WO2001027295A1

WO2001027295A1 - Recombinant attenuated listerias for immunotherapy

Info

Publication number: WO2001027295A1
Application number: PCT/DE2000/003629
Authority: WO
Inventors: Dirk Schadendorf; Annette Paschen; Trinad Chakraborty; Eugen Domann
Original assignee: Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
Priority date: 1999-10-14
Filing date: 2000-10-13
Publication date: 2001-04-19
Also published as: AU1993701A; EP1228224A1; DE19949594A1

Abstract

The invention relates to listeria expression vectors for expressing the human tumor antigens tyrosinase, Trp-1, MelanA/MART-1 and Trp-2 or antigen epitopes derived therefrom, and to attenuated listeria bacteria containing these expression vectors, preferably bacteria of the listeria monocytogenes strain. These bacteria can be used for prophylactic, adjuvant or therapeutic immunotherapy, for example for treating malignant melanoma.

Description

Recombinant attenuated listeria for immunotherapy

The present invention relates to Listeria expression vectors which allow expression of the human tumor-associated antigens tyrosinase, Trp-1, MelanA / MART-1 and Trp-2, and to attenuated Listeria bacteria containing these expression vectors, these preferably being bacteria of the strain Listeria monocytogenes. These bacteria can be used for prophylactic, adjuvant or therapeutic immunotherapy, for example for the treatment of malignant melanoma.

Currently, tumor therapy is still essentially based on the three main pillars: surgery, chemo-, adjuvant chemo- and radiotherapy. However, these therapies have the following serious disadvantages: (a) They are hardly effective in the metastatic stage of the disease or as preventive therapy after removal of the primary tumor, i.e. healing at the metastatic stage is no longer possible (b) they are related to the clinical response to which

Duration of the recurrence-free interval, the overall survival as well as the patient's quality of life very inadequate and

(c) they have a number of partially serious ones

Side effects, for example considerable damage to normal tissue. In addition, there are currently no preventive options that could be based, for example, on "vaccination". However, this would be very desirable, especially with regard to malignant melanoma.

Thus, the invention is essentially based on the technical problem of providing means for tumor therapy, in particular for the therapy of malignant melanoma, which do not show the disadvantages of the current therapy methods described above, in particular allow preventive use, as an adjuvant after removal of a Primary tumors are effective or are of therapeutic benefit in the stage of distant etastasis.

This technical problem has been solved by providing the embodiments characterized in the patent claims.

The Listeria expression vectors according to the invention or the recombinant attenuated Listeria bacteria are constructs which are active in gene therapy and which can be used for prophylactic or in the context of adjuvant or therapeutic tumor control, for example in the case of malignant melanoma. A tumor-specific immune response can be generated by preferably oral immunization, i.e. The body's cellular immune system can be used to target the tumor cells. This treatment can also be combined with treatment methods such as chemotherapy or radiotherapy if necessary, but it preferably replaces the latter forms of therapy.

The present invention is based on the finding that prophylactic or therapeutic treatment of tumors is possible by means of immunization, preferably oral immunization, using the recombinant attenuated listeria according to the invention as a synthesis and transport vehicle for tumor-associated antigens. The expression of the individual tumor-associated antigens is preferably carried out as fusion proteins in which, for example, a Listeria-specific signal sequence is fused to the N-terminus of the antigen. After expression, these fusion proteins are exported from the bacterial cells into the environment. After oral administration, the bacteria pass through the mucosal epithelium of the intestinal tract in the area of Peyer's plaques and are absorbed by the antigen-presenting cells (APCs) of the immune system located there by phagocytosis. The Listeria are therefore initially in the phagosome of the infected cell into which they are placed Secretion fusion proteins, which are available for processing to generate HLA class I and HLA class II peptides. Due to the natural infection cycle, both the wild-type Listeria bacterium and defined attested mutants (provided they do not have a deletion of the hly gene) can pass into the cytosol of the cell. The fusion proteins secreted in the cytosol are in turn accessible to processing for the generation of HLA class I peptides. The peptides generated in both cell compartments (phagosome and cytosol) are thus available for loading HLA I or HLA class II molecules. The peptide-HLA complex is presented on the cell surface of the APCs and a specific T cell response (CD4 + and CD8 + T cells) is induced against the expressed tumor-associated antigens, ie a cellular cytotoxic immune response of the body's immune system against a tumor. As a result, the tumor cells are specifically identified as degenerate and killed. The advantages of this procedure include the fact that the body's own immune system is specifically mobilized in the direction of destruction of the tumor. It represents a simple immunization method since the APCs obtain the tumor-associated antigens by means of a bacterial infection, ie no labor-intensive ex vivo modification of autologous APCs is necessary in the procedure according to the invention, since a naturally occurring infection process is used to modify the cells of the immune system.

