Source: https://patents.google.com/patent/US6602506?oq=6%2C370%2C566
Timestamp: 2018-03-21 23:02:19
Document Index: 75157959

Matched Legal Cases: ['ART-1', 'ART-1', 'ART-1', 'ART-1', 'ART-1', 'ART-1', 'ART-1', 'ART-1']

US6602506B1 - Delivery of proteins into eukaryotic cells with recombinant Yersinia - Google Patents
Delivery of proteins into eukaryotic cells with recombinant Yersinia Download PDF
US6602506B1
US6602506B1 US09318141 US31814199A US6602506B1 US 6602506 B1 US6602506 B1 US 6602506B1 US 09318141 US09318141 US 09318141 US 31814199 A US31814199 A US 31814199A US 6602506 B1 US6602506 B1 US 6602506B1
US09318141
Pierre B. van der Bruggen
Anne M. Boland
Thierry R. Boon-Falleur
The present application is a divisional of Ser. No. 09/036,582 filed on Mar. 6, 1998, now U.S. Pat. No. 5,965,381.
Bacteria of the genus Yersinia cause diseases in humans and rodents ranging from enteritis and lymphadenitis to plague. The genus Yersinia encompasses three species: Yersinia enterocolitica, which is the most prevalent Yersinia species in humans and causes a broad range of gastro-intestinal syndromes; Yersinia pseudotuberculosis, which causes adenitis and septicaemia; and Yersinia pestis, which is the causative agent of plague.
Genetic studies revealed that the virulence of Yersinia is determined by a 70 kb plasmid (pYV), which encodes and governs the production of a set of proteins called Yops (for Yersinia outer proteins). These Yops form an integrated anti-host system that allows the extracellular adhesion of Yersinia to the surface of host cells and the subsequent injection of a set of toxic effector proteins into the host cell's cytosol. Recent studies further revealed that such an anti-host system, also called “Yersinia virulon”, is composed of the following four elements: (i) a contact or type III secretion system called Ysc, which is devoted to the secretion of Yop proteins out of the bacterial cells; (ii) a set of “translocators” for translocating the effector proteins into the eukaryotic host cells, which consist of YopB, YopD and possible other proteins such as LcrV; (iii) a control element and recognition system (YopN and LcrG); and (iv) a set of “effector proteins” including YopE, YopH, YopO/YpkA, YopM and YopP/YopJ, which are injected (or translocated) into the eukaryotic host cells and disrupt the functions of such host cells. Transcription of these genes is controlled both by temperature and by contact with a eukaryotic cell. See review by Cornelis et al. (1997).
The effector proteins disrupt the function of host cells in a number of ways. The 23 kd YopE is a cytotoxin that disrupts the actin-microfilament structure of cultured Hela cells (Rosqvist et al. (1990) Mol. Microbiol. 4: 657-667; Rosqvist et al. (1991) Infect. Immun. 59: 4562-4569). The 51 kd YopH is a protein tyrosine phosphatase (PTPase) related to eukaryotic PTPases, which acts on tyrosine-phosphorylated proteins of infected macrophages (Hartland et al. (1994) Infect. Immun. 62: 4445-4453). Presumably as a result of this action, YopH inhibits bacterial uptake and oxidative burst by cultured macrophages (Rosquvist et al. (1988) Infect. Immun. 56: 2139-2143; Bliska et al. (1995) Infect. Immun. 63: 681-685). YopO (or YpkA) is an 81 kd serine/threonine kinase, which is targeted to the inner surface of the plasma membrane of the eukaryotic cell and might function to interfere with the signal transduction pathway of the eukaryotic cell (Hakansson et al. (1996) Mol. Microbiol. 20: 593-603). YopM is an acidic 41 kd protein having 12 leucine-rich repeats, which suggests that YopM might bind thrombin and interfere with platelet-mediated events of the inflammatory response (Leung et al. (1989) J. Bacterial. 171: 4623-4632). YopP is involved in the induction of apoptosis in macrophages (Mills et al. (1997) Proc. Acad. Natl. Sci. USA 94: 12638-12643).
A number of genes have been identified that encode tumor rejection antigen precursors (or TRAPs), which are processed into TRAs in tumor cells. Such TRAP-encoding genes include members of the MAGE family, the BAGE family, the DAGE/Prame family, the GAGE family, the RAGE family, the SMAGE family, NAG, Tyrosinase, Melan-A/MART-1, gp100, MUC-1, TAG-72, CA125, mutated proto-oncogenes such as p21ras, mutated tumor suppressor genes such as p53, tumor associated viral antigens such as HPV16 E7. See, e.g., review by Van den Eynde and van der Bruggen (1997) in Curr. Opin. Immunol. 9:684-693, Sahin et al. (1997) in Curr. Opin. Immunol. 9:709-716, and Shawler et al. (1997) Advances in Pharmacology 40: 309-337 Academic Press, Inc.: San Diego, Calif. The identification of these genes has allowed recombinant production of TRAs or TRAPs which may be subsequently used as vaccines to treat various cancerous conditions.
One embodiment of the present invention provides mutant Yersinia strains deficient in producing functional effector proteins. A preferred mutant Yersinia strain-of the present invention is a quintuple-mutant strain designated as yopEHMOP.
Another embodiment of the present invention provides expression vectors for delivery of heterologous proteins to eukaryotic cells. In accordance with the present invention, such an expression vector is characterized by (in the 5′ to 3′ direction) a promoter, a first nucleic acid sequence encoding a delivery signal, a second nucleic acid sequence fused thereto coding for a heterologous protein to be delivered.
FIG. 1 illustrates the plasmid map of the expression vector pMS111-MAGE-1 (YopE130-MAGE1).
FIG. 2 (A) depicts the procedure for stimulating CTL 82/30 with EBV-transformed human B cells (HLA-A1) mixed with recombinant Yersinia; (B) depicts the quantitation of IFN-γ released by activated CTLs.
FIG. 3 depicts the sequence of the Yersinia enterocolitica YopM gene (SEQ ID NO: 33).
FIG. 4 depicts the sequence of the Yersinia enterocolitica YopE gene (SEQ ID NO: 34).
FIG. 5 depicts the sequence of the Yersinia enterocolitica YopH gene (SEQ ID NO: 35).
FIG. 6 depicts the sequence of the Yersinia enterocolitica YopP gene (SEQ ID NO: 36).
FIG. 7 depicts the sequence of the Yersinia enterocolitica YopP gene (SEQ ID NO: 37).
The term “Yersinia” as used herein means all species of Yersinia, including Yersinia enterocolitica, Yersinia pseudotuberculosis and Yersinia pestis.
For the purpose of the present invention, the term “recombinant Yersinia” used herein refers to Yersinia genetically transformed with the expression vectors of the present invention.
The term “delivery” used herein refers to the transportation of a protein from a Yersinia to a eukaryotic cell, including the steps of expressing the protein in the Yersinia, secreting the expressed protein(s) from such Yersinia and translocating the secreted protein(s) by such Yersinia into the cytosol of the eukaryotic cell. Accordingly, a “delivery signal” refers to a polypeptide sequence which can be recognized by the secretion and translocation system of Yersinia and directs the delivery of a protein from Yersinia to eukaryotic cells.
As used herein, the “secretion” of a protein refers to the transportation of such protein outward across the cell membrane of a Yersinia. The “translocation” of a protein refers to the transportation of such protein across the plasma membrane of a eukaryotic cell into the cytosol of such eukaryotic cell.
“Eukaryotic cells” as used herein, the surface of which Yersinia adhere to, are also referred to as “target cells” or “target eukaryotic cells”.
The effector proteins of Yersinia, i.e., the Yersinia virulon proteins which are normally translocated into the cytosol of the target eukaryotic cells, are toxic to the target cell. Thus, a “functional effector protein” refers to an effector protein having a defined catalytic activity and which is capable of eliciting specific toxicity toward the target cells.
The term “mutation” is used herein as a general term and includes changes of both single base pair and multiple base pairs. Such mutations may include substitutions, frame-shift mutations, deletions and truncations.
