or K- Ras, H-Ras, N-Ras, 12-K-Ras, Mesothelin, PSCA, NY-ESO-1, WT-1, Survivin, gp100, PAP, Proteinase 3, SPAS-1, SP- And antigens derived from antigens selected from the group consisting of 17, PAGE-4, TARP, and CEA.

In some embodiments, the polypeptide encoded by a polynucleotide in a recombinant nucleic acid molecule, expression cassette, and / or vector is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY- Antigens selected from the group consisting of ESO-1, WT-1, survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, or CEA. In some embodiments, the polypeptide comprises K-Ras. In some embodiments, the polypeptide comprises H-Ras. In some embodiments, the polypeptide comprises N-Ras. In some embodiments, the polypeptide comprises K-Ras. In some embodiments, the polypeptide comprises mesothelin (eg, human mesothelin). In some embodiments, the polypeptide comprises PSCA. In some embodiments, the polypeptide comprises NY-ESO-1. In some embodiments, the polypeptide comprises WT-1. In some embodiments, the polypeptide comprises survivin. In some embodiments, the polypeptide comprises gp100. In some embodiments, the polypeptide comprises PAP. In some embodiments, the polypeptide comprises proteinase 3. In some embodiments, the polypeptide comprises SPAS-1. In some embodiments, the polypeptide comprises SP-17. In some embodiments, the polypeptide comprises PAGE-4. In some embodiments, the polypeptide comprises TARP. In some embodiments, the polypeptide comprises CEA.

In some embodiments, the antigen encoded by a polynucleotide in a recombinant nucleic acid molecule, expression cassette, and / or vector is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY- Antigen, selected from the group consisting of ESO-1, WT-1, survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, or CEA. In some embodiments, the antigen is K-Ras. In some embodiments, the antigen is H-Ras. In some embodiments, the antigen is N-Ras. In some embodiments, the antigen is K-Ras. In some embodiments, the antigen is mesothelin. In some embodiments, the antigen is PSCA. In some embodiments, the antigen is NY-ESO-1. In some embodiments, the antigen is WT-1. In some embodiments, the antigen is survivin. In some embodiments, the antigen is gp100. In some embodiments, the antigen is PAP. In some embodiments, the antigen is proteinase 3. In some embodiments, the antigen is SPAS-1. In some embodiments, the antigen is SP-17. In some embodiments, the antigen is PAGE-4. In some embodiments, the antigen is TARP. In some embodiments, the antigen is CEA. In some embodiments, the antigen is human mesothelin.

In some embodiments, the antigen is an antigen derived from mesothelin, SPAS-1, proteinase-3, EphA2, SP-17, gp100, PAGE-4, TARP, or CEA, or one of its proteins. In some embodiments the antigen is mesothelin or is derived from mesothelin. In other embodiments, the antigen is EphA2 or an antigen derived from EphA2. In some embodiments, the antigen encoded by a polynucleotide in a recombinant nucleic acid molecule, expression cassette, or expression vector described herein is not Epha2 (or an antigen derived from Epha2). In some embodiments, the antigen is a tumor-associated antigen other than Epha2. In some embodiments, the antigen is derived from a tumor-associated antigen other than Epha2. In some embodiments, a polypeptide encoded by a polynucleotide in a recombinant nucleic acid molecule, expression cassette, and / or expression vector comprises an antigen except Epha2. In some embodiments, a polypeptide encoded by a polynucleotide in a recombinant nucleic acid molecule, expression cassette, and / or expression vector comprises an antigen other than Epha2 or an antigen derived from Epha2.

In some embodiments, the polynucleotides in the recombinant nucleic acid molecule, expression cassette, and / or expression vector are K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, It encodes a polypeptide comprising an antigen derived from WT-1, survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, or CEA. In some embodiments, the polypeptide comprises an antigen derived from K-Ras. In some embodiments, the polypeptide comprises an antigen derived from H-Ras. In some embodiments, the polypeptide comprises an antigen derived from N-Ras. In some embodiments, the polypeptide comprises an antigen derived from 12-K-Ras. In some embodiments, the polypeptide comprises an antigen derived from mesothelin. In some embodiments, the polypeptide comprises an antigen derived from PSCA. In some embodiments, the polypeptide comprises an antigen derived from NY-ESO-1. In some embodiments, the polypeptide comprises an antigen derived from WT-1. In some embodiments, the polypeptide comprises an antigen derived from survivin. In some embodiments, the polypeptide comprises an antigen derived from gp100. In some embodiments, the polypeptide comprises an antigen derived from PAP. In some embodiments, the polypeptide comprises an antigen derived from proteinase 3. In some embodiments, the polypeptide comprises an antigen derived from SPAS-1. In some embodiments, the polypeptide comprises an antigen derived from SP-17. In some embodiments, the polypeptide comprises an antigen derived from PAGE-4. In some embodiments, the polypeptide comprises an antigen derived from TARP. In some embodiments, the polypeptide comprises an antigen derived from CEA.

In some embodiments, the polynucleotides in the recombinant nucleic acid molecule, expression cassette, and / or expression vector are K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, Encodes an antigen derived from WT-1, survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, or CEA. In some embodiments, the antigen is derived from K-Ras. In some embodiments, the antigen is derived from H-Ras. In some embodiments, the antigen is derived from N-Ras. In some embodiments, the antigen is derived from 12-K-Ras. In some embodiments, the antigen is an antigen derived from mesothelin. In some embodiments, the antigen is an antigen derived from PSCA. In some embodiments, the antigen is an antigen derived from NY-ESO-1. In some embodiments, the antigen is an antigen derived from WT-1. In some embodiments, the antigen is an antigen derived from survivin. In some embodiments, the antigen is an antigen derived from gp100. In some embodiments, the antigen is an antigen derived from PAP. In some embodiments, the antigen is an antigen derived from proteinase 3. In some embodiments, the antigen is an antigen derived from SPAS-1. In some embodiments, the antigen is an antigen derived from SP-17. In some embodiments, the antigen is an antigen derived from PAGE-4. In some embodiments, the antigen is an antigen derived from TARP. In some embodiments, the antigen is an antigen derived from CEA.

In some embodiments, the antigen is mesothelin, or an antigen fragment or antigen variant thereof. Thus, in some embodiments, polypeptides encoded by polynucleotides in recombinant nucleic acid molecules, expression cassettes and / or vectors include mesothelin, or antigen fragments or antigen variants thereof. In some embodiments, the polypeptide encoded by the polynucleotide is mesothelin, or an antigen fragment or antigen variant thereof.

In some embodiments, the antigen is mesothelin (eg, human mesothelin) lacking a mesothelin signal peptide and / or a GPI (glycosylphosphatidylinositol) anchor. Thus, in some embodiments, polypeptides encoded by polynucleotides include mesothelin signal peptides and / or mesothelins that lack GPI anchors. In some embodiments, the polypeptides encoded by the polynucleotides are mesothelins lacking mesothelin signal peptides and / or GPI anchors. In some embodiments, the polypeptide encoded by the polynucleotide is human mesothelin lacking a mesothelin signal peptide and / or a GPI anchor. In some embodiments, the polypeptide encoded by the polynucleotide is human mesothelin that lacks the mesothelin signal peptide and the GPI anchor.

In some embodiments, the antigen is NY-ESO-1, or an antigen fragment or antigen variant thereof. Thus, in some embodiments, a polypeptide encoded by a polynucleotide in a recombinant nucleic acid molecule, expression cassette, or vector comprises an antigen that is NY-ESO-1, or an antigen fragment or antigen variant thereof. In some embodiments, the polypeptide is an antigen that is NY-ESO-1, or an antigen fragment or antigen variant thereof.

In some embodiments, a polypeptide encoded by a polynucleotide in a recombinant nucleic acid molecule, expression cassette, or vector comprises at least one antigenic fragment of a tumor-associated antigen, eg, human prostate stem cell antigen (PSCA; Genbank Accession No. AF043498 ), Human testicular antigen (NY-ESO-1; Genbank Accession No. M_001327), human fetal antigen (CEA; Genbank Accession No. M29540), human mesothelin (Genbank Accession No. U40434), human survivin (Genbank Accession No. U75285) , Human proteinase 3 (Genbank No. X55668), human K-Ras (Genbank Accession No. M54969 & P01116), Human H-Ras (Genbank Accession No. P01112), Human N-Ras (Genbank Accession No. P01111), and Human 12-K-Ras (K-Ras comprising Gly12Asp mutations) (see, eg, Genbank Accession No. K00654). In some embodiments, a polypeptide encoded by a polynucleotide in a recombinant nucleic acid molecule, expression cassette, or expression vector comprises an antigenic fragment of a tumor-associated antigen having at least one conservatively substituted amino acid. In some embodiments, a polypeptide encoded by a polynucleotide in a recombinant nucleic acid molecule, expression cassette, or expression vector comprises an antigen fragment having at least one deleted amino acid residue. In some embodiments, a recombinant nucleic acid molecule, expression cassette, or polypeptide encoded by a polynucleotide in an expression vector is derived from a combination of one or more types of tumor-associated antigens, eg, antigen fragments derived from mesothelin and Ras. Combination of antigenic sequences.

Representative regions of tumor antigens that are predicted to be antigenic include: amino acids 25-35 of the PSCA amino acid sequence in Genbank Accession No. AF043498; 70-80; And 90-118; Amino acids 40-55, 75-85, 100-115, and 128-146 of NY-ESO-1 in Genbank Accession No. NM001327; Amino acids 70-75, 150-155, 205-225, 330-340, and 510-520 of the CEA amino acid sequence of Genbank Accession No. M29540; Amino acids 90-110, 140-150, 205-225, 280-310, 390-410, 420-425, and 550-575 of the mesothelin polypeptide sequence of Genbank Accession No. U40434; Amino acids 12-20, 30-40, 45-55, 65-82, 90-95, 102-115, and 115-130 of the survivin polypeptide sequence of Genbank Accession No. U75285; Amino acids 10-20,30-35, 65-75, 110-120, and 160-170 of the amino acid sequence of Proteinase-3 found in Genbank Accession No. X55668; Amino acids 10-20, 30-50, 55-75, 85-110, 115-135, 145-155, and 160-185 of Genbank accession number P01117 or M54968 (human K-Ras); Genbank accession number P01112 (human H -Ras) amino acids 10-20, 25-30, 35-45, 50-70, 90-110, 115-135, and 145-175; Amino acids 10-20, 25-45, 50-75, 85-110, 115-135, 140-155, and 160-180 of Genbank Accession No. P01111 (human N-Ras); And the first 25-amino acid of 12-K-Ras (sequences disclosed in Genbank Accession No. K00654). These antigenic regions were predicted by Hopp-Woods and Welling antigenic plots.

In some embodiments, a polypeptide encoded by a polynucleotide of the present invention as an individual polypeptide, as a fusion protein with a selected signal peptide, or as a protein chimera in which a polypeptide is inserted into another polypeptide, comprises at least one of the following peptides of human mesothelin: A polypeptide comprising: SLLFLLFSL (amino acids 20-28; (SEQ ID NO: 64)); VLPLTVAEV (amino acids 530-538; (SEQ ID NO: 65)); ELAVALAQK (amino acids 83-92; (SEQ ID NO: 66)); ALQGGGPPY (amino acids 225-234; (SEQ ID NO: 67)); FYPGYLCSL (amino acids 435-444; (SEQ ID NO: 68)); And LYPKARLAF (amino acids 475-484; (SEQ ID NO: 69)). For example, in some embodiments, the antigens encoded by the polynucleotides of the invention are (antigenic) fragments of human mesothelin comprising one or more of these peptides. Further information regarding these mesothelin peptide sequence wheat medically related immune responses and their interactions can be found in PCT Publication WO 2004/006837.

Alternatively, the polynucleotide in a recombinant nucleic acid molecule, expression cassette, or expression vector may encode an autoimmune disease-specific antigen (or polypeptide comprising an autoimmune disease-specific antigen). In T cell mediated autoimmune diseases, T cell responses to autoantigens result in autoimmune diseases. The type of antigen for use in treating an autoimmune disease comprising the vaccine of the invention can target specific T cells that elicit an autoimmune response. For example, the antigen may be part of a T cell receptor, genotype, specific for those T cells that give rise to an autoimmune response, and antigens incorporated into the vaccine of the invention may be specific for those T cells that give rise to an autoimmune response. Will induce an immune response. Removing those T cells would be a therapeutic mechanism to alleviate autoimmune disease. Another possibility is to incorporate the polynucleotide encoding the antigen into a recombinant nucleic acid molecule, which in autoimmune disease will result in an immune response that targets the antibody produced against the self antigen or targets specific B cells that secrete the antibody. For example, polynucleotides encoding idiotype antigens can be incorporated into recombinant nucleic acid molecules, resulting in anti-idiotype immune responses against antibodies that react with self antigens in such B cells and / or autoimmune diseases. Will bring. Autoimmune diseases treatable with a vaccine comprising a bacterium comprising the expression cassette of the invention and a recombinant nucleic acid molecule include rheumatoid arthritis, multiple sclerosis, Crohn's disease, lupus, severe myasthenia, vitiligo, skin sclerosis, psoriasis, normal pemphigus, fibers Myalgia, colitis and diabetes, but are not limited to these. Similar approaches may be taken to treat allergic reactions, and antigens incorporated into the vaccine bacteria target T cells, B cells or antibodies, which are effective in modulating allergic responses. In some autoimmune diseases, such as psoriasis, the disease also results in excessive proliferative cell growth with expression of antigens that may be targeted. Such antigens are contemplated which result in an immune response to excessive proliferative cells.

In some embodiments, the antigen is an antigen that targets unique diseases associated with protein structure. One example of this is the targeting of antibodies, B cells or T cells using an idiotype antigen as discussed above. Another possibility is to target unique protein structures that result from specific diseases. An example of this is incorporating antigens that will generate an immune response to proteins that cause amyloid plaques observed in diseases such as Alzheimer's disease, Creutzfeldt-Jakob disease (CJD) and mad cow disease (BSE). This approach may only provide a reduction in plaque formation, but it may be possible to provide a therapeutic vaccine in the case of diseases such as CJD. This disease is caused by the infected form of prion protein. In some embodiments, the polynucleotides of the present invention may encode antigens for infectious forms of prion proteins to remove, reduce, or inhibit infectious proteins in which the immune response generated by the vaccine causes CJD.

In some embodiments, the polypeptide encoded by the recombinant nucleic acid molecule, expression cassette, and / or polynucleotide of the expression vector comprises an infectious disease antigen or comprises an antigen derived from an infectious disease antigen. In some embodiments, the polypeptide comprises an infectious disease antigen. In some embodiments, the polypeptide comprises an antigen derived from an infectious disease antigen. In some embodiments, the polypeptide encoded by the recombinant nucleic acid molecule, expression cassette, and / or polynucleotide of the expression vector is an infectious disease antigen or an antigen derived from an infectious disease antigen. In some embodiments, the polypeptide encoded by the recombinant nucleic acid molecule, expression cassette, and / or expression vector is an infectious disease antigen. In some embodiments, the polypeptide encoded by the recombinant nucleic acid molecule, expression cassette, and / or expression vector is derived from an infectious disease antigen.

In another embodiment of the invention, the antigen is derived from a human or animal pathogen. The pathogen is optionally a virus, bacterium, fungus, or protozoa. For example, the antigen may be a virus or fungal or bacterial antigen. In one embodiment, the antigen encoded by a recombinant nucleic acid molecule, expression cassette, and / or expression vector derived from a pathogen is a protein produced by, or derived from, a protein produced by the pathogen. For example, in some embodiments, polypeptides encoded by recombinant nucleic acid molecules, expression cassettes, and / or expression vectors are fragments and / or variants produced by a pathogen.

For example, in some embodiments the antigen is derived from a human immunodeficiency virus (gp 120, gp 160, gp41, p24gag and p55gag) and gag antigens, and pol, env, tat, vif, rev, nef, vpr, vpu Protein and the LTR region of HIV), feline immunodeficiency virus, or human or animal herpes virus. For example, in some embodiments, the antigen is gp120. In one embodiment, the antigen is Herpes simplex virus (HSV) types 1 and 2 (eg, direct initial proteins such as gD, gB, gH, ICP27), cytomegalovirus (eg, gB and gH), meta It is derived from the neumo virus, Epstein-Barr virus or varicella zoster virus (eg gpI, II or III). (See, eg, Chee et al. (1990) Cytomegaloviruses (JK McDougall, ed., Springer Verlag, pp. 125-169; McGeoch et al. (1988) J. Gen. Virol. 69: 1531-1574; US Pat No. 5,171,568; Baer et al. (194) Nature 310: 207-211; and Davison et al. (1986) J. Gen. Virol. 67: 1759-1816.)

In another embodiment, the antigen is a hepatitis B virus (eg, hepatitis B surface antigen), hepatitis A virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, or hepatitis G virus. Derived from hepatitis virus. See, for example, WO 89/04669; WO 90/11089; And WO 90/14436. The hepatitis antigen can be a surface, core, or other related antigen. The HCV genome encodes several viral proteins, including El and E2. See, for example, Houghton et al., Hepatology 14: 381-388 (1991).

Antigens which are viral antigens are optionally derived from viruses from any of the following families: Picornaviridae (eg, polioviruses, rhinoviruses, etc.); Caliciviridae; Togaviridae (eg, Rubella virus, dengue virus, etc.); Flaviviridae; Coronaviridae; Reoviridae (eg, rotaviruses, etc.); Birnaviridae; Rhabodoviridae (eg, rabies virus, etc.); Orthomyxoviridae (eg, influenza A, B, and C viruses, etc.); Filoviridae; Paramyxoviridae (eg, mumps virus, measles virus, respiratory cell fusion virus, etc.); Bunyaviridae; Arenaviridae; Retroviradae (eg, HTLV-I; HTLV-11; HIV-1 (known as HTLV-111, LAV, ARV, hTLR, etc.) (this is isolate HIVI11b, HIVSF2, HTVLAV, HIVLAI, HIVMN); HIV-1CM235, HIV-1, including but not limited to antigens from HIV-2); Apes immunodeficiency virus (SIV); Papilloma virus, tick-borne encephalitis, etc. For example, for the description of these and other viruses, Virology, 3rd Edition (WK Joklik ed. 1988); See Fundamental Virology , 3rd Edition (BN Fields, DM Knipe, and PM Howley, Eds. 1996). In one embodiment, the antigen is Flu-HA (Morgan et al. J. Immunol. 160: 643 (1998)).

In some alternative embodiments, the antigen is Mycobacteria, Bacillus, TYersinia, Salmonella, Neisseria, Borrelia (eg, OspA or OspB or derivatives thereof), chlamydia, or Bordetella (eg, p. 69, PT and FHA), or from parasites such as heat protozoa or toxoplasma. In one embodiment, the antigen is from Mycobacterium tuberculosis (eg ESAT-6, 85A, 85B, 85C, 72F), Bacillus anthracis (eg PA), or Yersinia pestis (eg F1, V) Induced. In addition, suitable antigens for use in the present invention include diphtheria, pertussis, tetanus, tuberculosis, bacteria or fungal pneumonia, otitis media, gonorrhea, cholera, typhoid fever, meningitis, mononucleosis, plague, dysentery or salmonellosis, legioner's disease, Lyme disease, leprosy, Malaria, hookworm, gonadotropia, schistosomiasis, wave flagellosis, rishman flagellosis, giardia, amoebiasis, filamentosis, borelia, and trichinosis. It can be obtained or derived from non-traditional pathogens, such as Kru, Creutzfeldt-Jakob disease (CJD), sheep trembling disease, infectious mink encephalopathy, and chronic wasting disease, or from protein-infected particles such as prions associated with mad cow disease.

In another embodiment, the antigen is from a biological pathogen involved in the onset or progression of neurodegenerative diseases (such as Alzheimer's disease), metabolic diseases (such as type 1 diabetes), and drug addiction (such as nicotine poisoning). Acquired or derived. Alternatively, the antigens encoded by the recombinant nucleic acid molecules are used for pain management and the antigens are pain receptors or other pathogens involved in the transmission of pain signals.

In some embodiments, the antigen is or is derived from a human protein. In other embodiments, the antigen is a non-human protein or is derived from a non-human protein (fragments and / or variants thereof). In some embodiments, the antigenic portion of the fusion protein encoded by the expression cassette is a protein from a non-human animal or a protein derived from a non-human animal. For example, even when antigens are expressed in Listeria-based vaccines to be used in humans, in some embodiments, the antigens can be murine mesothelin or derived from murine mesothelin.

V. Codon-Optimization

In some embodiments, one or more of the polynucleotides (ie, polynucleotide sequences) in the recombinant nucleic acid molecule, expression cassette, and / or expression vector are codon-optimized (relative to natural coding sequences). In some embodiments, the polynucleotides within the recombinant nucleic acid molecules (and / or expression cassettes and / or expression vectors) described herein that encode the signal peptides are codon-optimized for expression in bacteria. In some embodiments, polypeptides other than signal peptides, such as polynucleotides encoding antigens or other therapeutic proteins, are codon-optimized for expression in bacteria. In some embodiments, the polynucleotide encoding the signal peptide and the polynucleotide encoding another polypeptide fused with the signal peptide are both codon-optimized for expression in bacteria. In some embodiments, polynucleotides encoding secreted proteins (or fragments thereof) used as scaffolds or polynucleotides encoding autolysis (or fragments or variants thereof) are codon-optimized.

A polynucleotide comprising a coding sequence is wherein at least one codon of the natural coding sequence of the polynucleotide is replaced with a codon that is used more frequently by an organism ("target organism") in which the coding sequence is expressed than the original codon of the native coding sequence. "Codon-optimized". For example, a polynucleotide encoding a non-bacterial antigen expressed in certain bacterial species is substituted with at least one of the codons from the native bacterial polynucleotide with a codon that is preferentially expressed in certain bacterial species expressing the non-bacterial antigen. In that case codon-optimized. As another example, a polynucleotide encoding a human cancer antigen that is part of an expression cassette in a recombinant Listeria monocy-togenes is more frequently encountered by Listeria monocytogenes for that amino acid than at least one codon in that polynucleotide sequence than the codon in the original human sequence. Codon-optimized when substituted with the codon used. Similarly, a polynucleotide encoding a natural signal peptide (LLO signal peptide, such as Listeria monocytogenes in the origin) of Listeria monocytogenes part of an expression cassette coding for a fusion protein comprising a human cancer antigen in a recombinant Listeria monocytogenes is a signal peptide Codon-optimized when at least one codon in the polynucleotide sequence encoding is substituted with a codon used more frequently by Listeria monocytogenes for that amino acid than the codon in the original (natural) sequence. In some embodiments, at least one codon substituted in a codon-optimized sequence is substituted with a codon most frequently used by the target organism to encode the same amino acid.
In some embodiments, at least two codons of the natural coding sequence of the polynucleotide have been replaced with codons that are used more frequently by the organism to which the coding sequence will be expressed than the original codons of the native coding sequence. In some embodiments, at least about 5 codons, at least about 10 codons, or at least about 20 codons of the polynucleotide have been replaced with codons that are used more frequently by the organism to which the coding sequence will be expressed than the original codons of the native coding sequence. .

In some embodiments, at least about 10% of the codons of the codon-optimized polynucleotides have been replaced with codons used more frequently (or most frequently) by the target organism (than the original codons of the native sequence). In other embodiments, at least about 25% of the codons of the codon-optimized polynucleotides have been replaced with codons used more frequently (or most frequently) by the target organism. In other embodiments, at least about 50% of the codons of the codon-optimized polynucleotides have been replaced with codons used more frequently (or most frequently) by the target organism. In another embodiment, at least about 75% of the codons of the codon-optimized polynucleotides have been replaced with codons that are used more frequently (or most frequently) by the target organism.

Codon preferences of different organisms have been extensively studied by those skilled in the art. See, eg, Sharp et al., Nucleic Acids Res. 15: 1281-95 (1987) and Uchijima et al., The Journal of Immunology, 161: 5594-9 (1998). As a result, codon usage tables for a wide variety of organisms are publicly available. For example, codon usage tables for a wide variety of organisms can be found at www.kazusa.or.jp/codon/ on the Internet and in other publicly available sites (Nakamura et al. (2000) Nucleic Acids Research 28). See: 292). Typical codon usage table from www.kazusa.or.jp/codon/, codon usage table of Listeria monocytogenes (http://www.kazusa.or.jp/codon/cgibin/showcodon.cgi?species=Listeria+monocytogenes+ [ gbbct]) is reproduced in Table 2A for convenience. Typical codon usage tables for Bacillus anthracis, Mycobacterium tuberculosis, Salmonella typhimurium, Mycobacterium bovis BCG , and Shigella flexneri are provided in Tables 2B, 2C, 2D, 2E, and 2F below, respectively.

In some embodiments of the invention, at least about 10%, at least about 25%, at least about 50% or at least about 75% of the codons in the codon-optimized coding sequence are the most preferred codons for that amino acid in the target organism. In another embodiment, 100% of the codons in the codon-optimized coding sequence are the most preferred codons for that amino acid in the target organism (ie, the sequence is “fully codon-optimized”). For example, in the embodiments shown below, all of the codons in the sequence characterized by codon-optimized were the codons most frequently used in the target organism; However, any codon substitution that results in codons used more frequently than the original (natural) sequence can be considered "codon-optimized." Table 3 below shows the optimal codon usage in Listeria monocytogenes for each amino acid.

In some embodiments, the codon-optimized polynucleotides encode a signal peptide. In some embodiments, the signal peptide is exogenous to the bacteria whose sequence is codon-optimized. In another embodiment, the signal peptide is a natural signal peptide of the bacteria whose sequence is codon-optimized. For example, in some embodiments the codon-optimized polynucleotide is a LLO signal peptide from Listeria monocytogenes , a Usp45 signal peptide from Lactococcus Lactis , a protective antigen signal peptide from Bacillus anthracis , a p60 signal peptide from Listeria monocytogenes and a PhoD from B. subtilis Tat signal peptide Code a signal peptide selected from the group consisting of signal peptides. In some embodiments, the codon-optimized polynucleotide encodes a signal peptide other than a protective antigen signal peptide from Bacillus anthracis . In some embodiments, the polynucleotide encoding the signal peptide is codon-optimized for expression in Listeria monocytogenes .

In some embodiments, the codon-optimized polynucleotides encode a (non-signal peptide) protein that is exogenous to the bacteria whose polynucleotide sequence is codon-optimized. In some embodiments, the codon-optimized polynucleotides encode a polypeptide comprising an antigen. For example, in some embodiments, the codon-optimized polynucleotide encodes a polypeptide comprising an antigen that is a tumor-associated antigen or an antigen derived from a tumor-associated antigen.

In some embodiments, the codon-optimization of polynucleotides encoding signal peptides and / or other polypeptides comprises a polypeptide (recombinant) comprising a signal peptide and / or other polypeptide in bacteria as compared to the corresponding polynucleotide that is not codon-optimized. Expression of nucleic acid molecules, expression cassettes, or fusion proteins, protein chimeras and / or exogenous polypeptides encoded by expression vectors. In some embodiments, codon-optimization of the polynucleotides enhances expression by at least about 2 times, at least about 5 times, at least about 10 times, or at least about 20 times (relative to corresponding polynucleotides without codon-optimization). In some embodiments, the codon-optimization of polynucleotides encoding signal peptides and / or other polypeptides may comprise polypeptides comprising signal peptides and / or other polypeptides derived from bacteria as compared to corresponding polynucleotides that are not codon-optimized. Enhance the secretion of fusion proteins, such as protein chimeras and / or exogenous polypeptides. In some embodiments, codon-optimization enhances secretion by at least about 2 times, at least about 5 times, at least about 10 times, or at least about 20 times (relative to corresponding polynucleotides without codon-optimization). In some embodiments, both levels of expression and secretion are enhanced. Levels of expression and / or secretion can be readily assessed using standard techniques in the art, such as western blots of various related bacterial culture portions.

VI. Expression cassette

Expression cassettes are also provided by the present invention. For example, in some embodiments, the present invention comprises any of the recombinant nucleic acid molecules described herein and comprises a coding sequence (eg, a first polynucleotide and a remaining polypeptide encoding a signal peptide) within the recombinant nucleic acid molecule. Provided is an expression cassette further comprising a promoter sequence operably linked with a second polynucleotide encoding). In some embodiments, the expression cassette is isolated. In some other embodiments, the expression cassette is contained in an expression vector, and the expression vector may be isolated and contained in bacteria. In another embodiment, the expression cassette is located in the chromosomal DNA of the bacterium. For example, in some embodiments, the expression cassette is integrated into the genome of the bacterium. In some embodiments, the expression cassette integrated into the genome of the bacterium comprises one or more elements from genomic DNA. For example, in some embodiments, a recombinant nucleic acid molecule is inserted at a site within a bacterial genomic DNA (eg, by site-specific integration or homologous recombination) such that the recombinant nucleic acid molecule is already present in the genomic DNA. It is operably linked to a promoter, resulting in a new expression cassette integrated into genomic DNA. In some other embodiments, the expression cassette is integrated into genomic DNA (eg, by site-specific integration or homologous recombination) as an intact unit comprising both a promoter and a recombinant nucleic acid molecule.

In some embodiments, the expression cassette is designed for expression of a polypeptide in a bacterium. In some embodiments, the expression cassette is designed for the expression of heterologous polypeptides, such as heterologous antigens, in bacteria. In some embodiments, the expression cassette provides for enhanced expression and / or secretion of the polypeptide.

In general, an expression cassette comprises the following organized elements: (1) a promoter and (2) a polynucleotide encoding a polypeptide. In some embodiments, the expression cassette comprises the following elements: (1) a promoter; (2) polynucleotides encoding signal peptides; And (3) a polynucleotide encoding a polypeptide (such as a heterologous protein). In another embodiment, the expression cassette comprises the following elements: (1) a prokaryotic promoter; (2) Shine-Dalgarno sequence; (3) polynucleotides encoding signal peptides; And (4) a polynucleotide encoding a polypeptide (such as a heterologous protein). In some embodiments, the expression cassette comprises one or more promoters.

In some embodiments, the expression cassette may also contain a transcription termination sequence inserted downstream of the C-terminus of the stop codon associated with the heterologous polypeptide. For example, in some embodiments, transcription termination sequences can be used in constructs designed for stable integration within bacterial chromosomes. Although not required, the inclusion of a transcription termination sequence as a final arranged element in a heterologous gene expression cassette may prevent the polar effect on the regulation of expression of neighboring genes due to trans read transcription. Thus, in some embodiments, suitable sequence elements known to those skilled in the art that promote rho-dependent or rho-independent transcription termination can be in a heterologous protein expression cassette.

In one aspect, the invention provides a kit comprising: (a) a first polynucleotide encoding a signal peptide, codon-optimized for expression in bacteria; (b) a second polynucleotide encoding a polypeptide that is within the same translation reading frame as the first polypeptide; And (c) a promoter operably linked to the first and second polynucleotides, wherein the expression cassette encodes a fusion protein comprising a signal peptide and a polypeptide.

In another aspect, the invention provides a polypeptide comprising (a) a first polynucleotide encoding a codon-optimized bacterial natural signal peptide for expression in a bacterium, and (b) within the same translation reading frame as the first polynucleotide. And a (c) promoter that is operably linked to the first and second polynucleotides of the expression cassette, wherein the recombinant nucleic acid molecule is a fusion comprising a signal peptide and a polypeptide. Code the protein. In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous with the signal peptide. In some embodiments, the second polynucleotide is heterologous to the first polynucleotide. In some embodiments, the polypeptide is heterologous (ie, exogenous to bacteria) where the signal peptide is a natural signal peptide. In some embodiments, the bacteria from which the signal peptide is derived are intracellular bacteria. In some embodiments, the bacteria are selected from the group consisting of Listeria, Bacillus, Yersinia pestis, Salmonella, Shigella, Brucella , Mycobacteria and E. coli . In some embodiments, the second polynucleotide is codon-optimized for expression in bacteria.

In another aspect, the invention encodes a polypeptide comprising (a) a first polynucleotide encoding a signal peptide, codon-optimized for expression in Listeria bacteria, (b) within the same translation reading frame as the first polynucleotide And a (c) promoter that is operably linked to the first and second polynucleotides of the expression cassette, the recombinant nucleic acid molecule comprising a fusion protein comprising a signal peptide and a polypeptide. Coding In some embodiments, the expression cassette is a polycistronic expression cassette. In some embodiments, the second polynucleotide is codon-optimized for expression in Listeria bacteria. In some embodiments, the polypeptide encoded by the second polynucleotide is foreign to the Listeria bacterium (i. E., Heterologous to the Listeria bacterium). In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous with the signal peptide. In some embodiments, the expression cassette comprises one or more promoters.

In another aspect, the invention provides a kit comprising: (a) a first polynucleotide encoding a non-secA1 bacterial signal peptide; (b) a second polynucleotide encoding a polypeptide that is within the same translation reading frame as the first polynucleotide; And a promoter operably linked with the first and second polynucleotides, wherein the expression cassette encodes a fusion protein comprising a signal peptide and a polypeptide. In some embodiments, the first and / or second polynucleotides are codon-optimized for expression in bacteria such as Listeria , Bacillus, Yersinia pestis, Salmonella, Shigella, Brucella , Mycobacteria or E. coli . In some embodiments, the polynucleotide (s) are codon-optimized for expression in Listeria , such as Listeria monocytogenes . In some embodiments, the signal peptide encoded by the codon-optimized first polynucleotide is a natural signal peptide of the bacterium that is codon-optimized for it. In some embodiments, the first polynucleotide encoding the signal peptide is heterologous to the second polynucleotide. In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous with the signal peptide. In some embodiments, the expression cassette is a polycistronic expression cassette. In some embodiments, the first polynucleotide, the second polynucleotide, or both the first and second polynucleotides are codon-optimized for expression in Listeria bacteria (eg, Listeria monocytogenes ). In some embodiments, the first and second polynucleotides are heterologous to one another. In some embodiments, the polypeptide and signal peptide encoded by the second polynucleotide are heterologous to one another. In some embodiments, the second by a polynucleotide encoding an exogenous polypeptide is a Listeria bacterium (i. E., A Listeria bacteria and heterologous). In some embodiments, the expression cassette comprises one or more promoters.

In addition, the present invention is (a) codons for expression in Listeria - optimized, polynucleotide encoding an exogenous polypeptide in Listeria, and (b) comprises a promoter is operably linked to a polynucleotide encoding an exogenous polypeptide An expression cassette is provided. In some embodiments, the polypeptide encoded by the expression cassette is an antigen (see, eg, description of any possible antigen above). In some embodiments, the expression cassette further comprises a polynucleotide encoding a signal peptide. Polynucleotides encoding a signal peptide are also operably linked with a promoter such that the expression cassette expresses a fusion protein comprising both the exogenous polypeptide and the signal peptide. Polynucleotides encoding suitable signal peptides for use in this expression cassette include, but are not limited to, those described above. In some embodiments, the polynucleotide encoding the signal peptide included in the expression cassette is codon-optimized for expression in bacteria such as Listeria (eg, Listeria monocytogenes bacteria) as described above.

