Novel VEGFR-2 Targeted Immunotherapy Approach

Abstract

The present invention relates to an attenuated Salmonella strain comprising at least one copy of a DNA molecule comprising an expression cassette encoding a VEGF receptor protein for use in cancer immunotherapy, wherein the cancer is a cancer cell expressing the VEGF receptor protein. The present invention also relates to an attenuated Salmonella strain comprising at least one copy of a DNA molecule comprising an expression cassette encoding a VEGF receptor protein for use in cancer immunotherapy, wherein the cancer is a cancer cell expressing the VEGF receptor protein and the cancer is selected from the group consisting of glioblastoma, carcinoid tumor, kidney cancer, particularly renal cell carcinoma, thyroid cancer, lung cancer, particularly non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, prostate cancer, gastrointestinal cancer, particularly colorectal cancer, more particularly colon cancer, and skin cancer, particularly melanoma. The present invention further relates to an attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising an expression cassette encoding a VEGF receptor protein for use in cancer immunotherapy in patients comprising at least one VEGF receptor protein-expressing cancer cell. [Selection diagram] Figure 9

Description

Technical Field

[0001] The present invention relates to an attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising an expression cassette encoding a VEGF receptor protein for use in cancer immunotherapy, wherein the cancer is characterized by cancer cells expressing the VEGF receptor protein. The present invention further relates to an attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising an expression cassette encoding a VEGF receptor protein for use in cancer immunotherapy, wherein the cancer is characterized by cancer cells expressing the VEGF receptor protein and the cancer is selected from the group consisting of glioblastoma, carcinoid cancer, renal cancer, particularly renal cell carcinoma, thyroid cancer, lung cancer, particularly non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, prostate cancer, gastrointestinal cancer, particularly colorectal cancer, more specifically colon cancer, and skin cancer, particularly melanoma. The present invention further relates to an attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising an expression cassette encoding a VEGF receptor protein for use in cancer immunotherapy in a patient having cancer cells expressing at least one VEGF receptor protein.

Background Art

[0002] Angiogenesis is an important factor contributing to the growth and metastasis of solid tumors. Vascular endothelial growth factor receptor (VEGFR) 2, also known as KDR or Flk-1, is a receptor with high affinity for vascular endothelial growth factor (VEGF) and is considered a major mediator of angiogenesis in solid tumors because it is involved in all important endothelial functions such as proliferation, migration, and angiogenesis. Tumor angiogenesis overexpresses VEGFR-2 and is surrounded by endothelial cells that are readily accessible via the bloodstream. Because of the genetic stability of these cells and their ability to support hundreds of tumor cells per endothelial cell, these cells have become a major target for anticancer therapies carried out via antibodies, tyrosine kinase inhibitors, or vaccines (Augustin, Trends Pharmacol Sci 1998, 19:216-222). To date, the VEGF / VEGFR2 signaling pathway has been targeted in numerous anti-angiogenic therapy approaches. Compounds such as bevacizumab and small molecules such as sunitinib and axitinib that specifically target tumor angiogenesis have shown efficacy in a range of tumor indications (Powles et al., Br J Cancer 2011, 104(5): 741-5); Rini et al., Lancet 2011, 378:1931-1939).

[0003] WO 2014 / 005683 discloses an attenuated Salmonella mutant comprising a recombinant DNA molecule encoding a VEGF receptor protein for cancer immunotherapy, particularly for the treatment of pancreatic cancer.

[0004] WO 2016 / 202459 discloses an attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising an expression cassette encoding a VEGF receptor protein for cancer treatment, wherein the treatment further comprises administration of at least one additional anticancer agent.

[0005] WO 2013 / 09189 discloses a method for growing an attenuated mutant Salmonella typhi strain lacking galactose epimerase activity and possessing a recombinant DNA molecule.

[0006] VEGF receptors have long been thought to be restricted to the malignant vasculature, i.e., the tumor stroma. However, recent expression analyses have revealed the expression of vascular endothelial growth factor receptors, particularly VEGFR-2, on the tumor cells themselves. Tumor-specific VEGF receptor expression has been observed in cancer cells of various origins. This suggests that VEGF may have an additional role in tumor formation as well as promoting angiogenesis.

[0007] 〔Object of the Invention〕 An object of the present invention is to provide a novel, safe, and efficient cancer immunotherapy approach targeting VEGF receptors. Such a novel cancer therapy approach would provide beneficial advantages for improving treatment options for cancer patients.

Summary of the Invention

[0008] 〔Summary of the Invention〕 Recent expression analyses have revealed tumor-specific expression of vascular endothelial growth factor receptors, particularly VEGFR-2, in cancer cells of various origins. However, the biological role of tumor-specific VEGF receptor expression remains unclear. The available data regarding the effect of tumor-specific VEGFR-2 expression on glioblastoma have generated significant controversy. Kessler et al. (Oncotarget, 2015) reported that VEGFR-2 expression in glioma cells promotes glioma cell proliferation and increases glioma cell resistance to various chemotherapeutic drugs, while Lu et al. (Cancer Cell, 2012) discovered that VEGF directly and negatively regulates tumor cell invasion via VEGFR-2.

[0009] The present invention is based on the surprising discovery that a Salmonella - based DNA vaccine targeting the VEGF receptor is particularly effective against tumors that exhibit tumor - specific VEGF receptor expression (optionally in addition to VEGF receptor expression in the tumor vasculature) compared to tumors that show VEGF receptor expression only in the tumor vasculature. Within the scope of the present invention, the term "tumor - specific VEGF receptor expression" refers to the expression of the VEGF receptor in the tumor cells themselves as opposed to the tumor vasculature.

[0010] Thus, in a first aspect, the present invention relates to an attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising an expression cassette encoding a VEGF receptor protein for use in cancer immunotherapy, wherein said cancer is characterized by cancer cells that express the VEGF receptor protein.

[0011] In a second aspect, the present invention relates to an attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising an expression cassette encoding a VEGF receptor protein for use in cancer immunotherapy, wherein said cancer is characterized by cancer cells that express the VEGF receptor protein, and wherein said cancer is selected from the group consisting of glioblastoma, carcinoid tumor, renal cancer, particularly renal cell carcinoma, thyroid cancer, lung cancer, particularly non - small cell lung cancer (NSCLC), breast cancer, ovarian cancer, prostate cancer, gastrointestinal cancer, particularly colorectal cancer, more specifically colon cancer, and skin cancer, particularly melanoma.

