Salmonella-based DNA vaccines combined with antibiotics

Abstract

The present invention relates to an orally administered Salmonella typhi Ty21a strain, optionally in combination with a checkpoint inhibitor, comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen, for use in the treatment of cancer in a human subject following treatment with an antibiotic.

Description

[Technical Field]

[0001] The present invention relates to an orally administered Salmonella typhi Ty21a strain, optionally in combination with a checkpoint inhibitor, comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen, for use in the treatment of cancer in a human subject following treatment with an antibiotic. [Background technology]

[0002] The discovery that tumors can elicit immunity has led to the development of numerous cancer immunotherapies designed to harness the immune system to selectively eliminate malignant cells while sparing normal tissue. However, vaccination with tumor antigens alone has yet to confer a significant survival benefit. Anticancer vaccines face many challenges, including the immunosuppressive microenvironment and optimally targeting host proteins for immune stimulation without eliciting an autoimmune response. Aberrant tumor vasculature creates a hypoxic microenvironment, which directs inflammatory cells toward immunosuppression. Furthermore, tumors alter the proliferation, differentiation, and function of immune cells throughout the body through the secretion of growth factors and cytokines.

[0003] There are several methods for immunizing against cancer, but one very promising method is to use bacteria such as Salmonella as a carrier for DNA vaccines against tumor or stromal antigens. For example, WO 2014 / 005683 discloses an attenuated strain of Salmonella containing a recombinant DNA molecule encoding a VEGF receptor protein for use in cancer immunotherapy, particularly for the treatment of pancreatic cancer. This vaccine, which expresses human VEGFR-2, is also called VXM01.

[0004] Furthermore, WO 2014 / 173542, WO 2015 / 090584, WO 2016 / 2020458, and WO 2018 / 167290 disclose attenuated strains of Salmonella containing recombinant DNA molecules encoding Wilms tumor protein, mesothelin, CMVpp65, or PD-L1, respectively, for use in cancer immunotherapy.

[0005] WO 2013 / 09189 discloses a method for growing an attenuated mutant Salmonella typhi strain lacking galactose epimerase activity and carrying a recombinant DNA molecule, and WO 2018 / 011289 discloses a rapid and effective method for producing a personalized cancer vaccine comprising an attenuated strain of Salmonella.

[0006] Glioblastoma is the most aggressive cancer originating in the brain, with WHO grade IV being the most aggressive form of glioma. Median patient survival after initial diagnosis remains less than 15 months in multiple study cohorts, with nearly all patients suffering from tumor recurrence, and only 25% surviving beyond one year. Since 2005, surgery followed by radiation therapy in combination with temozolomide has been the standard first-line treatment for glioblastoma. Further treatment options are limited after initial treatment failure. There is no standard treatment for recurrent glioblastoma. Novel and more effective immunotherapeutic approaches are greatly needed to prolong patient survival. VXM01 is a DNA vaccine encoding VEGFR-2, delivered orally in a Salmonella Ty21a carrier. High expression of VEGFR-2 on the surface of glioblastoma tumor tissue and tumor vasculature serves as a promising target for VEGFR-2-primed T cells. In a phase I / II VXM01 study of glioblastoma, administration of VXM01 to 14 patients with recurrent tumors demonstrated an acceptable safety profile. Objective clinical responses (CR and PR) in two patients and prolonged overall survival could be associated with VEGFR-2-specific immune responses. J Clin Oncol 36, 2018 (suppl; abstr. 2017).

[0007] Because treatment options for recurrent glioblastoma are particularly limited and the prognosis for patients with this particular solid tumor is poor, improved cancer therapy approaches are needed in general, including combination therapies, particularly in further improving therapeutic vaccinations against tumors. Checkpoint inhibitors have previously been described to improve vaccination with Salmonella-based DNA vaccines, such as in WO 2016 / 202459 and WO 2018 / 083209.

[0008] The host microbiota, or resident microorganisms, has recently attracted interest. The gut microbiome, in particular, contains a rich and highly diverse array of microorganisms that perform important and beneficial functions, including nutrient metabolism, maintenance of intestinal homeostasis, and modulation of intestinal mucosal immunity. Given the growing list of ways the microbiome may influence the immune system, it may be surprising to discover that it also influences vaccine responses, although evidence is currently relatively limited (Lynn and Pulendran, J. Leukoc. Biol., 2018; 103(2): 225-231). While the microbiome is speculated to act as a vaccine adjuvant and be important for antibody responses, its interactions and effects are far from understood. Even less is known about T cell responses, which would be detrimental to therapeutic vaccines against solid tumors. Furthermore, only a small fraction of the microbiome's role in the human immune response remains.

[0009] One study reported a positive effect of antibiotics on B-cell responses to a vaccine containing live-attenuated Salmonella typhi Ty21a in mice (Woo et al., Clinical and Diagnostic Laboratory Immunology, 1999; 6(6): 832-837). In this study, Salmonella typhi Ty21a was transformed with pBR322 to be resistant to ampicillin and doxycycline and transiently resistant to clarithromycin, and then administered intraperitoneally to mice along with ampicillin, doxycycline, or clarithromycin. No effect on T-cell responses was reported.

[0010] The inventors have surprisingly found that pretreatment with antibiotics enhances the therapeutic efficacy of a Salmonella-based oral DNA vaccine against human cancer. Summary of the Invention

[0011] The present invention relates to an orally administered Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen for use in the treatment of cancer in a human subject following antibiotic treatment.

[0012] In one embodiment, the Salmonella Typhi Ty21a strain comprises a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one antigen selected from the group consisting of human Wilms tumor protein (WT1), human mesothelin (MSLN), human CEA, CMV pp65, human PD-L1, human VEGFR-2, and human fibroblast activation protein (FAP). In another embodiment, the Salmonella Typhi Ty21a strain comprises a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one neoantigen (preferably, at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens).

[0013] Optionally, the S. Typhi strain Ty21a can be administered in combination with at least one checkpoint inhibitor, preferably simultaneously with or prior to said at least one checkpoint inhibitor. In one embodiment, the S. Typhi strain Ty21a is administered in combination with at least one checkpoint inhibitor, preferably an immunomodulatory antibody 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.

[0014] In one embodiment, the S. Typhi Ty21a strain is administered at least three days after the completion of antibiotic treatment. In one embodiment, the S. Typhi Ty21a strain is administered within one month after the completion of antibiotic treatment, preferably the first dose of the S. Typhi Ty21a strain is administered approximately between three days and one month after the completion of antibiotic treatment. Preferably, the antibiotic is one to which the S. Typhi Ty21a strain containing the DNA molecule comprising at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen is not resistant.

[0015] The antibiotic to be administered may be a combination preparation. In certain embodiments, the antibiotic selected may be a penicillin (e.g., amoxicillin, ampicillin, piperacillin, or flucloxacillin), a cephalosporin, a polymyxin (e.g., colistin), a rifamycin (e.g., rifaximin), a lipiarmycin, a quinolone (e.g., ciprofloxacin), a sulfonamide (e.g., sulfamethoxazole), a macrolide (e.g., erythromycin), a linocosamide, a tetracycline (e.g., tetracycline), or a combination of penicillins (e.g., penicillin, ampicillin, piperacillin, or flucloxacillin). The antibiotic may be selected from the group consisting of cyclosporine, cyclohexyl benzoate ...

[0016] The Salmonella typhi strain Ty21a for use according to the present invention may be accompanied by chemotherapy or radiotherapy.

[0017] In a preferred embodiment, the cancer to be treated is a solid tumor, examples of which are 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. In one embodiment, the solid tumor is a glioblastoma (preferably a recurrent glioblastoma).

[0018] The Salmonella typhi Ty21a strain for use according to the present invention is about 10 6 ~about 10 9 , more precisely about 10 6 ~about 10 8 , most precisely about 10 6 ~about 10 7The S. Typhi Ty21a strain may be administered as a single dose containing colony-forming units (CFUs); and / or the S. Typhi Ty21a strain may be administered two to four times during the first week, followed by single booster doses every two to four weeks. The S. Typhi Ty21a strain may be in the form of a pharmaceutical composition, further comprising at least one pharmaceutically acceptable excipient. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 shows the plasmid map of pVAX10.VR2-1, which expresses VEGFR-2 as a representative antigen.

[0020] [Figure 2A] Figure 2A shows a schematic overview of the phase I / II combination clinical trial in patients with recurrent glioblastoma treated with VXM01 and the anti-PD-L1 checkpoint inhibitor avelumab, including the timeline of this trial and the individual responses of participating patients, including partial response (PR), stable disease (SD), and progressive disease (PD).

[0021] [Figure 2B] Figure 2B shows a schematic overview of a Phase I / II combination clinical trial in patients with recurrent glioblastoma treated with VXM01 and the anti-PD-L1 checkpoint inhibitor avelumab concomitantly with antibiotics, including the timeline of the trial and individual responses of participating patients (e.g., partial response (PR), stable disease (SD), and progressive disease (PD)). The duration of treatment of these four patients with Cotrim forte® is indicated by the black bars.

[0022] [Figure 3] Figure 3 shows the tumor responses of nine patients treated with VXM01 and the anti-PD-L1 checkpoint inhibitor avelumab shown in Figure 2. Individual patient numbers are shown on the x-axis, and tumor diameter as a percentage of the tumor diameter at baseline (d0) is given on the y-axis for the months indicated.

[0023] [Figure 4] Figure 4 shows VEGFR-2-specific T cell responses and tumor responses to treatment with VXM01 and avelumab in patient #0104, who was pre-treated with Cotrim forte®. A) Enzyme-Linked Immuno Spot Assay (ELISpot) results (pooled - negative control) on blood samples from patient #0104 at day 0 of the clinical trial and approximately 3, 6, and 9 months into the clinical trial are shown as VEGFR-2 pooled-specific spot counts per 4 x 10 peripheral blood mononuclear cells (PBMCs). B) Change in tumor volume compared to baseline is shown for patient #0104 at approximately 3, 6, and 9 months into the clinical trial.