In addition, the present invention provides a kit comprising: (a) a first polynucleotide encoding a non- Listeria signal peptide; (b) a second polynucleotide encoding a polypeptide that is within the same translation reading frame as the first polynucleotide; And (c) a promoter operably linked with the first and second polynucleotides, wherein the expression cassette encodes a fusion protein comprising both a non- Listeria signal peptide and a polypeptide. In some embodiments, the expression cassette is a polycistronic expression cassette. In some embodiments, the first polynucleotide, second polynucleotide, or both first and second polynucleotides are codon-optimized for expression in Listeria (eg, Listeria monocytogenes ). In some embodiments, the first and second polynucleotides are heterologous to one another. In some embodiments, the polypeptide and signal peptide encoded by the second polynucleotide are heterologous to one another. In some embodiments, the polypeptide encoded by the second polynucleotide is foreign to the Listeria bacterium (i. E., Heterologous to the Listeria bacterium). In some embodiments, the expression cassette comprises one or more promoters.

Furthermore, the present invention provides a kit comprising: (a) a first polynucleotide encoding bacterial autolysis, or a catalytically active fragment or catalytically active variant thereof; And (b) a second polynucleotide encoding a polypeptide that is within the same translation reading frame as the first polynucleotide; And (c) a promoter operably linked with the first and second polynucleotides, the expression cassette comprising a polypeptide encoded by the second polynucleotide and self-dissolving, or a catalytically active fragment thereof Encodes a protein chimera comprising a catalytically active variant, wherein in the protein chimera, the polypeptide is self-dissolved, or fused with a catalytically active fragment or catalytically variant thereof, or self-dissolved, or inserted into a catalytically active fragment or catalytically active variant thereof do. In some embodiments, the protein chimera is catalytically active like autodissolution. In some embodiments, the polypeptide is autolytic and heterologous. In some embodiments, the bacterial autolysis is from intracellular bacteria (eg, Listeria ). In some embodiments, a second polynucleotide encoding a polypeptide is inserted into a first polynucleotide encoding autolytic, or a catalytically fragment or catalytically active fragment thereof, and an expression cassette is used to autolyze the polypeptide, or a catalyst thereof. Encodes a protein chimera inserted into an active fragment or catalytically active variant (ie, the polypeptide is autolysed or embedded within its catalytic fragment or catalytically active variant). In another embodiment, the second polynucleotide is located outside of the first polynucleotide encoding autolytic, or catalytically fragment or catalytically active variant thereof, and the expression cassette is characterized in that the polypeptide autolyzes, or catalytically fragments or catalyst thereof. It encodes a protein chimera fused with an active variant. In some embodiments, the polypeptide is autolytic and heterologous. In some embodiments, the first polynucleotide and the second polynucleotide are heterologous to one another. In some embodiments, the autolysis is SecA2-dependent autolysis. In some embodiments, the autolysis is peptidoglycan hydrolase (eg, N-acetylmuramidase or p60). In some embodiments, the vesicle cassette further comprises a polynucleotide encoding a signal peptide (eg, a signal peptide normally associated with autolysis or a signal peptide heterologous to the signal peptide). For example, in some embodiments, an expression cassette encodes a protein chimera comprising a p60 signal peptide, a p60 protein (or a catalytic fragment or catalytic variant thereof), and a polypeptide heterologous to p60 embedded in a p60 sequence. . In some embodiments, the polypeptide encoded by the second polynucleotide is a non- Listeria polypeptide.

In another aspect, the invention provides a kit comprising: (a) a first polynucleotide encoding a signal peptide, (b) a second polynucleotide encoding a secreted protein or fragment thereof that is within the same translation reading frame as the first polynucleotide, (c) a third polynucleotide encoding a polypeptide heterologous to the secreted protein or fragment thereof that is within the same translational reading frame as the first and second polynucleotides, and (d) the first, second and third polys An expression cassette comprising a promoter operably linked to a nucleotide, wherein the recombinant nucleic acid molecule encodes a protein chimera comprising a signal peptide, a polypeptide encoded by a second polynucleotide, and a secreted protein or fragment thereof The polypeptide encoded by the third polynucleotide may be a protein secreted from a protein chimera or And the fused fragment, is positioned within the secreted protein, or its fragments.

In some embodiments, the promoter in the expression cassette (or recombinant nucleic acid molecule described herein) described herein is a prokaryotic promoter. For example, the prokaryotic promoter may be a Listeria promoter. In some embodiments, the Listeria promoter is a hly promoter. In some embodiments, the promoter is a prfA-dependent promoter (eg, actA promoter). In some embodiments, the promoter is a constitutive promoter (eg, a p60 promoter). In some embodiments, an expression cassette comprising a recombinant nucleic acid molecule described herein comprises a hly, actA, or p60 promoter operably linked to a polynucleotide of the recombinant nucleic acid molecule. Those skilled in the art will readily be able to identify suitable additional prokaryotic and / or Listeria promoters for use in the expression cassette in view of the intended use of the expression cassette and the host bacteria in which it will be placed.

For example, many suitable mycobacterial promoters are known for use in recombinant expression cassettes in mycobacteria and other bacteria. These include Mycobacterium bovis BCG promoters HSP60 and HSP70, and also mycobactin promoters, α-antigen promoters of M. tuberculosis and BCG and 45 KDa antigen promoters, superoxide dasmutase promoters, MBP-70, mycobacterial asd promoters, Promoters such as the mycobacterial 14 KDa and 12 KDa antigen promoters, the mycobacterial phage promoters such as the Bxb1, Bxb2 and Bxb3 promoters, the L1 and L5 promoters, the D29 promoter and the TM4 promoter (see, eg, US Pat. No. 6,566,121). . Suitable promoters for use with Bacillus anthracis include, but are not limited to, the pagA promoter, alpha-amylase promoter (Pamy) and Pntr (see, eg, Gat et al., Infect. Immun. 71: 801-13 (2003)). ). Suitable promoters for use in recombinant Salmonella expression cassettes and vaccines are also known and include the nirB promoter, osmC promoter, P (pagC), and P (tac) (eg, Bumann, Infect. Immun. 69: 7493). -500 (2001); Wang et al. Vaccine, 17: 1-12 (1999); McSorley et al., Infect. Immun. 65: 171-8 (1997). Various E. coli promoters are also known to those skilled in the art.

In some embodiments, the promoter used in the expression cassette described herein is a constitutive promoter. In other embodiments, the promoter used in the expression cassettes described herein is an inducible promoter. Inducible promoters can be induced by molecules (eg, proteins) that are endogenous to the bacteria in which the expression cassette will be used. Alternatively, the inducible promoter can be induced by a molecule (eg, a small molecule or protein) that is heterologous to the bacterium in which the expression cassette will be used. Various inducible promoters are known to those skilled in the art.

In some embodiments of the expression cassette, at the 3'-end of the promoter is a poly-purine Shine-Dalgarno sequence, which is the element necessary for the 30S ribosomal subunit to engage (via 16S rRNA) into the heterologous gene RNA transcript to initiate translation. The Shine-Dalgarno sequence typically has the following sequence: 5'-NAGGAGGU-N _5-10 -AUG (start codon) -3 '(SEQ ID NO: 85). There are also variants of the poly-purine Shine-Dalgarno sequence. In particular, the Listeria hly gene encoding Listerilisin O (LLO) has the following Shine-Dalgarno sequence: AAGGAGA GTGAAACCC ATG (SEQ ID NO: 70) (Shine-Dalgarno sequence underlined, translation start codon bold Letters).

The construction of expression cassettes used for bacteria and the construction of expression cassettes used specifically for recombinant bacterial vaccines are known in the art. For example, descriptions of the preparation and use of various bacterial expression cassettes and / or recombinant bacterial vaccines can be found in the following references, each of which is incorporated herein by reference in its entirety: Horwitz et al., Proc. . Natl. Acad. Sci. USA, 97: 13853-8 (2000); Garmory et al., J. Drug Target, 11: 471-9 (2003); Kang et al., FEMS Immunol. Med. Microbiol. 37: 99-104 (2003); Garmory et al., Vaccine, 21: 3051-7 (2003); Kang et al., Infect. Immun., 1739-49 (2002); Russman, et al., J. Immunol., 167: 357-65 (2001); Harth et al., Microbiology, 150: 2143-51 (2004); Varaldo et al., Infect. Immun., 72: 3336-43 (2004); Goonetilleke et al., J. Immunol., 171: 1602-9 (2003); Uno-Furuta et al., Vaccine, 21: 3149-56 (2003); Biet et al., Infect. Immun., 71: 2933-7 (2003); Bao et al., Infect. Immun., 71: 1656-61 (2003); Kawahara et al., Clin. Immunol., 105: 326-31 (2002); Anderson et al., Vaccine, 18: 2193-202 (2000); Bumann, Infect. Immun., 69: 7493-500 (2001); Wang et al., Vaccine, 17: 1-12 (1999); McSorley et al., Infect. Immun., 65: 171-8 (1997); Gat et al., Infect. Immun., 71: 801-13 (2003); U.S. Patent No. 5,504, 005; U.S. Patent No. 5,830,702; U.S. Patent No. 6,051,237; United States Patent Publication No. 2002/00 25323; US Patent Publication No. 2003/0202985; WO 04/062597; U.S. Patent No. 6,566,121; And US Patent No. 6,270,776.

In some embodiments, it is desirable to construct an expression cassette that utilizes bicistronic, polycistronic (also called multicistronic) expression of heterologous coding sequences. Such expression cassettes may, for example, utilize a single promoter operably linked to two or more independent coding sequences. These coding sequences may, for example, correspond to each gene or otherwise correspond to the desired and / or selected sub-fragments of the entire designated gene. In this later example, the gene may contain a sequence encoding a hydrophobic trans-membrane domain, which can potentially inhibit effective secretion from Listeria . Thus, it may be desirable to separate the coding sequence of this gene from the hydrophobic domain into two sub-fragments; In this example two sub-fragments are then expressed as bicistronic messages. The use of polycistronic expression requires 30 ribosomal subunits to stay on the polycistronic RNA message after the translation termination of the first coding sequence and the release of the 50 ribosomal subunits, followed by the RNA message being "read" to the next initiation codon. During which 50 ribosomal subunits are combined with 30 ribosomal subunits to resume translation.

Like other bacteria, Listeria monocytogenes takes advantage of the polycistronic expression of its genome repertoire. For example, the sequence of the Listeria monocytogenes intergenic region from selected polycistronic messages can be used to construct a polycistronic expression cassette for the expression of heterologous proteins selected from recombinant Listeria species. For example, some of the prfA-dependent virulence factors from Listeria monocytogenes are expressed from the polycistronic message. For example, Listeria monocytogenes ActA and PlcB proteins are expressed as bicistronic messages. Shown below is a DNA sequence corresponding to Listeria monocytogenes ActA and PlcB intergenic sequence (5'-3 '):

5'-TAAAAACACAGAACGAAAGAAAAA GTGAGG TGAATGA-3 '(SEQ ID NO: 71)

(The Shine-Dalgarno sequence for initiating translation of plcB is shown in bold. The first three nucleotides in the sequence correspond to the Ocher stop codon.) A non-limiting example of a bistronic expression vector is a bistronic used in Listeria monocytogenes . There is an hEphA2 expression vector and see Example 28 below.

Alternatively, other known intergenic or synthetic sequences can be used to construct a polycistronic expression cassette for use in Listeria or other bacteria. The construction of intergenic regions leading to substantial secondary RNA structure should be prevented, thereby avoiding unwanted transcription termination by rho-independent mechanisms.

Importantly, if secretion of any or all translated proteins expressed from polycistronic messages is desired, the signal peptide must be functionally linked to each coding region. In some embodiments, these signal peptides are different from each other.

Thus, in some embodiments, the expression cassettes described herein for use in Listeria or other bacteria are polycistronic (eg, biscistronic). Two or more polypeptides are encoded by bicistronic or polycistronic expression cassettes as isolated polypeptides. In some embodiments, the bicistronic or polycistronic expression cassette comprises an intergenic sequence (eg, derived from a biscistronic or polycistronic gene) located between the coding sequences of these two polypeptides. In some embodiments, the intergenic sequence comprises a sequence that facilitates ribosomal entry and translation initiation. In some embodiments, the intergenic sequence comprises a Shine-Dalgarno sequence. In some embodiments, the intergenic sequence is Listeria monocytogenes actA-plcB intergenic sequence. Typically, the intergenic sequence is a polynucleotide sequence encoding a first polypeptide (or a first fusion protein comprising a first polypeptide and a signal peptide) and a second fusion comprising a second polypeptide (or a second polypeptide and a signal peptide) Proteins) are located between the polynucleotide sequences encoding the protein.

Thus, in one aspect, the present invention provides a kit comprising: (a) a first polynucleotide encoding a first polypeptide; (b) a second polynucleotide encoding a second polypeptide; (c) an intergenic sequence located between the first and second polynucleotides; And (f) a promoter operably linked with the first and second polynucleotides, wherein the expression cassette encodes the first and second polypeptides as two separate polypeptides. In some embodiments, the first and second polypeptides are polypeptides selected from any of the polypeptides described herein (eg, in Section IV above). In some embodiments, at least one of the first or second polypeptides comprises an antigen. In some embodiments, the first and second polynucleotides each comprise a (different or identical) fragment of the same antigen. In some embodiments, the antigen is a tumor-associated antigen or is derived from a tumor-associated antigen.

Furthermore, the present invention provides a kit comprising: (a) a first polynucleotide encoding a first signal peptide; (b) a second polynucleotide encoding the first (non-signal) polypeptide, which is in the same translation reading frame as the first polynucleotide; (c) a third polynucleotide encoding a second signal peptide; (d) a fourth polynucleotide encoding a second (non-signal) polypeptide, which is in the same translation reading frame as the third polynucleotide; (e) intergenic sequences (typically between the second and third polynucleotides); And (f) a promoter operably linked with a first polynucleotide, a second polynucleotide, a third polynucleotide, and a fourth polynucleotide, wherein the expression cassette comprises a first signal peptide and a first polypeptide. Encodes both a first fusion protein comprising a and a second fusion protein comprising a second signal peptide and a second polypeptide. In some embodiments, at least one of the polynucleotides encoding the signal peptide is codon-optimized for expression in bacteria. In some embodiments, the third and / or fourth polynucleotides are codon-optimized for expression in bacteria (preferably in addition to codon-optimization of polynucleotides encoding signal peptides). In some embodiments, the first and / or second signal peptide is a non-secA1 bacterial signal peptide. In some embodiments, the second and third polypeptides are polypeptides selected from any of the polypeptides described herein, such as polypeptides comprising an antigen (see, eg, Section IV above). In some embodiments, the first and second polypeptides are polypeptides selected from any of the polypeptides described herein (eg, see Section IV above). In some embodiments, at least one of the first and second polypeptides comprises an antigen. In some embodiments, the first and second polynucleotides each comprise a fragment of the same antigen. In some embodiments, the antigen is a tumor-associated antigen or is derived from a tumor-associated antigen.

For example, the present invention provides a polycistronic expression cassette for the expression of heterologous polypeptides in Listeria , wherein the expression cassette encodes at least two isolated non- Listeria polypeptides. In some embodiments, the polycistronic expression cassette is a biscistronic expression cassette encoding two separate non- Listeria polypeptides. In some embodiments, the expression cassette comprises: (a) a first polynucleotide encoding a first non- Listeria polypeptide; (b) a second polynucleotide encoding a second non- Listeria polypeptide; (c) an intergenic sequence located between the first polynucleotide and the second polynucleotide; And (d) a promoter operably linked to the first and second polynucleotides, wherein the expression cassette encodes the first and second polypeptides as two separate polypeptides. If the expression cassette is a polycistronic expression cassette that encodes three polypeptides as separate polypeptides, the expression cassette is a third polynucleotide operably linked to a promoter and a second located between the second polynucleotide and the third polynucleotide. It will include intergenic sequences. In some embodiments, at least one of the non- Listeria polypeptides comprises an antigen. In some embodiments, at least two of the non- Listeria polypeptides each comprise a fragment of the same antigen.

In some embodiments, the expression cassette comprises: (a) a first polynucleotide encoding a first signal peptide; b) a second polynucleotide encoding a first (non-signal) non- Listeria polypeptide; (c) a third polynucleotide encoding a second signal peptide; (d) a fourth polynucleotide encoding a second (non-signal) non- List-eria polypeptide, which is in the same translation reading frame as the third polynucleotide; (e) an intergenic sequence located between the second polynucleotide and the third polynucleotide; And (f) a promoter operably linked with a first polynucleotide, a second polynucleotide, a third polynucleotide, and a fourth polynucleotide, wherein the expression cassette comprises a first comprising a first signal peptide and a first polypeptide. It encodes both a fusion protein and a second fusion protein comprising a second signal peptide and a second polypeptide. In some embodiments, at least one of the non- Liste-ria polypeptides comprises an antigen. In some embodiments, at least two of the non- Listeria polypeptides are each fragment of the same antigen.

The present invention also provides a method for producing recombinant bacteria (eg, recombinant Listeria bacteria) using any of the expression cassettes described herein. In some embodiments, a method of making a recombinant bacterium using the expression cassette described herein comprises introducing the expression cassette into a bacterium. In some embodiments, the expression cassette is integrated into the genome of the bacterium. In some other embodiments, the expression cassette is in a plasmid incorporated into the bacterium. In some embodiments, the incorporation of the expression cassette into the bacteria occurs by conjugation. Introduction of expression cassettes into bacteria can be done by any standard technique known in the art. For example, incorporation of expression cassettes into bacteria can occur by conjugation, transduction (transfection), or transformation.

VII. vector

The invention further provides vectors, such as expression vectors, which comprise any of the expression cassettes and / or recombinant nucleic acid molecules described herein. In some embodiments the vector is a plasmid. In some embodiments the vector is linear. In some embodiments, the vector is annular. In some embodiments, the vector is an integrated or homologous recombinant vector. In some embodiments the vector is pAM401. In some embodiments, the vector is pPL2. In some embodiments, the vectors are isolated.

As indicated above, in some embodiments, the expression cassettes described herein are contained in an expression vector. In some embodiments the vector is a plasmid. In some embodiments the vector is linear. In other embodiments, the expression cassette is inserted (ie integrated) into the bacterial genomic DNA using the expression vector. In some embodiments the expression vector is linear. In other embodiments the expression vector is annular.

Suitable expression vectors for use in bacteria such as Listeria are known to those skilled in the art. There are a variety of suitable vectors used as plasmid construct backbones for expression cassette assemblies. The particular plasmid construct backbone is selected based on whether the expression of polynucleotides (ie, polynucleotides encoding heterologous antigens) from bacterial chromosomes or extrachromosomal episomes is desired.

In some embodiments, the incorporation of the expression cassette (and / or recombinant nucleic acid molecule) of the Listeria monocytogenes ( Listeria ) into the bacterial chromosome is for Listeriphage integrase that catalyzes sequence-specific integration of the vector into the Listeria chromosome. This is accomplished using an integrated vector containing an expression cassette. For example, it has been described in the literature that integration vectors known as pPL1 and pPL2 program stable single-copy integration of heterologous protein expression cassettes into non-toxic regions of the bacterial genome (Lauer et al. 2002 J. Bacteriol. 184: 4177-4178; US Patent Publication No. 2003-0203472). This integration vector is stable as a plasmid in E. coli and introduced by conjugation into the desired Listeria background. Each vector lacks Listeria -specific origin of replication and encodes phage integrase, which makes the vector stable only upon integration into the chromosomal phage attachment site. Starting with the desired plasmid construct, the process of generating a recombinant Listeria strain that expresses the desired protein (s) takes approximately one week. The pPL1 and pPL2 integration vectors are based on U153 and PSA Listeriphages, respectively. The pPL1 vector is integrated into the open reading frame of the comK gene and pPL2 is integrated into the tRNAArg gene in such a way that upon successful integration, the natural sequence of the tRNAArg gene is restored, thereby maintaining its native expressed function. pPL1 and pPL2 integration vectors contain multiple cloning site sequences to facilitate the construction of plasmids containing expression cassettes such as recombinant nucleic acid molecules or heterologous protein expression cassettes. Certain specific examples of the use of the pPL2 integration vector are described in Examples 2 and 3 below.

Alternatively, incorporation of the expression cassette (and / or recombinant nucleic acid molecule) into the Listeria chromosome can be accomplished through allelic exchange methods known to those skilled in the art. In particular, a composition in which alleles that do not incorporate a gene encoding an antibiotic resistance protein as part of a construct containing an expression cassette is preferred. For example, the pKSV7 vector (Camilli et al. Mol. Microbiol. (1993) 8, 143-157) contains a temperature-sensitive Listeria Gram-positive replication origin, which indicates a non-acceptable temperature that represents a pKSV7 plasmid recombined with the Listeria chromosome. Is used to select the recombinant clone. The pKSV7 allele exchange plasmid vector contains a number of cloning site sequences and chloramphenicol resistance genes to facilitate the construction of plasmids containing protein expression cassettes. Expression cassette constructs for insertion into the Listeria chromosome can be flanked by about 1 Kb of chromosomal DNA sequence corresponding to the exact location of the desired integration. The pKSV7-expressing cassette plasmid can be introduced into the desired bacterial strain by electroporation according to standard methods for electroporation of gram positive bacteria. A non-limiting example of a method for allele exchange using the pKSV7 vector is provided in Example 16 below.

In other embodiments, it may be desirable to express polypeptides (including fusion proteins comprising polypeptides) from stable plasmid episomes. The maintenance of plasmid episomes through generations requires co-expression of proteins, which confers a selective advantage over plasmid-containing bacteria. As a non-limiting example, a protein co-expressed from a plasmid with a polypeptide may be an antibiotic resistant protein, eg chloramphenicol, or a bacterial protein (expressed from a chromosome of a wild type bacterium) that may also confer a selective advantage. Can be. Non-limiting examples of bacterial proteins are the enzymes required for purine or amino acid biosynthesis (selected using a regulatory medium without related amino acids or other necessary precursor macromolecules), or genes that confer selective benefits in vitro or in vivo. Transcription factors required for the expression of Gunn et al., 2001, J. Immuol. 167: 6471-6479. As a non-limiting example, pAM401 is a suitable plasmid for episomal expression of selected polypeptides in various Gram-positive bacteria (Wirth et al., 1986, J. Bacteriol 165: 831-836). See Examples 3 and 13 below for further explanation of typical uses of pAM 401.

B. Expression of the transfer cassette into the bacterial chromosome of anthracis, for example, B. anthracis sequence of a vector in a chromosome-specific integration can be achieved by using the integrated vector containing the expression cassette for phage integrase catalyzes have. The site of attachment of integrase and B. anthracis phage can be used to derive an integration vector and incorporate the desired antigen expression cassette into the vaccine composition. As a non-limiting example, the attachment sites of integrase and B. anthracis latent phage w-alpha can be used to derive B. anthracis specific integration vectors (McCloy, EW, 1951, Studies on a lysogenic Bacillus stain. I. A Bacteriophage specific for Bacillus anthracis.J. Hyg. 49: 114-125).

Alternatively, the incorporation of the antigen expression cassette into the B. anthracis saltbody can be accomplished through allelic exchange methods known to those skilled in the art. For example, Gat et al., Infect. See Immun., 71: 801-13 (2003). In particular, a composition in which alleles that do not incorporate a gene encoding an antibiotic resistance protein as part of the construct containing the expression cassette is preferred. For example, the pKSV7 vector (Camilli et al., Mol. Microbiol. 1993, 8: 143-157) is a temperature-selective Listeria -used to select recombinant clones at a non-acceptable temperature that represents a pKSV7 plasmid recombined with bacterial chromosomes. Derived gram positive replication origin. The pKSV7 allele exchange plasmid vector contains a number of cloning site sequences and chloramphenicol resistance genes to facilitate the construction of plasmids containing protein expression cassettes. Expression cassette constructs for insertion into the Bacillus anthracis chromosome can be flanked by about 1 Kb of chromosomal DNA sequence corresponding to the exact location of the desired integration. The pKSV7-expressing cassette plasmid can be introduced into the desired bacterial strain by electroporation according to standard methods for electroporation of gram positive bacteria. Non-limiting examples of methods for allele exchange in B. anthracis are described in US Patent Application No. Provided at 10 / 883,599. In particular, allele exchange using the pKSV7 vector can be used in strains of B. anthracis to add the desired antigen expression cassette at any desired position in the bacterial chromosome.

The allele exchange method using pKSV7 described above is widely used to use gram positive bacteria. In addition, various expression vectors useful in bacteria, including recombinant bacterial vectors, are known to those skilled in the art. Examples include the vectors described in the following references, each of which is incorporated herein by reference in its entirety: Horwitz et al., Proc. Natl. Acad. Sci. USA, 97: 13853-8 (2000); Garmory et al., J. Drug Target, 11: 471-9 (2003); Kang et al., FEMS Immunol. Med. Microbiol., 37: 99-104 (2003); Garmory et al. Vaccine, 21: 3051-7 (2003); Kang et al., Infect. Immun. 1739-49 (2002); Russman et al. J. Immunol. 167: 357-65 (2001); Harth et al. Microbiology, 150: 2143-51 (2004); Varaldo et al., Infect. Immun., 72: 3336-43 (2004); Goonetilleke et al., J. Immunol. 171: 1602-9 (2003); Uno-Furuta et al., Vaccine, 21: 3149-56 (2003); Biet et al., Infect. Immun., 71: 2933-7 (2003); Bao et al., Infect. Immun., 71: 1656-61 (2003); Kawahara et al., Clin. Immunol, 105: 326-31 (2002); Anderson et al., Vaccine, 18: 2193-202 (2000); Bumann, Infect. Immun. 69: 7493-500 (2001); Wang et al., Vaccine, 17: 1-12 (1999); McSorley et al., Infect Immun., 65: 171-8 (1997); Gat et al., Infect. Immun., 71: 801-13 (2003); U.S. Patent No. 5,504,005; U.S. Patent No. 5,830,702; U.S. Patent No. 6,051,237; United States Patent Publication No. 2002/0025323; United States Patent Publication No. 2003/0202 985; WO 04/062597; U.S. Patent No. 6,566,121; And US Patent No. 6,270,776.

Furthermore, the present invention provides a kit comprising: (a) a first polynucleotide encoding a first signal peptide; (b) a second polynucleotide encoding the first polypeptide that is within the same translation reading frame as the first polynucleotide; (c) intergenic sequences; (d) a third polynucleotide encoding a second signal peptide; (e) a fourth polynucleotide encoding the second polypeptide, which is in the same translation reading frame as the third polynucleotide; And f) an expression cassette comprising a promoter that is operably linked with a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, and an intergenic sequence, the expression cassette comprising: It encodes both a first fusion protein comprising a first signal peptide and a first polynucleotide and a second fusion protein comprising a second signal peptide and a second polypeptide.

Furthermore, the present invention provides a method for producing recombinant bacteria (eg, recombinant Listeria bacteria) using any of the expression vectors described herein. In some embodiments, a method of making a recombinant bacterium using the expression vector described herein includes introducing the expression vector into the bacterium.

VIII. Bacteria and other host cells

Further, the present invention is directed to recombinant nucleic acid molecules, expression cassettes, and / or described herein (e.g., see the overview of the invention, sections I, II, VI and VII of the detailed description, and certain examples below). A host cell comprising a vector is provided. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an antigen-presenting cell, such as a dendritic cell. In some embodiments, the cell is a bacterial cell. In some embodiments, the cells are isolated.

For example, the present invention is directed to recombinant nucleic acid molecules, expression cassettes, and / or described herein (e.g., see the overview of the invention, Sections I, II, VI and VII of the detailed description, and certain examples below). Or a bacterium comprising the vector. Bacteria comprising these polynucleotides are also referred to as “recombinant bacteria,” and bacteria including recombinant nucleic acid molecules, expression cassettes, or vectors described herein are also referred to as “recombinant bacteria”. In some embodiments, bacteria comprising recombinant nucleic acid molecules, expression cassettes, and / or expression vectors are isolated. In some embodiments, a recombinant bacterium comprising a recombinant nucleic acid molecule, expression cassette, and / or expression vector expresses a polypeptide or fusion protein encoded by the recombinant nucleic acid molecule, expression cassette, and / or expression vector contained therein. In some embodiments, the recombinant bacterium secretes a polypeptide or fusion protein encoded by a recombinant nucleic acid molecule, expression cassette, and / or expression vector contained therein. In some embodiments, the recombinant bacterium expresses and secretes an amount of polypeptide and / or fusion protein sufficient to elicit an immune response in the host upon administration of the bacterium (or composition comprising the bacterium) to the host (eg, a human subject). do.

In some embodiments, the bacteria are selected from the group consisting of gram positive bacteria, gram negative bacteria, intracellular bacteria and mycobacteria. In some embodiments, the bacteria are gram positive bacteria. In some embodiments of the invention, the bacterium is an intracellular bacterium (eg, an intracellular bacterium). In some embodiments, the bacteria belong to the Listeria genus. In another embodiment, the bacteria is a member of the Listeria monocytog-enes species. In some other embodiments, the bacteria are members of Listeria ivanovii, Lis-teria seeligeri , or Listeria innocua species. In some embodiments, the bacteria are members of the genus Bacillus . In another embodiment the bacterium is Bacillus anthracis . In another embodiment, the bacterium is Yersinia pestis . In another embodiment of the invention, the bacteria are from the genus Salmonella . In some embodiments, the bacterium is Salmonella typhimurium . In some embodiments, the bacteria belong to the genus Shigella . For example, in some embodiments, the bacterium is Shigella flexneri . In some embodiments, the bacteria are members of the genus Brucella . In another embodiment, the bacterium is mycobacteria. Mycobacteria are optionally members of the Mycobacterium tuberculosis species. In some embodiments, the bacterium is Bacillus Calmette-Guerin (BCG). In some embodiments, the bacterium is E. coli, for example, Shin-less terry duck O (LLO) an E. coli strain to express. Thus, in some embodiments, the bacterium comprising the recombinant nucleic acid molecule, expression cassette, and / or vector described herein is selected from the group consisting of Listeria, Bacillus anthracis, Yersinia pestis, Salmonella , and mycobacteria. In some other embodiments, the bacterium comprising the recombinant nucleic acid molecule, expression cassette, and / or vector described herein is from the group consisting of Listeria, Bacillus, Yersinia pestis, Salmonella, Shigella, Brucella, Mycobacteria and E. coli . Is selected.

In some embodiments, recombinant nucleic acid molecules, expression cassettes, and / or vectors described herein (eg, see the overview of the invention, sections I, II, VI, and VII of the detailed description, and certain examples below). Bacteria of the invention that have been modified to express a polypeptide through insertion of and, at least in some embodiments, to secrete a polypeptide, are wild type bacteria. For example, in some embodiments, the recombinant bacteria are wild type Listeria bacteria, such as Listeria monocytogenes bacteria, which include recombinant nucleic acid molecules, expression cassettes, and / or vectors. However, in some embodiments of the invention, the bacterium comprising the expression cassette and / or the vector is a mutant strain of bacteria. In some embodiments, the bacteria are weakened. In some embodiments, the bacterium is a weakened mutant strain of the bacterium. Mutations in which the gene “xyz” has been deleted are referred to herein as Δxyz or xyz ⁻ or xyz deletion mutations. For example, the bacterial strain is deleted uvrA gene uvrA mutation, ΔuvrA, or uvrA herein ^- is called. In addition, it will be understood by those skilled in the art that referring to a particular mutation or strain as a “xyz” mutation or “xyz” strain sometimes refers to a mutation or strain from which the xyz gene is deleted.

In a mutant bacterium comprising an expression cassette and / or an expression vector, the mutation may be any kind of mutation. For example, mutations may constitute point mutations, frame-shift mutations, insertions, deletions of part or all of a gene. In addition, in some embodiments of the modified strains, portions of the bacterial genome have been replaced with heterologous polynucleotides. In some embodiments the mutation is spontaneous. In another embodiment, the mutation is the result of artificial mutation pressure. In another embodiment, the mutation in the bacterial genome is the result of genetic engineering.

In some embodiments, bacteria comprising any of the recombinant nucleic acid molecules, expression cassettes and / or vectors described herein are attenuated for intercellular propagation, penetration into non-phagocytic cells, or proliferation (relative to wild type bacteria). . Bacteria can be weakened by mutations or other modifications. In some embodiments, bacteria comprising any of the recombinant nucleic acid molecules, expression cassettes, and / or expression vectors described herein are attenuated for intercellular propagation (eg, Listeria monocytogenes actA mutations). In some embodiments, bacteria comprising any of the recombinant nucleic acid molecules, expression cassettes, and / or expression vectors described herein are attenuated for infiltration into non-phagocytic cells (eg, Listeria monocytogenes such as inlB deletion mutations). Internalin mutations). In some embodiments, bacteria comprising any of the recombinant nucleic acid molecules, expression cassettes, and / or expression vectors described herein are attenuated for proliferation. In some embodiments, the bacteria are attenuated for intercellular propagation and penetration into non-phagocytic cells.

In some embodiments, the bacteria comprising the expression cassettes and / or expression vectors described herein are attenuated for intercellular propagation. In some embodiments, the bacteria (eg Listeria ) are defective (relative to non-mutant or wild type bacteria) with respect to ActA , or its equivalent (depending on the organism). In some embodiments, the bacterium comprises one or more mutations in actA . For example, the bacterium (eg, Listeria ) can be an actA deletion mutant. ActA is an actin polymerase encoded by the actA gene (Kocks et al., Cell, 68: 521-531 (1992); Genbank Accession No. AL591974, nts 9456-11389). This actin polymerase protein is involved in the recruitment and polymerization of host F-actin in a poly of Listeria bacteria. As a result of the subsequent polymerization and dissociation of actin, Listeria protrudes into neighboring cells through the cytosol. This motility allows bacteria to spread directly from cell to cell without exposure to the extracellular environment, thereby avoiding host defenses such as antibody development. In some embodiments, the attenuated Listeria optionally contains mutations in both the internalin gene, such as inlB , and actA . In this embodiment of the invention the Listeria strain is not only weakened for infiltration into non-phagocytic cells but also weakly for intercellular propagation.

In some embodiments, the ability of the attenuated bacterium to intercellular propagation is at least about 10%, at least about 25%, at least about 50%, at least about 75% compared to bacteria without attenuating mutations (eg wild type bacteria). , Or by at least about 90%. In some embodiments, the ability of attenuated bacteria to intercellular propagation is reduced by at least about 25% compared to bacteria without attenuating mutations. In some embodiments, the ability of attenuated bacteria to intercellular propagation is reduced by at least about 50% compared to bacteria without attenuating mutations.

