JP3993898B2 - Methods and compositions for stimulating an immune response to a differentiation antigen stimulated by a modified differentiation antigen - Google Patents

Description

Background of the Invention
The present application relates to methods and compositions for stimulating an immune response against differentiation antigens.
Differentiation antigens are tissue-specific antigens that are common to self and several allogeneic tumors of the same origin and are on normal tissue equivalents at the same stage of differentiation. Differentiation antigens have been shown to be expressed by a variety of tumor types, including melanoma, leukemia, lymphoma, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer and lung cancer. For example, differentiation antigens expressed by melanoma cells include Melan-A / MART-1, Pmel17, tyrosinase and gp75. Differentiation antigens expressed by lymphomas and leukemias include CD19 and CD20 / CD20 B lymphocyte differentiation markers. An example of a differentiation antigen expressed by colorectal cancer, breast cancer, pancreatic cancer, prostate cancer, ovarian cancer and lung cancer is mucin polypeptide muc-1. The differentiation antigen expressed by breast cancer is her2 / neu. The her2 / neu differentiation antigen is also expressed by ovarian cancer. Differentiation antigens expressed by prostate cancer include prostate specific antigen, prostatic acid phosphatase and prostate specific membrane antigen.
Melanocyte differentiation antigens have been shown to be recognized and associated with human autoantibodies and T cells with melanoma (Wang et al., J. EXP. Med. 183: 799-804 ( 1996); Vijayasaradhi et al., J. Exp. Med. 171: 1375-1380 (1990)). Unfortunately, in most cases, the individual's immune system becomes tolerant to these antigens and does not have an effective immune response. It is therefore desirable to have a method of stimulating an immune response against a differentiation antigen in vivo for the treatment of cancer characterized by the tumor expressing the differentiation antigen. It is an object of the present invention to provide such a method.
Summary of the present invention
It has been found that immune system tolerance to self differentiation antigens can be overcome and immune responses can be stimulated by administration of therapeutic differentiation antigens. The therapeutic differentiation antigen is modified in one of three ways in relation to the targeted differentiation antigen in the individual being treated (ie, the differentiation antigen for which an immune response is desired). First, under conditions where the therapeutic differentiation antigen is expressed in a different cell type than the individual being treated, the therapeutic differentiation antigen may be syngeneic even if it is syngeneic with the target differentiation antigen. Good. For example, human differentiation antigens expressed in insect cells and other non-human host cells can be used to stimulate an immune response to the differentiation antigen of a human subject. Second, the therapeutic differentiation antigen may be a variant of a syngeneic differentiation antigen, such as a glycosylation variant. Third, the therapeutic differentiation antigen may be the same type (wild type or mutant) differentiation antigen from a different species than the individual receiving treatment. For example, mouse differentiation antigens can be used to stimulate an immune response to the corresponding differentiation antigen in a human subject. Administration of an antigen modified according to the present invention exhibits effective immunity against the unique antigen expressed by the cancer in the individual being treated.
Another aspect of the present invention is specific compositions and cell lines useful for performing the methods of the present invention. In particular, the invention includes non-human cell lines that express human differentiation antigens, such as insect cell lines, and expression vectors useful for generating such cell lines.
[Brief description of the drawings]
Figure 1 summarizes the results of tumor protection experiments using mice immunized with human gp75 expressed in Sf9 insect cells.
Figure 2 summarizes the results of tumor protection using mice immunized with a gene gun with DNA encoding xenogeneic human gp75.
Detailed Description of the Invention
The present invention provides a method for stimulating an immune response against a tissue expressing a target differentiation antigen in a test individual. Although it is desirable that the test individual is a human, the present invention is also provided for veterinary applications such as animal species, preferably mammals or birds.
As used in the specification and claims of this application, the term “immune response” includes cellular and humoral immune responses. The immune response desirably provides sufficient immune protection against the growth of tumors that express the targeted differentiation antigen. The term “stimulate” is devoted to the initial stimulation of a new immune response or an increase in an already existing immune response.
According to the present invention, a test individual is treated by administering a therapeutic differentiation antigen of the same type as the targeted differentiation antigen in an amount effective to stimulate an immune response. Thus, for example, if the targeted differentiation antigen is a gp75 antigen found in melanoma cells and melanocytes, the therapeutic antigen is also a gp75 antigen. However, it has been experimentally found that administration of syngeneic differentiation antigens expressed in cells of the same species as the test individual is not effective in stimulating the immune response (see Examples 1 and 2). Therefore, in order to be effective in the method of the present invention, the therapeutic differentiation antigen must be modified in relation to the target differentiation antigen.
