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DESCRIPTION

The present invention relates to a compound selected from a polynucleotide coding for Glypican-1 , a vector, and a host cell genetically engineered so as to express Glypican-1. The present invention further relates to the use of the compound for the prevention or treatment of a tumour.

STATE OF THE ART

The development of successful oral vaccines using specific “tumour-associated antigens” (TTAs) requires, besides the incorporation of molecules that may enhance the immunogenic effect, a careful design of vectors that carry and direct the drug towards the target. The oral administration of a vaccine vector has various advantages where the aim is to improve the effectiveness of a vaccination. However, there is a need to protect peptides or immunogenic proteins, their release, and the adjuvant capacity of their vectors during their passage through the gastrointestinal tract until reaching the main site of operation. In fact, though the physiological and biological structure of the gastrointestinal system has been amply studied, questions regarding the interactions of the various potential vaccine vectors that may be used through the oral route remain largely unanswered. In the case of vaccine approaches using other mucosal routes, it has been demonstrated that there exists a direct correlation between the prolonged presentation of the immunogen and the production of long-term protective immunity. Because of its nature and role, the gastrointestinal tract shows differences in this context due to the fact that, given its nature, it is possible for there to be phenomena of immune tolerance. Therefore, in the case of oral administration and passage through the gastrointestinal tract it is of extreme importance to achieve a better understanding of the dose and release kinetics of the most suitable antigen (TAA). Furthermore, it is extremely important to correctly choose the appropriate materials (biomaterials) to be used as effective administration systems. SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a genetically engineered host cell comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 1 , or a vaccine composition comprising said host cell, for use in eliciting an immune response against Glypican-1 (GPC-1 ) in an individual and/or in the treatment or prevention of a tumour, wherein the host cell is a bacterial cell and wherein the host cell or the composition is administered orally. Preferably, the nucleotide sequence is identical to SEQ ID NO:1.

In one embodiment, the host cell is a probiotic bacterium.

Preferably, the host cell belongs to the genus Escherichia and/or Bifidobacterium and/or Lactobacillus', more preferably, the host cell belonging to the genus Escherichia belongs to the species E. Coli Nissle, wherein the host cell belonging to the genus Bifidobacterium belongs to the species B. longum or B. infantis or B. clausii, and wherein the host cell belonging to the genus Lactobacillus belongs to the species L reuteri, L acidophilus, L casei, L lactis, L plantarum and combinations thereof.

In one embodiment, the tumour is a solid tumour; it is preferably a tumour that expresses Glypican-1 and, more preferably, it is a tumour that overexpresses Glypican-1 .

In one embodiment, the tumour is selected from: glioblastoma, prostate cancer, oesophageal cancer, pancreatic cancer, and breast cancer; the tumour is preferably pancreatic carcinoma and, more preferably, it is pancreatic ductal adenocarcinoma.

In one embodiment, the host cell or vaccine composition for the medical uses specified above is administered in association or in combination with at least one drug or a therapy to treat or prevent the tumour.

Preferably, the at least one drug is selected from temozolamide, paclitaxel, nab- paclitaxel, folfirinox, epirubicin, capecitabine, oxaliplatin, prostate hormone therapy, and EGFR inhibitor drugs and wherein the at least one therapy is surgery or radiotherapy. Preferably, the composition is formulated for oral administration; more preferably, it is formulated as a solution, tablet, capsule, pill, granular powder, hard-shelled capsule, orodispersible granules, sachets, lozenges, or drinkable vials.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a Ponceau red staining of a blot with transferred proteins of E. Coli Nissle (A); and an immunoblotting with anti-GPC1 antibody diluted 1 :5000 (B). The marker was loaded into well 1 ; the total lysates of E. Coli Nissle transformed with the vector containing the gene of interest were loaded into wells 2 and 3, and the lysates of E. Coli Nissle WT were loaded into wells 4 and 5.

Figure 2 shows a histogram with the mean count of mouse serum positivity for BxPC3 and Jurkat cells assessed by flow cytometry. Basal and after 19 days of treatment.

Figure 3 shows a histogram with the response of the mouse sera tested by ELISA to the antigen GPC1 .