In vitro assays are known to those skilled in the art to determine whether bacteria, such as Listeria bacteria, are attenuated for intercellular propagation. For example, the diameter of plaques formed over time after infection of selected culture cell monolayers can be measured. Plaque assays in L2 cell monolayers are described in Skoble, J., DAPortnoy, and MDWelch. 2000, Three regions within ActA promote Arp2 / 3 complex-mediated actin nucleation and Listeria monocytogenes motility. Sun, A., A. Camilli, and DA Portnoy 1990 Isolation of Listeria monocytogenes small-plaque mutants defective for intracellular growth and cell-to, using variations on the measurements described in J. Cell Biol. 150: 527-538. -cell spread. Infect.Immun. 58: 3770-3778, as already described. In short, L2 cells are grown to a confluence in a 6-well tissue culture dish and then infected with bacteria for 1 hour. Post-infection cells are covered with medium warmed to 40 ° C. consisting of 0.8% agarose, fetal bovine serum (eg 2%), and the desired concentration of gentamicin. Gentamicin concentration in the medium has a profound effect on plaque size and is a measure of the ability of selected Listeria strains to intercellularly propagate (Glomski, IJ, MM Gedde, AW Tsang, JA Swanson, and DA Portnoy. 2002. J. Cell Biol. 156: 1029-1038). For example, three days after monolayer infection, the plaque size of a Listeria strain with a phenotype of defective intercellular propagation was at least 50% compared to wild type Listeria when covered with a medium containing 50 μg / ml gentamicin. Until it is reduced. On the other hand, when the infected monolayer was covered with medium + agarose containing only 5 μg / ml gentamycin, the plaque size of the wild type Listeria and the Listeria strain with the phenotype of defective intercellular propagation were similar. Thus, the relative ability of selected strains to effect intercellular propagation in infected cell monolayers as compared to wild type Listeria can be measured by varying the concentration of gentamicin in the medium containing agarose. Optionally, visualization and measurement of plaque diameter can be facilitated by adding medium containing Neutral Red (GIBCO BRL; 1: 250 dilution in DME + Agarose medium) to the covered layer 48 hours after infection. In addition, plaque assays can be performed on monolayers derived from other primary cells or continuous cells. For example, HepG2 cells, hepatocyte-derived cell lines, or primary human hepatocytes can be used to assess the ability of selected Listeria mutations to effect intercellular propagation compared to wild type Listeria . In some embodiments, Listeria comprising mutations or other modifications of weakening for the propagation between the Listeria cell generates a "pinpoint" plaques at high concentrations of gentamicin (about 50㎍ / ml).

In some embodiments, the bacterium comprising the expression cassette and / or expression vector described herein is a mutant bacterium that is attenuated for nucleic acid repair (relative to wild type, such as a bacterium without a weakened genetic mutation). For example, in some embodiments, the bacterium is defective with respect to at least one DNA repair enzyme (eg, Listeria monocytogenes uvrAB mutation). In some embodiments, the bacteria are defective with respect to one of PhrB, UvrA, UvrB, UvrC, UvrD, and / or RecA , or their equivalents (depending on the genus and species of the bacterium). In some embodiments, the bacteria are defective with respect to UvrA, UvrB, and / or UvrC. In some embodiments, the bacterium comprises a weakening mutation in the phrB, uvrA, uvrB, uvrC, uvrD, and / or recA genes. In some embodiments, the bacterium comprises one or more mutations in the uvrA, uvrB , and / or uvrC genes. In some embodiments, the bacteria are functionally deleted in UvrA , UvrB , and / or UvrC . In some embodiments, the bacteria are deleted in functional UvrA and UvrB . In some embodiments, the bacterium is a uvrAB deletion mutation. In some embodiments, the bacterium is a ΔuvrABΔactA mutation. In some embodiments, the nucleic acid of a bacterium that is attenuated for nucleic acid repair and / or is defective with respect to at least one DNA repair enzyme is modified by reaction with a nucleic acid targeting compound (see below). Nucleic acid repair mutations, such as the ΔuvrAB Listeria monocytogenes mutations, and methods of making these mutations are described in US Pat. 2004/0197343 (see, eg, Example 7 of US 2004/0197343).

In some embodiments, the ability of a weakened bacterium to repair a nucleic acid is at least about 10%, at least about 25%, at least about 50%, at least about 75% compared to a bacterium without attenuating mutations (eg, wild type bacteria). , Or by at least about 90%. In some embodiments, the ability of attenuated bacteria to nucleic acid repair is reduced by at least about 25% compared to bacteria without attenuating mutations. In some embodiments, the ability of attenuated bacteria to nucleic acid repair is reduced by at least about 50% compared to bacteria without attenuating mutations.

Confirmation of the presence of specific mutations in bacterial strains can be obtained through various methods known to those skilled in the art. For example, relevant portions of the genome of the strain can be cloned and sequenced. Alternatively, specific mutations can be identified by PCR using a pair of primers encoding regions adjacent to deletions or other mutations. In addition, Southern blots can be used to detect changes in the bacterial genome. In addition, standard techniques in the art, such as western blotting, can be used to analyze whether a particular protein is expressed by this strain. In addition, identification of strains containing mutations in the desired gene can be obtained by comparing the phenotype of this strain with previously reported phenotypes. For example, the presence of nucleotide ablation repair mutations, such as the deletion of uvrAB , uses assays to test the bacteria's ability to repair its nucleic acids using the nucleotide ablation repair (NER) machine, Can be evaluated by comparison. Such functional analysis is known in the art. For example, cyclobutane dimer ablation or ablation of UV-induced (6-4) products can be measured to determine NER enzyme deficiency in mutations (eg, Franklin et al., Proc. Natl. Acad. Sci. USA, 81: 3821-3824 (1984)). Alternatively, survival measurements can be made to assess the lack of nucleic acid repair. For example, bacteria can be subjected to Solaren / UVA treatment and then assessed for their ability to proliferate and / or survive as compared to wild type.

In some embodiments, the bacteria are attenuated for penetration into non-phagocytic cells (relative to non-mutant or wild type bacteria). In some embodiments, the bacterium (eg, Listeria ) is defective with respect to one or more internalins (or equivalents). In some embodiments, the bacteria are defective with respect to Internalin A. In some embodiments, the bacteria are defective with respect to internalin B. In some embodiments, the bacterium comprises a mutation in inlA . In some embodiments, the bacterium comprises a mutation in inlB . In some embodiments, the bacterium comprises a mutation in both actA and inlB . In some embodiments, the bacteria are deleted in functional ActA and internalin B. In some embodiments, the bacterium is a ΔactAΔinlB double deletion mutation. In some embodiments, the bacteria are defective with respect to both ActA and internalin B.

In some embodiments, the ability of attenuated bacteria to penetrate into non-phagocytic cells is at least about 10%, at least about 25%, at least about 50%, as compared to bacteria without attenuating mutations (eg, wild type bacteria), Reduced by at least about 75%, or at least about 90%. In some embodiments, the ability of attenuated bacteria to penetrate into non-phagocytic cells is reduced by at least about 25% compared to bacteria without attenuating mutations. In some embodiments, the ability of attenuated bacteria to penetrate into non-phagocytic cells is reduced by at least about 50% compared to bacteria without attenuating mutations. In some embodiments, the ability of attenuated bacteria to penetrate into non-phagocytic cells is reduced by at least about 75% compared to bacteria without attenuating mutations.

In some embodiments, a weakened bacterium, such as a mutant Listeria strain, is not attenuated for penetration into one or more types of non-phagocytic cells. For example, a weakened strain may be weakened for penetration into hepatocytes, but not weakened for penetration into epithelial cells. As another example, the weakened strain may be weakened for infiltration into epithelial cells but not for hepatocytes. In addition, the weakening of the penetration of certain modified bacteria into non-phagocytic cells results in mutations in designated genes such as deletion mutations, coding for invading proteins that interact with specific cellular receptors, and facilitating infection of non-phagocytic cells. Is understood. For example, the Listeria ΔinlB mutant strain is attenuated for infiltration into hepatocyte growth factor receptor (c-met), including hepatocyte cell lines (eg HepG2), and non-phagocytic cells expressing primary human hepatocytes.

In some embodiments, even if bacteria (eg mutant Listeria ) are weakened for infiltration into non-phagocytic cells, Listeria can still be taken up by at least dendritic cells and / or macrophages such as macrophages. In one embodiment, the ability of attenuated bacteria to penetrate into phagocytes is not reduced by modifications made to the strain, eg, by invading mutations (ie, about 95 of the measured ability of the strain to be taken up by phagocytes). More than% is maintained after deformation). In other embodiments, the ability of the attenuated bacteria to penetrate into the phagocytes is reduced by up to about 10%, up to about 25%, up to about 50%, or up to about 75%.

In some embodiments, the amount of attenuation of the ability of bacteria (eg Listeria bacteria) to penetrate into non-phagocytic cells ranges from two-fold reductions to even higher levels of attenuation. In some embodiments, the weakening of the bacterial ability to penetrate into non-phagocytic cells is at least about 0.3 log, about 1 log, about 2 log, about 3 log, about 4 log, about 5 log, or at least about 6 log. In some embodiments, the attenuation is about 0.3 to> 8 log, about 2 to> 8 log, about 4 to> 8 log, about 6 to> 8 log, about 0.3 to 8 log, and about 0.3 to 7 log, and about 0.3 to 6 logs, about 0.3 to 5 logs, about 0.3 to 4 logs, about 0.3 to 3 logs, about 0.3 to 2 logs, and about 0.3 to 1 log. In some embodiments, the attenuation ranges from about 1 to> 8 logs, 1 to 7 logs, 1 to 6 logs, yet about 2 to 6 logs, yet about 2 to 5 logs, and about 3 to 5 logs.

Internalin numbers were identified in L. monocytogenes (Boland et al., Clinical Micro biology Reviews, 2001, 14: 584-640). These internalins include, but are not limited to, InlA, InlB, InlC, InlC2, InlD, InlE, InlF, InlG and InlH (Dramsi et al., Infection and Immunity, 65: 1615-1625 (1997); Rolelsbauer et al Mol) Gen. Genet. 260: 144-158 (1998). Gene sequences encoding these proteins have been previously reported. For example, the sequences for both inlA and inlB have been reported in Gaillard et al., Cell, 65: 1127-1141 (1991) as GenBank Accession No. M67471. Genes encoding additional members of the internalin-associated protein family, including lmo0327, lmo0331, lmo0514, lmo0610, lmo0732, lmo1136, lmo1289, lmo2396, lmo0171, lmo0333, lmo0801, lmo1290, lmo2026 and lmo2821, are described in Glaser et al. Science, 294 : See Web Table 2 (www.sciencemag.org/cgi/content/full/294/5543/849/DC1) of Supplementary Web Materials, 849-852 (2001). Sequences can be found in the L. mono-cytogenes strain EGD genome, GenBank Accession No. AL591824, and / or L. monocytogenes strain EGD-e genome, GenBank Accession No. NC_003210. do).

InlA (Internalin A) (Gaillard et al., Cell, 65: 1127-1141 (1991); Genbank Accession No. NC — 003210) is involved in the uptake of Listeria by epithelial cells, such as intestinal epithelial cells.

InlB (Internaline B) (Gaillard et al., Cell, 65: 1127: 1141 (1991); Genbank Accession No. AL591975 ( Listeria monocytogenes strain EGD, complete genome, segment 3/12, inlB gene region: nts. 97008-98963); And Genbank Accession No. NC_003210 ( Listeria mono-cytogenes strain EGD, complete genome, inlB gene region: nts. 457008-458963), each of which is incorporated herein by reference in its entirety), comprises hepatocyte or blood brain barriers. It is related to the uptake of Listeria by endothelial cells, such as vascular endothelial cells of the brain microvascular structure (for further description of Internaline B, see Ireton et al., J. of Biological Chemistry, 274: 17025-17032 (1999); Dramsi; Molecular Microbiology 16: 251-261 (1995); Mansell et al., J. of Biological Chemistry, 276: 43597-43603 (2001); and Bierne et al., J. of Cell Science 115: 3357-3367 (2002). All of which are incorporated herein by reference).

In some embodiments, the bacteria are defective with respect to ActA, internalin B, or both ActA and internalin B. In some embodiments, the bacteria are defective in functional ActA, internalin B, or both ActA and internalin B. In some embodiments, the bacteria is deleted in functional ActA. In some embodiments, the bacteria is deleted in functional internalin B. In some embodiments, the bacteria are deleted in functional ActA and internalin B.

In vitro assays are known to those of skill in the art to determine whether bacteria (eg, mutant Listeria strains) have been weakened for infiltration into non-phagocytic cells. For example, Dramsi et al., Molecular Microbiology 16: 251-261 (1995) and Gaillard et al., Cell 65: 1127-1141 (1991) are all assays capable of screening the ability of L. monocytogenes strains to enter certain cell lines. Explain. For example, to determine whether a Listeria bacterium with a specific strain is weakened against invasion of a particular type of non-phagocytic cell, the ability of the weakened Listeria bacterium to penetrate a particular type of non-phagocytic cell is measured and no modification is made. Compared to the ability of the same Listeria bacteria to penetrate into non-phagocytic cells. Similarly, the Listeria strain having the particular mutation in order to weaken the measurement gone for penetration of a specific type of non-phagocytic cells, the ability of the mutant Listeria strain is measured to penetrate to a specific type of non-phagocytic cells, the Listeria strain without the mutation Compared to the ability to infiltrate non-phagocytic cells. In addition, confirmation of whether a strain has a defect related to internalin B can be obtained by comparing the phenotype of this strain with the phenotype already reported for the internalin B mutation.

In some embodiments, the weakening of bacteria can be measured in terms of the biological effect of Listeria on the host. Pathogenicity of bacterial strains can be assessed by measuring LD ₅₀ in mice or other vertebrates. LD ₅₀ is the amount or dose of Listeria injected into the vertebrate required to cause death in 50% of the vertebrate. The LD ₅₀ value is a measure of the level of attenuation and can be compared to bacteria with specific modifications (eg, mutations) and bacteria without specific modifications. For example, if a bacterial strain without a specific mutation has an LD ₅₀ of 10 ³ bacteria and a bacterial strain with a specific mutation has an LD ₅₀ of 10 ⁵ bacteria, the strain is attenuated so that the LD ₅₀ is 100-fold or 2 log It is increased to.

Alternatively, the degree of attenuation of the bacteria's ability to infect non-phagocytic cells can be assessed even more directly in vitro. For example, the ability of a modified Listeria bacterium to infect non-phagocytic cells, such as hepatocytes, can be compared with the ability to infect phagocytes of non-modified Listeria or wild type Listeria . In such assays, modified Listeria and non-modified Listeria are typically added to non-phagocytic cells in vitro for a limited time period (e.g., 1 hour), and then the cells are washed with gentamicin-containing solution to remove any cells. The titer is evaluated by killing extra bacteria, lysing the cells and then reputing. Examples of such assays are described in US Pat. It is found in 2004/0228877.

As a further example, the degree of weakening can also be determined qualitatively by other biological effects, such as the extent of histopathology or serum liver enzyme levels. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin and bilirubin levels in serum are measured for mice injected with Listeria (or other bacteria) in clinical laboratories. As with the manner of assessing the weakening of Listeria, comparisons of these effects in mice or other vertebrates can be made for Listeria with and without Listeria with certain modifications / mutations. Attenuation of Listeria can also be measured by histopathology. In addition, the amount of Listeria that can be recovered from various tissues of infected vertebrates, such as the liver, spleen and nervous system, can be used as a measure of the level of weakening by comparing these values in vertebrates mutated against non-mutant Listeria . . For example, the amount of Listeria that can be recovered from infected tissues such as the liver or spleen as a function of time can be used as a measure of attenuation by comparing these values in mice mutated for non-mutant Listeria .

Thus, the weakening of the Listeria can be measured as an item on the bacterial load in particular selected organs in mice known as targeted by the wild-type Listeria. For example, weakening of Listeria can be measured by counting colonies (colony forming units; CFUs) resulting from plating a dilution of liver or spleen homogenate (homogenized in H ₂ O + 0.2% NP40) on BHI agar medium. Can be. Liver or spleen cfu can be measured over time after administration of the modified Listeria by a number of routes including, for example, intravenous, intraperitoneal, intramuscular, and subcutaneous. In addition, Listeria can be measured for drug-tolerance in the liver and spleen (or any other selected organ) over time after administration by the described competitive index assay and compared with wild type Listeria (or any other selected Listeria strain). have.

The degree of weakening in the uptake of the weakened bacteria by non-phagocytic cells need not be complete weakening to provide a safe and effective vaccine. In some embodiments, the degree of weakening is such that it provides a reduction in toxicity sufficient to prevent or reduce symptoms of toxicity to a level that does not endanger life.

In some embodiments of the invention, the bacterium comprising the recombinant nucleic acid molecule, expression cassette and / or expression vector described herein is a mutant strain of Listeria . In a further embodiment, the bacteria is a weakened mutant strain of Listeria monocytogenes . Several typical mutant strains of attenuated Listeria are described in US Pat. 60 / 446,051 (submit February 6, 2003), 60 / 449,153 (submit February 21, 2003), 60 / 511,719 (submit October 15, 2003), 60 / 511,919 (submit October 15, 2003) ), 60 / 511,869 (filed October 15, 2003), 60 / 541,515 (filed February 2, 2004), and 10 / 883,599 (filed June 30, 2004), as well as US Patent Publication No. 2004/0197343 and US2004 / 0228877, each of which is incorporated herein by reference. In addition, the mutant strains of Listeria are described in International Application No. PCT / US2004 / 23881 is described. For example, a bacterium comprising an expression cassette and / or vector is a mutant strain of Listeria monocytogenes , which is optionally defective for both ActA or internalin B, or both ActA and internalin B. In some embodiments, the bacteria are actA ^- (e.g., such as Skoble, J. of Cell Biology, 150 included in DP-L4029 (DP-L3078 strains, reference herein: as described in the 527-537 (2000) ., etc. This Lauer, J. Bacteriol 184: 4177 (2002 ); been hardened to its pro phage, as described in U.S. Patent Publication ^{No. 2003/0203472)), actA - inlB} - ( for example, DP-L4029 inlB , deposited on October 3, 2003, to the American Type Culture Collection (ATCC) (10801 University Blvd., Manassas, Virginia 20110-2209, USA) under the Budapest Treaty under the International Convention on the Deposit of Microorganisms in the Patent Process. Accession No. ^{PTA-5562), actA - uvrAB} - ( for example, DP-L4029 uvrAB, under the Budapest Treaty of the International code for the microorganisms deposited with the process of Patent American Type Culture Collection (ATCC) ( 10801 University Blvd. Manassas, Virginia Deposited on Oct. 3, 2003, 20110-2209, USA, accession number PTA-5563), or actA ^- inlB ^- uvrAB ^Is a mutant strain of Listeria monocytogenes . In some embodiments, the attenuated Listeria bacterium (eg, Listeria monocytogenes bacterium) is a ΔactAΔinlB double deletion mutation.

Bacterial mutations can be achieved through conventional mutagenesis methods such as mutagenesis chemicals or mutation selection after irradiation. Bacterial mutations can also be accomplished by those skilled in the art through recombinant DNA techniques. For example, Camilli et al., Molecular Micro. Allelic exchange methods using the pKSV7 vector described above in connection with the introduction of heterologous expression cassettes into the bacterial genome, as described in 8: 143-157 (1993), are suitable for use in generating mutations including deletion mutations (Camilli et al. 1993) is incorporated herein by reference. One typical example of the preparation of Listeria monocytogenes internalin B using the pKSV7 vector is provided in Example 24 below. Or otherwise, Biswas et al., J. Bacteriol. The gene replacement protocol described in 175: 3628-3635 (1993) can be used. Other similar methods are known to those skilled in the art.

The composition of various bacterial mutations is described in US Pat. 10 / 883,599, US Patent Publication No. 2004/0197343, and US Patent Publication No. 2004/0228877, all of which are incorporated herein by reference.

In some embodiments of the invention, the bacterium comprising the recombinant nucleic acid molecule, expression cassette and / or expression vector is a mutant strain of Bacillus anthracis . In some embodiments, the bacterium is a Sterne strain. In some embodiments, the bacterium is an Ames strain. In some embodiments, the Bacillus anthracis bacterium is a uvrAB mutation. In some embodiments, the Bacillus anthracis strain is uvrC mutation. In some embodiments, the Bacillus anthracis mutation is a recA mutation. In some embodiments, the bacterium is a ΔuvrAB mutation of Bacillus anthracis (eg, the Bacillus anthracis Sterne ΔuvrAB mutation, the American Type Culture Collection (ATCC) (108011 University Blvd. Manassas, Virginia, 20110-2209, US, dated February 20, 2004, accession number PTA-5825.

Methods of altering the genome of Bacillus anthracis are known to those skilled in the art. One way to generate mutations in Bacillus anthracis is by allele exchange using an allele exchange vector known to those skilled in the art. A typical allele exchange plasmid is pKSV7 as described in Camilli et al., Molecular Microbiology, 8: 143-147 (1993). As a first step in generating the mutant Bacillus anthracis , PCR-amplification of approximately 1000 bp in both the genomic region to be deleted or mutated and upstream and downstream of B. anthracis is then cloned into the pKSV7 plasmid vector (or analog vector) ( Bacillus genus). -Specific or B. anthracis -specific temperature (ts) replicons may be substituted with Listeria ts replicons present in the pKSV7 allele exchange plasmid vector). A restriction endonuclease recognition site in the region to be deleted or mutated is used to delete the desired portion of the target gene in this region. Alternatively, a portion of the target gene in this region is removed and replaced with a sequence containing the desired mutation or other alteration. The region of the B. anthracis genome to be amplified can be altered before or after cloning into an allele exchange plasmid using, for example, a restriction enzyme or a combination of restriction enzyme and synthetic gene sequence. In some embodiments, the sequence can be altered as a PCR amplicon and then cloned into pKSV7. In another embodiment, the amplicon is first inserted into another plasmid and then altered and excised to pKSV7. Alternatively, PCR amplicons can be inserted directly into pKSV7 and then modified, for example using convenient restriction enzymes. Next, the pKSV7 plasmid containing the altered sequence is introduced into B. anthracis . This can be done by electroporation. The bacteria are then selected on the medium at the allowable temperature in the presence of chloramphenicol. Subsequently, the choice for single crossover integration into bacterial chromosomes by passing several generations at non-acceptable temperatures in the presence of chloramphenicol is followed. Finally, colonies are passed through several generations in an antibiotic-free medium at acceptable temperatures, thereby achieving plasmid ablation and solidification (double crossover). US Provisional Application No., which is incorporated herein by reference. 60/584, 886 and 60 / 599,522, and US Patent Publication No. 2004/0197343 provides further explanation regarding the construction of various types of Bacillus anthracis mutations.

In some embodiments of the invention, the bacterium comprising the recombinant nucleic acid molecule, expression cassette, and / or expression vector is a bacterium that has been modified such that the bacterium is weakened for proliferation (relative to non-modified bacteria). Preferably, the modified bacteria maintain sufficient gene expression level despite the modification. For example, in some embodiments, the gene expression level is substantially unaffected by the modification, so that the antigen is expressed at a level sufficient to stimulate an immune response to the antigen upon administration of a bacterium that expresses the antigen to the host. . In some embodiments, the bacterial nucleic acid is modified by reaction with a nucleic acid targeting compound. In some embodiments, the modified bacterial nucleic acid is modified by reaction with a nucleic acid targeting compound that reacts directly with the nucleic acid, thereby attenuating the growth of the bacteria. In some embodiments, the nucleic acid targeting compound is a nucleic acid alkylating agent, for example β-alanine, N- (acridin-9-yl), 2- [bis (2-chloroethyl) amino] ethyl ester. In some embodiments, the bacterium is treated with a soraren compound. For example, in some embodiments, the bacterium is selected from solarenes, such as 4 '-(4-amino-2-oxa) butyl-4,5', 8-trimethylsorrene ("S-59"), and UVA light. It is deformed by the processing to. In some embodiments, the nucleic acid of the bacterium is modified by treatment with a soraren compound and UVA irradiation. A description of using a nucleic acid targeting compound to modify a bacterium to attenuate the bacterium for proliferation is described in the following U.S. Patent Application and Publications, both incorporated herein by reference: 60 / 446,051 (2 2003) Submitted March 6), 60 / 449,153 (submitted February 21, 2003), 60 / 490,089 (submitted July 24, 2003), 60 / 511,869 (submitted October 15, 2003), 60 / 541,515 (2004 February 2, 2004), 10 / 883,599 (filed June 30, 2004), and US 2004/01 97343. July 23, 2004, wherein the modified bacteria and their use are also incorporated herein by reference. International Application No. PCT / US2004 / 23881 is described.

For example, for ΔactAΔuvrAB L. monocytogenes , in some embodiments, S-59 soraren can be added (approximately) to a log-phase culture of (approximately) OD ₆₀₀ = 0.5 up to 200 nM, after which the culture has one optical density. When reached, it is inactivated with ⁶ J / m ² of UVA light. Inactivation conditions are optimized by varying the concentration of S-59, UVA dose, S-59 exposure time before UVA treatment and by varying treatment time during bacterial growth of Listeria actA / uvrAB strains. Parent Listeria strains are used as controls . Inactivation (log-killing) of Listeria is measured by the inability of bacteria to form colonies on BHI (brain heart stock) agar plates. In addition, ³⁵ S-pulse-tracking experiments were used to confirm the expression of heterologous proteins of S-59 / UVA inactivated Listeria and virulence factors such as LLO and p60, thus newly expressed proteins after S-59 / UVA inactivation. Synthesis and secretion can be measured. Expression of LLO and p60 using ³⁵ S-metabolic labeling can be routinely measured. S-59 / UVA Inactivation Listeria actA / uvrAB can be incubated for 1 hour in the presence of ³⁵ S-methionine. Antigen expression and expression of heterologous proteins, endogenous LLOs, and p60 can all be measured for total cell lysate, and TCA precipitates of bacterial culture fluids. LLO-, p60- and heterologous protein-specific monoclonal antibodies can be used for immunoprecipitation to demonstrate continuous expression and secretion from recombinant Listeria after inactivation.

In some embodiments, the bacteria weakened for proliferation are also weakened for nucleic acid repair and / or are defective with respect to at least one DNA repair enzyme. For example, in some embodiments, the bacterium whose nucleic acid has been modified (in combination with UVA treatment) with a nucleic acid targeting compound such as soraren is a uvrAB deletion mutation.

In some embodiments, bacterial growth is attenuated by at least about 0.3 log, and at least about 1 log, about 2 log, about 3 log, about 4 log, about 6 log, or at least about 8 log. In other embodiments, bacterial growth is about 0.3 to> 10 log, about 2 to> 10 log, about 4 to> 10 log, about 6 to> 10 log, about 0.3 to 8 log, about 0.3 to 6 log, about 0.3 To 5 log, about 1 to 5 log, or about 2 to 5 log. In some embodiments, the expression of the antigen by the bacteria is at least about 10%, about 25%, about 50%, about 75%, or at least about 90% of the antigen expression by bacteria that have not been modified by the bacterial nucleic acid.

In some embodiments, the nucleic acid of the bacteria is not modified by reaction with the nucleic acid targeting compound. In some embodiments, the recombinant bacteria are not attenuated for proliferation. In some embodiments, recombinant bacteria do not weaken their ability to repair nucleic acids. In some embodiments, the recombinant bacterium is not defective with respect to at least one DNA repair enzyme.

In some embodiments, the signal peptide encoded by the recombinant nucleic acid molecule, expression cassette, and / or first polynucleotide of the expression vector contained within the recombinant bacterium is the native signal peptide of the recombinant bacterium. In some embodiments, polynucleotides encoding natural signal peptides of recombinant bacteria are codon-optimized for expression in recombinant bacteria. In some embodiments, the polynucleotides are fully codon-optimized. In some embodiments, the signal peptide encoded by the recombinant nucleic acid molecule, expression cassette, and / or first polynucleotide of the expression vector in the recombinant bacterium is exogenous to the host recombinant bacterium. In some embodiments, polynucleotides encoding signal peptides that are exogenous to host recombinant bacteria are codon-optimized for expression in recombinant bacteria.

In some embodiments, the second polynucleotide of the recombinant nucleic acid molecule, expression cassette, and / or expression vector in the recombinant bacterium is codon-optimized for expression in the recombinant bacterium. In some embodiments, the second polynucleotide is fully codon-optimized for expression in recombinant bacteria. In some embodiments, both the first polynucleotide and the second polynucleotide in the recombinant bacterium are codon-optimized for expression in the recombinant bacterium. In some embodiments, both the first polynucleotide and the second polynucleotide in the recombinant bacterium are fully codon-optimized for expression in the recombinant bacterium.

In one aspect, the invention provides a bacterium comprising an expression cassette, the expression cassette comprising: (a) a first polynucleotide encoding a signal peptide, codon-optimized for expression in a bacterium; (b) a second polynucleotide encoding a polypeptide that is within the same translation reading frame as the first polynucleotide; And a promoter operably linked with the first and second polynucleotides, wherein the expression cassette encodes a fusion protein comprising a signal peptide and a polypeptide. In some embodiments, as described herein, eg, in Section III, the signal peptide to be encoded is derived from a bacterium. In some embodiments, the bacterial signal peptide encoded by the expression cassette is derived from bacteria of the same genus and / or species as the bacteria comprising the expression cassette. In some embodiments, the signal peptide is a natural signal peptide of the host recombinant bacterium. In another embodiment, the bacterial signal peptide encoded by the expression cassette is derived from bacteria of a different genus and / or species than the bacteria comprising the expression cassette. In some embodiments, the signal peptide is exogenous to bacteria comprising an expression cassette. In some embodiments, the signal peptide is secA1, secA2, or Tat signal peptide. In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous to the bacterium (ie, exogenous).

In another aspect, the invention provides a kit comprising: (a) a first polynucleotide encoding a codon-optimized bacterial natural signal peptide for expression in a bacterium, and (b) within the same translation reading frame as the first polynucleotide, Provided are bacteria comprising a recombinant nucleic acid molecule comprising a second polynucleotide encoding a polypeptide, said recombinant nucleic acid molecule encoding a fusion protein comprising a signal peptide and a polypeptide. In some embodiments, the bacteria are intracellular bacteria. In some embodiments, the recombinant nucleic acid molecule is part of an expression cassette further comprising a promoter operably linked to both the first polynucleotide and the second polynucleotide. In some embodiments, the bacteria are selected from the group consisting of Listeria, Bacillus, Yersinia pestis, Salmonella, Shigella, Brucella , Mycobacteria and E. coli . In some embodiments, the bacteria are Listeria (eg, Listeria monocytogenes ).

In another aspect, the invention provides a recombinant Listeria bacterium (eg, Listeria monocytogenes ) comprising a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising (a) a codon optimized signal peptide for expression in Listeria bacteria. A second polynucleotide encoding a polypeptide that is within the same translational reading frame as the first polynucleotide, and wherein the recombinant nucleic acid molecule comprises a fusion protein comprising a signal peptide and a polynucleotide. Coding In some embodiments, the recombinant nucleic acid molecule is part of an expression cassette further comprising a promoter operably linked to both the first polynucleotide and the second polynucleotide. In some embodiments, the second polynucleotide is codon-optimized for expression in Listeria bacteria. In some embodiments, the polypeptide encoded by the second polynucleotide is foreign to the Listeria bacterium (i. E., Heterologous to the Listeria bacterium). In some embodiments, Listeria bacteria are attenuated. For example, Listeria may be weakened for intercellular propagation, penetration into non-phagocytic cells, or proliferation. In some embodiments, the recombinant Listeria bacterium is defective with respect to ActA, internalin B, or both ActA and internalin B (eg, a ΔactAΔinlB double deletion mutant). In some embodiments, the nucleic acid of a recombinant bacterium is modified by reaction with a nucleic acid targeting compound (eg, a soraren compound).

In another aspect, the invention provides a bacterium comprising a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising a first polynucleotide encoding a non-secA1 bacterial signal peptide, and an agent encoding a polypeptide (eg, an antigen). 2 polynucleotides, wherein the second polynucleotide is in the same translation reading frame as the first polynucleotide, and the recombinant nucleic acid molecule encodes a fusion protein comprising a signal peptide and a polypeptide. In some embodiments, the polypeptide encoded by the second polynucleotide is heterologous with the signal peptide. In some embodiments, the recombinant nucleic acid molecule is part of an expression cassette further comprising a promoter operably linked to both the first polynucleotide and the second polynucleotide. In some embodiments, the bacteria are bacteria selected from the group consisting of Listeria, Bacillus, Yersinia pestis, Salmonella, Shigella, Brucella , Mycobacteria and E. coli . In some embodiments, the polypeptide encoded by the second polynucleotide is exogenous to the bacterium (ie, heterologous to the bacterium).

In another aspect, the invention provides a bacterium comprising an expression cassette, the expression cassette comprising: (a) a first polynucleotide encoding a non-secA1 bacterial signal peptide; (b) a second polynucleotide encoding a polypeptide (eg, a polypeptide heterologous to bacteria) within the same translation reading frame as the first polynucleotide; And (c) a promoter operably linked to both the first polynucleotide and the second polynucleotide, wherein the expression cassette encodes a fusion protein comprising a signal peptide and a polypeptide. Herein, for example, as described in section III above, in some embodiments, the non-secA1 bacterial signal peptide is a secA2 signal peptide. In some other embodiments, the non-secA1 bacterial signal peptide is a Tat signal peptide. In some embodiments, the bacterial signal peptide encoded by the expression cassette is derived from bacteria of the same genus and / or species as the bacteria comprising the expression cassette. In another embodiment, the bacterial signal peptide encoded by the expression cassette is derived from bacteria of a different genus and / or species than the bacteria comprising the expression cassette.

In another aspect, the invention provides a recombinant Listeria bacterium comprising a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising (a) a first polynucleotide encoding a non-secA1 bacterial signal peptide, and (b) a first polynucleotide And a second polynucleotide encoding the polypeptide, which is within the same translation reading frame as, wherein the recombinant nucleic acid molecule encodes a fusion protein comprising the signal peptide and the polypeptide. In some embodiments, the recombinant nucleic acid molecule is part of an expression cassette further comprising a promoter operably linked to both the first polynucleotide and the second polynucleotide. In some embodiments, the Listeria bacteria are attenuated. In some embodiments, the Lis-teria bacteria is Listeria monocytogenes bacteria. For example, Listeria may be weakened for intercellular propagation, penetration into non-phagocytic cells, or proliferation. In some embodiments, the recombinant Listeria bacterium is defective with respect to ActA, internalin B, or both ActA and internalin B (eg, a ΔactAΔinlB double deletion mutation). In some embodiments, the nucleic acid of a recombinant bacterium is modified by reaction with a nucleic acid targeting compound (eg, a soraren compound).

In another aspect, the invention provides a recombinant Listeria bacterium comprising a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising a polynucleotide encoding a polypeptide that is exogenous to the Listeria bacterium, the polynucleotide for expression in Listeria . Codon-optimized.

As a further aspect, the present invention provides a recombinant Listeria bacterium comprising an expression cassette, the expression cassette includes: (a) a codon for expression in Listeria - optimized, polynucleotide encoding an exogenous polypeptide to the Listeria bacterium; And (b) a promoter operably linked to the polynucleotide encoding the exogenous polypeptide. In some embodiments, Listeria is Listeria monocytogenes . In another embodiment, the Listeria bacteria belong to Listeria ivanovii, Listeria seeligeri, or Listeria innocua species. In some embodiments, the Listeria bacteria are attenuated strains of the Listeria bacteria described above.