[0012] In a third aspect, the present invention relates to an attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising an expression cassette encoding a VEGF receptor protein for use in cancer immunotherapy in a patient comprising cancer cells that express at least one VEGF receptor protein.

[0013] In a particular embodiment, the attenuated strain of Salmonella is a strain of the Salmonella enterica species. In particular, the attenuated strain of Salmonella is the attenuated strain of Salmonella typhi Ty21a.

[0014] In certain embodiments, the expression cassette is a eukaryotic expression cassette. In particular, the expression cassette comprises a CMV promoter.

[0015] In certain embodiments, the VEGF receptor protein is particularly human VEGFR-2. In particular, the VEGF receptor protein is selected from the group consisting of VEGFR-2 having the amino acid sequence shown in SEQ ID NO: 1 and proteins having at least 80% sequence identity therewith. In particular, the VEGF receptor protein has the amino acid sequence shown in SEQ ID NO: 1.

[0016] In certain embodiments, the DNA molecule comprises a kanamycin antibiotic resistance gene, pMB1 ori and a CMV promoter. In particular, in such embodiments, the DNA molecule comprises a DNA sequence as shown in SEQ ID NO: 2.

[0017] In certain embodiments, the cancer immunotherapy is used in combination with chemotherapy, radiotherapy or biological cancer therapy. In particular, in such embodiments, an attenuated strain of Salmonella is administered before, during or after chemotherapy or radiotherapy treatment or biological cancer therapy, or before and during chemotherapy or radiotherapy treatment or biological cancer therapy.

[0018] In certain embodiments, the biological cancer therapy comprises administration of at least one additional DNA vaccine encoding a tumor antigen and / or a tumor stromal antigen. In particular, in such embodiments, the at least one additional DNA vaccine encoding the tumor antigen and / or the tumor stromal antigen is selected from at least one copy of an additional DNA molecule comprising an additional expression cassette encoding the tumor antigen and / or the tumor stromal antigen, from at least one additional attenuated strain of Salmonella. In particular, the at least one additional attenuated strain of Salmonella is Salmonella typhi Ty21a comprising an additional eukaryotic expression cassette.

[0019] In certain embodiments, the tumor antigen encoded by the at least one additional DNA vaccine is selected from the group consisting of human Wilms' Tumor protein (WT1), human mesothelin (MSLN), CEA, and CMV pp65. In particular, the tumor antigen encoded by the at least one additional DNA vaccine is a human Wilms' Tumor protein (WT1) having an amino acid sequence as set forth in SEQ ID NO: 3 and a protein having at least about 80% sequence identity thereto, a human mesothelin (MSLN) having an amino acid sequence as set forth in SEQ ID NO: 4 and a protein having at least about 80% sequence identity thereto, a human CEA having an amino acid sequence as set forth in SEQ ID NO: 5 and a protein having at least about 80% sequence identity thereto, a CMV pp65 having an amino acid sequence as set forth in SEQ ID NO: 6 and a protein having at least about 80% sequence identity thereto, a CMV pp65 having an amino acid as set forth in SEQ ID NO: 7 and a protein having at least about 80% sequence identity thereto, and a CMV pp65 having an amino acid sequence as set forth in SEQ ID NO: 8 and a protein having at least about 80% sequence identity thereto, and is selected from the group consisting thereof. In particular, the human Wilms' Tumor protein (WT1) has the amino acid sequence set forth in SEQ ID NO: 3, the human mesothelin (MSLN) has the amino acid sequence set forth in SEQ ID NO: 4, the human CEA has the amino acid sequence set forth in SEQ ID NO: 5, and the CMV pp65 has the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In certain embodiments, the tumor stromal antigen encoded by the at least one additional DNA vaccine is selected from the group consisting of human fibroblast activation protein (FAP).

[0020] In certain embodiments, the attenuated strain of Salmonella is administered orally.

[0021] In certain embodiments, the single dose of the attenuated strain of Salmonella is about 10 5 ~ about 10 11 , particularly about 10 6 ~ about 10 10, more specifically about 10 6 ~ about 10 9 , more specifically about 10 6 ~ about 10 8 , even more specifically about 10 6 ~ about 10 7 colony forming units (CFU).

[0022] In certain embodiments, the attenuated Salmonella strain is used in an individualized immunotherapy that includes the step of assessing the expression pattern of at least one VEGF receptor protein, particularly VEGFR-2, and / or the pre-immune response to the protein in a patient.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0024] 〔Detailed Description of the Invention〕 In a first aspect, the present invention relates to an attenuated strain of Salmonella containing at least one copy of a DNA molecule comprising an expression cassette encoding a VEGF receptor protein, particularly VEGFR-2, for use in cancer immunotherapy, wherein the cancer is characterized by cancer cells expressing a VEGF receptor protein, particularly VEGFR-2.

[0025] In the context of the present invention, the term "cancer characterized by cancer cells expressing a VEGF receptor protein" refers to cancer symptoms characterized by the presence of cancer cells expressing at least one VEGF receptor protein, particularly VEGFR-2, at the mRNA level and / or at the protein level. In certain embodiments, the expression of at least one VEGF receptor protein, particularly VEGFR-2, at the mRNA level and / or at the protein level is increased compared to non-cancerous cells of the same tissue type. For example, the expression of at least one VEGF receptor protein, particularly VEGFR-2, may be increased compared to non-cancerous cells of the same tissue type in the same patient. In another embodiment, the expression of at least one VEGF receptor protein, particularly VEGFR-2, may be increased compared to the average expression in non-cancerous cells of the same tissue in a representative population of healthy subjects. Cancer symptoms characterized by VEGF receptor protein expression include, in particular, glioblastoma, carcinoid tumors, kidney cancer, particularly renal cell carcinoma, thyroid cancer, lung cancer, particularly non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, prostate cancer, gastrointestinal cancer, particularly colorectal cancer, more specifically colon cancer, and skin cancer, particularly melanoma.

