DNA vaccine for use in pancreatic cancer patients

Abstract

The present invention relates to an attenuated mutant strain of Salmonella comprising a recombinant DNA molecule encoding a VEGF receptor protein. In particular, the present invention relate to the use of said attenuated mutant strain of Salmonella in cancer immunotherapy.

Claims

1. A method of providing cancer immunotherapy comprising administering to a patient suffering from a cancer, a DNA vaccine comprising an attenuated mutant strain of Salmonella typhi Ty21a comprising an expression cassette encoding a human VEGFR-2 having the amino acid sequence of SEQ ID NO 1, wherein a single dose of the vaccine is 10.sup.9 colony forming units (CFU) or less than 10.sup.9 CFU.

2. The method of claim 1, wherein the expression cassette is a eukaryotic expression cassette.

3. The method of claim 2, wherein the cancer immunotherapy is an anti-angiogenic cancer immunotherapy and wherein the DNA vaccine comprises the attenuated Salmonella typhi strain Ty21a transformed by a plasmid that contains the DNA encoding the VEGFR-2 protein of SEQ ID NO 1.

4. The method of claim 3, wherein the plasmid is the 7580 bp pVAX10.VR2-1 having the sequence of SEQ ID NO 3.

5. The method of claim 1, wherein the cancer is pancreatic cancer.

6. The method of claim 5, wherein said pancreatic cancer is stage IV or locally advanced pancreatic cancer.

7. The method of claim 1, wherein the cancer includes metastases.

8. The method of claim 1, further comprising administering to the patient chemotherapy and/or radiotherapy.

9. The method of claim 8, wherein the chemotherapeutic agent is gemcitabine.

10. The method of claim 8, wherein the administration of the DNA vaccine is carried out during the chemotherapy treatment cycle.

11. The method of claim 1, wherein the DNA vaccine is administered orally.

12. The method of claim 1, wherein the single dose of the vaccine is a dose selected from the group of: 1?10.sup.5, 1?10.sup.6, 1?10.sup.7, 1?10.sup.8, and 1?10.sup.9 CFU.

13. The method of claim 1, wherein the single dose of the vaccine is less than 1?10.sup.9 CFU.

14. The method of claim 1, wherein the single dose of the vaccine is less than 1?10.sup.8 CFU.

15. The method of claim 1, wherein the single dose of the vaccine comprises from 10.sup.6 to 10.sup.9 CFU.

16. A method of providing cancer immunotherapy to a cancer patient comprising administering to the patient a DNA vaccine VXM01, comprising the attenuated Salmonella typhi strain Ty21a transformed by a 7580 bp plasmid, the plasmid comprising a DNA encoding the VEGFR-2 protein of SEQ ID NO 1 under the control of a CMV promoter, the kanamycin resistance gene, and the pMB1 ori, and is designated as pVAX10.VR2-1, and wherein a single dose of the vaccine is 10.sup.9 CFU or less than 10.sup.9 CFU.

17. The method of claim 1, wherein the single dose of the vaccine comprises from 10.sup.6 to 10.sup.8 CFU.

18. The method of claim 1, wherein the method further comprises administering to the patient one or more further attenuated mutant strains(s) of Salmonella typhi Ty21a comprising a eukaryotic expression cassette encoding a tumor antigen and/or a tumor stroma antigen.