In one embodiment of the invention, both the therapeutic differentiation antigen and the target are from the same species. A therapeutic differentiation antigen is produced by expression in a cell of another species different from the first species. In another embodiment of the invention, the therapeutic differentiation antigen is a variant from a syngeneic differentiation antigen. In yet another embodiment of the present invention, it is a heterologous differentiation antigen. Each of these aspects is discussed in turn below.
Administration of therapeutic differentiation antigens can be accomplished by several routes. First, a therapeutic differentiation antigen, one or more adjuvants such as alum, QS21, TITERMAX or derivatives thereof, incomplete or complete Freund's and related adjuvants to increase the strength of the immune response, And may be administered as part of a vaccine composition that may include cytokines such as granulocyte-macrophage colony stimulating factor, flt-3 ligand, interleukin-2 and interleukin-12. The vaccine composition may be in the form of a differentiation antigen for treatment in the form of a solution or suspension, and the differentiation antigen for treatment may be introduced into a lipid carrier such as a liposome. Such compositions are generally administered by routes such as subcutaneous, intradermal or intramuscular. The vaccine composition comprising the expressed therapeutic differentiation antigen is administered in an amount effective to stimulate an immune response against the targeted differentiation antigen in the test individual. The preferred amount to be administered depends on the species of target individual and the specific antigen, but given in increasing doses, the extent of antibody formation or T cell response is measured by ELISA or a similar test. Can be determined through routine testing. T cell responses are also measured by cellular immunoassays such as cytotoxicity, cytokine release assays and proliferation assays.
Differentiated antigens for the treatment of syngeneic or heterologous mutants are also introduced according to the present invention using DNA immunization techniques in which the DNA encoding the antigen is introduced into a subject expressing the antigen itself. Also good.
Syngeneic antigens expressed in cells of different species
According to the present invention, an immune response against a target differentiation antigen can be stimulated by administration of a syngeneic differentiation antigen expressed in cells of different species. In general, the subject to be treated is a human or other mammal. Thus, insect cells are a preferred type of cell for the expression of syngeneic differentiation antigens. Suitable insect cell lines include Sf9 cells and Schneider 2 Drosophila cells. Therapeutic differentiation antigens can also be expressed in bacteria, yeast or mammalian cell lines such as COS and Chinese hamster ovary cells. For mammalian subjects, host cells that are evolutionarily distant from the subject being treated, such as insects, yeast or bacteria, are unlikely to produce proteins that are expressed in the same manner as the subject. It can be said that it is preferable.
In order to provide expression of the differentiation antigen in the selected system, a DNA encoding a part of the differentiation antigen sufficient to provide the differentiation antigen or immunologically effective expression product is converted into an appropriate expression vector. Insert into. There are many known vector systems that are used to express gene products incorporated into host cells, baculovirus vectors used with insect cells, bacterial and yeast expression vectors, or plasmids used with mammalian cells Contains vectors (such as psvk3). The use of these systems is well known in the art.
For the treatment of humans with syngeneic differentiation antigens, a cDNA encoding the human differentiation antigen to be targeted must be useful. cDNA can be generated by reverse transcription of mRNA, and specific cDNA encoding the targeted differentiation antigen can be identified from a human cDNA library using probes derived from the protein sequence of the differentiation antigen. A variety of human differentiation antigen cDNA sequences have been derived by these methods and are known in the art. For example, the sequence of human gp75 (also known as tyrosinase-related protein-1) is described in Vijayasaradhi, S .; , Bouchard, B. Houghton, A .; N, “The melanoma antigen gp75 is the human homologue of the mouse b (brown) locus gene product.” Exp. Med. 171: 1375-1380 (1990); Exp. Med. 169: Known from 2029-2042 (1989). Other human differentiation antigens with known cDNA sequences include gp100 (also known as tyrosinase-related protein-2) (Kawakami et al., Proc. Nat'l. Acad. Sci. (USA) 91: 6458-6462 (1994); Adema et al., J. Biol. Chem. 269: 20126-20133 (1994)), and mart-1 / melan-a for malignant melanoma; CD19 and CD20 for non-Hodgkin's lymphoma; 2 / neu (King et al., Science 229: 874-976 (1985)); muc-1 for breast, colorectal, lung, and pancreatic tumors (Spicer et al., J. Biol. Chem. 266: 15099-15109 (1991) ) Prostate specific membrane antigen, prostate specific antigen and prostatic acid phosphatase for prostate cancer (Israeli et al., Cancer Res. 54: 6344-6347 (1994); Monne et al., Cancer Res. 54: 6344-6437 (1994); Vihko Et al., FEBS Lett. 236: 275-281 (1988)).