[0026] Thus, in a second aspect, the present invention relates to an attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising an expression cassette encoding a VEGF receptor protein, particularly VEFGR-2, for use in cancer immunotherapy, wherein the cancer is characterized by cancer cells expressing a VEGF receptor protein, particularly cancer cells expressing VEGFR-2, and wherein the cancer is selected from the group consisting of glioblastoma, carcinoid tumors, kidney cancer, particularly renal cell carcinoma, thyroid cancer, lung cancer, particularly non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, prostate cancer, gastrointestinal cancer, particularly colorectal cancer, more specifically colon cancer, and skin cancer, particularly melanoma.

[0027] One particularly promising indication for VEGFR-2-targeted immunotherapy is glioblastoma. Glioblastoma exhibits very high levels of tumor angiogenesis. Furthermore, VEGFR-2 can target both the tumor vasculature and tumor cells. Approximately 20% - 50% of glioblastoma patients show tumor-specific VEGFR-2 expression, which is particularly observed at the invasive front. Additionally, VEGFR-2 expression has been observed in glioma-like stem cells. To date, treatment options for glioblastoma have remained inadequate. For example, the monoclonal antibody Avastin, which targets only VEGF, showed utility in progression-free survival but not in overall survival.

[0028] In a third aspect, the invention relates to an attenuated strain of Salmonella comprising at least one copy of a DNA molecule encoding a VEGF receptor protein, particularly VEGFR-2, for use in immunotherapy in cancer patients having cancer cells expressing at least one VEGF receptor protein, particularly cancer cells expressing at least one VEGFR-2.

[0029] In certain embodiments of the invention, a patient is determined to have cancer characterized by cancer cells expressing a VEGF receptor protein or to have cancer cells expressing at least one VEGF receptor protein. In a first step, the patient's tumor-specific VEGF receptor protein expression, such as tumor-specific expression of VEGFR-2, can be evaluated at the mRNA or protein level, preferably in vitro. For this purpose, tumor tissue samples (e.g., biopsies) can be stained, for example, by immunohistochemical staining or subjected to in situ hybridization procedures. Methods for evaluating tissue-specific antigen expression are well known in the art.

[0030] According to the present invention, an attenuated Salmonella strain functions as a bacterial carrier for a recombinant DNA molecule containing an expression cassette encoding a VEGF receptor protein for delivery of the recombinant DNA molecule to target cells. Such a delivery vector containing a DNA molecule encoding a heterologous antigen (e.g., VEGF receptor protein) is called a DNA vaccine. Thus, the terms "DNA vaccine encoding ~" and "attenuated strain of Salmonella containing at least one copy of a DNA molecule containing an expression cassette encoding ~" are used interchangeably herein.

[0031] In the context of the present invention, the term "vaccine" refers to an agent that, when administered, can induce an immune response in a subject. The vaccine can preferably prevent, mitigate or treat a disease.

[0032] The live attenuated Salmonella strain according to the present invention stably carries a recombinant DNA molecule encoding a VEGF receptor protein. It can be used as a vehicle for oral administration of this recombinant DNA molecule.

[0033] Genetic immunization appears to be more advantageous than conventional vaccination. Since the target DNA can be detected for a significant period of time, it can act as an antigen depot. Sequence motifs in some plasmids, such as CpG islands, are immunostimulatory and can function as adjuvants that are promoted by immune stimulation with LPS and other bacterial components.

[0034] Live attenuated Salmonella vectors produce their own immunomodulatory factors in situ, such as lipopolysaccharide (LPS), which can confer advantages over other forms of administration, such as microvesicles. Furthermore, the mucosal vaccines according to the invention have a mode of action within lymphatic vessels that has proven to be beneficial. After inoculation with the attenuated vaccine of the invention, macrophages and other cells in the Peyer's patches of the gastrointestinal tract are invaded by the mutant bacteria. The bacteria are taken up by phagocytes. Due to their attenuated mutations, the bacteria of the S. typhi Ty21 strain are unable to survive in these phagocytes and die at this point. Recombinant DNA molecules are released and subsequently migrate into the cytosol of phagocytic immune cells via a special transport system or by endosomal leakage. Finally, the recombinant DNA molecules enter the nucleus where they undergo transcription, causing a large amount of VEGF receptor protein expression in the cytosol of phagocytes. The infected cells undergo apoptosis, are carried on the VEGF receptor protein antigen, and are taken up and processed by the immune system of the gastrointestinal tract. The danger signals of bacterial infection act as potent adjuvants in this process, targeting antigen-specific CD8 + T cells and inducing a strong antibody response at both the systemic and mucosal compartment levels. The immune response peaks around day 10 after vaccination. The absence of an anti-carrier response allows for boosting with the same vaccine multiple times.

[0035] In the context of the present invention, the term "attenuated" refers to a strain with reduced pathogenicity compared to the parental strain without attenuating mutations. The attenuated strain preferably has lost its pathogenicity but retains the ability to induce protective immunity. Attenuation can be achieved by deletion of various genes such as pathogenicity genes, regulatory genes, and metabolic genes. Attenuated bacteria are also found in nature but can be artificially produced in the laboratory, for example, by adaptation to a new medium or cell culture, or can be produced by recombinant DNA. Administration of about 10 11 CFU of the attenuated Salmonella strain of the invention causes salmonellosis in less than 5%, more preferably less than 1%, and most preferably less than 1% of the subjects.

[0036] In the context of the present invention, the term "comprising" or "containing" means "including but not limited to". This term is open-ended and identifies the presence of any recited feature, element, integer, step or component, but does not exclude the presence of one or more other features, elements, integers, steps or components or groups thereof. Thus, the term "comprising" encompasses the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" as used throughout this specification and particularly in the claims may be replaced by the term "consisting of".

[0037] A DNA molecule comprising an expression cassette encoding a VEGF receptor protein is suitably a recombinant DNA molecule, i.e. a genetically engineered DNA construct, preferably a DNA construct consisting of DNA fragments of different origins. The DNA molecule can be a linear nucleic acid or, preferably, a circular DNA plasmid produced by introducing an open reading frame encoding a VEGF receptor protein into an expression vector plasmid.

[0038] In the context of the present invention, the term "expression cassette" refers to a nucleic acid unit comprising at least one open reading frame (ORF) under the control of regulatory sequences that control its expression. The expression cassette preferably can mediate the transcription of an included open reading frame encoding an antigen, such as a VEGF receptor protein, in a target cell. An expression cassette typically includes a promoter, at least one open reading frame and a transcription termination signal.