The present invention thus relates to a Listeria expression vector for immunotherapy, the Listeria expression vector comprising the following DNA sequences functionally linked: (a) a promoter active in Listeria; and (b) a DNA sequence coding for human tyrosinase, Trp-1, MelanA / MART-1 or Trp-2 or antigenic epitopes derived therefrom.

The term "promoter active in Listeria" used here refers to all promoters which allow the expression of tumor-associated antigens in Listeria. These are preferably promoters of Listeria monocytogenes genes, for example constitutive promoters or promoters activated under the conditions of the infection. Promoters which lead to strong expression of the desired antigen are particularly preferred. This term also refers to promoter fragments or promoters with modified sequences that are still biologically active. In a preferred embodiment, the promoter for the Listeria expression vectors is a promoter of the hly, actA, plcA, plcB or mpl gene, which are each active under the conditions of the infection and which are the Listeria proteins haemolysin, ActA , Encode phosphotidylinositol-specific phospholipase C, phosphatidylcholine-specific phospholipase C or metalloprotease. These promoters have already been described in detail in the literature: actA / plcB promoter: Domann et al. , (1992), EMBO J.

11: 1981-1990 (The transcription of the actA and the plcB gene is controlled by a common promoter) - hly promoter: Domann et al. , (1989), Nucleic Acids

Res. 17: 6406 plcA promoter: Domann et al. , (1991), Mol.

Microbiol. 5: 361-366 mpl promoter: Domann et al. , (1991) Infect. Immune. 59: 65-72

The term "used for human tyrosinase, Trp-1, MelanA / MART-1 or Trp-2 encoding DNA sequence" refers to any DNA sequence that encodes all or part of the native protein. These DNA sequences and the derived amino acid sequences are described, for example: human tyrosinase gene: Genbank Accession No.: M27160 human trp-1 gene: Genbank Accession No.: AF001295 human trp-2 gene: Genbank Accession No.: D17547 human MelanA / MART -l Gen: Genbank Accession No .: U06452 Please also refer to Figures 1-4.

For the antigens tyrosinase, Trp-1, Trp-2 and MelanA / MART-l are differentiation antigens of melanocytic origin. Since these antigens are exclusively expressed in melanocytic cells (melanocytes) in the course of melanogenesis, they are extremely well suited to generate a specific immune response against melanoma cells. These differentiation antigens also have the advantage that they are expressed by up to 100% of the cells of a pigmented tumor (melanoma), whereas only approx. 50% of the melanoma cancer testis express antigens such as MAGE-1. The enzymes tyrosinase, Trp-1, Trp-2 catalyze the process of pigment formation (malanine biosynthesis). The biosynthesis takes place in the specific organelles, the melanosomes, in the matrix of which the MelanA / MART-1 protein is also located.