A therapeutic differentiation antigen expressed in cells of different species is administered to the test individual in an amount sufficient to induce an immune response. The composition to be administered may be a lysate of cells expressing the therapeutic differentiation antigen, or may be a purified or partially purified preparation of the therapeutic differentiation antigen.
Variants of syngeneic differentiation antigen
In a second embodiment of the present invention, a variant of a cognate differentiation antigen expressed by the target tumor was used to stimulate an immune response directed against the tumor. Thus, for example, if the tumor is a human tumor that expresses gp75, a variant of human gp75 is used as a therapeutic differentiation antigen.
One skilled in the art will recognize that not all variants will produce useful antigens in the methods of the present invention. For example, a large-scale deletion body that has lost an important epitope cannot be expected to show an action, and is a differentiation antigen for treatment in the sense used in the specification and claims of the present application. Is unthinkable. However, wider variations, particularly variations that alter the tertiary and / or quaternary structure of the expressed differentiation antigen, are within the scope of the present invention.
A preferred type of therapeutic differentiation antigen variant is a glycosylation variant. In any given membrane protein, there will generally be one or multiple glycosylation sites, each of which varies in importance in its effect on protein transport and degradation. For example, mouse gp75 has six N-glycosylation sites, one of which is highly resistant to protease digestion and the other two are important for transporting proteins out of the endoplasmic reticulum. It is. Glycosylation variants that are modified at these sites (Asn96, Asn104, Asn181, Asn304, Asn350, Asn385) are prepared using site-directed mutations. These mutations convert a cognate protein, which is usually non-immunogenic, into a modified antigen with immunity.
Genetic immunization with a cognate glycosylated gp75 variant modified to reduce the glycosylation at this site at amino acid position 350, asparagine, produces autoantibodies against early processed forms of gp75 in the cell. I found it stimulating. These autoantibodies did not recognize mature gp75. We have produced antibodies similar to these by immunization with cells expressing this modified protein, ie by immunization with a modified protein with the same effect.
Heterogeneous differentiation antigen
According to the present invention, an immune response to a target differentiation antigen can be stimulated by administration of the same type of a different type of differentiation antigen. Thus, for example, an immune response against a tumor expressing gp75 can be stimulated by immunization with gp75 from a different species that breaks tolerance to self-antigens. For human therapy, the preferred xenoantigen is the rodent antigen, but also from other mammals such as dogs, cats, cows and sheep, or from birds, fish, amphibians, reptiles, insects or other more distant species. Obtainable.
Heterologous differentiation antigens may be administered as purified differentiation antigens derived from the source organism. Proteins can also be purified from cell lysates for this purpose using column chromatography. Proteins can also be purified from recombinant drug substances such as bacteria, yeast clones, mammalian and insect cell lines that express the desired product for this purpose. Nucleic acid sequences of many differentiation antigens from many non-human actives are known, mouse tyrosinase (gp75) (Yamamoto et al., Japanese J. Genetics 64: 121-135 (1989)); mouse gp100 (Bailin et al., J Invest. Dermatol. 106: 24-27 (1996)); and rat prostate specific membrane antigen (Bzdega et al., J. Neurochem. 69: 2270-2277 (1997)).
The heterologous antigen may also be administered indirectly via genetic immunization of the subject with DNA encoding the differentiation antigen. The cDNA encoding the differentiation antigen is combined with a promoter effective for expression of the nucleic acid polymer in mammalian cells. This is done by digesting the nucleic acid polymer with a restriction endonuclease and cloning into a plasmid containing a promoter such as the SV40 promoter, cytomegalovirus (CMV) promoter or Rous sarcoma virus (RSV) promoter. The resulting construct is then used as a vaccine for genetic immunization. Nucleic acid polymers can also be cloned into plasmids and viral vectors known to be transduced into mammalian cells. These vectors include retrovirus vectors, adenovirus vectors, vaccinia virus vectors, poxvirus vectors, and adenovirus-related vectors.