[0039] In certain embodiments, the attenuated Salmonella strain is of the Salmonella enterica species. S. enterica strains can potentially be delivered by the mucosal route of immunization, i.e., orally or nasally, which offers advantages of simplicity and safety compared to parenteral administration. Thus, attenuated derivatives of Salmonella bacteria are attractive vehicles for delivering heterologous antigens to the mammalian immune system. Furthermore, Salmonella strains elicit strong humoral and cellular immune responses at both systemic and mucosal sites. Batch production costs are low and formulations of live bacterial vaccines are very stable. Attenuation can be achieved by deletion of various genes, including virulence genes, regulatory genes, and metabolic genes.

[0040] Several Salmonella typhimurium strains attenuated by aro mutations have been demonstrated to be safe and efficient delivery vehicles for heterologous antigens in animal models.

[0041] In certain embodiments, the attenuated Salmonella strain and at least one additional attenuated Salmonella strain are Salmonella typhi Ty21a. The live attenuated Salmonella typhi Ty21a strain is the active ingredient of Typhoral L® (which is also known as Viotif®) (manufactured by Berna Biotech Ltd., Crucell Company, Switzerland). It is the only currently approved oral live vaccine against typhoid fever. This vaccine has been extensively tested and proven to be safe with respect to toxicity to patients and transmission to third parties (Wahdan et al., J. Infectious Diseases 1982, 145:292-295). The vaccine is approved in over 40 countries and has been used to prevent typhoid fever in millions of individuals, including thousands of children. It has an unparalleled safety record. There is no available data indicating that Salmonella typhi Ty21a can enter the bloodstream systemically. The attenuated Salmonella typhi Ty21a live vaccine strain thus enables specific targeting of the immune system in the gastrointestinal tract while being safe and well tolerated. The manufacturing approval number for Typhoral L® is PL 1574 / 0001 dated December 16, 1996. A single dose of the vaccine contains at least 2×10 9 viable Salmonella typhi Ty21a colony forming units and at least 5×10 9 non-viable Salmonella typhi Ty21a cells.

[0042] The well-tolerated oral live vaccine against typhoid fever is induced by chemical mutagenesis of the wild-type pathogenic bacterial isolate S. typhi Ty2, and the vaccine has a loss-of-function mutation in the galE gene that causes a defect in the ability to metabolize galactose. This attenuated strain is unable to reduce sulfate to sulfide, a property that distinguishes it from the wild-type Salmonella typhi Ty2 strain. With regard to its serological characteristics, the typhoid bacterium Ty21a strain contains the O9 antigen, which is a polysaccharide that constitutes the outer membrane of the bacterium, and lacks the O5 antigen, which is a characteristic component of typhoid bacteria. This serological property is the basis for including each test in the panel of identity tests for batch release.

[0043] In certain embodiments, the expression cassette is a eukaryotic cell expression cassette. In particular, the expression cassette comprises a CMV promoter. As used herein, the term "eukaryotic cell expression cassette" refers to an expression cassette that enables the expression of an open reading frame in a eukaryotic cell. It has been demonstrated that the amount of heterologous antigen required to induce an appropriate immune response is toxic to the bacterium and would lead to cell death, excessive attenuation, and loss of expression of the heterologous antigen. It is possible to overcome this toxicity problem by using a eukaryotic cell expression cassette that is not expressed in the bacterial vector but only in the target cells, and the expressed protein typically exhibits a eukaryotic glycosylation pattern.

[0044] A eukaryotic expression cassette contains regulatory sequences, preferably a promoter and a polyadenylation signal, which can control the expression of an open reading frame in eukaryotic cells. The promoter and polyadenylation signal inserted into the recombinant DNA molecule contained in the attenuated strain of Salmonella of the present invention are preferably selected to be functional in the cells of the subject to be immunized. Examples of suitable promoters for the production of DNA vaccines, particularly for humans, include, but are not limited to, cytomegalovirus (CMV), such as the strong CMV immediate early promoter, simian virus 40 (SV40), mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV), such as the HIV long terminal repeat (LTR) promoter, Moloney virus, Epstein - Barr virus (EBV), and promoters from Rous sarcoma virus (RSV), the synthetic CAG promoter consisting of the CMV early enhancer element, the chicken β - actin gene, the first exon and the first intron, the splice acceptor of the rabbit β - globin gene, and promoters from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein. In certain embodiments, the eukaryotic expression cassette contains a CMV promoter. In the present invention, the term "CMV promoter" refers to the strong immediate early cytomegalovirus promoter.

[0045] Examples of suitable polyadenylation signals for the production of DNA vaccines, particularly for humans, include, but are not limited to, the bovine growth hormone (BGH) polyadenylation site, the SV40 polyadenylation signal, and the LTR polyadenylation signal. In certain embodiments, the eukaryotic expression cassette contained in the recombinant DNA molecule by the attenuated strain of Salmonella of the present invention contains the BGH polyadenylation site.

[0046] In addition to regulatory elements necessary for the expression of VEGF receptor proteins, such as promoters and polyadenylation signals, other elements can also be included in the recombinant DNA molecule. Such additional elements include enhancers. Enhancers are, for example, enhancers of human actin, human myosin, human hemoglobin, human muscle creatine, as well as viral enhancers, such as those from CMV, RSV, and EBV.

[0047] Regulatory sequences and codons are generally species-dependent to maximize protein production, and the regulatory sequences and codons are preferably selected to be efficient in the species to be immunized. Those skilled in the art can produce recombinant DNA molecules that are functional in a particular subject's species.

[0048] In certain embodiments, the VEGF receptor protein is VEGFR-2, particularly human VEGFR-2. In particular, the VEGF receptor protein is selected from the group consisting of VEGFR-2 having the amino acid sequence shown in SEQ ID NO: 1, and proteins having at least 80% sequence identity therewith. In particular, the VEGF receptor protein has the amino acid sequence shown in SEQ ID NO: 1.

[0049] In this context, the term "about" or "approximately" means within 80% to 120%, or within 90% to 110%, particularly within 95% to 105% of a given numerical value or range.