The expression "for human coding DNA sequence" also relates to DNA sequences which encode such forms of human tyrosinase, Trp-1, MelanA / MART-1 or Trp-2, the changes compared to the native form, ie for example Deletions, additions or exchanges of one or more amino acids and / or modified amino acid (s) or the attachment of an ubiquitin residue or modified oligosaccharide side chains, their antigenic properties remaining in whole or in part or in the desired manner, ie they have, for example, the properties described in the examples below with regard to the treatment of a tumor. The exchanges preferably include "conservative" exchanges of amino acid residues, ie exchanges for biologically similar residues, for example the substitution of a hydrophobic residue (for example isoleucine, valine, leucine, methionine) for another hydrophobic residue, or the substitution of one polar residue for another polar residue (e.g. arginine against lysine, glutamic acid against aspartic acid etc.). The exchanges also include "non-conservative" exchanges, which can maintain or even enhance the antigenic properties of the proteins or individual derived protein fragments (peptides). This can be biological or enzymatic Change the activity of the native protein. Deletions can lead to the generation of molecules which are significantly smaller in size, ie which lack amino acids at the N or C terminus, for example. The above variants also relate to variants which have a better effectiveness in combating tumors compared to the original form. Methods for generating the above changes in the amino acid sequence or corresponding nucleic acid sequence are known to the person skilled in the art and are described in standard works in molecular biology, for example in Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989). The person skilled in the art is also able to determine whether a protein or peptide encoded by a nucleic acid sequence modified in this way still has the desired antigenic properties of tyrosinase, Trp-1, MelanA / MART-1 or Trp-2 (in whole or in part). features. These antigenic properties can be determined for the proteins / peptides by stimulating antigen-specific cytotoxic T cell lines. In the case of peptides, it is also advisable to check their HLA binding properties in the context of FACS analyzes. The DNA sequences encoding the tyrosinase, Trp-1, MelanA / MART-1, Trp-2 or the above variants should preferably have one transcription termination sequence and one

Have translation termination sequence to ensure stable and correct transcription or translation.

General methods known in the art can be used to construct the L i, steria expression vectors according to the invention, inter alia for ligation of the fragments for the promoter and the tumor-associated antigens and insertion into the vector. These methods include, for example, in vitro recombination techniques, synthetic methods, and in vivo recombination methods, as described, for example, in Ausubel and Frederick (1991), Current Protocols in Molecular Biology (J.Wiley & Sons, New York) are described.

Most preferred are Listeria expression vectors in which the DNA sequence coding for human tyrosinase, Trp-1, MelanA / MART-1 or Trp-2 is linked to a DNA sequence coding for a Listeria protein (fragment). that a fusion protein is encoded. The portion derived from the Listeria protein preferably represents the N-terminal portion of the fusion protein. Various Listeria proteins or fragments thereof are suitable for the production of this fusion protein, for example the genes disclosed above with regard to the Listeria promoters. Even more preferred are Listeria expression vectors in which the Listeria protein portion of the fusion protein comes from a protein involved in the lysis of the host vacuoles or in the movements of the bacteria in the host cell, preferably Listeriolysin 0 (Lyse), ActA (intracellular movement) or PI-PLC (lysis). The advantage of listeriolysin 0 to use a Listeria phospholipase or the ActA protein for the construction of fusion proteins is that these proteins are secreted. In the event of infection, these fusion proteins therefore preferentially get into the phagolysosome or cytosol of the infected cell, i.e. into the cell compartments in which the generation of HLA class I and HLA class II presented peptides takes place. The fusion proteins can preferably look as follows: a) only the secretory signal sequence of a Listeria-specific protein is fused with the antigen b) the secretory signal sequence including one

Excerpt of the subsequent native

Protein sequence is fused to the antigen, c) a short fragment of the antigen is added

Maintenance of the reading frame built into the sequence of the Listeria protein mentioned.

In addition, the present invention relates to Listeria expression vectors in which the Listeria protein portion of the Fusion protein comprises a signal sequence of a secreted Listeria protein. The signal sequence is preferably derived from Listeria hemolysin, a Listeria phospholipase or the ActA protein.

The starting vector for the production of the Listeria expression vector according to the invention is any vector which leads to expression of the desired antigens in Listeria. This can be an autosomal or stably inserting vector into the Listeria genome. The output vector is preferably a "shuttle" vector which can be propagated in another host, for example E. coli. Examples of such vectors are pKSV7 (Frankel et al., 1995, J. Immunol. 155: 4775-4782), pCGU34 (Paglia et al., 1997, Eur. J. Immunol. 27: 1570-1575), pAUL-A ( Niebuhr et al., 1997, EMBO J. 16: 5444-5445) and pLIGAl60.