Nucleic acid constructs containing a promoter, antigen-coding region and cell sorting region can be administered directly, embedded in liposomes prior to administration, or coated with colloidal gold particles. Techniques for embedding DNA vaccines in liposomes are known in the art from Murray, “Gene Transfer and Expression Protocols” Humana Pres, Clifton, NJ (1991). Similarly, the technique of coating naked DNA with gold particles is also described in Yang “Gene transfer into mammalian somatic cells in vivo”, Crit. Rev. Biotech. 12: 335-356 (1992), and techniques for protein expression using viral vectors can be found in Adolph, K. “Viral Genome Methods” CRC Press, Florida (1996).
In genetic immunization, the vaccine composition is preferably administered intradermally, subcutaneously or intramuscularly by injection or gas delivered by particle bombardment and is effective to stimulate an immune response in the host organism The right amount is delivered. This composition uses liposome transfection, particle bombardment or viral infection (including co-cultivation techniques), ex vivo blood or bone marrow derived cells (including APCs) May be administered. The treated cells are then returned to the subject to be immunized. The amount of substance required depends on the immunogenicity of each individual and is believed to be unpredictable in advance, but the method of determining the appropriate amount for any individual subject is straightforward. Specifically, a series of increasing doses starting at about 0.1 μg, measuring antibody titration using ELISA, detecting CTL using chromium release assay, or cytokine release assay The resulting immune response is observed by detecting a TH (helper T cell) response using.
The present invention will be further detailed with reference to the following non-limiting examples.
Example 1
C57BL / 6 mice, a) a similar gp75⁺B16 melanoma cells (non-mutated b locus protein; b) syngeneic B16 cells expressing IL-2, GM-CSF and IFN-γ; c) syngeneic gp75^-From a syngeneic fibroblast transduced with cDNA expressing the B16 melanoma variant, B78H.1 and the mouse b allele; d) a hydrophilic peptide of gp75 conjugated to a carrier protein; and e) from the syngeneic melanoma cell Immunized with purified, full-length gp75 glycoprotein. Cells, purified glycoproteins or peptides were combined with an adjuvant containing Freund's adjuvant, a mixture of bacterial cell wall scaffolds and endotoxin derivatives (DETOX), and a saponin construct (QS21). Immunization was tested by intraperitoneal, subcutaneous and intradermal routes. After immunization, mice are evaluated for antibodies to gp75 by ELISA, immunoprecipitation and Western blot, and for cytotoxic T lymphocytes (CTL) against B16,⁵¹Cr release cell-mediated cytotoxicity assay was used to evaluate. As summarized in Table 1, no antibody or CTL against gp75 was detected after either immunization method, supporting C57BL / 6 to remain tolerant to gp75 glycoprotein.
Example 2
As shown in Example 1, syngeneic C57BL / 6 immunized with cell-bound or purified gp75 protein did not produce autoantibodies against gp75. We next evaluated whether gp75 encoded by cDNA delivered to the dermis of syngeneic C57BL / 6 mice by particle bombardment induces an autoantibody response.
C57BL / 6 mice were genetically immunized with a cDNA encoding the full length of syngeneic gp75 under the control of the CMV promoter once a week for 5 weeks. Sera from these mice were then evaluated by immunoprecipitation for autoantibodies against gp75 as described in Materials and Methods. None of the mice detected antibodies (0/28), indicating that C57BL / 6 mice were tolerant to syngeneic proteins.
Example 3
A baculovirus vector encoding the full-length rodent gp75 was obtained from Dr. In collaboration with Charles Tackney (Iclone, New York, NY), constructed and isolated using standard techniques (Summers & Smith, “Manual for Methods on Baculovirus Vectors and Insect Cell Culture”, Texas Agricultural Experiment Station Bulletin No 1555 (1987); Lucklow & Summers, Biotechnology 6: 47-55 (1988)). In summary, the 1.8 kb EcoRI fragment of pHOMERB2 encoding rodent gp75 is subclone into a pBbac-related baculovirus expression vector from Stratagene and the expression vector is baculovirus. Introduced. Spodoptera frugiperda Sf9 insect cells were co-infected with this virus body and wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) to produce recombinant AcNPV expressing mouse gp75 by homologous recombination. After plaque purification, Sf9 cells were infected with recombinant virus and clones expressing high levels of gp75 were identified by screening with antibodies against gp75. These cell lines were used for immunization studies.