In another aspect, the present invention provides a recombinant Listeria bacterium (eg, Listeria monocytogenes) comprising a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising (a) a first polynucleotide encoding a non- Listeria signal peptide; And (b) a second polynucleotide encoding a polypeptide within the same translation reading frame as the first polynucleotide, wherein the recombinant nucleic acid molecule encodes a fusion protein comprising both the non- Listeria signal peptide and the polypeptide. In some embodiments, Listeria bacteria are attenuated. For example, Listeria may be weakened for intercellular propagation, penetration into non-phagocytic cells, or proliferation. In some embodiments, the recombinant Listeria bacterium is defective with respect to ActA, internalin B, or both ActA and internalin B (eg, a ΔactAΔinlB double deletion mutation). In some embodiments, the nucleic acid of a recombinant bacterium is modified by reaction with a nucleic acid targeting compound (eg, a soraren compound).

In another aspect, the invention provides a polynucleotide encoding a non- Listeria signal peptide, a second polynucleotide encoding a polypeptide (eg, a non- Listeria polypeptide) that is within the same translation reading frame as the first polynucleotide. , And a recombinant Listeria bacterium (eg, from Listeria monocytogenes species) comprising an expression cassette comprising a promoter operably linked to both the first polynucleotide and the second polynucleotide. The expression cassette encodes a fusion protein comprising both non- Listeria signal peptides and polypeptides. In some embodiments, Listeria bacteria are attenuated for intercellular propagation, penetration into non-phagocytic cells, or proliferation. In some embodiments, the Listeria bacteria are defective with respect to ActA, internalin B, or both ActA and internalin B. In some embodiments, the nucleic acid of a recombinant bacterium is modified by reaction with a nucleic acid targeting compound (eg, a soraren compound). In some embodiments, the first polynucleotide, the second polynucleotide, or both the first polynucleotide and the second polynucleotide are codon-optimized for expression in Listeria . In some embodiments, the first polynucleotide and / or the second polynucleotide are codon-optimized for expression in Listeria monocytogenes . In some embodiments, the polypeptide encoded by the second polynucleotide is an antigen and in some embodiments the antigen may be a non-bacterial antigen. For example, the polypeptide is in some embodiments a tumor-associated antigen or is derived from such a tumor-associated antigen. For example, in some embodiments, the polypeptide is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP , Proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, or CEA, K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY- Derived from ESO-1, WT-1, survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, or CEA. For example, in some embodiments, the polypeptide is mesothelin or is derived from fragments or variants of mesothelin. In some other embodiments, the polypeptide is NY-ESO-1 or is derived from a fragment or variant of mesothelin. In some embodiments, the antigen is an infectious disease antigen or is derived from an infectious disease antigen. In a preferred embodiment, the signal peptide is a bacterial signal peptide. In some embodiments, the signal peptide is from a bacterium belonging to the genus Bacillus, Staphylococcus, or Lactococcus . For example, in some embodiments, the signal peptide is derived from Bacillus anthracis, Bacillus subtilis, Staphylococcus aureus or Lactococcus lactis . In some embodiments, the signal peptide is a secA1 signal peptide, such as a Usp45 signal peptide from Lactococcus lactis or a protective antigen signal peptide from Bacillus anthracis . In some embodiments, the signal peptide is a secA2 signal peptide. In another embodiment, the signal peptide is a Tat signal peptide, such as a B. subtilis Tat signal peptide (eg, PhoD).

Furthermore, the present invention provides a recombinant bacterium comprising a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising (a) a first polynucleotide encoding bacterial autolysis, or a catalytically active fragment or catalytically active variant thereof; And (b) a second polynucleotide encoding a polypeptide that is within the same translation reading frame as the first polynucleotide, wherein the recombinant nucleic acid molecule autolyzes, or catalyzes, the polypeptide encoded by the second polynucleotide. A protein chimera comprising a fragment or a catalytically active variant, wherein the polypeptide in the protein chimera is autolysed, or fused with a catalytically active fragment or catalytically variant thereof, or autolysed, or a catalytically active fragment or catalytically variant thereof Is located within. In some embodiments, the recombinant bacteria are intracellular bacteria, such as Listeria bacteria (eg, Listeria monocytogenes ). In some embodiments, the polypeptide encoded by the second polynucleotide is exogenous to recombinant bacteria.

In another aspect, the present invention provides a recombinant Listeria bacterium comprising a polycistronic expression cassette, wherein the polycistronic expression cassette encodes at least two isolated non- Listeria polypeptides. For example, in some embodiments, the expression cassette operates on a first polynucleotide encoding a first non- Listeria polypeptide, a second polynucleotide encoding a second non- Listeria polypeptide, and a first and second polynucleotide. Possibly linked promoters. In some embodiments, the recombinant Listeria bacteria belongs to the Listeria monocytogenes species. In some embodiments, the first and / or second non- Listeria polypeptide comprises an antigen (or fragment thereof).

In some embodiments, the present invention provides a kit comprising: (a) a first polynucleotide encoding a first signal peptide; (b) a second polynucleotide encoding the first polypeptide that is within the same translation reading frame as the first polynucleotide; (c) intergenic sequences; (d) a third polynucleotide encoding a second signal peptide; (e) a fourth polynucleotide encoding the second polypeptide, which is in the same translation reading frame as the third polynucleotide; And (f) an expression cassette comprising a promoter that is operably linked with a first polynucleotide, a second polynucleotide, a third polynucleotide, a fourth polynucleotide, and an intergenic sequence. Listeria ), wherein the expression cassette encodes both a first fusion protein comprising a first signal peptide and a first polypeptide and a second fusion protein comprising a second signal peptide and a second polypeptide. In some embodiments, at least one of the polynucleotides encoding the signal peptide is codon-optimized for expression in bacteria. In some embodiments, the third and / or fourth polynucleotides are codon-optimized for expression in bacteria. In some embodiments, the first and / or second polypeptide is heterologous to a recombinant bacterium (eg, a heterologous antigen). In some embodiments, the first and / or second signal peptide is a non-secA1 bacterial signal peptide. The first and / or second signal peptide may be a native signal peptide of recombinant bacteria or may be exogenous. In some embodiments, the recombinant bacterium is a Listeria bacterium and the first and / or second signal peptide is a non- Listeria . In some embodiments, the intergenic sequence is Listeria monocytogenes actA-plcB intergenic sequence. In some embodiments, the bacteria are Listeria monocytogenes .

In another aspect, the invention provides a second polynucleotide encoding (a) a first polynucleotide encoding a signal peptide, (b) a secreted protein, or fragment thereof, within the same translational reading frame as the first polynucleotide. And (c) a recombinant nucleic acid molecule comprising a third polynucleotide encoding a secreted protein, or fragment thereof, that is within the same translational reading frame as the first and second polynucleotides. Wherein the recombinant nucleic acid molecule encodes a protein chimera comprising a signal peptide, a polypeptide encoded by a second polynucleotide, and an isolated protein, or fragment thereof, wherein the polypeptide is a protein secreted from the protein chimera, or Is located within a protein fused to, or isolated from, a fragment thereof. In some embodiments, the bacterium is a Listeria bacterium. In some embodiments, where the bacterium is a Listeria bacterium, the polypeptide encoded by the third polynucleotide is exogenous to Listeria . In some embodiments, the bacteria are Listeria monocytogenes .

In some embodiments (eg, in some embodiments of each of the foregoing embodiments), the expression cassette contained within the bacterium is integrated into the genome of the bacterium. In another embodiment, the expression cassette contained in the bacterium is on a plasmid in the bacterium.

In general, the promoter used in the expression cassette will be an expression cassette suitable for performing heterologous expression using a particular bacterial host of choice. One skilled in the art can readily identify a promoter suitable for use in bacteria. In some embodiments, the promoter is a bacterial promoter. In a further embodiment, the promoter on the expression cassette in the bacterium is a promoter from a bacterium belonging to the same genus as the bacterium containing the expression cassette. In another embodiment, the promoter on the expression cassette in the bacterium is a promoter from a bacterium belonging to the same species as the bacterium containing the expression cassette. For example, if the bacteria containing the expression cassette belong to Listeria monocytogenes species, then the promoter used in the expression cassette is optionally derived from a Listeria gene such as hly . In other embodiments, the promoter is a constitutive promoter (eg, p60 promoter) or a prfA-dependent promoter (eg, actA promoter). Again, as described above, the promoter of the expression cassette is, in some embodiments, a constitutive promoter. In other embodiments, the promoter of the expression cassette is an inducible promoter, which is also described above.

In some embodiments (eg, in certain embodiments of each of the foregoing embodiments), a fusion protein comprising a polypeptide comprising a polypeptide encoded by an expression cassette in a polypeptide or bacterium is an antigen, or for example the IV above. Other proteins of therapeutic value, described in the section. In some embodiments, the polypeptide or protein comprising the polypeptide is secreted from bacteria. In some embodiments, the polypeptides expressed and / or secreted from the bacteria are heterologous to the bacteria.

In some embodiments, the invention provides recombinant Listeria comprising an expression cassette, the expression cassette includes: (a) a codon for expression in the Listeria-optimized, bacteria (Listeria or non-Which of Listeria) signal peptide A first polynucleotide encoding a; (b) a second polynucleotide encoding a non- Listeria antigen that is in the same translation reading frame as the first polynucleotide; And (c) a promoter operably linked with the first and second polynucleotides, wherein the expression cassette encodes a fusion protein comprising a signal peptide and an antigen. In a further embodiment, Listeria is a strain of Listeria monocytogenes such as actA ^- inlB ^- strain. In some embodiments, the expression cassette is integrated into the genome of the recombinant Listeria . In some embodiments, the second polynucleotide is codon-optimized for expression in Listeria .

The present invention also provides a Listeria comprising an expression cassette, the expression cassette comprising: (a) a first polynucleotide encoding a secA2 or Tat bacterial signal peptide; (b) a second polynucleotide encoding an antigen that is within the same translation reading frame as the first polynucleotide; And (c) a promoter operably linked with the first and second polynucleotides, wherein the expression cassette encodes a fusion protein comprising a signal peptide and an antigen. In some embodiments, the bacterial signal peptide is a Listeria signal peptide. In another embodiment, the bacterial signal peptide is a non- Listeria signal peptide. In a further embodiment, Listeria is a strain of Listeria monocytogenes , such as actA ^- inlB ^- strain. In some embodiments, the expression cassette is integrated into the genome of the recombinant Listeria . In some embodiments, the polynucleotide encoding the signal peptide (even if the signal peptide is the signal peptide of Listeria ) and / or the polynucleotide encoding the antigen is codon-optimized for expression in Listeria .

Optimized, non-In further embodiments, the present invention produced the recombinant Listeria comprising an expression cassette, wherein the expression cassette includes: (a) a codon for expression in Listeria Listeria polynucleotide encoding an antigen; And (b) a promoter operably linked with the polynucleotide encoding the exogenous polypeptide. In some embodiments, the expression cassette further comprises a polynucleotide encoding a bacterial signal peptide, wherein the bacterial signal peptide is also codon-optimized for expression in Listeria . In one embodiment, the bacterial signal peptide is a Listeria signal peptide. In another embodiment, the bacterial signal peptide is a non- Listeria signal peptide. In some embodiments, the bacterial signal peptide is a secA1 signal peptide, secA2 signal peptide, or Tat signal peptide. In a further embodiment, Listeria is a strain of Listeria monocytogenes such as actA ^- inlB ^- strain. In some embodiments, the expression cassette is integrated into the genome of the recombinant Listeria .

In another embodiment, the present invention provides a kit comprising: (a) a first polynucleotide encoding a codon-optimized, bacterial ( Listeria or non- Listeria ) signal peptide for expression in Listeria ; (b) a second polynucleotide encoding a non- Listeria antigen that is codon-optimized for expression in Listeria and is within the same translation reading frame as the first polynucleotide; And first and second, and provides a recombinant Listeria bacterium comprising a second polynucleotide and operably linked promoter, this expression cassette encodes a fusion protein comprising the signal peptide and the antigen. In some embodiments, the Listeria bacteria belong to the Listeria monocytogenes species. In some embodiments, the Listeria bacteria are actA ^- inlB ^- mutant strains of Listeria monocytogenes .

The present invention also provides a bacterium, such as Listeria , which comprises one or more expression cassettes described herein. In certain compositions, the molecular mass of a given protein can inhibit the expression of the protein from recombinant bacteria such as recombinant Listeria . One approach to addressing this problem is to molecularly "split" the gene encoding the protein of interest and to divide each segment into a sequence whose secretion from the bacterium (eg, secA1, secA2, or Tat elements) is programmed. Is possible to fuse. One approach is to individually induce recombinant Listeria expressing each fragment of the heterologous gene. Alternatively, each component of the molecularly divided gene (or including a suitable element for secretion) may be transgenic across the bacterial chromosome using, for example, allele exchange, using methods well established in the art. Can be introduced into the area. Another example is the expression of molecularly divided genes as a single polycistronic message. According to this composition, what is interspersed between the protein-coding sequences of the molecularly divided genes may be the Shine-Dalgarno ribosomal binding sequence, thereby resuming protein synthesis on the polycistronic message.

In a further aspect, the present invention provides methods for improving expression and / or secretion of heterologous polypeptides in recombinant bacteria such as Listeria . Any of the polynucleotides, expression cassettes and / or expression vectors described herein can be used in these methods. For example, the present invention provides for expression of heterologous polypeptides fused with signal peptides in Listeria , including codon-optimizing a polypeptide-coding sequence on an expression cassette, a signal peptide-coding sequence of an expression cassette, or both, and Provides a way to improve secretion. The present invention also provides a method for improving expression and / or secretion of heterologous polypeptides fused with signal peptides in Listeria , including the use of signal peptides from non- Listeria sources and / or secretion pathways other than secA1. .

The present invention also provides a method for producing a recombinant bacterium (eg, a recombinant Listeria bacterium) comprising introducing a recombinant nucleic acid molecule, an expression cassette, and / or an expression vector described herein into a bacterium to produce a recombinant bacterium. do. For example, in some embodiments, (a) a codon-optimized first polynucleotide encoding a native signal peptide of a bacterium, and (b) within the same translation reading frame as the first polynucleotide A recombinant nucleic acid molecule comprising a second polynucleotide encoding the polypeptide and encoding a fusion protein comprising the signal peptide and the polypeptide is introduced into the bacterium to produce the recombinant bacterium. In some embodiments, (a) a first polynucleotide encoding a non-secA1 bacterial signal peptide, and (b) a second polynucleotide encoding a polypeptide within the same translational reading frame as the first polynucleotide, Recombinant nucleic acid molecules encoding fusion proteins comprising signal peptides and polynucleotides are introduced into the bacteria to produce recombinant bacteria. In some embodiments, the recombinant nucleic acid molecule introduced into the bacteria to produce the recombinant bacterium is (a) a first polynucleotide encoding a non- Listeria signal peptide, and (b) is in the same translation reading frame as the first polynucleotide. A recombinant nucleic acid molecule encoding a fusion protein comprising a second polynucleotide encoding a polypeptide and comprising both a non- Listeria signal peptide and a polypeptide. In some embodiments, a recombinant nucleic acid molecule used to prepare a recombinant bacterium comprises: (a) a bacterial autolysis, or a first polynucleotide encoding a catalytically active fragment or catalytically variant thereof, and (b) a first polynucleotide A recombinant nucleic acid molecule comprising a second polynucleotide encoding a polypeptide within the same translational reading frame, wherein the non- Listeria polypeptide is autolysed, or fused with a catalytically active fragment or catalytically variant thereof. Or encodes a protein chimera which is self-dissolved, or inserted into a catalytically active fragment or catalytically active variant thereof. In certain other embodiments, the poly-cis-tronic by introducing an expression cassette is provided a method for manufacturing a recombinant Listeria bacterium, comprising producing a recombinant Listeria bacterium, wherein the expression cassette comprises at least two separate non with Listeria bacteria - Listeria Encode the polypeptide.

IX. Pharmaceutical, Immunogenic, and / or Vaccine Compositions

In addition, various different compositions are provided by the present invention, such as pharmaceutical compositions, immunogenic compositions, and vaccines. These compositions include any of the recombinant bacteria described herein (eg, in the Summary of the Invention, Sections I and VIII of the Detailed Description, and various places in the specification, including the examples below). In some embodiments, the composition is isolated.

For example, the present invention provides a pharmaceutical composition comprising (i) a pharmaceutically acceptable carrier; And (ii) a recombinant bacterium described herein.

For example, the present invention provides a pharmaceutical composition comprising (i) a pharmaceutically acceptable carrier; And (ii) a first polynucleotide encoding a signal peptide, codon-optimized for expression in bacteria, a second polynucleotide encoding a polypeptide, within the same translation reading frame as the first polynucleotide, and the first and A pharmaceutical composition is provided comprising an expression cassette comprising a promoter operably linked with a second polynucleotide, the expression cassette comprising a recombinant bacterium encoding a fusion protein comprising a signal peptide and a polypeptide.

In addition, the present invention provides a pharmaceutical composition comprising (i) a pharmaceutically acceptable carrier; And (ii) a first polynucleotide encoding a non-secA1 bacterial signal peptide, a second polynucleotide encoding a polypeptide in the same translational reading frame as the first polynucleotide, and operably with the first and second polynucleotides. Provided are pharmaceutical compositions comprising a recombinant bacterium comprising an expression cassette comprising a linked promoter, the expression cassette encoding a fusion protein comprising a signal peptide and a polypeptide.

In addition, the present invention provides a pharmaceutical composition comprising (i) a pharmaceutically acceptable carrier; And (ii) (a) codons for expression in Listeria - optimized, polynucleotide encoding an exogenous polypeptide in Listeria; And (b) is to provide a pharmaceutical composition comprising a recombinant Listeria bacterium comprising an expression cassette comprising a promoter operably linked to a polynucleotide encoding an exogenous polypeptide.

In addition, the present invention provides a pharmaceutical composition comprising (i) a pharmaceutically acceptable carrier; And (ii) (a) a first polynucleotide encoding a non- Listeria signal peptide; (b) a second polynucleotide encoding a polypeptide that is within the same translation reading frame as the first polynucleotide; And (c) an expression cassette comprising a promoter operably linked to both the first and second polynucleotides, wherein the expression cassette encodes a recombinant Listeria encoding a fusion protein comprising both a non- Listeria signal peptide and a polypeptide. A pharmaceutical composition comprising bacteria is provided.

As used herein, "carrier" includes any and all solvents, dispersion media, excipients, coatings, diluents, antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. Pharmaceutically acceptable carriers are well known to those skilled in the art and include any substance which, when combined with an active ingredient, allows the ingredient to maintain biological activity and is non-reactive with the subject's immune system. For example, pharmaceutically acceptable carriers include, but are not limited to, water, buffered saline solutions (eg, 0.9% saline), emulsions such as oil / water emulsions, and various types of wetting agents. Possible carriers also include, but are not limited to, oils (eg mineral oil), dextrose solution, glycerol solution, lime powder, starch, salts, glycerol, and gelatin.

Any suitable carrier known to those skilled in the art can be used in the pharmaceutical composition, and the type of carrier will vary depending on the mode of administration. The compositions of the present invention may be formulated for any suitable mode of administration, including, for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. In some embodiments, for parenteral administration such as subcutaneous injection, the carrier comprises water, saline, alcohol, fat, wax or buffer. In some embodiments, any of the above or solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate are used for oral administration.

Compositions comprising such carriers are formulated by well known conventional methods (eg, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, edited, Mack Publishing Co., Easton, PA, 1990; and Remington, The Science). and Practice of Pharmacy 20th Edition, Mack Publishing, 2000).

In addition to pharmaceutical compositions, immunogenic compositions are provided. For example, the present invention includes the recombinant bacteria described herein (see, eg, the recombinant bacteria described elsewhere in the specification, including the outline of the invention, Sections I and VIII of the detailed description, and examples below). It provides an immunogenic composition. In some embodiments, the immunogenic composition comprises a recombinant bacterium, wherein the immunogenic composition is expressed as a separate protein, either as part of a fusion protein or embedded in a protein chimera (depending on the recombinant nucleic acid molecule or expression cassette used). A polypeptide sequence that is part of a polypeptide that has been incorporated may or may be an antigen. In other words, in some embodiments, an immunogenic composition comprises a recombinant bacterium comprising a recombinant nucleic acid molecule or expression cassette encoding a polypeptide comprising an antigen. Suitable antigens include, but are not limited to, any of those described herein (eg, in Section IV above). In some embodiments, the recombinant bacteria in the immunogenic composition expresses a polypeptide comprising the antigen at a level sufficient to induce an immune response to the antigen upon administration of the composition to a host (eg, a mammal such as a human). In some embodiments, the immune response stimulated by the immunogenic composition is a cell-mediated immune response. In some embodiments, the immune response stimulated by the immunogenic composition is a humoral immune response. In some embodiments, the immune response stimulated by the immunogenic composition includes both humoral and cell-mediated immune responses.

For example, in one aspect, the present invention provides an immunogenic composition comprising recombinant bacteria, wherein the bacteria comprises (a) a first polynucleotide encoding a signal peptide, codon-optimized for expression in the bacterium; (b) a second polynucleotide encoding an antigen that is within the same translation reading frame as the first polynucleotide; And (c) an expression cassette comprising a promoter operably linked with the first and second polynucleotides, the expression cassette encoding a fusion protein comprising a signal peptide and an antigen.

In another aspect, the present invention provides an immunogenic composition comprising recombinant bacteria, wherein the bacteria comprises (a) a first polynucleotide encoding a non-secA1 bacterial signal peptide; (b) a second polynucleotide encoding an antigen that is within the same translation reading frame as the first polynucleotide; And (c) an expression cassette comprising a promoter operably linked with the first and second polynucleotides, the expression cassette encoding a fusion protein comprising a signal peptide and an antigen.

In another aspect, the present invention provides an immunogenic composition comprising recombinant Listeria bacteria, wherein the recombinant Listeria bacteria comprises an expression cassette, wherein the expression cassette encodes (a) a non- Listeria antigen and expresses in Listeria . Codon-optimized polynucleotides for; And (b) a promoter operably linked with the polynucleotide encoding the antigen.

In addition, the present invention provides a kit comprising: (a) a first polynucleotide encoding a non- Listeria signal peptide; (b) a second polynucleotide encoding an antigen that is within the same translation reading frame as the first polynucleotide; And (c) a recombinant Listeria bacterium comprising an expression cassette comprising a promoter operably linked with the first and second polynucleotides, wherein the expression cassette comprises a non- Listeria signal peptide and an antigen. Code for all-inclusive fusion proteins.

In another aspect, the invention provides an immunogenic composition (or vaccine) comprising a recombinant bacterium comprising the recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule is (a) codon-optimized for expression in the bacterium. A first polynucleotide encoding a natural signal peptide, and (b) a second polynucleotide encoding a polypeptide comprising an antigen that is within the same translational reading frame as the first polynucleotide, wherein the recombinant nucleic acid molecule comprises a signal peptide Encode a fusion protein comprising a polypeptide.

In another aspect, the invention provides an immunogenic composition (or vaccine) comprising a recombinant Listeria bacterium, wherein the recombinant bacterium is (a) a first poly encoding a signal peptide that is codon-optimized for expression in Listeria A recombinant nucleic acid molecule comprising a nucleotide, and (b) a second polynucleotide encoding a polypeptide comprising an antigen that is within the same translational reading frame as the first polynucleotide, wherein the recombinant nucleic acid molecule comprises a signal peptide and the polypeptide. The fusion protein is encoded.

In another aspect, the invention provides an immunogenic composition (or vaccine) comprising a recombinant bacterium comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule comprises a first polynucleotide encoding a non-secA1 bacterial signal peptide, and A second polynucleotide encoding a polypeptide comprising an antigen that is within the same translational reading frame as the first polynucleotide, wherein the recombinant nucleic acid molecule encodes a fusion protein comprising a signal peptide and the polypeptide.

In another aspect, the invention provides an immunogenic composition (or vaccine) comprising a recombinant Listeria bacterium comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule is (a) a first encoding a non-secA1 bacterial signal peptide. A polynucleotide, and (b) a second polynucleotide encoding a polypeptide that is heterologous to the signal peptide or exogenous to bacteria, within the same translational reading frame as the first polynucleotide, wherein the recombinant nucleic acid molecule comprises the signal peptide and the polypeptide The fusion protein. In some embodiments, the polypeptide encoded by the first polynucleotide is an antigen.

In another aspect, the invention provides an immunogenic composition comprising a recombinant Listeria bacterium (or vaccines), wherein the recombinant Listeria bacterium comprises a recombinant nucleic acid molecule, a recombinant nucleic acid molecule is a polynucleotide encoding an exogenous polypeptide to Listeria Wherein the polynucleotide encoding this exogenous polypeptide is codon-optimized for expression in Listeria . In some embodiments, the exogenous polypeptide comprises an antigen.

In another aspect, there is provided an immunogenic composition (or vaccine) comprising a recombinant Listeria bacterium of the invention, wherein the recombinant bacterium comprises (a) a first polynucleotide encoding a non- Listeria signal peptide, and (b) a first A recombinant nucleic acid molecule comprising a second polynucleotide encoding a polypeptide comprising an antigen that is within the same translational reading frame as the polynucleotide, wherein the recombinant nucleic acid molecule is a fusion protein comprising both a non- Listeria signal peptide and a polypeptide Code

The present invention also provides an immunogenic composition (or vaccine) comprising a recombinant bacterium, wherein the recombinant bacterium comprises (a) bacterial autolysis, or a first polynucleotide encoding a catalytically active fragment or catalytically active variant thereof, And (b) a recombinant nucleic acid molecule comprising a second polynucleotide encoding a polypeptide that is within the same translational reading frame as the first polynucleotide, wherein the recombinant nucleic acid molecule is for use with the polypeptide encoded by the second polynucleotide. Resolves, or encodes a protein chimera comprising a catalytically active fragment or catalytically variant thereof, wherein the polypeptide in the protein chimera is fused to, or fused to, or located in a catalytically active fragment or catalytically active variant thereof. In some embodiments, the polypeptide encoded by the second polynucleotide comprises an antigen.

In another aspect, the invention provides an immunogenic composition comprising a recombinant Listeria bacterium (or vaccines), wherein the recombinant Listeria bacterium comprises a recombinant nucleic acid molecule, a recombinant nucleic acid molecule has at least two separate non - Listeria polypeptide And at least one of them comprises an antigen.

In another aspect, the invention provides an immunogenic composition (or vaccine) comprising a recombinant bacterium, wherein the recombinant bacterium comprises: (a) a first polynucleotide encoding a signal peptide, (b) a translational reading identical to the first polynucleotide A second polynucleotide encoding a secreted protein, or fragment thereof, within the frame, and (c) heterologous to the secreted protein, or fragment thereof, within the same translational reading frame as the first and second polynucleotides A protein nucleic acid comprising a recombinant nucleic acid molecule comprising a third polynucleotide encoding a polypeptide, the recombinant nucleic acid molecule comprising a signal peptide, a polypeptide encoded by a third polynucleotide, and a secreted protein, or a fragment thereof And the polypeptide encoded by the third polynucleotide is in the protein chimera Fused with, or located within, the secreted protein, or fragment thereof. In some embodiments, the heterologous polypeptide encoded by the third polynucleotide comprises an antigen.

By testing the ability of recombinant bacteria to stimulate an immune response in vitro or in a model system, one can determine whether a particular type of recombinant bacterium (and / or a specific expression cassette) is useful in an immunogenic composition (or as a vaccine).

These immune cell responses can be measured by in vitro and in vivo methods to determine whether the immune response of a particular recombinant bacterium (and / or a particular expression cassette) is effective. One possibility is to specify the presentation of a protein or antigen of interest by antigen-presenting cells mixed with a recombinant bacterial population. Recombinant bacteria can be mixed with suitable antigen-presenting cells or cell lines, for example dendritic cells, and antigen presentation of dendritic cells to T cells that recognize proteins or antigens can be measured. If the recombinant bacterium expresses a protein or antigen at sufficient levels, it is processed by the dendritic cells into peptide fragments and appears on T cells in the environment of MHC class I or class II. To detect a given protein or antigen, T cell clones or T cell lines that respond to a particular protein or antigen can be used. T cells can also be T cell hybridomas, where T cells are immortalized by the fusion of cancer cell lines. Such T cell hybridomas, T cell clones or T cell lines may comprise CD8 + or CD4 + T cells. Dendritic cells can appear on CD8 + or CD4 + T cells depending on the pathway by which the antigen is processed. CD8 + T cells recognize antigens in the environment of MHC class I and CD4 + recognize antigens in the environment of MHC class II. T cells will be stimulated by specific antigens by T cell receptors of the antigens presented, resulting in IL-2, tumor necrosis factor-α (TNF-α), or interferon-γ (IFN-γ). Any such protein is produced and the protein produced can be measured quantitatively (eg, using an ELISA assay, ELISPOT assay, or intracellular cytokine staining (ICS)). These are techniques well known in the art and are also illustrated in the examples below (see, eg, Example 21 below).

Alternatively, the hybridomas can be designed to include receptor genes such as β-galactosidase that are activated upon stimulation of T cell hybridomas by a given antigen. Increased production of β-galactosidase can be readily determined by its activity on a substrate, such as chlorophenol red-B-galactoside, resulting in a color change. Color change can be measured directly as an indicator for specific antigen presentation.

Additional in vitro and in vivo methods for assessing antigen expression of the recombinant bacterial vaccines of the invention are known to those skilled in the art. It is also possible to directly measure the expression of specific heterologous antigens by recombinant bacteria. For example, radioactively labeled amino acids can be added to a cell population and the amount of radioactivity incorporated into a particular protein can be measured. Proteins synthesized by the cell population can be isolated, for example, by gel electrophoresis or capillary electrophoresis, and the expression levels of specific proteins can be assessed by quantitatively measuring the amount of radioactivity. Alternatively, the protein can be expressed without radioactivity and visualized by various methods, such as ELISA assays, or by gel electrophoresis and Western blot, and detected using enzyme bound antibodies or fluorescent labeled antibodies.

Example 21 below provides certain specific examples of how immunogenicity can be assessed using some of the techniques known to those skilled in the art. For example, Elispot assays, intracellular cytokine staining assays (ICS), cytokine expression measurement of stimulated splenocytes, and assessment of cytotoxic T cell activity in vitro and in vivo are techniques that assess immunogenicity known to those of skill in the art. It is all. A typical description of these techniques using model antigens is provided in Example 21. In addition, typical assays are described in Examples 31A and 31B below.

In addition, the therapeutic efficacy of the vaccine composition can be assessed even more directly by administering the immunogenic composition or vaccine to an animal model, such as a mouse model, and then assessing survival or tumor growth. For example, survival may be measured after administration and challenge (eg, as a tumor or pathogen). See, for example, the assays described in Examples 20 and 31B-D below.

Mouse models useful for testing the immunogenicity of immunogenic compositions or vaccines that express specific antigens may first modify tumor cells so that they express the antigen or model antigen of interest, and then tumor cells expressing the antigen of interest to the mouse. It can be prepared by implantation. Mice express polypeptides comprising antigens or model antigens of interest prior to tumor cell transplantation into the mouse (to test the prophylactic efficacy of the candidate composition) or after tumor cell transplantation (to test the therapeutic efficacy of the candidate composition). The vaccine may be vaccinated with a candidate immunogenic composition or vaccine comprising recombinant bacteria.

As an example CT26 mouse murine colon carcinoma cells can be transfected using a suitable vector comprising an expression cassette encoding a desired antigen or model antigen using standard techniques in the art. Standard techniques such as flow cytometry and western blot can then be used to identify clones that express the antigen or model antigen to a level sufficient for use in immunogenicity and / or efficacy analysis.

Or alternatively corresponds to, or is derived from, an antigen that is endogenous to a tumor cell line (eg, retrovirus gp70 tumor antigen AH1, or heteroclinic epitope AH1-A5, which is endogenous to CT26 mouse murine colon carcinoma cells) Candidate compositions comprising recombinant bacteria expressing the antigen can be tested. In such assays tumor cells can be transplanted into an animal model without further modification to express additional antigens. Candidate vaccines comprising the antigen can then be tested.

As indicated, there is also provided a vaccine composition comprising the bacteria described herein. For example, the present invention includes the recombinant bacteria described herein (see, eg, the recombinant bacteria described elsewhere in the specification, including the outline of the invention, Sections I and VIII of the detailed description, and examples below). Wherein the polypeptide sequence is part of a fusion protein or is embedded in a protein chimera (depending on the recombinant nucleic acid molecule or expression cassette used) and part of a polypeptide expressed by a recombinant bacterium as an isolated protein is an antigen. . Suitable antigens include any of those described herein (eg, in Section IV above).

In one aspect, the invention provides a vaccine comprising a bacterium, wherein the bacterium comprises (a) a first polynucleotide encoding a signal peptide, codon-optimized for expression in the bacterium; (b) a second polynucleotide encoding an antigen that is within the same translation reading frame as the first polynucleotide; And (c) an expression cassette comprising a promoter operably linked with the first and second polynucleotides, the expression cassette encoding a fusion protein comprising a signal peptide and an antigen.

In another aspect, the invention provides a vaccine comprising a bacterium, wherein the bacterium comprises (a) a first polynucleotide encoding a non-secA1 bacterial signal peptide; (b) a second polynucleotide encoding an antigen that is within the same translation reading frame as the first polynucleotide; And (c) an expression cassette comprising a promoter operably linked with the first and second polynucleotides, the expression cassette encoding a fusion protein comprising a signal peptide and an antigen.

In another aspect, the invention provides a vaccine comprising a recombinant Listeria bacterium comprising a nucleic acid molecule, wherein the nucleic acid molecule (a) encodes a non- Listeria antigen and is codon-optimized polynucleotide for expression in Listeria ; And (b) a promoter operably linked with the polynucleotide encoding the antigen.

In another aspect, the invention provides a kit comprising: (a) a first polynucleotide encoding a non- Listeria signal peptide; (b) a second polynucleotide encoding an antigen that is within the same translation reading frame as the first polynucleotide; And (c) a recombinant Listeria bacterium comprising an expression cassette comprising a promoter operably linked with both the first polynucleotide and the second polynucleotide, wherein the expression cassette comprises a non- Listeria signal peptide and an antigen. Encode a fusion protein containing all of them.

In some embodiments, the vaccine composition comprises antigen-presenting cells (APCs) infected with any of the recombinant bacteria described herein. In some embodiments the vaccine (or immunogenic or pharmaceutical composition) does not comprise antigen-presenting cells (ie, the vaccine or composition is a bacteria-based vaccine or composition, not an APC-based vaccine or composition).

Suitable methods of administration for administration of vaccine compositions (and pharmaceutical and immunogenic compositions) are known in the art and include oral, intravenous, intradermal, intraperitoneal, intramuscular, lymphatic, intranasal and subcutaneous routes of administration. .

Vaccine formulations are known in the art, and in some embodiments, a number of additives, such as preservatives (eg thimerosal, 2-phenoxyethanol), stabilizers, adjuvants (eg aluminum hydroxide, Aluminum phosphate, cytokines), antibiotics (eg neomycin, streptomycin) and other substances. In some embodiments, stabilizers, such as lactose or monosodium glutamate (MSG), are added to stabilize the vaccine formulation against various conditions, such as temperature changes or lyophilization processes. In some embodiments, the vaccine formulation may also include suspension fluids or diluents, such as sterile water, saline or isotonic buffered saline (eg, phosphate buffered to physiological pH). The vaccine may also contain small amounts of residual material from the manufacturing process.

