Neoantigen targeting DNA vaccines for combination therapy

Abstract

The present invention relates to a Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens, and at least one engineered T cell, NKT cell, or NK cell comprising at least one tumor antigen-binding cell surface receptor for use in combination in the treatment of a solid tumor in a subject.

Description

[Technical field]

[0001] The present invention relates to a Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens, and at least one engineered T cell, NKT cell or NK cell comprising at least one tumor antigen-binding cell surface receptor for use in combination in the treatment of a solid tumor in a subject. [Background technology]

[0002] The knowledge that tumors can be immunogenic has led to the development of many cancer immunotherapies designed to harness the immune system to selectively eliminate malignant cells while sparing normal tissue. However, the survival benefit obtained from vaccination against tumor antigens alone remains modest. Anticancer vaccines face many challenges, one of which is the immunosuppressive microenvironment. The abnormal vasculature of tumors creates a hypoxic microenvironment that polarizes inflammatory cells toward immunosuppression. Furthermore, tumors systemically alter the proliferation, differentiation, and function of immune cells through the secretion of growth factors and cytokines.

[0003] For the cure of cancer, complete eradication of cancer stem cells is crucial. Many immune escape mechanisms of human tumors remain a major challenge in cancer immunotherapy. Thus, there is a great need for improved cancer treatment approaches, including combination approaches for cancer treatment, which have so far not been met.

[0004] Recently, adoptive cell therapy of cancer using reprogrammed T cells, such as CAR-T cells and CAR-NKT cells, as well as reprogrammed NK cells (CAR-NK cells), has shown promise. Early attempts have treated patients with a variety of solid and liquid tumors, but breakthroughs with CAR-T cell therapy have been achieved targeting B-cell hematological tumors. Two immunotherapies using anti-CD19 modified T cells, KYMRIAH (tisagenlecleucel) and YESCARTA (axicabtageneciloleucel), have recently been approved by the FDA. However, although CAR-T cell therapy is effective against some hematological cancers, its efficacy against common solid tumors is modest, and especially durable complete responses are rare.

[0005] While the main focus so far has been on improving CAR-T cells, the introduction of CARs into cell types other than traditional αβ T cells, such as γδ T cells, natural killer T (NKT) cells, and natural killer (NK) cells, is gaining importance.

[0006] Another immunotherapy approach that has attracted interest in cancer treatment is vaccination against cancer.There are several methods for immunization against cancer, but a very promising method is the use of bacteria such as Salmonella as a carrier of DNA vaccine against tumor antigens or stromal antigens.For example, WO2014 / 005683 discloses an attenuated strain of Salmonella that contains a recombinant DNA molecule that codes for VEGF receptor protein for use in cancer immunotherapy, particularly in the treatment of pancreatic cancer.

[0007] Furthermore, WO2014 / 173542 and WO2015 / 090584 disclose attenuated strains of Salmonella comprising recombinant DNA molecules encoding Wilms tumor protein or mesothelin for use in cancer immunotherapy.

[0008] WO2013 / 09189 discloses a method for propagating an attenuated mutant Salmonella typhi strain lacking galactose epimerase activity and carrying a recombinant DNA molecule, and WO2018 / 011289 discloses a rapid and effective method for generating personalized cancer vaccines comprising attenuated Salmonella strains. Summary of the Invention

[0009] Object of the invention In light of the prior art, it is an object of the present invention to provide a novel cancer treatment, which would offer major advantages for improving treatment options for cancer patients with solid tumors.

[0010] Summary of the Invention In one embodiment, the present invention relates to a Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens for use in treating a solid tumor in a subject, wherein the subject has been or will be treated with at least one engineered T cell, NKT cell or NK cell comprising at least one tumor antigen-binding cell surface receptor.

[0011] In another aspect, the present invention relates to a Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens in combination with at least one engineered T cell, NKT cell or NK cell comprising at least one tumor antigen-binding cell surface receptor for use in treating a solid tumor in a subject.

[0012] According to the present invention, the at least five or more neoantigens are tumor-specific antigens identified in a solid tumor of the subject. Preferably, the five or more neoantigens comprise CD8 T cell antigens or CD8 and CD4 T cell antigens. In one embodiment, the at least one polypeptide comprises 10 or more, preferably 20 or more, preferably 30 or more, preferably 50 or more neoantigens. In another embodiment, the at least one polypeptide comprises 5-300 neoantigens, 10-300 neoantigens, 20-300 neoantigens, preferably 30-300 neoantigens, preferably 50-300 neoantigens. In yet another embodiment, the at least one polypeptide comprises 10-200 neoantigens, 10-200 neoantigens, 20-200 neoantigens, preferably 30-200 neoantigens, preferably 50-200 neoantigens.

[0013] The Salmonella typhi Ty21a strain can further comprise a DNA molecule encoding at least one polypeptide comprising a tumor-specific and / or tumor-associated antigen that is not a neoantigen, wherein said tumor-specific and / or tumor-associated antigen that is not a neoantigen is expressed in said solid tumor, and wherein the at least one polypeptide comprising the tumor-specific and / or tumor-associated antigen that is not a neoantigen is (a) encoded by the same DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens or is encoded by a further separate DNA molecule, (b) encoded by at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens or is encoded by a further separate expression cassette, or (c) at least one polypeptide comprising five or more neoantigens or is a further separate polypeptide.

[0014] According to the present invention, the Salmonella typhi strain Ty21a can be administered simultaneously with at least one checkpoint inhibitor, preferably selected from the group consisting of antibodies against PD-1, PD-L1, CTLA-4, IDO, GITR, OX40, TIM-3, LAG-3, KIR, CSF1R and CD137.

[0015] The at least one engineered T cell, NKT cell, or NK cell comprising at least one tumor antigen-binding cell surface receptor may be a T cell, NKT cell, or NK cell comprising a chimeric antigen receptor (CAR), also referred to as a chimeric antigen receptor (CAR)-T cell, a CAR-NKT cell, or a CAR-NK cell, respectively. In one embodiment, the Salmonella typhi Ty21a strain is administered after adoptive cell transfer of at least one engineered T cell, NKT cell, or NK cell comprising at least one tumor antigen-binding cell surface receptor. Preferably, the Salmonella typhi Ty21a strain is administered about 2 weeks to 4 months, preferably 2 to 3 months, after the first adoptive cell transfer of at least one engineered T cell, NKT cell, or NK cell comprising at least one tumor antigen-binding cell surface receptor.

[0016] In one embodiment, the subject has undergone lymphodepleting chemotherapy prior to adoptive cell transfer of at least one engineered T cell, NKT cell, or NK cell comprising at least one tumor antigen-binding cell surface receptor, where the subject in need of treatment is immunocompromised and requires normalization of lymphocyte and / or white blood cell counts prior to administration of Salmonella Typhi Ty21a strain.

[0017] According to the present invention, a Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens is co-administered with at least one Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding a tumor antigen, a tumor stromal antigen and / or a checkpoint inhibitor antigen, preferably an antigen selected from the group consisting of human Wilms tumor protein (WT1), human mesothelin (MSLN), human CEA, CMV pp65, human PD-L1, VEGFR-2 and human fibroblast activation protein (FAP). More specifically, VEGFR-2 may comprise the amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:1; WT1 may comprise the amino acid sequence of SEQ ID NO:3, or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:3; MSLN may comprise the amino acid sequence of SEQ ID NO:4, or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:4; human CEA may comprise the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:5; CMV pp65 may comprise the amino acid sequence of SEQ ID NO:6, 7 or 8, or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:6, 7 or 8; and / or human PD-L1 may comprise the amino acid sequence of SEQ ID NO:9, or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:9.

[0018] In one embodiment, the Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens is in the form of a pharmaceutical composition and may further comprise at least one pharma- ceutically acceptable excipient. The pharmaceutical composition may further comprise at least one Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding a tumor antigen and / or a tumor stromal antigen, preferably an antigen selected from the group consisting of WT1, MSLN, CEA, CMV pp65, PD-L1, VEGFR-2 and FAP.

[0019] The solid tumor to be treated can be any solid tumor, such as colon cancer, pancreatic cancer, lung cancer, ovarian cancer, mesothelioma, glioblastoma, gastric cancer, hepatocellular carcinoma, renal cell carcinoma, prostate cancer, cervical cancer, breast cancer, or melanoma.

[0020] A single dose of the Salmonella typhi Ty21a strain according to the present invention is about 10 6 ~about 10 10 , more specifically, about 10 6 ~about 10 9 , more specifically, about 10 6 ~about 10 8 , most specifically about 10 7 ~about 10 8 In one embodiment, the Salmonella typhi Ty21a strain of the present invention is administered 2-4 times in the first week, preferably 4 times in the first week, followed by single booster doses every 2-4 weeks, particularly on days 1 and 7, preferably on days 1, 3, 5 and 7, followed by single booster doses every 2-4 weeks. [Brief description of the drawings]

[0021] [Figure 1A-B] Frequencies of the indicated epitope-specific CD8+ T cell populations in splenocytes from C57BL / 6 mice orally immunized with (A) empty vector and (B) VXMNeo1m. Percentages of epitope-specific pentamer-positive CD8+ T cells among total CD8+ T cells for the indicated epitope are shown. [Figure 1C] (C) Frequency of the indicated epitope-specific CD8+ T cell populations in splenocytes of C57BL / 6 mice orally immunized with VXM06m. Percentages of epitope-specific pentamer-positive CD8+ T cells among total CD8+ T cells for the indicated epitope are shown. [Diagram 2] Drug Product Stability Testing. Final drug products (Product 1, Product 2 and Product 3) manufactured from three constructs encoding three target antigens based on the Salmonella Typhi Ty21a delivery platform at 104 (P4), 105 (P5), 106 (P6) or 107 (P7) CFU / ml were incubated at ≤70°C for the indicated times. Viable counts (CFU / ml) are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present invention may be understood more readily by reference to the following detailed description of the invention.

[0023] In one embodiment, the present invention relates to a Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens. The Salmonella typhi Ty21a strain encoding at least one polypeptide comprising five or more neoantigens can be used for treating a solid tumor in a subject. In particular, the at least five or more neoantigens are tumor-specific antigens identified in a solid tumor of said subject.

[0024] The present invention further relates to a Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens for use in treating a solid tumor in a subject, wherein the subject has been treated or is treated with at least one engineered T cell, NKT cell or NK cell comprising at least one tumor antigen-binding cell surface receptor. In particular, the at least five or more neoantigens are tumor-specific antigens identified in the solid tumor of the subject. Furthermore, the at least one tumor antigen-binding cell surface receptor binds to at least one tumor antigen identified to be expressed or overexpressed in the solid tumor of the subject. Thus, preferably, at least one tumor antigen is identified to be expressed or overexpressed in the solid tumor of the subject, and the subject has been treated or is treated with at least one engineered T cell, NKT cell or NK cell comprising at least one tumor antigen-binding cell surface receptor targeting at least one tumor antigen identified to be expressed or overexpressed in the solid tumor of the subject.

[0025] Furthermore, the present invention relates to a Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens in combination with at least one engineered T cell, NKT cell or NK cell comprising at least one tumor antigen-binding cell surface receptor for use in treating a solid tumor in a subject. Alternatively, a Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens is used in combination with at least one engineered T cell, NKT cell or NK cell comprising at least one tumor antigen-binding cell surface receptor for treating a solid tumor in a subject. In particular, the at least five or more neoantigens are tumor-specific antigens identified in the solid tumor of said subject. Furthermore, the at least one tumor antigen-binding cell surface receptor binds to at least one tumor antigen identified as expressed or overexpressed in the solid tumor of the subject. Thus, preferably, at least one tumor antigen is identified as being expressed or overexpressed in a solid tumor of the subject, and the subject has been treated or is to be treated with at least one engineered T cell, NKT cell or NK cell that comprises at least one tumor antigen-binding cell surface receptor that targets at least one tumor antigen identified as being expressed or overexpressed in a solid tumor of the subject.