[0050] Within the scope of the present invention, the term "a protein having at least about 80% sequence identity with a predetermined protein, for example, VEGFR-2 having the amino acid sequence shown in SEQ ID NO: 1" refers to a protein that may be different from the amino acid sequence encoding the amino acid sequence of the reference protein, for example, VEGFR-2 having the amino acid sequence of SEQ ID NO: 1. Such a protein can be of natural origin, for example, a mutant of a wild-type protein, for example, a mutant of wild-type VEGFR-2, or a homolog of a different species, or an engineered protein, for example, engineered VEGFR-2. It is known that codon usage varies between species. Thus, when expressing a heterologous protein in a target cell, it may be necessary or at least useful to adapt the nucleic acid sequence to the codon usage of the target cell. Methods for designing and constructing derivatives of a given protein are well known to those skilled in the art.

[0051] A protein having at least about 80% sequence identity with a predetermined protein, such as VEGFR-2 having the amino acid sequence shown in SEQ ID NO: 1, can include one or more mutations including additions, deletions and / or substitutions of one or more amino acids as compared to the reference protein, such as VEGFR-2 having the amino acid sequence shown in SEQ ID NO: 1. According to the teachings of the present invention, the amino acids to be deleted, added and / or substituted may be contiguous amino acids, or may be scattered throughout the length of the amino acid sequence of a protein having at least 80% sequence identity with the reference protein, such as VEGFR-2 having the amino acid sequence shown in SEQ ID NO: 1. According to the teachings of the present invention, any number of amino acids can be added, deleted and / or substituted as long as the amino acid sequence identity with the reference protein is at least 80% and the mutant protein is immunogenic. Preferably, the immunogenicity of a protein having at least 80% sequence identity with a predetermined reference protein, such as VEGFR-2 having the amino acid sequence shown in SEQ ID NO: 1, is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1% compared to the reference protein (such as VEGFR-2 having the amino acid sequence shown in SEQ ID NO: 1) when measured by ELISA. Methods for designing and constructing protein homologs and methods for testing such homologs for their immunogenicity are well known to those skilled in the art. In certain embodiments, the amino acid sequence identity with the reference protein (such as VEGFR-2 having the amino acid sequence of SEQ ID NO: 1) is at least about 80%, at least about 85%, at least about 90%, or most preferably at least about 95%. Methods and algorithms for determining sequence identity, including comparison of a parent protein with its derivative having deletions, additions and / or substitutions with respect to the parent protein, are well known to those skilled in the art. At the DNA level, a nucleic acid sequence encoding a protein having at least about 80% sequence identity with a predetermined reference protein, such as VEGFR-2 having the amino acid sequence shown in SEQ ID NO: 1, may differ to an even greater extent due to the degeneracy of the genetic code.

[0052] In certain embodiments, the DNA molecule comprises a kanamycin antibiotic resistance gene, pMB1 ori, and a CMV promoter. In certain embodiments, the recombinant DNA molecule is derived from the commercially available pVAX1™ expression plasmid (Invitrogen, San Diego, California). This expression vector was modified by replacing the high-copy pUC origin of replication with the low-copy pMB1 origin of replication of pBR322. The low-copy modification was performed to reduce the metabolic burden and make its constructs more stable. The resulting expression vector backbone was named pVAX10.

[0053] In certain embodiments, the DNA molecule comprises the DNA sequence shown in SEQ ID NO: 2 (vector backbone pVAX10).

[0054] The expression plasmid pVAX10.VR2-1 was prepared by inserting the ORF encoding human VEGFR-2 having the amino acid sequence of SEQ ID NO: 1 into the expression vector backbone via NheI / XhoI (WO 2013 / 091898). The expression plasmid pVAX10.VR2-1 is depicted schematically in FIG. 9. The DNA vaccine comprising the attenuated Salmonella strain Ty21a containing the expression plasmid pVAX10.VR2-1 is designated VXM01 (WO 2013 / 091898).

[0055] In certain embodiments, the cancer immunotherapy is used in combination with chemotherapy, radiotherapy, or biological cancer therapy. In particular, in such embodiments, the attenuated strain of Salmonella is administered before, during, or after chemotherapy, radiotherapy, or biological cancer therapy, or before and during chemotherapy, radiotherapy, or biological cancer therapy. Complete eradication of cancer stem cells is essential for the cure of cancer. To obtain maximum efficacy, it would be beneficial to combine different treatment approaches.

[0056] In the context of the present invention, the term "biological cancer therapy" refers to cancer therapies involving the use of organisms, such as viruses, substances obtained from organisms, or variants of such substances produced in the laboratory. Some biological cancer therapies aim to stimulate the body's immune system to fight cancer cells (so-called biological cancer immunotherapy). Examples of biological cancer therapy approaches include the delivery of tumor antigens and tumor stromal antigens by Salmonella - based DNA vaccines, particularly DNA vaccines based on Salmonella typhi Ty21a, the delivery of therapeutic antibodies as drugs, the administration of immunostimulatory cytokines, and the administration of immune cells including engineered T cells. Therapeutic antibodies include antibodies that target tumor antigens or tumor stromal antigens.

[0057] In certain embodiments, biological cancer therapy includes the administration of at least one additional DNA vaccine (at least one additional attenuated Salmonella strain comprising at least one copy of a DNA molecule comprising an expression cassette encoding said antigen) encoding a tumor antigen and / or a tumor stromal antigen. In certain such embodiments, said at least one additional DNA vaccine encoding a tumor antigen and / or a tumor stromal antigen is selected from at least one additional attenuated strain of Salmonella comprising at least one copy of an additional DNA molecule comprising an additional expression cassette encoding a tumor antigen and / or a tumor stromal antigen. In particular, said at least one additional attenuated strain of Salmonella is Salmonella typhi Ty21a comprising an additional eukaryotic expression cassette.