For example, in some embodiments the vaccine composition is lyophilized. Lyophilized formulations may be combined with sterile solutions (eg, citrate-bicarbonate buffer, buffered water, 0.4% saline, etc.) prior to administration.

In some embodiments, the vaccine composition may further comprise additional ingredients known in the art to improve the immune response to the vaccine, such as an adjuvant or co-stimulatory molecule. In addition to those described above, possible adjuvants include bacterial nucleic acid sequences such as chemokines and CpG. In some embodiments, the vaccine comprises an antibody, eg CTLA4, which improves the immune response to the vaccine. In some embodiments, the co-stimulatory molecule is comprised of GM-CSF, IL-2, IL-12, IL-14, IL-15, IL-18, B7.1, B7.2, and B7-DC One or more factors selected from, which are optionally included in the vaccine composition of the present invention. Other co-stimulatory molecules are also known to those skilled in the art.

In a further aspect, the present invention provides a method for improving a vaccine or immunogenic composition comprising Listeria expressing an antigen. Any of the polynucleotides, expression cassettes, and / or expression vectors described herein can be used in these methods. For example, the present invention provides a vaccine or immunogenic composition comprising Listeria bacteria, comprising codon-optimizing a polypeptide-coding sequence on an expression cassette, or a signal peptide-coding sequence of an expression cassette, or both. A method of improving is provided wherein the Listeria bacterium expresses a heterologous antigen fused with a signal peptide. The present invention provides a method for ameliorating a vaccine or immunogenic composition comprising Listeria bacteria, comprising using signal peptides from non- Listeria sources and / or secretion pathways other than secA1, wherein the Listeria bacteria are Expresses a heterologous antigen fused with a peptide.

Also provided is a vaccine production method of the present invention. For example, in one embodiment, a method of making a vaccine comprising recombinant bacteria (eg, recombinant Listeria bacteria) includes introducing a recombinant nucleic acid molecule, expression cassette, or expression vector described herein into a bacterium. Wherein the recombinant nucleic acid molecule, expression cassette, or expression vector encodes an antigen. For example, in some embodiments, (a) a codon-optimized first polynucleotide encoding a native signal peptide of a bacterium, and (b) within the same translation reading frame as the first polynucleotide The vaccine is prepared by introducing into a bacterium a recombinant nucleic acid molecule comprising a second polynucleotide encoding an antigen, and encoding a fusion protein comprising a signal peptide and an antigen. In some embodiments, (a) a first polynucleotide encoding a non-secA1 bacterial signal peptide, and (b) a second polynucleotide encoding an antigen that is within the same translational reading frame as the first polynucleotide, A recombinant nucleic acid molecule encoding a fusion protein comprising a signal peptide and an antigen is introduced into a bacterium to prepare a vaccine. In some embodiments, the recombinant nucleic acid molecule introduced into the bacteria to prepare a vaccine comprises: (a) a first polynucleotide encoding a non- Listeria signal peptide, and (b) an antigen in the same translation reading frame as the first polynucleotide. And a recombinant nucleic acid molecule that encodes a fusion protein comprising a second polynucleotide encoding a non- Listeria signal peptide and an antigen. In some embodiments, a recombinant nucleic acid molecule used to prepare a vaccine comprises (a) a bacterial autolysate, or a first polynucleotide encoding a catalytically active fragment or catalytically active variant thereof, and (b) the first polynucleotide. A second polynucleotide encoding the polypeptide, within the same translation reading frame, wherein the non- Listeria polypeptide is autolysed, or fused with a catalytically fragment or catalytically active variant thereof, or autolysed, or a catalytic activity thereof Recombinant nucleic acid molecules encoding protein chimeras inserted into fragments or catalytically active variants. In certain other embodiments, the poly-cis-tronic introducing an expression cassette and which comprises producing a vaccine, there is provided a method of producing a vaccine comprising a recombinant Listeria bacterium, wherein the poly-cis-tronic expression cassette has at least two separated by Listeria bacteria Non- Listeria polypeptide, wherein at least one of the polypeptides is an antigen.

Also provided are kits comprising any of the recombinant nucleic acid molecules, expression cassettes, vectors, bacteria and / or compositions of the invention.

X. How to use

Various methods are provided using the recombinant bacteria or pharmaceutical, immunogenic, or vaccine compositions described herein to induce an immune response in a host and / or to prevent or treat a condition. In some embodiments, the condition being treated or prevented is a disease. In some embodiments, the disease is cancer. In some embodiments, the disease is an infectious disease. In addition, recombinant bacteria are also useful for the production and isolation of heterologous proteins, such as mammalian proteins.

As used herein, "treatment" or "treating" (with respect to a condition or disease) is an approach for obtaining advantageous or desirable results, preferably including clinical results. For the purposes of the present invention, an advantageous or preferred outcome with respect to a disease is to improve the condition associated with the disease, cure the disease, reduce the severity of the disease, delay the progression of the disease, alleviate one or more symptoms associated with the disease, disease One or more of an increase in quality of life, and / or prolongation of survival in a person suffering from the disease. Likewise, for the purposes of the present invention, an advantageous or desirable outcome with respect to a condition is to improve the condition, heal the condition, reduce the severity of the condition, delay the progression of the condition, alleviate one or more symptoms associated with the condition, Including but not limited to one or more of an increase in quality of life, and / or prolongation of survival in a suffering person. For example, in embodiments in which the compositions described herein are used in the treatment of cancer, advantageous or desirable results include reduced proliferation (or destruction of these cells) of neoplasia or cancerous cells, of neoplastic cells found in cancer. Reduced metastasis, reduced tumor size, reduced symptoms from cancer, increased quality of life in people suffering from cancer, decreased dose of other medications needed to treat the disease, delayed cancer progression, and / or cancer One or more of the prolongation of survival of the patient with the disease.

As used herein, the term “preventing” a disease or “defending the host” from a disease (used interchangeably) refers to the interruption, probation, interruption, delay, delay, and / or delay of the onset or progression of the disease, And one or more of stabilizing disease progression and / or delaying disease development. The term "preventing" a state or "defending a host" from a state (used interchangeably) means stopping, suspending, suspending, delaying, delaying, and / or delaying the initiation or progression of a state, And / or one or more delays of state occurrence. The duration of this prophylaxis can vary in length of time depending on the history of the disease or condition and / or the individual to be treated. For example, if the vaccine is designed for the prophylaxis or protection against infectious diseases caused by a pathogen, the term "preventing" or "defending the host" from a disease means infection by the host's pathogen, host's pathogen. Progression of infection by, initiation or interruption of the onset or progression of a disease associated with infection of the host by the pathogen, suspend, interruption, delay, delay, and / or delay, and / or stabilization of progression of the disease associated with infection of the host by the pathogen It includes but is not limited to one or more of them. Also, for example, where the vaccine is an anti-cancer vaccine, the term "preventing" or "defending the host" from a disease refers to the occurrence of cancer or metastasis, the progression of cancer, or the cessation of cancer recurrence, suspension, interruption, It includes, but is not limited to, one or more of delay, delay, and / or delay.

In one aspect, the present invention includes an effective amount of a recombinant bacterium described herein or a recombinant bacterium described herein (see, eg, the Summary of the Invention, Sections I, VIII and IX of the Detailed Description, or Examples below). Provided is a method of inducing an immune response in a host against an antigen, comprising administering to the host an effective amount of a composition (eg, a pharmaceutical composition, an immunogenic composition, or a vaccine). In some embodiments, the polypeptide encoded by a recombinant nucleic acid, expression cassette, and / or expression vector in a recombinant bacterium comprises an antigen or is a fusion protein or protein chimera comprising the antigen.

For example, in one aspect, the invention provides a method of inducing an immune response in a host against an antigen comprising administering to the host an effective amount of a composition comprising the recombinant bacterium, wherein the recombinant bacterium is (a) a bacterium. A first polynucleotide encoding a signal peptide, codon-optimized for expression in; (b) a second polynucleotide encoding an antigen that is within the same translation reading frame as the first polynucleotide; And (c) an expression cassette comprising a promoter operably linked with the first and second polynucleotides, the expression cassette encoding a fusion protein comprising a signal peptide and an antigen.

In another aspect, the present invention provides a method of inducing an immune response in a host against an antigen comprising administering to the host an effective amount of a composition comprising a recombinant bacterium comprising the expression cassette, wherein the expression cassette comprises (a) a first polynucleotide encoding a secA1 bacterial signal peptide; (b) a second polynucleotide encoding an antigen that is within the same translation reading frame as the first polynucleotide; And (c) a promoter operably linked with the first and second polynucleotides, wherein the expression cassette encodes a fusion protein comprising a signal peptide and an antigen.

In another aspect, the invention provides a method of inducing an immune response in a host against a non- Listeria antigen comprising administering to the host an effective amount of a composition comprising a recombinant Listeria bacterium comprising a nucleic acid molecule, wherein the nucleic acid The molecule may comprise (a) a codon-optimized polynucleotide encoding a non- Listeria antigen and for expression in Listeria ; And (b) a promoter operably linked with the polynucleotide encoding the antigen.

In another aspect, the invention provides a kit comprising: (a) a first polynucleotide encoding a non- Listeria signal peptide; (b) a second polynucleotide encoding an antigen that is within the same translation reading frame as the first polynucleotide; And (c) administering to the host an effective amount of a recombinant Listeria bacterium comprising an expression cassette comprising a promoter operably linked to both the first and second polynucleotides. Provided, wherein the expression cassette encodes a fusion protein comprising both non- Listeria signal peptides and polypeptides.

In some embodiments of the methods of inducing an immune response described herein, the bacteria is administered in the form of pharmaceutical compositions, immunogenic compositions and / or vaccine compositions.

In some embodiments, the immune response is an MHC class I immune response. In another embodiment, the immune response is an MHC class II immune response. In another embodiment, the immune response induced by administration of a bacterium or composition is both an MHC Class I and MHC Class II response. Thus, in some embodiments, the immune response comprises a CD4 + T-cell response. In some embodiments, the immune response comprises a CD8 + T-cell response. In some embodiments, the immune response includes both a CD4 + T-cell response and a CD8 + T-cell response. In some embodiments, the immune response comprises a B-cell response and / or a T-cell response. B-cell responses can be measured by measuring the titer of the antibody against the antigen using methods known to those skilled in the art. In some embodiments, the immune response induced by the compositions described herein is a humoral response. In another embodiment, the induced immune response is a cellular immune response. In some embodiments, the immune response includes both cellular and humoral immune responses. In some embodiments, the immune response is antigen-specific. In some embodiments, the immune response is an antigen-specific T-cell response.

In addition to providing a method for inducing an immune response, the invention also provides a method for preventing or treating a condition in a host (eg, a subject, such as a human patient). In some embodiments the condition is a disease. The method provides an effective amount of a recombinant bacterium described herein (e.g., an overview of the invention, Sections I, VIII and IX of the detailed description, or examples below), or a composition comprising a recombinant bacterium described herein. Administering to the host. In some embodiments the disease is cancer. In some embodiments the disease is an infectious disease.

For example, in one aspect, the present invention provides a method of preventing or treating a disease (or condition) in a host comprising administering to the host an effective amount of a composition comprising the bacterium, wherein the bacterium is (a) bacteria A first polynucleotide encoding a signal peptide, codon-optimized for expression in (b) a polypeptide (eg, an antigen and / or therapeutic mammalian protein) within the same translational reading frame as the first polynucleotide A second polynucleotide encoding a); And (c) an expression cassette comprising a promoter operably linked with the first and second polynucleotides, the expression cassette encoding a fusion protein comprising a signal peptide and an antigen.

In another aspect, the present invention provides a method of preventing or treating a disease (or condition) in a host comprising administering to the host an effective amount of a composition comprising a recombinant bacterium, wherein the bacterium is (a) a non-secA1 bacterium. A first polynucleotide encoding a signal peptide; (b) a second polynucleotide encoding a polypeptide (eg, an antigen and / or a mammalian protein) within the same translation reading frame as the first polynucleotide; And (c) an expression cassette comprising a promoter operably linked to the first and second polynucleotides, wherein the expression cassette encodes a fusion protein comprising a signal peptide and an antigen.

In another aspect, the invention provides a method of preventing or treating a disease (or condition) in a host comprising administering to the host an effective amount of a composition comprising a recombinant Listeria bacterium comprising the nucleic acid molecule, wherein the nucleic acid molecule (A) a polynucleotide encoding a non- Listeria polypeptide (eg, an antigen and / or therapeutic mammalian protein) and codon optimized for expression in Listeria ; And (b) a promoter operably linked to the polynucleotide encoding the antigen.

In another aspect, the invention provides a kit comprising: (a) a first polynucleotide encoding a non- Listeria signal peptide; (b) a second polynucleotide encoding a polypeptide (eg, an antigen and / or therapeutic mammalian protein) within the same translation reading frame as the first polynucleotide; And (c) administering to the host an effective amount of a composition comprising a recombinant Listeria bacterium comprising an expression cassette comprising a promoter operably linked to both the first and second polynucleotides. A method of preventing or treating a protein, wherein the expression cassette encodes a fusion protein comprising both a non- Listeria signal peptide and a polypeptide.

In some embodiments the disease is cancer. In some embodiments, where the condition to be treated or prevented is cancer, the disease is melanoma, breast cancer, pancreatic cancer, liver cancer, colon cancer, colorectal cancer, lung cancer, brain cancer, testicular cancer, ovarian cancer, squamous cell cancer, gastrointestinal cancer, cervical cancer, Kidney cancer, thymic cancer or prostate cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is metastatic.

In other embodiments the disease is an autoimmune disease. In another embodiment, the disease is an infectious disease or other disease caused by a pathogen such as a virus, bacteria, fungus, or protozoa. In some embodiments, the disease is an infectious disease.

In some embodiments, the use of recombinant bacteria in the prophylaxis or treatment of cancer delivers the recombinant bacteria to cells of the individual's immune system, thereby being present in the individual, or increasing the risk of the individual being exposed to the environment and / or family predisposition. Preventing or treating cancer with factors. In another embodiment, the use of recombinant bacteria in the prophylaxis or treatment of cancer includes delivering the recombinant bacteria to an individual who has had a tumor removed in the past or has a cancer that is now alleviated.

In some embodiments, administration of a composition comprising a recombinant bacterium described herein to a host elicits a CD4 + T-cell response in the host. In some other embodiments, administration of a composition comprising a recombinant bacterium described herein to a host elicits a CD8 + T-cell response in the host. In some embodiments, administration of a composition comprising a recombinant bacterium described herein elicits both a CD4 + T-cell response and a CD8 + T-cell response in a host.

The efficacy of a vaccine or other composition for the treatment of a condition can be assessed in an individual, for example in a mouse. Mouse models are recognized as models for efficacy in humans and are useful for evaluating and defining vaccines of the invention. The mouse model is used to demonstrate the potential for efficacy of the vaccine in any individual. Vaccines can be evaluated for their ability to provide a prophylactic or therapeutic effect on certain diseases. For example, in the case of an infectious disease, the mouse population can be vaccinated with the desired amount of a suitable vaccine of the invention, wherein the recombinant bacterium expresses an infectious disease related antigen. Mice can then be infected with infectious agents associated with vaccine antigens and assessed for protection against infection. Progression of infectious disease can be observed compared to the control population (vaccinated only with bacteria that are not vaccinated or contain no excipients or suitable antigens).

For cancer vaccines, tumor cell models can be used, in which case tumor cell lines expressing the desired tumor antigen are before or after vaccination (therapeutic model) with a tumor comprising the bacterium of the invention containing the desired tumor antigen. (Prevention Model) Can be injected into mouse populations. Vaccinations using recombinant bacteria containing tumor antigens can be compared to a control population vaccinated with non-vaccinated, excipients or with recombinant bacteria expressing irrelevant antigens. The effectiveness of the vaccine in such models can be assessed in terms of tumor volume as a function of time after tumor injection or in terms of surviving population as a function of time after tumor injection (eg, Example 31D). In one embodiment, the tumor volume in a mouse vaccinated with a composition comprising the recombinant bacteria is about the tumor volume in a mouse vaccinated with a recombinant bacterium expressing an unvaccinated, excipient or unrelated antigen. Less than 5%, about 10%, about 25%, about 50%, about 75%, about 90% or about 100%. In other embodiments, this difference in tumor volume is observed at least about 10 days, about 17 days, or about 24 days after tumor implantation in mice. In one embodiment, the average survival time in mice vaccinated with the composition comprising the recombinant bacteria is at least about 2 than in mice vaccinated with a recombinant bacterium expressing an unvaccinated, excipient or unrelated antigen. Days, about 5 days, about 7 days or at least about 10 days longer.

In the methods described herein the host (ie the subject) is any vertebrate, preferably a mammal, includes livestock, race animals and primates, and includes humans. In some embodiments, the host is a mammal. In some embodiments, the host is human.

Delivery of a recombinant bacterium, or a composition comprising this strain, may be by any suitable method, such as intradermal, subcutaneous, intraperitoneal, intravenous, intramuscular, lymphatic, intraoral or intranasal, and any given malignant or infectious disease or other It can be achieved by any path appropriate to the condition.

Compositions comprising recombinant bacteria and immunostimulants can be administered to the host simultaneously, sequentially or separately. Examples of immunostimulatory agents include, but are not limited to, IL-2, IL-12, GMCSF, IL-15, B7.1, B7.2, and B7-DC and IL-14. Further examples of stimulants are provided in section IX above.

As used herein, an “effective amount” of a bacterium or composition (pharmaceutical composition or immunogenic composition) is an amount sufficient to achieve an advantageous or desirable result. For prophylactic use, advantageous or desirable outcomes may be used to eliminate the risk of a disease, including biochemical, histological and / or behavioral symptoms of the disease, complications of the disease, and intermediate pathological phenotypes, Results such as reducing, reducing severity, or delaying onset of disease. For therapeutic use, advantageous or desirable outcomes include one or more symptoms (biochemical, histological and / or behavioral) resulting from the disease, including inhibition of the disease, complications that occur during the course of the disease, and intermediate pathological phenotypes. ), An increase in the quality of life of a person suffering from the disease, a decrease in the dose of other medications necessary to treat the disease, an enhancement of the effects of other medications, a delay in the progression of the disease, and / or an extension of the patient's survival. Include clinical results. An effective amount can be administered in one or more administrations. For the purposes of the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment. As will be understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved with other drugs, compounds, or pharmaceutical compositions. Thus, an effective amount may be contemplated in terms of administering one or more therapeutic agents, and it may be contemplated that a single agent is provided in an effective amount if the desired result can be achieved or achieved with one or more other agents.

In some embodiments, for therapeutic treatment of cancer, the effective amount includes an amount that produces a desired immune response, wherein the immune response slows the growth of the target tumor, reduces the size of the tumor, or preferably Completely remove the tumor. Administration of the vaccine may be repeated at appropriate intervals and may be administered simultaneously to multiple different sites in the vaccinated individual. In some embodiments, for the prophylactic treatment of cancer, the effective amount includes a dose that results in a protective immune response such that the likelihood of the individual developing the cancer is significantly sensitive. The vaccination regimen may be in one dose or repeated at appropriate intervals until a protective immune response is established.

In some embodiments, the therapeutic treatment of an individual for cancer can be initiated for an individual diagnosed with cancer as an initial treatment, or used in conjunction with other treatments. For example, an individual who has had surgical removal of the tumor or who has been treated with radiotherapy or chemotherapy may be treated with a vaccine to reduce or reduce any tumor remaining in the individual or to reduce the risk of cancer recurring. Can be. In some embodiments, prophylactic treatment of an individual for cancer will commence for an individual with an increased risk of contact with any cancer due to environmental conditions or genetic predisposition.

The dosage of the pharmaceutical composition or vaccine provided to the host will vary depending on the species of the host, the size of the host, and the condition or disease of the host. In addition, the dosage of the composition will depend on the frequency and route of administration of the composition. The correct dosage is chosen by the attending physician in terms of the treated patient.

In some embodiments, pharmaceutical compositions, immunogenic compositions, or vaccines used in the methods comprise recombinant bacteria comprising the recombinant nucleic acid molecules, expression cassettes and / or expression vectors described herein. In some embodiments, recombinant bacteria are described in Thomas W. Dubensky, Jr., filed June 30, 2004, which is incorporated herein by reference. US Patent Application No. entitled “Modified Pre-Living Microbe, Vaccine Composition and Method of Use” 10 / 883,599, US Patent Publication No. 2004/0228877 and US Patent Publication No. Modified and / or mutant bacteria, such as those described in 2004/0197343. In some embodiments, a single dose of a pharmaceutical composition or vaccine comprising such modified and / or mutant bacteria or any of the other recombinant bacteria described herein comprises a bacterial organism of about 10 ² to about 10 ¹² . . In another embodiment, the single dose comprises about 10 ⁵ to about 10 ¹¹ bacterial organisms. In another embodiment, the single dose comprises about 10 ⁶ to about 10 ¹¹ bacterial organisms. In another embodiment, the single dose of the pharmaceutical composition or vaccine comprises about 10 ⁷ to about 10 ¹⁰ bacterial organisms. In another embodiment, the single dose of the pharmaceutical composition or vaccine comprises about 10 ⁷ to about 10 ⁹ bacterial organisms.

In some embodiments, the single dose comprises at least about 1 × 10 ² bacterial organisms. In some embodiments, the single dose of the composition comprises at least about 1 × 10 ⁵ organisms. In other embodiments, the single dose of the composition or vaccine comprises at least about 1 × 10 ⁶ bacterial organisms. In another embodiment, the single dose of the composition or vaccine comprises at least about 1 × 10 ⁷ bacterial organisms.

In some embodiments, a single dose of a pharmaceutical composition, immunogenic composition, or vaccine comprising recombinant, modified and / or mutant bacteria described herein is about 1 CFU / kg to about 1 × ^{10 10} CFU / kg (CFU = Colony forming unit). In some embodiments, the single dose of the composition comprises about 10 CFU / kg to about 1 × 10 ⁹ CFU / kg. In another embodiment, the single dose of the composition or vaccine comprises about 1 × 10 ² CFU / kg to about 1 × ^{10 8} CFU / kg. In another embodiment, the single dose of the composition or vaccine comprises about 1 × 10 ³ CFU / kg to about 1 × ^{10 8} CFU / kg. In another embodiment, the single dose of the composition or vaccine comprises about 1 × 10 ⁴ CFU / kg to about 1 × 10 ⁷ CFU / kg. In some embodiments, the single dose of the composition comprises at least about 1 CFU / kg. In some embodiments, the single dose of the composition comprises at least about 10 CFU / kg. In another embodiment, the single dose of the composition or vaccine comprises about 1 × 10 ² CFU / kg. In another embodiment, the single dose of the composition or vaccine comprises at least about 1 × 10 ³ CFU / kg. In another embodiment, the single dose of the composition or vaccine comprises at least about 1 × 10 ⁴ CFU / kg.

In some embodiments, a suitable (ie effective) dosage for one host, such as a human, can be estimated from LD ₅₀ data for another host, such as a mouse, using methods known to those skilled in the art.

In some embodiments, the pharmaceutical composition, immunogenic composition, or vaccine comprises antigen-presenting cells, eg, dendritic cells, infected with recombinant bacteria, including the recombinant nucleic acid molecules, expression cassettes, and / or expression vectors described herein. do. In some embodiments, the bacterium is disclosed in US Patent Application No. 30, 2004, filed June 30, 2004, incorporated herein by reference. 10 / 883,599, and US Patent Publication No. And may be modified and / or mutated, such as those described in 2004/0228877 and US 2004/0197343. Such antigen-presenting cell based vaccines are described, for example, in Thomas W. Dubensky, Jr., filed June 23, 2004, which is incorporated herein by reference. International Application No. entitled "Antigen-presenting Cell Vaccine and Method of Use thereof", et al. PCT / US2004 / 23881; United States Patent Application No. filed June 30, 2004. 10 / 883,599; United States Patent Publication No. 2004/0228877; And US Patent Publication No. US 2004/0197343. In some embodiments, each dose of an antigen-presenting cell based vaccine comprising bacteria such as those described herein comprises between about 1 × ^{10 3} and about 1 × ^{10 10} antigen-presenting cells. In some embodiments, each dosage of the vaccine comprises about 1 × 10 ⁵ to about 1 × ^{10 9} antigen-presenting cells. In some embodiments, each dosage of the vaccine comprises between about 1 × ^{10 7} and about 1 × ^{10 9} antigen-presenting cells.

In some embodiments, multiple administrations of dosage units are preferred per day or over a week or month or year or year. In some embodiments, the dosage unit is administered daily for several days or once a week for several weeks.

Furthermore, the present invention relates to any recombinant bacterium described herein (ie, any bacterium comprising a recombinant nucleic acid molecule, expression cassette, or vector described herein) in the manufacture of a medicament for inducing an immune response in a host against an antigen. A use is provided wherein the polypeptide encoded by a recombinant nucleic acid molecule, expression cassette, and / or vector in a bacterium comprises an antigen. In some embodiments, the antigen is a heterologous antigen. The present invention also provides the use of the recombinant bacteria described herein in the manufacture of a medicament for preventing or treating a condition (eg, a disease such as cancer or an infectious disease) in a host. Furthermore, the present invention provides a recombinant bacterium described herein for use in inducing an immune response in a host to an antigen, wherein the polypeptide encoded by the recombinant nucleic acid molecule, expression cassette, and / or vector in the bacterium Include. Furthermore, the present invention provides the recombinant bacteria described herein for use in preventing or treating a condition (such as a disease) in a host.

The invention also provides a method of inducing MHC class I antigen presentation or MHC class II antigen presentation on antigen-presenting cells comprising contacting the antigen-presenting cells with the bacteria described herein.

Furthermore, the present invention provides a method for producing an antigen-presenting cell, comprising the steps of (a) contacting an antigen-presenting cell from a host with a recombinant bacterium described herein for a time sufficient to load the antigen-presenting cell under suitable conditions; And (b) administering the antigen-presenting cell to the host.

Recombinant nucleic acid molecules, expression cassettes, and other possible uses of bacteria will also be recognized by those skilled in the art. For example, the recombinant nucleic acid molecules, expression cassettes, vectors, and recombinant bacteria (and other host cells) described herein are useful for the preparation and isolation of heterologous polypeptides. Thus, in another aspect, the present invention provides a method for preparing a bacterium comprising (a) introducing into an bacterium the expression cassette or vector described herein (eg, by transfection, transformation, or conjugation); And (b) growing the bacteria in culture under conditions suitable for protein expression. In another aspect, the present invention provides a method for preparing a bacterium comprising (a) introducing into an bacterium the expression cassette or vector described herein (eg, by transfection, transformation, or conjugation); (b) growing the bacteria in cell culture under conditions suitable for protein expression; And (c) separating the protein from the bacterial cell culture. Suitable methods of transformation, transfection, and conjugation are well known to those skilled in the art, and methods for culturing and growing bacteria and for separating secreted or non-secreted proteins from cell culture are also well known to those skilled in the art.

The following examples are provided to illustrate but not limit the invention.

Example 1. Preparation of Representative Mutant Listeria Strains.

Listeria strains were derived from 10403S [Bishop et al., J. Immunol. 139: 2005 (1987). Listeria strains in which the indicated genes were deleted in-frame were generated via allele exchange by SOE-PCR and established methods [Camilli, et al, Mol. Microbiol. 8: 143 (1993)]. Mutant strain LLO L461T (DP-L4017) is described by Glomski, et al, J of Cell. Biol. 156: 1029 (2002). actA ^_ mutant (DP-L4029) is the DP-L3078 strain described in Skoble et al., J. of Cell Biology, 150: 527-537 (2000), which is incorporated herein by reference in its entirety. Preserved as phage; (Prophage preservation is described in (Lauer et al., J. Bacteriol. 184: 4177 (2002); US Patent Publication No. 2003/0203472). ActA ^_ uvrAB ^- The construction of the strain is L4029 / uvrAB 2 US Provisional Application No. 60 / 446,051, filed May 6, (see, eg, Example 7 of the above application), as well as US Patent Publication No. 2004/0197343. DP-L4029uvrAB ( Listreria monocytogenes deletion mutant) The American Type Culture Collection (ATCC) located at Boulevard 10801, Manassas, Virginia, USA 20110-2209, October 3, 2003, under the provisions of the Budapest Convention on the International Recognition of Microbial Deposits for the purposes of the patent process, for the purposes of the patent process. Further description of the mutant Listeria is provided in the following applications or patent publications, each of which is incorporated herein by reference in its entirety: US Patent Publication No. 2004/0228877; US Patent Publication No. 2004/0 197343; PCT International Application PCT / US2004 / 23881, filed July 23, 2004; and US Patent Application No. 10/883, filed June 30, 2004. Also, representative Listeria monocytogenes ΔactAΔin1B double deletion. The mutant is designated as PTA-5562 and is an American type culture located at Boulevard 10801, Manassas University, Virginia, USA 20110-2209, October 3, 2003, under the provisions of the Budapest Treaty on the International Recognition of Microbial Deposits for the purposes of the patent process. It was deposited in the collection (ATCC).

One non-limiting example of a method for removing genes of Listeria monocytogenes to generate attenuated mutants is provided in Example 24 below.

Example 2 Construction of Listeria Strains Expressing AH1 / OVA or AH1-A5 / OVA

Truncated form of model antigen ovalbumin (OVA), immunodominant epitope from mouse colon cancer known as AH1 (SPSYVYHQF (SEQ ID NO: 72) and altered epitope AH1-A5 (SPSYAYHQF (SEQ ID NO: 73); Slansky et al ., Immunity, 13: to produce a mutant Listeria strain expressing the 529-538 (2000)) pPL2 integration vector (Lauer et al 184: 4177 ( 2002.);. with the United States Patent Publication No. 2003/0203472 No.) OVA and AH1-A5 / OVA recombinant Listeria strains containing a single copy integrated into the non-irritating site of the Listeria genome were derived.

A. Construction of OVA Expression Listeria (DP-L4056).

An antigen expression cassette is first prepared which is composed of hemolytic-deleted LLO (pPL2 / LLO-OVA) fused with the cleaved OVA and contained in the pPL2 integration vector. The Listeria-OVA vaccine strain is derived by introducing pPL2 / LLO-OVA into the L. monocytogenes strain DP-L4056 at the PSA (phage from ScottA) attachment site tRNA ^Arg- attBB '.

Amplify hemolytic-deleted LLO by PCR using the following templates and primers:

Source: DP-L4056 genomic DNA

Primer: Forward (KpnI-LLO nts. 1257-1276):

5'-CTCT GGTACC TCCTTTGATTAGTATATTC (SEQ ID N0: 74)

(T _m : LLO-spec: 52 ° C. Total: 80 ° C.)

          Reverse (BamHI-XhoI-LLO nts. 2811-2792):

5'-CAAT GGATCCCTCGAG ATCATAATTTACTTCATCCC (SEQ ID NO: 75)

(T _m : LLO-spec: 52 ° C. Total: 102 ° C.)

PCR is also used to amplify the cleaved OVA using the following templates and primers:

Source: pDP3616 plasmid DNA from DP-E3616 Escherichia coli [Higgins et al., Mol. 31: 1631-1641 (1999).

primer:

        Forward (Xhol-NcoI OVA cDNA nts. 174-186):

5'- ATTT CTCGAG T CCATGG GGGGTTCTCATCATC (SEQ ID NO: 76)

(T _m : OVA-spec: 60 ° C. Total: 88 ° C.)

       Reverse (XhoI-NotI-HindIII):

5'-GGTG CTCGAG T GCGGCCGCAAGCTT (SEQ ID N0: 77)

(T _m : total: 82 ° C.)

One protocol for completing the construction process involves first cutting the LLO amplicon into KpnI and BamHI and inserting the KpnI / BamHI vector into the pPL2 vector (pPL2-LLO). The OVA amplicon is then cut into XhoI and NotI and inserted into pPL2-LLO truncated to XhoI / NotI. (Note: pPL2 vector does not contain any XhoI site; pDP-3616 contains 1 XhoI site, which is used in OVA reverse primer design. Construct pPL2 / LLO-OVA is a restriction enzyme assay (KpnI-LLO). -XhoI-OVA-NotI) and sequencing The plasmid pPL2 / LLO-OVA is introduced into E. coli by transformation and then by conjugation, exactly as Listeria (DP-L4056) as described by Lauer et al. ) (Or into other predetermined strains of Listeria , such as inlB- mutants or inlB-actA- double mutants).

B. Construction of Listeria strains expressing AH1 / OVA or AH1-A5 / OVA.

To prepare Listeria expressing AH1 / OVA or AH1-A5 / OVA, an insert with antigen is first prepared from oligonucleotides and then ligated into vector pPL2 / LLO-OVA (prepared as described above). do.

The following oligonucleotides are used to prepare AH1 or AH1-A5 inserts:

AHI Epitope Inserts (Clal-Pstl Compatible Terminals):

      Raise the top strand (above AH1):

      5'-CGATTCCCCTAGTTATGTTTACCACCAATTTGCTGCA (SEQ ID NO: 78)

      Strand up (under AH1):

      5'-GCAAATTGGTGGTAAACATAACTAGGGGAAT (SEQ ID N0: 79)

AHI-A5 epitope insert (ClaI-AvaII compatible ends):

The sequence of the AH1-A5 epitope is SPSYAYHQF (SEQ ID N0: 73) (5'-AGT CCA AGT TAT GCA TAT CAT CAA TTT-3 '(SEQ ID NO: 80)).

Top: 5'-CGATAGTCCAAGTTATGCATATCATCAATTTGC (SEQ ID NO: 81)

Below: 5'-GTCGCAAATTGATGATATGCATAACTTGGACTAT (SEQ ID N0: 82)

Oligonucleotide pairs for a given epitope are mixed with each other in the same molar ratio and heated at 95 ° C. for 5 minutes. The oligonucleotide mixture is then cooled slowly. The annealed oligonucleotide pair is then ligated with the relevant restriction enzyme at a 200: 1 molar ratio with the prepared pPL2-LLO / OVA plasmid. The identity of the new construct can be confirmed through restriction enzyme analysis and / or sequencing.

The plasmid is then introduced into E. coli by transformation and then by conjugation, precisely by Lauer. into Listeria (DP-L4056) or other predetermined strains of Listeria, as described by et al., for example actA ^_ mutant strain (DP-L0429), LLO L461T strain (DP-L4019) or actA ^_ luvrAB ^_ strain It can be introduced and integrated into (DP-L4029uvrAB).