[0024] [Figure 5] Figure 5 shows the levels of intratumoral immune biomarkers in immunohistochemistry sections of tumor samples obtained from patient 0104 at baseline. A) Levels of CD8+ T cells, FoxP3+ T cells, and CD68+ T cells per mm2 are shown. B) PD-1 and PD-L1 staining are shown as histo-scores.

[0025] [Figure 6] Figure 6 shows baseline intratumoral immune biomarker levels and tumor response after treatment with VXM01 and avelumab in patient #0109, who was pretreated with Cotrim forte® from study days 3 through 7 (i.e., covering the time points of the second through fourth initial study drug doses). A) Baseline levels of CD8+ T cells, FoxP3+ T cells, and CD68+ T cells per mm2 are shown. B) PD-1 and PD-L1 staining are shown as histo-scores.

[0026] [Figure 7]Figure 7 shows tumor responses in three evaluable patients treated with VXM01 and the anti-PD-1 checkpoint inhibitor nivolumab in a previous clinical trial. Tumor size shrank in patients 2611 and 2603, who showed PR after 30 months and CR after 6 months of treatment with VXM01 and nivolumab, respectively. In patient 2603, a partial response (tumor size shrank from 15 × 11 mm at baseline to 2 × 2 mm at 3 months) was already observed at 3 months with VXM01 monotherapy. DETAILED DESCRIPTION OF THE INVENTION

[0027] In one aspect, the present invention relates to an orally administered Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen for use in treating cancer in a human subject following antibiotic treatment.

[0028] In another aspect, the present invention relates to a method of treating cancer in a human subject, comprising administering an antibiotic to the human subject followed by oral administration of a Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen.

[0029] In yet another aspect, the present invention relates to an orally administered Salmonella typhi strain Ty21a comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen for use in treating cancer in a human subject who has been or is being treated with at least one antibiotic.

[0030] The tumor antigen can be a tumor-specific antigen or a tumor-associated antigen. Tumor-specific antigens include neoantigens. Thus, in one embodiment, the Salmonella Typhi Ty21a strain comprises a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one neoantigen. Alternatively, the Salmonella Typhi Ty21a strain may be referred to throughout this application as comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen. In one embodiment, the Salmonella Typhi Ty21a strain comprises a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one neoantigen (preferably at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens).

[0031] The live attenuated Salmonella strain, more specifically the S. Typhi Ty21a strain of the present invention, stably harbors a recombinant DNA molecule comprising at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen. The term "S. Typhi Ty21a strain," as used herein, refers to an attenuated strain of Salmonella, more specifically an attenuated strain of S. Typhi, which attenuated strain is Ty21a, and is used herein synonymously with "attenuated strain S. Typhi Ty21a."

[0032] According to the present invention, the Salmonella typhi Ty21a strain functions as a bacterial carrier for a DNA molecule containing at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen and delivers the DNA molecule to target cells. Therefore, the DNA molecule is a recombinant DNA molecule. Preferably, the DNA molecule is a plasmid containing at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen. Such a bacterial carrier or delivery vector containing a DNA molecule containing at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen can also be referred to as a DNA vaccine. Therefore, the present invention further relates to an orally administered DNA vaccine comprising a Salmonella typhi Ty21a strain containing a DNA molecule containing at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen for use in treating cancer in a human subject following antibiotic treatment.

[0033] Genetic immunization may have advantages over conventional vaccination: the target DNA can be detected for a considerable period of time and thus act as a depot for antigens; sequence motifs within some plasmids (e.g., CpG islands) are immunostimulatory and can function as adjuvants, enhanced by the immune stimulation caused by LPS and other bacterial components.

[0034] In the context of the present invention, the term "vaccine" refers to an agent capable of inducing an immune response in a subject when administered. Preferably, the vaccine can prevent, ameliorate, or treat a disease. In the context of the present invention, the vaccine is an oral vaccine. The Salmonella Typhi Ty21a strain of the present invention comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen can be abbreviated as "S. Typhi Ty21a strain encoding at least one antigen" or "cancer vaccine." In a preferred embodiment, the Salmonella Typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encodes at least one tumor antigen and / or stromal antigen, more preferably a tumor antigen or stromal antigen.

[0035] Live attenuated Salmonella vectors produce their own immunomodulatory factors (e.g., lipopolysaccharide (LPS)) in situ, which may constitute an advantage over other forms of administration (e.g., microencapsulation). Furthermore, the mucosal vaccine of the present invention has an intralymphatic mode of action, which has proven beneficial. After inoculation with the attenuated vaccine of the present invention, the modified bacteria invade macrophages and other cells in the intestinal Peyer's patches. The bacteria are ingested by these phagocytes. Due to the attenuating mutation, Salmonella Typhi Ty21 bacteria are unable to persist in these phagocytes and die. After release, the recombinant DNA molecule is transferred into the cytosol of phagocytes via specific transport systems or endosomal leakage. Finally, the recombinant DNA molecule enters the nucleus, where it is transcribed, resulting in the abundant expression of the at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen in the cytosol of phagocytes. Infected cells loaded with at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen undergo apoptosis and are internalized and processed by the intestinal immune system. Danger signals from bacterial infection act as powerful adjuvants in this process, leading to strong target antigen-specific CD8+ T cell and antibody responses at both the systemic and mucosal tissue levels. This immune response peaks approximately 10 days after vaccination. The absence of anti-carrier responses allows for multiple boosts with the same vaccine.

[0036] In the context of the present invention, the term "attenuated" refers to a bacterial strain that, due to an attenuating mutation, has reduced pathogenicity compared to a parent bacterial strain that does not carry the attenuating mutation. Preferably, an attenuated bacterial strain has lost its pathogenicity but retains the ability to induce protective immunity. Attenuation can be achieved by deletion of various genes, including virulence, regulatory, and metabolic genes. Attenuated bacteria can be found in nature or can be artificially created in the laboratory, for example by adapting to new media or cell culture conditions, or by recombinant DNA technology. The attenuated strains of Salmonella according to the present invention can be cultured for approximately 10 11Preferably, administration of CFU results in less than 5% of subjects experiencing salmonellosis, more preferably less than 1%, and most preferably less than 1%.Strain Ty21a of the present invention is an attenuated strain of Salmonella typhi.

[0037] The terms "comprise" or "comprising" mean "including, by way of non-limiting example." This term is intended to be open-ended and specifies the presence of any stated feature, element, integer, step, or component, but does not preclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" encompasses the more restrictive terms "consisting of" and "essentially consisting of." In one embodiment, the term "comprising" can be individually replaced with the term "consisting of." The term "one" as used herein may include a plurality, and therefore includes, but is not limited to, "one."

[0038] The term "antigen" as used herein refers to any protein or peptide suitable for inducing an immune response. However, in the context of the Salmonella Typhi Ty21a strain of the present invention, the term "antigen" relates to a tumor antigen, a tumor stromal antigen, or a checkpoint inhibitor antigen, which can be a tumor-specific antigen (including a neoantigen) or a tumor-associated antigen. The term "tumor antigen" as used herein refers to an antigen that is expressed or overexpressed only in tumors (preferably solid tumors). Thus, in one embodiment, the Salmonella Typhi Ty21a strain comprises a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one neoantigen. In a preferred embodiment, the at least one neoantigen is expressed as at least one polypeptide comprising five or more neoantigens.

[0039] In one embodiment, the Salmonella Typhi Ty21a strain comprises a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one antigen selected from the group consisting of human Wilms' tumor protein (WT1), human mesothelin (MSLN), human CEA, CMV pp65, human PD-L1, human VEGFR-2, and human fibroblast activation protein (FAP), preferably human VEGFR-2. The Salmonella Typhi Ty21a strain of the present invention can also comprise a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising one, two, three, four, five, or more antigens selected from the group consisting of tumor antigens, stromal antigens, and checkpoint inhibitor antigens, preferably human VEGFR-2.

[0040] In another embodiment, or in addition, the Salmonella Typhi Ty21a strain comprises a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neo-antigens, which in one embodiment are tumor-specific antigens identified in a solid tumor of the subject.

[0041] Non-limiting examples of tumor antigens, particularly human tumor antigens, include human Wilms tumor protein (WT1), human mesothelin (MSLN), human carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), folate-binding protein (FBP), ganglioside GD2, ganglioside GD3, human programmed death-ligand 1 (PD-L1), vascular endothelial growth factor receptor 2 (VEGFR-2), human fibroblast activation protein (FAP), melanoma antigen A1 (MAGE-A1), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), mucin-1 (MUC1), glypican-3 (GPC3), epithelial cell adhesion molecule (EpCAM), B-cell maturation antigen (BCMA), and tyrosine-protein kinase transmembrane receptor (ROR1), and anti-cytomegalovirus pp65 (CMV). pp65), and solid tumors, for example, may express these tumor antigens, as listed in Table 1: [Table 1]

[0042] In one particular example, the Salmonella typhi Ty21a strain of the present invention comprises a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one antigen selected from the group consisting of WT1, MSLN, CEA, CMVpp65, PD-L1, VEGFR-2, and FAP. In a further example, the Salmonella typhi Ty21a strain of the present invention can comprise a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising one, two, three, four, five, or more antigens. In one particular example, the Salmonella typhi Ty21a strain comprises a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neo-antigens.

[0043] In a particular embodiment, human VEGFR-2 comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1. In a particular embodiment, human Wilms tumor protein (WT1) comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 3. In a particular embodiment, human mesothelin (MSLN) comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 4. In a particular embodiment, human CEA comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 5. In a particular embodiment, CMV pp65 comprises the amino acid sequence of SEQ ID NO: 6, 7, or 8, or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 6, 7, or 8. In particular embodiments, human PD-L1 comprises the amino acid sequence of SEQ ID NO: 9 or 10, or an amino acid sequence that shares at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 9, 10, or 11.

[0044] 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.