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention relates to a compound selected from: a) a polynucleotide coding for Glypican-1 or for a portion of Glypican-1 , preferably comprising a nucleotide sequence at least 80% identical to SEQ ID NO: 1 ; b) a vector comprising said polynucleotide; and c) a genetically engineered host cell comprising said polynucleotide.

In one embodiment, the polynucleotide comprises a nucleotide sequence that is substantially homologous or identical to SEQ ID NO.1 (Table 1 ). For example, the polynucleotide comprises a nucleotide sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to SEQ ID NO. 1 .

Preferably, the polynucleotide comprises a nucleotide sequence at least 80%, more preferably at least 90%, homologous or identical to SEQ ID NO. 1 . In one embodiment, the polynucleotide consists in a nucleotide sequence that is substantially homologous or identical to SEQ ID NO.1. For example, the polynucleotide consists in a nucleotide sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to SEQ ID NO. 1 .

Preferably, the polynucleotide consists in a nucleotide sequence at least 80%, more preferably at least 90%, homologous or identical to SEQ ID NO. 1 .

In one embodiment, the polynucleotide comprises at least one sequence coding for a protein portion which enables the transfer and anchorage of the protein Glypican-1 on the outer part of the host cell.

In one embodiment, the polynucleotide comprises at least one sequence coding for at least one portion of a bacterial flagellum, preferably coding for a flagellin. In one embodiment, the at least one portion of a bacterial flagellum is a portion of a flagellin of Salmonella enterica.

In one embodiment, the vector comprising the above-described polynucleotide is selected from: viral vector, plasmid, viral particles, and phage.

Preferably, the vector is a plasmid vector.

In one embodiment of the invention, the genetically engineered host cell which expresses Glypican-1 or a portion of Glypican-1 is selected from: bacterial cell, fungal cell, animal cell, insect cell and plant cell; the host cell is preferably a bacterial cell. Preferably, the bacterial cell is a probiotic bacterium.

Preferably, the host cell comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 1 , more preferably identical to SEQ ID NO: 1.

In one embodiment, the host cell is a bacterial cell belonging to the genus Escherichia and/or Bifidobacterium and/or Lactobacillus or another probiotic bacterium. Preferably, the host cell belonging to the genus Escherichia belongs to the species E. Coli Nissle, the host cell belonging to the genus Bifidobacterium belongs to the species B. longum or B. infantis or B. clausii, and the host cell belonging to the genus Lactobacillus belongs to the species L reuteri, L acidophilus, L casei, L lactis, L plantarum and combinations thereof. In a preferred embodiment, the host cell belonging to the genus Escherichia is the strain E. Coli Nissle 1917, the host cell belonging to the genus Bifidobacterium is B. longum, and the host cell belonging to the genus Lactobacillus is L acidophilus.

In a preferred embodiment of the invention, the host cell, preferably a bacterial cell, more preferably a probiotic, comprises the polynucleotide described above and expresses the human protein Glypican-1 . In other words, the host cell is a bacterial cell comprising the polynucleotide described above and which expresses the protein Glypican-1 or a portion thereof on the bacterial membrane.

In another embodiment, the host cell comprises the vector described above.

In one embodiment, the polynucleotide described above is inserted into the vector and/or host cell with techniques known to the person skilled in the art, such as, for example, cloning techniques or techniques including the use of CRISPR/CAS technology.

A second aspect of the present invention relates to a pharmaceutical composition comprising the compound as described above in detail. In a preferred embodiment, the composition is a vaccine composition, i.e. a vaccine. Preferably, the composition of the present invention can further comprise at least one pharmacologically acceptable excipient, i.e. a compound acceptable for pharmaceutical use which is useful in the preparation of the composition and is generally biologically safe and nontoxic.

In a preferred embodiment of the invention, the composition comprises the host cell which expresses Glypican-1 (or “GPC-1”) or a portion of Glypican-1 as described above.

According to a preferred embodiment of the invention, the composition is formulated for oral administration. Preferably, the composition is formulated as a solution, tablet, capsule, pill, granular powder, hard-shelled capsule, orodispersible granules, sachets, lozenges, drinkable vials or in the form of suspensions in oil.