[0026] In another aspect, the present invention relates to a method of treating a solid tumor in a subject, the method comprising administering to the subject a Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens, the subject being treated or being treated with at least one engineered T cell, NKT cell or NK cell comprising at least one tumor antigen-binding cell surface receptor. More specifically, the method comprises administering to the subject at least one engineered T cell, NKT cell or NK cell comprising at least one tumor antigen-binding cell surface receptor, and then administering to the subject a Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens. The at least one engineered T cell, NKT cell or NK cell comprising at least one tumor antigen-binding cell surface receptor is administered to the subject by adoptive cell transfer. The method may further include identifying at least five or more neoantigens as tumor-specific antigens in the solid tumor of the subject, and generating a Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising the five or more neoantigens. The method may further include identifying at least one tumor antigen identified as expressed or overexpressed in the solid tumor of the subject, and administering at least one engineered T cell, NKT cell, or NK cell comprising at least one tumor antigen-binding cell surface receptor that targets the at least one tumor antigen identified as expressed or overexpressed in the solid tumor of the subject.

[0027] According to the present invention, the attenuated Salmonella strain serves as a bacterial carrier of a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens for the delivery of said DNA molecule to a target cell. Preferably, the DNA molecule is part of a plasmid comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens. Such a bacterial carrier or delivery vector comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens may also be referred to as a DNA vaccine.

[0028] In the context of the present invention, the term "vaccine" refers to an agent capable of inducing an immune response in a subject upon administration. The vaccine is preferably capable of preventing, ameliorating or treating a disease. In the context of the present invention, the vaccine is preferably an oral vaccine. The Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens according to the present invention can be abbreviated as "Salmonella typhi Ty21a strain encoding at least one polypeptide comprising five or more neoantigens" or "neoantigen cancer vaccine".

[0029] Neoantigen cancer vaccine The live attenuated Salmonella strain, more specifically S. typhi Ty21a strain, according to the present invention stably carries a recombinant DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens. It is used as a carrier for oral delivery of said recombinant DNA molecule. The term "attenuated strain of S. typhi Ty21a" as used herein refers to an attenuated strain of Salmonella, more specifically S. typhi, where the attenuated strain is Ty21a and is used interchangeably herein with "S. typhi Ty21a".

[0030] Genetic immunization may have advantages over conventional vaccination: the target DNA can be detected for a significant period of time, thus acting as a depot of antigens. Some plasmid sequence motifs, such as CpG islands, are immunostimulatory and can function as adjuvants that are enhanced by immune stimulation by LPS and other bacterial components.

[0031] Live attenuated Salmonella vectors produce autoimmune regulators such as lipopolysaccharide (LPS) in situ, which is an advantage over other forms of administration such as microencapsulation. Furthermore, the mucosal vaccine according to the invention has an intralymphatic mode of action that has proven beneficial. After ingestion of the attenuated vaccine according to the invention, macrophages and other cells in the intestinal Peyer's patches are invaded by the modified bacteria. The bacteria are taken up by these phagocytes. Due to their attenuating mutations, the bacteria of the Salmonella Typhi Ty21 strain cannot persist in these phagocytes and die. The recombinant DNA molecule is released and then transferred to the cytoplasm of phagocyte immune cells by a specific transport system or endosomal leakage. Finally, the recombinant DNA molecule enters the nucleus, where it is transcribed and expresses a large amount of a polypeptide containing five or more neoantigens in the cytoplasm of the phagocyte. The infected cells undergo apoptosis and are loaded with the polypeptide containing five or more neoantigens, which are taken up and processed by the intestinal immune system. Danger signals from bacterial infection act as a powerful adjuvant in this process, eliciting strong target antigen-specific CD8+ T cell and antibody responses at the level of the systemic and mucosal compartments. The immune response peaks 10 days after vaccination. The lack of an anti-carrier response allows for multiple boosts with the same vaccine.

[0032] In the context of the present invention, the term "attenuated" refers to a bacterial strain that has a reduced virulence due to an attenuating mutation compared to a parental bacterial strain that does not have the attenuating mutation. Preferably, the attenuated bacterial strain has lost virulence but retains the ability to induce protective immunity. Attenuation can be achieved by deletion of various genes, including virulence genes, regulatory genes, and metabolic genes. Attenuated bacteria may be found in nature or may be artificially produced in the laboratory, for example, by adaptation to a new medium or cell culture, or may be produced by recombinant DNA technology. Approximately 10% of the attenuated strains of Salmonella according to the present invention have been isolated. 11 Administration of CFU preferably causes Salmonellosis in less than 5%, more preferably less than 1%, and most preferably less than 1% of subjects. The Ty21a strain of the present invention is an attenuated strain of Salmonella typhi.

[0033] In the context of the present invention, the term "comprise" or "comprising" means "including, but not limited to." The term is intended to be open-ended to specify the presence of any stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups. Thus, the term "comprising" includes the more restrictive terms "consisting of" and "consisting essentially of." In one embodiment, the term "comprising" as used throughout the application, and in particular in the claims, can be replaced with the term "consisting of." As used herein, the term "a" can include a plurality, including, but not limited to, "one."

[0034] The term "neoantigens" as used herein refers to peptides generated from somatically mutated genes that are expressed only in cancer cells but not in normal tissues of the same patient. Genes and chromosomes can undergo mutations in both somatic and germline tissues. Contrary to germline mutations, somatic mutations are not transmitted to offspring. Thus, somatic mutations of genes are acquired in cancer cells and during cancer development. Typically, the mutations are tumor-specific point mutations that generate neoepitopes, also called mutant epitopes or point mutant peptides. These are highly immunogenic because they are not present in normal tissues and therefore bypass central thymic tolerance. Neoantigens preferably consist of neoepitopes that are presented as peptides by MHC I or MHC II. The mutations may also be frameshift mutations resulting in frameshift peptide (FSP) antigens. FSP neoantigens encompass long antigenic amino acid stretches that are caused by the insertion or deletion of a single nucleotide but can contain multiple immunologically relevant neoepitopes. In certain embodiments, the term "neoantigen" further includes T cell epitopes associated with peptide processing (TEIPP). TEIPP is derived from a ubiquitously expressed non-mutated "self" protein that is not loaded on MHC I in healthy cells. In immune-evading cancers, antigen processing components such as transporters associated with antigen processing (TAP) are often downregulated. Therefore, TEIPP can be presented on the surface of cancer cells only in cells with defects in antigen processing equipment, such as the absence of TAP due to mutation or epigenetic silencing (Marjit et al., Journal of Experimental Medicine, 2018, 215(9):2325).

[0035] During cancer progression, mutations accumulating in the cancer genome can affect protein-coding genes, leading to changes in the protein sequence. Mutant proteins are proteolytically cleaved into short peptides and presented on the tumor cell surface by the human MHC (human leukocyte antigen (HLA)). These somatically mutated genes, i.e. neoantigens, present in malignant cells but not in normal cells, can be recognized as foreign by tumor-infiltrating lymphocytes (TILs). The term "neoantigen" therefore preferably refers to peptides consisting of peptides containing somatic mutations presented by MHC I or II. Antigens presented by MHC I are also called CD8 T cell antigens. Antigens presented by MHC II are also called CD4 T cell antigens (or T helper antigens). As neoantigens can be recognized as foreign by TILs, they can induce a strong tumor-specific immune response. Neoantigens released after tumor cell death initiate many processes that ultimately lead to T cells recognizing cancer cells through the interaction of specific T cell receptors (TCRs) with specific neoantigen-MHC complexes.

[0036] The term "at least one polypeptide comprising five or more neoantigens" as used herein refers to one polypeptide or one or more polypeptides that together comprise five or more neoantigens. It is not important whether the five or more neoantigens are part of the same polypeptide or different polypeptides. Thus, the five or more neoantigens may be expressed as one polypeptide or as two or more polypeptides. Preferably, the neoantigens contained within the at least one or more polypeptides are 10 or more, 20 or more, 30 or more, 50 or more, or more than 50 neoantigens. In the context of Salmonella typhi Ty21a strain as used herein, an insert encoding at least one polypeptide may contain up to 300 neoantigens, preferably up to 200 neoantigens. Antigens presented as peptides on MHC class I or II (human HLA) are typically 11-30 amino acids for MHC II (CD4 antigen) and 8-10 amino acids for MHC I (CD8 antigen). Thus, preferred ranges of neoantigens contained within at least one polypeptide are 5-300, 10-300, 20-300, 30-300, 50-300, or more than 50-300 neoantigens. More preferred ranges of neoantigens contained within at least one polypeptide are 5-200, 10-200, 20-200, 30-200, 50-200, or more than 50-200 neoantigens. Each polypeptide containing the fused neoantigen is proteolytically cleaved into neoantigens in antigen presenting cells and presented via HLA to induce T cell responses.

[0037] According to the present invention, the five or more neoantigens may include CD8 T cell antigens and / or CD4 T cell antigens. Preferably, the five or more neoantigens include CD8 T cell antigens and CD4 T cell antigens.

[0038] It is hypothesized that vaccination with neoantigens could expand existing neoantigen-specific T cell populations and induce a broader repertoire of new T cell specificities in cancer patients.

[0039] Neoantigens are typically peptides having 8 to 30 amino acids, preferably 8 to 20 amino acids, and more preferably 8 to 12 amino acids.

[0040] In neoantigen cancer vaccines, it is beneficial if the vaccine targets multiple neoantigens, thereby reducing the risk of immune evasion due to loss of expression of a subset of neoantigens. Also encompassed by the present invention is sequential treatment of patients with different Salmonella typhi Ty21a strains comprising DNA molecules comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens, including targeting selected neoantigens or new subsets of neoantigens during tumor progression.

[0041] The advantage of the attenuated strain of Salmonella typhi Ty21a (also called "S. typhi Ty21a") as a carrier of at least one polypeptide containing five or more neoantigens is the absence of plasmid variability, the need for propagation and the need for sterility testing by oral administration, only in the insert coding for one or more neoantigens, which is an established quality control assay. Furthermore, the expression plasmid suitable for transformation, as well as the Salmonella typhi Ty21a strain as a carrier, allows a large number (up to 300) of epitopes (neoantigens). The neoantigens can be inserted into the plasmid as strings of beads (expressed as one or more polypeptides) separated by linkers, if desired. The linkers can be, but are not limited to, GS linkers, 2A cleavage sites, or IRES sequences. Due to the need for rapid production and limited quality control, the time to generate a Salmonella typhi Ty21a strain containing a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens is short and can be achieved, for example, within 15 days, preferably within 14 days after identification of the neoantigens. 11 Due to the high yield of bacteria with a net yield of colony forming units (CFU), overnight fermentation is sufficient and no upscaling is necessary. This allows for short production times and low manufacturing costs. Furthermore, each lot is sufficient for years of treatment and the formulation has been shown to be stable for at least three years. Thus, no batch variation occurs as one batch lasts for the entire treatment of a subject with a solid tumor.

[0042] A method for generating a S. typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette comprising five or more neoantigens for an individual subject having a solid tumor comprises the steps of: (a) providing a tumor cell sample and a control sample from said subject; (b) identifying five or more neoantigens present in the tumor cell sample that are not present in the control sample; (c) selecting cDNAs encoding the five or more neoantigens; (d) synthesizing cDNAs comprising the five or more neoantigens; (e) cloning the cDNAs into at least one eukaryotic expression cassette; (f) transforming a recipient S. typhi Ty21a strain with a DNA molecule comprising at least one eukaryotic expression cassette comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising the five or more neoantigens; (f) fermenting the strain obtained in step (f) and diluting to a target concentration based on CFU; and (g) analyzing the transformed S. typhi Ty21a including sequencing the cDNAs encoding at least one polypeptide comprising the five or more neoantigens. The control sample can be any sample of normal tissue or blood from the subject to be treated. Preferably, the control sample is a blood sample. The blood sample can further be used for HLA typing of the patient. The sample of tumor cells can be a tumor biopsy.