Description

SHORT DESCRIPTION OF FIGURES AND TABLES

(1) FIG. 1: Amino acid sequence of VEGFR-2 cDNA cloned into plasmid pVAX10.VR2-1

(2) FIG. 2: Plasmid map of pVAX10.VR2-1

(3) FIG. 3: Dose escalating design of vaccine VXM01

(4) FIG. 4: Overall study scheme

(5) FIG. 5: VXM01 specific T cell responses

(6) FIG. 6: Effects of VXM01 on tumor perfusion

(7) FIG. 7: Effects of VXM01 on VEGF A serum levels

(8) FIG. 8: Effects of VXM01 on collagen IV serum levels

(9) FIG. 9: Effects of VXM01 on blood pressure

(10) FIG. 10: VXM01-induced anti-carrier immunity

(11) FIG. 11: Analysis cascade of VXM01 excretion

(12) FIG. 12: VXM01 excretion

(13) Table 1: Patient selection criteria

(14) Table 2: VMX01 specific T cell responses

EXAMPLES

Example 1 Salmonella typhi Ty21a Strain Preparation and Plasmid Preparation

(15) The first step in the preparation of the research seed lot (RSL) consisted of the isolation of the attenuated Salmonella typhi Ty21a strain followed by the transformation of the attenuated bacteria with the plasmid DNA (pVAX10.VR2-1).

(16) Liquid culture medium was inoculated with a Salmonella typhi Ty21a isolate and the liquid culture was then plated onto an agar medium for the purpose of isolating single bacterial colonies. Single colonies were isolated and grown in liquid culture medium. Two cultures, namely VAX.Ty21-1 and VAX.Ty21-2, were then formulated with glycerol, aliquoted (1 ml) and stored at ?75? C.?5? C. pending use. Identity of each of the two cultures was further confirmed.

(17) The principle of plasmid synthesis was based on double strand in vitro gene synthesis with the following steps: The whole pVAX10-VR2.1 plasmid sequence of 7.58 kb was subdivided (by software analysis) in 5 sections of ?1.5 kb. Each section was subdivided into 40-50 bp oligonucleotides each having overlapping regions between oligonucleotides of both strands The in vitro synthesized oligonucleotides were then phosphorylated by incubation with T4 polynucleotide kinase After the annealing process of overlapping oligonucleotides under appropriate conditions, the Taq DNA ligase enzyme connected the aligned oligonucleotides Upon completion of the ligation step, PCR was performed using primers annealed at outward positions, to increase the yield of the ligated plasmid fragments (?1.5 kb) A preparative agarose gel electrophoresis was performed to isolate the PCR products The isolated PCR products were cloned into TOPO vectors (Invitrogen K#4575-40) and transformed into TOP10 E. coli cells for propagation After TOPO plasmid isolation, a restriction and sequence verification was performed The isolated aligned fragments were assembled via overlap PCR. This process was followed by linearly assembling the pVAX10.VR2-1 plasmid After XhoI restriction digest (single restriction site is present in the pVAX10.VR2-1 plasmid, see FIG. 2) and covalent binding via T4 ligase, E. coli was transformed with the circular plasmid for propagation After final plasmid sequence verification, the pVAX10.VR2-1 plasmid was transformed into the S. typhi Ty21a bacterial strain.

(18) The plasmid pVAX10.VR2-1 was thus successfully synthesized (no deviation to reference sequence). This plasmid was further used to transform the S. typhi Ty21a bacterial strain.

Example 2 VXM01Phase I Clinical Trial; Study Description

(19) This phase I trial examined the safety, tolerability, and immunological and clinical responses to VXM01. The randomized, placebo-controlled, double blind dose-escalation study included 45 patients with locally advanced or stage IV pancreatic cancer. The patients received four doses of VXM01 or placebo in addition to gemcitabine as standard of care. Doses from 10.sup.6 CFU up to 10.sup.10 CFU of VXM01 were evaluated in the study. An independent data safety monitoring board (DSMB) was involved in the dose-escalation decisions. In addition to safety as primary endpoint, the VXM01-specific immune reaction, as well as clinical response parameters were evaluated.

(20) Preclinical Efficacy Assessment:

(21) The efficacy and safety of this approach in animals has been validated multiple times by the inventors. Further experiments by the inventors showed an activity of this vaccine in two different models of pancreatic cancer.

(22) VXM01, the vaccine used in this trial, is a humanized version of the anti-VEGFR-2 vaccine previously tested in mice. It encodes the human full-length VEGFR-2 and uses the licensed Salmonella typhi strain Ty21a instead of Salmonella typhimurium as a carrier. The vaccine is assumed to lead to VEGFR-2 protein expression in monocytes and dendritic cells after entry of VXM01 in the Peyer's patches via M cells of the gut, and internalization by antigen-presenting cells followed by translation of the encoded DNA.