[0045] VEGFR-2, also known as the kinase insert domain-containing receptor (KDR), appears to mediate nearly all known cellular responses to VEGF. For example, the role of VEGF in angiogenesis appears to be mediated through the interaction of this protein with VEGFR-2. VEGFR-2 is a high-affinity receptor for VEGF as well as VEGF-C and VEGF-D. It is 1,356 amino acids in length and has a molecular mass of 200–230 kDa. VEGFR-2 was identified in humans through a screen of endothelial cDNAs 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 and hematopoietic precursors, as well as endothelial cells, neoplastic hematopoietic stem cells, and umbilical cord stroma. However, VEGFR-2 mRNA appears to be downregulated in the quiescent adult vasculature.

[0046] The extracellular domain of VEGFR-2 contains 18 potential N-linked glycosylation sites. VEGFR-2 is initially synthesized as a 150 kDa protein, rapidly glycosylated to a 200 kDa intermediate form, and then further glycosylated at a slower rate to the mature 230 kDa protein, which is expressed on the cell surface. In one embodiment, at least one tumor antigen, tumor stromal antigen, and / or checkpoint inhibitor antigen comprises or is the extracellular domain of VEGFR-2. The Salmonella typhi Ty21a strain containing a DNA molecule comprising at least one eukaryotic expression cassette encoding VEGFR-2 is also referred to as VXM01. More specifically, VXM01 contains the plasmid shown in FIG. 1.

[0047] VEGF receptors have long been thought to be restricted to the vasculature of malignant tumors, i.e., the tumor stroma. However, recent expression analyses have revealed that vascular endothelial growth factor receptors, particularly VEGFR-2, are expressed on the surface of tumor cells themselves. Tumor-specific VEGF receptor expression has been observed on the surface of cancer cells of various origins, indicating that VEGF may have additional effects on tumorigenesis beyond promoting angiogenesis. Non-limiting examples of cancers characterized by VEGFR-2-expressing cancer cells include glioblastoma, carcinoid cancer, kidney cancer (particularly renal cell carcinoma), thyroid cancer, lung cancer (particularly non-small cell lung cancer [NSCLC]), breast cancer, ovarian cancer, prostate cancer, gastrointestinal cancer (particularly colorectal cancer, more specifically colon cancer), and skin cancer (particularly melanoma).

[0048] One particularly promising target for VEGFR-2-targeted immunotherapy is glioblastoma. Glioblastoma exhibits extensive tumor angiogenesis. Furthermore, VEGFR-2 may be targeted on both the tumor vasculature and the surface of tumor cells. Approximately 20%–50% of glioblastoma patients exhibit tumor-specific VEGFR-2 expression, which is particularly observed at the invasive front. Furthermore, VEGFR-2 expression has been observed in glioma-like stem cells. To date, treatment options for glioblastoma remain inadequate. For example, the monoclonal antibody Avastin, which targets only VEGF, has demonstrated benefits in terms of progression-free survival (PFS) but not overall survival (OS).

[0049] Therefore, the present invention also encompasses a case in which a human subject has been determined to have a cancer characterized by cancer cells expressing VEGFR-2 or to have cancer cells expressing at least one VEGFR-2. As a first step, tumor-specific VEGFR-2 expression (e.g., tumor-specific expression of VEGFR-2) in the subject can be assessed at the mRNA or protein level, preferably in vitro. For this purpose, tumor tissue samples (e.g., biopsies) can be stained, for example, by immunohistochemistry or in situ hybridization. Methods for assessing tumor-specific antigen expression are well known in the art. The same applies to determining whether a human subject has a cancer characterized by cancer cells expressing tumor antigens (particularly cancer cells expressing human WT1, human MSLN, human CEA, CMV pp65, human PD-L1, and human FAP), or whether a human subject has cancer cells expressing at least one tumor antigen (particularly cancer cells expressing human WT1, human MSLN, human CEA, CMV pp65, human PD-L1, and human FAP). Those skilled in the art will readily appreciate that the Salmonella typhi Ty21a strain containing a DNA molecule comprising at least one eukaryotic expression cassette encoding human WT1, human MSLN, human CEA, CMV pp65, human PD-L1, and / or a FAP may be used to treat cancer in a human subject determined to have a cancer characterized by cancer cells expressing WT1, MSLN, CEA, CMV pp65, PD-L1, and / or a FAP, respectively, or to have cancer cells expressing at least one of WT1, MSLN, CEA, CMV pp65, PD-L1, and / or a FAP.

[0050] Mesothelin is a 40-kDa cell surface glycoprotein present on the surface of normal mesothelial cells and is overexpressed in several human tumors, including mesothelioma and adenocarcinomas of the ovary and pancreas. The mesothelin gene encodes a 71-kDa precursor protein, which is processed to produce a 31-kDa cleaved 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 present at low levels on the surface of a limited set of normal adult tissues (e.g., mesothelium), but is a tumor differentiation antigen that is aberrantly overexpressed in a variety of human tumors, including mesothelioma, ovarian cancer, and pancreatic cancer; squamous cell carcinomas 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 in mesothelin knockout mice have revealed no detectable phenotype, and both male and female mice produce healthy offspring. Studies of pancreatic cancer suggest that mesothelin plays a role in tumorigenesis by increasing cell proliferation, migration, and the S-phase cell population. Furthermore, there is evidence that mesothelin is an immunogenic protein. The tumor antigen mesothelin is a promising candidate for cancer vaccine development due to its expression profile, its oncogenic function, and its immunogenicity.

[0051] 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. Numerous well-characterized transcript variants resulting from alternative splicing at two coding exons have been well characterized. WT1 plays a crucial role in the development of the urogenital system and is involved in cell proliferation and differentiation. The WT1 gene was isolated as the gene responsible for Wilms tumor, a pediatric renal tumor. It is highly expressed in a variety of malignancies, including several types of hematologic malignancies and various solid tumors. In contrast, normal tissue expression of WT1 in adults is restricted to the gonads, uterus, kidney, mesothelium, and progenitor cells in various tissue types. WT-1 negatively influences differentiation and promotes the proliferation of progenitor cells. Furthermore, overexpressed WT1 is immunogenic; WT1-specific T cells as well as IgG anti-WT1 antibodies have been observed in cancer patients. Tumor antigen WT1 is a promising candidate for developing cancer vaccines due to its expression profile, its oncogenic function, and its immunogenicity.In a specific embodiment, WT1 is truncated.In a specific embodiment, the zinc finger domain of WT1 is deleted.In a specific embodiment, the truncated WT1 has the amino acid sequence of SEQ ID NO: 3.

[0052] The zinc finger domain at the C-terminus of WT1 contains four zinc finger motifs. Truncated WT1, having the amino acid sequence of SEQ ID NO: 3, represents amino acids 1-371 of UniProt ref P19544-7. Deletion of the zinc finger domain minimizes the risk of immunological cross-reactivity with other zinc finger-containing transcription factors. Furthermore, truncated WT1 lacking the zinc finger domain has greater immunogenic potential than full-length WT1. Additionally, deletion of the zinc finger motifs, which are essential for DNA binding, eliminates the oncogenic potential of WT1, thereby minimizing the risk of oncogenicity.

[0053] The tegument protein CMV pp65 is a major immunodominant protein of human cytomegalovirus (CMV). Although the biological function of CMV pp65 is unclear, it is thought to be involved in cell cycle regulation. CMV pp65 is a nucleotropic protein that exhibits protein kinase activity and can bind to polo-like kinase (PLK-1). Human CMV pp65 is expressed in over 90% of glioblastoma samples but not in surrounding normal brain. Therefore, this viral protein is a promising candidate as a tumor-specific target for developing novel cancer immunotherapies.

[0054] The CMV pp65 protein contains two bipartite nuclear localization signals (NLSs) near the carboxy terminus, located at amino acids 415-438 and 537-561, and a phosphate-binding site at lysine-436 that is involved in kinase activity. Mutation of lysine-436 to asparagine and deletion of amino acids 537-561 result in a protein without kinase activity and significantly reduced nuclear localization. This mutant protein exhibits unchanged immunogenicity.

[0055] In a particular embodiment, CMV pp65 has the amino acid sequence of SEQ ID NO: 6. SEQ ID NO: 6 represents the amino acid sequence of wild-type human CMV pp65. In another particular embodiment, CMV pp65 has the amino acid sequence of SEQ ID NO: 7. SEQ ID NO: 7 represents the amino acid sequence of human CMV pp65, which has the mutation K436N compared to wild-type human CMV pp65 having the amino acid sequence of SEQ ID NO: 6. In another particular embodiment, CMV pp65 has the amino acid sequence of SEQ ID NO: 8. SEQ ID NO: 8 represents the amino acid sequence of a truncated version of CMV pp65 having the amino acid sequence of SEQ ID NO: 7, lacking the second, more C-terminal NLS (nuclear localization sequence) (i.e., amino acids 537-561 of CMV pp65 of SEQ ID NO: 7).

[0056] Carcinoembryonic antigen (CEA) (also known as CEACAM5 and CD66e) is a member of a family of closely related glycosylphosphatidylinositol (GPI) cell surface-anchored glycoproteins involved in cell adhesion. CEA is normally produced in gastrointestinal tissues during embryonic development; protein expression ceases before birth. Therefore, CEA is normally present at very low levels in the blood of healthy adults. However, serum levels are elevated in several types of cancer (especially colorectal cancer), making it a useful tumor marker. CEA levels may also be elevated in gastric, pancreatic, lung, breast, and medullary thyroid cancer, as well as in several non-neoplastic conditions (e.g., ulcerative colitis, pancreatitis, liver cirrhosis, COPD, Crohn's disease, and hypothyroidism).

[0057] Programmed cell death 1 (PD-1) is expressed on the surface of T cells and transmits inhibitory signals that maintain T cell quiescence against cognate antigens. Its ligand, PD-L1, is normally expressed on the surface of antigen-presenting cells, placental cells, and non-hematopoietic cells in the inflammatory microenvironment. PD-L1 has been reported to be expressed on the surface of immunosuppressive myeloid-derived suppressor cells (MDSCs). In addition, PD-L1 is widely expressed on the surface of various types of cancer cells, and cancer cells utilize the PD-1 / PD-L1 signaling axis to evade the host immune system. PD-L1 expression by cancer cells has been shown to correlate with disease stage and poor patient prognosis.