Example 3 Construction of Listeria Polynucleotide and Expression Cassette Elements

A. Cloning Vector

Selected heterologous antigen expression cassette molecular constructs were selected from pPL2 [Lauer et. al. J. Bacteriol. 2002, or PAM401 [Wirth et. al., J. Bacteriol. 165: 831-836, modified to contain multiple cloning sequences of pPL2 (Aat II bovine fragment, 171 bps), and blunted Xba I and Nru I in tetracycline resistance gene (pAM401-MCS, FIG. 32) Insert between recognition sites In general, the hly promoter and (selected) signal peptide sequences were inserted between the only Kpn I and Bain HI sites in the pPL2 or pAM401-MCS plasmid vectors. The selected EphA2 gene (sometimes modified to contain N-terminal and C-terminal epitope tags; see description below) was cloned into this construct between the only Bam HI and Sac I sites. Molecular constructs based on the pAM401-MCS plasmid vector were introduced by electroporation into selected Listeria monocytogenes strains that were also treated with lysozyme using methods conventional to those skilled in the art. The expected plasmid structure in the Listeria transfectant was confirmed by DNA isolation from colonies formed on chloramphenicol containing BHI agar plates (10 ug / ml) by restriction enzyme analysis. Heterologous protein expression and secretion was measured as described below using recombinant Listeria transformed with various pAM401-MCS based heterologous protein expression cassette constructs.

pPL2 based heterologous protein expression cassette constructs were prepared according to the methods described above [Laurer et. al., J. Bacteriol. 184, 4177-4186 (2002)], were incorporated into the tRNA ^Arg of the genome of the selected Listeria strain. In brief, the pPL2 heterologous protein expression cassette construct plasmid was first subjected to E. coli host strain SM10 [Simon et. al., Bio / Technology 1: 784-791 (1983). The pPL2 based plasmid was then transferred to Listeria strains selected from SM10 transformed by conjugation. After incubation in drug-selective BHI agar plates containing 7.5 ug chloramphenicol per ml and 200 ug streptomycin per ml, the selected colonies are purified by passage three times in plates of the same composition. To determine whether the pPL2 vector was integrated at the phage adhesion site, select each colony to select forward primer NC16 (5'-gtcaaaacatacgctcttatc-3 '(SEQ ID NO: 94)) and reverse primer PL95 (5'-acataatcagtccaaagtagatgc-3' (SEQ ID). Screen by PCR using primer pairs of NO: 95)). Selected colonies with pPL2 based plasmids incorporated into the tRNA ^Arg gene of the genome of the selected Listeria strain yielded a 499bps diagnostic DNA amplicon.

B. Promoter

The heterologous protein expression cassette contained a prfA dependent hly promoter that encodes the transcription of Listeriricin O (LLO) and is activated within the microenvironment of infected cells. Nucleotide 205586-206000 (414 bps) was amplified by PCR from Listeria monocytogenes , strain DP-L4056, using the primer pair shown below. The amplified site contains the hly promoter and also the first 28 amino acids of LLO, including the secA1 signal peptide (see below) and the PEST domain. Listeria monocytogenes , the expected sequence of the strain EGD region can be found in GenBank (Accession No. gi│6802048│ref│NC_003210. 1) [16802048]). Primers used in PCR are as follows:

Primer Pairs:

         Forward (Kpnl-LLO nts. 1257-1276):

5'-CTCT GGTACC TCCTTTGATTAGTATATTC (SEQ ID NO: 74)

         Reverse (Bam HI-LLO nts.):

5'-CTCT GGATCC ATCCGCGTGTTTCTTTTCG (SEQ ID NO: 84)

(The restriction enzyme recognition site is underlined.)

The 422 bp PCR amplicon was cloned into plasmid vector pCR-XL-TOPO (Invitrogen, Carlsbad, Calif.) According to the manufacturer's instructions. The nucleotide sequence of the Listeria specific base in the pCR-XL-TOPO-hly promoter plasmid clone was determined. Listeria monocytogenes strain DP-L4056 contained 8 nucleotide base changes flanking the prfA box in the hly promoter compared to the EGD strain. The hly promoter alignment for Listeria monocytogenes DP-L4056 and EGD strains is shown in Figure 1 below.

The 422 bp DNA corresponding to the hly promoter and secA1 LLO signal peptide was digested with Kpn I and Bam HI to liberate from the pCR-XL-TOPO-hly promoter plasmid clone and according to conventional methods known to those skilled in the art [Lauer et al. . al. 2002 J. Bact. This plasmid is known as pPL2-hlyP (natural).

C. Shine-Dalgano Sequence

The poly-purine shine-dalgano sequence contains elements necessary for the 30S ribosomal subunit (via 16S rRNA) to intervene at the 3 'end of the promoter to heterologous gene RNA transcripts and initiation of translation. Typically the shine-dalgano sequence has the following consensus sequence: 5'-NAGGAGGU-N5-10-AUG (starting codon) -3 '(SEQ ID NO: 85). There is a variation of the poly-purine shine-dalgano sequence. Notably, the Listeria hly gene encoding Listeriolisin O (LLO) has the following Shine- Dalgano sequence: A AGGAGA GTGAAACCC ATG (SEQ ID NO: 70) (The Shine-Dalgano sequence is underlined and translation begins Codons are bold).

Example 4 Polynucleotides Encoding Fusion Proteins Including secA1 Signal Peptide (LLO) and Human EphA2

The sequence of the expression cassette encoding the full-length human EphA2 antigen fused to the secA1 signal peptide (LLO signal peptide) and the LLO PEST sequence is shown in FIG. 2. The amino acid sequence of the fusion protein encoded by this expression cassette is shown in FIG.

Example 5. Codon Optimization of the Extracellular Domain of Human EphA2 (EX2)

The sequence encoding the extracellular domain of human EphA2 (amino acids 25-526) was codon optimized for the expression of Listeria monocytogenes . The native nucleotide sequence encoding the extracellular domain of human EphA2 is shown in FIG. 4. Nucleotide sequences for optimal codon usage in Listeria are shown in FIG. 5. The amino acid sequence of the extracellular domain of human EphA2 is shown in FIG. 6.

Example 6. Polynucleotides encoding a fusion protein comprising the extracellular domains of secA1 signal peptide (LLO) and huEph2 (EX2).

A. Codon-Unoptimized Polynucleotides

The sequence of the polynucleotide encoding the extracellular domain of human EphA2 fused to the secA1 signal peptide (LLO signal peptide) and the LLO PEST sequence is shown in FIG. 7. The amino acid sequence of the fusion protein encoded by this expression cassette is shown in FIG.

B. Expression cassette with codon-optimized extracellular domain of human EphA2.

Codon-optimized LLO PEST sequences for the expression of the expression cassette encoding the extracellular domain of human EphA2 fused to the secA1 signal sequence (LLO signal peptide) and the sequence encoding the extracellular domain of EphA2 in Listeria monocytogenes are shown in FIG. It is shown in 9. The amino acid sequence of the fusion protein encoded by this expression cassette is shown in FIG.

C. Expression cassette with codon-optimized secA1 signal peptide and codon-optimized Sepponite domain of human EphA2.

Codon- both the sequence of the expression cassette encoding the extracellular domain of human EphA2 fused to the secA1 signal sequence (LLO signal peptide) and the sequence encoding the extracellular domain, signal peptide and PEST sequence of EphA2 are expressed in Listeria monocytogenes . The optimized LLO PEST sequence is shown in FIG. The amino acid sequence of the fusion protein encoded by this expression cassette is shown in FIG.

Example 7 Codon-Optimized Expression Cassette Encoding a Fusion Protein Including Extracellular Domains of Tat Signal Peptide ( B. subtilis phoD) and HuEphA2 (EX2)

The sequence of the expression cassette encoding the extracellular domain of the EphA2 antigen fused to the codon-optimized Tat signal peptide ( B. subtilis phoD), both of which encodes the extracellular domain of EphA2 and the signal peptide in Listeria monocytogenes 13 is shown. The amino acid sequence of the fusion protein encoded by this expression cassette is shown in FIG.

Example 8. Codon-Optimization of Intracellular Domain of Human EphA2 (CO).

The sequence encoding the intracellular domain of human EphA2 (amino acids 558-975) was codon-optimized for expression in Listeria monocytogenes . The unique nucleotide sequence encoding the extracellular domain of human EphA2 is shown in FIG. 15. Nucleotide sequences for optimal codon usage in Listeria are shown in FIG. 16. The amino acid sequence of the extracellular domain of human EphA2 is shown in FIG. 17.

Example 9. A polynucleotide encoding a fusion protein comprising the intracellular domains of secA1 signal peptide (LLO) and huEphA2 (CO).

A. Codon-Unoptimized Polynucleotides.

The intracellular domain and LLO PEST sequence of human EphA2 antigen fused to secA1 signal peptide (LLO) are shown in FIG. 18. The amino acid sequence of the fusion protein encoded by this expression cassette is shown in FIG.

B. Expression cassette with codon-optimized intracellular domain of human EphA2.

The codon-optimized LLO PEST sequence for the expression cassette encoding the intracellular domain of huEphA2 antigen fused to secA1 signal peptide (LLO signal peptide) and the sequence encoding the intracellular domain of EphA2 in Listeria monocytogenes are shown in FIG. It is shown in 20. The amino acid sequence of the fusion protein encoded by this expression cassette is shown in FIG.

C. Expression cassette with codon-optimized secAl signal peptide and codon-optimized intracellular domain of human EphA2.

The sequence of the expression cassette encoding the intracellular domain of the huEphA2 antigen fused to the secA1 signal peptide (LLO signal peptide) and the sequence, signal peptide and PEST sequence encoding the intracellular domain of EphA2 are both codon-expressed for expression in Listeria monocytogenes . The optimized LLO PEST sequence is shown in FIG. 22. The amino acid sequence of the fusion protein encoded by this expression cassette is shown in FIG.

Example 10. Codon-optimized expression cassette encoding a fusion protein comprising a B. subtilis phoD signal peptide and an intracellular domain of huEphA2 (CO).

The sequence of the expression cassette encoding the intracellular domain of EphA2 and the intracellular domain of the EphA2 antigen fused to a codon-optimized Tat signal peptide ( B. subtilis phoD) for expression in Listeria monocytogenes is It is shown in FIG. The amino acid sequence of the fusion protein encoded by this expression cassette is shown in FIG.

Example 11 Codon-Optimized Expression Cassette Encoding a Fusion Protein comprising an LLO Signal Peptide and NY-ESO-1.

The expression cassette was designed for the expression of testicular cancer antigen NY-ESO-1 (Genbank Accession No. NM_001327) in Listeria monocytogenes . The sequence of the expression cassette encoding NY-ESO-1 fused to secA1 signal peptide (LLO) and the LLO PEST sequence are shown in FIG. 26. The sequences encoding signal peptides in the expression cassette as well as the antigen were codon-optimized for expression in Listeria monocytogenes . The amino acid sequence of the fusion protein encoded by this expression cassette is shown in FIG.

Example 12 Codon-Optimized Expression Cassette for Encoding Antigen Fused to a Non- Listeria secA1 Signal Peptide ( L. Lactis usp45)

Expression cassettes were designed for the expression of heterologous antigens in Listeria monocytogenes using non- Listeria secA1 signal peptides.

The amino acid sequence of the usp45 signal peptide of Lactococcus lactis (Steidler et al., Nature Biotechnology, 21: 785-9 (2003)), its own coding sequence and coding sequences optimized for expression in Listeria monocytogenes are shown below.

Amino acid sequence:

MKKKIISAILMSTVILSAAAPLSGVYA'DT (SEQ ID NO: 46)

Signal peptidase recognition site: VYA-DT (SEQ ID NO: 55)

Unique nucleotide sequence:

5'ATGAAAAAAAAGATTATCTCAGCTATTTTAATGTCTACAGTGATACTTTCTGCTGCAGCCCCGTTGTCAGGTGTTTACGCTGACACA3 '(SEQ ID N0: 86)

Codons optimized for expression in Listeria monocytogenes :

5'ATGAAAAAAAAAATTATTAGTGCAATTTTAATGAGTACAGTTATTTTAAGTGCAGCAGCACCATTAAGTGGTGTTTATGCAGATACA3 '(SEQ ID NO: 87)

The sequence of the partial expression cassette containing the hly promoter of Listeria monocytogenes operably linked to the codon-optimized sequence encoding the Usp45 signal peptide is shown in FIG. 28. This sequence can bind to a codon-optimized or non-codon-optimized antigen sequence for expression of a fusion protein comprising a Usp45 signal peptide and a given antigen.

Example 13. Codon-optimized expression cassettes and vectors encoding antigen fused to secA2 signal peptide (p60).

 A. Design of Codon-Optimized Expression Cassettes.

Expression cassettes were designed for the expression of heterologous antigens in Listeria monocytogenes using the secA2 secretion pathway. The amino acid sequence of the p60 signal peptide of Listeria monocytogenes , its native coding sequence and the coding sequence optimized for expression in Listeria monocytogenes are shown below.

Amino acid sequence:

MNMKKATIAATAGIAVTAFAAPTIASA'ST (SEQ ID NO: 48)

Signal peptidase recognition site: ASA-ST (SEQ ID NO: 57)

Natural nucleotide sequence:

5'ATGAATATGAAAAAAGCAACTATCGCGGCTACAGCTGGGATTGCGGTAACAGCATTTGCTGCGCCAACAATCGCATCCGCAAGCACT3 '(SEQ ID NO: 90)

Codons optimized for expression in Listeria monocytogenes :

5'ATGAATATGAAAAAAGCAACAATTGCAGCAACAGCAGGTATTGCAGTTACAGCATTTGCAGCACCAACAATTGCAAGTGCAAGTACA3 '(SEQ ID NO: 91)

The sequence of the partial expression cassette containing the hly promoter of Listeria monocytogenes operably linked to the native sequence encoding the p60 signal peptide is shown in FIG. 29. The sequence of the partial expression cassette containing the hly promoter of Listeria monocytogenes operably linked to the codon-optimized sequence encoding the p60 signal peptide is shown in FIG. 30.

B. Configuration of pPL2-hlypro_p60.

It is also possible to construct an expression cassette in which the antigen coding sequence is inserted into the frame of one or more sites in the coding sequence of the p60 gene. Description of the construction of partial expression cassettes useful for inserting antigen sequences in a frame within the p60 sequence is described below. This partial expression cassette contains a hly promoter.

Each primary PCR reaction using Pfx or Vent polymerase is performed using the following primers and pPL2-hlyP-OVA (same as pPL2 / LLO-OVA described above in Example 2A) as the first template:

pPL2-5F: 5'-GACGTCAATACGACTCACTATAG (SEQ ID NO: 92)

p60-hlyP-237R: 5'-CTTTTTTCATATTCATGGGTTTCACTCTCCTTCTAC (SEQ ID NO: 93) The final empricon is 285 bps in size.

In addition, each primary PCR reaction using Pfx or Vent polymerase is carried out using the following primer and pCR-TOPO-p60 as the second template. (The vector pCR-TOPO-p60 is made of a pCR-TOPO vector purchased from Invitrogen, Carlsbad, Calif., In which the genomic p60 sequence of Listeria monocytogenes has been inserted. The primers used in this PCR reaction are as follows:

hlyP-p60-1F: 5'-AAGGAGAGTGAAACCCATGAATATGAAAAAAGCAAC (SEQ ID NO: 88)

pCR-TOPO-2283R: 5'-GTGTGATGGATATCTGCAGAATTC (SEQ ID NO: 89)

The final amplicon is 1510 bps. The PCR reaction is then washed with an S6 column (Bio-Rad Laboratories, Hercules, California).

PCR reactions are then performed using about 5 ul of each primary PCR reaction as a template. Secondary PCR reaction using the following primers:

Kpnl-LLO 1257F (previously used primer):

5'CTCTGGTACCTCCTTTGATTAGTATATTC (SEQ ID NO: 74) and pCR-TOPO-2258R: 5'-CCCTTGGGGATCCTTAATTATACG (SEQ ID NO: 83).

The final amplicon is 1715 bps. The expected amplicon size for all PCR reactions is confirmed by agarose gel analysis. The secondary PCR reaction is washed, digested with BamHI, rewashed and digested with KpnI. The hlyP-p60 gene fragment (KpnI-BamHI) (FIG. 30) is then ligated between the BamHI and KpnI sites of both pPL2 and modified pAM401 (pAM401-MCS; FIG. 32) plasmids.

The construction of the pPL2-p60 plasmid was then confirmed by BamHI / KpnI (1697,6024 bps) and HindIII (210,424,3460,3634 bps) degradation. In addition, the PstI site of the pPL2-p60 plasmid is identified as unique. (KpnI / PstI digestion will also yield fragments of 736 and 6985 bps.) The construction of the pAM401-p60 plasmid (KpnI / PstI and KpnI / BamHI fragments at the p60 site) is the same as for the pPL2 construct.

The overall preliminary separation of each plasmid is then prepared using methods known to those skilled in the art.

Certain antigen coding sequences can then be inserted into the same conventional frame as the p60 sequence and the p60 sequence using techniques known to those skilled in the art. Typically, the insertion or insertions should leave the p60 intact N-terminal signal peptide sequence. In addition, the C-terminal autolysis sequence of p60 should be left intact.

Example 14. Codon-Optimization of Human Mesothelin Coding Sequence for Expression in Listeria monocytogenes .

Codon-optimized polynucleotides encoding human mesothelin, cancer antigens are shown in FIG. 33. The sequence shown in FIG. 32 was codon-optimized for expression in Listeria monocytogenes . The polypeptide sequence encoded by the sequence of FIG. 33 is shown in FIG. 34.

Example 15 Codon-Optimization of Murine Mesothelin Coding Sequence for Expression in Listeria monocytogenes .

Codon-optimized polynucleotide sequences encoding human mesothelin, cancer antigens are shown in FIG. 35. The sequence shown in FIG. 35 was codon-optimized for expression in Listeria monocytogenes . The polypeptide sequence encoded by the sequence of FIG. 35 is shown in FIG. 36.

Example 16 Integration of Expression Cassettes into Listeria Chromosomes via Allele Exchange

As a possible alternative to using an integration vector such as pPL2 to insert a heterologous gene expression cassette into the chromosome of Listeria , allele exchange can be used.

Briefly, bacteria into which the pKSV7 heterologous protein expression cassette plasmid has been electroporated are selected by plate culture on BHI agar medium containing chloramphenicol (10 ug / ml) and incubated at 30 ° C. tolerance. Single cross-integration into the chromosome of bacteria is selected by passing several individual colonies of multiple generations at 41 ° C. unacceptable temperature of medium containing chloramphenicol. Finally, plasmid ablation and curing (double crossover) is accomplished by passing some individual colonies of multiple generations at an acceptable temperature of 30 ° C. in BHI medium without chloramphenicol. Confirmation of integration of the heterologous protein expression cassette into the bacterial chromosome is confirmed by PCR using primer pairs that amplify a defined region from inside the heterologous protein expression cassette to the bacterial chromosomal targeting sequence not contained in the pKSV7 plasmid vector construct.

Example 17 Cloning and Insertion of EphA2 into pPL2 Vector for Expression in Selected Recombinant Listeria monocytogenes Strains

The outer (EX2) and cytoplasmic (CO) domains of EphA2 flanking the EphA2 penetrating membrane helix were cloned separately for insertion into various pPL2 signal peptide expression constructs. Codon-optimized genes were used to correspond to native mammalian sequences or to express EphA2 EX2 and EphA2 CO domains in Listeria monocytogenes . The optimal codons of Listeria (see Table 3 above) for each of the 20 amino acids were used for codon-optimized EphA2 EX2 and EphA2 CO. Codon-optimized EphA2 EX2 and CO domains were synthesized by extending overlapping oligonucleotides using techniques common to those skilled in the art. The expected sequence of all synthetic EphA2 constructs was confirmed by nucleotide sequencing.

Primary amino acid sequences along with the native and codon-optimized nucleotide sequences for the EX2 and CO domains of EphA2 are shown in FIGS. 4-6 (EX2 sequence) and FIGS. 15-17 (CO domain sequence).

In addition, the amino acids of the EphA2 EX2 and CO genes synthesized to detect and secrete EphA2 expressed in Western blot using FLAG (Strata gene, La Jolla, CA) and myc epitopes using antibodies specific for FLAG or protein, respectively. And inserted into the frame at the carboxy terminus. Thus, the expressed protein has the following ordered components: NH2--signal peptide-FLAG-EphA2-myc-CO2. FLAG and myc epitope tag amino acids and codon-optimized nucleotide sequences:

FLAG:

     5'-GATTATAAAGATGATGATGATAAA (SEQ ID NO: 96)

     NH2-DYKDDDDK-CO2 (SEQ ID NO: 97)

Myc:

    5'-GAACAAAAATTAATTAGTGAAGAAGATTTA (SEQ ID NO: 98)

    NH2-EQKLISEEDL-CO2 (SEQ ID NO: 99)

Example 18 Detection of Synthetic and Secreted Heterologous Proteins by Western Blot Analysis.

Synthesis and secretion of EphA2 protein from various selected recombinant Listeria -EphA2 strains was determined by Western blot analysis of trichloroacetic acid (TCA) precipitated bacterial culture liquid. Briefly, medium-log stage cultures of Listeria grown in BHI medium are collected in 50 mL conical centrifuge tubes, bacteria are precipitated, and ice-cooled TCAs are added to the bacterial culture supernatant at final [6%] concentration. Add and incubate on ice for at least 90 minutes or overnight. TCA-precipitated protein was collected by centrifugation at 2400 × g for 20 minutes at 4 ° C. The precipitate was then resuspended in 300-600 ul booty of TE, pH 8.0, containing 15 ug / ml phenol red. Sample dissolution was promoted by vortexing. Sample pH was adjusted by addition of NH ₄ OH if necessary until the color was pink. All samples were prepared for electrophoresis by adding 4 × SDS loading buffer and incubating at 90 ° C. for 10 minutes. The samples were then centrifuged at 14,000 rpm for 5 minutes in a microcentrifuge and the supernatant collected and stored at -20 ° C. For Western blot analysis, 20 ul (equivalent of culture liquid of 1-4 × ^{10 9} bacteria) of the fractions prepared according to conventional methods used by those skilled in the art were loaded onto a 4-12% SDS-PAGE gel, electrophoresed, protein Was transferred to a PDDF membrane. The transported membranes were prepared for incubation with antibodies by incubating in 5% milk powder in PBS for 2 hours at room temperature with stirring. The antibody was used under the following dilution of PBST buffer (0.1% Tween 20 in PBS): (1) 1: 10,000 rabbit anti-Myc polyclonal antibody (ICL laboratories, Newberg, Oregon); (2) 1: 2,000 murine anti-FLAG monoclonal antibody (Stratagene, La Jolla, Calif.); And (3) rabbit anti-EphA2 (carboxy end-specific) polyclonal antibodies (sc-924, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Specific binding of the antibody to protein targets was detected by exposure to the film and detection through a second incubation and ECL chemiluminescence assay kit (Amersham) with goat anti-rabbit or anti-mouse conjugated with horseradish peroxidase. Was evaluated.

Example 19 Secretion of EphA2 Protein by Recombinant Listeria Encoding Various Forms of EphA2

A. Listeria : [Strain DP-L4029 (actA) or DP-L4017 (LLO L461T)]

Expression Cassette Construct: LLOss-PEST-CO-EphA2

The native sequence of the EphA2 CO domain was fused to the native secA1 LLO sequence and a heterologous antigen expression cassette was inserted between pPL2 plasmids KpnI and SacI described above under the control of the Listeria hly promoter. pPL2-EphA2 plasmid constructs were introduced by conjugation into Listeria strains DP-L4029 (actA ^_ ) and DP-L4017 (L461TLLO) as described above. 37 shows the results of Western blot analysis of TCA-precipitated bacterial cultures of 4029-EphA2 CO and 4017-EphA2 CO. This analysis demonstrated that recombinant Listeria engineered to contain heterologous protein expression cassettes composed of unique sequences corresponding to secA1 and EphA2 CO fusion proteins secrete multiple EphA2 specific fragments lower than the 52 kDa expected molecular weight. Prove the necessity of the transformation.

B. Listeria : [DP-L029 (actA ^_ ]

Expression cassette constructs:

Natural LLOss-PEST-FLAG-EX2_EphA2-myc-codon Op

2. (Codon Op) LLOss-PEST- (CodonOp) FLAG-EX2_EphA2-myc

Genetic fusion of the codon-optimized secA1 LLO signal peptide sequence for expression in Listeria or the native secA1 LLO signal peptide sequence for expression in Listeria , and a heterologous antigen under the control of the Listeria hly promoter The expression cassette was inserted between pPL2 plasmids KpnI and SacI described above. The pPL2-EphA2 plasmid construct was introduced by conjugation into the Listeria strain DP-L4029 (actA) described above. 38 shows the results of Western blot analysis of TCA-precipitated bacterial cultures of Listeria actA encoding a natural signal peptide or codon-optimized secA1 LLO signal peptide fused with a codon-optimized EphA2 EX2 domain. This analysis demonstrated that the combination of the use of sequences of both heterologous proteins optimized for the desired codon usage in signal peptides and Listeria monocytogenes resulted in the expression of the expected full-length EphA2 EX2 domain protein. Expression of the full-length EphA2 EX2 domain protein was insufficient only by codon-optimization of the EphA2 coding sequence. Heterologous protein expression levels (short or full-length) codon for expression in Listeria monocytogenes - was the highest when using the optimized Listeria monocytogenes LLO secA1 signal peptide.

C. Listeria : [DP-L4029 (actA)]

Expression cassette constructs:

3. Natural LLOss-PEST- (Codon 0p) FLAG-EphA2_CO-myc

4.Codon Op LLOss-PEST- (Codon Op) FLAG-EphA2_CO-myc

5.Codon Op PhoD- (Codon Op) FLAG-EphA2_CO-myc

To expression in Listeria the Tat signal peptide of the phoD gene of an optimized Bacillus subtilis codon-native secA1 LLO signal peptide sequence or codons to expression in Listeria-optimized secA1 LLO signal peptide sequence or, alternatively, codons to expression in Listeria Genetically fused with the optimized EphA2 CO domain sequence and a heterologous antigen expression cassette was inserted between the pAM401-MCS plasmid KpnI and SacI sites described above under the control of the Listeria hly promoter. The pAM401-EphA2 plasmid construct was introduced by electroporation into the Listeria strain DP-L4029 (actA) described above. FIG. 39 shows Westerns of TCA-precipitated bacterial cultures of Listeria actA encoding a natural signal peptide or codon-optimized secA1 LLO signal peptide, or a codon-optimized Bacillus subtilis phoD Tat signal peptide fused with a codon-optimized EphA2 CO domain. The results of the blot analysis are shown. This analysis demonstrated that combining the use of sequences of both heterologous proteins optimized for the desired codon usage in signal peptides and Listeria monocytogenes resulted in the expression of the full-length EphA2 CO domain protein expected. In addition, the expression and secretion of the expected full-length EphA2 CO domain protein was due to recombinant Listeria encoding a codon-optimized Bacillus subtilis phoD Tat signal peptide fused with a codon-optimized EphA2 CO domain. This result represents a novel and unexpected finding that signal peptides of distinct bacterial species can be used to program the secretion of heterologous proteins of recombinant Listeria . Expression of the full-length EphA2 CO domain protein was insufficient only by codon-optimization of the EphA2 sequence. The level of heterologous protein expression was highest when using codon-optimized signal peptides for expression in Listeria monocytogenes .

D. Transfection of 293 cells with a pCDNA4 plasmid encoding the full length EphA2 expression cassette construct.

6. pCDNA4-EphA2

The native full length EphA2 gene was cloned into the eukaryotic CMV promoter basal expression plasmid pCDNA4 (Invitrogen, Carlsbad, Calif.). FIG. 40 shows the results of Western blot analysis of lysates prepared from 293 cells transfected with pCDNA4-EphA2 plasmid, demonstrating mass expression of full length EphA2 protein in mammalian cells.

Example 20 Therapeutic Efficacy of Balb / C Mice Carrying CT26 Tumors Encoding Human EphA2 Immunized with Recombinant Listeria Encoding Codon-Optimized EphA2.

The following data shown in FIGS. 41-44 demonstrated the following:

CT26.24 (huEphA2 +) lung tumors with recombinant Listeria encoding OVA.AH1 (MMTV gp70 immunodominant epitope) or OVA.AH1-A5 (irregularly altered MMTV gp70 immunodominant epitope) for increased T cell receptor binding. Immunization of bearing Balb / C mice confers long term survival (FIG. 41).

The EphA2 CO domain is highly immune, resulting in a significant long-term increase in survival of Balb / C mice carrying CT26.24 (huEphA2 +) lung tumors when immunized with recombinant Listeria encoding a codon-optimized or native EphA2 CO domain sequence. Was observed (FIG. 43).

The EphA2 EX2 domain is weakly immune and Balb / C with CT26.24 (huEphA2 +) lung tumors only when immunized with recombinant Listeria encoding a codon-optimized secA1 signal peptide fused to a codon-optimized EphA2 CO domain sequence Increased survival of the mice was observed. No therapeutic efficacy was observed in mice when immunized with recombinant Listeria encoding a codon-optimized secA1 signal peptide fused with a codon-optimized EphA2 EX2 domain sequence (FIG. 42). The preference of using both codon-optimized secA1 signal peptide and EphA2 EX2 domain sequences was supported by the statistically significant therapeutic antitumor efficacy shown in Table 4 below.

Table 4. Comparison by log-grade test of survival curves shown in FIG. 42.

Significantly, immunization of Balb / C mice with pCDNA4-EphA2 plasmid with CT26.24 (huEphA2 +) lung tumors despite treatment with 293 cells transfected with pCDNA4-EphA2 plasmid resulted in very high levels of protein expression. No antitumor efficacy was observed (FIG. 44).

For therapeutic in vivo tumor studies, female Balb / C mice were implanted intravenously with ⁵ × 10 ⁵ CT26 cells stably expressing EphA2. After 3 days, mice were randomly picked and vaccinated intravenously with various recombinant Listeria strains encoding EphA2. In some cases, mice were vaccinated with either pCDNA4 plasmid or pCDNA4-EphA2 plasmid in the forearm muscle. As a positive control, mice were vaccinated intravenously with recombinant Listeria strains encoding OVA.AHI or OVA.AH1-A5 protein chimeras. Mice injected with Hanks Balanced Salt Solution (HBSS) buffer or unmodified Listeria served as negative controls. All experimental groups included 5 mice. When the mice began to show any signs of stress or difficulty breathing, they were killed for survival studies.

Example 21.Evaluation of Antigen-Specific Immune Responses after Vaccination

Vaccines of the invention can be evaluated using a variety of in vitro and in vivo methods. Some assessments relate to the analysis of antigen specific T cells in the spleen of vaccinated mice. Non-limiting examples of methods for assessing immune responses in vitro and in vivo are provided in this example. The antigens listed in this representative description of the assay are necessarily model antigens, not antigens produced using recombinant nucleic acid molecules, expression cassettes, and / or expression vectors described herein. Those skilled in the art will readily appreciate that the assays described in this example can be readily used to assess in vitro or in vivo immune responses of bacteria comprising recombinant nucleic acid molecules, expression cassettes, and / or expression vectors described herein. Will recognize.

For example, C57B1 / 6 or Balb / c were vaccinated intravenously with 0.1LD ₅₀ of Listeria strain expressing OVA (or other suitable antigen). Seven days after the vaccine, spleen cells of mice are collected by placing the spleen in ice-cold RPMI 1640 medium and preparing a single cell suspension therefrom (typically 3 mice per group). Alternatively, lymph nodes of mice can be collected in a similar manner and prepared in a single cell suspension to replace spleen cells in the assay described below. Typically, splenic cells are evaluated by intravenous or intraperitoneal administration of the vaccine, while cells of spleen cells and lymph nodes are evaluated by intramuscular, subcutaneous or intradermal administration of the vaccine.

Unless stated otherwise, all antibodies used in this example are available from Pharmingen, San Diego, California.

ELISPOT Assay: As an example, when using Listeria strains with OVA antigens, the quantitative frequency of antigen specific T cells resulting from immunization in a mouse model is assessed using ELISPOT assays. The antigen specific T cells evaluated are OVA specific CD8 + or LLO specific CD8 + or CD4 + T cells. This OVA antigen model evaluates the immune response to heterologous tumor antigens inserted into the vaccine and can be substituted with any antigen of interest. LLO antigens are specific for Listeria . Specific T cells are assessed by detection of cytokinin secretion (eg IFN-γ) following recognition of specific antigens. PVDF based 96 well plates (BD Biosciences, San Jose, Calif.) Are coated overnight at 4 ° C. with anti-rat IFN-γ monoclonal antibody (mAb R4; 5 ug / ml). Plates are washed and blocked for 2 hours at room temperature with 200 ul of complete RPMI. Spleen cells of vaccinated mice (or vaccinated control mice) are added at 2 × 10 5 cells per well and incubated for 20 to 22 hours at 37 ° C. in the presence of varying concentrations of peptides ranging from 0.01 to 10 uM. Peptides used for OVA and LLO include SL8, MHC class I epitope for OVA, LL0190 (NEKYAQAYPNVS (SEQ ID NO: 100) Invitrogen) MHC class II epitope for Listeriricin O (Listeria antigen), LL0296 (VAYGRQVYL (SEQ ID NO: 101) MHC Class I Epitope for Listeriricin O or LLO91 (GYKDGNEYI (SEQ ID NO: 102)) MHC Class I Epitope for Listeriricin O. LLO1go and LL0296 are C57B1 / 6 While used in the model, LL091 is used in the Balb / c model: After washing, the plates were coated with a biotin attached secondary antibody specific for IFN-γ (XMG1.2) diluted to 0.5 ug / ml in PBS. After incubation for 2 hours at room temperature, the plates were washed and diluted in PBS containing 1% BSA in 1 nm gold chlorine anti-biotin conjugate (GAB-1; 1: 200 dilution; Ted Pella, Redding, Incubate at 37 ° C. for 1 hour. The plate is then incubated with the substrate (silver enrichment kit; 30 ml / well; Ted Pella) at room temperature for 2 to 10 minutes to spot develop the plate. The plate is then distilled water to stop the substrate reaction. After air drying the plates, the dots of each well are counted using an automatic ELISPOT plate reader (CTL, Cleveland, OH) Cytokine responses are 2x10 ⁵ cells for OVA specific T cells or Listeria specific T cells. Expressed as the number of IFN-γ dot forming cells (SFC) per cell.

Intracellular Cytokine Staining Assay (ICS): To further evaluate the number of antigen specific CD8 + or CD4 + T cells and to correlate the results with those obtained from the ELISPOT assay, ICS was performed and the cells were subjected to flow cytometry analysis. Evaluate by Splenocytes of the vaccinated and control mice are incubated with SL8 (stimulating OVA specific CD8 + cells) or LL0190 (stimulating LLO specific CD4 + cells) for 5 hours in the presence of Brefeldin A (Pharmingen). Brefeldin A inhibits the secretion of cytokines produced upon stimulation of T cells. Spleen cells incubated with unrelated MHC class I peptides are used as controls. Splenocytes stimulated with 20 ng / ml PMA (forball-12-myristate-13-acetate, Sigma) and 2 pg / ml ionomycin (Sigma) were positive for cytokine staining in IFN-γ and TNF-α cells. Use as a control. For detection of cytoplasmic cytokine expression, cells are stained with FITC-anti CD4 mAb (RM 4-5) and PerCP-anti CD8 mAb (53-6.7), fixed and permeable with Cytofix / CytoPerm solution (Pharmingen) And stain with ice-conjugated anti-TNF-α mAb (MP6-XT22) and APC-conjugated anti-IFN-γ mAb (XMG1.2) for 30 minutes on ice. The percentage of cells expressing intracellular IFN- [gamma] and / or TNF- [alpha] is determined by flow cytometry (FACScalibur, Mountain View Becton Dickinson, CA), and data are obtained using CELLQuest software (Becton Dickinson Immunocytometry System). And analyzed. Since fluorescent labels on various antibodies can all be distinguished by FACScalibur, suitable cells are identified by opening and closing the gate for CD8 + and CD4 + stained with either or both of anti-IFN-γ or anti-TNF-α.