C57BL / 6 mice were immunized with lysates of Sf9 insect cells (gp75 / Sf9 or wt / Sf9, respectively) expressing either syngeneic gp75 in the baculovirus vector or wild type baculovirus. gp75 / Sf9 lysate (1 or 5 × 10⁶Mice immunized with (cells) expressed autoantibodies to gp75 with (120/120 mice) or without (25/28 mice) Freund's adjuvant. No antibody was detected after immunization with wt / Sf9 (0 out of 46 mice). Autoantibodies appeared after 2-4 immunizations, persisted over 4 months after the last immunization, and reacted with gp75 expressed in syngeneic melanocyte cells (B16F10 and JBRH melanoma). The antibodies were of the IgG class based on reactivity with rabbit anti-mouse IgG and protein A, and copurification of antibody reactivity with IgG fractions from serum.
The difference in immunogenicity between gp75 / Sf9 and mouse gp75 is 8 × 10⁶B16 melanoma cells contain 20 μg of gp75, whereas 1 × 10⁶Compared to only 14 μg in gp75 / Sf9 cells, it was not simply due to quantitative differences in the amount of gp75 between the two preparations. Also, 10 μg of purified mouse gp75 mixed with wt / Sf9 lysate did not induce autoantibodies. Although it is clear that Sf9 cells give an adjuvant effect (Prehaud et al., Virology 173: 390-399 (1989); Ghiasi et al., J. Gen. Virology 73: 719-722 (1992)), these results are Other differences between gp75 produced in insect cells and gp75 produced in mouse cells (eg, carbohydrate structure) have been shown to be necessary to induce autoantibodies.
Example 4
In contrast to immunization with gp75 / Sf9 lysate, gp75. Immunization with purified gp75 (12 μg) produced in Sf9 insect cells plus Freund's adjuvant induced autoantibodies that recognize the early processed form of gp75 at 68/70 kDa. This type of gp75 contains only immature high mannose N-linked carbohydrate, and as a result, this gp75 is located in the endoplasmic reticulum or Golgi apparatus.
Example 5
Mouse, gp75⁺We immunized with human melanoma cell line SK-MEL-19 and Freund's adjuvant and evaluated the expression of autoantibodies against rodent gp75. All mice (20/20) expressed autoantibodies. There was no response without adjuvant (0/5 mice) and antibodies against gp75^-It was not detected in the serum of 12 mice immunized with human melanoma SK-MEL-131 or SK-MEL-37 plus Freund's adjuvant. Three of the five mice immunized with purified human gp75 (10 μg / dose immunized 5 times) with Freund's adjuvant expressed autoantibodies against gp75, but compared to the amount of gp75 in the SK-MEL-19 lysate Thus, although likely due to the small amount of purified gp75 used, the antibody response was generally weak. Thus, human gp75 administration abolished tolerance to gp75 in C57BL / 6 mice.
Example 6
B16 melanoma cells and normal melanocytes in C57BL / 6 mice express GP75, the wild type b allele of the Brown locus. As described above, the product of this locus is recognized by sera from mouse gp75 expressed in gp75 / Sf9 cells and syngeneic mice immunized with human gp75. We have previously shown that passive transfer of mouse monoclonal antibodies against gp75 to mice implanted with B16F10 tumors leads to tumor rejection (Hara et al., Int. J. Cancer 61: 253-260 (1995)). In order to determine whether the observed autoimmune response provided similar protection against tumors, the in vivo effects of gp75 immune recognition were studied using syngeneic tumor models.
Mice (5 per group) were treated with gp75 / Sf9 lysate (5 × 10⁶gp75 / Sf9 cells) subcutaneously at the same time 10^FiveB16F10 melanoma cells were administered intravenously and the development of lung metastases 14 days after tumor induction was monitored. Mice immunized with wt / Sf9 cells and non-immunized mice were used as controls. The results are shown in Fig. Summarize in one. As shown, mice immunized with gp75 / Sf9 lysate well protected against the formation of lung metastases compared to controls. Significant protection (53% reduction in lung metastases) was also observed when immunization was performed 4 days after tumor induction when metastasis would be established. Less protective effect was observed in mice immunized with wt / Sf9 lysate compared to non-immunized controls.