[0058] In particular embodiments, the PD-L1 is selected from the group consisting of full-length PD-L1 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 that shares at least 80% sequence identity with amino acids 19-238 of SEQ ID NO: 11, SEQ ID NO: 11, or SEQ ID NO: 10. In particular embodiments, the PD-L1 is selected from the group consisting of PD-L1 having the amino acid sequence of SEQ ID NO: 9 and proteins that share at least 80% sequence identity therewith. In another particular embodiment, the PD-L1 is selected from the group consisting of PD-L1 having the amino acid sequence of SEQ ID NO: 10 and proteins that share at least 80% sequence identity therewith. In another particular embodiment, the PD-L1 is selected from the group consisting of PD-L1 having the amino acid sequence of SEQ ID NO: 11 and proteins that share at least 80% sequence identity therewith. In another particular embodiment, the PD-L1 is selected from the group consisting of PD-L1 having the amino acid sequence of amino acids 19 to 238 of SEQ ID NO: 11, and proteins sharing at least 80% sequence identity thereto. In particular, the PD-L1 has the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. Preferably, the PD-L1 comprises the amino acid sequence of amino acids 19 to 238 of SEQ ID NO: 11. In one embodiment, the PD-L1 comprises at least the extracellular domain, with or without its signaling peptide.

[0059] As used herein, the term "about" or "approximately" means within 80% to 120%, or within 90% to 110%, including within 95% to 105%, of a given value or range.

[0060] In the context of the present invention, the phrase "a protein having at least 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 provided amino acid sequence. The protein can be of natural origin (e.g., a mutant version of a wild-type protein, e.g., a mutant version of a wild-type VEGFR-2 protein), or a homologue from a different species, or an engineered protein (e.g., an engineered VEGFR-2 protein). Methods for designing and constructing derivatives of a given protein are well known to those skilled in the art.

[0061] A protein that is at least 80% identical to 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 can be consecutive amino acids or can be scattered throughout the length of the amino acid sequence of a protein that is at least 80% identical to a given reference protein. According to the teachings of the present invention, any number of amino acids can be added, deleted, and / or substituted, as long as the amino acid sequence is at least 80% identical to the reference amino acid sequence and the mutated protein is immunogenic. Preferably, the immunogenicity of a protein that is at least 80% identical to the reference amino acid sequence is reduced by less than 50%, 40%, 30%, 20%, 10%, 5%, or 1% compared to the reference amino acid sequence, as measured by ELISA. Methods for designing and constructing protein homologs and testing the immunogenic potential of such homologs are well known to those skilled in the art. In particular embodiments, the sequence identity with the reference amino acid sequence is at least 85%, at least 90%, at least 95%, and most particularly at least 99%. Methods and algorithms for determining sequence identity (including comparison of a parent protein with its derivatives that have deletions, additions, and / or substitutions compared to the parent sequence) are well known to those skilled in the art. At the DNA level, nucleic acid sequences encoding proteins that have at least 80% sequence identity with a reference amino acid sequence may differ more significantly due to the degeneracy of the genetic code.

[0062] A tumor antigen is an antigen that is expressed or overexpressed only in tumors, preferably solid tumors. Thus, a tumor antigen can be a tumor-specific antigen or a tumor-associated antigen. Tumor-specific antigens include neoantigens. Thus, in one embodiment, the Salmonella typhi Ty21a strain comprises a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one neoantigen. In a preferred embodiment, the at least one neoantigen is expressed as at least one polypeptide comprising five or more neoantigens. Preferably, the five or more neoantigens are tumor-specific antigens identified in the subject's solid tumor.

[0063] The term "neoantigen" 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 mutate in somatic or germline tissues. Unlike germline mutations, somatic mutations are not transmitted to offspring. Therefore, somatic mutations in genes are acquired during cancer development in cancer cells. Typically, mutations are tumor-specific point mutations, generating neoepitopes (also called mutant epitopes or point mutant peptides). Because mutations are not present in normal tissues, they are highly immunogenic and thus circumvent central thymic tolerance. Neoantigens include, and preferably consist of, neoepitopes presented as peptides by MHC I or MHC II. Mutations can also be frameshift mutations, resulting in frameshift peptide (FSP) antigens. FSP neoantigens arise from the insertion or deletion of a single nucleotide but encompass long antigenic amino acid stretches that can contain multiple immunologically significant neoepitopes. In a particular embodiment, the term "neoantigen" further includes T cell epitopes associated with peptide processing (TEIPPs). TEIPPs are derived from ubiquitously expressed, non-mutated "self" proteins that are not loaded onto MHC I in healthy cells. In immune-evading cancers, antigen processing components (such as transporters associated with antigen processing (TAPs)) are often downregulated. Therefore, TEIPPs may be presented on the surface of cancer cells only in cells with defective antigen processing mechanisms, such as the absence of TAP due to mutation or epigenetic silencing (Marjit et al., Journal of Experimental Medicine, 2018, 215(9): 2325).

[0064] During cancer progression, mutations accumulate in the cancer genome, potentially affecting protein-coding genes and resulting in altered protein sequences. Mutant proteins are proteolytically cleaved into short peptides and presented on the surface of tumor cells by MHC (human leukocyte antigens (HLA) in humans). These somatically mutated genes, or neoantigens, are presented by malignant T cells but not by normal cells and may be recognized as foreign by tumor-infiltrating lymphocytes (TILs). Therefore, the term neoantigen refers to peptides containing, or preferably consisting of, peptides containing somatic mutations presented by MHC I or II. Neoantigens presented by MHC I can also be referred to as CD8 T cell antigens. Neoantigens presented by MHC II can also be referred to as CD4 T cell antigens (or T helper antigens). Because neoantigens can be recognized as foreign by TILs, they can induce potent tumor-specific immune responses. Neoantigens released after tumor cell death initiate multiple processes that ultimately lead to T cell recognition of cancer cells through the interaction of distinct T cell receptors (TCRs) with specific neoantigen-MHC complexes.

[0065] The phrase "at least one polypeptide comprising five or more neoantigens" herein refers to one polypeptide or two or more polypeptides collectively comprising five or more neoantigens. Whether the five or more neoantigens are part of the same or different polypeptides is immaterial. Thus, the five or more neoantigens can be expressed as one polypeptide or as two or more polypeptides. Preferably, the neoantigens contained in 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 the Salmonella typhi Ty21a strain used herein, an insert encoding the at least one polypeptide can contain up to 300 neoantigens, preferably up to 200 neoantigens. Antigens presented as peptides on the surface of MHC class I or II (in human HLA) are typically 11-30 amino acids in length for MHC II (CD4 antigen) and 8-10 amino acids in length for MHC I (CD8 antigen). Thus, the five or more neoantigens can preferably include a CD8 T cell antigen or a CD8 T cell antigen and a CD4 T cell antigen. Furthermore, preferred ranges of neoantigens contained within the at least one polypeptide can be 5-300, 10-300, 20-300, 30-300, 50-300, or greater than 50-300. More preferred ranges of neoantigens contained within the at least one polypeptide can be 5-200, 10-200, 20-200, 30-200, 50-200, or greater than 50-200. Each polypeptide comprising the fusion neoantigen is proteolytically cleaved into neoantigens within antigen-presenting cells, which are presented via HLA to elicit T cell responses.

[0066] According to the present invention, the five or more neo-antigens may include CD8 T cell antigens and / or CD4 T cell antigens. Preferably, the five or more neo-antigens include CD8 T cell antigens and CD4 T cell antigens.

[0067] It is hypothesized that vaccination with neoantigens will both expand pre-existing neoantigen-specific T cell populations and induce a broader repertoire of new T cell specificities in cancer patients.

[0068] Neoantigens are typically peptides having 8 to 30 amino acids, preferably 8 to 20 amino acids, and more preferably 8 to 12 amino acids.

[0069] It would be beneficial for a neoantigen cancer vaccine if the vaccine could target multiple neoantigens, thus reducing the risk of immune escape due to loss of expression of a subset of neoantigens. The invention also encompasses treating a human subject sequentially with different Salmonella typhi Ty21a strains containing a DNA molecule that includes at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens, including new neoantigens or new subsets of neoantigens that are selected and targeted during tumor progression.

[0070] The advantages of using an attenuated strain of Salmonella typhi Ty21a (also referred to as "S. typhi Ty21a") as a carrier of at least one polypeptide containing five or more neoantigens are: a) established quality control assays; b) individual plasmids differ only in the insert encoding one or more neoantigens; c) no propagation requirements; and d) no need for sterility testing due to oral administration. Furthermore, the use of an expression plasmid suitable for transformation and the S. typhi Ty21a strain as a carrier allows for a large number (up to 300) of epitopes (neoantigens). Neoantigens can be inserted into the plasmid as a string of beads (expressed as one or more polypeptides), optionally separated by a linker. Non-limiting examples of linkers include a GS linker, a 2A cleavage site, or an IRES sequence. Because of the rapid production and limited need for 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, e.g., within 15 days, preferably within 14 days or less, from the identification of the neoantigens. Overnight fermentation is sufficient, and the bacteria are high-yielding, so scale-up is not necessary, with a net yield of 10 in a 1 L culture. 11 The production volume is approximately colony-forming units (CFUs), which allows for short production times and low manufacturing costs. Furthermore, each batch is sufficient for years of treatment, and the drug product has been shown to be stable for at least three years. Therefore, batch variability is eliminated, as one batch lasts for the entire treatment period of human subjects with solid tumors.