Cytokine Expression in Stimulated Spleen Cells: Levels of cytokine secretion by spleen cells in mice can also be assessed for control and vaccinated C57B1 / 6 mice. Spleen cells are stimulated with SL8 or LLO190 for 24 hours. Stimulation with the irrelevant peptide HSV-gB2 (Invitrogen, SSIEFARL, SEQ ID NO: 4) was used as a control. Supernatants of stimulated cells are collected and the levels of T Adjury-1 and T Adjuvant 2 cytokines are determined using ELISA assay (Biosciences, CO) or Cytometry Bead Array Kit (Pharmingen).

Evaluation of Cytotoxic T Cell Activity: OVA specific CD8 + T cells can be further assessed by measuring their cytotoxic activity in vitro or directly in C57B1 / 6 mouse in vivo. CD8 + T cells recognize and lyse their respective target cells in an antigen-specific manner. In vitro cytotoxicity is determined using chromium secretion assays. Splenocytes from mice vaccinated with Listeria- OVA (internal) without administration were irradiated EG7.OVA cells (EL-4 transfected to express OVA) to elongate OVA-specific T cells in the splenic cell population. Tumor cell line, Virginia Manassas) or 100 nM SL8 at 10: 1 ratio. After 7 days of culture, the cytotoxic activity of effector cells was assayed for standard 4-hour 51Cr secretion using only EL-4 cells excited with EG7.OVA or SL8 as target cells (ATCC, Manassas, VA) and EL-4 cells as negative controls. Decide on The YAC-1 cell line (ATCC, Virginia Manassas) uses as a target to determine NK cell activity in order to distinguish the activity attributable to T cells from the activity attributable to NK cells. The percentage of specific cytotoxicity is calculated as 100 x (experimental-voluntary secretion) / (maximal secretion-voluntary secretion). Spontaneous secretion is determined by incubation of target cells without effector cells. Maximal secretion is determined by lysing the cells with 0.1% Triton X-100. Experiments are considered valid for analysis if spontaneous secretion is <20% of maximal secretion.

For evaluation of cytotoxic activity of OVA-specific CD8 + T cells in vivo, spleen cells of C57B1 / 6 mice that have never been tested are divided into two equal aliquots. Each group is excited with 0.5 ug / ml of specific peptide, target (SL8) or control (HSV-gB2) at 37 ° C. for 90 minutes. Cells are then washed three times in medium and twice in PBS + 0.1% BSA. Cells are resuspended at 1 × 10 ⁷ per ml in warm PBS + 0.1% BSA (10 ml or less) to label with carboxyfluorescein diacetate succinimidyl ester (CFSE, molecular probe, Eu gene, OR). To target cell suspension 1.25 uL of a 5 mM stock of CFSE was added and the sample was mixed by vortexing. A 10-fold dilution of CFSE stock was added to the control cell suspension and the samples were vortexed and mixed. Cells are incubated at 37 ° C. for 10 minutes. Staining is stopped by adding PBS chilled with a large amount of ice (> 40 ml). Cells are washed twice with PBS at room temperature, then resuspended and counted. Each cell suspension is diluted to 50 × 10 ⁶ per ml and 100 ul of each population is mixed and injected through the tail vein of untested or vaccinated mice. After 12-24 hours, the spleen is harvested and the entire 5 x 10 ⁶ cells are analyzed by flow cytometry. High (target) and low (control) fluorescence peaks are listed and the ratio of the two is used to determine the percentage of target cell lysis. In vivo cytotoxicity assays can assess the lytic activity of antigen specific T cells without the need for in vitro restimulation. In addition, this assay evaluates T cell function in the natural environment.

Example 22. Human EphA2-specific immunity induced by vaccination of Balb / c mice with Listeria strains expressing EphA2.

Codon-optimized sequence for expressing Balb / c mice (n = 3) in L. monocytogenes ( Listeria hEphA2-ECD in FIG. 45) two weeks away from Listeria L461T expressing hEphA2 (Listeria hEphA2-ICD in FIG. 45) From ΔactA (actA ^_ ) strains expressing the extracellular domain of hEphA2. (Alternatively the intracellular domain of hEphA2 is referred to herein as hEphA2-ICD, hEphA2 ICD, EphA2 CO or CO. Alternatively, the extracellular domains of hEphA2 are referred to herein as hEphA2-ECD, hEphA2 ECD, EphA2 EX2, Or as EX2.) The mice were euthanized and spleens were harvested and pooled 6 days after the last immunization. For ELISPOT analysis, cells were restimulated in vitro with P815 cells expressing full-length hEphA2 or cell lysates prepared from these cells. Parental P815 cells or cell lysates served as negative controls. In addition, cells were stimulated with recombinant hEphA2 Fc fusion protein. IFN-gamma positive spot forming colonies (SFC) were measured using a 96 well spot reader. As shown in FIG. 45, increased IFN-gamma SFC was observed with splenic cells derived from mice vaccinated with Listeria -hEphA2. Both hEphA2 expressing cells or cell lysate stimuli increased IFN-gamma SFCs presenting CD4 + T cell responses as well as EphA2-specific CD8 +. Spleen cells from mice vaccinated with the parent Listeria control did not demonstrate an increase in IFN-gamma SFC.

Example 23 CD4 + and CD8 + T Cell Responses are Required for EphA2 Specific Antitumor Efficacy

Balb / c mice (n = 10) were inoculated intravenously with 2 × 10 ⁵ CT26-hEphA2 on day 0. CD4 + cells and CD8 + T cells were depleted by injection of 200 ug anti-CD4 (ATCC hybridoma GK1.5) or anti-CD8 (ATCC hybridoma 2.4-3) on days 1 and 3, which was analyzed by FACS analysis (data Not shown). Mice were then immunized intravenously with 0.1LD50 Listeria L461T expressing hEphA2 ICD on day 4 and monitored for survival.

As shown in FIG. 46, the groups depleted of CD4 + and CD8 + did not exhibit the antitumor response observed in animals that did not deplete T cells. The data is summarized in Table 5 below.

Table 5

The foregoing data indicate the need for both CD4 + and CD8 + T cells for optimal inhibition of tumor growth.

Example 24 Deletion of inlB from Listeria by Allele Exchange

In some embodiments, the bacterium comprising the recombinant nucleic acid molecule and expression cassette described herein is a mutant Listeria . For example, in some embodiments, the bacterium comprising the recombinant nucleic acid molecule and the expression cassette is a Listeria monocytogenes strain that lacks the actA gene, the inlB gene, or both. One representative method of generating deletion mutants in Listeria is described below.

Deletion of the internalin B gene (inlB) from Listeria DP-L4029 (or from other selected mutant strains or from wild type Listeria ) is described by Camilli et al., Mol. Microbiol. 8: 143-147 (1993)]. Conjugation overlap extension (SOE) PCR can be used to prepare constructs used in the allele exchange process. The source of the internalin B gene is the sequence listed as Genebank Accession No. 591975 ( Listeria monocytogenes strain EGD, whole genome, fragment 3/12; inlB gene site: nts. 97008-98963), which is incorporated herein by reference in its entirety; Or Genbank Accession No. NC — 003210 ( Listeria monocytogenes strain EGD, whole genome, inlB gene site: nts. 457008-458963, which is incorporated herein by reference in its entirety.

In the first PCR reaction, about 1000 bps sequences upstream and downstream from Listeria inlB genes 5 'and 3' are amplified using the following templates and primers, respectively:

Template: DP-L4056 or DP-L4029 Genomic DNA

Primer pair 1 (for amplification upstream from the 5 'end of inlB):

         Lm-96031F: 5'-GTTAAGTTTCATGTGGACGGCAAAG (SEQ ID NO: 103)

(T _m : 72 ° C)

         Lm- (3'inlB-R +) 9702OR: 5'-

AGGTCTTTTTCAGTTAA CTATCCTCTCCTTGATTCTAGTTAT

(SEQ ID NO: 104) (T _m : 114 ℃)

        (Underlined sequence complementary to the downstream side of the InlB carboxy terminus)

        Emblecon Size (bps): 1007

Primer pair 2 (for amplifying downstream sites from the 3 'end of InlB):

        Lm- (5'inlB-F +) 98911F: 5'-

CAAGGAGAGGATAGT TAACTGAAAAAGACCTAAAAAAGAAGGC

(SEQ ID NO: 105) (T _m : 118 ℃)

        (Underlined sequence complementary to downstream side of InlB amino terminus)

       Lm-99970R: 5'-TCCCCTGTTCCTATAATTGTTAGCTC (SEQ ID N0: 106)

(T _m : 74 ℃)

Emblecon size (bps): 1074

In the secondary PCR reaction, the primary PCR amplicon is fused via SOE PCR using complementarity between the forward primer from pair 1 and the reverse primer from pair 2. This leads to the correct deletion of the inlB coding sequence: nts. 97021-98910 = 1889 bps. The following templates and primers were used in the secondary PCR reaction:

Template: Washed Primary PCR Reaction

Primer Pairs:

         Lm-96043F: 5'-GTGGACGGCAAAGAAACAACCAAAG (SEQ ID NO: 107)

(T _m : 74 ℃)

         Lm-99964R: 5'-GTTCCTATAATTGTTAGCTCATTTTTTTC (SEQ ID NO: 108)

(T _m : 74 ℃)

        (Amplicon Size (bps): 2033)

The protocol for completing the configuration process is as follows:

The first PCR reaction (3 temperature cycles) is performed using Vent DNA polymerase (NEB) and 10 ul of overnight 30 ° C. Listeria DP-L4056 OR DP-L4029 overnight culture. The expected size of the Listeria amplicon is confirmed by 1% agarose gel (1007 bps and 1074 bps). The primary PCR reaction is gel purified and the DNA is eluted with a gene Clean (BIO 101).
Secondary PCR reactions are performed using approximately 1 μl of each primary reactant as a template. The expected size of Listeria amplicons from the secondary PCR reaction is confirmed by 1% agarose gel (2033 bps). Adenosine residues are added to the 3 'end of the Listeria dl inlB amplicon using Taq polymerase.

The Listeria dl inlB amplicon is then inserted into the pCR2.1-TOPO vector. The pCR2.1-TOPO-dlinlB plasmid DNA was digested with XhoI and KpnI and gel purified 2123 bp fragment. KpnI / XhoI 2123 bp fragment is digested with KpnI and XhoI and inserted into pKSV7 vector prepared by treatment with CIAP (pKSV7-dlinlB). Thereafter, the suitability of the inlB sequence in pKSV7-dl inlB is confirmed. The inlB gene is removed from a given Listeria strain by allele exchange with the pKSV7-dlinlB plasmid.

Example 25 Codon-Optimized Signal Peptides for the Construction of Recombinant Listeria

Some representative codon-optimized signal peptides that can be used in expression cassettes of recombinant Listeria are provided in Table 6 below:

Table 6. Representative Signal Peptides for the Construction of Recombinant Listeria

¹ shows the PEST sequence of LLO.

Example 26. Codon-Optimized Expression Cassettes Containing Protective Antigen (PA) Signal Peptides

The expression cassette was designed for the expression of heterologous antigens in Listeria monocytogenes using non- Listeria secA1 signal peptides. The amino acid sequence of the protective antigen (PA) signal peptide of Bacillus anthracis (Ba) (GenBank Accession No. NC — 007322), its native coding sequence and coding sequences optimized for expression in Listeria monocytogenes are shown below:

Amino acid sequence:

MKKRKVLIPLMALSTILVSSTGNLEVIQAEV (SEQ ID N0: 47)

Signal peptidase recognition site: IQA'EV (SEQ ID N0: 56)

Native nucleotide sequence:

ATGAAAAAACGAAAAGTGTTAATACCATTAATGGCATTGTCTACGATATTAGTTTCAAGCACAGGTAATTTAGAGGTGATTCAGGCAGAAGTT (SEQ ID NO: 111)

Codons optimized for expression in Listeria monocytogenes :

ATGAAAAAACGTAAAGTTTTAATTCCATTAATGGCATTAAGTACAATTTTAGTTAGTAGTACAGGTAATTTAGAAGTTATTCAAGCAGAAGTT (SEQ ID N0: 114)

The sequence of the partial expression cassette containing the hly promoter of Listeria monocytogenes operably linked to the codon-optimized sequence encoding the Ba PA signal peptide is shown in FIG. 47. This sequence can be combined with codon-optimized or non-codon-optimized antigen sequences for expression of fusion proteins including Bacillus anthracis PA signal peptide and certain antigens.

Example 27 Expression and Secretion of Antigens from Recombinant Listeria Including Codon-Optimized Expression Cassettes.

Codon optimization of both signal peptides and tumor antigens provides efficient expression and secretion from recombinant Listeria : Codon-optimization of both signal peptides and heterologous protein coding genetic elements from recombinant Listeria based vaccines of human tumor antigens containing hydrophobic domains Provides optimal secretion Efficient antigen secretion from cytoplasmic bacteria is required for efficient presentation through the MHC class I pathway and CD8 + T cell ignition and thus directly links to the efficacy of Listeria based vaccines. The secretion of immune targets, two malignant membrane-bound human tumor antigens, mesothelin and NY-ESO-1 from recombinant Listeria is associated with pancreatic and ovarian cancer (mesothelin), and melanoma (NY-ESO-) among other solid tumors 1) was optimized through codon-optimization of both antigen and signal peptide coding sequences.

Alternatives including secA2 and Twin-Arg translocation (Tat) fused in frame with untested or codon-optimized sequence encoding selected signal peptides associated with secA1 or framed fusion with selected human tumor antigen-human NY-ESO-1 or human mesothelin Various expression cassettes were constructed that included hly promoters linked to the integrative secretion pathway. (See Examples 11-14 and 25 for antigen sequences and / or signal sequences). Western blot analysis of TCA precipitate cultures of Listeria grown in BHI broth was used to evaluate the synthesis and secretion of recombinant heterologous proteins. (We used a similar method as described in Example 18 above for Western blot analysis.)

The results of this experiment are shown in Figures 48A-C. When both the signal peptide coding sequence and operably linked foreign antigen encoding sequence, including when derived from Listeria monocytogenes optimized for codon usage in Listeria monocytogenes, it was observed an efficient expression and secretion of full-length tumor antigens from recombinant Listeria. 48A shows expression / secretion of human mesothelin by ΔactA Listeria monocytogenes with constructs comprising LLO signal peptide fused with human mesothelin using native codons of both LLO and mesothelin. According to Western blot of TCA-precipitated bacterial cultures, the expected secretion of full-length mesothelin (62 kDa) was not observed with this construct and only a few small fragments were secreted (FIG. 48A).

FIG. 48B shows Western blot analysis of expression / secretion of human mesothelin by Listeria monocytogenes ΔactA containing a plasmid (pAM401) containing constructs encoding various signal peptides fused with human mesothelin. In each construct, the mesothelin coding sequence was codon-optimized for expression in Listeria monocytogenes . When indicated, the signal peptide coding sequences used were codon-optimized ("CodOp") to include the native sequence ("native") or to express in Listeria monocytogenes . Secreted mesothelin was detected using affinity-purified polyclonal anti-human / mouse antibodies prepared by injecting rabbits with selected peptides with IFA.

Importantly, as shown in lanes 3-5 and 8-9 of Figure 48B, when both signal peptide and mesothelin coding sequences were codon-optimized for expression in Listeria , the secretion of full-length mesothelin (62 kDa) was observed. It became. Importantly this result also includes Listeria derived signal peptides of the bacterial LLO and p60 proteins involved in the secA1 and secA2 secretion pathways respectively, all of which contain the rarely used codons. (The LLO PEST sequence is also included as an LLO signal peptide and this coding sequence is codon-optimized.) Efficient secretion of full length mesothelin (62 kDa) was achieved when the native coding sequence of Listeria LLO signal peptide was used (lane 7). , Codon-optimized Listeria LLO signal peptide, rather than FIG. 48B), was associated with codon-optimized mesothelin (8 lanes, FIG. 48B). In addition, the secretion of full-length mesothelin (62 kDa) was not associated with the codon-optimized Listeria p60 signal peptide codon-optimized mesothelin, but when the native coding sequence of the Listeria p60 signal peptide was used (lane 6, FIG. 48B). Was observed (lane 3, Fig. 48B). Finally, secretion of full-length mesothelin (62 kDa) was observed when codon-optimized signal peptides of bacterial species other than Listeria monocytogenes were operably linked to codon-optimized mesothelin (FIG. 48B). Signal peptides of Bacillus anthracis protective antigen (Ba PA) or signal peptides of Lactococcus lactis Usp45 protein (Ll Usp45) programmed the efficient secretion of full-length mesothelin (62 kDa) from recombinant Listeria strains (Figure 48B, lanes 4 and 5). . Bacillus subtilis phoD signal peptide (Bs phoD) also programmed the efficient secretion of full length mesothelin from Listeria (FIG. 48B, lane 9). Bands having a molecular weight of about 62,000 correspond to mesothelin and double band pairs probably correspond to undivided and split mesothelin polypeptides (ie, partial cleavage).

FIG. 48C shows expression / secretion of NY-ESO-1 from Listeria moocytogenes ΔactΔinlB with a construct comprising a sequence encoding an LLO signal peptide fused with a sequence encoding human NY-ESO-1, all of which are Listeria Codon-optimized to express in. Secreted NY-ESO-1 was detected using NY-ESO-1 monoclonal antibody.

In this example, signal peptides and tumor antigens were synthesized using the most preferred codons for each amino acid defined as frequency of occurrence per 1000 codons in the coding sequence of the Listeria genome (http://www.kazusa.or.jp/ codon / cgi-bin / showcodon.cgi? species = Listeria + monocytogenes + [gbbct]). Signal peptides associated with secAl, secA2 or twin-Arg translocation (Tat) secretion pathways from the Listeria and other Gram-positive bacteria genes have programmed efficient secretion of human tumor antigens from recombinant Listeria . Surprisingly, the signal peptides of the Listeria proteins LLO and p60 each contain rare codons (frequency <10 per 1000 codons), and optimization of this sequence was necessary for efficient secretion of mesothelin and NY-ESO-1 from recombinant Listeria . (Figure 48B). Mesothelin secretion is also associated with the secA1 signal peptide of B. anthracis protective antigen (pagA) and Lactococcus lactis Usp45 and the Tat signal peptide of phosphodiesterase / alkaline phosphatase D gene (PhoD) of B. subtilis . Was observed.

Signal peptides of different secretion pathways were used to determine if a particular pathway was favored for optimal secretion of heterologous proteins. For example, the Tat pathway is used for the secretion of folded proteins in bacteria and the B. subtilis phoD protein is secreted through this mechanism. It has been hypothesized that the secretion of tumor antigens containing important hydrophobic domains, such as native NY-ESO-1, can be facilitated by folding before transport. However, these results indicated that codon-optimization of both signal peptide and tumor antigen coding sequences, but not the secretory pathway, is the primary prerequisite for efficient secretion of mammalian proteins.

Importantly, the phenotype of recombinant vaccines using any route for tumor antigen secretion was not significantly affected compared to the parent Listeria Δact / ΔinlB strain. The median lethality (LD50) of Listeria Δact / ΔinlB is 1 × 10 ⁸ cfu in C57BL / 6 mice. Stable single copy site-specific incorporation of the tumor antigen expression cassette into harmless sites on the chromosome of Listeria Δact / ΔinlB was performed using a pPL2 integration vector. Listeria act △ / △ LD50 of tumor antigen encoding is inlB Listeria act △ / △ inlB it was within 5 times.

 Example 28 Construction of a Bicistronic hEphA2 Expression Vector.

 As a non-limiting example, the construction of an antigen expression cassette is provided in which the outer (EX2) and inner (CO; kinase inactive) domains of hEphA2 arise from bicistronic transmission codes. Secretion of the EX2 and CO domains is effected by the functional linkage of the EX2 and CO domains with the Ba PA and Bs PhoD signal peptides, respectively.

Codon-optimized human EphA2 kinase inactive plasmids known as phEphA2KD are used in the construction of bicistronic hEphA2 expression vectors. (EphA2 is a receptor tyrosine kinase but kinase activity is removed by mutations from K to M of the active site of the enzyme.) The coding sequence of phEphA2KD is shown in FIG. 49. The phEphA2KD sequence of FIG. 49 contains the codon-optimized coding sequence of hEphA2 with the penetrating membrane domain removed, and contains only 5 'and 3' Bam HI and Sac I restriction enzyme sites to facilitate construction of a functional antigen expression cassette. Include. The Mlu I recognition sequence is shown in bold in the sequence shown in FIG. 49.

Sub-fragments of human EphA2 (penetrating domain removed, kinase inactivation) between two Mlu I restriction enzyme recognition sequences (filling the gene synthesis methods known in the art such as oligonucleotide synthesis, PCR and / or Klenow fill) Synthesized). During synthesis, the actA-plcB intergenic site is correctly inserted at the junction between the EphA2 extracellular and intracellular domains, which are separated by the hydrophobic penetrating membrane domain of the original protein. The sequence of the Mlu I sub-fragment of codon-optimized human EphA2 containing the actA-plcB intergenic site is shown in FIG. 50 (intergenic sites are shown in bold). Additionally, codon-optimized Bs phoD signal peptide is placed at the 3 'end of the actA-plcB intergenic sequence and in-frame fused with the downstream EphA2 CO domain coding sequence.

The functional human EphA2 bicistronic cassette is assembled by substituting a Mlu I fragment comprising an actA-plcB intergenic site and a Bs phoD signal peptide instead of the corresponding site in the kinase inactive human EhpA2 sequence from which the transmembrane shown in FIG. 49 has been removed. This final sequence contains a unique Bam HI and Sac I restriction enzyme recognition site at its 5 'and 3' ends, respectively, to facilitate insertion and functional linkage to the hly promoter and the original signal peptide, eg Ba PA.

Thus, the seven ordered functional elements of the bicistronic human EphA2 antigen expression cassette are as follows: hly promoter-Ba PA signal peptide-EX domain EphA2-termination codon-actA-plcB intergenic site (shine-dalgano) Together with sequence) -Bs PhoD signal peptide-CO domain EphA2-termination codon. Preferably all EphA2 and signal peptide coding sequences are codon-optimized.

Recombinant Listeria strains expressing and secreting the EphA2 EX and CO domains can be derived by the methods described herein using pAM401, pKSV7 or pPL1 and pPL2 integration vectors. Expression and secretion of EphA2 protein are detected by Western analysis of certain bacterial fractions using the methods described herein and / or methods known to those skilled in the art.

Example 29 Expression and Secretion of Antigens from Recombinant Listeria Including Antigen-Bacterial Protein Chimeras.

In some embodiments of the invention, all of the sequences encoding the signal peptide and its heterologous protein fusion partner are codon-optimized. In some embodiments, it is desirable to place the codon-optimized heterologous protein sequence within defined regions of the protein whose native form is secreted from Listeria . The heterologous protein sequence is functionally placed within the defined sequence of the selected secretory Listeria protein sequence to synthesize and secrete protein chimeras corresponding to the combined molecular weight of the secreted protein.

The secretion of heterologous proteins can be facilitated by using a host Listeria bacterium device that is required for optimal secretion of its bacterial protein. Molecular chaperones promote the secretion of selected bacterial proteins.

As a non-limiting example, the protein chimera, mesothelin, between the L. monocytogenes protein p60 and the human tumor antigen was generated. The human tumor antigen, mesothelin, was generated by precise placement in the L. monocytogenes protein p60 at amino acid position 70 (although it can be appreciated that any desired heterologous protein coding sequence can be selected to generate a protein chimera). Protein chimeras contained optimal codons for expression in p60 amino acids 1-70 and Listeria of the entire mesothelin coding sequence. In addition, the p60-human mesothelin protein chimera is functionally linked to the L. monocytogenes hly promoter incorporated into the pPL2 vector, which then generates recombinant L. monocytogenes that express and secrete human mesothelin as described herein. Used to. The experimental method used to construct a recombinant Listeria that optimally expresses and secretes the p60-human mesothelin protein chimera is described below.

In some embodiments, an important feature of the protein chimera between the selected L. monocytogenes gene and the selected heterologous protein sequence is that protein chimeras as well as maintain optimal secretion of the protein chimeras through interaction of the secretory mechanism with L. monocytogenes expressing proteins with bacterial chaperones. In order to maintain the functional activity of L. monocytogenes, the heterologous protein sequence is functionally placed in the selected L. monocytogenes gene. In some embodiments, functionally placing the heterologous sequence within the L. monocytogenes secA dependent proteins NamA and p60 is desirable to retain the peptidoglycan cell wall hydrolase activity of the protein. (See, eg, Lenz et. Al. (2003 PNAS, 100: 12432-12437) for a description of the SecA2-dependent proteins NamA and p60 proteins). In some embodiments, it is desirable to functionally place the heterologous protein coding sequence between the signal sequence (SS) and the cell wall binding domain (LySM) and the catalytic domains Lyz-2 (NamA) and p60-dom (p60) [Lenz et al. . al. (2003)].

In some embodiments, expression of an antigen or heterologous protein is functionally linked to a prfA-dependent promoter. As such, expression of the heterologous protein is induced in the microformatting environment of recombinant Listeria infected cells.

The first step in the construction of the p60-mesothelin protein chimera involves the DNA synthesis of a prfA-dependent hly promoter functionally linked to the DNA sequence encoding the first 70 amino acids of p60 with a codon for optimal secretion in Listeria . (In some embodiments, codon usage can be further modified to avoid sites of excessive RNA secondary structure that can inhibit protein translation efficacy.) DNA sub-fragments corresponding to hly promoter-70 N-terminal p60 amino acids Was synthesized. (Alternatively this can be done by gene synthesis methods known in the art such as oligonucleotide synthesis, PCR and / or Klenow fill-in, etc.)

The first 70 amino acid sequence of L. monocytogenes , strain 10403S is shown below:

Those skilled in the art can understand that there are many laboratories and fields of isolation for L. monocytogenes encoding genes, including p60, which may include variability at both nucleotide sequence and amino acid levels but nevertheless are essentially the same genes and proteins. . One skilled in the art can also understand that protein chimeras can be constructed using the genes of any laboratory or field isolate (including food-producing or clinical strains) of L. monocytogenes .

The synthetic DNA sequence corresponding to hly promoter-70 N-terminal p60 amino acid is shown in FIG. 51. In addition, codons encoding p60 amino acid residues 69 (L) and 70 (Q) were modified to contain a unique Pst I enzyme recognition sequence to facilitate functional insertion of the heterologous sequence. In addition, the 5 'end of the synthesized sub-fragment contains a unique KpnI enzyme recognition sequence.

Sub-fragments digested with 447 bp KpnI and PstI were ligated to the corresponding KpnI and Pstl sites of the pPL2 vector, digested with KpnI and PstI enzymes and digested with bovine visceral alkaline phosphatase (CIAP). This plasmid is known as pPL2-hlyP-Np60 CodOp. The residue of the native p60 gene was then cloned between the pPL2-hlyP-Np60 CodOp plasmid, only Pst I and BamHI sites. Residues of the p60 gene were cloned by PCR using heat-resistant polymerase and proof-reading containing the following primer pairs:

Forward primer:

5'-CGC CTGCAGGTAAATAATGAGGTTGCTG (SEQ ID NO: 117)

Reverse primer:

5'-CGCGGATCCTTAATTATACGCGACCGAAG (SEQ ID NO: 118)

The 1241 bp amplicon was digested with PstI and BamHI and purified 1235 bp was digested with PstI and BamHI and ligated into a pPL2-hlyP-Np60 CodOp plasmid treated with CIAP. This plasmid contains the entire L. monocytogenes p60 gene with an optimal codon corresponding to amino acids 1-77 and a natural codon corresponding to amino acids 78-478 and is functionally linked to the L. monocytogenes hly promoter. This plasmid is known as pPL2-hlyP-Np60 CodOp (1-77) and the KpnI-BamHI sub-fragment containing hly p functionally linked with the p60 coding sequence is shown in FIG. 52. The expected sequence of the pPL2-hlyP-Np60 CodOp (1-77) plasmid was confirmed by sequencing.

The next step in construction is between the N-terminal signal sequence of p60 and the first LysM cell wall binding domain, so that the only PstI site of the pPL2-hlyP-Np60 CodOp (1-77) plasmid that maintains the normal biological function of the L. monocytogenes protein Functional insertion of the heterologous protein coding sequence of.

As a non-limiting example, codon-optimized human mesothelin was inserted into the only PstI site of the pPL2-hlyP-Np60 CodOp (1-77) plasmid for optimal expression in L. monocytogenes . Specifically, full-length mesothelin or signal peptide and mesothelin (mesothelin ΔSP / ΔGPI) from which the GPI linker domain has been removed are used for expression in L. monocytogenes using a thermostable polymerase with corrective activity and the following primer pairs. Cloning from the plasmid described in Example 27, containing full length human mesothelin containing the optimal codon:

1. Battlefield

Forward primer (huMeso 3F):

5'-AAACTGCAGGCATTGCCAACTGCACGTCC (SEQ ID NO: 119)

Reverse primer (hMeso 1935R):

5'-AAACTGCAGAGCTAATGTACTGGCTAATAATAATGCTAAC (SEQ ID NO: 120)

2. △ signal peptide, △ GPI fixation

Forward Primer (huMeso133F):

5'-CGCCTGCAGCGTACATTAGCAGGTGAAACAGG (SEQ ID NO: 121)

Reverse primer (huMeso 1770R):

5'-CGCCTGCAGGCCTTGTAAACCTAAACCTAATGTATC (SEQ ID NO: 122)

Purify PCR amplicons of 1932 bps (full length mesothelin) and 1637 bps (mesothelin ΔSP / ΔGPI), digest with PstI, and recombine to the only PstI site of plasmid pPL2-hlyP-Np60 CodOp (1-77) Gated, digested with PstI and treated with CIAP. The matched N-CO direction of p60 and mesothelin domain was confirmed by restriction map. This plasmid is known as pPL2-hlyP-Np60 CodOp (1-77) -mesothelin and pPL2-hlyP-Np60 CodOp (1-77)-mesothelin ΔSP / ΔGPI, and through the examples included herein The selected L. monocytogenes strains described (eg, ΔactAΔinlB double deletion mutations) were introduced.

The sequence of the KpnI-BamHI sub-fragment of plasmid pPL2-hlyP-Np60 CodOp (1-77) -mesothelin containing hly promoter functionally linked to the p60-human mesothelin ΔSP / ΔGPI protein chimeric coding gene is shown in FIG. 53. It is shown in.

KpnI-BamHI sub-fragment of plasmid pPL2-hlyP-Np60 CodOp (1-77) -mesothelin ΔSP / ΔGPI containing hly promoter functionally linked to the p60-human mesothelin ΔSP / ΔGPI protein chimeric coding gene The sequence of is shown in FIG.

Western analysis of expression and secretion of p60-mesothelin protein chimera:

As described throughout the examples, expression and secretion of selected heterologous antigens results in a potent ignition of MHC class I-restricted CD8 + T cell responses. TRNA-Arg chromosome insertion of pPL2-hlyP-Np60 CodOp (1-77) -mesothelin or pPL2-hlyP-Np60 CodOp (1-77) -mesothelin ΔSP / ΔGPI plasmids generated by the methods described herein Expression and secretion of the protein chimera into the medium by the recombinant L. monocytogenes ΔactAΔinlB double deletion mutants contained were carried out by the methods described in the Examples included herein using mesothelin-specific polyclonal antibodies. Test by Western analysis.

The indicated engineered deletions of hmesothelin (ΔSP / ΔGPI, also referred to as ΔSS / ΔGPI, ΔSP / ΔGPI, ΔSS / ΔGPI, etc.) for the proteins shown in some lanes are as follows: : The deleted signal sequence (ΔSP) corresponds to the N-terminal 34 amino acid of h mesothelin (see, eg, FIG. 34 or GenBank Accession No. BC009272 for the sequence of human mesothelin). The deleted GPI (ΔGPI) domain corresponds to the C-terminal 42 amino acids starting with amino acid residues GLy-Ile-Pro and ending with amino acid residues Thr-Leu-Ala (see, eg, FIG. 34).

This assay consisted of human mesothelin or p60 precisely inserted human mesothelin ΔSP / ΔGPI (inserted in frame of amino acid 70 of P60 between the N-terminal signal sequence and the first of the two LysM cell wall binding domains). Protein chimeras demonstrated that they are efficiently expressed and secreted from recombinant L. monocytogenes . See FIG. 55. (The Y axis in FIG. 55 represents the molecular weight (kDa) of the protein in the ladder running in the leftmost lane.) Specifically, lanes 1-4 in FIG. 55 contain human mesothelin or human mesothelin ΔSP / ΔGPI. One predicts the expression and secretion of protein chimeras. The efficiency of expression and secretion of human mesothelin ΔSP / ΔGPI compared to full-length mesothelin is evident in lanes 2 and 4. For the protein chimeras shown in lanes 3 and 4, native N-terminal p60 amino acids were used. In the chimeras running in lanes 1 and 2 of FIG. 55, nucleotides encoding amino acids T and V at positions 29 and 64 were removed, respectively. Lane 5 shows the expression and secretion of Bacillus anthracis PA signal peptide fused to human ΔSPΔGPI-mesothelin (where both signal peptide and mesothelin coding sequence are codon-optimized for expression in L. monocytogenes ), lanes 6 shows the expression and secretion of LLO fused to full-length human mesothelin (where both the signal peptide and the mesothelin coding sequence are codon-optimized for expression in L. monocytogenes ). Lane 8 shows protein expression by J293, human cell line, while lane 7 shows protein expressed and secreted by J293 containing a plasmid encoding full length human mesothelin (“J293 / full length”). Lane 10 shows expression and secretion from Listeria with endogenous p60 removed. The lower panel of FIG. 55 shows Western analysis of L. monocytogenes p60 secretion using polyclonal α-p60 antibody. This result indicates that an equal amount of Lm-secreted protein was loaded into the gel.

This result indicates that p60 can be used as a molecular chaperone to secrete heterologous proteins and to promote their presentation to the MHC class I pathway.

Example 30 Additional Examples of Antigen Expression and Secretion by Recombinant Listeria monocvtogenes .

A. Expression of the intracellular domain (ICD) of EphA2 from bicistronic constructs using non- Listeria signal peptides.

FIG. 56 shows Western blot analysis of expression and secretion of intracellular domains (ICD) of EphA2 from bicistronic transcoding using non- Listeria , non-secA1 signal sequences.

EphA2 is a protein comprising an extracellular domain (ECD) and an intracellular domain (ICD). Listeria ΔactAΔinlB was engineered to express bicistronic mRNA, which encoded the extracellular and intracellular domains of Epha2 as an isolated polypeptide. All of the sequences encoding the signal sequences used for constructs ( B. subtilis phoD signal peptide, B. anthracis protective antigen signal peptide and L. lactis Usp45 signal peptide) were codon-optimized for expression in L. monocytogenes . In addition, sequences encoding ECD and ICD domains were codon-optimized for expression in L. monocytogenes . The Listeria promoter hly of the LLO gene was used as a promoter of these constructs.