(23) Preclinical Safety Assessment:

(24) Preclinical toxicity studies in mice included, but were not restricted to a single dose toxicity study in mice conducted with the human vaccine VXM01. As VXM01 is specific for the human host, the study of the human vaccine in mice focused on possible effects of process-related impurities and related signs and symptoms of possible relevance for cardiovascular, respiratory, or central nervous system impairment. In order to investigate the toxicity profile of an anti-VEGFR-2 T-cell response, a repeated dose toxicity study was conducted using the murine analog construct of VXM01 which induced a dose-dependent T-cell response in mice. In accordance to the inventors' previous observations, no treatment-related deaths and no toxicologically important clinical signs were observed throughout these studies, which were conducted according to Good Laboratory Practice (GLP).

(25) The vector Salmonella typhi Ty21a used here is a live, attenuated bacterial carrier that allows for the oral delivery of the vaccine VXM01. It is itself an approved vaccine against typhoid fever (Vivotif?, Crucell, formerly Berna Biotech Ltd., Switzerland) that has been extensively tested and has demonstrated its safety regarding patient toxicity as well as transmission to third parties (Wandan et al., J. Infectious Diseases 1982, 145:292-295). VXM01 is classified as a gene transfer medicinal product and subject to the respective guidance and regulations.

(26) Study Descriptions and Objectives:

(27) The conducted study was a monocenter, placebo controlled, double blind dose escalation study of the experimental vaccine VXM01 in patients with inoperable or stage IV pancreatic cancer. The vaccine was given as add-on to a standard of care gemcitabine treatment.

(28) The objectives were to examine the safety and tolerability, and immunological and clinical responses to the investigational anti-VEGFR-2 vaccine VXM01, as well as to identify the maximum tolerated dose (MTD) of VXM01. The MTD is defined as the highest dose level at which less than two of up to six patients under VXM01 treatment experience a dose-limiting toxicity (DLT).

(29) Primary endpoints for safety and tolerability were as follows: Number of DLTs defined as any adverse event (AE) related to study drug of grade 4 or higher, or grade 3 or higher for gastrointestinal fistula, diarrhea, gastrointestinal perforation, multi-organ failure, anaphylaxis, any auto-immune disorder, cytokine-release syndrome, intestinal bleeding, renal failure, proteinuria, thromboembolic events, stroke, heart failure, or vasculitis according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE).

(30) Secondary endpoints, which assess the efficacy of the experimental vaccine to elicit a specific immune response to VEGFR-2, included the number of immune positive patients.

(31) A further secondary endpoint was the clinical response: Tumor staging according to the response evaluation criteria in solid tumors (RECIST), overall response rate, progression free survival, overall survival, and changes in tumor perfusion. Tumor perfusion was determined by dynamic contrast-enhanced Magnetic Resonance Imaging (DCE-MRI) on a 1.5 Tesla system (Magnetom Aera, Siemens, Erlangen, Germany).

(32) VXM01 had been manufactured according to Good Manufacturing Practice (GMP) and was given in a buffered solution. The placebo control consisted of isotonic sodium chloride solution.

(33) Patient Selection and Clinical Study Design:

(34) The study included a maximum of 45 patients with either locally advanced and inoperable or stage IV pancreatic cancer. The eligibility criteria are summarized in Table 1.