[0071] A method for generating 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 an individual subject with a solid tumor includes: (a) providing a tumor cell sample and a control sample from the subject; (b) identifying five or more neoantigens present in the tumor cell sample but absent in the control sample; (c) selecting the five or more neoantigens; (d) synthesizing cDNA encoding at least one polypeptide comprising the five or more neoantigens; (e) cloning the cDNA into at least one eukaryotic expression cassette; (f) transforming a recipient Salmonella typhi Ty21a strain with the DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising the five or more neoantigens; (g) fermenting the strain obtained in step (f) and diluting it to a target concentration based on CFU; and (h) analyzing the transformed Salmonella typhi Ty21a strain, including sequencing the cDNA 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. The term "normal tissue" refers to the same tissue, i.e., non-cancerous tissue, preferably of the same origin. The control sample is preferably a blood sample. The blood sample can further be used to determine the patient's HLA type. The tumor cell sample can be a tumor biopsy.

[0072] Methods for detecting (any) coding mutations in tumors and reliably predicting or identifying mutated peptides that autologous human leukocyte antigen (HLA) molecules bind with high affinity are known in the art. For example, whole-exome sequencing (WES) of matched tumor and normal cell DNA from individual patients can be performed. Identified somatic mutations are then validated without antibodies, and the expression of mutated alleles is assessed by RNA sequencing of the tumor. Peptides predicted to have a high potential for binding to the patient's autologous HLA-A or HLA-B proteins are then selected. This can be confirmed, for example, by ex vivo interferon-γ 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.

[0073] A polypeptide can contain several neoantigens fused to each other, preferably 5 or more, 10 or more, 20 or more, 30 or more, or 50 or more. Typical plasmids used to transfect Salmonella Typhi Ty21a strains (e.g., the pVAX1™ expression plasmid (Invitrogen, San Diego, CA) or its derivative pVAX10) can express up to approximately 300 neoantigens. Thus, the polypeptide can contain approximately 5-300, 10-300, 20-300, 30-300, or 50-300 neoantigens, preferably 10-200, 20-200, 30-300, or 50-200 neoantigens. The polypeptide is cleaved intracellularly into peptides, which are presented on the surface of MHC I or MHC II molecules, depending on the type of neoantigen. Individual neoantigens can be separated by linkers (e.g., GS linkers, specifically designed linkers, or 2A cleavage sites). The DNA molecules encoding the neoantigens can also be separated by an IRES sequence, resulting in separate polypeptides.

[0074] 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 can further comprise a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising at least one non-neoantigen tumor antigen and / or tumor stromal antigen, wherein said at least one non-neoantigen tumor antigen is expressed in a solid tumor of the patient being treated. The phrase "at least one non-neoantigen tumor antigen and / or tumor stromal antigen" as used herein means at least one tumor antigen and / or stromal antigen (provided that the tumor antigen is not a neoantigen). The at least one polypeptide comprising at least one non-neoantigen tumor antigen and / or tumor stromal antigen can be (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 by an additional DNA molecule; (b) encoded by at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens, or by an additional expression cassette; or (c) encoded by at least one polypeptide comprising five or more neoantigens, or by an additional polypeptide. Thus, the Salmonella typhi Ty21a strain can be transformed with two DNA molecules, the first encoding five or more neoantigens and the second encoding at least one non-neoantigen tumor antigen and / or tumor stromal antigen. Alternatively, the Salmonella typhi Ty21a strain can be transformed with a single DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens and at least one additional eukaryotic expression cassette encoding at least one non-neoantigen tumor antigen and / or tumor stromal antigen. Alternatively, the Salmonella typhi Ty21a strain can be transformed with a single DNA molecule containing at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens, and further containing at least one non-neoantigen tumor antigen and / or tumor stromal antigen.Non-limiting examples of non-neoantigen tumor antigens in this context include WT1, MSLN, CEA, HER2, EGFR, FBP, GD2, GD3, MAGE-A1, PSCA, PSMA, PD-L1, MUC1, GPC3, and CMV pp65. Tumor antigens can be tumor-specific or tumor-associated antigens. 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 expressed in tumor stroma (non-limiting examples of which include VEGFR-2 and FAP). A Salmonella typhi Ty21a strain containing a DNA molecule containing at least one eukaryotic expression cassette encoding at least one polypeptide containing five or more neoantigens can also contain a DNA molecule containing at least one eukaryotic expression cassette encoding at least one polypeptide containing a checkpoint inhibitor antigen, wherein the at least one checkpoint inhibitor antigen or its ligand is overexpressed in the solid tumor of the patient being treated. Therefore, the checkpoint inhibitor antigen can also be a tumor antigen (e.g., PD-L1), which is often upregulated on the surface of tumor cells. The same applies to the expression of a checkpoint inhibitor antigen in a Salmonella typhi Ty21a strain containing a DNA molecule containing at least one eukaryotic expression cassette encoding at least one polypeptide containing five or more neoantigens as to at least one non-neoantigen tumor antigen and / or tumor stromal antigen. An example of a checkpoint inhibitor antigen is PD-1 or PD-L1; other examples are CTLA-4, IDO, GITR, OX40, TIM-3, LAG-3, KIR, CSF1R, and CD137. The DNA molecule used in this context is preferably an expression plasmid.PD-L1 can also be considered a tumor antigen or a tumor-associated antigen.

[0075] A DNA molecule containing at least one eukaryotic expression cassette can also be called a recombinant DNA molecule, i.e., an engineered DNA construct, and is preferably composed of DNA fragments of different origins. The DNA molecule can be a linear nucleic acid or a circular DNA plasmid (preferably the latter), which is produced by introducing an open reading frame encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen into a eukaryotic expression cassette in a plasmid. A plasmid containing a eukaryotic expression cassette can also be called a eukaryotic expression plasmid.

[0076] 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. Preferably, the expression cassette also comprises a transcription termination signal. Preferably, the expression cassette is capable of mediating transcription in a target cell of the included open reading frame encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen. Eukaryotic expression cassettes typically comprise a promoter, at least one open reading frame, and a transcription termination signal, which allow expression in eukaryotic target cells.

[0077] In a particular embodiment, a single dose of Salmonella Typhi Ty21a strain comprises about 10 6 ~about 10 9 , more precisely about 10 6 ~about 10 8 , most precisely about 10 6 ~about 10 7 Contains colony forming units (CFU).

[0078] More specifically, a single dose of Salmonella typhi Ty21a strain contains approximately 1 × 10 6 ~Approx. 1×10 9 , more precisely, about 1 × 10 6 ~Approx. 1×10 8 , most precisely about 1 × 10 6 ~Approx. 1×10 7Contains colony forming units (CFU).

[0079] In this context, the term "about" or "approximately" means within 3 times or within 2 times, including within 1.5, of a given value or range.

[0080] 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 a single boost dose every 2 to 4 weeks.The Salmonella Typhi Ty21a strain of the present invention is preferably administered on days 1 and 7 (preferably days 1, 3, 5, and 7), followed by a single boost dose every 2 to 4 weeks.

[0081] In a specific embodiment, the treatment comprises a single or multiple administrations of the Salmonella Typhi Ty21a strain of the present invention, or a pharmaceutical composition or DNA vaccine comprising the Salmonella Typhi Ty21a strain of the present invention. The single doses of the multiple administrations may be the same or different, but are preferably the same and within the ranges disclosed herein. In one embodiment, the treatment comprises a prime vaccination and a boost vaccination. The term "prime vaccination" refers to an initial vaccination to prime the immune system, typically comprising two to four single-dose vaccinations within the first week. The term "boost vaccination" refers to subsequent periodic repeated single-dose vaccinations to further stimulate the primed immune system, typically comprising single-dose booster vaccinations every two to four weeks. In particular, the treatment can include priming vaccinations with a Salmonella Typhi Ty21a strain encoding at least one tumor antigen (including at least one polypeptide comprising five or more neoantigens), tumor stromal antigen, and / or checkpoint inhibitor antigen, or a pharmaceutical composition comprising a Salmonella Typhi Ty21a strain of the present invention, administered 2-4 times in the first week of treatment, followed by single boost doses every 2-4 weeks.

[0082] The Salmonella typhi Ty21a strain encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen is for use in treating cancer in a human subject, preferably treating a solid tumor in a human subject, wherein the subject has been or has been treated with at least one antibiotic. In a preferred embodiment, the at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen is used in treating cancer, preferably treating a solid tumor in a human subject, following antibiotic treatment (i.e., the subject has been treated with at least one antibiotic).

[0083] The Salmonella Typhi Ty21a strain can be administered an appropriate time after the completion of antibiotic treatment. For example, the first dose of the Salmonella Typhi Ty21a strain can be administered about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 1 month, or about 2 months after the completion of antibiotic treatment, preferably about 3 days, 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, or about 1 month. In one embodiment, the Salmonella Typhi Ty21a strain is administered at least 3 days after the completion of antibiotic treatment, i.e., 3 days or more than 3 days after the completion of antibiotic treatment. In one embodiment, the (first dose) Salmonella Typhi Ty21a strain is administered about 3 days after the completion of antibiotic treatment. In another embodiment, the (initial dose) of the Salmonella Typhi Ty21a strain is administered within one month after the completion of antibiotic treatment. In yet another embodiment, the (initial dose) of the Salmonella Typhi Ty21a strain is administered approximately one day to one month, preferably three days to one month, more preferably three days to two weeks after the completion of antibiotic treatment. The phrase "after antibiotic treatment" as used herein means after the completion of antibiotic treatment, i.e., after the last dose of antibiotic. The times or periods or ranges indicated relate to the first dose of Salmonella Typhi Ty21a administered, although treatment or administration can continue as long as necessary.

[0084] Without being bound by theory, antibiotics may affect the gut microbiome, which may facilitate the uptake of orally administered Salmonella Typhi Ty21a of the present invention. Increased uptake of Salmonella Typhi Ty21a likely increases the expression of at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen in host cells, thereby increasing the presentation of at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen to the immune system and enhancing immune responses, particularly T cell-mediated immune responses, which are particularly important in cancer treatment. Therefore, any antibiotic suitable for reducing or influencing the gut microbiome is suitable in the context of the present invention. Antibiotics can be single compounds or combinations (e.g., combination preparations including sulfonamides or β-lactamase inhibitors). Non-limiting examples of such β-lactamase inhibitors include sulbactam or tazobactam.