Passive transfer of sera from mice immunized with wt / Sf9 lysate to 5 non-immunized mice resulted in a 68% reduction in lung metastases compared to mice treated with the same amount of normal mouse serum ( p = 0.02), supporting the conclusion that tumor protection is at least partially mediated by humoral mechanisms.
Human gp75⁺Mice immunized with SK-MEL-19 were also significantly protected against B16F10 melanoma compared to unimmunized mice (4 mice in immunized mice against 275 ± 77 lung metastases in control mice). ± 7 metastases-6 mice in 1 group). Recognition of other foreign antigens other than gp75 cannot be fully evaluated, but gp75^-Immunization with melanoma SK-MEL-131 did not induce tumor protection against B16F10 melanoma.
Mice immunized against immature, early processed forms of gp75 with gp75 purified from gp75 / Sf9 cells were not well protected against B16F10 metastasis (5 immunized) Not 366 ± 78 metastases in 4 immunized mice versus 412 ± 94 metastases in control mice). Eventually, however, one animal in this group expressed autoantibodies against mature gp75 and was protected against lung metastases (only 21 metastases).
Example 7
C57BL / 6 mice were genetically immunized with cDNA encoding full-length human gp75 under the control of the CMV promoter for 5 weeks once a week by gene gun injection. As controls, mice were injected with full length mouse gp75 under control of the CMV promoter, gp75 glycosylated mutant (gly31) or null (null, meaningless) DNA. Four weeks after the last immunization, the mice are 2 × 10 6 from the tail vessel^FiveB16F10LM3 melanoma cells were injected. One group of treated mice was again induced with melanoma cells. Twenty days after tumor induction, mice were sacrificed and surface metastatic lung nodules were counted. There were 10 untreated groups, 9 null and mouse gp75 groups, 8 gly31 groups, and 19 human gp75 groups. The significance of CD4, CD8 and NK cells was also tested by depletion using monoclonal antibodies (rat mAb GK1.5 against CD4; mAb 53.6.7 against CD8; mAbPK1.36 against NK1.1). The need for CD4 T cells was also assessed by exploring tumor rejection in CD4 knockout mice following in vivo transfer of the human gp75 gene with a gene gun.
Fig. 2, mice immunized with xenogeneic human gp75 are significantly protected from lung metastases (mean 41 ± 15 metastases) when induced with B16F10LM3 melanoma (p <0.0001) compared to control mice Lung nodules were reduced by 84%. Syngeneic mice that received in vivo gene transfer of glycosylated mouse gp75 mutants were not well protected against B16F10LM3 tumor induction (mean 300 ± 12 metastases) and delivered control DNA by particle bombardment ( The same was true for 292 ± 15 transitions on average and those that were untreated (average 307 ± 20 transitions) (p> 0.45). CD8 deletion did not change tumor rejection but CD4 (by mAb or knockout)⁺And NK1.1⁺Depletion reduced the level of protection achieved. Thus, the latter two cells may play a role in the tumor protection achieved using genetic immunization with heterologous DNA.

Claims (5)

A pharmaceutical composition for stimulating an immune response against a self-tumor antigen in a subject individual of the first mammalian species comprising as an active ingredient an immunologically effective amount of a nucleic acid sequence encoding a therapeutic tumor antigen There,
The therapeutic tumor antigen is a mammalian tumor antigen that is a homologue of a self-tumor antigen;
The therapeutic tumor antigen is in a heterogeneous relationship with the first mammalian species,
A non-viral plasmid wherein the nucleic acid sequence encoding a therapeutic tumor antigen comprises a nucleic acid sequence encoding the therapeutic tumor antigen under the control of a promoter that promotes expression of the therapeutic tumor antigen in an individual of the first mammalian species Is a vector,
Pharmaceutical composition . What first mammalian species is a human der, tumor antigen for the treatment is a non-human antigen, pharmaceutical composition of claim 1. The pharmaceutical composition according to claim 1 or 2, wherein the nucleic acid sequence encoding a therapeutic tumor antigen is conjugated to a liposome carrier . The pharmaceutical composition according to claim 1 or 2, wherein the nucleic acid sequence encoding the therapeutic tumor antigen coats the colloidal gold particles . The group consisting of gp75, gp100, mart-1 / melan-A for malignant melanoma, CD19, CD20, her-2 / neu, muc-1, prostate specific membrane antigen, prostate specific antigen and prostate acid phosphatase The pharmaceutical composition according to any one of claims 1 to 4, which is selected from:

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