The mutation can also be generated in the coding region of an effector-encoding gene such that the catalytic activity of the encoded effector protein is abolished. The “catalytic activity” of an effector protein refers to the anti-target cell function of an effector protein, i.e., toxicity. Such activity is governed by the catalytic motifs in the catalytic domain of an effector protein. The approaches for identifying the catalytic domain and/or the catalytic motifs of an effector protein are well within the ken of those skilled in the art. See, for example, Sory et al. (1995), Boland et al. (1996) and Cornelis et al. (1997).
Accordingly, one preferred mutation of the present invention is a deletion of the entire catalytic domain. Another preferred mutation is a frameshift mutation in an effector-encoding gene such that the catalytic domain is not present in the protein product expressed from such “frameshifted” gene. A most preferred mutation is a mutation with the deletion of the entire coding region.
The mutations that are generated in the Yop genes may be introduced into Yersinia by a number of methods. One such method involves cloning a mutated Yop gene (i.e., a yop gene), into a “suicide” vector which is capable of introducing the mutated yop sequence into Yersinia via allelic exchange. Such “suicide” vectors are described by Kaniga et al. (1991) Gene 109: 137-141 and by Sarker et al. (1997) Mol. Microbiol 23: 409-411.
A further aspect of the present invention is directed to an expression vector for use in combination with the instant mutant Yersinia strains to deliver a desired protein into eukaryotic cells. In accordance with the present invention, such a vector is characterized by (in the 5′ to 3′ direction) a promoter, a first nucleic acid sequence encoding a delivery signal, a second nucleic acid sequence fused thereto coding for a heterologous protein to be delivered.
In accordance with present invention, the promoter of the expression vector is preferably from a Yersinia virulon gene. A “Yersinia virulon gene” refers to genes on the Yersinia pYV plasmid, the expression of which is controlled both by temperature and by contact with a target cell. See review by Cornelis et al. (1997). Such genes include genes coding for elements of the secretion machinery (the Ysc genes), genes coding for translocators (YopB, YopD, and LcrV), genes coding for the control elements (YopN and LcrG), and genes coding for effectors (YopE, YopH, YopO/YpkA, YopM and YopP/YopJ).
In a preferred embodiment of the present invention, the promoter is from an effector-encoding gene selected from any one of YopE, YopH, YopO/YpkA, YopM and YopP/YopJ. More preferably, the promoter is from YopE.
“A delivery signal”, as described hereinabove, refers to a polypeptide which can be recognized by the secretion and translocation system of Yersinia and therefore directs the secretion and translocation of a protein into a eukaryotic cell.
The term “heterologous protein” used herein refers to a protein other than a Yersinia Yop protein. “Yop proteins” refer to Yersinia virulon proteins that are secreted, including the translocators and the effectors.
According to the present invention, “a heterologous protein” includes naturally occurring proteins or parts thereof. The term “part of a protein” includes a peptide or polypeptide fragment of a protein that is of sufficient length to be antigenic. Preferably, such a fragment consists of at least 8 or 9 contiguous amino acids of a protein. “A heterologous protein” as used in the present invention also includes artificially engineered proteins or parts thereof, such as fusion of two or more naturally occurring proteins or parts thereof, polyepitopes (in-frame fusion of two or more peptide epitopes) as exemplified by Thompson et al. (1995) in Proc. Natl. Acad. Sci. USA 92: 5845-5849.
The protein expressed from the fused first and second DNA sequences is also termed as a “fusion protein” or a “hybrid protein”, i.e., a hybrid of Yersinia delivery signal and a heterologous protein.
A “tumor associated protein” refers to a protein that is specifically expressed in tumors or expressed at an abnormal level in tumors relative to normal tissues. Such tumor associated proteins include, but are not limited to, members of the MAGE family, the BAGE family (such as BAGE-1), the DAGE/Prame family (such as DAGE-1), the GAGE family, the RAGE family (such as RAGE-1), the SMAGE family, NAG, Tyrosinase, Melan-A/MART-1, gp100, MUC-1, TAG-72, CA125, mutated proto-oncogenes such as p21ras, mutated tumor suppressor genes such as p53, tumor associated viral antigens (e.g., HPV16 E7), HOM-MEL-40, HOM-MEL-55, NY-COL-2, HOM-HD-397, HOM-RCC-1.14, HOM-HD-21, HOM-NSCLC-11, HOM-MEL-2.4, and HOM-TES-11. Members of the MAGE family include, but are not limited to, MAGE-1, MAGE-2, MAGE-11. Members of the GAGE family include, but are not limited to, GAGE-1, GAGE-6. See, e.g., review by Van den Eynde and van der Bruggen (1997) in Curr. Opin. Immunol. 9:684-693, Sahin et al. (1997) in Curr. Opin. Immunol. 9:709-716, and Shawler et al. (1997). These proteins have been shown to associate with certain tumors such as melanoma, lung cancer, prostate cancer, breast cancer, renal cancer and others.
A number of coding sequences for small antigenic peptides can also be employed in the present invention. One skilled in the art can readily determine the length of the fragments required to produce immunogenic peptides. Alternatively, the skilled artisan can also use coding sequences for peptides that are known to elicit specific T cell responses, such as tumor-associated antigenic peptides (TRAs) as disclosed by U.S. Pat. No. 5,462,871, U.S. Pat. No. 5,558,995, U.S. Pat. No.5,554,724, U.S. Pat. No. 5,585,461, U.S. Pat. No.5,591,430, U.S. Pat. No. 5,554,506, U.S. Pat. No.5,487,974, U.S. Pat. No. 5,530,096, U.S. Pat. No.5,519,117. Examples of TRAs are provided in Table 1. See also review by Van den Eynde and van der Bruggen (1997) and Shawler et al. (1997). Antigenic peptides of a pathogen origin can also be used, such as those disclosed by Gilbert et al. (1997).
As described herein above, sequences coding for a full-length naturally occurring protein, a part of a naturally occurring protein, combinations of parts of a naturally occurring protein, or combinations of different naturally occurring proteins or parts from different proteins, may all be employed in the present invention. For example, a sequence coding for multiple epitopes may be used, such as those described by Thomson et al. (1995). Preferably, the second DNA sequence of the present expression vector codes for at least one epitope of a protein. An “epitope” refers to a peptide of at least 8 or 9 amino acids.
The vectors of the instant invention may include other sequence elements such as a 3′ termination sequence (including a stop codon and a poly A sequence), or a gene conferring a drug resistance which allows the selection of Yersinia transformants having received the instant vector.
The expression vectors of the present invention may be transformed by a number of known methods into Yersinia. For the purpose of the present invention, the methods of transformation for introducing an expression vector include, but are not limited to, electroporation, calcium phosphate mediated transformation, conjugation, or combinations thereof. For example, a vector can be transformed into a first bacteria strain by a standard electroporation procedure. Subsequently, such a vector can be transferred from the first bacteria strain into Yersinia by conjugation, a process also called “mobilization”. Yersinia transformant (i.e., Yersinia having taken up the vector) may be selected, e.g., with antibiotics. These techniques are well known in the art. See, for example, Sory et al. (1994).
One preferred embodiment of the present invention is directed to a Yersinia of the above-described mutant Yersinia strain transformed with an expression vector for delivery of a heterologous protein as hereinabove described into a eukaryotic cell.
By “target”, is meant the extracellular adhesion of Yersinia to a eukaryotic cell.
In particular, the present invention contemplates antigen-presenting cells. “Antigen presenting cells” as referred herein express at least one class I or class II MHC determinant and may include those cells which are known as professional antigen-presenting cells such as macrophages, dendritic cells and B cells. Other professional antigen-presenting cells include monocytes, marginal zone Kupffer cells, microglia, Langerhans' cells, interdigitating dendritic cells, follicular dendritic cells, and T cells. Facultative antigen-presenting cells can also be used according to the present invention. Examples of facultative antigen-presenting cells include astrocytes, follicular cells, endothelium and fibroblasts. As used herein, “antigen-presenting cells” encompass both professional and facultative types of antigen-presenting cells.