In another embodiment, the composition is formulated as a suppository.

In one embodiment, the composition is formulated for administration by inhalation. In one embodiment, the composition is in lyophilised form in order to be reconstituted and obtain a liquid formulation.

In one embodiment, the composition is formulated for parenteral administration, preferably as a solution, suspension, sterile emulsion, or powder to be 5 resuspended before use. Preferably, the composition is formulated for subcutaneous administration.

A third aspect of the present invention relates to the compound or composition as described above in detail for use as a medicament.

A fourth aspect of the present invention relates to the compound or composition as described above in detail or the human protein Glypican-1 or a portion thereof, for use in eliciting an immune response against Glypican-1 (GPC-1 ) in an individual and/or in the prevention or treatment of a tumour, preferably a malignant tumour.

In one embodiment, the human protein Glypican-1 comprises an amino acid sequence that is substantially identical to SEQ ID NO.2. For example, the human protein Glypican-1 comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO. 2.

In a preferred embodiment, the human protein Glypican-1 comprises an amino acid sequence at least 80%, more preferably at least 90%, identical to SEQ ID NO. 2.

In one embodiment, the human protein Glypican-1 consists in an amino acid sequence substantially identical to SEQ ID NO. 2. For example, the human protein Glypican-1 consists in an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO. 2.

In a preferred embodiment, the human protein Glypican-1 consists in an amino acid sequence at least 80%, more preferably at least 90%, identical to SEQ ID NO. 2.

In one embodiment of the invention, the human protein Glypican-1 comprises modifications to the N-terminal and/or C-terminal region, for example such as to increase the immunogenicity of the protein. Said modifications are preferably selected from deletions, additions, alterations of amino acids and combinations thereof. Alternatively, the protein Glypican-1 can be modified, preferably in its primary structure, by acetylation, carboxylation, phosphorylation, and combinations thereof.

In a further embodiment, said Glypican-1 is conjugated/bound with a molecule, a metal, or a marker, for example proteins, for the preparation of fusion proteins. In a further embodiment of the invention, said Glypican-1 is modified by means of molecular biology techniques to improve its resistance to proteolytic degradation and/or to optimise its solubility or improve its pharmacokinetic characteristics.

According to one embodiment of the invention, said Glypican-1 can be modified for the purpose of facilitating or improving delivery, preferably by PEGylation, or using container/shuttle/carrier systems, preferably of the liposome, micelle, capsule, emulsion, matrix, or gel type and the like.

In a further embodiment, said Glypican-1 is coated with a structure capable of improving its stability and/or half-life and/or water solubility and/or immunological characteristics. Said structure is for example a pH-sensitive microsphere, a microsphere, a micro-pill, a liposome, or nano-delivery systems with conductive particles mediated by pulsed electric fields (PEFs) or fusion with cell-penetrating peptides (CPPs). In one embodiment, the protein Glypican-1 is obtained by means of recombinant DNA techniques known to the person skilled in the art, preferably by cloning of the polynucleotide (cDNA) coding for Glypican-1 in a plasmid vector for the expression of the recombinant enzyme in bacteria.

In a preferred embodiment of the invention, Glypican-1 is obtained by cloning of the polynucleotide coding for Glypican-1 comprising SEQ ID NO. 1 , as described above in detail.

In a further embodiment of the invention, said Glypican-1 is synthesised by means of conventional protein synthesis techniques known to the skilled person. For example, the protein can be synthesised by chemical synthesis using solid-phase peptide synthesis.

In a further embodiment of the invention, the protein Glypican-1 is isolated or purified with methods known to the person skilled in the art. For example, the protein Glypican-1 can be purified by means of biochemical methods, such as filtration, affinity, or immunoaffinity, or by means of high-performance liquid chromatography (HPLC, RP-HPLC, ion-exchange HPLC, size-exclusion HPLC). In one embodiment, the tumour, preferably a malignant tumour, expresses the human protein Glypican-1. Preferably, the tumour is a tumour that overexpresses the human protein Glypican-1 .