[0043] Methods are known in the art to detect (all) mutations in tumors and reliably predict or determine mutant peptides with high affinity binding of self-human leukocyte antigen (HLA) molecules. For example, whole exome sequencing (WES) of matched tumor and normal cell DNA from individual patients can be performed. Identified somatic mutations are then orthogonally validated and assessed for expression of mutant alleles by RNA sequencing of tumors. Peptides predicted to bind to the patient's self-HLA-A or HLA-B proteins are then selected. This can be confirmed, for example, by ex vivo interferon gamma enzyme-linked immunospot (ELISPOT). Alternatively, HLA-peptide ligands can be isolated from cell culture media and identification can be performed by LC-MS / MS analysis.

[0044] A polypeptide may contain multiple neoantigens fused to each other, preferably 5 or more, 10 or more, 20 or more, 30 or more, or 50 or more neoantigens. In a typical plasmid used to transfect Salmonella typhi Ty21a strain, such as the pVAX1™ expression plasmid (Invitrogen, San Diego, California) or its derivative pVAX10, up to about 300 neoantigens can be expressed. Thus, a polypeptide may contain about 5-300, 10-300, 20-300, 300-300, or 50-300 neoantigens, preferably 10-200, 20-200, 30-300, or 50-200 neoantigens. The polypeptide is cleaved into peptides in the cell and presented on MHC I or MHC II molecules depending on the type of neoantigen. Individual neoantigens can be separated by linkers, such as GS linkers, specifically designed linkers or 2A cleavage sites. DNA molecules encoding neoantigens can also be separated by IRES sequences, resulting in separate polypeptides.

[0045] The Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens further comprises a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising at least one tumor antigen that is not a neoantigen and / or a tumor stromal antigen, wherein said at least one tumor antigen that is not a neoantigen is expressed in a solid tumor of the patient to be treated. The at least one polypeptide comprising at least one tumor antigen that is not a neoantigen and / or a tumor stromal antigen may be (a) encoded by the same DNA molecule comprising at least one eukaryotic expression cassette comprising at least one polypeptide comprising five or more neoantigens or by a further separate DNA molecule, (b) encoded by at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens or by a further separate expression cassette, or (c) at least one polypeptide comprising five or more neoantigens or a further separate polypeptide. Thus, the S. typhi Ty21a strain can be transformed with two DNA molecules, the first one encoding five or more neoantigens and the second one encoding at least one tumor antigen that is not a neoantigen and / or tumor stromal antigen. Alternatively, the S. typhi Ty21a strain can be transformed with one DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens and at least one further eukaryotic expression cassette encoding at least one tumor antigen that is not a neoantigen and / or tumor stromal antigen. Alternatively, the S. typhi Ty21a strain can be transformed with one DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens and further comprising at least one tumor antigen that is not a neoantigen and / or tumor stromal antigen.Examples of tumor antigens in this context include, but are not limited to, WT1, MSLN, CEA, HER2, EGFR, FBP, GD2, GD3, MAGE-A1, PSCA, PSMA, MUC1, GPC3 and CMVpp65. The tumor antigen may be a tumor-specific antigen or a tumor-associated antigen. The term "tumor-specific antigen" as used herein refers to an antigen that is expressed in tumors but not in normal tissues. The term "tumor-associated antigen" as used herein refers to an antigen that is overexpressed in tumors compared to normal tissues. The term "tumor stromal antigen" as used herein refers to an antigen that is expressed in tumor stroma, including, but not limited to, VEGFR-2 and FAP. The Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens may comprise a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising a checkpoint inhibitor antigen, wherein said at least one checkpoint inhibitor antigen or its ligand is overexpressed in the solid tumor of the patient to be treated. The same applies to the expression of checkpoint inhibitor antigens in the Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens, as well as to at least one tumor antigen that is not a neoantigen and / or a tumor stromal antigen. An example of a checkpoint inhibitor antigen is PD-1 or PD-L1. The DNA molecule used in this context is preferably an expression plasmid.

[0046] A DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens may also be referred to as a recombinant DNA molecule, i.e. an engineered DNA construct, preferably composed of DNA fragments of different origins. The DNA molecule may be a linear nucleic acid or, preferably, a circular DNA plasmid, generated by introducing an open reading frame encoding at least one polypeptide comprising five or more neoantigens into a eukaryotic expression cassette of a plasmid. A plasmid comprising a eukaryotic expression cassette may also be referred to as a eukaryotic expression plasmid.

[0047] 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 is preferably capable of mediating the transcription in a eukaryotic target cell of the enclosed open reading frame encoding at least one polypeptide comprising five or more neoantigens. Eukaryotic expression cassettes typically comprise a promoter, at least one open reading frame, and a transcription termination signal that allows expression in a eukaryotic target cell.

[0048] In a particular embodiment, a single dose of Salmonella typhi Ty21a strain is about 10 6 ~about 10 10 , more specifically, about 10 6 ~about 10 9 , more specifically, about 10 7 ~about 10 9 , more specifically, about 10 6 ~about 10 8 , most specifically about 10 6 ~about 10 7 1000 colony forming units.

[0049] More specifically, a single dose of Salmonella typhi Ty21a strain is approximately 1×10 6 ~Approx. 1×10 10 , more specifically, about 1 × 10 6 ~Approx. 1×10 9, more specifically, about 1 × 10 7 ~Approx. 1×10 9 , more specifically, about 1 × 10 6 ~Approx. 1×10 8 , especially in detail, about 1 × 10 6 ~Approx. 1×10 7 Contains colony forming units (CFU).

[0050] Furthermore, the S. typhi strain S. typhi Ty21a of the present invention is preferably administered 2-4 times in the first week, preferably 4 times in the first week, followed by booster doses every 2-4 weeks, particularly on days 1 and 7, preferably on days 1, 3, 5 and 7, followed by booster doses of a single dose every 2-4 weeks.

[0051] In this context, the term "about" or "approximately" means within a factor of 3, or within a factor of 2, including within a factor of 1.5, of a given value or range.

[0052] In a particular embodiment, the treatment comprises a single or multiple administrations of the Salmonella typhi Ty21a strain encoding at least one polypeptide comprising five or more neoantigens or the pharmaceutical composition according to the present invention. The single doses of administration may be the same or different as disclosed herein, preferably the same, and preferably within the ranges disclosed herein. In particular, the treatment comprises 2-4 primary vaccinations in the first week of treatment, followed by a single booster vaccination every 2-4 weeks of the Salmonella typhi Ty21a strain encoding at least one polypeptide comprising five or more neoantigens or the pharmaceutical composition according to the present invention.

[0053] The Salmonella typhi Ty21a strain encoding at least one polypeptide comprising five or more neoantigens is for use in treating a solid tumor in a subject, where the subject has been treated or will be treated with at least one engineered T cell, NKT cell or NK cell comprising at least one tumor antigen-binding cell surface receptor.

[0054] The solid tumor treated according to the present invention may be any solid tumor, in particular a solid tumor selected from colon cancer, pancreatic cancer, lung cancer, ovarian cancer, mesothelioma, glioblastoma, gastric cancer, hepatocellular carcinoma, renal cell carcinoma, prostate cancer, cervical cancer, breast cancer and melanoma.

[0055] Engineered T cells, NKT cells or NK cells A Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens is administered to a subject having a solid tumor for use in treating the solid tumor. In one embodiment, the subject has been treated or is further treated with at least one engineered T cell, NKT cell or NK cell comprising at least one tumor antigen-binding cell surface receptor. Preferably, at least one engineered T cell, NKT cell or NK cell comprising at least one tumor antigen-binding cell surface receptor is provided to a subject having a solid tumor by adoptive cell transfer (ACT), i.e., intravenous injection of at least one engineered T cell, NKT cell or NK cell. As used herein, the term "adoptive cell transfer" refers to the transfer of cells into a patient or subject. In the context of at least one engineered T cell, NKT cell or NK cell, it is used synonymously with "administered" or "to be administered." The term "at least one engineered T cell, NKT cell or NK cell" as used herein means "at least one engineered T cell comprising at least one tumor antigen-binding cell surface receptor, at least one engineered NKT cell comprising at least one tumor antigen-binding cell surface receptor, at least one engineered NK cell comprising at least one tumor antigen-binding cell surface receptor" and can be abbreviated to "at least one engineered T cell, NKT cell or NK cell, or "at least one engineered T cell, at least one engineered NKT cell, or at least one engineered NK cell". More specifically, the at least one engineered T cell comprising at least one tumor antigen-binding cell surface receptor can be an engineered conventional αβ T cell or an engineered γδ T cell, and preferably, the at least one engineered T cell comprising at least one tumor antigen-binding cell surface receptor is an engineered conventional αβ T cell.

[0056] At least one engineered T cell, NKT cell or NK cell comprising at least one tumor antigen-binding cell surface receptor is usually custom manufactured. This requires identifying the tumor antigen expressed in the solid tumor of the subject to be treated. Identifying the tumor antigen expressed in the solid tumor of the subject to be treated comprises (a) providing a tumor cell sample and a control sample from said subject, and (b) identifying the tumor antigen expressed in the tumor cell sample that is not present in the control sample. The tumor cell sample may be a tumor biopsy. Furthermore, it should be understood that the tumor cell sample includes solid tumor tissue as well as tumor stromal tissue. Identifying the tumor antigen expressed in the solid tumor of the subject to be treated to generate at least one engineered T cell, NKT cell or NK cell and identifying the five or more neoantigens expressed in the solid tumor of the subject to be treated to generate the Salmonella typhi Ty21a strain encoding at least one polypeptide comprising five or more neoantigens can be performed simultaneously or at different times, and / or can be performed in the same tumor cell sample or different tumor cell samples. Preferably, identifying five or more neoantigens and tumor antigens and / or tumor stromal antigens expressed in a solid tumor or in the stroma of a solid tumor of a subject to be treated comprises the steps of: (a) providing a tumor cell sample from said subject and a control sample; and (b) identifying five or more neoantigens and / or tumor stromal antigens and / or tumor stromal antigens present in the tumor cell sample that are not present in the control sample. The tumor antigen targeted by the at least one tumor antigen-binding cell surface receptor may be identical to or different from at least one of the neoantigens targeted by the Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens.

[0057] At least one engineered T cell, NKT cell or NK cell comprising at least one tumor antigen-binding cell surface receptor can be an autologous or allogeneic T cell, NKT cell or NK cell. The term "autologous" means that the T cell, NKT cell or NK cell is obtained from a patient, genetically engineered by introducing a nucleic acid molecule encoding at least one tumor antigen-binding cell surface receptor, expanded in vitro and returned to the subject to be treated. Preferably, the engineered T cell is an autologous T cell, i.e. originates from the subject to be treated. The T cells, in particular the engineered conventional αβ T cells, may be allogeneic to the subject to be treated, i.e. originates from another healthy donor. The engineered NKT cell as well as the engineered NK cell can be an autologous or allogeneic NKT cell or NK cell, respectively. The adoptive cell transfer of T cells, in particular conventional αβ T cells, carries the risk of graft-versus-host disease (GvHD), which is less than in the case of adoptive cell transfer of NKT cells, NK cells or γδ T cells. The allogeneic engineered T cells, NKT cells, or NK cells containing at least one tumor antigen-binding cell surface receptor can be pre-made or commercially manufactured. Furthermore, for NK cells, the use of allogeneic cells has the advantage that NK cells are less likely to be inhibited by KIR signaling induced by self-MHC molecules.

[0058] The term "engineered" as used herein means "modified" to express at least one tumor antigen-binding cell surface receptor on the cell surface. A gene or mRNA encoding at least one tumor antigen-binding cell surface receptor on the cell surface is introduced into the T cell, NKT cell or NK cell, preferably by transfection or transduction. The T cell, NKT cell or NK cell may be transfected or transduced with RNA or DNA encoding at least one tumor antigen-binding cell surface receptor. The engineered T cell, NKT cell or NK cell is then grown or expanded ex vivo and then reinjected into the subject. Suitable vectors for gene delivery are known in the art and include, for example, viral vectors such as retroviral and lentiviral vectors, or transposons such as Piggy-Bac (PB) and Sleeping Beauty (SB). RNA transiently engineered T cell, NKT cell or NK cell, preferably CAR-T cell, CAR-NKT cell or CAR-NK cell, are also envisioned by the present invention.