Those skilled in the art are able, through the extensive teachings in the art, to determine the MHC molecule for presentation of a particular antigen. For example, U.S. Pat. No. 5,405,940 teaches the determination of HLA-A1 as the presenting molecule for a peptide of MAGE-1, EADPTGHSY (SEQ ID NO: 1); U.S. Pat. No. 5,558,995 teaches the determination of HLA-Cw 1601 for presenting another peptide of MAGE-1, SAYGEPRKL (SEQ ID NO: 2); U.S. Pat. No. 5,530,096 teaches the determination of HLA-A2 as the presenting molecule for a peptide of Tyrosinase, MLLAVLYCL (SEQ ID NO: 3). In the event the eukaryotic cells being targeted do not express a desired HLA or MHC molecule, the gene encoding such molecule may be introduced into the eukaryotic cells by well known transformation or transfection procedures.
Further in accordance with the present invention, the delivery of a protein can be achieved by contacting a eukaryotic cell with a recombinant Yersinia under appropriate conditions. Various references and techniques are conventionally available for those skilled in the art regarding the conditions for inducing the expression and translocation of virulon genes, including the desired temperature, Ca++ concentration, manners in which Yersinia and target cells are mixed, and the like. See, for example, Cornelis, Cross talk between Yersinia and eukaryotic cells, Society for General Microbiology Symposium, 55; MoCRAE, SAUNDERS, SMYTH, STOW (eds), Molecular aspects of host-pathoge interactions, Cambridge University Press, 1997. The conditions may vary depending on the type of eukaryotic cells to be targeted, e.g., the conditions for targeting human epithelial carcinoma Hela cells (Sory et al. (1994)); the conditions for targeting mouse thymoma or melanoma cells (Starnbach et al. (1994) J. Immunol. 153: 1603); the conditions for targeting mouse macrophages (Boland et al. (1996)). Such variations can be addressed by those skilled in the art using conventional techniques.
According to the present invention, a mixture of a recombinant Yersinia and an antigen presenting cell can be used as the “stimulator cell” in such an in vitro procedure for producing CTLs specific for the protein being delivered. The MHC determinants expressed by the antigen presenting cell used are compatible with those expressed by the mammal from which PBLs are isolated, and at least one of these MHC molecules is capable of presenting, to T cells, one or more epitopes derived from the protein being delivered. CTL cells generated as such can be administered, in a therapy regimen of adoptive transfer, to a mammal a pathological condition characterized by an abnormal expression of the protein used in the delivery system. See teachings by Greenberg (1986) J. Immunol. 136 (5): 1917; Riddel et al. (1992) Science 257: 238; Lynch et al. (1991) Eur. J. Immunol. 21: 1403; and Kast et al. (1989) Cell 59: 603 for adoptive transfer. CTLs, by lysing the cells abnormally expressing such antigens, can alleviate or treat the pathological condition at issue such as a tumor, an infection with a parasite or a virus.
By “treating”, is meant alleviating or inhibiting a pathological condition, e.g., inhibiting tumor growth or metastasis, reducing the size of tumor, or diminishing symptoms of a pathogen infection.
A safe recombinant Yersinia may be employed in an immunogenic composition to induce an immune response for treating various pathological conditions in mammals.
The pathological conditions contemplated by the present invention include tumors and pathogen infections, as disclosed herein.
The immunogenic compositions can include, in addition to a recombinant Yersinia, other substances such as cytokines, adjuvants and pharmaceutically acceptable carriers. Cytokines can also be included in such immunogenic compositions using additional recombinant Yersinia of the present invention capable of delivering a cytokine, for example.
EXAMPLE 1 Bacterial Strains, Plasmids and Growth Conditions
The work was carried out with Y. enterocolitica E40(pYV40) (see, M. P. Sory et al. (1995) “Identification of the YopE and YopH domains required for secretion and internalization into the cytosol of macrophages, using the cyaA gene fusion approach” Proc. Nat'l Acad. Sci. USA 92: 11998-12002), its isogeneic ampicillin sensitive derivative MRS40(pYV40) (see, M. R. Sarker et al., and their various non-polar mutants. Plasmids are listed in Table 1. Bacteria were grown in Brain Heart Infusion (BHI) (Difco, Detroit, Mich.). After overnight preculture, bacteria were diluted 1/20 in fresh BHI, allowed to grow for 30 minutes at room temperature, and synthesis of the Yop virulon was induced by incubation for 150 minutes at 37° C. before infection.
MAGE-1 HLA-A1 EADPTGHSY 161-169 1
HLA-Cw16 SAYGEPRKL 230-238 2
MAGE-3 HLA-A1 EVDPIGHLY 168-176 3
HLA-A2 FLWGPRALV 271-279 4
HLA-B44 MEVDPIGHLY 167-176 5
BAGE HLA-Cw16 AARAVFLAL 2-10 6
GAGE-1,2 HLA-Cw16 YRPRPRRY 9-16 7
RAGE HLA-B7 SPSSNRIRNT 11-20 8
GnT-V HLA-A2 VLPDVFIRC(V) 2-10/11 9
MUM-1 HLA-B44 EEKLIVVLF exon 2/ 10
EEKLSVVLF 11
CDK4 HLA-A2 ACDPHSGHFV 23-32 12
ARDPHSGHFV 13
β-catenin HLA-A24 SYLDSGIHF 29-37 14
SYLDSGIHS 15
Tyrosinase HLA-A2 MLLAVLYCL 1-9 16
HLA-A2 YMNGTMSQV 369-377 17
HLA-A2 YMDGTMSQV 369-377 18
HLA-A24 AFLPWHRLF 206-214 19
HLA-B44 SEIWRDIDF 192-200 20
HLA-B44 YEIWRDIDF 192-200 21
HLA-DR4 QNILLSNAPLGPQ 56-70 22
HLA-DR4 DYSYLQDSDPDSF 448-462 23
Melan-AMART-1 HLA-A2 (E)AAGIGILTV 26/27-35  24
HLA-A2 ILTVILGVL 32-40 25
gp100Pme1117 HLA-A2 KTWGQYWQV 154-162 26
HLA-A2 ITDQVPFSV 209-217 27
HLA-A2 YLEPGPVTA 280-288 28
HLA-A2 LLDGTATLRL 457-466 29
HLA-A2 VLYRYGSFSV 476-485 30
DAGE HLA-A24 LYVDSLFFL 301-309 31
MAGE-6 HLA-Cw16 KISGGPRISYPL 292-303 32
EXAMPLE 2 Construction of the Polymutant Strains
To construct the yopHOPEM polymutant strain, the yopE, yopH, yopO, yopM and yopP genes were successively knocked out by allelic exchange in the MRS40 strain using the suicide vectors pMRS101 and pKNG101. See, K. Kaniga et al. (1991) “A wide-host range suicide vector for improving reverse genetics in gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica” Gene 109: 137-141 and M. R. Sarker et al. (1997) “An improved version of suicide vector pKNG101 for gene replacement in Gram-negative bacteria” Mol. Microbiol. 23: 409-411. The various deletions are described in Table 2 in the “suicide vectors and mutators” section. The YopE gene was first mutated using the mutator pPW52 (see, P. Wattiau et al. (1993) “SycE, a chaperone-like protein of Yersinia enterocolitica involved in the secretion of YopE” Mol. Microbiol. 8: 123-131), giving strain MRS40(pAB4052). Mutation of the YopH gene in this strain with the mutator pAB31 (see, S. D. Mills et al. (1997) “Yersinia enterocolitica induces apoptosis in macrophages by a process requiring functional type III secretion and translocation mechanisms and involving YopP, presumably acting as an effector protein” Proc. Natl. Acad. Sci. USA 94: 12638-12643) gave the double yopEH mutant MRS40(pAB404). The triple yopEHO mutant MRS40(pAB405) was then obtained by allelic exchange with the mutator pAB34 (see, S. D. Mills et al., 1997). The YopP gene was then mutated with mutator pMSK7 (see S. D. Mills et al. (1997)), leading to the yopEHOP mutant MRS40(pMSK46). The yopHOPEM strain MRS40(pABL403) was finally obtained by allelic exchange with the yopM mutator pAB38 (see, S. D. Mills et al., 1997).
Plasmids Characteristics References
pABL403 pYV40 yopE21, yopHΔ1-352 see Example 2 of the
yopOΔ65-558, yopP23, yopM23 present specification
Suicide Vectors and
pKNG101 oriR6K sacBR+ onTRK2 strAB+ K. Kaniga et al. (1991)
Gene 109: 137-141.
pMRS101 oriR6K sacBR+ onTRK2 strAB+ M. R. Sarker and G. R.
oriColE1 bla + Cornelis (1997) Mol.