The expression cassette encoding the bicistronic mRNA was integrated into the Listeria genome using the integration vector pPL2. Western blot analysis of various bacterial fractions using standard techniques was used to detect and measure the accumulated intracellular EphA2 domain. The results demonstrated that the intracellular domain of EphA2 was expressed and secreted from bicistronic constructs using non- Listeria signal peptides encoded by codon-optimized sequences.

Expression constructs included: (1) a codon-optimized sequence and an EphA2 (codon-optimized sequence encoding a L. lactis Usp45 secretion sequence operably (functionally) linked to the coding sequence of the extracellular domain of EphA2 (the first polypeptide)); Codon-optimized sequence (lane 1) encoding a B. subtilis phoD secretion signal operably linked to the intracellular domain of the second polypeptide); And (2) a codon-optimized sequence encoding a B. anthracis protective antigen secretion sequence operably linked with the coding sequence of the extracellular domain of EphA2 (first polypeptide) and the coding of the intracellular domain of EphA2 (second polypeptide). Codon-optimized sequence encoding lane B. subtilis phoD secretion sequence operably linked to the sequence (lanes 2-3 (two different clones); see description of constructs of this expression cassette in Example 28 above). Control experiments with weakened parent Listeria ΔactAΔinlB strains (lane 4) showed a detectable change in cross-reactivity in some control blots. Lanes 1-3 represent the slow and fast shifting bands, which correspond to intracellular domains (ICDs). Expressed intracellular domains of EphA2 from both constructs (lanes 1-3) were observed in all three bacterial fractions. Lane 4 (control) shows only slow moving bands. Since no antibody to the extracellular domain was available, expression / secretion of the extracellular domain was not analyzed.

B. Plasmid-based expression and secretion of murine mesothelin as a function of N-terminal fusion with various codon-optimized signal peptides.

FIG. 57 shows plasmid basal expression and secretion of murine mesothelin expressed from codon-optimized mesothelin coding sequences using various signal peptides, including non- Listeria signal sequences and non-secA1 signal sequences. Plasmid based expression and secretion of murine mesothelin is shown as a function of N-terminal fusion with various signal peptides encoded by codon-optimized sequences. In all cases, not only the sequences encoding the signal peptides of the mesothelin fusion protein were codon-optimized but also the murine mesothelin coding sequences were codon-optimized for expression in L. monocytogenes . Expression and secretion of murine mesothelin from L. monocytogenes were measured. Listeria was loaded into the pAM401 plasmid and the plasmid encoded mesothelin. Various plasmid base constructs were tested, with signal sequences varying. Western blots were performed with proteins recovered from various fractions of secreted protein (A), cell wall (B) and cell lysate (C). Explaining each fraction, lanes 1-2 represent mouse mesothelin expressed by fusion with B. anthracis protective antigen signal sequence, lanes 3-4 represent mouse mesothelin expressed by fusion with Lactococcus lactis Usp45 signal sequence, Lanes 5-6 represent murine mesothelin expressed by fusion with the B. subtilis phoD signal sequence, lanes 7-8 represent murine mesothelin expressed by fusion with the p60 signal sequence, and lanes 9-10 represent the LLO signal sequence The mouse mesothelin expressed by fusion is shown, and lane 11 shows the protein expressed by the control host Listeria ΔactAΔinlB. The results demonstrated that the highest expression and secretion was observed where the signal sequence included the B. anthracis protective antigen signal sequence (lanes 1-2) and the B. subtilis phoD signal sequence (lanes 5-6).

C. Listeria monocytogenes chromosome-based expression and secretion of human mesothelin.

58 shows Western blot analysis of Listeria monocytogenes chromosome-based expression and secretion of human mesothelin in various bacterial cell fractions (ie secretory proteins, cell walls and lysates). Expression and secretion of human mesothelin were tested when fused to non- Listeria secA1 and non-secAl signal peptides. All Listeria bacteria tested were ΔactA / ΔinlB Listeria and were as follows: Listeria ΔactA / ΔinlB (control Listeria not manipulated to express mesothelin) (lane 1); Listeria (lanes 2-3) encoding the B. anthracis protective antigen signal sequence fused to ΔSS / ΔGPIhmesothelin; Listeria (lanes 4-5) encoding the B. subtilis phoD signal sequence fused to ΔSS / ΔGPIhmesothelin; Listeria (lanes 6-7) encoding the B. arthracis protective antigen signal sequence fused with full length hmesothelin; Listeria (lanes 8-9) encoding the B. subtilis phoD signal sequence fused to full length hmesothelin (lanes 8-9).

Sequences encoding signal sequences fused to mesothelin of all of the Listeria were codon-optimized for expression in L. monocytogenes . In addition, mesothelin coding sequences (ΔSS / ΔGPI and full length) were codon-optimized for expression in L. monocytogenes of each of the constructs. In each of the above Listeria expressing mesothelins, a mesothelin expression cassette was inserted into the Listeria chromosome via integration with pPL2.

When human mesothelin was engineered to remove its signal sequence and remove the hydrophobic site (gpi site), the highest expression of the B. subtilis phoD secretion sequence occurred (lanes 4-5).

Example 31 Additional Example of Immunogenicity and Antitumor Efficacy of Recombinant Listeria monocytogenes Vaccine.

The following examples show, for example, vaccine-dependent stimulation of cytokine expression, vaccine-dependent survival of animals with tumors, vaccine-dependent reduction of tumor metastasis and vaccine-dependent tumor volume as a result of vaccination with Listeria of the present invention. Initiate reduction.

A. Immunogenicity of Listeria including P-60-model antigen chimera.

59A and B show delivery of heterologous antigens to the MHC class I pathway by Listeria expressing p60-antigen chimera or LLO signal peptide antigen fusion protein. The heterologous antigen used in this experiment is AH1-A5. Vaccination N- terminal p60 p60 polypeptide signal sequence inserted into the p60 protein chimeric expression cassette Listeria engineered to contain ( "p60- based composition" encoding (SL8 OVA fused to peptide) AH1-A5, including the peptide sequence ) Or Listeria (“LLO-based constructs”) engineered to encode LLO signal peptides linked to nucleic acids encoding AHI-A5 within the same antigen, OVA. All of these constructs used used Listeria promoter hly. p60 is the peptidoglycan autolysate of Listeria secreted by the secA pathway, while LLO is Listeriolicin .

To make the p60-based construct, the nucleic acid encoding p60 was engineered to include a PstI cloning site, which had no change in the silent mutation, ie the amino acid sequence encoded. The PstI site is located between the N-terminal signal sequence and the first of two LysM cell wall binding domains of the p60 sequence. Polynucleotides encoding heterologous polypeptides, including AH1-A5 epitopes (SPSYAYHQF (SEQ ID NO: 73)) and SL8 epitopes (SIINFEKL (SEQ ID NO: 123)), were inserted into the PstI cloning site in frame. The coding sequence of this epitope was isolated with unique XhoI and codon-optimized for expression in L. monocytogenes . Insertion into the PstI site occurred with an equivalent amount of p99 nucleotide bases of 199. The first 1-70 amino acids of the p60 coding sequence were codon-optimized for expression in L. monocytogenes . Thus, the first 27 amino acids corresponding to the signal peptide were expressed from optimal codons for expression in L. monocytogenes . The antigen expression cassette further included unique 5 'and 3' KpnI and SacI sites for site-specific integration adjacent to the tRNA ^Arg gene of the L. monocytogenes genome for insertion into the MCS of the pPL2 plasmid, respectively. LLO-based constructs included sequences encoding LLO signal sequences operably linked to nucleic acids encoding AH1-A5 in OVA) without the use of any codon optimization. Thus, in this study, the signal peptide was from Listeria LLO or Listeria p60.

The composition genomwa Listeria site-specific recombination vector mediating, disposed pPL2 was inserted into the Listeria genome.

59A and B show the immune response to vaccination (tail vein) of Listeria expressing AH1-A5 antigen with p60 signal sequence / autolysis as p60 chimera, and Listeria expressing AH1-A5 antigen linked to LLO signal sequence. Represents an immune response to vaccination. "Unstim" in the x-axis of the figure means that no peptide was added to the wells (ie cells were not stimulated), "AH1" means that AH1 nonapeptide was added to the wells, and "AH1-A5 "Means that AH1-A5 nonapeptide was added to the wells. All bacterial vaccines were engineered to contain integrated nucleic acids encoding AH1-A5 (bacterial vaccines did not encode AH1) (see, eg, Slansky et al. (2000) Immunity 13: 529-538). When vaccinated with Listeria containing p60-based constructs, this strain is indicated as "p60" on the x-axis of the figure. When vaccinated with Listeria containing LLO-based constructs, this strain is indicated as "LLO" on the x-axis of the figure.

The overall protocol for vaccination with Listeria expressing a p60-based construct was as follows: (1) Mice were vaccinated (tail vein (intravenous)) with Listeria containing the integrated nucleic acid, wherein the integrated nucleic acid was p60. P60d containing a nucleic acid encoding AH1-A5 inserted at nucleotide 199 of was encoded. In other words, the nucleic acid encoding the AH1-A5 antigen was in frame and operably linked with the p60 signal sequence and p60 autolysis. Nucleic acids encoding AH1-A5 have been codon-optimized for expression in L. monocytogenes ; (2) spleens were removed 7 days after infection; (3) Splenocytes were isolated and placed in the wells, and then splenocytes were not added with peptides as shown on the x-axis (FIGS. 59A and 59B) and AH1 was added (FIG. 59A), or AH1-A5 was added. Incubated by addition (FIG. 59B); (4) Cells were incubated for 5 hours after peptide addition, and then the percentage of IFN gamma expressing CD8 + T cells was assessed by FACS analysis. Similar protocols were used for vaccination with Listeria expressing LLO-based constructs.

The results indicate that Listeria vaccine stimulated CD8 + T cell expression of IFN gamma when the added peptide was AH1 (FIG. 59A) or when the added peptide was AH1-A5 (FIG. 59B). The stimulation was somewhat higher when the integrated AH1-A5 was operably linked to the LLO signal sequence and the stimulation was somewhat lower when the integrated AH1-A5 was operably linked to the p60 signal sequence (FIGS. 59A and B).

60A and B show experiments performed with the same two Listeria vaccines described above, ie, shown in FIGS. 59A and B. Figure 60A shows a case where the mouse is p60--based composition ( "p60") with Listeria engineered to contain, or LLO--based composition to prevent Listeria engineered to contain ( "LLO") inoculation. As indicated on the x-axis of FIG. 60A, cell based analytes may be devoid of peptide (not stimulated; "unstim"), or LLO91-99 peptide ("LLO91"; Badovinac and Harty (2000) J. Immunol. 164 (6444-6452). The results showed a similar immune response (IFNgamma expression) when the Listeria vaccine contained either p60-based or LLO-based constructs. As reflected in the results of the cell-based assay, the stimulated immune response of FIG. 60A is due to the Listeria endogenous expression of the native LLO.

Figure 60B shows a Listeria engineered mouse to contain a p60-based construct operably linked to a hly promoter and signal peptide sequence encoding a nucleic acid encoding AH1-A5, or a nucleic acid wherein a hly promoter and signal peptide encode AH1-A5. Results are shown when vaccinated with Listeria engineered to contain LLO-based constructs operably linked to. The added peptides may be free of peptide (not stimulated; "unstim") or p60 _217-225 ("p60 ₂₁₇ "; Sijts et al. (1997) J. Biol. Chem. 272: 19261), as indicated on the x-axis. -19268). As reflected in the results of the cell based assay, the stimulated immune response in FIG. 60B is due to the combination of Listeria expression of endogenous p60 against LLO-based constructs and the combination of expressed p60 protein chimeric sequences for endogenous p60 and p60-based constructs.

B. Therapeutic Efficacy of Listeria Expressing Human Mesothelin

The results shown in FIG. 61 reveal that vaccination with Listeria expressing human mesothelin prolongs survival in mice with tumors, in which tumor cells in the mouse are engineered to express human mesothelin. Tumor cells were CT26 cells expressing human mesothelin and mice were Balb / c mice (all CT26 tumor studies described herein included Balb / c mice). In one of the expression cassettes, the sequence encoding the non- Listeria signal sequence is operably linked in frame with the coded-optimized sequence encoding human mesothelin, which lacks its signal sequence and GPI anchor. In studying the effect of the fusion protein on the immune response to tumors, an expression cassette encoding a signal peptide fused with human mesothelin (ΔGPIΔSS) was administered to mice bearing tumors with Listeria vaccine. The expression cassette encoding the mesothelin fusion protein is integrated into the Listeria chromosome. On day 0 Balb / c mice were injected intravenously with CT26 cells 2 × 10 ⁵ expressing human mesothelin (CT.26 huMeso +). On day 3, 1e7 colony forming unit (CFU) Listeria was inoculated (intravenously).

FIG. 61 shows the percent survival of mice (indicated on the y-axis) for CT26 tumors expressing human mesothelin, wherein the vaccine is Hank's Balanced Salt Solution (HBSS) (mock vaccine; “HBSS”); Listeria ΔactAΔinlB (positive control vaccine; “SF-AH1A5”) expressing SF-AH1A5 from the integrated expression cassette; Or Listeria ΔactAΔinlB comprising an expression cassette encoding a B. anthracis protective antigen signal sequence (coded by a non-coded optimized sequence) fused with huMesothelin (coded by a codon-optimized sequence), huMesothelin Had a deleted signal sequence and a deleted region encoding a hydrophobic gpi-anchor peptide (“BaPA-huMeso ΔgpiΔss”). Listeria with SF-AH1A5 constructs and BaPA-huMeso ΔgpiΔss constructs contained these constructs as chromosomalally integrated constructs. Nucleic acid molecules encoding SF-AH1A5 and nucleic acid molecules encoding BaPA-huMeso ΔgpiΔss constructs are integrated into the Listeria genome using pPL2. SF is an abbreviation of eight amino acid peptides derived from ovalbumin, which is also known as SL8 (see, eg, Shastri and Ganzalez (1993) J. Immunol. 150: 2724-2736). The abbreviations "SF-AH1A5", "SF-AH1-A5" and "OVA / AH1-A5" refer to AH1-A5 linked to an ovalbumin scaffold. "SF AH1-A5" refers to an SF peptide fused at the N-terminus of 386 at amino acids 138 of AH1-A5 (SPSYAYHQF (SEQ ID NO: 73)) and GenBank Accession No. P01012 (Orbalbumin). Polynucleotides encoding “SF-AH1A5” in this example included codon-optimized nucleic acids encoding AH1-A5 and non-codon-optimized nucleic acids encoding ovalbumin-derived sequences.

A single immunization with Listeria expressing huMesothelin shows the results sikindaneun prolong the survival of mice containing huMesothelin expressing tumor. Percent survival was highest in the chromosome integrated B. anthracis protective antigen signal sequence fused with Δ signal sequence / Δgpi huMesothelin (BaPA-huMeso ΔgpiΔss; closed square). Survival was lowest when “vaccinated” using a control salt solution.

C. Reduction of lung tumor nodule levels in mice with tumors vaccinated with List-eria expressing human mesothelin due to mesothelin-specific anti-tumor efficacy

The data in FIG. 62 show that the level of lung tumor nodules is reduced by vaccination with Listeria ΔactAΔinlB expressing human mesothelin , wherein the tumor cells were engineered to express human mesothelin . The mouse species was Balb / c and lung tumor cells were CT26 cells hiding vectors expressing human mesothelin. On day 0, 2x10 ⁵ CT26 cells expressing human mesothelin were administered intravenously to Bal / c mice. Sequences encoding the various signal sequences were operably linked within the frame with codon-optimized sequences encoding human mesothelin in the expression cassette. An expression cassette encoding various signal peptides fused with human mesothelin was administered to the tumor bearing mice via a Listeria vaccine comprising this expression cassette. On day 3, 1 × 10 ⁷ CFU / 100 μl of Listeria vaccine was administered intravenously to mice with tumors. Negative control immunizations were done with HBSS or Listeria ΔactAΔinlB . Positive control vaccinations used Listeria expressing OVA fusion proteins comprising AH1 A5 (along with OVA sequences in frame) (OVA fusion proteins comprising AH1 A5 were encoded by non-codon optimized expression cassettes). Became). On day 19 mice were killed and lungs collected to count lung tumor nodules.

Listeria vaccine reduced the number of metastases in the lungs. In the control vaccine containing only HBBS or Listeria ΔactAΔinlB , consistent 250 metastases and an average of 135 metastases were detected per lung, respectively. Listeria (“pAM-LLO-HuMeso”) with a plasmid (pAM401) encoding an LLO signal peptide fused with human mesothelin showed about 25 metastases per lung. The polynucleotide sequence of the pAM-LLO-HuMeso plasmid encoding the LLO signal peptide and human mesothelin sequence was codon-optimized for expression in L. monocytogenes . Listeria (“BaPA-huMeso ΔgpiΔss”) with integrated sequences encoding B. anthracis protective antigen signal sequence (BaPA) (ΔgpiΔ signal sequence) fused with huMesothelin also exhibited an average of about 25 metastases per lung. The polynucleotides in BaPA-huMeso ΔgpiΔss encoding the B. anthracis protective antigen signal sequence were not codon-optimized, whereas the polynucleotides encoding the deleted human mesothelin sequence of the mesothelin signal peptide and the GPI anchor were from L. monocytogenes . Codon-optimized for expression.

FIG. 63 shows the results of a control study using mice containing lung tumor nodules developed using CT26 parental target cells. Balb / c mice were used, but wild-type CT26 was injected instead (2x10 ⁵ cells on day 0 (intravenous). This study showed that the anti-tumor efficacy of vaccination with Listeria vaccine expressing the mesothelin fusion protein was mesothelin specific. The sequences encoding the various signal sequences were linked to codon-optimized sequences encoding human mesothelin in the expression cassette in frame (the constructs used in this experiment were described above to make the data shown in FIG. 62). Expression cassettes encoding the various signal peptides fused with human mesothelin were administered to the tumor-bearing mice via the Listeria vaccine comprising the expression cassette. (1x10 ⁷ CFU / 100㎕, intravenously on day 3). the tumor cells in a particular study did not express the human endothelin method. survive the side Made. If you have access to the data was also measured the number of lung metastasis. There were each vaccinated group of 5 mice in the voice verification standard vaccination is included HBSS or Listeria ΔactAΔinlB. Positive control standard vaccination is the AH1A5 Listeria (not codon-optimized) expressing the containing OVA fusion protein was included.

This result is shown in FIG. The cross indicates failure to survive and each vaccination group contained 5 mice. Mice survived the positive control inoculation and the average number of metastases detected in the lungs was about 25 per lung. Since tumor cells were not engineered to express human mesothelin, mice inoculated with Listeria ("pAM-LLO-HuMeso") hiding plasmids expressing LLO signal peptide fused with human mesothelin did not survive. Listeria (“BaPA-huMeso ΔgpiΔss”) having a B. anthracis protective antigen secretion sequence (BaPA; encoded by a non-codon optimized nucleotide sequence) (ΔgpiΔ signal sequence) in which the mouse is chromosomalally integrated fused with human mesothelin ), Some survived but others failed.

D. Vaccination with Listeria expressing codon-optimized human mesothelin reduces tumor volume.

64 shows that vaccination with Listeria ( ΔactAΔinlB ) expressing human mesothelin from an expression cassette comprising codon-optimized mesothelin codon sequences reduced tumor volume.

Sequences encoding the various signal sequences were operably linked to codon-optimized sequences encoding human mesothelin in the expression cassette in frame. Expression cassettes encoding the various signal peptides fused with human mesothelin were submitted to tumor-bearing mice via a Listeria vaccine comprising this expression cassette. Listeria vaccines expressing human mesothelin used for the immunization of tumor-bearing mice in this study included: LLO signal peptide fused with human mesothelin (codon-optimized sequence for expression in L. monocytogenes) Listeria ( ΔactAΔinlB L. monocytogenes ) (“pAM opt. LLO-opt.huMeso”) with a pAM401 plasmid that expresses and secretes ( encoded by ); Listeria ("pAM non-opt.BaPA-opt.huMeso") with a pAM401 plasmid expressing the B. anthracis protective antigen signal sequence (coded by a non-codon optimized expression cassette) fused with huMesothelin; And the B. anthracis Protective Antigen signal peptide fused with huMesothelin - (non-coded by the codon optimized sequence) as containing the integrated expression cassette encoding Listeria, wherein the deletion is huMesothelin signal sequence and a hydrophobic anchor peptide gpi- Having a deleted region of "Non-opt.BaPA-opt.huMesodelgpi-ss".

In this study, CT26 murine colon tumor cells 2x10 ⁵ cells engineered to express human mesothelin in Balb / c mice were implanted subcutaneously (day 0). Three days after injection of CT26 cells, mice were vaccinated intravenously with a Listeria vaccine of non- Listeria control or 1 × 10 ⁷ colony forming units (CFU). Negative control inoculations included HBSS. Positive control inoculum included Listeria (codon optimized) expressing SF-AH1A5 (SF is an abbreviated 8 amino acid peptide derived from ovalbumin, also known as SL8 (eg Shastri and Ganzalez (1993) J. Immunol. 150: 2724 -2736) Average tumor volumes were measured at various time points.

The results of this study are shown in FIG. 64. This result indicated that vaccination with Listeria expressing human mesothelin fused to various signal peptides reduced tumor volume. Vaccination with Listeria expressing B. anthracis protective antigen signal peptide fused with human mesothelin was protective (open circle with dashed line). Vaccination with Listeria expressing plasmid-encoding human mesothelin fused with LLO signal peptide was protective (open triangle). Immunization with Listeria containing a chromosomal integrated expression cassette encoding B. anthracis protective antigen (non-codon optimized nucleic acid) signal peptide (ΔgpiΔ signal sequence) fused with human mesothelin was also protective ( Open oval with solid line). With respect to the positive control, Listeria expressing chromosome integrated SF-AH1A5 was also protective (open square). The highest tumor volume, and the earliest time of tumor growth initiation, occurred in mice receiving mock vaccine (HBSS).

E. Immunogenicity of Listeria Vaccines Expressing Human Mesothelin Fused to Non- Listeria Signal Sequences

FIG. 65 shows immunogenicity of the Listeria ΔactA / ΔinlB- hMesothelin strain, where Listeria contained chromosomalally integrated nucleic acids encoding hMesothelin fused to Bacillus anthracis signal peptide (optimized Ba PA hMeso ΔGPIΔSS). Immune responses were assessed using ELISPOT assays sensitive to the expression of interferon-gamma.

This study included the following steps: (1) B. anthracis protective antigen signal peptide (non-) fused with huMesothelin (coded by a codon-optimized sequence that lacks a mesothelin signal sequence and a hydrophobic gpi-anchor sequence) Mice (Balb / c mice or C57BL / 6 mice) were vaccinated with Listeria containing an integrated expression cassette encoding the codon optimization sequence; (2) the spleen was removed after 7 days; (3) Cells removed from the spleen were dispersed in wells. Each well had about 200,000 splenocytes; (4) One of three kinds of medium was added to the well as shown. Splenocytes in the study with Balb / c mice received medium alone ("Unstimulated"), mesothelin peptide pool ("Meso pool"), or p60 _217-225 ("p60 ₂₁₇ "). Splenocytes in the study with C57BL / 6 received medium alone ("Unstimulated"), mesothelin peptide pool ("Meso pool") or LLO _296-304 ("LLO _296-304 "); (5) ELISPOT analysis was performed to determine the number of immune cells in response to the added peptide (s). The mesothelin peptide pool included 153 different peptides, which span the entire sequence of hMesothelin, each peptide is 15 amino acids long and overlaps adjacent peptides by 11 amino acids.

The result of ELISPOT is shown in FIG. This result indicated that Listeria vaccines expressing human mesothelin fused to B. anthracis signal peptide could induce an immune response against mesothelin in Balb / c mice. Higher IFN-gamma responses to Listeria -expressing hMesothelin were observed in Balb / c mouse immune system than C57BL / 6 immune system. The ELISPOT signal for p60 or LLO is a response to naturally occurring p60 and LLO proteins of Listeria .

All publications, patents, patent applications, Internet sites, and accession number / database sequences (including both polynucleotide and polypeptide sequences) cited herein may be incorporated in each publication, patent, patent application, Internet site, or accession number / database. The entirety of which is hereby incorporated by reference for all purposes to the same extent as the sequences are specifically and individually shown.

Claims (88)

(a) substituted with a first polynucleotide sequence, the codons for expression in Listeria monocytogenes bacteria that at least one of natural coding sequence, a codon of the polynucleotide more frequently used in Listeria monocytogenes encoding a natural signal peptide of the Listeria monocytogenes bacteria A codon-optimized first polynucleotide sequence; And (b) a second polynucleotide sequence encoding a polypeptide heterologous to the signal peptide, the second polynucleotide sequence being in the same translation reading frame as the first polynucleotide sequence A recombinant nucleic acid molecule comprising a recombinant nucleic acid molecule encoding a fusion protein comprising a signal peptide and a polypeptide. The method of claim 1, wherein the signal peptide is a recombinant nucleic acid molecule characterized in that the p60 signal peptide from the LLO signal peptide, or Listeria monocytogenes from Listeria monocytogenes. An expression cassette comprising the recombinant nucleic acid molecule of claim 1, further comprising a promoter operably linked to the first polynucleotide sequence and the second polynucleotide sequence of the recombinant nucleic acid molecule. A recombinant Listeria monocytogenes bacterium comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule is (a) substituted with a first polynucleotide sequence, the codons for expression in Listeria monocytogenes bacteria that at least one of natural coding sequence, a codon of the polynucleotide more frequently used in Listeria monocytogenes encoding a natural signal peptide of the Listeria monocytogenes bacteria A codon-optimized first polynucleotide sequence; And (b) a second polynucleotide sequence encoding a polypeptide heterologous to the signal peptide, the second polynucleotide sequence being in the same translation reading frame as the first polynucleotide sequence A recombinant Listeria monocytogenes bacterium comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule encodes a fusion protein comprising a signal peptide and a polypeptide. The method of claim 4, comprising an expression cassette comprising the recombinant nucleic acid molecule, wherein the expression cassette further comprises a promoter operably linked to both the first polynucleotide sequence and the second polynucleotide sequence of the recombinant nucleic acid molecule. Recombinant Listeria monocytogenes bacteria. 5. The antigen of claim 4, wherein the polypeptide encoded by the second polynucleotide sequence comprises an antigen selected from the group consisting of a tumor-associated antigen, a polypeptide derived from a tumor-associated antigen, an infectious disease antigen and a polypeptide derived from an infectious disease antigen. Recombinant Listeria monocytogenes bacteria, characterized in that it comprises. 7. The polypeptide of claim 6 wherein the polypeptide encoded by the second polynucleotide sequence is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, Or comprises an antigen selected from the group consisting of survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP and CEA, or K-Ras, H-Ras, N- Ras, 12-K-Ras, Mesothelin, PSCA, NY-ESO-1, WT-1, Survivin, gp100, PAP, Proteinase 3, SPAS-1, SP-17, PAGE-4, TARP and A recombinant Listeria monocytogenes bacterium comprising a polypeptide derived from an antigen selected from the group consisting of CEA. The polypeptide of claim 7 wherein the polypeptide encoded by the second polynucleotide sequence comprises mesothelin or an antigenic fragment or antigenic variant thereof, or comprises NY-ESO-1 or an antigenic fragment or antigenic variant thereof. Recombinant Listeria monocytogenes bacteria, characterized in that it comprises. 9. The recombinant Listeria monocytogenes bacterium of claim 8, wherein the polypeptide encoded by the second polynucleotide sequence comprises human mesothelin that has deleted the signal peptide and the GPI linker domain. The recombinant Listeria monocytogenes bacterium of claim 4, wherein the polypeptide encoded by the second polynucleotide sequence is heterologous to the signal peptide. The method of claim 4, wherein the signal peptide is a recombinant Listeria monocytogenes bacteria, characterized in that the signal peptide is selected from the group consisting of LLO signal peptide and p60 signal peptide from Listeria monocytogenes from Listeria monocytogenes. 5. The recombinant Listeria monocytogenes bacterium according to claim 4, which is weakened against intercellular propagation, penetration into non-phagocytic cells, or proliferation. 5. The recombinant Listeria monocytogenes bacterium of claim 4, wherein the recombinant Listeria monocytogenes bacterium is defective in relation to ActA, Internalin B, or both ActA and Internalin B. The recombinant Listeria monocytogenes bacterium of claim 4, wherein the nucleic acid of the recombinant bacterium is modified by reaction with a nucleic acid targeting compound. (a) substituted with a first polynucleotide sequence, the codons for expression in Listeria monocytogenes bacteria that at least one of natural coding sequence, a codon of the polynucleotide more frequently used in Listeria monocytogenes encoding a natural signal peptide of the Listeria monocytogenes bacteria A codon-optimized first polynucleotide sequence; And (b) a second polynucleotide sequence encoding a polypeptide heterologous to the signal peptide, the second polynucleotide sequence being in the same translation reading frame as the first polynucleotide sequence And optimized, as the recombinant nucleic acid molecule comprising a second polynucleotide sequence is Listeria monocytogenes is one or more of the natural coding sequence of the polynucleotides for expression in a bacterial codon a codon that replaced with codons that are more frequently used in Listeria monocytogenes The recombinant nucleic acid molecule is characterized by encoding a fusion protein comprising a signal peptide and a polypeptide. 16. The recombinant nucleic acid molecule of claim 15, wherein the signal peptide is a secA2 signal peptide. 16. The recombinant nucleic acid molecule of claim 15, wherein the signal peptide is a p60 signal peptide from Listeria monocytogenes . 16. The antigen of claim 15, wherein the polypeptide encoded by the second polynucleotide sequence comprises an antigen selected from the group consisting of a tumor-associated antigen, a polypeptide derived from a tumor-associated antigen, an infectious disease antigen and a polypeptide derived from an infectious disease antigen. Recombinant nucleic acid molecule, characterized in that it comprises. An expression cassette comprising the recombinant nucleic acid molecule of claim 15 and further comprising a promoter operably linked to the first polynucleotide sequence and the second polynucleotide sequence of the recombinant nucleic acid molecule. A recombinant Listeria monocytogenes bacterium comprising the recombinant nucleic acid molecule of claim 15. A recombinant Listeria monocytogenes bacterium comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule is (a) substituted with a codon that is a first polynucleotide sequence encoding a natural signal peptide of the Listeria monocytogenes, wherein the Listeria monocytogenes at least one of natural coding sequence of the polynucleotide codons for expression in the more frequently used in Listeria monocytogenes A codon-optimized first polynucleotide sequence; And (b) a second polynucleotide sequence encoding a polypeptide that is heterologous to the signal peptide, exogenous to the bacteria, or both, wherein the second polynucleotide sequence is in the same translation reading frame as the first polynucleotide sequence A recombinant Listeria monocytogenes bacterium comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule encodes a fusion protein comprising a signal peptide and a polypeptide. 22. The method of claim 21, comprising an expression cassette comprising the recombinant nucleic acid molecule, wherein the expression cassette further comprises a promoter operably linked to both the first polynucleotide sequence and the second polynucleotide sequence of the recombinant nucleic acid molecule. Recombinant Listeria monocytogenes bacteria. The method of claim 21, wherein the first polynucleotide sequence and the second polynucleotide sequence are such that one or more codons of the natural coding sequence of the polynucleotide are replaced with codons used more frequently in Listeria monocytogenes for expression in Listeria monocytogenes bacteria. Recombinant Listeria monocytogenes bacteria characterized by codon-optimized. The recombinant Listeria monocytogenes bacterium of claim 21, wherein the signal peptide is a secA2 signal peptide. The method of claim 24, wherein the recombinant nucleic acid molecule further comprises (c) a third polynucleotide sequence encoding secA2 autolysis or fragment thereof that is within the same translational reading frame as the first polynucleotide sequence and the second polynucleotide sequence; , wherein the second polynucleotide sequence is located within the third polynucleotide sequence, or polynucleotide sequences or the first and the third polynucleotide of recombinant Listeria monocytogenes bacteria, characterized in that located between the sequences. 23. The antigen of claim 21, wherein the polypeptide encoded by the second polynucleotide sequence comprises an antigen selected from the group consisting of a tumor-associated antigen, a polypeptide derived from a tumor-associated antigen, an infectious disease antigen and a polypeptide derived from an infectious disease antigen. Recombinant Listeria monocytogenes bacteria, characterized in that it comprises. The polypeptide of claim 26, wherein the polypeptide encoded by the second polynucleotide sequence is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, Or comprises an antigen selected from the group consisting of survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP and CEA, or K-Ras, H-Ras, N- Ras, 12-K-Ras, Mesothelin, PSCA, NY-ESO-1, WT-1, Survivin, gp100, PAP, Proteinase 3, SPAS-1, SP-17, PAGE-4, TARP and A recombinant Listeria monocytogenes bacterium comprising a polypeptide derived from an antigen selected from the group consisting of CEA. The recombinant Listeria monocytogenes bacterium of claim 27, wherein the polypeptide encoded by the second polynucleotide sequence comprises mesothelin or an antigenic fragment or antigenic variant thereof. 29. The recombinant Listeria monocytogenes bacterium of claim 28, wherein the polypeptide encoded by the second polynucleotide sequence comprises a human mesothelin lacking a signal peptide and a GPI anchor. 22. The recombinant Listeria monocytogenes bacterium of claim 21, wherein the recombinant Listeria monocytogenes bacterium is weakened for intercellular propagation, penetration into non-phagocytic cells, or proliferation. 22. The recombinant Listeria monocytogenes bacterium of claim 21, wherein the recombinant Listeria monocytogenes bacterium is defective in relation to ActA, Internalin B, or both ActA and Internalin B. The recombinant Listeria monocytogenes bacterium of claim 21, wherein the nucleic acid of the recombinant bacterium is modified by reaction with a nucleic acid targeting compound. (a) substituted with a first polynucleotide sequence, the codons for expression in Listeria monocytogenes bacteria that at least one of natural coding sequence, a codon of the polynucleotide more frequently used in Listeria monocytogenes encoding a natural signal peptide of the Listeria monocytogenes bacteria A codon-optimized first polynucleotide sequence; (b) a second polynucleotide sequence encoding a secreted protein or fragment thereof heterologous to the signal peptide, the second polynucleotide sequence being in the same translation reading frame as the first polynucleotide sequence; And (c) a third polynucleotide sequence encoding a polypeptide heterologous to the secreted protein or fragment thereof, the third polynucleotide sequence being in the same translation reading frame as the first and second polynucleotide sequences As a recombinant nucleic acid molecule comprising The recombinant nucleic acid molecule encodes a protein chimera comprising a signal peptide, a polypeptide encoded by a third polynucleotide sequence, and a secreted protein or fragment thereof, The polypeptide encoded by the third polynucleotide sequence is fused with a protein or fragment thereof secreted from the protein chimera, or is located in a secreted protein or fragment thereof. A recombinant Listeria monocytogenes bacterium comprising the recombinant nucleic acid molecule of claim 33. 7. The recombinant Listeria monocytogenes bacterium of claim 6, wherein the infectious disease antigen is derived from a virus selected from the group consisting of hepatitis virus, influenza virus and papilloma virus. 36. The recombinant Listeria monocytogenes bacterium of claim 35, wherein the infectious disease antigen is derived from hepatitis A virus, hepatitis B virus and hepatitis C virus. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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