The present invention also relates to the Listeria expression vectors according to the invention (autosomal or stably integrated into the genome, for example via homologous recombination) containing recombinant, attenuated Listeria bacteria, preferably Listeria monocytogenes or Listeria innocua, the latter being particularly suitable for enhancing an immune response. The person skilled in the art can select suitable listeria according to the usual criteria with regard to the use of bacteria for vaccination, ie the listeria which can be used for the purposes according to the invention should have immunogenicity, but be sufficiently attenuated to allow safe use in humans. For this it is necessary that the mutant phenotype of the Listeria is absolutely stable, which is usually only possible by creating chromosomal deletions. Examples of attenuated mutants include mutants that are deficient in cell-to-cell spread, actA-negative mutants that are deficient in intracellular growth, hly2- (listeriolysin) negative mutants, and mutants that are at least one phospholipase -Gens are deficient (Guz et al., Infect. Immun. 63 (1995), 3665-3673). Examples of suitable Listeria strains include the Δmpl2 mutant (Paglia et al., Eur. J. Immunol. 27: 1570-1575). Suitable attested Listeria strains are also described in the international patent application PCT / EP98 / 08096. Process for transforming the above listeria with the Listeria expression vectors according to the invention, for example electroporation, for phenotypically selecting transforms and for expressing the DNA sequences encoding the tumor-associated antigens (optionally as fusion proteins) using the Listeria expression vectors described above are known in the art.

Finally, the present invention further relates to a medicament (vaccine) containing the recombinant attenuated Listeria bacteria according to the invention and their use for immunotherapy. This immunotherapy is suitable for the treatment of pigmented tumor types, preferably for the therapy of malignant melanoma or malignant schwannoma

(Subset of neuroblastomas). These drugs may also contain a pharmaceutically acceptable one

Carrier. Suitable carriers and the formulation of such

Medicines are known to the person skilled in the art. Suitable carriers include, for example, phosphate-buffered saline solutions, water, emulsions, for example oil / water emulsions, wetting agents, sterile solutions, etc. The medicament according to the invention can be administered, for example, for oral administration in the form of an elixir, a capsule or suspension. The appropriate dosage and mode of administration, preferably oral, intravenous or intraperitoneal administration, will be determined by the attending physician and will depend on various factors including, for example, the age, gender, weight of the patient, stage and severity of the tumor Mode of administration etc. In any case, the administration must be in an effective amount, ie an amount that the tumor-associated antigen is expressed in an amount that an immune response in T- Cells against the tumor-associated antigen is induced, so that cells containing this antigen are destroyed. The medicament according to the invention can either be administered alone or in combination with other tumor therapies.

Expression vectors according to the invention were deposited at the DSMZ (German Collection for Microorganisms and Cell Cultures GmbH), Mascheroder Weg lb, Braunschweig on October 5, 1999 in accordance with the provisions of the Budapest Treaty. The deposited samples have the following deposit numbers:

Escherichia coli XL2-Blue pLIGA-trp2 DSM 13072 Escherichia coli XL2-Blue pLIGA-tyro DSM 13073 Escherichia coli XL2-Blue pLIGA-trpl DSM 13074

The figures further illustrate the invention.

Fig. 1: Representation of the cDNA coding region of human.

Tyrosinase and the derived protein sequence

Fig. 2: Representation of the cDNA coding region of human.

Trp-1 and the derived protein sequence

Fig. 3: Representation of the cDNA coding region of human.

Trp-2 and the derived protein sequence

Fig. 4: Representation of the cDNA coding region of human.

MART-1 / MelanA and the derived protein sequence

Fig. 5: Analysis of the surface markers of infected dentritic cells

The expression of the surface markers of dentritic cells not infected with L. monoycytogenic bacteria (thin lines) and the infected dentritic cells (thick lines) were analyzed. Shaded gray histograms represent the controls

The following examples illustrate the invention.