Microbiol. 23: 409-411.
pAB31 pMRS101 yopHΔ1-352+ S. D. Mills et al. (1997)
USA 94: 12638-12643.
pAB34 pMRS101 yopOΔ65-558+ S. D. Mills et al. (1997)
pAB38 PMRS101 yopM23+ S. D. Mills et al. (1997)
pMSK7 pMRS101 yopP23+ S. D. Mills et al. (1997)
pPW52 pKNG101 yopE21+ P. Waattiau and G. R.
Cornelis (1993) Mol.
Microbiol. 8: 123-131.
EXAMPLE 3 Construction of a Plasmid Encodina YoDE130-MAGE-1 and Introduction of this Plasmid into Yersinia
The sequence encoding protein MAGE-1 was inserted in frame with a sequence encoding a truncated YopE, YopE130, containing the first 130 amino acids of YopE. Such a plasmid is graphically depicted in FIG. 1.
The open reading frame of MAGE-1 was amplified by PCR using a MAGE-1 cDNA cloned in pcDNAI/Amp (Invitrogen, Carlsbad, Calif.) as template. The upstream primer, AAACTGCAGATGTCTCTTGAGCAGAGGAGTC (SEQ ID NO: 38), consisted of the first nucleotides of the open reading frame of MAGE-1 preceded by a PstI site. The downstream primer, AAACTGCAGTCAGACTCCCTCTTCCTCCTC (SEQ ID NO: 39), consisted of nucleotides complementary to the last nucleotides of the open reading frame of MAGE-1 followed by a PstI site. The PCR product was digested with PstI and inserted in frame with the truncated YopE at the PstI site of vector pMS111 (see, Sory et al. (1994) Molecular Microbiology 14: 583-594). pMS111-MAGE-1 was electroporated in bacteria strain DH5F′IQ. DNA was extracted from some clones and the DNA of a positive recombinant clone was electroporated in bacteria strain SM10. After mobilization of pMS111 from SM10 in Yersinia MRS40 (pABL403), recombinant clones were then selected on agar-containing medium, supplemented with nalidixic acid, sodium-arsenite and chloramphenicol. MRS40 is an isogeneic derivative of E40 sensitive to ampicillin (see, Sory et al. (1995) Proc. Natl. Acad. Sci. USA 92: 11998-12002).
EXAMPLE 4 Targeting EBV-Transformed B Cells
One colony of Yersinia MRS40 (pABL403) containing pMS111-MAGE-1 was then grown overnight at 28° C. in LB medium supplemented with nalidixic acid, sodium m-arsenite and chloramphenicol. The overnight culture was diluted in fresh medium in order to obtain an OD (optical density) of 0.2. The fresh culture was amplified at 28° C. for approximately 2 hours. The bacteria were washed in 0.9% NaCl and resuspended at 108 bacteria per ml in 0.9% NaCl. 50,000 EBV-transformed HLA-A1+ B cells (KASOII-EBV) were placed in microwells (96 wells round-bottomed) and pelleted by centrifugation. The supernatant was discarded and various dilutions of bacteria were added in 100 ul of complete RPMI 1640 (culture media was supplemented with 10% FCS and with L-arginine (116 mg/ml), L-asparagine (36 mg/ml), L-glutamine (216 mg/ml). Two hours after infection, gentamicin (30 μg/ml) was added for the next two hours, and the cells were finally washed three times.
EXAMPLE 5 Recognition of Taraeted B Cells by MZ2-CTL 82/30
MZ2-CTL 82/30 are specific for the MAGE-1 peptide EADPTGHSY (SEQ ID NO: 1) which is presented by HLA-A1 (U.S. Pat. No. 5,342,774). 5000 MZ2-CTL 82/30 cells were added in each microwell containing the Yersinia in a final volume of 100 μl of Iscove's complete medium (culture medium was supplemented with 10% human serum, L-arginine (116 mg/ml), L-asparagine (36 mg/ml), L-glutamine (216 mg/ml), streptomycine (0.1 mg/ml), penicillin (200 U/ml), IL-2 (25 U/ml) and gentamicin (15 μg/ml). After overnight incubation, the presence of IFN-gamma (that is produced by CTL upon activation) in the supernatant of the co-culture was tested in a standard ELISA assay (Biosource, Fleurus, Belgium). FIG. 2A graphically depicts such a procedure.
As indicated in FIG. 2B, the HLA-A1+ B cells infected with Yersinia encoding YopE130-MAGE-1 were recognized by the CTL 82/30, while the same cells infected with the control plasmid YopE130 were not. The optimal concentration of bacteria is around 1,000,000 per microwell.
39 1 9 PRT Human MAGE-1 peptide 1 Glu Ala Asp Pro Thr Gly His Ser Tyr 5 2 9 PRT Human MAGE-1 peptide 2 Ser Ala Tyr Gly Glu Pro Arg Lys Leu 5 3 9 PRT Human MAGE-3 peptide 3 Glu Val Asp Pro Ile Gly His Leu Tyr 5 4 9 PRT Human MAGE-3 peptide 4 Phe Leu Trp Gly Pro Arg Ala Leu Val 5 5 10 PRT Human MAGE-3 peptide 5 Met Glu Val Asp Pro Ile Gly His Leu Tyr 5 10 6 9 PRT Human BAGE peptide 6 Ala Ala Arg Ala Val Phe Leu Ala Leu 5 7 8 PRT Human GAGE-1,2 peptide 7 Tyr Arg Pro Arg Pro Arg Arg Tyr 5 8 10 PRT Human RAGE peptide 8 Ser Pro Ser Ser Asn Arg Ile Arg Asn Thr 5 10 9 10 PRT Human GnT-V peptide 9 Val Leu Pro Asp Val Phe Ile Arg Cys Val 5 10 10 9 PRT Human MUM-1 peptide 10 Glu Glu Lys Leu Ile Val Val Leu Phe 5 11 9 PRT Human MUM-1 peptide 11 Glu Glu Lys Leu Ser Val Val Leu Phe 5 12 10 PRT Human CDK4 peptide 12 Ala Cys Asp Pro His Ser Gly His Phe Val 5 10 13 10 PRT Human CDK4 peptide 13 Ala Arg Asp Pro His Ser Gly His Phe Val 5 10 14 9 PRT Human -catenin peptide 14 Ser Tyr Leu Asp Ser Gly Ile His Phe 5 15 9 PRT Human -catenin peptide 15 Ser Tyr Leu Asp Ser Gly Ile His Ser 5 16 9 PRT Human Tyrosinase peptide 16 Met Leu Leu Ala Val Leu Tyr Cys Leu 5 17 9 PRT Human Tyrosinase peptide 17 Tyr Met Asn Gly Thr Met Ser Gln Val 5 18 9 PRT Human Tyrosinase peptide 18 Tyr Met Asp Gly Thr Met Ser Gln Val 5 19 9 PRT Human Tyrosinase peptide 19 Ala Phe Leu Pro Trp His Arg Leu Phe 5 20 9 PRT Human Tyrosinase peptide 20 Ser Glu Ile Trp Arg Asp Ile Asp Phe 5 21 9 PRT Human Tyrosinase peptide 21 Tyr Glu Ile Trp Arg Asp Ile Asp Phe 5 22 15 PRT Human Tyrosinase peptide 22 Gln Asn Ile Leu Leu Ser Asn Ala Pro Leu Gly Pro Gln Phe Pro 5 10 15 23 15 PRT Human Tyrosinase peptide 23 Asp Tyr Ser Tyr Leu Gln Asp Ser Asp Pro Asp Ser Phe Gln Asp 5 10 15 24 10 PRT Human Melan-AMART-1 peptide 24 Glu Ala Ala Gly Ile Gly Ile Leu Thr Val 5 10 25 9 PRT Human Melan-AMART-1 