(35) TABLE-US-00001 TABLE 1 Inclusion Criteria 1. Written informed consent, signed and dated 2. Locally advanced, inoperable and stage IV pancreatic cancer patients according to UICC based on diagnostic imaging using computer-tomography (CT) or histological examinations 3. Male or post-menopausal female 4. Age ? 18 years 5. Chemotherapy naive within 60 days before screening visit except gemcitabine treatment 6. Karnofsky index > 70 7. Life expectancy > 3 months 8. Adequate renal, hepatic, and bone marrow function 9. Absolute neutrophil count > 1500/?L 10. Hemoglobin > 10 g/dL 11. Platelets > 75000/?L 12. Prothrombin time and international normalized ratio (INR) < 1.5 times upper limit of normal (ULN) (except under anticoagulant treatment) 13. Aspartate aminotransferase < 4 times ULN 14. Alanine aminotransferase < 4 times ULN 15. Total bilirubin < 3 times ULN 16. Creatinine clearance estimated according to Cockcroft-Gault > 30 mL/min 17. Proteinuria < 1 g protein on 24 h urine collection Exclusion Criteria 18. State after pancreas resection (complete or partial) 19. Resectable disease 20. Drug trial participation within 60 days before screening visit 21. Other previous or current malignancy except basal or squamous cell skin cancer, in situ cervical cancer, or any other cancer from which the patient has been disease-free for < 2 years 22. Prior vaccination with Ty21a State after pancreas resection (complete or partial) 23. Resectable disease 24. Drug trial participation within 60 days before screening visit 25. Other previous or current malignancy except basal or squamous cell skin cancer, in situ cervical cancer, or any other cancer from which the patient has been disease-free for < 2 years 26. Prior vaccination with Ty21a 27. Cardiovascular disease defined as: Uncontrolled hypertension (systolic blood pressure > 160 mmHg or diastolic blood pressure > 100 mmHg) Arterial thromboembolic event within 6 months before randomization including: Myocardial infarction Unstable angina pectoris Cerebrovascular accident Transient ischemic attack 28. Congestive heart failure New York Heart Association grade III to IV 29. Serious ventricular arrhythmia requiring medication 30. Clinically significant peripheral artery disease > grade 2b according to Fontaine 31. Hemoptysis within 6 months before randomization 32. Esophageal varices 33. Upper or lower gastrointestinal bleeding within 6 months before randomization 34. Significant traumatic injury within 4 weeks before randomization 35. Non-healing wound, bone fracture or any history of gastrointestinal ulcers within three years before inclusion, or positive gastroscopy within 3 months before inclusion 36. Gastrointestinal fistula 37. Thrombolysis therapy within 4 weeks before randomization 38. Bowel obstruction within the last 30 days before screening visit 39. Liver cirrhosis ? grade B according to Child-Pugh Score- Classification 40. Presence of any acute or chronic systemic infection 41. Radiotherapy within 4 weeks before randomization 42. Major surgical procedures, or open biopsy within 4 weeks before randomization 43. Fine needle aspiration within 7 days before randomization 44. Chronic concurrent therapy within 2 weeks before and during the double-blind study period with: Corticosteroids (except steroids for adrenal failure) or immunosuppressive agents Antibiotics Bevacizumab Any epidermal growth factor receptor inhibitor 45. Chemotherapy except gemcitabine before Day 10 Multi-drug resistant gram-negative germ 46. Pregnancy 47. Lactation 48. Inability to comply with study and/or follow-up procedures 49. History of other disease, metabolic dysfunction, physical examination finding, or clinical laboratory finding giving reasonable suspicion of a disease or condition that contraindicates the use of an investigational drug or that might affect the interpretation of the study results or render the patient at high risk for treatment complications 50. Women of childbearing potential 51. Any history of drug hypersensitivity 52. Any condition which results in an undue risk for the patient during the study participation according to the investigator

(36) A total of 371 patients were screened for the study. 326 patients were ineligible because of excluded medical therapies (179), preexisting medical conditions (129) in patient's medical history and personal reasons (18). 45 patients were enrolled, randomized and completed successfully the 10 days in-house study phase at the Clinical Research Unit at the University Clinics of Heidelberg (KliPS), in line with the study protocol. Demographic baseline disease characteristics of the patients were not significantly different in the two groups, but time since diagnosis was longer in the VXM01 group (8 vs. 6 months) and patients in the VXM01 group had a more advanced tumor stage at the time of inclusion (CA19.9>1000 in 40% vs. 20% and metastatic disease in 83% vs. 53%).