[0085] Alternatively, the S. Typhi Ty21a strain can be administered to a human subject currently undergoing or having been treated with antibiotics. In one embodiment, the S. Typhi Ty21a strain is administered subsequent to or after completion of antibiotic treatment. In another embodiment, at least an initial dose of the S. Typhi Ty21a strain can be administered during antibiotic treatment, preferably 2-4 doses in the first week (prime vaccination), during antibiotic treatment, with repeated single-dose boost doses, preferably every 2-4 weeks, administered after completion of antibiotic treatment.

[0086] Without being bound by theory, it is possible that the simultaneous administration of the S. Typhi Ty21a strain and antibiotics to patient number 0104, in addition to affecting the gut microbiome, may have resulted in killing or inactivation of the S. Typhi Ty21a strain containing the DNA molecule containing at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen, stimulating the immune system, and / or the DNA molecule containing at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen being taken up by host cells through phagocytosis, further expression of the at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen, resulting in effective priming. However, the decrease in tumor volume observed in patient 0104 at 6 months (after 5 months of combined vaccine and antibiotic treatment) compared with that at 3 months, and the further decrease in tumor volume compared with baseline at 9 months, along with the progression of the antigen-specific immune response, suggest that the vaccine was not effective during the first 5 months (i.e., while administered with antibiotics) and that antibiotic treatment followed by boosts every 4 weeks was sufficient to induce an antigen-specific immune response. The immune response may be further enhanced when the S. Typhi Ty21a strain is administered 2–4 times weekly as a prime vaccination followed by a single boost dose every 2–4 weeks, i.e., when the prime and boost are administered after antibiotic treatment is completed (e.g., when the first dose of the prime vaccination is administered approximately 3 days to approximately 1 month after antibiotic treatment is completed, followed by a boost vaccination).

[0087] The antibiotic can be a broad-spectrum antibiotic (such as the broad-spectrum penicillin antibiotics amoxicillin, ampicillin, or piperacillin) or a narrow-spectrum antibiotic (such as a macrolide antibiotic (azithromycin, erythromycin, clarithromycin, fidaxomicin, or roxithromycin) or vancomycin). The antibiotic can also be a bacteriostatic antibiotic, non-limiting examples of which include tetracyclines and sulfonamides, or a bactericidal antibiotic (such as a beta-lactam antibiotic, which includes a penicillin antibiotic).

[0088] In certain embodiments, the antibiotic of choice is selected from penicillins (e.g., amoxicillin, ampicillin, piperacillin, and flucloxacillin), cephalosporins, polymyxins (e.g., colistin), rifamycins (e.g., rifaximin), lipiarmycins, quinolones (e.g., ciprofloxacin), sulfonamides, macrolides (e.g., erythromycin), linocosamides, tetracyclines (e.g., tetracycline), aminoglycosides, benzodiazepines, benzocaine ... and selected from the group consisting of liposides (e.g., paromomycin and neomycin), cyclic lipopeptides (e.g., daptomycin), glycylcyclines (e.g., tigecycline), oxozolidinones (e.g., linezolid), nitrozimazoles (e.g., metronidazole), lipiarmycins (e.g., fidaxomicin), and dihydrofolate reductase inhibitors (e.g., diaminopyrimidines such as trimethoprim or tetroxoprim). The antibiotic is preferably selected from penicillins (such as amoxicillin, ampicillin, piperacillin, and flucloxacillin), polymyxins (such as colistin), rifamycins (such as rifaximin), quinolones (such as ciprofloxacin), sulfonamides (such as sulfometaxozole), macrolides (such as erythromycin), tetracyclines (such as tetracycline), aminoglycosides (such as paromomycin), cyclic lipopeptides (such as daptomycin), nitrosimazoles (such as metronidazole), and diaminopyrimidines (such as trimethoprim). In a particular embodiment, the antibiotic is selected from amoxicillin, ampicillin, piperacillin, flucloxacillin, colistin, rifaximin, ciprofloxacin, sulfometaxozole, erythromycin, tetracycline, paromomycin, daptomycin, metronidazole, and trimethoprim. Those skilled in the art will appreciate that antibiotics are active in the intestine. One reason that antibiotics are preferentially active in the intestine may be, but is not limited to, poor absorption from the intestine. Therefore, one preferred route of administration of antibiotics is oral administration.

[0089] In certain embodiments, an antibiotic can be used in combination, for example, with another antibiotic or another (potentiating) drug. In one embodiment, the antibiotic is sulfamethoxazole or trimethoprim, or a combination thereof, with sulfamethoxazole and trimethoprim being preferred. In a preferred embodiment, the antibiotic is cotrimoxazole. In yet another embodiment, the antibiotic is a combination of a penicillin antibiotic (e.g., amoxicillin, ampicillin, piperacillin, or flucloxacillin) and a β-lactamase inhibitor (e.g., sulbactam or tazobactam). Preferably, the antibiotic is ampicillin and piperacillin combined with a β-lactamase inhibitor. In a preferred embodiment, the antibiotic is a combination of ampicillin and sulbactam, or a combination of piperacillin and tazobactam. If the S. Typhi Ty21a strain contains inherent antibiotic resistance or if the DNA molecule comprising at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen contains an antibiotic resistance gene, the antibiotic can be any antibiotic except for one or more antibiotics to which the S. Typhi Ty21a strain comprising the DNA molecule comprising at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen is resistant. In other words, the antibiotic is preferably an antibiotic to which the S. Typhi Ty21a strain comprising the DNA molecule comprising at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen is not resistant.

[0090] The antibiotic is preferably administered for at least three days, preferably for at least one week, more preferably for at least two weeks.

[0091] The cancer to be treated according to the present invention is preferably a solid tumor, preferably 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. In a preferred embodiment, the solid tumor is pancreatic cancer or glioblastoma, more preferably glioblastoma. In one embodiment, the cancer is recurrent glioblastoma. Combination containing Salmonella typhi Ty21a strain

[0092] The Salmonella typhi strain Ty21a for use according to the present invention may also be administered in combination with one or more other compounds or treatments.

[0093] In some embodiments, the S. Typhi Ty21a strain is administered in conjunction with chemotherapy or radiation therapy. The S. Typhi Ty21a strain can be administered before, during, or after chemotherapy or radiation therapy, or before and during chemotherapy or radiation therapy. To treat cancer, complete eradication of cancer stem cells may be important. Therefore, to maximize efficacy, a combination of different therapeutic approaches may be effective.

[0094] Examples of chemotherapeutic agents that can be used in combination with the Salmonella typhi Ty21a strain of the present invention include gemcitabine, amifostine (Ethyol), cabazitaxel, cisplatin, dacarbazine (DTIC), dactinomycin, docetaxel, mechlorethamine, streptozocin, cyclophosphamide, carnustine (BCNU), lomustine (CCNU), doxorubicin (Adriamycin), doxorubicin lipoprotein (Doxil), folinic acid, gemcitabine (Gemzar), daunorubicin, daunorubicin lipoprotein (Daunoxome), procarbazine, ketoconazole, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil (5-FU), vinblastine, vincristine, bleomycin, paclitaxel (Taxol), and docetaxel (Taxotere). , aldesleukin, asparaginase, busulfan, carboplatin, cladribine, camptothecin, CPT-11, 10-hydroxy-7-ethyl-camptothecin (SN38), dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, idarubicin, mesna, interferon alpha, interferon beta, irinotecan, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, oxaliplatin, plicamycin, mitotane, pegaspargase, pentastatins, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil, and combinations thereof are possible.

[0095] The most preferred chemotherapeutic agents according to the present invention are cabazitaxel, carboplatin, oxaliplatin, cisplatin, cyclophosphamide, docetaxel, gemcitabine, xorubicin, paclitaxel (Taxol), irinotecan, vincristine, vinblastine, vinorelbine, folinic acid, 5-fluorouracil, and bleomycin, especially gemcitabine.

[0096] In one embodiment, the Salmonella Typhi Ty21a strain used is accompanied by a biological cancer therapy. In the context of the present invention, the term "biological cancer therapy" refers to cancer therapy that involves the use of biopharmaceuticals, i.e., protein-based drugs (including antibodies) or vaccines, or the use of cell-based treatments (such as CAR-T cells, CAR-NK cells, or CAR-NKT cells, or ex vivo primed antigen-presenting cells (APCs)).

[0097] In a preferred embodiment, administration of the VEGFR-2-encoding Salmonella Typhi Ty21a strain is combined with administration of the VEGFR-2-encoding Salmonella Typhi Ty21a strain encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen selected from the group consisting of WT1, MSLN, CEA, CMV pp65, PD-L1, and FAP, optionally further combined with at least one checkpoint inhibitor. The VEGFR-2-encoding Salmonella Typhi Ty21a strain and the VEGFR-2-encoding Salmonella Typhi Ty21a strain encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen selected from the group consisting of WT1, MSLN, CEA, CMV pp65, PD-L1, and FAP can be administered simultaneously or separately.

[0098] In the context of the present invention, the term "simultaneously" means that different attenuated strains of S. typhi Ty21a are administered on the same day, more precisely within 12 hours, more precisely within 2 hours. The different attenuated strains of S. typhi Ty21a may, but need not, be in the same dosage form. The term "separately" as used in this context means that they are administered in different dosage forms, on different days, more precisely on different dosing schedules.

[0099] In a particularly preferred embodiment, the Salmonella Typhi Ty21a strain is administered in combination with at least one checkpoint inhibitor, preferably simultaneously with or prior to said at least one checkpoint inhibitor. The at least one checkpoint inhibitor may be an immunomodulatory antibody, 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.