[0058] In certain embodiments, the tumor antigen encoded by the at least one additional DNA vaccine is selected from the group consisting of human Wilms' tumor protein (WT1), human mesothelin (MSLN), human CEA, and CMV pp65. In particular, the tumor antigen encoded by the at least one additional DNA vaccine is human Wilms' tumor protein (WT1) having the amino acid sequence shown in SEQ ID NO: 3 and a protein having at least about 80% sequence identity therewith, human mesothelin (MSLN) having the amino acid sequence shown in SEQ ID NO: 4 and a protein having at least 80% sequence identity therewith, human CEA having the amino acid sequence shown in SEQ ID NO: 5 and a protein having at least 80% sequence identity therewith, CMV pp65 having the amino acid sequence shown in SEQ ID NO: 6 and a protein having at least 80% sequence identity therewith, CMV pp65 having the amino acid sequence shown in SEQ ID NO: 7 and a protein having at least 80% sequence identity therewith, and CMV pp65 having the amino acid sequence shown in SEQ ID NO: 8 and a protein having at least 80% sequence identity therewith, and is selected from the group consisting of. In particular, human Wilms' tumor protein (WT1) has the amino acid sequence shown in SEQ ID NO: 3, human CEA has the amino acid sequence shown in SEQ ID NO: 5, and CMV pp65 has the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In certain embodiments, the tumor stromal antigen encoded by the at least one additional DNA vaccine is selected from the group consisting of human fibroblast activation protein (FAP).

[0059] In certain embodiments, the attenuated strain of Salmonella encoding the VEGF receptor protein is administered before or simultaneously with at least one additional DNA vaccine encoding a tumor antigen and / or a tumor stromal antigen.

[0060] In the context of the present invention, the term "simultaneously with" means the administration on the same day, more specifically within 12 hours, and even more specifically within 2 hours, of an attenuated strain of Salmonella encoding a VEGF receptor protein and at least one additional DNA vaccine encoding a tumor antigen and / or a tumor stromal antigen.

[0061] In certain embodiments, the administration of an attenuated strain of Salmonella encoding a VEGF receptor protein and at least one additional DNA vaccine encoding a tumor antigen and / or a tumor stromal antigen is carried out for 8 consecutive weeks, more preferably within 3 to 6 consecutive weeks. The attenuated Salmonella strain encoding the VEGF receptor protein and at least one additional DNA vaccine encoding a tumor antigen or a tumor stromal antigen may be administered by the same route or by different routes. For example, in particular, when at least one additional DNA vaccine is an additional attenuated Salmonella strain, it may be administered orally.

[0062] The single dose of the additional attenuated strain of Salmonella may contain from about 10 5 to about 10 11 , in particular from about 10 6 to about 10 10 , more specifically from about 10 6 to about 10 9 , more specifically from about 10 6 to about 10 8 , most particularly from about 10 6 to about 10 7 colony forming units (CFU).

[0063] Chemotherapeutic agents that can be used in combination with the attenuated strain of Salmonella of the present invention include, for example, gemcitabine, amifostine (Ethyol), cabazitaxel, carboplatin, oxaliplatin, cisplatin, capecitabine, dacarbazine (DTIC), dactinomycin, docetaxel, mechlorethamine, streptozocin, cyclophosphamide, nimustine (ACNU), carmustine (BCNU), lomustine (CCNU), doxorubicin (Adriamycin), doxorubicin lipo (Doxil), folic acid, gemcitabine (Gemzar), daunorubicin, daunorubicin lipo (Daunoxome), epirubicin, procarbazine, ketoconazole, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil (5-FU), vinblastine, vincristine, bleomycin, paclitaxel (Taxol), docetaxel (Taxotere), pemetrexed, aldesleukin, asparaginase, busulfan, carboplatin, cladribine, camptothecin, CPT-11, 10-hydroxy-7-ethylcamptothecin (SN38), dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, idarubicin, mesna, interferon α, interferon β, irinotecan, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, oxaliplatin, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil, temozolomide, and combinations thereof.

[0064] The most preferred chemotherapeutic agents according to the present invention are cabazitaxel, carboplatin, oxaliplatin, cisplatin, cyclophosphamide, docetaxel, etoposide, gemcitabine, doxorubicin, lomustine, paclitaxel (Taxol), irinotecan, vincristine, vinblastine, vinorelbine, folic acid, 5-fluorouracil, bleomycin and temozolomide, particularly gemcitabine.

[0065] In certain embodiments, the cancer immunotherapy is used in combination with a combination of chemotherapy and radiotherapy. In such embodiments in particular, the chemotherapy includes administration of temozolomide.

[0066] In certain embodiments, an attenuated strain of Salmonella is administered orally. Oral administration is easier, safer, and less painful than parenteral administration. However, it should be noted that an attenuated strain of Salmonella encoding the VEGF receptor protein can also be administered by any other suitable route. Preferably, a therapeutically effective amount is administered to the patient, and this amount depends on the particular use, type of malignancy, weight, age, sex and health status of the patient, form of administration and prescription, etc., and the administration can be a single administration or multiple administrations as needed.

[0067] An attenuated strain of Salmonella encoding the VEGF receptor protein can be provided in solution, suspension, lyophilized form, enteric-coated capsule, or any other suitable dosage form. Typically, the attenuated strain of Salmonella is formulated as an oral liquid. This embodiment offers the advantage of improved patient compliance. Preferably, the oral liquid includes means for at least somewhat neutralizing gastric acid, i.e., means for neutralizing the pH of gastric juice to near pH 7. Preferably, the oral liquid is a buffered suspension containing an attenuated strain of Salmonella encoding the VEGF receptor protein. In certain embodiments, the buffered suspension is obtained by suspending the attenuated strain of Salmonella in a suitable buffer, preferably a buffer containing 2.6 g of sodium bicarbonate, 1.7 g of L-ascorbic acid, 0.2 g of lactose monohydrate, and 100 mL of drinking water.

[0068] An attenuated strain of Salmonella encoding the VEGF receptor protein is surprisingly effective at relatively low doses. The effectiveness of an attenuated strain of Salmonella encoding the VEGF receptor protein is particularly high in cancers having cancer-specific VEGF receptor protein expression. Administration of a low dose of live bacterial vaccine minimizes the risk of excretion and the risk of transmission to third parties.

[0069] In certain embodiments, the single dose of an attenuated strain of Salmonella encoding a VEGF receptor protein, particularly Salmonella typhi Ty21a encoding human VEGFR-2, is about 10 5 to about 10 11 , particularly about 10 6 to about 10 10 , more specifically about 10 6 to about 10 9 , particularly about 10 6 to about 10 8 , most particularly about 10 6 to about 10 7 colony forming units (CFU).

[0070] In this regard, the term "about" or "approximately" means within three times, or within two times, for example within 1.5 times, of a given numerical value or range.