(37) Male and postmenopausal female patients were enrolled in this study. However, differences between the two genders were not investigated. The average survival time of the patients participating in this trial was under 6 months. However, the follow-up period for the patients as defined per protocol was up to 24 months. The study treatment was given first-line as an add-on to standard of care. Taking further into account other factors, among them the multiple primary and secondary pharmacodynamic preclinical studies, the risk-benefit analysis was assumed to have a favorable result for the patient population selected.

(38) The starting dose consisted of a solution containing 10.sup.6 colony forming units (CFU) of VXM01 or placebo. This VXM01 dose was chosen for safety reasons and was assumed to be below the minimal effective dose to elicit an immune response. For comparison, one dose of Typhoral, the licensed vaccine against typhoid fever, contains 2?10.sup.9 to 6?10.sup.9 CFU of Salmonella typhi Ty21a, equivalent to approximately thousand times the VXM01 starting dose. The dose was escalated in factor-of-ten logarithmic steps, which appears to be justified for a live bacterial vaccine. The dose escalating design is depicted in FIG. 3.

(39) Complying with guidelines for first in human trials, the patients of one dose group were treated in cohorts. The first administration of VXM01 in any dose group was given to one patient only accompanied by one patient receiving placebo. The second cohort of each dose group consisted of two patients receiving VXM01 and one patient receiving placebo. This staggered administration with one front-runner, i.e. only one patient receiving VXM01 first, served to mitigate the risks.

(40) A third cohort of patients (three receiving VXM01 and one receiving placebo) were included in the 10.sup.8, 10.sup.9, and 10.sup.10 dose groups. This approach minimized exposure to VXM01 doses assumed to be sub-therapeutic. The third cohort and the first two cohorts of the next higher treatment group were treated in parallel based on a clearly defined randomization strategy. This strategy allowed for recruitment of available patients and avoided selection bias for patients treated in parallel in the lower and higher dose group. In the 10.sup.6 and 10.sup.7 dose groups, a third cohort of patients was included only if one patient out of the initial three patients receiving VXM01 of the respective dose group experienced a DLT and required confirmation by a decision of the Data Safety Monitoring Board (DSMB).

(41) All patients completed the seven day vaccination course of 4 doses every second day in line with the protocol without any dose reduction. Because of no observed dose limiting toxicities (DLT) the maximum tolerated dose was not reached. VXM01 was well tolerated at all dose levels. AEs and SAEs where equally distributed among both groups and there were no obvious signs for dose-dependent side effects among the groups.

(42) The environmental risk inherent to an oral vaccine is the potential of excretion to the environment and subsequent vaccination of people outside the target population. All study patients were confined in the study site (KliPS) for the period during which vaccinations took place plus three additional days. All feces of study patients were collected and incinerated. Body fluids and feces samples were investigated for VXM01 shedding. Fecal excretion of VXM01 was observed in two patients, one in the 10.sup.9 and one in the 10.sup.10 dose group. VXM01 excretion in feces in both patients was transient at one occasion after the first or second administration, respectively, and disappeared without antibiotic treatment. In other body fluids, no excretion was determined.

(43) Hygienic precautions were applied to protect study personnel from accidental uptake. Study personnel was trained specifically for this aspect of the study.

(44) Patients were only discharged from hospital, if they tested negative for excretion of the vaccine after the last administration of the study drug. In case a patient tested positive for excretion after the last administration, an antibiotic decontamination of the gastrointestinal tract was conducted before the patient was discharged. Excretion was followed up until results were negative. These measures appear to have been justified and sufficient to protect the environment and study personnel from exposure to VXM01 until the shedding profile had been elucidated.

(45) VXM01 was applied in parallel to the gemcitabine background therapy as shown in FIG. 4 (overall study scheme). In brief, gemcitabine was given on days 1, 8, and 15 of a 28 days chemotherapy cycle. The vaccine was given four times on days 1, 3, 5, and 7, starting three days after the last dose of gemcitabine. The double blinded phase of the study ended 31 days after the last patient had received the last administration.