peptide 25 Ile Leu Thr Val Ile Leu Gly Val Leu 5 26 9 PRT Human gp100Pmel117 peptide 26 Lys Thr Trp Gly Gln Tyr Trp Gln Val 5 27 9 PRT Human gp100Pmel117 peptide 27 Ile Thr Asp Gln Val Pro Phe Ser Val 5 28 9 PRT Human gp100Pmel117 peptide 28 Tyr Leu Glu Pro Gly Pro Val Thr Ala 5 29 10 PRT Human gp100Pmel117 peptide 29 Leu Leu Asp Gly Thr Ala Thr Leu Arg Leu 5 10 30 10 PRT Human gp100Pmel117 peptide 30 Val Leu Tyr Arg Tyr Gly Ser Phe Ser Val 5 10 31 9 PRT Human DAGE peptide 31 Leu Tyr Val Asp Ser Leu Phe Phe Leu 5 32 12 PRT Human MAGE-6 peptide 32 Lys Ile Ser Gly Gly Pro Arg Ile Ser Tyr Pro Leu 5 10 33 1330 DNA Yersinia enterocolitica 33 aaaaatggcc aaaaactttc aatggtagaa gagctaaatt tggataagta acgcataaaa 60 attttcgacg aaaaactata tatatatata tatttaatat gtatggtttc atttgcaatg 120 aaaaaaccga taataaagat attttcagaa aggcattcaa tatgtttata aacccaagaa 180 atgtatctaa tacttttttg caagaaccat tacgtcattc ttctgattta actgagatgc 240 cagttgaggc agaaaatgtt aaatctaagg ctgaatatta taatgcatgg tcggaatggg 300 aacgaaatgc ccctccgggg aatggtgaac agaggggaat ggcggtttca aggttacgcg 360 attgcctgga ccgacaagcc catgagctag aactaaataa tctggggctg agttctttgc 420 cggaattacc tccgcattta gagagtttag tggcgtcatg taattctctt acagaattac 480 cggaattgcc gcagagcctg aaatcacttc aagttgataa taacaatctg aaggcattat 540 ccgatttacc tcctttactg gaatatttag gtgccgctaa taatcagctg gaagaattac 600 cagagttgca aaactcgtcc ttcttgacat ctattgatgt tgataacaat tcactgaaaa 660 cattacctga tttacctcct tcactggaat ttcttgctgc tggtaataat cagctggaag 720 aattgtcaga gttgcaaaac ttgcccttct tgactgcgat ttatgctgat aacaattcac 780 tgaaaacatt acccgattta cccccttccc tgaaaacact taatgtcaga gaaaattatt 840 taactgatct gccagaatta ccgcagagtt taaccttctt agatgtttct gataatattt 900 tttctggatt atcggaattg ccaccaaact tgtataatct caatgcatcc agcaatgaaa 960 taagatcttt atgcgattta cccccttcac tggtagaact tgatgtcaga gataatcagt 1020 tgatcgaact gccagcgtta cctccacgct tagaacgttt aatcgcttca tttaatcatc 1080 ttgctgaagt acctgaattg ccgcaaaacc tgaaactgct ccacgtagag tacaacgctc 1140 tgagagagtt tcccgatata cctgagtcag tggaagatct tcggatggac tctgaacgtg 1200 taattgatcc atatgaattt gctcatgaga ctatagacaa acttgaagat gatgtatttg 1260 agtagtgcgc aagagcgttc ataattctgc gtcacgttaa aatatcatta caacgtaatc 1320 actttatcga 1330 34 1152 DNA Yersinia enterocolitica 34 gaattcccca actttgacac cgataaccgg ttcaatagta tctggaatag acagcgaaag 60 ttgttgaaat aattgagtga tagcttgttc aaatgaatac atttgatctc ctaatagtta 120 gataaaatat caacttaacc aaagcactct cggcagacca tcaattttag cctataattt 180 ttagttttta ttttgtctaa tataacaaca aaaacagcag cggtttttta tataaccacc 240 ggctattttc ccactaagat aaccttgttt taatagccaa gggaataaat agtcatgaaa 300 atatcatcat ttatttctac atcactgccc ctgccggcat cagtgtcagg atctagcagc 360 gtaggagaaa tgtctgggcg ctcagtctca cagcaaaaaa gtgatcaata tgcaaacaat 420 ctggccgggc gcactgaaag ccctcagggt tccagcttag ccagccgtat cattgagagg 480 ttatcatcaa tggcccactc tgtgattgga tttatccaac gcatgttctc ggaggggagc 540 cataaaccgg tggtgacacc agcactcacg cctgcacaaa tgccaagccc tacgtctttc 600 agtgatagta tcaagcaact tgctgctgag acgctgccaa aatacatgca gcagttgagt 660 agcttggatg cagagacgct gcagaaaaat catgaccagt tcgccacggg cagcggccct 720 cttcgtggca gtatcactca atgccaaggg ctgatgcagt tttgtggtgg ggaattgcaa 780 gctgaggcca gtgccatttt aaacacgcct gtttgtggta ttcccttctc gcagtgggga 840 actgttggtg gggcggccag cgcgtacgtc gccagtggcg ttgatctaac gcaggcagca 900 aatgagatca aagggctggg gcaacagatg cagcaattac tgtcattgat gtgatatgga 960 taaaaacaag ggggtagtgt ttcccccttt ttctatcaat attgcgaata tcttcgtccc 1020 tgatctttca ggggcgaatc gttttttagc atgctcattg ttagaatttc tgacttatct 1080 ctcttctgta ttactactca tactctggaa aatcctgagc atttatatct atggattgat 1140 gcagcactcg ag 1152 35 2000 DNA Yersinia enterocolitica 35 agggcattgg aattaaaaat atatttatct aaatgatgat gagtttaaat tacatttgcg 60 tattaaaatg aataacgcat tattaacgta ttaccatctg ttcccgctta attttttaaa 120 aaatttaagg taacaatgag tatatatctt atgggaaaag ccaaaaaact aacgaacact 180 ataataattc gattaacatc aatgaaaata cacggctcac ctattattaa aataatacga 240 ctagcattat aagaaaaaat attttttatg tttatagtat aggcgtgtat ttaattagtt 300 cttaatttaa ttaaggaggg aagcatgaac ttatcattaa gcgatcttca tcgtcaggta 360 tctcgattgg tgcagcaaga gagcggtgat tgtaccggga aattaagagg taacgttgct 420 gccaataaag aaactacctt tcaaggtttg accatagcca gtggtgccag agagtcagaa 480 aaagtatttg ctcaaactgt actaagccac gtagcaaata ttgttctaac tcaagaagat 540 accgctaagc tattgcaaag cacggtaaag cataatttga ataattatga attaagaagt 600 gtcggcaatg gtaatagtgt acttgtcagt ttacgtagtg accaaatgac actacaagac 660 gccaaagtgc tgttggaggc tgcattgcga caagagtcgg gagcgagggg gcatgtatca 720 tctcattcac attcagtcct tcacgcaccg ggaaccccgg tgcgtgaagg actgcgttca 780 catctagacc ccagaacacc accgttgcca ccgcgtgaac gaccacacac ttctggccat 840 cacggggctg gcgaagccag agccaccgca ccaagcactg tttctcctta tggcccagaa 900 gcgcgcgcag aactcagcag ccgcctcacc acattgcgca atacgctggc gccagcaacg 960 aatgatccgc gttacttaca agcctgcggc ggtgaaaagc taaaccgatt tagagatatt 1020 caatgctgtc ggcaaaccgc agtacgcgcc gatcttaatg ccaattacat ccaggtcggt 1080 aacactcgta ccatagcgtg ccagtatccg ctacaatctc aacttgaaag ccatttccgt 1140 atgctggcag aaaaccgaac gccagtgttg gctgttttag cgtccagttc tgagatagcc 1200 aatcaaagat tcggtatgcc agattatttc cgccagagtg gtacctatgg cagtatcact 1260 gtagagtcta aaatgactca gcaagttggt ctcggtgacg ggattatggc agatatgtat 1320 actttaacga ttcgtgaagc gggtcaaaaa acaatttctg ttcctgtggt tcatgttggc 1380 aattggcccg atcagaccgc agtcagctct gaagttacca aggcactcgc ttcactggta 1440 gatcaaacag cagaaacaaa acgcaatatg tatgaaagca aaggaagttc agcggtagca 1500 gatgactcca aattacggcc ggtaatacat tgccgtgcgg gtgttggccg tactgcgcaa 1560 ctgattggcg caatgtgcat gaatgatagt cgtaatagtc agttaagcgt agaagatatg 1620 gtcagccaaa tgcgagtaca aagaaatggt attatggtac aaaaagatga gcaacttgat 1680 gttctgatta agttggctga aggacaaggg cgaccattat taaatagcta atgtaaatat 1740 ttattcctat gagtaaataa aattactaag agatatacac cactttgcca atcaaagaaa 1800 