[0071] In certain embodiments, the attenuated Salmonella strain is used in personalized cancer immunotherapy, which includes the step of evaluating, in a patient, the expression pattern of at least one VEGF receptor protein, particularly VEGFR-2, and / or the pre-immune response thereto. Alternatively, the attenuated Salmonella strain is used in cancer immunotherapy in a patient determined to have cancer characterized by cancer cells expressing a VEGF receptor protein (e.g., VEGFR-2), or having cancer cells expressing at least one VEGF receptor protein (e.g., VEGFR-2), by evaluating the expression pattern of at least one VEGF receptor protein, particularly VEGFR-2, and / or the pre-immune response thereto. The patient's VEGF receptor protein expression and / or the pre-immune response to the patient's VEGF receptor protein can be evaluated in an initial step, for example, by a companion diagnostic. Methods for evaluating the expression of a target gene, such as VEGFR-2, at either the mRNA level or the protein level are well known to those skilled in the art. For example, immunohistochemical staining, flow cytometry, or RNA sequencing, or alternative methods using labels can be used to identify the target expression level in the tumor. Similarly, methods for evaluating the pre-immune response of a patient to a specific protein, such as VEGFR-2, are well known to those skilled in the art. The patient's existing VEGFR-2-specific T cell pool can be detected, for example, by ELISpot or multimer FACS analysis. The occurrence of high levels of tumor-specific VEGFR-2 expression and / or a pre-immune response to VEGFR-2 is a prognostic indicator for knowing the quality of patients who will respond particularly favorably to treatment with an attenuated Salmonella strain encoding VEGFR-2.

[0072] Depending on the occurrence of possible side effects, it may be desirable to include treatment with an antibiotic or an anti-inflammatory agent.

[0073] If adverse events occur that resemble hypersensitivity reactions mediated by histamine, leukotrienes, or cytokines, treatment options for heat, anaphylaxis, blood pressure instability, bronchospasm, and dyspnea are available. Treatment options for unwanted T cell-derived autoaggression are derived from the standard treatment schemes for acute and chronic graft-versus-host disease applied after stem cell transplantation. Cyclosporine and glucocorticoids are proposed as treatment options.

[0074] In rare cases of systemic Salmonella typhi Ty21a infection, appropriate antibiotic therapy, such as therapy with fluoroquinolones like ciprofloxacin or ofloxacin, is recommended. Bacterial infections of the gastrointestinal tract are treated with agents such as rifaximin.

[0075] An attenuated strain of Salmonella encoding a VEGF receptor protein can be provided as a pharmaceutical composition. The pharmaceutical composition can be in the form of a solution, suspension, enteric-coated capsule, lyophilized powder, or any other form suitable for the intended use.

[0076] The pharmaceutical composition may further contain one or more pharmaceutically acceptable excipients.

[0077] In the context of the present invention, the term "excipient" refers to natural or synthetic substances formulated together with the active ingredient of a medicament. Suitable excipients include antiadherents, binders, coating agents, disintegrants, flavoring agents, coloring agents, lubricants, glidants, absorbents, preservatives, and sweetening agents.

[0078] In the context of the present invention, the term "pharmaceutically acceptable" refers to molecular entities and other ingredients of pharmaceutical compositions that are physiologically tolerable and that typically do not cause an adverse reaction when administered to a mammal (e.g., a human). The term "pharmaceutically acceptable" also means approved by a regulatory agency of the Federal or a State government for use in mammals, particularly humans, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia.

[0079] In certain embodiments, the pharmaceutical composition is provided as a drinking solution. This embodiment provides the advantage of improved patient compliance and enables a rapid, feasible and inexpensive mass vaccination program.

[0080] In particular, a suitable drinking solution includes means for at least partially neutralizing gastric acid, i.e., means for bringing the pH of gastric juice to around pH 7. In certain embodiments, the drinking solution is a buffered suspension obtained by suspending an attenuated strain of Salmonella according to the present invention in a suitable buffer, preferably a buffer that at least partially neutralizes gastric acid, preferably a buffer containing 2.6 g of sodium bicarbonate, 1.7 g of L-ascorbic acid, 0.2 g of lactose monohydrate and 100 mL of drinking water.

[0081] In certain embodiments, the cancer immunotherapy includes single or multiple administrations of an attenuated strain of Salmonella encoding a VEGF receptor protein or a pharmaceutical composition containing the same. The dose for each administration may be the same or different. In particular, the cancer immunotherapy includes 1, 2, 3, 4, 5 or 6 administrations of an attenuated strain of Salmonella encoding a VEGF receptor protein, wherein preferably the multiple administrations are carried out within 3 to 6 consecutive months.

[0082] Example 1. Treatment of VXM01 in patients with surgically resectable recurrent glioblastoma The purpose of this study was to investigate the safety, tolerability, immunity and biomarker response to the DNA vaccine VXM01 encoding VEGFR-2.

[0083] This study was conducted in patients with surgically resectable recurrence of glioblastoma in whom at least one standard treatment including chemoradiotherapy with temozolomide had failed. All patients received the DNA vaccine VXM01 as an additional therapy to their standard treatment.

[0084] This study consisted of a screening period, a treatment and observation period of up to 3 months, a tumor progression observation period from the 3rd month to the 12th month, and a boosting period from the 8th week to the 48th week during the tumor progression observation period. After the study ended, the patients were followed up for up to 2 years.

[0085] The treatment and observation period included a single oral administration of VXM01 on each of the 1st, 3rd, 5th, and 7th days, and a reoperation at the 5 ± 1st week after inclusion. During the boosting treatment period, VXM01 was administered at the 8th, 12th, 16th, 20th, 24th, 28th, 32nd, 36th, 40th, 44th, and 48th weeks in single oral boosting doses every 4 weeks.

[0086] VXM01 was administered orally at a single dose of 10 6 and 10 7 colony - forming units (CFU) / mL. Five out of nine glioblastoma patients showed a favorable disease course.

[0087] Patient 2605: Patient 2605 is a 55 - year - old female patient with recurrent glioblastoma WHO grade IV. Previous cancer treatments included the first surgery for glioblastoma and first - line chemoradiotherapy with 60 Gy, followed by temozolomide at a dose of 75 mg / m 2 2.

[0088] The patient was treated with VXM01 at a dose of 10 6 CFU. The VXM01 treatment was started with four initial doses on the test days of the 1st, 3rd, 5th, and 7th days. After a normal surgery was performed on the 35th day, the boosting administration every 4 weeks starting from the 8th week was continued. At the 10th week, lomustine / etoposide chemotherapy was started on top of VXM01.