[0059] In some embodiments, at least one engineered T cell, NKT cell, or NK cell comprises at least one tumor antigen-binding cell surface receptor on its cell surface, where the tumor antigen is selected from carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), folate binding protein (FBP), GD2, GD3, human epidermal growth factor receptor 2 (HER2, erb-B2), melanoma antigen A1 (MAGE-A1), mesothelin (MSLN), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), mucin-1 (MUC1), glypican-3 (GPC3), Wilms tumor protein (WT1), epithelial cell adhesion molecule (EpCAM), B cell maturation antigen (BCMA), and tyrosine protein kinase transmembrane receptor (ROR1). The tumor antigen may be expressed by, for example, but not limited to, solid tumors listed in Table 1.

[0060] [Table 1]

[0061] The term "engineered T cells comprising at least one tumor antigen-binding cell surface receptor" refers to T cells with a recombinant T cell receptor that binds to a tumor antigen. In particular, this refers to T cells with a recombinant surface receptor that comprises at least one tumor antigen-binding domain, an activation domain, and a costimulatory domain. Preferably, this refers to T cells carrying a chimeric antigen receptor (CAR), so-called "CAR-T cells". As used herein, engineered T cells comprising at least one tumor antigen-binding cell surface receptor or CAR-T cells can be understood to refer to engineered conventional αβ T cells or CAR-αβ T cells, respectively. Additionally or alternatively, engineered T cells or CAR-T cells comprising at least one tumor antigen-binding cell surface receptor can be engineered conventional αβ T cells or CAR-αβ T cells, or engineered γδ T cells or CAR-γδ T cells. In the context of the present invention, engineered T cells or CAR-T cells can include small amounts of other T cell subsets, such as NKT cells or CAR-NKT cells. Typically, the engineered T cells or CAR-T cells according to the invention comprise less than 5%, less than 2%, less than 1% or less than 0.5% of NKT cells or CAR-NKT cells, respectively. The term "at least one engineered T cell comprising at least one tumor antigen-binding cell surface receptor" includes a population of engineered T cells, preferably a substantially pure population of engineered T cells, more preferably a mixture of cells comprising more than 50%, more than 60%, more than 70%, more than 80%, more than 90% or more than 95% T cells. The engineered T cells are preferably generated from peripheral blood (PB), bone marrow (BM), umbilical cord blood (CB), placenta or induced pluripotent stem cells (iPSCs) from different donors or subjects to be treated.

[0062] The term "engineered NKT cells comprising at least one tumor antigen-binding cell surface receptor" refers to NKT cells with a recombinant surface receptor that binds to a tumor antigen, in particular a recombinant surface receptor comprising at least one tumor antigen-binding domain, an activation domain and a costimulatory domain. Preferably, this refers to NKT cells carrying a chimeric antigen receptor (CAR), so-called "CAR-NKT cells". NKT cells have a T cell receptor that recognizes glycolipids and lipids presented via the non-classical MHC protein CD1d. The term "NKT cells" as used herein refers to CD1d-restricted T cells. NKT cells are a subset of T cells that co-express the αβ T cell receptor, but also express various molecular markers typically associated with NK cells, such as NK1.1. NKT cells are subdivided into NKT cells with invariant T cell receptors (invariant or type 1 NKT cells) and diverse NKT cells (type 2 NKT cells). CAR-NKT cells have the advantage that CAR activity can act synergistically with the intrinsic anti-tumor activity of NKT cells. The term "at least one engineered NKT cell comprising at least one tumor antigen-binding cell surface receptor" includes a population of engineered NKT cells, preferably a substantially pure population of engineered NKT cells, more preferably a mixture of cells comprising more than 50%, 60%, 70%, 80%, 90% or 95% NKT cells. The engineered NKT cells are preferably generated from peripheral blood (PB), bone marrow (BM), umbilical cord blood (CB), placenta or induced pluripotent stem cells (iPSCs) from different donors or subjects to be treated.

[0063] The term "engineered NK cells comprising at least one tumor antigen-binding cell surface receptor" refers to NK cells with a recombinant surface receptor that binds to a tumor antigen, in particular NK cells with a recombinant surface receptor comprising at least one tumor antigen-binding domain, an activation domain and a costimulatory domain. Preferably, this refers to NK cells carrying a chimeric antigen receptor (CAR), so-called "CAR-NK cells". NK cells are lymphocytes that contain a number of activating and inhibitory germline-encoded receptors. These receptors include the NKG2D receptor, which recognizes the stress ligands MIC-A and MIC-B on tumor cells, as well as the natural cytotoxic receptors NKp30, 46 and p44. On the other hand, the killer cell immunoglobulin-like receptor (KIR) family binds to class I MHC molecules and inhibits NK cell activation. There are two main mechanisms that trigger NK effector functions: (i) self-deficiency: lack of inhibitory ligands, such as the result of downregulated MHC presentation, and (ii) self-induction: pro-activating stimuli outweigh their inhibitory counterparts, such as upregulation of stress ligands or antibody-coated cells. The term "at least one engineered NK cell comprising at least one tumor antigen-binding cell surface receptor" includes a population of engineered NK cells, preferably a substantially pure population of engineered NK cells, more preferably a mixture of more than 50%, more than 60%, more than 70%, more than 80%, more than 90% or more than 95% NK cells. The engineered NK cells are preferably generated from peripheral blood (PB), bone marrow (BM), umbilical cord blood (CB), placenta or induced pluripotent stem cells (iPSCs), from different donors or subjects to be treated, or from NK cell lines (e.g., NK-92 cells). CAR-NK cells have the advantage that CAR activity can act synergistically with the intrinsic anti-tumor activity of NK cells.

[0064] In a preferred embodiment, the at least one engineered T cell, NKT cell, or NK cell comprising at least one tumor antigen-binding cell surface receptor is a CAR-T cell, a CAR-NKT cell, or a CAR-NK cell.

[0065] The term "tumor antigen-binding cell surface receptor" refers to a recombinant surface receptor that comprises at least one tumor antigen-binding domain, preferably a chimeric antigen receptor (CAR). CAR constructs typically consist of three components: an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain. The extracellular domain comprises the antigen recognition site and is usually composed of a single chain variable fragment (scFv). In some embodiments, the extracellular domain may also comprise two or more scFvs with the same or preferably different antigen specificities. This is typically linked to the transmembrane domain via a hinge region, which confers flexibility for proper orientation and binding to the antigen. The intracellular signaling domain comprises a stimulatory domain, such as the intracellular domain of an activating receptor (e.g., CD3ζ or FcRγ), preferably at least one co-stimulatory domain, such as the intracellular domain of a co-stimulatory receptor (e.g., CD38 or 4-1BB). CARs are artificial cell surface receptors, i.e. transmembrane proteins, that contain an extracellular ligand recognition domain, an intracellular signaling domain (e.g., CD3ζ or FcRγ) that activates the respective cell, and preferably at least one intracellular domain of a co-stimulatory receptor (e.g., CD28 or 4-1BB). They are called chimeric because moieties from different sources are fused together. The extracellular ligand recognition domain is preferably based on the specificity of a monoclonal antibody and is typically a single chain variable fragment.

[0066] CARs encode transmembrane chimeric molecules with dual functions: (a) immune recognition of tumor antigens expressed on the surface of tumor cells, and (b) active promotion and propagation of signaling events that control activation and / or lytic machinery. This system has several advantages: (1) providing "reprogrammed T cells", "reprogrammed NKT cells" or "reprogrammed NK cells" of ex vivo activation mechanisms, (2) breaking the tolerance acquired by tumor cells, and (3) circumventing the limitations of HLA-mediated antigen recognition, overcoming one of the barriers to the broader application of cellular immunotherapy.

[0067] For T cells, the CAR can be, for example, a chimeric fusion protein comprising an scFv as an extracellular ligand recognition domain, and an intracellular signaling domain comprising a CD3 ζ chain signaling domain or an FcR γ chain signaling domain. This confers antibody-type specificity to the T lymphocyte and activates all the functions of the effector cell, including the production of cytokines such as IL-2 and lysis of the target cell. The CAR can further comprise an intracellular CD28 costimulatory domain and / or additional transducer domains such as from CD27, 4-1BB, CD40L, PD-1 or OX40. Typically, the CAR is pre-designed and / or provided as a DNA or RNA molecule encoding the CAR. Thus, the tumor antigen targeted by the CAR-T cells is typically a tumor antigen that is expressed or overexpressed in a solid tumor and identified as being expressed in the solid tumor of the subject to be treated, although theoretically it could also be a neoantigen.

[0068] According to the present invention, engineered T cells containing at least one tumor antigen-binding cell surface receptor also include "excoordinating CAR-T cells". Excoordinating CAR-T cells are further optimized to express ligands that induce or constitutively secrete active cytokines such as interleukins or further coat the CAR-T cells to improve efficacy and persistence. The choice of "excoordinating" agents is based on knowledge of the tumor microenvironment and the role of other elements of the innate and adaptive immune systems. Examples are interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), CD40L and 4-1BBL. These agents have been shown to further enhance CAR T cell efficacy and persistence in the tumor microenvironment through different mechanisms. They are typically expressed as independent genes in the same CAR vector.

[0069] Two recently approved CAR-T cells, KYMRIAH (tisagenlecleucel) and YESCARTA (axicabtagenecilloreucel), both contain an anti-CD19 murine scFv, but they signal through different costimulatory domains fused to the CD3 ζ chain: 4-1BB for KYMRIAH and CD28 for YESCARTA.

[0070] For NKT cells, the CAR can be, for example, a chimeric fusion protein comprising an scFv as an extracellular ligand recognition domain, and an intracellular signaling domain comprising a CD3ζ chain signaling domain or an FcRγ chain signaling domain. This confers antibody-type specificity to the NKT cells and activates all the functions of effector cells. In NKT cells, this includes the production of IFNγ and granulocyte-macrophage colony-stimulating factor (GM-CSF), lysis of target cells based on perforin and Fas ligand killing. The CAR can further comprise an intracellular CD28 costimulatory domain and / or additional transducer domains, such as from CD27, CD40L, PD-1, 4-1BB or OX40. Typically, the CAR is pre-designed and / or provided as a DNA or RNA molecule encoding the CAR. Thus, the tumor antigen targeted by the CAR-NKT cells is typically a tumor antigen that is expressed or overexpressed in a solid tumor and identified as being expressed in the solid tumor of the subject to be treated, although theoretically it could also be a neoantigen.

[0071] For NK cells, the CAR can be, for example, a chimeric fusion protein that includes an scFv as an extracellular ligand recognition domain, and an intracellular signaling domain that includes a CD3 zeta chain signaling domain or an FcR gamma chain signaling domain. This gives the NK cell antibody-type specificity and activates all the functions of an effector cell. In NK cells, this includes the production of IFN gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF) and the lysis of target cells. The CAR can further include an intracellular CD28 costimulatory domain and / or additional transducer domains, such as from CD27, CD40L, PD-1, 4-1BB, 4-1BB, OX40 or 2B4 (CD244) or DNAX activation protein 12 (DAP12) domains. An important cytokine in the protective tumor microenvironment is transforming growth factor beta (TGF-β), which inhibits NK cells. Thus, fusing the extracellular domain of the TGF-β receptor to the intracellular domain of the NKG2D receptor may further improve the efficacy of CAR-NK cells. Typically, CARs are pre-designed and / or provided as DNA or RNA molecules encoding the CAR. Thus, the tumor antigens targeted by CAR-NK cells are typically tumor antigens that are expressed or overexpressed in certain solid tumors and identified as being expressed in the solid tumor of the subject to be treated, although in theory they could also be neoantigens.