ctttaaacct caactaaagt aagcaattag ttgaggttta tctgctatag aataattatt 1860 aacaaaaata taaacaacaa aattaaaagt tatgtgtcta cttttacttt atgtaaccaa 1920 acccattaat ggataccgta cgtttttctt ttatagaatt aaaccagtaa atgagatgat 1980 gaaggacgat gatcatcgtc 2000 36 867 DNA Yersinia enterocolitica 36 atgattgggc caatatcaca aataaacagc ttcggtggct tatcagaaaa agagacccgt 60 tctttaatca gtaatgaaga gcttaaaaat atcataatac agttggaaac tgatatagcg 120 gatggatcct ggttccataa aaattattca cgcctggata tagaagtcat gcccgcatta 180 gtaattcagg cgaacaataa atatccggaa atgaatctta attttgttac atctccccag 240 gacctttcga tagaaataaa aaatgtcata gaaaatggag ttggatcttc ccgcttcata 300 attaacatgg gggagggtgg aatacatttc agtgtaattg attacaaaca tataaatggg 360 aaaacatctc tgatattatt tgaaccagta aactttaata gtatggggcc agcgatactg 420 gcaataagta caaaaacggc cattgaacgt tatcaattac ctgattgcca tttttccatg 480 gtggaaatgg atattcagcg aagctcatct gaatgtggta tttttagttt ggcactggca 540 aaaaaacttt acaccgagag agatagcctg ttgaaaatac atgaagataa tataaaaggt 600 atattaagtg atagtgaaaa tcctttaccc cacaataagt tggatccgta tctcccggta 660 actttttaca aacatactca aggtaaaaaa cgtcttaatg aatatttaaa tactaacccg 720 cagggagttg gtactgttgt taacaaaaaa aatgaaacca tctttaatag gtttgataac 780 aataaatcca ttatagatgg aaaggaatta tcagtttcgg tacataaaaa gagaatagct 840 gaatataaaa cacttctcaa agtataa 867 37 2190 DNA Yersinia enterocolitica 37 atgaaaatca tgggaactat gccaccgtcg atctccctcg ccaaagctca tgagcgcatc 60 agccaacatt ggcaaaatcc tgtcggtgag ctcaatatcg gaggaaaacg gtatagaatt 120 atcgataatc aagtgctgcg cttgaacccc cacagtggtt tttctctctt tcgagaaggg 180 gttggtaaga tcttttcggg gaagatgttt aacttttcaa ttgctcgtaa ccttactgag 240 acactccatg cagcccagaa aacgacttcg caggagctaa ggtctgatat ccccaatgct 300 ctcagtaatc tctttggagc caagccacag accgaactgc cgctgggttg gaaagggaag 360 cctttgtcag gagctccgga tcttgaaggg atgcgagtgg ctgaaaccga taagtttgcc 420 gagggcgaaa gccatattag tataatagaa actaaggata atcagcggtt ggtggctaag 480 attgaacgct ccattgccga ggggcatttg ttcgcagaac tggaggctta taaacacatc 540 tataaaaccg cgggcaaaca tcctaatctt gccaatgtcc atggcatggc tgtggtgcca 600 tacggtaacc gtaaggagga agcattgctg atggatgagg tggatggttg gcgttgttct 660 gacacactaa gaagcctcgc cgatagctgg aagcaaggaa agatcaatag tgaagcctac 720 tggggaacga tcaagtttat tgcccatcgg ctattagatg taaccaatca ccttgccaag 780 gcagggatag tacataacga tatcaaaccc ggtaatgtgg tatttgaccg cgctagcgga 840 gagcccgttg tcattgatct aggattacac tctcgttcag gggaacaacc taaggggttt 900 acagaatcct tcaaagcgcc ggagcttgga gtaggaaacc taggcgcatc agaaaagagc 960 gatgtttttc tcgtagtttc aacccttcta catggtatcg aaggttttga gaaagatccg 1020 gagataaagc ctaatcaagg actgagattc attacctcag aaccagcgca cgtaatggat 1080 gagaatggtt acccaatcca tcgacctggt atagctggag tcgagacagc ctatacacgc 1140 ttcatcacag acatccttgg cgtttccgct gactcaagac ctgattccaa cgaagccaga 1200 ctccacgagt tcttgagcga cggaactatt gacgaggagt cggccaagca gatcctaaaa 1260 gatactctaa ccggagaaat gagcccatta tctactgatg taaggcggat aacacccaag 1320 aagcttcggg agctctctga tttgcttagg acgcatttga gtagtgcagc aactaagcaa 1380 ttggatatgg gggtggtttt gtcggatctt gataccatgt tggtgacact cgacaaggcc 1440 gaacgcgagg ggggagtaga caaggatcag ttgaagagtt ttaacagttt gattctgaag 1500 acttacagcg tgattgaaga ctatgtcaaa ggcagagaag gggataccaa gagttccagt 1560 gcggaagtat ccccctatca tcgcagtaac tttatgctat cgatcgccga accttcactg 1620 cagaggatcc aaaagcatct ggaccagaca cactcttttt ctgatatcgg ttcactagtg 1680 cgcgcacata agcacctgga aacgctttta gaggtcttag tcaccttgtc accgcaaggg 1740 cagcccgtgt cctctgaaac ctacagcttc ctgaatcgat tagctgaggc taaggtcacc 1800 ttgtcgcagc aattggatac tctccagcag cagcaggaga gtgcgaaacg gcaactatct 1860 attctgatta atcgttcagg ttcttgggcc gatgttgctc gtcagtccct gcagcgtttt 1920 gacagtaccc ggcctgtagt gaaattcggc actgagcagt ataccgcaat tcaccgtcag 1980 atgatggcgg cccatgcagc cattacgcta caggaggtat cggagtttac tgatgatatg 2040 cgaaacttta cagcggactc tattccacta ctgattcgac ttggacgaag cagtttaata 2100 gatgagcatt tggttgaaca gagagagaag ttgcgagacg tgacgaccat cgccgagcga 2160 ctgaaccggt tggagcggga atggatgtga 2190 38 31 DNA Yersinia enterocolitica 38 aaactgcaga tgtctcttga gcagaggagt c 31 39 30 DNA Yersinia enterocolitica 39 aaactgcagt cagactccct cttcctcctc 30
1. An immunogenic composition, comprising a recombinant Yersinia, wherein said Yersinia comprises at least one mutation in at least one Yersinia effector-encoding gene; and said Yersinia is deficient in the production of at least one functional effector protein and is transformed with an expression vector which comprises, in the 5′ to 3′ direction:
a promoter from a Yersinia virulon gene;
a first DNA sequence encoding a delivery signal from a Yersinia effector protein, operably linked to said promoter; and
a second DNA sequence coding for a heterologous protein, fused in frame to the 3′ end of said first DNA sequence, wherein said heterologous protein is a tumor-associated protein.
2. The immunogenic composition according to claim 1, wherein said effector encoding gene is selected from the group consisting of YopE, YopH, YopO, YopM and YopP of Y. enterocolitica; and YopE, YopH, YpkA, YopM and YopJ of Y. pseudotuberculosis.
3. The immunogenic composition according to claim 1, wherein said mutation is a mutation of the promoter sequence of said effector gene.
4. The immunogenic composition according to claim 1, wherein said mutation is a mutation of the coding sequence of said effector gene.
5. The immunogenic composition according to claim 1, wherein said Yersinia is Y. enterocolitica yopEHOMP transformed with said expression vector or Y. pseudotuberculosis yopEHAOJ transformed with said expression vector.
6. The immunogenic composition according to claim 1, wherein said Yersinia is Y. enterocolitica MRS40 (pABL403) transformed with said expression vector.
7. The immunogenic composition according to claim 1, wherein said Yersinia virulon gene is a Yersinia effector-encoding gene.