Example 1: Production of two different human

Tyrosinase expressing Listeria expressions

At the 5 'and at the 3' end of the cDNA coding for the human tumor-associated tyrosinase antigen (Genbank accession number: M27160) were PCR (see Table 1) using the specific primers given in Tables 1 and 2 (tyr -5 / 2- LIGA + tyr / 3-LIGA; tyr / 5-LIGA + tyr / 3-LIGA) a Ndeel or Sall recognition sequence was introduced. The primer combination tyr-5/2-LIGA and tyr / 3-LIGA (Tab. 1) was used to amplify the complete tyrosinase cDNA sequence. The primer combination tyr / 5-LIGA and tyr / 3-LIGA (Table 2) was used to amplify a 5 '-deleted tyrosinase cDNA sequence. Both PCR amplificates were then treated as follows: Using the restriction sites mentioned, the fragment was cloned into the vector PLIGA160 in frame downstream of the plasmid-encoded actA signal sequence. The expression of the resulting fusion gene is controlled by the plasmid-encoded actA promoter (Domann et al., 1992, EMBO J. 11: 1981-1990; Genbank Accession: X59723). The inserted DNA sequence and its transition points to the vector were sequenced for control. The resulting expression vector was named pLIGA-tyrof (encodes entire tyrosinase cDNA) or pLIGA-tyro (encoded 5 '-deleted tyrosinase cDNA). The latter was deposited with the DSMZ on October 5 under the number DSM 13073.

Example 2: Preparation of two different Listeria expressing the human trp-1 protein

expression vectors

At the 5 'and 3' ends, the one associated with human tumors TrD-1 antigen coding cDNA (Genbank accession number: AF001295) were PCR (see Table 1) using the specific primers given in Tables 1 and 2 (trpl-5/2-LIGA + trpl / 3-LIGA; trpl / 5-LIGA + trpl / 3-LIGA) introduced a Ndel or a Bgl II recognition sequence. The primer combination trpl-5/2-LIGA and trpl / 3-LIGA (Tab. 1) was used to amplify the complete trp-1 cDNA sequence. The primer combination trpl / 5-LIGA and trpl / 3-LIGA (Tab. 2) was used for the amplification of a 5 '-deleted trp-1 cDNA sequence. Both PCR amplificates were then treated as follows: using the restriction sites mentioned, the fragment was cloned into the vector pLIGA160 in frame downstream of the plasmid-encoded actA signal sequence. The expression of the resulting fusion gene is controlled by the plasmid-encoded actA promoter (Domann et al., 1992, EMBO J. 11: 1981-1990; Genbank Accession: X59723). The inserted DNA sequence and its transition points to the vector were sequenced for control. The resulting expression vector was called pLIGA-trplf (encodes the entire trp-1 cDNA) or pLIGA-trpl (encoded 5 '-related trp-1 cDNA). The latter was deposited on October 5, 1999 with the DSMZ Braunschweig under DSM 13074.

Example 3: Provision of two different Listeria expression vectors expressing the human trp-2 protein

At the 5 'and 3' ends of the cDNA coding for the human tumor-associated Trp-2 antigen (Genbank accession number: D17547) were PCR (see Table 1) using the specific primers given in Tables 1 and 2 (trp2 -5 / 2- LIGA + trp-2/3-LIGA; trp2 / 5-LIGA + trp2 / 3-LIGA) a Ndel or a Sal I recognition sequence was introduced. The primer combination trp2-5 / 2-LIGA and trp2 / 3-LIGA (Tab. 1) was used to amplify the complete trp-2 cDNA sequence. The primer combination trp2 / 5-LIGA and trp2 / 3-LIGA (Tab. 2) was used to amplify a 5 'deleted trp-2 cDNA sequence. Both PCR amplificates were then treated as follows: using the restriction sites mentioned, the fragment was cloned into the vector pLIGAl60 in frame downstream of the plasmid-encoded actA signal sequence. The expression of the resulting fusion gene is controlled by the plasmid-encoded actA promoter (Domann et al., 1992, EMBO J. 11: 1981-1990; Genbank Accession: X59723). The inserted DNA sequence and its transition points to the vector were sequenced for control.

The resulting expression vector was called pLIGA-trp2f (encodes the entire trp-2 cDNA) or pLIGA-trp2

(encodes 5'-deleted trp-2 cDNA). The latter were

October 1999 at the DSMZ Braunschweig under DSM 13072.