[0072] NK cells or CAR-NK cells comprising at least one tumor antigen-binding cell surface receptor may further comprise a CAR comprising NKG2D fused to the signaling domain of CD3ζ and further expressing DAP10. NKG2D is a C-type lectin-like receptor. Human NKG2D receptor monomers assemble into a hexameric structure through the association of the transmembrane domain with DAP10 dimers. DAP10 functions as an adaptor protein and transmits a signal after the ligand binds to NKG2D. NKG2D ligands are induced self-proteins that are completely absent or present only at low levels on the surface of normal cells, but are overexpressed, for example, by transformed cells (tumor antigens). Thus, the CAR can combine the extracellular domain and the transmembrane domain from the native receptor fused to the CD3ζ signaling domain and further bind to DAP10. Because the NKG2D receptor recognizes several different ligands, which are often upregulated during cellular stress, NKG2D-CAR-NK cells are also multispecific and less prone to antigen loss of tumor cells. This receptor was developed for CAR-NK cells, but is also suitable for CAR-T and CAR-NKT cells.

[0073] According to the present invention, engineered NKT cells or NK cells that contain at least one tumor antigen binding protein on their cell surface also include "excoordinating CAR-NK cells" or "excoordinating CAR-NKT cells". Excoordinating CAR-NK cells are further optimized to induce or constitutively secrete active cytokines or to express ligands that further excoordinating the CAR cells to improve efficacy and persistence. The choice of "excoordinating" agents is based on knowledge of the tumor microenvironment and the role of other elements of the innate and adaptive immune systems. Examples are interleukins such as IL-2 and IL-15 for NK cells and IL-15 for NKT cells. These agents have been shown to further enhance the efficacy and persistence in the tumor microenvironment of CAR-NKT cells and CAR-NK cells through different mechanisms. They are typically expressed as independent genes in the same CAR vector.

[0074] NK cells do not persist after adoptive transfer without cytokine support. Although the shorter life span of NK cells is advantageous, allowing antitumor activity while lowering the probability of long-term adverse events such as prolonged cytopenias caused by on-target / off-tumor toxicity to normal tissues, NK cells may also limit their efficacy. For survival and proliferation in vivo, NK cells require sustained cytokine support, without which they can only be detected in the circulation for 1-2 weeks. The two most commonly used cytokines to support the persistence of adoptively transferred NK cells are IL-2 and IL-15. Thus, genes for IL-2 and / or IL-15 can be incorporated within the CAR construct of CAR-NK cells. This allows constant cytokine support to be provided to the CAR-transduced cells.

[0075] Interleukins may be administered exogenously, but infusion of IL-2 or IL-15 has significant side effects. An alternative or additional approach to exogenous administration of cytokines is lymphodepleting chemotherapy prior to adoptive cell transfer of NK cells, such as cyclophosphamide and fludarabine. This provides a favorable environment for NK cell proliferation by depleting mature lymphocytes (which consume IL-15), resulting in a significant increase in endogenous IL-15 levels. Besides the selection of the target epitope, the CAR design, and the applied dose and administration regimen, efficient tumor homing and long-term survival in the tumor environment are important for the success of CAR-T cell, CAR-NKT cell, or CAR-NK cell therapy of solid tumors. In most cases, the subject is lymphopenic before administration of CAR-T cell, CAR-NKT cell, or CAR-NK cell, making the possibility of subsequent cytokine support even more important.

[0076] A common side effect of CAR-T cells in liquid tumors is the abundant production of cytokines, which causes severe cytokine release syndrome (CRS). This risk is less pronounced in solid tumors. Without being bound by theory, this may be explained by the different effector-target cell stoichiometry, which is typically higher in liquid tumors compared to solid tumors. Moreover, this risk is generally considered to be lower in adoptive cell transfer of NKT cells or NK cells compared to T cells, particularly conventional αβT cells.

[0077] Moreover, most tumor antigens are not tumor-selective (tumor-specific antigens), especially in solid tumors, but are often simply overexpressed (tumor-associated antigens). Therefore, there is a risk of extratumoral toxicity. However, methods to reduce extratumoral toxicity are known in the art. For example, extratumoral toxicity can be controlled by administering the required number of cells in two, three or more doses. Also, extratumoral toxicity can be minimized using transiently engineered RNACAR-T cells, CAR-NKT cells or CAR-NK cells. Also, when starting treatment at an early stage of tumor development, starting treatment before the number of cancer cells becomes excessive is beneficial for treatment outcome and reduces extratumoral toxicity.

[0078] Furthermore, target selectivity can be ensured by recognition of two tumor antigens expressed on the same cell. This can be achieved using two chimeric receptors designed to deliver stimulatory and costimulatory signals in separate CARs, e.g., tandem CARs that mediate bispecific activation of T cells, NKT cells, or NK cells through binding of the CD3 ζ chain signaling domain and the CD28 costimulatory domain, requiring independent binding of two different tumor antigens for efficient signaling. Inhibitory chimeric antigen receptors (iCARs) can also be used to divert CAR-T cells, CAR-NKT cells, or CAR-NK cell activity from normal tissues. iCARs bind native antigens (i.e., only presented on normal tissues) and contain inhibitory signaling domains, such as from PD-1 or CTLA-4, to stop the activation of active CARs. Thus, both approaches, tandem CARs and iCARs, combine the activity of two chimeric antigen receptors.

[0079] One problem that is particularly relevant for CAR-T cell immunotherapy, but also for CAR-NKT cell or CAR-NK cell immunotherapy, is antigen escape, which can make CAR-T cells, CAR-NKT cells or CAR-NK cells ineffective against cancer cells. Moreover, CAR-T cells, CAR-NKT cells or CAR-NK cells are typically not administered repeatedly. Thus, there is a need for follow-up therapies that allow targeting other tumor antigens, preferably multiple tumor antigens, such as neoantigens.

[0080] For safety reasons, CAR-modified T cells, and possibly NKT and NK cells, may further comprise a suicide system such as inducible caspase-9 (iCasp9) or truncated epidermal growth factor receptor (EGFR, which lacks the signaling domain and can be targeted with anti-EGFR antibodies for rapid elimination of transgenic cells). This may be particularly relevant for CAR-T and CAR-NKT cells. Mature CAR-NK cells have limited persistence, whereas umbilical cord blood or hematopoietic stem cell-derived NK cells have a higher risk for long-term toxicity, and thus suicide systems may also be utilized in these cells.

[0081] The inventors have found that the Salmonella typhi Ty21a strain encoding at least one polypeptide comprising five or more neoantigens is particularly suitable as a follow-up therapy because it can target multiple neoantigens expressed in solid tumors. Thus, the Salmonella typhi Ty21a strain encoding at least one polypeptide comprising five or more neoantigens is administered after adoptive cell transfer of at least one engineered T cell, NKT cell, or NK cell comprising at least one tumor antigen-binding cell surface receptor. If the subject has not undergone lymphodepleting chemotherapy prior to adoptive cell transfer of at least one engineered T cell, NKT cell, or NK cell comprising at least one tumor antigen-binding cell surface receptor, the Salmonella typhi Ty21a strain encoding at least one polypeptide comprising five or more neoantigens may be administered together with or immediately after (e.g., within hours or days) the adoptive cell transfer of at least one engineered T cell, NKT cell, or NK cell comprising at least one tumor antigen-binding cell surface receptor.

[0082] At least one engineered T cell, NKT cell or NK cell that comprises at least one tumor antigen-binding cell surface receptor is typically administered in one adoptive cell transfer and not repeatedly administered.However, the required number of engineered T cell, NKT cell or NK cell can be administered as a split dose in two or three subsequent adoptive cell transfers.Thus, treatment can include a first, and optionally a second, third, possibly further administration within a period of two or more days.The remaining period of adoptively transferred cells can be up to 3 months, up to 4 months, or up to 6 months.Some researchers claim that they are living cells that exist for life.

[0083] Prior to adoptive cell transfer of at least one engineered T cell, NKT cell, or NK cell comprising at least one tumor antigen-binding cell surface receptor, the subject can be treated with Salmonella typhi Ty21a comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding VEGFR-2 (e.g., as disclosed in WO2014 / 005683). In one embodiment, VEGFR-2 comprises the amino acid sequence of SEQ ID NO: 1. This vaccine (VXM01) is known to enhance the number of tumor infiltrating lymphocytes (TILs). Thus, this vaccine can enhance the efficacy of at least one engineered T cell, NKT cell, or NK cell comprising at least one tumor antigen-binding cell surface receptor. Furthermore, since at least one engineered T cell, NKT cell, or NK cell comprising at least one tumor antigen-binding cell surface receptor is typically produced on demand, this vaccine provides cancer immunotherapy while at least one engineered T cell, NKT cell, or NK cell comprising at least one tumor antigen-binding cell surface receptor is prepared. Thus, in certain embodiments, a subject is first treated with S. typhi Ty21a comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding VEGFR-2, followed by adoptive cell transfer of at least one engineered T cell, NKT cell or NK cell comprising at least one tumor antigen-binding cell surface receptor (with or without lymphodepleting chemotherapy), and S. typhi Ty21a encoding at least one polypeptide comprising five or more neoantigens.

[0084] In certain embodiments, the Salmonella typhi Ty21a strain encoding at least one polypeptide comprising five or more neoantigens is administered simultaneously or about 2 weeks to 4 months, preferably 2 to 3 months, after the initial adoptive cell transfer of at least one engineered T cell, NKT cell, or NK cell comprising at least one tumor antigen-binding cell surface receptor, where contemporaneous means within a few hours or days, preferably the same day or within a week.

[0085] In certain embodiments, the subject undergoes lymphodepletion, particularly lymphodepleting chemotherapy, prior to adoptive cell transfer of at least one engineered T cell, NKT cell, or NK cell comprising at least one tumor antigen-binding cell surface receptor. The term "lympho-depleting chemotherapy" as used herein refers to chemotherapy that results in lymphopenia in the subject prior to adoptive cell transfer. It also includes non-myeloablative lymphodepleting chemotherapy, which may also improve the efficacy of adoptive cell transfer therapy. Lympho-depleting chemotherapy is also referred to as "conditioning". Lympho-depleting chemotherapy is known in the art and may include, for example, the use of cyclophosphamide (CTX) or CXT and fludarabine for 7 days. CTX does not affect early hematopoietic bone marrow progenitor cells.

[0086] In order to induce an immune response against neoantigens in a subject who has undergone lymphodepleting chemotherapy prior to adoptive cell transfer, lymphocytes must be replenished, or in other words, the subject must recover immunocompetence, before administering the Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens. Depending on the type of lymphodepleting chemotherapy, lymphocytes are replenished about 1 to 2 months after lymphodepleting chemotherapy. Typically, lymphocytes are replenished about 2 to 16 weeks, 4 to 16 weeks, 2 to 16 weeks, or 8 to 12 weeks after lymphodepleting chemotherapy. Preferably, the Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens is administered to the subject after lymphodepleting chemotherapy, more preferably after the lymphocyte count has normalized after lymphodepleting chemotherapy, more preferably after the subject has recovered immunocompetence. A normal lymphocyte count is 1000 / mm 3 A normal white blood cell count is 4000 / mm 3 The immune system was functioning normally with a white blood cell count of 2000 / mm3. 3 The decision will be based on the above.

[0087] In cases where lymphodepletion has been performed prior to adoptive cell transfer, the Salmonella typhi Ty21a strain encoding at least one polypeptide comprising five or more neoantigens is administered approximately 2 weeks to 4 months, preferably 1 month to 4 months, preferably 2 months to 4 months, more preferably 2 months to 3 months, after the initial adoptive cell transfer of at least one engineered T cell, NKT cell or NK cell comprising at least one tumor antigen-binding cell surface receptor.

[0088] Combination therapy with neoantigen cancer vaccine According to the present invention, the S. typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens can further be co-administered with at least one checkpoint inhibitor. The term "checkpoint inhibitor" is used synonymously with "immune checkpoint inhibitor" herein. Typically, checkpoint therapy blocks inhibitory checkpoints and restores immune system function. In particular, the at least one checkpoint inhibitor can be selected from the group consisting of antibodies, in particular antibodies against PD-1, PD-L1, CTLA-4, IDO, GITR, OX40, TIM-3, LAG-3, KIR, CSF1R and CD137. The checkpoint inhibitor can be administered simultaneously or separately with the at least one S. typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens.