(46) For this phase I trial (advanced or stage IV pancreatic cancer patients) a patient population with dismal prognosis and the relatively gentle standard of care with regard to immunosuppression was chosen. Co-regimes of the chemotherapeutic agent gemcitabine with tumor vaccination may be synergistic. In addition, specific T-cell activation was measured in this patient setting demonstrating effectiveness of the vaccine VXM01. A placebo control was included in the present trial, in order to gain further knowledge on specific safety issues related to the active vaccine vs. the background treatment. In addition, the pooled placebo patients served as a sound comparator for assessing specific immune activation and other signs of clinical efficacy. If and when moving into phase II, a different patient entity with a longer life expectancy can be envisaged depending on the observed safety profile. Such studies will also include tumor types that have shown to be more susceptible to anti-angiogenic treatment.

Example 3 VXM01 Specific T-Cell Responses

(47) Responses to VXM01 were assessed by monitoring the frequencies of VEGFR2 specific T-cells in peripheral blood of VXM01 and placebo treated patients, detected by INF? ELISpot, at different time points prior during and post vaccination.

(48) Firstly, T-cells and peptide pulsed DC were added to wells coated with anti-INF? antibodies. After a period of incubation, cells were removed with secreted INF? left binding with the coat antibodies. Then detection antibody was added to detect the bound INF?, and after a signal amplification, the final yield could be viewed as color spots representing single activated and specific T-cells.

(49) Positivity of ELISpot samples was graded according to predefined rules defining signal increase resulting in grade 0 to 3 per sample:

(50) No increase: grade 0

(51) Clear increase but <3?: grade 1

(52) ?3? but <5? increase: grade 2

(53) ?5? increase: grade 3

(54) The ELISpot immune response of study patients is depicted in Table 2:

(55) TABLE-US-00002 TABLE 2 VEGFR-2 specific T-cell response (Patients w/grading score ? 3) 10.sup.6 10.sup.7 10.sup.8 10.sup.9 10.sup.10 CFUs/ CFUs/ CFUs/ CFUs/ CFUs/ Placebo admin admin admin admin admin 1/11 2/6 3/5 1/6 0/6 2/6

(56) The results of the ELISpot immune response of study patients is graphically depicted in FIG. 5.

Example 4 Effects on Tumor Perfusion

(57) Tumor perfusion was evaluated by contrast media transit time (Ktrans) during dynamic contrast enhanced magnetic resonance imaging (DCEMRI) to characterize treatment response. Dynamic Contrast-Enhanced T1weighted imaging was performed. DCEMRI was assessed on a 1.5 Tesla System (Magnetom Aera, Siemens, Erlangen, Germany) on day 0, 38 and 3 months after treatment. Dynamic contrastenhanced MR-imaging was performed with VIBE (volumetric interpolated breath-hold) sequences. For that purpose, a dose of 8 ml Gadovist was injected.

(58) For every examination, regions of interest were manually drawn within the tumor-tissue followed by pixel-by-pixel analysis using a Siemens software package (Tissue 4D). ROI-modeling was based on the Tofts model with assumed T10 (1000 ms) and Parker AIF. For the estimation of tumor-perfusion Ktrans was regarded as primary endpoint.

(59) Mean changes in tumor perfusion were ?9% in the VXM01 group (n=26) vs. +18% in the placebo group (n=11). A greater than 33% drop in tumor perfusion was detected in 35% of evaluable VXM01 treated patients vs. 10% in the placebo group. The strongest responders were further analyzed in a subgroup analysis. Maximum average effects were detected at the d38 time point. The effects of various doses of VXM01 on tumor perfusion are graphically depicted in FIG. 6.

Example 5 Biomarkers of Angiogenesis

(60) In order to further characterize the VEGFR-2 specific T-cell mediated, anti-angiogenic activity of VXM01, accompanying changes in angiogenesis biomarkers VEGF A, human collagen IV and blood pressure were monitored.