In one embodiment, the tumour is a solid tumour, preferably selected from: glioblastoma, prostate cancer, oesophageal cancer, pancreatic cancer, and breast cancer, preferably pancreatic carcinoma, even more preferably pancreatic ductal adenocarcinoma,

In a preferred embodiment of the invention, the compound is/the composition comprises the host cell that expresses Glypican-1 (or “GPC-1”) or a portion of Glypican-1 as described above.

Preferably, the compound or composition or human protein Glypican-1 as described above in detail is administered orally or by inhalation.

In a preferred embodiment of the invention, the compound or composition or human protein Glypican-1 as described above in detail is administered orally, for example formulated as a spray for oral use or as a tablet or as orodispersible granules.

In another embodiment, the compound or composition or human protein Glypican-1 as described above in detail is administered parenterally, preferably subcutaneously.

The Applicant has in fact surprisingly found that, by inserting a specific immunogenic sequence into a probiotic bacterium one generates an immunogenic response towards a specific tumour-associated antigen (TAA), Glypican-1. The immunogenic response against the antigen Glypican-1 makes the compound, composition and protein Glypican-1 described above in detail useful in combating and preventing some solid tumours, such as glioblastoma and pancreatic ductal adenocarcinoma. The immune response against Glypican-1 generated with oral administration is wholly comparable to the immune response obtained with a subcutaneous administration of the compound and composition described above. Furthermore, the protein Glypican-1 is not expressed in benign tumours or in non-cancerous tissues; therefore, the immune response generated by the vaccine is specific for the treatment of a tumour that expresses or overexpresses Glypican-1 .

In a further embodiment, the compound or composition or human protein Glypican-1 as described above in detail is used individually or in combination with at least one therapeutic treatment for the treatment of a tumour, preferably a malignant tumour. Preferably, the at least one therapeutic treatment for the treatment of a tumour is selected from: at least one chemotherapy drug, a surgical intervention for the removal and/or reduction of the tumour, immunotherapy, radiotherapy, thermal ablation of the tumour and combinations thereof.

In one embodiment of the invention, the at least one chemotherapy drug is selected from: temozolamide, paclitaxel, nab-paclitaxel, folfirinox, epirubicin, capecitabine, oxaliplatin, prostate hormone therapy, and EGFR inhibitor drugs. Preferably, the at least one therapy is surgery or radiotherapy.

A fifth aspect of the present invention relates to a method for preventing or treating a tumour, preferably a malignant tumour, which expresses the human protein Glypican-1 . Preferably, the tumour is a tumour that overexpresses the human protein Glypican-1.

In one embodiment, the tumour is selected from: glioblastoma, prostate cancer, oesophageal cancer and pancreatic cancer, preferably pancreatic carcinoma, even more preferably pancreatic ductal adenocarcinoma.

Preferably, said method comprises at least a step of administering a compound or composition or human protein Glypican-1 as described above in detail to an individual who has a need therefor.

In a preferred embodiment of the invention, the compound or composition or human protein Glypican-1 as described above in detail is administered orally, for example formulated as a spray for oral use or as a tablet or as orodispersible granules.

In another embodiment, the compound or composition or human protein Glypican-1 as described above in detail is administered parenterally, preferably subcutaneously.

A sixth aspect of the present invention relates to a method for stimulating the immune system of an individual against the cancer antigen Glypican-1. Preferably, said method comprises at least a step of administering a compound or composition or human protein Glypican-1 as described above in detail to an individual who has a need therefor. In a preferred embodiment of the invention, the compound or composition or human protein Glypican-1 as described above in detail is administered orally, for example formulated as a spray for oral use or as a tablet or as orodispersible granules.

In another embodiment, the compound or composition or human protein Glypican-1 as described above in detail is administered parenterally, preferably subcutaneously.