[0089] Preferably, at least one checkpoint inhibitor is administered in an approved galenic formulation of a commercially available product.

[0090] In the context of the present invention, the term "simultaneously" refers to administration on the same day, more particularly within 12 hours, more particularly within 2 hours, of an attenuated strain of Salmonella typhi Ty21a comprising at least one polypeptide comprising five or more neoantigens and at least one eukaryotic expression cassette encoding a checkpoint inhibitor. The term "separately" as used in this context refers to administration on different days, more particularly in different dosing regimens, and in different dosage forms.

[0091] Surprisingly, a relatively low dose of the S. typhi Ty21a strain encoding at least one polypeptide comprising five or more neoantigens and at least one checkpoint inhibitor in a subject who has been or will be treated with at least one engineered T cell, NKT cell or NK cell comprising at least one tumor antigen-binding cell surface receptor shows a synergistic effect on T cell, NKT cell or NK cell responses and / or overall survival. Administration of a low dose of the live vaccine minimizes the risk of shedding and therefore the risk of transmission to third parties.

[0092] According to the present invention, a subject receiving a S. typhi strain Ty21a comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens can be further treated with at least one S. typhi Ty21a comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one tumor antigen, tumor stromal antigen and / or checkpoint inhibitor antigen. In one embodiment, the treatment further comprises at least one S. typhi Ty21a comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding an antigen selected from the group consisting of WT1, MSLN, CEA, CMV pp65, PD-L1, VEGFR-2 and FAP administered to the subject. At least one Salmonella typhi Ty21a comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding an antigen selected from the group consisting of WT1, MSLN, CEA, CMV pp65, PD-L1, VEGFR-2 and FAP can be administered simultaneously or separately with at least one Salmonella typhi Ty21a comprising a DNA molecule comprising at least one eukaryotic expression cassette comprising five or more neoantigens.

[0093] In the context of the present invention, the term "simultaneously" means administration of different attenuated strains of S. typhi Ty21a on the same day, more particularly within 12 hours, more particularly within 2 hours. The different attenuated strains of S. typhi Ty21a can be, but do not necessarily have to be, in the same dosage form. The term "separately" as used in this context means administration on different days, more particularly in different dosage regimens, and in different dosage forms.

[0094] In certain embodiments, human VEGFR-2 comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, human Wilms tumor protein (WT1) comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 3. In certain embodiments, human mesothelin (MSLN) comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 4. In certain embodiments, human CEA comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 5. In certain embodiments, CMV pp65 comprises the amino acid sequence of SEQ ID NO: 6, 7, or 8, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 6, 7, or 8. In certain embodiments, human PD-L1 comprises the amino acid sequence of SEQ ID NO: 9 or 10, or an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 9, 10, or 11.

[0095] Preferably, VEGFR-2 has the amino acid sequence of SEQ ID NO: 1, WT1 has the amino acid sequence of SEQ ID NO: 3, MSLN has the amino acid sequence of SEQ ID NO: 4, CEA has the amino acid sequence of SEQ ID NO: 5, CMV pp65 has the amino acid sequence of SEQ ID NO: 6, 7 or 8, and / or PD-L1 has the amino acid sequence of SEQ ID NO: 9, 10 or 11.

[0096] VEGFR-2, also known as the kinase insert domain-containing receptor (KDR), appears to mediate almost all known cellular responses to VEGF. For example, the role of VEGF in angiogenesis appears to be through the interaction of this protein with VEGFR-2. VEGFR-2 is a 1356 amino acid long, 200-230 kDa molecular weight high affinity receptor for VEGF, a receptor for VEGF-C and VEGF-D, identified in humans by screening endothelial cDNA for tyrosine kinase receptors and shares 85% sequence identity with the previously discovered mouse fetal liver kinase 1 (Flk-1). VEGFR-2 is normally expressed in endothelial cells, hematopoietic progenitor cells, as well as endothelial cells, neonatal hematopoietic stem cells, and umbilical cord stroma. However, in the quiescent adult vasculature, VEGFR-2 mRNA appears to be downregulated.

[0097] The extracellular domain of VEGFR-2 contains 18 potential N-linked glycosylation sites. VEGFR-2 is initially synthesized as a 150 kDa protein that is rapidly glycosylated to a 200 kDa intermediate and then further glycosylated at a slower rate to the mature 230 kDa protein that is expressed on the cell surface.

[0098] Mesothelin is a 40 kDa cell surface glycoprotein present on normal mesothelial cells and is overexpressed in several human tumors, including mesothelioma, ovarian and pancreatic adenocarcinoma. The mesothelin gene encodes a 71 kDa precursor protein that is processed to produce a 31 kDa small protein named megakaryocyte potentiating factor (MPF) and a 40 kDa cell-binding fragment, mesothelin. Mesothelin has been shown to exhibit megakaryocyte colony-forming activity in the presence of interleukin-3. Mesothelin is a tumor differentiation antigen that is present at low levels on restricted normal adult tissues such as mesothelium, but is aberrantly overexpressed in a wide range of human tumors, including mesothelioma, ovarian and pancreatic cancer, squamous cell carcinoma of the cervix, head and neck, vulva, lung and esophagus, lung adenocarcinoma, endometrial carcinoma, biphasic synovial sarcoma, desmoplastic small round cell tumor and gastric adenocarcinoma. The normal biological function of mesothelin is unknown. Studies with mesothelin knockout mice showed no detectable phenotype and produced healthy offspring of both sexes. Studies in pancreatic cancer suggest that mesothelin plays a role in tumorigenesis by increasing cell proliferation, migration, and S-phase cell populations. Furthermore, there is evidence that mesothelin is an immunogenic protein. Because of its expression profile, oncogenic function, and immunogenicity, the tumor antigen mesothelin is a promising candidate for cancer vaccine development.

[0099] Wilms tumor gene 1 (WT1) encodes a zinc finger transcription factor involved in cell proliferation and differentiation. The WT1 protein contains four zinc finger motifs at the C-terminus and a proline / glutamine-rich DNA binding domain at the N-terminus. Multiple transcript variants resulting from alternative splicing in the two coding exons have been well characterized. WT1 plays an essential role in the development of the urogenital system and is involved in cell proliferation and differentiation. The WT1 gene was isolated as the causative gene for Wilms tumor, a pediatric renal neoplasm. It is highly expressed in a wide range of malignancies, including several types of hematological malignancies and various solid tumors. In contrast, normal tissue expression of WT1 in adults is restricted to gonads, uterus, kidney, mesothelium, and progenitor cells in various types of tissues. WT-1 negatively affects differentiation and promotes proliferation of progenitor cells. Moreover, overexpressed WT1 is immunogenic, and WT1-specific T cells and IgG anti-WT1 antibodies have been observed in cancer patients. Due to its expression profile, its oncogenic function and its immunogenicity, tumor antigen WT1 is a promising candidate for cancer vaccine development. In certain embodiments, WT1 is truncated. In certain aspects, the zinc finger domain of WT1 is deleted. In certain embodiments, the truncated WT1 has the amino acid sequence shown in SEQ ID NO:3.

[0100] The zinc finger domain at the C-terminus of WT1 contains four zinc finger motifs. The amino acid sequence of truncated WT1 found in SEQ ID NO: 3 represents amino acids 1-371 of UniProtref P19544-7. Deletion of the zinc finger domain minimizes the risk of immunological cross-reactivity with other zinc fingers, including transcription factors. Furthermore, truncated WT1 lacking the zinc finger domain is more immunogenic than full-length WT1. Furthermore, deletion of the zinc finger motifs essential for DNA binding abolishes the oncogenic potential of WT1, minimizing the risk of oncogenicity.

[0101] The tegument protein CMV pp65 is the major immunodominant protein of human cytomegalovirus (CMV). The biological function of CMV pp65 is unknown, but it is thought to be involved in cell cycle regulation. CMV pp65 is a nucleophilic protein that exhibits protein kinase activity that can bind to polo-like kinase 1 (PLK-1). HCMV pp65 is expressed in over 90% of glioblastoma specimens but not in the surrounding normal brain. Thus, this viral protein is a promising candidate as a tumor-specific target for novel development of cancer immunotherapy.

[0102] The CMV pp65 protein contains two bipartite nuclear localization signals (NLS) at amino acids 415-438, near the carboxy terminus at amino acids 537-561, and a phosphate-binding site associated with its kinase activity at lysine 436. Mutation of the lysine at position 436 to asparagine and deletion of amino acids 537-561 results in a protein without kinase activity and with severely reduced nuclear localization. This mutant protein exhibits unaltered immunogenicity.

[0103] In certain embodiments, CMV pp65 has the amino acid sequence found in SEQ ID NO: 6. SEQ ID NO: 6 represents the amino acid sequence of wild-type CMV pp65. In certain other embodiments, CMV pp65 has the amino acid sequence found in SEQ ID NO: 7. SEQ ID NO: 7 represents the amino acid sequence of CMV pp65 having the mutation K436N relative to wild-type human CMV pp65 of SEQ ID NO: 6. In certain other embodiments, CMV pp65 has the amino acid sequence found in SEQ ID NO: 8. SEQ ID NO: 8 represents the amino acid sequence of a truncated version of CMV pp65 of SEQ ID NO: 7, lacking the second, more C-terminal NLS (nuclear localization sequence) (i.e., amino acids 537-561 of CMV p65 of SEQ ID NO: 7).

[0104] Carcinoembryonic antigen (also known as CEACAM5 and CD66e) is a member of a family of highly related glycosylphosphatidylinositol (GPI) cell surface anchored glycoproteins involved in cell adhesion. CEA is normally produced in gastrointestinal tissues during fetal development, and protein expression terminates before birth. Thus, CEA is usually present only at very low blood levels in healthy adults. However, serum concentrations are elevated in some cancers, particularly colorectal cancer, making it a tumor marker. CEA levels are also elevated in gastric, pancreatic, lung, breast, and medullary thyroid cancers, as well as in non-neoplastic diseases such as ulcerative colitis, pancreatitis, liver cirrhosis, COPD, Crohn's disease, and hypothyroidism.

[0105] Programmed cell death 1 (PD-1) is expressed on the surface of T cells and transmits inhibitory signals that maintain the functional silence of T cells against cognate antigens. Its ligand, PD-L1, is normally expressed on antigen-presenting cells, placental cells, and non-hematopoietic cells in the inflammatory microenvironment. PD-L1 has been reported to be expressed on immunosuppressive myeloid-derived suppressor cells (MDSCs). In addition, PD-L1 is widely expressed on the surface of various types of cancer cells that use the PD-1 / PD-L1 signaling axis to evade the host immune system. Expression of PD-L1 by cancer cells has been shown to correlate with disease stage and poor patient prognosis.

[0106] In certain embodiments, the PD-L1 is selected from the group consisting of full length PDL1 and a truncated PD-L1 comprising the extracellular domain of PD-L1. The truncated PD-L1 may comprise the amino acid sequence of amino acids 19-238 of SEQ ID NO:11, the amino acid sequence of SEQ ID NO:11, the amino acid sequence of SEQ ID NO:10, or an amino acid sequence of SEQ ID NO:11 that shares at least 80% sequence identity with amino acids 19-238 of SEQ ID NO:11, or an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO:10. In certain embodiments, the PD-L1 is selected from the group consisting of PD-L1 having the amino acid sequence found in SEQ ID NO:9, and proteins that share at least 80% sequence identity therewith. In certain other embodiments, the PD-L1 is selected from the group consisting of PD-L1 having the amino acid sequence found in SEQ ID NO:10, and proteins that share at least 80% sequence identity therewith. In certain other embodiments, the PD-L1 is selected from the group consisting of PD-L1 having the amino acid sequence found in SEQ ID NO: 11, and proteins which share at least 80% sequence identity therewith. In certain other embodiments, the PD-L1 is selected from the group consisting of PD-L1 having the amino acid sequence of amino acids 19-238 of SEQ ID NO: 11, and proteins which share at least 80% sequence identity therewith. In particular, the PD-L1 has the amino acid sequence found in SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, and preferably the PD-L1 comprises the amino acid sequence of amino acids 19-238 of SEQ ID NO: 11. In one embodiment, PD-L1 comprises at least the extracellular domain with or without a signaling peptide.