Example 4: Preparation of a human MelanA / MART-1

G e n e x p r i m i e r e n s e L i s t e ria - expression vector

At the 5 'and at the 3' end of the cDNA coding for the human tumor-associated MelanA antigen (Genbank accession number: U06452) were PCR (see Table 1) using the primers given in Table 1 (melanA-5/2 -LIGA, melanA / 3- LIGA) introduced a Ndel or a Bgl II recognition sequence. Using the restriction sites, the fragment was cloned into the vector pLIGA160 in frame downstream of the plasmid-encoded actA signal sequence. The expression of the resulting fusion gene is controlled by the plasmid-encoded actA promoter (Domann et al., 1992, EMBO J. 11: 1981-1990; Genbank Accession: X59723). The inserted DNA sequence and its transition points to the vector were sequenced for control. Example 5: Antigen expression in Listeria monocytogenes

The Listeria expression vectors described in Examples 1 to 4 above were amplified in the E. coli strain XL2-Blue in each case in the L. monocytogenes strain EGD and in the attested mutants Δhly2 and ΔactA (Guzmann et al., 1995, Infect Immun. 63: 3665-3573) by electroporation. The technique used for this is well known to the person skilled in the art. Plasmid-bearing listeria were identified from the plasmid-mediated erythromycin resistance. The expression of the tumor-associated antigens is then determined using immunological detection methods (eg immunological staining, Western blot) using specific antibodies (MelanA-AK available from Novocastra; Tyrosinase-AK available from BioTrend, Cologne; Trp-1 AK described in Thomsen et al, 1985, J. Invest. Dermatol. 85: 169-174). Each of the tumor-associated antigens could be detected, i.e. the chosen expression path was successful.

Example 7: Immunization of mice of the transgenic mouse strain HLA-A2

The Listeria expression vector pLIGA-MelanA described in Example 4 above was, after amplification in the E.coli strain XL2-Blue, the L. monocytogenes strain EGD (Gύzman et al., 1995, Infect. Immun. 63: 3665-3573) Electroporation introduced. These bacteria were grown overnight at 37 ° C. in BHI (brain heart infusion) medium (manufacturer: Difco). A 1:50 dilution culture was set up and the growth of the bacteria continued until the middle log phase. The bacteria were harvested by centrifugation at 3000xg. The cells were washed three times with PBS and resuspended in PBS. As a control, the EGD bacteria carrying the unchanged plasmid pLIGAl60 were also treated and cultivated. Mice from the transgenic strain HLA-A2k ^b (Vitiello et al., 1991, J. Exp. Med. 173: 1007-1015) were treated with the bacteria resuspended in PBS (EGD-pLIGA-MelanA and EGD-pLIGA160) in the 7-day period Interval immunized. The mice each received oral administration of lxlO ⁶ bacteria on day 0 and 1X10 ⁷ bacteria on day 7, 14, 21, etc.

The primary goal of the immunization experiments is to generate a cellular cytotoxic T cell response. The antigen-specific activity of cytotoxic T cells is considered the basis of an efficient antitumor immune response.

The spleens from 3 immunized mice are removed 7 days after the last immunization, pooled and mechanically processed so that a single cell suspension is present. The

Spleen cells are transformed into a HLA-A2k ^b cell line

(C1R-A2k ^b ) restimulated. This cell line is with the MelanA

Antigen coding gene is stably transfected and can therefore be used

Stimulation of the cytotoxic activity of antigen-specific T cells can be used. After 5-7 days, the living cells are harvested and checked for their ability to lyse the mentioned antigen-expressing target cells in the ⁵¹ Cr release experiment which is well known to the person skilled in the art.

L. monocytogenes

% Specific lysis of the MdanA-expressing CIR-Alk * target cells EGD effect target ratio 50: 1 25: 1 10: 1 pLIGA-MelanA 30 25 18

PUGA160

The MHC class I Melan A restricted lysis of MelanA expressing C1R-A2k ^b target cells is shown by lymphocytes which were primarily stimulated in vivo with the recombinant L. monocytogenes EGD pLIGA-MelanA vaccine strain. 7 days after the last immunization with EGD-pLIGA-MelanA or EGD-pUGA160 (negative control), the spleen cells were restimulated on day 5 after removal in vitro. The C1R-A2kb cell line syngeneic with the immunized HLA-A2k transgenic mouse strain, which was stably transfected with the human MelanA coding gene, was used for in vitro restimulation. At the end of the culture, the lymphocytes in the ^s, Cr-

Release experiment tested for their ability to lyse the CIR-A∑k "MelanA transfected cell line.