8. The immunogenic composition according to claim 7, wherein said effector-encoding gene is selected from the group consisting of YopE, YopH, YopO, YopM and YopP of Y. enterocolitica; and YopE, YopH, YpkA, YopM and YopJ of Y. pseudotuberculosis.
9. The immunogenic composition according to claim 8, wherein said effector-encoding gene is Y. enterocolitica YopE.
10. The immunogenic composition according to claim 1, wherein said effector protein is selected from the group consisting of YopE, YopH, YopO, YopM and YopP of Y. enterocolitica; and YopE, YopH, YpkA, YopM and YopJ of Y. pseudotuberculosis.
11. The immunogenic composition according to claim 10, wherein said effector protein is a Y. enterocolitica YopE or Y. pseudotuberculosis YopE.
12. The immunogenic composition according to claim 1, wherein said delivery signal is Y. enterocolitica YopE130.
13. The immunogenic composition according to claim 1, wherein said tumor-associated protein is selected from the group consisting of members of the MAGE family, the BAGE family, the DAGE/Prame family, the GAGE family, the RAGE family, the SMAGE family, NAG, Tyrosinase, Melan-A/MART-1, gp100, MUC-1, TAG-72, CA125, p21ras, p53, HPV16 E7, HOM-MEL-40, HOM-MEL-55, NY-COL-2, HOM-HD-397, HOM-RCC-1.14, HOM-HD-21, HOM-NSCLC-11, HOM-MEL-2.4, and HOM-TES-11.
14. The immunogenic composition according to claim 13, wherein said tumor-associated protein is MAGE-1.
15. An immunogenic composition, comprising a recombinant Yersinia enterocolitica MRS40 (pABL403), wherein said Yersinia enterocolitica is deficient in the production of any functional effector protein and is transformed with an expression vector which comprises, in the 5′ to 3′ direction:
a second DNA sequence coding for a heterologous protein, fused in frame to the 3′ end of said first DNA sequence, wherein said heterologous protein comprises at least one epitope of a tumor-associated protein.
16. The immunogenic composition according to claim 15, wherein said heterologous protein comprises multiple epitopes of tumor-associated proteins.
17. The immunogenic composition according to claim 15, wherein said tumor-associated protein is selected from the group consisting of members of the MAGE family, the BAGE family, the DAGE/Prame family, the GAGE family, the RAGE family, the SMAGE family, NAG, Tyrosinase, Melan-A/MART-1, gp100, MUC-1, TAG-72, CA125, p21ras, p53, HPV16 E7, HOM-MEL-40, HOM-MEL-55, NY-COL-2, HOM-HD-397, HOM-RCC-1.14, HOM-HD-21, HOM-NSCLC-11, HOM-MEL-2.4, and HOM-TES-11.
18. The immunogenic composition according to claim 17 wherein said tumor-associated protein is MAGE-1.
19. The immunogenic composition according to claim 15, wherein said Yersinia virulon gene is a Yersinia effector-encoding gene.
20. The immunogenic composition according to claim 15, wherein said delivery signal is from a Yersinia effector protein selected from the group consisting of YopE, YopH, YopO, YopM and YopP of Y. enterocolitica; and YopE, YopH, YpkA, YopM and YopJ of Y. pseudotuberculosis.
21. The immunogenic composition according to claim 20, wherein said delivery signal is from Y. enterocolitica YopE or Y. pseudotuberculosis YopE.
22. The immunogenic composition according to claim 21, wherein said delivery signal is Y enterocolitica YopE130.
23. A method of inducing a cytotoxic T-lymphocyte (CTL) response specific for a heterologous protein in a subject in need of such response, comprising the steps of:
(a) obtaining from said subject an antigen-presenting cell expressing an MHC molecule;
(b) forming a cell mixture by contacting said antigen-presenting cell with a recombinant Yersina, wherein said Yersinia comprises at least one mutation in at least one Yersinia effector-encoding gene; and said Yersinia is deficient in the production of at least one functional effector protein and is transformed with an expression vector, wherein said expression vector comprises in 5′ to 3′ direction:
a second DNA sequence fused in frame to the 3′ end of said first DNA sequence, wherein said second DNA sequence codes for at least one epitope of said heterologous protein which is presented by said MHC molecule of said antigen-presenting cell;
(c) contacting a sample containing peripheral blood lymphocytes taken from said subject, with the cell mixture formed in step (b), thereby producing CTLs specific for said heterologous protein; and
(d) administering CTLs produced in step (c) to said subject thereby inducing a CTL response specific for said heterologous protein in said subject.
24. A method for treating a tumor in a subject, comprising administering a recombinant Yersinia to said subject, wherein said Yersinia is deficient in the production of any functional effector protein and is transformed with an expression vector which comprises, in the 5′ to 3′ direction:
a second DNA sequence coding for a heterologous protein, fused in frame to the 3′ end of said first DNA sequence; and wherein said heterologous protein is a tumor-associated protein and elicits an immune response specific to said tumor.
25. The method of claim 24, wherein the Yersinia deficient in the production of any functional effector protein is Y. enterocolitica MRS40 (pABL403).
26. A method for inducing in a subject a cytotoxic T-lymphocyte (CTL) response specific to a tumor-associated antigen, comprising administering a recombinant Yersinia to said subject, wherein said Yersinia comprises at least one mutation in at least one Yersinia effector-encoding gene; and said Yersinia is deficient in the production of at least one functional effector protein and is transformed with an expression vector which comprises, in the 5′ to 3′ direction:
a second DNA sequence coding for said tumor-associated antigen, fused in frame to the 3′ end of said first DNA sequence; and wherein said tumor-associated antigen elicits a CTL response specific to said tumor-associated antigen.
27. The method of claim 26, wherein said tumor-associated protein is selected from the group consisting of members of the MAGE family, the BAGE family, the DAGE/Prame family, the GAGE family, the RAGE family, the SMAGE family, NAG, Tyrosinase, Melan-A/MART-1, gp100, MUC-1, TAG-72, CA125, p21ras, p53, HPV16 E7,HOM-MEL-40, HOM-MEL-55, NY-COL-2, HOM-HD-397, HOM-RCC-1.14, HOM-HD-21, HOM-NSCLC-11, HOM-MEL-2.4, and HOM-TES-11.
28. The method of claim 27, wherein said tumor-associated protein is MAGE-1.
US09318141 1998-03-06 1999-05-25 Delivery of proteins into eukaryotic cells with recombinant Yersinia Expired - Fee Related US6602506B1 (en)
US09036582 US5965381A (en) 1998-03-06 1998-03-06 Delivery of proteins into eukaryotic cells with recombinant yersinia
US09318141 US6602506B1 (en) 1998-03-06 1999-05-25 Delivery of proteins into eukaryotic cells with recombinant Yersinia
US09036582 Division US5965381A (en) 1998-03-06 1998-03-06 Delivery of proteins into eukaryotic cells with recombinant yersinia
US6602506B1 true US6602506B1 (en) 2003-08-05
ID=21889413
US09036582 Expired - Fee Related US5965381A (en) 1998-03-06 1998-03-06 Delivery of proteins into eukaryotic cells with recombinant yersinia
US09318141 Expired - Fee Related US6602506B1 (en) 1998-03-06 1999-05-25 Delivery of proteins into eukaryotic cells with recombinant Yersinia
US (2) US5965381A (en)
EP (1) EP1058723A1 (en)
JP (1) JP2002508939A (en)
CN (1) CN1304442A (en)
CA (1) CA2322665A1 (en)
WO (1) WO1999045098A3 (en)
US20100285068A1 (en) * 2007-06-29 2010-11-11 Universidad Del Pais Vasco Method for the internalization of non-invasive bacteria in eukaryote cells
US20110183908A1 (en) * 2008-03-17 2011-07-28 Rueter Christian Yopm as delivery vehicle for cargo molecules and as biological therapeutic for immunomodulation of inflammatory reactions
DK0668350T4 (en) * 1994-02-16 2009-02-23 Us Gov Health & Human Serv Melanoma associated antigen, epitopes thereof, vaccines against melanoma, and
WO2001042267A1 (en) * 1999-12-10 2001-06-14 Epimmune Inc. Inducing cellular immune responses to mage2/3 using peptide and nucleic acid compositions
WO2002077249A3 (en) * 2001-03-26 2002-12-12 Univ Louvain Type iii bacterial strains for use in medicine
FR2862312B1 (en) 2003-11-13 2006-02-17 Univ Grenoble 1 transfer tool and for producing proteins employing the type III secretion system of Pseudomonas
JP4926168B2 (en) 2005-05-13 2012-05-09 オックスフォード バイオメディカ（ユーケー）リミテッド peptide
EP2152890A1 (en) * 2007-05-23 2010-02-17 MannKind Corporation Multicistronic vectors and methods for their design
WO1989010137A1 (en) 1988-04-29 1989-11-02 Universite Catholique De Louvain Vaccine derived from bacteria of the genus yersinia
WO1993022423A1 (en) 1992-04-29 1993-11-11 Microcarb Inc. Nutrient phospholipids for pathogenic bacteria
US5405940A (en) 1992-08-31 1995-04-11 Ludwig Institute For Cancer Research Isolated nonapeptides derived from MAGE genes and uses thereof
US5530096A (en) 1992-12-22 1996-06-25 Ludwig Institute For Cancer Research Isolated, tyrosinase derived peptides and uses thereof
US6407063B1 (en) * 1998-10-02 2002-06-18 Ludwig Institute For Cancer Research Tumor antigens and CTL clones isolated by a novel procedure
A. Boland et al. (1996) "Status of YopM and YopN in the Yersinia Yop Virulon . . . " The EMBO Journal 15 (19): 5191-5201.