[0100] According to the present invention, the Salmonella typhi Ty21a strain containing a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen (preferably encoding at least one tumor antigen and / or stromal antigen) can further be administered with at least one checkpoint inhibitor. The term "checkpoint inhibitor" is used herein as a synonym for "immune checkpoint inhibitor." Typically, checkpoint therapy blocks inhibitory checkpoints to restore immune system function. Specifically, the at least one checkpoint inhibitor may be an antibody specifically selected from the group consisting of antibodies against programmed cell death protein 1 (PD-1), programmed cell death 1 ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), indoleamine-2,3-dioxygenase (IDO), glucocorticoid-induced TNFR-related protein (GITR), tumor necrosis factor receptor superfamily member 4 (OX40), T-cell immunoglobulin and mucin domain-containing 3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), killer cell immunoglobulin-like receptor (KIR), colony-stimulating factor 1 receptor (CSF1R), and CD137. Thus, in a particularly preferred embodiment, the S. Typhi Ty21a strain is administered in combination with at least one checkpoint inhibitor, wherein the at least one checkpoint inhibitor is an immunomodulatory antibody 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, preferably antibodies against PD-1, PD-L1, and / or CTLA-4, more preferably antibodies against PD-1 or PD-L1. The checkpoint inhibitor can be administered simultaneously with or separately from the at least one S. Typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen.

[0101] In one embodiment, a Salmonella Typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least VEGFR-2 is administered orally for use in treating cancer in a human subject following antibiotic treatment, and the Salmonella Typhi Ty21a strain is administered in combination 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, more preferably antibodies against PD-1, PD-L1, and / or CTLA-4, even more preferably antibodies against PD-1 or PD-L1. The checkpoint inhibitor can be administered simultaneously or separately with the at least one Salmonella Typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding VEGFR-2.

[0102] Preferably, the at least one checkpoint inhibitor is administered in a commercially approved galenical formulation.

[0103] In the context of the present invention, the term "concurrently" means that the attenuated strain of Salmonella typhi Ty21a comprising at least one eukaryotic expression cassette encoding one tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen and the checkpoint inhibitor are administered on the same day, more precisely within 12 hours, more precisely within 2 hours. The term "separately," when used in this context, means administration on different days, more precisely in different dosage forms with different dosing regimens. Salmonella typhi Ty21a

[0104] Attenuated strains of Salmonella, especially the species Salmonella enterica, are attractive vehicles for delivering heterologous antigens to the mammalian immune system because S. enterica strains can potentially be delivered via the mucosal route of immunization, i.e., through the mouth or nose. This offers the advantage of being simple and safe compared to parenteral administration. Furthermore, Salmonella strains induce strong humoral and cellular immune responses at both the systemic and mucosal tissue levels. Batch preparation costs are low, and live bacterial vaccine formulations are highly stable. Attenuation can be achieved by deletion of various genes, including virulence, regulatory, and metabolic genes.

[0105] Several Salmonella typhimurium strains attenuated by allomutation have been shown to be safe and effective delivery vehicles for heterologous antigens in animal models.

[0106] According to the present invention, the attenuated strain of Salmonella is the typhoid strain Ty21a, also referred to as Salmonella Typhi Ty21a. The live attenuated Salmonella Typhi Ty21a strain is the active ingredient in Typhoral L® (also known as Vivotif®) (manufactured by Berna Biotech Ltd., a Crucell Company, Switzerland). This is currently the only live oral vaccine licensed against typhoid fever. This vaccine has been vigorously tested and proven safe with respect to toxicity to the patient and transmission to others (Wahdan et al., J. Infectious Diseases 1982, 145:292-295). The vaccine is licensed in over 40 countries and has been used in millions of individuals, including thousands of children, for preventive vaccination against typhoid fever. It has an unparalleled safety track record. There is no available data indicating that Salmonella Typhi Ty21a can enter the systemic bloodstream. The live attenuated Salmonella typhi Ty21a vaccine strain therefore allows for specific targeting of the immune system in the gut while being safe and well tolerated. The marketing authorization number for Typhoral L® is PL 15747 / 0001 dated December 16, 1996. One dose of the vaccine contains at least 2 x 109 viable Salmonella Typhi Ty21a colony-forming units and at least 5 x 10 9 Contains non-viable Salmonella typhi Ty21a cells.

[0107] This well-tolerated live oral vaccine against typhoid fever was derived by chemical mutagenesis of the wild-type pathogenic bacterial isolate Salmonella Typhi Ty2, which is unable to metabolize galactose as a result of a loss-of-function mutation in the galE gene. This attenuated strain is also unable to reduce sulfate to sulfide, which distinguishes it from wild-type Salmonella Typhi Ty2 strains. Serologically, the Salmonella Typhi Ty21a strain contains the O9-antigen, the outer membrane polysaccharide of this bacterium, but instead lacks the O5-antigen, a characteristic component of Salmonella Typhimurium. This serological characteristic supports the rationale for including each test in the panel of identification tests for batch release.

[0108] The expression cassette used in the Salmonella typhi Ty21a strain of the present invention is a eukaryotic expression cassette, particularly comprising a CMV promoter. In the context of the present invention, the term "eukaryotic expression cassette" refers to an expression cassette that allows for the expression of an open reading frame in a eukaryotic cell. It has been shown that the amount of heterologous antigen required to induce a sufficient immune response can be toxic to bacteria, resulting in cell death, excessive attenuation, or loss of heterologous antigen expression. Using a eukaryotic expression cassette that is not expressed in the bacterial vector but only in the target cell may overcome this toxicity issue, and the expressed protein typically exhibits a eukaryotic glycosylation pattern.

[0109] The eukaryotic expression cassette comprises regulatory sequences capable of controlling the expression of an open reading frame, preferably a promoter and polyadenylation signal, in a eukaryotic cell. The promoter and polyadenylation signal contained in the eukaryotic expression cassette contained in the Salmonella typhi Ty21a strain of the present invention are preferably selected to function in the cells of the subject to be immunized. Non-limiting examples of promoters particularly suitable for the production of human DNA vaccines include 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), the synthetic CAG promoter consisting of the CMV early enhancer element, the promoter, first exon, and first intron of the chicken beta-actin gene, and the splice acceptor of the rabbit beta-globin gene, as well as promoters from human genes (such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein). In a particular embodiment, the eukaryotic expression cassette contains a CMV promoter. In the context of the present invention, the term "CMV promoter" refers to the strong immediate-early cytomegalovirus promoter.

[0110] Non-limiting examples of polyadenylation signals particularly suitable for the production of human DNA vaccines include the bovine growth hormone (BGH) polyadenylation site, the SV40 polyadenylation signal, and the LTR polyadenylation signal. In one particular embodiment, the eukaryotic expression cassette contained in the Salmonella typhi Ty21a strain of the present invention comprises a BGH polyadenylation site.

[0111] In addition to regulatory elements, such as a promoter and polyadenylation signal, required for expression of a heterologous polypeptide, other elements can be included in a eukaryotic expression cassette. Such additional elements can include enhancers, such as those from human actin, human myosin, human hemoglobin, and human muscle creatine, as well as viral enhancers, such as those from CMV, RSV, and EBV.

[0112] Regulatory sequences and codons are generally species-dependent, and therefore, in order to maximize protein production, regulatory sequences and codons are preferably selected to be effective in the species to be immunized. One skilled in the art can generate recombinant DNA molecules that function in a given subject species (e.g., a human subject).

[0113] In particular embodiments, the DNA molecule, or the 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 a pMB1 ori or pUC), and a strong promoter (such as a CMV promoter). In particular embodiments, the recombinant DNA molecule, or the 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, CA)).

[0114] This expression vector can be modified by replacing the high-copy pUC origin of replication of pBR322 with the low-copy pMB1 origin of replication. The low-copy modification was made to reduce metabolic burden and make the construct more stable. The resulting expression vector backbone was designated pVAX10.

[0115] In a particular embodiment, the expression plasmid comprises the DNA molecule of SEQ ID NO: 2, which correlates to the sequence of the expression vector pVAX10 without the part of the multiple cloning site located between the restriction sites NheI and XhoI (vector backbone pVAX10).

[0116] The Salmonella typhi Ty21a strain is administered orally. Oral administration is easier, 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. A therapeutically effective dose is preferably administered to the subject, and this dose depends on the specific application, the type of malignant tumor, the subject's weight, age, sex, and health condition, the method of administration, and the formulation. Administration can be single or multiple, as needed.

[0117] The S. Typhi Ty21a strain encoding at least one polypeptide containing five or more neoantigens can be provided in the form of a solution, suspension, lyophilizate, 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. The drinking solution preferably includes a means for at least partially neutralizing gastric acid, i.e., adjusting the pH of the gastric acid to a pH close to 7. The drinking solution is preferably a buffered suspension containing the S. Typhi Ty21a strain of the present invention. In a particular embodiment, the buffered 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.

[0118] In a particular embodiment, a single dose of Salmonella Typhi Ty21a strain is about 10 6 ~about 10 9 , preferably about 10 6 ~about 10 8 , more preferably about 10 6 ~about 10 7 Contains colony forming units (CFU).

[0119] More specifically, a single dose of Salmonella Typhi Ty21a strain is approximately 1 × 10 6 ~Approx. 1×10 9 , preferably about 1 x 10 6 ~Approx. 1×10 8 , more preferably about 1×106 ~Approx. 1×10 7 Contains colony forming units (CFU).

[0120] Furthermore, the Salmonella Typhi Ty21a strain of the present invention is administered 2 to 4 times in the first week, preferably 4 times in the first week, followed by a single boost dose every 2 to 4 weeks, particularly on days 1 and 7, preferably days 1, 3, 5, and 7, followed by a single boost dose every 2 to 4 weeks.

[0121] In this context, the term "about" or "approximately" means within 3 times or within 2 times, including within 1.5, of a given value or range.

[0122] Depending on the occurrence of possible side effects, it may be preferable to include treatment with antibiotics or anti-inflammatory agents.