[0107] As used herein, the term "about" or "approximately" means 95% to 105%, inclusive, 80% to 120%, alternatively 90% to 110% of a given value or range.

[0108] In the context of the present invention, the term "protein sharing at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:X" refers to a protein having an amino acid sequence that has more than 80% amino acid identity when aligned with the amino acid sequence provided. The protein can be, for example, a mutant of a wild-type protein, for example, a mutant of a wild-type VEGFR-2 protein, or a homologue of a different species, or an engineered protein, for example, an engineered VEGFR-2 protein. Methods for designing and constructing derivatives of a given protein are well known to those skilled in the art.

[0109] A protein that shares at least about 80% sequence identity with a given amino acid sequence may contain one or more mutations, including addition, deletion and / or substitution of one or more amino acids, compared to the reference amino acid sequence. According to the teachings of the present invention, the deleted, added and / or substituted amino acids may be consecutive amino acids or may be interspersed throughout the length of the amino acid sequence of the protein that shares at least about 80% sequence identity with a given reference protein. According to the teachings of the present invention, any number of amino acids may be added, deleted and / or substituted, so long as the amino acid sequence identity with the reference amino acid sequence is at least about 80% and the mutant protein is immunogenic. Preferably, the immunogenicity of a protein that shares at least about 80% sequence identity with a reference amino acid sequence is reduced by 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 amino acid sequence, as measured by ELISA. Methods for designing and constructing protein homologs and testing such homologs for their immunogenicity are well known to those skilled in the art. In certain embodiments, the sequence identity with the reference amino acid sequence is at least about 85%, at least about 90%, at least about 95%, and most particularly at least about 99%. Methods and algorithms for determining sequence identity, including comparison of parent proteins and their derivatives with deletions, additions and / or substitutions to the parent protein and parent sequence, are well known to those skilled in the art. At the DNA level, nucleic acid sequences encoding proteins that share at least about 80% sequence identity with the reference amino acid sequence may differ more due to the degeneracy of the genetic code.

[0110] In a specific embodiment, administration of a Salmonella Typhi Ty21a strain encoding at least one polypeptide comprising five or more neoantigens is combined with administration of an attenuated strain of Salmonella encoding a tumor or tumor stromal antigen selected from WT1, MSLN, CEA, CMV pp65, PD-L1, VEGFR-2, and FAP, and a checkpoint inhibitor.

[0111] In a particular embodiment, the treatment may involve chemotherapy or radiation therapy. For the cure of cancer, it is essential to completely eradicate cancer stem cells. Therefore, it is beneficial to combine different therapeutic approaches to obtain the maximum effect.

[0112] Chemotherapeutic agents that can be used in combination with the Salmonella typhi Ty21a strain of the present invention include, for example, amifostine (ethyol), cabazitaxel, cisplatin, dacarbazine (DTIC), dactinomycin, docetaxel, mechlorethamine, streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), doxorubicin fatty (doxil), folinic acid, gemcitabine (gemzar), daunorubicin, daunorubicin fatty (daunoxome), procarbazine, ketoconazole, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil (5-FU), vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxo tere), aldesleukin, asparaginase, busulfan, carboplatin, cladribine, camptothecin, CPT-11, 10-hydroxy-7-ethylcamptothecin (SN38), dacarbazine, floxiridine, fludarabine, hydroxyurea, ifosfamide, idarubicin, mesna, interferon alpha, interferon beta, irinotecan, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, oxaliplatin, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil and combinations thereof.

[0113] The most preferred chemotherapeutic agents according to the invention are cabazitaxel, carboplatin, oxaliplatin, cisplatin, cyclophosphamide, docetaxel, gemcitabine, doxorubicin, paclitaxel (taxol), irinotecan, vincristine, vinblastine, vinorelbine, folinic acid, 5-fluorouracil and bleomycin, especially gemcitabine.

[0114] In particular, the Salmonella typhi Ty21a strain is administered before or during chemotherapy or radiotherapy. In another particular embodiment, the Salmonella typhi Ty21a strain is administered before and during chemotherapy or radiotherapy treatment.

[0115] Salmonella typhi Ty21a Attenuated strains of Salmonella strains, especially Salmonella enterica species, are attractive vehicles for the delivery of heterologous antigens to the mammalian immune system, due to the possibility of delivering S. enterica strains via mucosal routes of immunization, i.e. oral or nasal, which offer advantages of simplicity and safety compared to parenteral administration. Furthermore, Salmonella strains induce strong humoral and cellular immune responses at the level of both the systemic and mucosal compartments. Batch preparation costs are low and formulations of live vaccines are very stable. Attenuation can be achieved by deletion of various genes, including virulence, regulatory, and metabolic genes.

[0116] Several Salmonella typhimurium strains attenuated by aro mutations have been shown to be safe and effective delivery vehicles for heterologous antigens in animal models.

[0117] According to the present invention, the attenuated strain of Salmonella is Salmonella enterica serovar Typhi strain Ty21a, also known as Salmonella typhi Ty21a. Live attenuated Salmonella typhi Ty21a is the active ingredient of Typhoral L®, also known as Vivotif® (manufactured by Berna Biotech Ltd., a Crucell Company, Switzerland). It is currently the only licensed live oral vaccine against typhoid fever. This vaccine has been extensively tested and proven safe with respect to toxicity in patients and transmission to third parties (Wahdan et al., J. Infectious Diseases 1982, 145:292-295). This vaccine is licensed in over 40 countries and is used to vaccinate millions of individuals, including thousands of children, against typhoid fever. It has an unparalleled safety record. No data are available showing that Salmonella typhi Ty21a enters the bloodstream systemically. Thus, the live attenuated Salmonella typhi Ty21a vaccine strain makes it possible to specifically target the immune system in the intestine while being safe and well tolerated. The marketing authorization number for Typhoral L® is PL15747 / 0001 dated 16 December 1996. A single dose of the vaccine contains at least 2 x 10 9 of viable Salmonella typhi Ty21a colony-forming units and at least 5 × 10 9 containing nonviable Salmonella typhi Ty21a cells.

[0118] This well-tolerated live oral vaccine against typhoid fever was obtained by chemical mutagenesis of the wild-type pathogenic bacterial isolate Salmonella typhi Ty2, which has a loss-of-function mutation in the galE gene and is therefore unable to metabolize galactose. The attenuated bacterial strain is also unable to reduce sulfate to sulfide, which distinguishes it from the wild-type Salmonella typhi Ty2 strain. With regard to serological characteristics, the Salmonella typhi Ty21a strain contains the O9 antigen, a polysaccharide of the bacterial outer membrane, and lacks the O5 antigen, a characteristic component of Salmonella typhi. This serological characteristic supports the rationale for including the respective test in the panel of confirmatory tests at batch release.

[0119] The expression cassette used in the Salmonella typhi Ty21a strain according to the invention is a eukaryotic expression cassette, in particular comprising a CMV promoter. In the context of the present invention, the term "eukaryotic expression cassette" refers to an expression cassette that allows the expression of an open reading frame in a eukaryotic cell. It has been shown that the amount of heterologous antigen required to induce an appropriate immune response can be toxic to the bacterium, resulting in cell death, over-attenuation or loss of expression of the heterologous antigen. This toxicity problem can be overcome by using a eukaryotic expression cassette that is not expressed in the bacterial vector but only in the target cell, and the expressed protein typically exhibits a eukaryotic glycosylation pattern.

[0120] The eukaryotic expression cassette comprises regulatory sequences, preferably a promoter and a polyadenylation signal, capable of controlling the expression of the open reading frame in a eukaryotic cell. The promoter and polyadenylation signal contained in the eukaryotic expression cassette comprised by the Salmonella typhi Ty21a strain of the invention are preferably selected so as to be functional in the cells of the subject to be immunized. Examples of suitable promoters include, but are not limited to, promoters from 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 Rous Sarcoma Virus (RSV), CMV early enhancer element, promoter, synthetic CAG promoter consisting of the first exon and first intron of chicken β-actin gene and splice acceptor of rabbit β-actin gene, as well as promoters from human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein. In certain embodiments, the eukaryotic expression cassette comprises a CMV promoter. In the context of the present invention, the term "CMV promoter" refers to the strong immediate early cytomegalovirus promoter.

[0121] In particular, for the production of DNA vaccines for humans, examples of suitable polyadenylation signals include, but are not limited to, the bovine growth hormone (BGH) polyadenylation site, the SV40 polyadenylation signal, and the LTR polyadenylation signal. According to a specific embodiment, the eukaryotic expression cassette contained in the recombinant DNA molecule contained by the attenuated mutant Salmonella strain of the present invention contains the BGH polyadenylation site.

[0122] In addition to the regulatory elements required for expression of the VEGF receptor protein, such as a promoter and a polyadenylation signal, other elements may also be included in the recombinant DNA molecule. Such additional elements include enhancers, such as enhancers from human actin, human myosin, human hemoglobin, human muscle creatine, and viral enhancers, such as CMV, RSV, and EBV.

[0123] Regulatory sequences and codons are generally species dependent, so that to maximize protein production, regulatory sequences and codons are preferably selected to be effective in the species being immunized. One skilled in the art can prepare recombinant DNA molecules that are functional in a given subject species, e.g., a human subject.

[0124] In certain embodiments, the DNA molecule or DNA molecule comprising at least one eukaryotic expression cassette comprises an antibiotic resistance gene, such as a kanamycin antibiotic resistance gene, an ori, such as the pMB1 ori or pUC, and a strong promoter, such as the CMV promoter. In certain embodiments, the recombinant DNA molecule or DNA molecule comprising at least one eukaryotic expression cassette is a plasmid, such as a plasmid based on or derived from the commercially available pVAX1™ expression plasmid (Invitrogen, San Diego, California).

[0125] This expression vector can be 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 done to reduce the metabolic burden and make the construct more stable. The resulting expression vector backbone was named pVAX10.

[0126] In a particular embodiment, the expression plasmid comprises a DNA molecule of SEQ ID NO:2 (vector backbone pVAX10), which correlates with the sequence of the expression vector pVAX10 without a portion of the multiple cloning site located between the restriction sites NheI and XhoI.

[0127] In a particular embodiment, the Salmonella typhi Ty21a strain is administered orally. Oral administration is simpler, safer and more comfortable than parenteral administration. However, it should be noted that the Salmonella typhi Ty21 strain of the present invention can also be administered by any other suitable route. Preferably, a therapeutically effective dose is administered to the subject, which depends on the specific application, the type of malignancy, the subject's weight, age, sex and health condition, the method of administration and the formulation, etc. Administration may be single or multiple, as required.

[0128] The S. typhi Ty21a strain encoding at least one polypeptide comprising five or more neoantigens can be provided in a solution, suspension, lyophilisate, enteric coated capsule or any other suitable form. Typically, the S. typhi Ty21a strain is formulated as a drinking solution. This embodiment offers the advantage of improved patient compliance. Preferably, the drinking solution comprises a means for at least partially neutralising the gastric acid, i.e. for bringing the pH of the gastric juices closer to a pH of 7. Preferably, the drinking solution is a buffer suspension comprising the S. typhi Ty21a strain according to the invention. In a particular embodiment, the buffer suspension is obtained by suspending the S. typhi Ty21a strain in a suitable buffer, preferably containing 2.6 g sodium bicarbonate, 1.7 g L-ascorbic acid, 0.2 g lactose monohydrate and 100 ml drinking water.