Example

The immunogenic sequence (SEQ ID NO:1 ) as described above was inserted into an expression vector of the immunogenic peptide in the probiotic bacterium E. Coli Nissle. The vector was then used for the transformation of E. Coli Nissle by electroporation. After appropriate bacterial growth (OD=1.5), the ability of the probiotic vector to express the immunogenic protein of interest was evaluated by western blot. This analysis made it possible to check both that the immunogenic protein had the expected size, i.e. that it had the right conformation, and that the immunogenic protein was expressed in relevant quantities and thus useful for a possible use of the probiotic for a vaccine approach with oral administration. For the purpose of performing the western blot, the bacteria were centrifuged and then the precipitate was lysed using Hepes 25 mM, 0.2 M NaCI 5% glycerol. Sonication was then carried out and the preparations were loaded into a 10% polyacrylamide gel (Figure 1 ). As may be seen in Figure 1 , the probiotic bacterium E. Coli Nissle transformed with the vector into which the immunogenic sequence in question was inserted is capable of producing large quantities of the protein of interest. As expected, the wells corresponding to the preparation of the untransformed E. Coli Nissle bacterium do not show any band (Figure 1 ).

For the purpose of evaluating the immunogenic capacity of the sequence of interest, in vivo experiments were performed in immunocompetent C57 mice (C57BL/6JOIaHsd). Two groups of mice were used. In a first series of mice, the ability of the vaccine vector to induce an anti-Glypican-1 immunogenic response with oral administration of the vaccine vector was evaluated. Of these mice, a first group of mice had the administration of E. Coli Nissle bacteria transformed to express the immunogenic peptide in order to generate an anti-Glypican-1 response; another group of mice, representing the control, had oral administration of untransformed bacteria. The plasma of the mice in both groups was tested by flow cytometry on cells belonging to the pancreatic ductal adenocarcinoma cell line BXPC3 expressing GPC1 and on cells belonging to the T-cell leukaemia Jurkat cell line used as a control. As shown in figure 2, the serum of mice to which the E. Coli Nissle bacteria expressing the immunogenic peptide of interest were administered showed positivity for cells expressing Glypican-1 and were thus able to recognise the protein of eukaryotic origin Glypican-1. The serum of the mice in this group did not show positivity for Jurkat cells, cells that do not express the protein Glypican-1 . These data are consistent with the presence of Glypican-1 antibodies in the serum of mice to which the E. Coli Nissle bacterium expressing the protein immunogenic towards Glypican-1 was administered. On the other hand, the mice in the group to which untransformed E. Coli Nissle bacteria were administered did not show an immune response that was specific for the protein Glypican-1 (Figure 2). These data were also confirmed through enzyme-linked immunosorbent assay (ELISA) experiments, in which an evaluation was made of the ability of the serum of mice in which the vaccine vector was administered to recognise the human protein GPC1. In such ELISA experiments it was observed that there is an increase in the concentration of anti-GPC1 antibodies in the serum of mice injected subcutaneously with E. Coli Nissle bacterial cells expressing the immunogenic peptide of interest, when the levels in the collected sera were evaluated at different times (T14 days, T30 days and T45 days compared to the basal value prior to treatment (time 0 (TO)). The ability of the vaccine vector consisting of E. Coli Nissle bacterial cells expressing the immunogenic peptide of interest to produce a concentration of antibodies specific for human Glypican- 1 using the oral route of administration was further evaluated by ELISA experiments. This evaluation was carried out in a group of mice, a first group of which received the vaccine vector consisting of 2*10 ⁹ E. Coli Nissle bacterial cells expressing the immunogenic peptide (OS GPC1-Flag), administered orally through the uptake of feed. A second group of mice representing the negative control received, again through the uptake of feed, 2*10 ⁹ of E. Coli Nissle WT bacterial cells (i.e. cells not expressing the immunogenic peptide) (OS WT). A third group of mice was tested by subcutaneous vaccination with 5*10 ⁸ of transformed bacteria lysed by heat (SC GPC1-Flag). The ELISA results show that through oral administration it was possible to obtain an anti-Glypican-1 antibody concentration. In particular, it was possible to observe an increase in the concentration of anti-GPC1 antibodies in mice in which the vaccine vector was orally administered by evaluating the levels in sera collected at different times. This increase in orally treated mice (OS GPC1-Flag) is comparable to the increase in mice treated subcutaneously (SC GPC1-Flag). This increase was not detected, however, in mice to which the untransformed E. Coli Nissle bacterium (OS WT) was administered (Figure 3).