[0123] If adverse events resembling histamine-, leukotriene-, or cytokine-mediated hypersensitivity reactions occur, treatment options are available for fever, anaphylaxis, blood pressure instability, bronchospasm, and dyspnea. In the case of unwanted T cell-derived autologous attacks, treatment options are derived from the standard treatment schemes applied in acute and chronic graft-versus-host disease after stem cell transplantation. Cyclosporine and glucocorticoids are suggested as treatment options.

[0124] In the unlikely event of a systemic Salmonella Typhi Ty21a infection, appropriate antibiotic therapy, for example with fluoroquinolones (including ciprofloxacin or ofloxacin), is recommended. Bacterial infections of the gastrointestinal tract should be treated with the respective agent (e.g., rifaximin). Pharmaceutical Composition

[0125] 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 tumor antigen, stromal antigen, and / or checkpoint inhibitor antigen.

[0126] The pharmaceutical compositions of the present invention can be in the form of a solution, suspension, enteric-coated capsule, lyophilized powder, or any other form suitable for oral use as envisioned. The pharmaceutical compositions of the present invention can further comprise one or more pharmaceutically acceptable excipients.

[0127] In the context of the present invention, the term "excipient" refers to a natural or synthetic substance that is formulated with an active ingredient of a pharmaceutical. Suitable excipients include anti-adherents, binders, coatings, disintegrants, flavors, colorants, lubricants, glidants, adsorbents, preservatives, solvents, and sweeteners.

[0128] In the context of the present invention, the term "pharmaceutically acceptable" refers to molecular compounds and other components of pharmaceutical compositions that are physiologically tolerable and typically do not produce undesirable reactions when administered to a mammal (e.g., a human). "Pharmaceutically acceptable" can also mean approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeia for use in mammals, or more specifically, in humans.

[0129] In particular, suitable drinking solutions typically comprise means for at least partially neutralizing gastric acid, i.e., for bringing the pH of the gastric acid closer to pH 7. In one particular embodiment, the drinking solution is a buffered suspension obtained by suspending the Salmonella typhi Ty21a strain of the invention in a suitable buffer, preferably a buffer that neutralizes gastric acid at least to some extent, preferably a buffer containing 2.6 g sodium bicarbonate, 1.7 g L-ascorbic acid, 0.2 g lactose monohydrate, and 100 ml drinking water.

[0130] In particular embodiments, the pharmaceutical composition is used in accordance with the present invention as a medicine, in particular for the treatment of cancer (preferably solid tumors) in a human subject. In particular embodiments, the pharmaceutical composition is used as a medicine, preferably following treatment with an antibiotic, in particular for the treatment of cancer (preferably solid tumors) in a human subject who has been treated or is currently being treated with at least one antibiotic. [Example]

[0131] Example 1: Phase I / II combination clinical trial of VXM01 and avelumab, optionally with the antibiotic Cotrim forte®

[0132] This multicenter, open-label, phase I / II study (EudraCT.gov no. 2017-003076-31, NCT03750071) will evaluate the efficacy and safety, as well as clinical and immunogenic responses, of VXM01 in combination with the checkpoint inhibitor antibody avelumab (anti-PD-L1) in patients with unresectable advanced glioblastoma (n=24) and patients with resectable advanced glioblastoma (n=6) after tumor resection and temozolomide-containing chemoradiotherapy. Thirty patients will be enrolled at eight study centers in Germany, the Netherlands, and France. Nine unresectable patients have been enrolled and analyzed to date. All patients had their primary tumors surgically removed, and these patients experienced recurrences with standard therapy, i.e., radiation therapy plus temozolomide.

[0133] 10 patients 6 or 10 7 Patients were treated with CFU of VXM01 and avelumab. Patients received oral VXM01 vaccine on days 1, 3, 5, and 7, followed by boosts every 4 weeks until progression. Avelumab was administered intravenously at a fixed dose of 800 mg every 2 weeks until progression. The end of the study was 60 weeks. Post-study follow-up visits included 1, 3, 6, 12, and 24 months. Samples for biomarker and immunogenicity testing were collected before, during, and at several time points after treatment.

[0134] As shown in Figures 2A and 3, six of the nine analyzed patients showed disease progression, and three showed partial responses (PRs) (patients 0104, 0109, and 2210), with tumor shrinkage of more than 50% (Figure 3). One of the partial responders even achieved a progression-free survival (PFS) of more than 9 months (patient 0104). Tumor size was determined using MRI according to the Response Assessment in Neuro-Oncology (RANO) criteria. Two of these partial responders (including the patient with a PFS of more than 9 months) received the antibiotic Cotrim forte® (sulfamethoxazole 800 mg and trimethoprim 160 mg) before and at the time of vaccination early in the study due to low lymphocyte counts and associated risk of pneumonia (Figure 2B). This antibiotic therapy was discontinued due to potential interactions with VXM01. In particular, patient number 0104 received Cotrim forte® for approximately 20 weeks from day 0 of the study, and patient number 0109 received Cotrim forte® for approximately 4 days.

[0135] Furthermore, VEGFR-2-specific T cell responses were analyzed in blood samples from patient 0104 using an Enzyme-Linked ImmunoSpot (ELISpot) assay with cryopreserved peripheral blood mononuclear cells at baseline and after 3, 6, and 9 months of treatment. The time course of VEGFR-2-specific immune responses (Figure 4A) and tumor responses (Figure 4B) in this patient indicates that VXM01 primarily contributes to the therapeutic effect of this treatment. The data demonstrate a clear increase in VEGFR-2-specific immune responses after the end of antibiotic treatment. Furthermore, tumor size decreased following the increase in VEGFR-2-specific immune responses. Intratumoral immune biomarker analysis using tumor tissue immunohistochemistry on samples obtained before treatment demonstrated high levels of tumor-infiltrating CD8+ T cells and low levels of Treg cells (FoxP3+ cells) and myeloid-derived suppressor cells (CD68+ cells) (Figure 5A). Furthermore, PD1 or PD-L1 expression was not detected in histological sections of tumor samples from patient 0104 before treatment (Figure 5B). Overall, the data suggest a beneficial effect of antibiotic pretreatment before inoculation with VXM01 alone or in combination with checkpoint inhibitors (e.g., avelumab).

[0136] In patient 0109, who achieved a partial response at 3 months (Figures 2 and 3 and Table 2), baseline intratumoral immune biomarker analysis revealed high levels of tumor-infiltrating CD8+ T cells and MDCS, but no Treg cells (FoxP3+ cells) (Figure 6A). Patient 0109 was treated with antibiotics for only a few days, which may have contributed to this patient's favorable outcome. [Table 2] Example 2: Phase I combination clinical trial of VXM01 and nivolumab

[0137] The beneficial effect of VXM01 in combination with nivolumab (anti-PD-1) was also observed in a phase I clinical trial in patients with refractory glioblastoma (Figure 9). Furthermore, the synergistic effect of VXM01 and anti-CTLA-4 antibodies has already been reported in mice in WO 2016 / 202459. Therefore, the beneficial effect of antibiotic pretreatment before VXM01 vaccination is expected when used in combination with other checkpoint inhibitors (anti-PD-1, anti-CTLA-4, or other).

Claims

1. 1. A pharmaceutical composition for the treatment of solid tumors in a human subject after completion of antibiotic therapy, comprising a Salmonella typhi Ty21a strain comprising a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one tumor antigen and / or VEGFR-2, the Salmonella typhi Ty21a strain is not resistant to the antibiotic; The pharmaceutical composition is for oral administration and is administered in combination with at least one checkpoint inhibitor.

2. 2. The pharmaceutical composition of claim 1, wherein the at least one tumor antigen is selected from the group consisting of human Wilms tumor protein (WT1), human mesothelin (MSLN), human CEA, and CMV pp65.

3. 2. The pharmaceutical composition of claim 1, wherein the Salmonella typhi Ty21a strain comprises a DNA molecule comprising at least one eukaryotic expression cassette encoding at least one polypeptide comprising five or more neoantigens.

4. 4. The pharmaceutical composition of claim 3, wherein the five or more neoantigens are tumor-specific antigens identified in a solid tumor of the subject.

5. A pharmaceutical composition described in any one of claims 1 to 4, wherein the pharmaceutical composition is administered simultaneously with or prior to administration of the at least one checkpoint inhibitor.

6. 6. The pharmaceutical composition of claim 5, wherein the at least one checkpoint inhibitor is an immunomodulatory antibody 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.

7. The pharmaceutical composition according to any one of claims 1 to 6, which is administered at least three days after completion of antibiotic treatment and / or within one month after completion of antibiotic treatment.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the antibiotic is a combined preparation.

9. 9. The pharmaceutical composition of claim 1, wherein the antibiotic is selected from the group consisting of penicillins, cephalosporins, polymyxins, rifamycins, lipiarmycins, quinolones, sulfonamides, macrolides, linocosamides, tetracyclines, aminoglycosides, cyclic lipopeptides, glycylcyclines, oxozolidinones, nitrozimazoles, and dihydrofolate reductase inhibitors.

10. 10. The pharmaceutical composition of claim 9, wherein the antibiotic is sulfamethoxazole or trimethoprim, or a combination thereof.

11. 11. The pharmaceutical composition of claim 10, wherein the antibiotic is cotrimoxazole.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein said treatment is accompanied by chemotherapy or radiation therapy.

13. 2. The pharmaceutical composition of claim 1, 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.

14. 14. The pharmaceutical composition of claim 13, wherein the solid tumor is glioblastoma.

15. 15. The pharmaceutical composition of claim 14, wherein the solid tumor is recurrent glioblastoma.

16. (a) a single dose of said pharmaceutical composition comprises 10 6 to 10 9 colony forming units (CFU) of Salmonella typhi Ty21a strain; and / or (b) the pharmaceutical composition of any one of claims 1 to 15, wherein the pharmaceutical composition is administered 2 to 4 times initially weekly, followed by a single boost dose every 2 to 4 weeks.

17. 17. The pharmaceutical composition according to any one of claims 1 to 16, further comprising at least one pharmaceutically acceptable excipient.

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