[0129] In a particular embodiment, a single dose of Salmonella typhi Ty21a strain is about 10 6 ~about 10 10 , more specifically, about 10 6 ~about 10 9 , more specifically, about 10 7 ~about 10 9 , more specifically, about 10 6 ~about 10 8 , most specifically about 10 6 ~about 10 6 1000 colony forming units.

[0130] More specifically, a single dose of Salmonella typhi Ty21a strain is approximately 1×10 6 ~Approx. 1×10 10 , more specifically, about 1 × 10 6 ~Approx. 1×10 9 , more specifically, about 1 × 10 7 ~Approx. 1×10 9 , more specifically, about 1 × 10 6 ~Approx. 1×10 8 , especially about 1 × 10 6 ~Approx. 1×10 6 1000 colony forming units.

[0131] Furthermore, the Salmonella typhi Ty21a strain of the present invention is preferably administered 2 to 4 times in the first week, preferably 4 times in the first week, followed by additional single doses every 2 to 4 weeks, particularly on days 1 and 7, preferably on days 1, 3, 5 and 7, followed by additional single doses every 2 to 4 weeks.

[0132] In this context, the term "about" or "approximately" means within a factor of 3, or within a factor of 2, including within a factor of 1.5 of a given value or range.

[0133] In a particular embodiment, the treatment comprises a single or multiple administrations of the Salmonella typhi Ty21a strain encoding at least one polypeptide comprising five or more neoantigens or the pharmaceutical composition according to the invention. The single doses of administration may be the same or different, preferably within the ranges disclosed herein. In particular, the treatment comprises 2-4 primary vaccinations in the first week of treatment, followed by a single booster vaccination every 2-4 weeks of the Salmonella typhi Ty21a strain encoding at least one polypeptide comprising five or more neoantigens or the pharmaceutical composition according to the invention, preferably the multiple administrations are administered consecutively for 3-6 months.

[0134] Depending on the occurrence of possible side effects, it may also be preferable to include treatment with antibiotics or anti-inflammatory drugs.

[0135] In the event of adverse events similar to histamine, leukotriene or cytokine mediated hypersensitivity reactions, treatment options are available for fever, hypersensitivity, blood pressure instability, bronchospasm and dyspnea. Treatment options for unwanted T cell derived autoaggression are derived from standard therapy schemes for acute and chronic graft-versus-host disease applied after stem cell transplantation. Cyclosporine and glucocorticoids are proposed as treatment options.

[0136] In unlikely cases of systemic S. Typhi Ty21a infection, appropriate antibiotic therapy with a fluoroquinolone, including ciprofloxacin or ofloxacin, is recommended. Bacterial infections of the gastrointestinal tract are treated with agents such as rifaximin.

[0137] Pharmaceutical Compositions In a further aspect, the present invention relates to a pharmaceutical composition comprising a Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens.

[0138] The pharmaceutical compositions of the present invention may be in the form of a solution, a suspension, an enteric coated capsule, a lyophilized powder, or any other form suitable for the intended use. The pharmaceutical compositions of the present invention may further comprise one or more pharma- ceutically acceptable excipients.

[0139] In the context of the present invention, the term "excipient" refers to a natural or synthetic substance that is formulated with the active ingredient of a drug. Suitable excipients include anti-adherents, binders, coatings, disintegrants, flavors, colorants, lubricants, glidants, adsorbents, preservatives and sweeteners.

[0140] In the context of the present invention, the term "pharmaceutical acceptable" refers to molecular entities and other components of a pharmaceutical composition that are physiologically tolerable and typically do not produce untoward reactions when administered to a mammal (e.g., a human). The term "pharmaceutical acceptable" can also mean approved by a federal or state government regulatory agency, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias, for use in mammals, and more particularly, in humans.

[0141] In particular, a suitable drinking solution comprises a means for at least partially neutralizing the gastric acid, i.e. for bringing the pH of the gastric juices closer to a pH of 7. In a particular embodiment, the drinking solution is a buffer suspension obtained by suspending the Salmonella typhi strain Ty21a of the invention in a suitable buffer, preferably in a buffer that neutralizes the gastric acid at least to a certain extent, preferably in a buffer containing 2.6 g sodium bicarbonate, 1.7 g L-ascorbic acid, 0.2 g lactose monohydrate and 100 ml drinking water.

[0142] In a particular aspect, the pharmaceutical composition is used as a medicament, in particular for use in treating a solid tumor in a subject according to the invention. In a particular embodiment, the pharmaceutical composition is for use as a medicament, in particular for use in treating a solid tumor in a subject, the subject being or being treated with at least one engineered T cell, NKT cell or NK cell comprising at least one tumor antigen-binding cell surface receptor, or any other use or method disclosed herein. EXAMPLES

[0143] Example 1: Proof of concept for inducing an immune response using Salmonella typhi Ty21a strain encoding a polypeptide containing multiple antigens Construction of a Salmonella typhi Ty21a strain encoding a polypeptide containing multiple antigens. Nine dominant CD8 epitopes, two on VEGFR-2 (called KDR2 and KDR3), two on MSLN (called MSLN GSL, MSLN IQL), one epitope on WT-1, and three epitopes on CEA (CEA-CSA, CEA CSV, CEA Constructs containing one epitope of CD8 (termed LTL) and OVA were cloned. Epitopes were selected as model antigens based on literature or on predicted binding properties. It is important to note that the model epitopes selected for proof of concept were not mutations or neoantigens but dominant CD8 epitopes from known tumor-associated antigens such as mesothelin (MSLN), Wilms' tumor 1 (WT-1), carcinoembryonic antigen (CEA), tumor stromal antigen (VEGFR-2) or ovalbumin (OVA).

[0144] Assessment of T cell responses to multiple antigens following vaccination In this initial animal study, C57BL / 6 mice were injected with VXMNeo1m or VXM empty vector vaccine for 10 min. 10CFU / dose were administered orally twice (q7dx2) within 1 week (orally). As a positive control, a group of mice was primed (q7dx2) with VXM06m, encoding the entire Wilms tumor (WT1) protein. The negative control group received the VXMNeo1m vector vaccine (10 10 The mice were administered po on the same schedule and dose of empty vector (CFU / dose, q7dx2). On study day 17, 10 days after the last vaccination, mice were euthanized and spleens were removed for pentamer analysis by flow cytometry (FC).

[0145] [Table 2]

[0146] The test article was administered orally via a gavage tube. Regardless of animal group, each animal was gavaged with a pre-treatment buffer (100 μL / animal / treatment) to neutralize gastric acid. This buffer consisted of 2.6 g sodium bicarbonate, 1.7 g L-ascorbic acid, and 0.2 g lactose monohydrate dissolved in 100 mL drinking water and applied within 30 minutes of applying the test article. A new application document was prepared on the day of application.

[0147] Epitope-specific CD8 T cells were analyzed by flow cytometry on splenocytes harvested 10 days after the last vaccination using a set of specific pentamers. A background threshold was set using an irrelevant pentamers, i.e., HPV16 E7 49-57.

[0148] The results of CD8 T cell responses measured using nine identical peptide pentamer flow cytometry reagents and an additional HPV reagent as a negative control are shown in FIG.

[0149] Example 2: Stability testing of pharmaceuticals The final drug product was produced from three constructs encoding three different target antigens based on the same Salmonella Typhi Ty21a delivery platform in the same formulation and container / closure system. 4 , 10 5 , 10 6 , 10 7 CFU / mL strengths were produced. Vials were stabilized at ≤70°C. At different stability evaluation time points, identity, content, potency, pH and microbiological purity were tested as per predefined stability testing protocol. Predefined specifications were met at all time points and samples from all three constructs were obtained confirming the stability of Vaximm platform constructs over a period of 3 years when stored at ≤70°C regardless of cloned insert. Viable count test results are shown in Figure 2.

[0150] Sequence Listing SEQ ID NO: 1 Amino acid sequence of human VEGFR-2 SEQ ID NO:2 Nucleotide sequence of the expression vector pVAX10 without the multiple cloning site located between the restriction sites NheI and XhoI SEQ ID NO: 3: Amino acid sequence of truncated (zinc finger domain deleted) human WT1 SEQ ID NO: 4 Amino acid sequence of human MSLN SEQ ID NO: 5 Amino acid sequence of human CEA SEQ ID NO: 6: Amino acid sequence of wild-type CMVpp65 SEQ ID NO: 7 Amino acid sequence of CMV pp65 with mutation K436N SEQ ID NO:8 Amino acid sequence of truncated CMV pp65 with the mutation K436N and lacking the C-terminal NLS (aa537-561 of SEQ ID NO:7) SEQ ID NO: 9. Amino acid sequence of human full-length human PD-L1 SEQ ID NO: 10 Amino acid sequence of human PD-L1 lacking the signal peptide SEQ ID NO: 11. Amino acid sequence of truncated human PD-L1 including the extracellular domain and signaling peptide

Claims

1. 1. A pharmaceutical composition for treating a solid tumor in a subject following adoptive cell transfer of engineered T cells, NKT cells or NK cells comprising a tumor antigen-binding cell surface receptor, the pharmaceutical composition comprising a Salmonella typhi Ty21a strain comprising a DNA molecule comprising a eukaryotic expression cassette encoding a polypeptide comprising five or more neoantigens, and the tumor antigen-binding cell surface receptor is a chimeric antigen receptor (CAR).

2. 2. The pharmaceutical composition of claim 1, wherein the five or more neoantigens are tumor-specific antigens identified in a solid tumor of the subject.

3. Five or more neoantigens (a) CD8 T cell antigen; or (b) CD8 and CD4 T cell antigens The pharmaceutical composition according to claim 1 or 2, comprising:

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the treatment further comprises administration of a checkpoint inhibitor.

5. 5. The pharmaceutical composition of claim 4, wherein the checkpoint inhibitor is selected from the group consisting of antibodies against PD-1, PD-L1, CTLA-4, IDO, GITR, OX40, TIM-3, LAG-3, KIR, CSF1R, and CD137.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the engineered T cell, NKT cell or NK cell comprising a tumor antigen-binding cell surface receptor is a chimeric antigen receptor (CAR)-T cell, a CAR-NKT cell or a CAR-NK cell.

7. Salmonella typhi strain Ty21a (a) about 2 weeks to 4 months after the initial adoptive cell transfer of engineered T cells, NKT cells, or NK cells containing a tumor antigen-binding cell surface receptor; or (b) about 2-3 months after the initial adoptive cell transfer of engineered T cells, NKT cells, or NK cells containing a tumor antigen-binding cell surface receptor. The pharmaceutical composition according to any one of claims 1 to 6, wherein the composition is administered to

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the subject has undergone lymphodepleting chemotherapy prior to adoptive cell transfer of engineered T cells, NKT cells or NK cells comprising a tumor antigen-binding cell surface receptor.

9. 9. The pharmaceutical composition according to claim 8, wherein the lymphocyte and / or white blood cell counts are standardized before the Salmonella typhi strain Ty21a is administered.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the Salmonella typhi Ty21a strain is administered orally.

11. 11. The pharmaceutical composition of any one of claims 1 to 10, wherein the treatment further comprises administration of a Salmonella typhi Ty21a strain comprising a DNA molecule comprising a eukaryotic expression cassette encoding an antigen selected from the group consisting of human Wilms tumor protein (WT1), human mesothelin (MSLN), human CEA, CMV pp65, human PD-L1, VEGFR-2 and human fibroblast activation protein (FAP).

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the solid tumor is selected from colorectal cancer, pancreatic cancer, lung cancer, ovarian cancer, mesothelioma, glioblastoma, gastric cancer, hepatocellular carcinoma, renal cell carcinoma, prostate cancer, cervical cancer, breast cancer and melanoma.

13. (a) A single dose of Salmonella typhi Ty21a strain is about 10 ６ ~about 10 ９ and / or (b) the pharmaceutical composition according to any one of claims 1 to 12, wherein the Salmonella typhi strain Ty21a is administered 2 to 4 times during the first week, followed by a single booster dose every 2 to 4 weeks.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the Salmonella typhi strain Ty21a is in the form of a pharmaceutical composition and further comprises at least one pharma- ceutically acceptable excipient.

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