(61) VEGF A:

(62) VEGF A was measured in human serum samples by ELISA using a commercial assay kit (ELISA Kit Quantikine Human VEGF A Immunoassay, R&D Systems, Cat.-No.: DVE00). The assay was used as described in the package insert and as modified as part of this study plan according to the foregoing validation study 580.132.2786.

(63) This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for human VEGF A had been pre-coated onto a microplate. Standards, quality controls (commercially obtained) and samples were pipetted into the wells and any VEGF A present was bound by the immobilized antibody. Calibrator, quality control samples, and samples were analyzed as duplicates. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for VEGF A was added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells and color developed in proportion to the amount of VEGF A bound in the initial step. The color development was stopped and the intensity of the color was measured using a spectrophotometric microtiter plate reader at 450 nm. A standard curve was generated by plotting the absorbance versus the respective VEGF A concentration for each standard. The concentration of VEGF A in the sample was determined directly from this curve.

(64) VEGF-A serum levels increased in the VXM01 group by 235% on both d38 and m3 vs. 17% and 31% in the placebo group (p=0.05 at m3). The quantification of VEGF A in patient serum samples is graphically depicted in FIG. 7.

(65) Collagen IV:

(66) Human Collagen IV was measured in human serum samples by ELISA using a commercial assay kit (Human Collagen IV ELISA, Serum, KAMIYA BIOMEDICAL COMPANY, Cat.-No.: KT-035). The assay was used as described in the package insert and as modified as part of this study report according to the foregoing validation study 580.132.3645.

(67) The human Collagen IV ELISA was a solid phase one-step sandwich ELISA. Collagen IV in the sample was bound simultaneously by a solid phase monoclonal antibody and a monoclonal antibody-enzyme conjugate, each directed at different antigenic sites. This resulted in the collagen IV molecule being sandwiched between the solid phase and enzyme-labeled antibodies. After removing unbound enzyme-labeled antibody and sample, the plate was incubated with chromogenic substrate (TMB). The resultant color development was directly proportional to the amount of collagen IV in the sample.

(68) Serum levels of collagen IV increased on d38 and m3 in average by 7% and 22%, respectively, in the VXM01 group vs. changes of 2% and ?7% in the placebo group (p=0.02 at m3). The quantification of collagen IV in patient serum samples is graphically depicted in FIG. 8.

(69) Blood Pressure:

(70) Blood pressure (systolic and diastolic) and pulse rate as pharmacodynamic markers of anti-angiogenic efficacy were measured after 5 minutes rest in supine position. Average systolic blood pressure changes were +3.6 mmHg and +3.9 mmHg in the treatment group vs. ?8.8 mHg and 9.1 mmHg under placebo (p=0.08 at d38). Effects on average blood pressure after the first vaccination dose (up to day 38) are graphically depicted in FIG. 9.

Example 6 Anti-Carrier Immunity

(71) In order to assess immune responses to the bacterial vehicle, anti-Salmonella typhi IgG and IgM immunoglobulins were detected by ELISA using two commercial assay kits (Salmonella typhi IgG ELISA, Cat. No. ST0936G and Salmonella typhi IgM ELISA, Cat. No. ST084M, Calbiotech. Inc., 10461 Austin Dr, Spring Valley, Calif. 91978, USA). These assays were qualitative assays. The assays were used as described in the package inserts respectively App. I/I) and as modified as part of the study plan according to the foregoing validation study 580.132.2785.

(72) Both assays employed the enzyme-linked immunosorbent assay technique. Calibrator, negative control, positive control and samples were analyzed as duplicates. Diluted patient serum (dilution 1:101) was added to wells coated with purified antigen. IgG or IgM specific antibody, if present, bound to the antigen. All unbound materials were washed away and the enzyme conjugate was added to bind to the antibody-antigen complex, if present. Excess enzyme conjugate was washed off and substrate was added. The plate was incubated to allow for hydrolysis of the substrate by the enzyme. The intensity of the color generated was proportional to the amount of IgG or IgM specific antibody in the sample. The intensity of the color was measured using a spectrophotometric microtiter plate reader at 450 nm. The cut off was calculated as follows:
Calibrator OD?Calibrator Factor (CF).