[0089] The reference target lesion of the tumor was 25×10 mm at screening. The growth of the tumor size is summarized in Table 1.

[0090]

Table 1

[0091] The baseline and MRI images at day 35, 12 weeks, 20 weeks, and 76 weeks are depicted in Figure 10.

[0092] The tumor size tended to decrease from 28×13 mm to 25×12 mm between the 10th test day and the 35th definitive surgery day. According to the RANO criteria, this was determined to be stable disease (SD). At week 12, 7 weeks after the regular reoperation performed on day 35, the determination according to the RANO criteria was progressive disease (PD) due to the occurrence of new target lesions. No visually detectable "target lesions" were seen in the MRI report at 12 weeks after surgery. Lomustine / etoposide chemotherapy was initiated on top of VXM01. At 20 weeks (i.e., 15 weeks after reoperation), the tumor was determined to be stable (SD) according to the RANO criteria. At 36 weeks, the lomustine / etoposide chemotherapy was stopped, and the patient continued to receive VXM01 treatment every 4 weeks, and the treatment was not discontinued until the filing date of this application.

[0093] The Karnofsky index was 100% at screening and 90% at 12 weeks.

[0094] Immunohistochemical staining of the primary tumor specimen obtained before the test revealed that the tumor cells of this patient expressed VEGFR-2. In the recurrent tumor specimen on day 35, after treatment with VXM01, it was found that the tumor cells did not express VEGFR-2.

[0095] In tumor tissue immunohistochemistry, CD8 + T cells increased 2.3-fold in the recurrent tumor after VXM01 treatment compared to the primary tumor.

Claims

**Claim 1**: A pharmaceutical composition for the treatment of cancer in a patient, containing an attenuated strain of Salmonella containing at least one copy of a DNA molecule comprising a eukaryotic expression cassette encoding a VEGFR-2 protein, wherein the cancer is characterized by cancer cells expressing the VEGFR-2 protein, and the patient is a patient determined to have the cancer, and wherein the attenuated strain of Salmonella is Salmonella typhi Ty21a. **Claim 2** The pharmaceutical composition according to claim 1, wherein the cancer is selected from the group consisting of glioblastoma, carcinoid cancer, renal cancer, renal cell carcinoma, thyroid cancer, lung cancer, non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, prostate cancer, gastrointestinal cancer, colorectal cancer, colon cancer, skin cancer, and melanoma. **Claim 3** **Claim 4**: A pharmaceutical composition for the treatment of cancer in a patient containing cancer cells expressing at least one VEGFR-2 protein, containing an attenuated strain of Salmonella containing at least one copy of a DNA molecule comprising a eukaryotic expression cassette encoding a VEGFR-2 protein, and the patient is a patient determined to have the cancer, and wherein the attenuated strain of Salmonella is Salmonella typhi Ty21a. **Claim 5** The pharmaceutical composition according to any one of claims 1 to 3, wherein the eukaryotic expression cassette contains a CMV promoter. **Claim 7** The pharmaceutical composition according to any one of claims 1 to 4, wherein the VEGFR-2 protein is selected from the group consisting of VEGFR-2 having the amino acid sequence shown in SEQ ID NO: 1 and proteins having at least 90% sequence identity therewith. **Claim 9** The pharmaceutical composition according to any one of claims 1 to 5, wherein the DNA molecule contains a kanamycin antibiotic resistance gene, pMB1 ori, and a CMV promoter. **Claim 11** The pharmaceutical composition according to any one of claims 1 to 5, wherein the DNA molecule contains the DNA sequence shown in SEQ ID NO:

2. **Claim 13** The pharmaceutical composition according to any one of claims 1 to 7, wherein the cancer immunotherapy is used in combination with chemotherapy, radiotherapy, or biological cancer therapy. **Claim 15** The pharmaceutical composition according to claim 8, wherein the biological cancer therapy comprises administration of at least one additional DNA vaccine encoding a tumor antigen and / or a tumor stroma antigen, the additional DNA vaccine being selected from at least one attenuated strain of Salmonella comprising at least one copy of an additional DNA molecule comprising an additional expression cassette encoding a tumor antigen and / or a tumor stroma antigen, and the at least one attenuated strain of Salmonella being Salmonella typhi Ty21a comprising an additional eukaryotic expression cassette.

10. The pharmaceutical composition according to claim 9, wherein the tumor antigen encoded by the at least one additional DNA vaccine is selected from the group consisting of human Wilms' tumor protein (WT1), human mesothelin (MSLN), human CEA, and CMV pp65.

11. The pharmaceutical composition according to claim 10, wherein the tumor antigen is selected from the group consisting of human Wilms' tumor protein (WT1) having the amino acid sequence shown in SEQ ID NO: 3 and a protein having at least 90% sequence identity therewith, human mesothelin (MSLN) having the amino acid sequence shown in SEQ ID NO: 4 and a protein having at least 90% sequence identity therewith, human CEA having the amino acid sequence shown in SEQ ID NO: 5 and a protein having at least 90% sequence identity therewith, CMV pp65 having the amino acid sequence shown in SEQ ID NO: 6 and a protein having at least 90% sequence identity therewith, CMV pp65 having the amino acid sequence shown in SEQ ID NO: 7 and a protein having at least 90% sequence identity therewith, and CMV pp65 having the amino acid sequence shown in SEQ ID NO: 8 and a protein having at least 90% sequence identity therewith.

12. The pharmaceutical composition according to claim 9, wherein the tumor stroma antigen encoded by the at least one additional DNA vaccine is human fibroblast activation protein (FAP).

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the pharmaceutical composition containing the attenuated strain of Salmonella is administered orally.

14. The single-dose administration amount of the attenuated strain of Salmonella is 10 6 to 10 9 The pharmaceutical composition according to any one of claims 1 to 13, comprising colony forming units (CFU).

15. The single-dose administration amount of the attenuated strain of Salmonella is 10 6 to 10 8 CFU, and the pharmaceutical composition according to claim 14.

16. The pharmaceutical composition according to any one of claims 1 to 15 for use in personalized cancer immunotherapy, comprising the step of evaluating the expression pattern of VEGFR-2 protein and / or the immune response against VEGFR-2 protein in a patient.

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