Cornelius et al. Folia Microbiologica Prague 14: 583-594, 1998.* *
D.L. Shawler et al. (1997) "Gene Therapy Approaches to Enhance Antitumor Immunity" Advances in Pharmacology 40: 309-337.
E.L. Hartland et al. (1994) "Essential Role of YopD in Inhibition of the Respiratory Burst of Macrophages by Yersinia enterocolitica" Infect. Immun. 62 (10): 4445-4453.
G.R. Cornelis and H. Wolf-Watz (1997) "The Yersinia Yop Virulon: A Bacterial System for Subverting Eukaryotic Cells" Molecular Microbiology 23 (5): 861-867.
J. Bliska and D.S. Black (1995) "Inhibition of the Fc Receptor-mediated Oxidative Burst in Macrophages by the Yersinia pseudotuberculosis Tyrosine Phosphatase" Infect. Immun. 63 (2): 681-685.
J.B. Van den Eynde and P. van der Bruggen (1997) "T cell Defined Tumor Antigens" Current Opinion in Immunology 9: 684-693.
K. Kaniga et al. (1991) "A Wide-host-range Suicide Vector for Improving Reverse Genetics in Gram-nega Bacteria: Inactivation of the blaA gene of Yersinia enterocolitica" Gene 109: 137-141.
K.Y. Leung and S.C. Straley (1989) "The yopM Gene of Yersinia pestis Encodes a Released Protein Having Homology With the Human Platelet Surface Protein GPIb" J. Bacterial. 171 (9): 4623-4632.
M. Sory et al. (1995) "Identification of the YopE and YopH Domains Required for Secretion . . . " Proc. Natl. Acad. Sci. USA 92: 11998-12002.
M.P. Sory and G.R. Cornelis (1994) "Translocation of a Hybrid YopE-adenylate Cyclase From Yersinia Enterocolitica into HeLa Cells" Molecular Microbiology 14 (3): 583-594.
M.R. Sarker and G.R. Cornelis (1997) "Direct Repeat Sequences in the cagA Gene of Helicobacter pylori: A Ghost of a Chance Encounter?" Molecular Microbiology 23 (2): 409-411.
Michiels, Thomas, et al., (1991) "Secretion of Hybrid Proteins by the Yersinia Yop Export System", Journal of Bacteriology, 173(5): 1677-1685.
O. Türeci et al. (1997) "Serological Analysis of Human Tumor Antigens: Molecular Definition and Implications" Molecular Medicine Today 3 (8): 342-349.
R. Rosqvist et al. (1988) "Inhibition of Phagocytosis in Yersinia pseudotuberculosis: A Virulence Plasmid-encoded Ability Involving to Yop2b Protein" Infect. Immun. 56 (8): 2139-2143.
R. Rosqvist et al. (1991) "Intracellular Targeting of the Yersinia YopE Cytotoxin in Mammalian Cells Induces Actin Microfilament Disruption" Infect. Immun. 59 (2): 4562-4569.
S. Hakansson et al. (1996) "The Yersinia YpkA Ser/Thr Kinase is Translocated and Subsequently Targeted to the Inner Surface of the HeLa Cell Plasma Membrane" Mol. Microbiol. 20 (3): 593-603.
S.A Thompson et al. 1995) "Minimal Epitopes Expressed in a Recombinant Polyepitope Protein . . . " Proc. Natl. Acad. Sci. USA 92: 5845-5849.
S.C. Gilbert et al. (1997) "A Protein Particle Vaccine Containing Multiple Malaria Epitopes" Nature Biotechnology 15: 1280-1284.
S.D. Mills et al. (1997) "Yersinia enterocolitica Induces Apoptosis in Macrophages . . . " Proc. Natl. Acad. Sci. USA 94: 12638-12643.
Sory, Marie-Paule, et al., (1992) "Expression of the Eukaryotic Trypanosoma cruzi CRA Gene in Yersinia enterocolitica and Induction of an Immune Response against CRA in Mice", Infection and Immunity, 60(9):3830-3836.
U. Sahin et al. (1997) "Serological Identification of Human Tumor Antigens" Current Opinion in Immunology 9: 709-716.
V.L. Miller and S. Falkow (1988) "Evidence for Two Genetic Loci in Yersinia Enterocolitica That Can Promote Invasion of Epithelial Cells" Infect. Immun. 56 (5): 1242-1248.
US8840901B2 (en) * 2008-03-17 2014-09-23 Universitaetsklinikum Muenster YopM as delivery vehicle for cargo molecules and as biological therapeutic for immunomodulation of inflammatory reactions
US20150132367A1 (en) * 2008-03-17 2015-05-14 Universitaetsklinikum Muenster Yopm as delivery vehicle for cargo molecules and as biological therapeutic for immunomodulation of the inflammatory reactions
WO1999045098A3 (en) 1999-12-23 application
EP1058723A1 (en) 2000-12-13 application
US5965381A (en) 1999-10-12 grant
WO1999045098A2 (en) 1999-09-10 application
JP2002508939A (en) 2002-03-26 application
CN1304442A (en) 2001-07-18 application
CA2322665A1 (en) 1999-09-10 application
Mukherji et al. 1995 Induction of antigen-specific cytolytic T cells in situ in human melanoma by immunization with synthetic peptide-pulsed autologous antigen presenting cells
Moors et al. 1999 Expression of listeriolysin O and ActA by intracellular and extracellular Listeria monocytogenes
US6685935B1 (en) 2004-02-03 Vectors for the diagnosis and treatment of solid tumors including melanoma
US20040013690A1 (en) 2004-01-22 Attenuated Listeria spp. and methods for using the same
De Smet et al. 1999 Alteration of a single amino acid residue reverses fosfomycin resistance of recombinant MurA from Mycobacterium tuberculosis
Dellagostin et al. 1995 Activity of mycobacterial promoters during intracellular and extracellular growth
Aldovini et al. 1993 The uraA locus and homologous recombination in Mycobacterium bovis BCG.
US6703492B1 (en) 2004-03-09 Staphylococcus epidermidis nucleic acids and proteins
US6991797B2 (en) 2006-01-31 M. tuberculosis antigens
WO1998023631A1 (en) 1998-06-04 Novel bacterial polypeptides and polynucleotides
Härtlein et al. 1983 Transport of hemolysin by Escherichia coli
Abe et al. 1997 Characterization of two virulence proteins secreted by rabbit enteropathogenic Escherichia coli, EspA and EspB, whose maximal expression is sensitive to host body temperature.
US6423545B1 (en) 2002-07-23 Unmarked deletion mutants of mycobacteria and methods of using same
Reed et al. 2002 The Salmonella typhimurium flagellar basal body protein FliE is required for flagellin production and to induce a proinflammatory response in epithelial cells
WO1995022561A2 (en) 1995-08-24 Peptides recognized by melanoma-specific cytotoxic lymphocytes, and uses therefor