EGD-p IGA160: negative control Table 1

Primer for lrfizieruno On ^p full Antiqen-encode en c-DNA

Tab. 1: Primer for the amplification of the c-DNA sequences coding for antigen. The recognition sequence of specific restriction enzymes, which were used for the cloning of the PCR amplificate in the vector pLIGA160, is marked in bold. The portion of a primer that binds to the complementary sequence of the c-DNA mentioned is underlined

PCR conditions for the amplification of the c-DNA fragments mentioned:

• Initial denaturation: 3 minutes at 94 "C

• Cycle (40 X): 0.5 min at 94 ^* C

1 min at 55 * C 1.5 min at 68 ^β C

• Final polymerization: 7 min. At 68 ^β C Table 2

Primer for amplification of the δ'-shortened anti-α-coding c-DNA

Tab. Primer for the amptification of the antigen-encoding 5'-deieted c-DNA

Sequences The recognition sequence of specific restriction enzymes, which were used to clone the PCR amplificate into the vector pUGA160, is marked in bold. The portion of a primer that binds to the complementary sequence of the c-DNA mentioned is underlined

PCR conditions for the amplification of the c-DNA fragments mentioned:

• Initial denaturation: 3 minutes at 94 ° C

• Cycle (40 X): 0.5 min at 94'C

1 min at 55 ^* C 1.5 min at ββ'C

• Final polymerization: 7 minutes at 68 ^# C. Example 8: Infection of human dendritic cells with

L. monocytogenes EGD and the attested mutant. monocytogenes Δhly2

Since recombinant listeria are to be used as a vector system in the context of an anti-melanoma vaccine in humans, it is necessary to analyze the interaction of these bacteria with the human cells of the immune system in addition to studies in animal models. For this reason, the interaction of Listeria monocytogenes EGD (wild-type strain) and of other attested strains with human dendritic cells (DC) was investigated. Only DZ are able to initiate a primary immune response against the relevant antigen after ingestion of an antigen vaccine (Banchereau et al., (1998), Nature 392, pp. 245-252). For an efficient supply of the DC with the anti-melanoma vaccine, it is therefore necessary that these cells can be infected by the listeria. The following experiment was carried out to determine the ef fectiveness. According to a standard protocol (Thurner et al. (1999), J. Immunol. Methods 223, pp. 1-15), the cells of the peripheral blood became immature in vitro DZ generated that were infected with the bacteria in a ratio of 1 (DZ): 5 (bacteria) (ie 1 x 10 ⁵ DZ were incubated with 5 x 10 ⁵ bacteria). In order to increase the infection efficiency, centrifugation was carried out at 1200 rpm for 5 minutes. After an hour of incubation, 10 µg / ml gentamycin was added to the cultures to kill the extracellular bacteria. Another 3 hours later, the DCs were washed twice with PBS and lysed by incubation with 1% NP-40 PBS to release the phagocytotic bacteria. To determine the number of bacteria, aliquots of the lysate were plated on bacterial growth agar ("Brain Heart Infusion Agar"). Since each intact bacterium forms a colony on the agar after incubation, the number of intracellular bacteria is defined as the number of colony-forming units (CFU). Table 3 below shows the result of the infection experiments, which demonstrate that immature human Double rooms can be infected efficiently by listeria.

Table 3

L. monocytogenes EGD L. monocytogenes Δhly2 (wild type) (deletion of

Listeriolysingens)

CFU 2.94 x 10 ⁵ 4.82 x 10 ⁴

Example 9: Determination of the Influence of Infection on the

Phenotype of dendritic cells

The interaction of immature DC with bacteria can lead to their maturation and thus have a positive influence on their ability to stimulate T cells. The maturation of the DC is accompanied by changes in the expression pattern of specific surface markers, i.e. the expression of specific surface molecules, which are essential for the stimulation of a cellular immune response, is increased. These are costimulatory molecules such as CD40, CD80, CD86 or adhesion molecules such as CD54. The CD83 surface molecule is a specific marker for mature dendritic cells, the function of which is still unclear. The expression detection of these surface markers is carried out by means of immunofluorescence in a flow cytometer (FACS).