(73) The antibody index of each determination was determined by dividing the OD value of each sample by cut-off value.

(74) Antibody Index Interpretation:

(75) TABLE-US-00003 <0.9 No detectable antibody to Salmonella typhi IgG or IgM by ELISA 0.9-1.1 Borderline positive >1.1 Detectable antibody to Salmonella typhi IgG or IgM by ELISA

(76) The number of patients with detectable anti-Salmonella typhi IgG immunoglobulins are depicted in FIG. 10.

Example 7 Excretion

(77) The shedding of bacteria in stool and body fluids, tears, saliva, urine and blood was monitored in the study VXM01-01-DE according to methods validated transferred as formerly validated according to GLP at an established central service laboratory (Huntingdon Life Sciences, Huntingdon, UK). Shedding and biodistribution in body fluids of VXM01 were determined by plate and enrichment cultivation. Identity of the VXM01 carrier bacterium was determined by serological agglutination and PCR methods.

(78) Test samples (blood, tears, urine, saliva and faeces) were collected at the site in Heidelberg and same-day delivery of post-vaccination samples took place to MicroMol GmbH located in Karlsruhe, Germany. The bacterial vector shedding and biodistribution analysis cascade was designed to detect live CFU of VXM01 or horizontal plasmid transfer. It consists of two separate analysis branches (Branch I and Branch II):

(79) Branch I: Plating method to detect any horizontal plasmid transfer

(80) Branch II: Liquid enrichment culture to detect live CFU of VXM01

(81) The analysis cascade is followed by a matrix decision in order to determine the excretion of live bacteria VXM01 or observation of a horizontal plasmid transfer. The cascade is outlined in FIG. 11.

(82) Analysis Branch I for detection of horizontal plasmid transfer:

(83) Day 0: Plating of the 5 test samples on 3 TSA (+kanamycin) plates each, incubation over night at 37? C. Day 1: Visual discrimination between VXM01 (Ty21a) and non-VXM01 morphotypes on the selective plates. Selection of non-VXM01 morphotypes (9 colonies each), streaking on agar plates (+kanamycin) for agglutination and parallel liquid culture (+kanamycin) for each pooled morphotype for PCR analysis the following day Day 2: PCR of each liquid morphotype pool
Analysis Branch II for detection of VXM01: Day 0: Preparation of liquid enrichment cultures (+kanamycin) for each of the 5 test samples Day 1: Direct PCR on each liquid enrichment culture. Streaking of each enrichment culture on agar plates (+kanamycin) for serological analysis the following day in case PCR is positive for plasmid Day 2: Serological confirmation of presence of VXM01 (Ty21a)

(84) Due to the fact that PCR of any test sample would not be discriminative between live CFU and/or free-floating plasmid or Ty21a genomic DNA and as agglutination cannot be applied directly on test samples, PCR as well as agglutination methods were used as second-line methods after plating method was applied. Identified live colonies grown on kanamycin-containing plates were further characterized by these methods. Only the plating method enables discrimination between live and dead cells (either VXM01 or foreign non-VXM01 plasmid transformants due to horizontal plasmid transfer).

(85) Fecal excretion of VXM01 was observed in two patients, one in the 10.sup.9 and one in the 10.sup.10 dose group. VXM01 excretion in feces in both patients was transient at one occasion after the first or second administration, respectively, and disappeared without antibiotic treatment. The numbers of VMX01 excreting patients in the various dose groups are graphically depicted in FIG. 12.

(86) In summary, VXM01 has the potential to target a variety of tumor types and to overcome multiple hurdles encountered by other present cancer vaccine approaches. A tempting vision is the possibility of combining the vaccine of the present invention with a multitude of other anti-cancer and immune-modulatory agents. The results of the here presented study motivate the inventors to move forward this highly interesting approach.