In order to determine the influence of Listeria infection on the phenotype of human DC, the procedure was as follows: According to a standard protocol (Thurner et al. (1999), J. Immunol. Methods 223, pp. 1-15), the cells of the peripheral blood in vitro immature DC generated with L. monocytogenes EGD

Wild type strain bacteria in a ratio of 1 (DZ): 5

(Bacteria) were infected (ie 1 x 10 ⁵ double rooms were infected with 5 x

10 ⁵ bacteria incubated). In order to increase the infection efficiency, centrifugation was carried out at 1200 rpm for 5 minutes. After an hour of incubation, 10 μg / ml genta ycin was added to the Cultures added to kill the extracellular bacteria. The batches were then incubated for an additional 20 hours. Parallel cultures that were left uninfected were used as controls. The double rooms were then harvested and washed. The cells were incubated indirectly or indirectly with the following monoclonal antibodies: FITC-conjugated anti-HLA-DR (Becton Dickinson, Heidelberg), anti-CD54 and PE-conjugated anti-CD83 (Coulter-Immunotech, Hamburg), PE-conjugated anti -CD80 (Pharmingen, Hamburg), FITC- conjugated anti-CD40 and FITC-conjugated anti-CD86 (Cymbus Biotechnology, Dianova, Hamburg). FITC-conjugated anti-mouse IgG (Dianova, Hamburg) was used as a secondary reagent for anti-CD54 detection. Mouse IgG was used as an isotype control. The analysis of the expression of the surface markers on the dendritic cells was carried out by means of cytofluorometry (FACScan, Becton Dickinson). The result is shown in FIG. 5. This figure shows that the infection leads to an increased expression of specific costimulatory molecules. Furthermore, the infection leads to a maturation of the DC, which can be seen from the CD83 expression. These data show that infection of the DC with bacterial vaccine vectors has a positive effect on the phenotype of the antigen presenting cells.

Claims

claims

1. Listeria expression vector for immunotherapy, the Listeria expression vector comprising the following DNA sequences functionally linked:

(a) a promoter active in Listeria; and

(b) a DNA sequence coding for human tyrosinase, Trp-1, MelanA / MART-1 or Trp-2.

2. Listeria expression vector according to claim 1, wherein the promoter active in Listeria is the promoter of the hly, actA, plcA, plcB or mpl gene.

3. Listeria expression vector according to claim 1 or 2, additionally containing a protein encoding DNA so that a fusion protein comprising a Listeria protein and human tyrosinase, Trp-1, MelanA / MART-1 or Trp-2 is encoded.

4. Expression vector according to claim 3, wherein the Listeria protein is an enzyme involved in the lysis of the host vacuoles or in the migration of the host cell.

5. Expression vector according to claim 4, wherein the Listeria protein is Listeriolysin 0, PI-PLC or ActA.

6. The expression vector of claim 3, wherein the Listeria protein comprises a signal sequence of a secreted Listeria protein.

7. Expression vector according to claim 6, wherein the signal sequence is derived from hemolysin (listeriolysin 0), a phospholipase (PI-PLC) or the ActA protein.

8. Expression vector according to one of claims 1 to 7, which is derived from pKSV7, pAUL-A or pLIGAlβO.

9. Recombinant attenuated Listeria bacterium containing the Listeria expression vector according to any one of claims 1 to 8.

10. A recombinant attenuated Listeria bacterium according to claim 9 which is Listeria monocytogenes.

11. Vaccine containing a recombinant attenuated Listeria bacterium according to claim 9 or 10.

12. Use of the recombinant attenuated Listeria bacteriu s according to claim 9 or 10 for immunotherapy.

13. Use according to claim 12, wherein the immunotherapy is the therapy of malignant melanoma.