Patent Application
20210275680
The invention presents the use of the TAS1R3 protein as a new marker and molecular target in epithelial tumors and other human pathologies. In particular, the use of the detection of the TAS1R3 protein as well as its expression products, as a marker for therapeutic, diagnostic or prognostic purposes, for tumors that normally express said protein is part of the present invention.
Significant progress has been made in the chemotherapy of neoplastic diseases over the past 30 years. This includes some progress in the development of new chemotherapeutic agents, and more particularly, in the development of regimens for the concurrent administration of drugs. A significant understanding of the neoplastic processes at the cellular and tissue level, and the mechanism of action of the basic antineoplastic agents, has also made it possible to obtain advances in the chemotherapy of a number of neoplastic diseases, including choriocarcinoma, Wilm's tumor, acute leukemia, rhabdomyosarcoma, retinoblastoma, Hodgkin's disease, and Burkitt's lymphoma. However, despite the progress made so far in the field of oncology, and in particular in chemotherapy, many of the most prevalent forms of human cancer are still resistant to the current chemotherapeutic treatments.
In this sense, in any oncological treatment regimen and for it to be effective, the concept of “total cell annihilation” is crucial. This concept holds that, in order to have an effective treatment regimen, either through a surgical or chemotherapeutic approach, or both, there must be a total cellular annihilation of all so-called “clonogenic” malignant cells, that is, of the cells that have the capacity to grow in an uncontrolled way, and to replace any tumor mass that could be eliminated. Due to the ultimate need to develop therapeutic agents and regimens that achieve total cell elimination, certain types of tumors have been more susceptible than others to therapy. For example, soft tissue tumors (e.g., lymphomas), and tumors of blood and blood-forming organs (e.g., leukemia) have generally responded more satisfactorily to chemotherapeutic therapy than solid tumors such as carcinomas. One reason for the susceptibility of soft tumors to chemotherapy is the greater physical accessibility to the lymphoma and leukemia cells in the chemotherapeutic intervention. Simply put, it is much more difficult for most chemotherapeutic agents to reach all the cells of a solid tumor mass, than to reach the soft tumors, and therefore, it is much more difficult to achieve total cell destruction in the case of solid tumors. In this regard, to treat such tumors, solid tumors, the doses of chemotherapeutic agents are significantly increased resulting in side effects, which generally limits the overall effectiveness of the therapy.
The strategy to develop satisfactory antitumor agents capable of treating solid tumors, therefore, involves the design of agents that selectively kill tumor cells, while exerting relatively few, if any, adverse effects against normal tissues. This objective has been difficult to achieve, because there are few qualitative differences between neoplastic and normal tissues. Due to this, in recent years, many efforts have been devoted to trying to identify tumor specific “marker antigens,” which can serve as immunological targets, both for chemotherapy and for diagnosis. In this sense, the present invention addresses this aspect by providing a new biomarker that is differentially expressed in the cell membrane of tumor cells in both primary tumors and disseminated cells. Additionally, the expression of this biomarker is related to a greater invasive and tumorigenic capacity of the tumor cells, increasing this expression especially under conditions of absence of nutrients, being able, therefore, to use this biomarker for the diagnosis/prognosis of cancer. Finally, since it is a membrane receptor, it is possible to design targeted therapies, for example ligands such as small molecules, peptides or antagonists, that stop cell proliferation and the ability of cells to migrate, invade and disseminate. So, in the past, immunotoxins have been used to selectively target cancer cells of solid tumors. Immunotoxins are conjugates of a specific target agent, typically an antibody or fragment thereof directed to the tumor, with a cytotoxic agent, such as a toxin moiety. The target agent is designed to direct the toxin to the cells that carry the target antigen, and kill such cells. On the other hand, “second generation” immunotoxins, such as those that use deglycosylated ricin A chain, have been developed to prevent hepatic entrapment of the immunotoxin and reduce hepatotoxicity (Blakey et al., 1987a; b), and those with new crosslinks to provide the immunotoxins with a higher in vivo stability (Thorpe et al., 1988). Immunotoxins have been shown to be effective in the treatment of lymphomas and leukemia in mice (Thorpe et al., 1988, Ghetie et al., 1991, Griffin et al., 1988a; b) as well as in humans (Vitetta et al., 1991). The present invention therefore provides specific target agents against the biomarker of the present invention, useful for the synthesis of immunotoxins.
In addition, the identification of new biomarkers, such as the one detailed in the present invention, are extremely useful for the development of personalized medicine, both for diagnostic and prognostic purposes, as well as for the selection of targeted therapies, and monitoring of the treatment. Personalized medicine is based on the premise that each tumor is unique, and undergoes dynamic changes over time, so it is important to have tools that allow determining the best therapeutic approach at all times. In the field of personalized medicine, liquid biopsy stands out for its determining role. Today, we know that tumors originate due to the appearance of genetic alterations that affect the proliferation and survival mechanisms of cells, as well as being complex and heterogeneous in their molecular composition. On the other hand, once the cancer is diagnosed, the primary tumor, or its metastases, must be characterized by analyzing the DNA, RNA and protein profile. Within the same tumor different tumor clones coexist that mark the evolution of the tumor and the resistance to the various therapies, hence, the term “dynamic tumors” has been coined. These are susceptible to variations over time, the determination of the dominant clone at each moment being key to select the most effective therapeutic strategy. The diagnosis of non-small-cell lung cancer (NSCLC) is usually performed by CT scan, a sensitive but not specific technique, which requires additional follow-up through invasive procedures (biopsy), which is why clinical practice requires new biomarkers that provide information attached to diagnostic imaging, as well as biomarkers that are able to provide information without the need to apply such aggressive procedures as a biopsy.
In the present invention, the use of the TAS1R3 receptor as a biomarker for application in cancer diagnosis, monitoring, and therapy is described for the first time. In this sense, the authors of the present invention have demonstrated that TAS1R3 is a biomarker of interest in oncology, useful for the diagnosis of the disease, and capable of providing relevant information thereupon, to monitor the evolution, select the treatment, and selectively direct therapeutic molecules. It has been determined that it is possible to identify therapies against this receptor, and that it is also possible to direct conjugates and controlled drug-release systems, such as nanoparticles, very efficiently observing an intracellular accumulation thereof in primary, disseminated and metastatic tumor cells. On the other hand, the presence of TAS1R3 in circulating tumor cells (CTCs) has also been demonstrated. These are tumor cells released into the bloodstream by the primary tumor and are considered key factors in the creation of metastases, so that their detection in early stages will serve as an early detector of metastasis, and are also useful in monitoring the disease and evaluating the response to drugs.
Overall, this discovery opens the doors to the functionalization of any type of molecule with a compound that acts as a ligand against the TAS1R3 receptor, preferably a ligand selected from the group consisting of antibodies, antibody fragments, aptamers, peptides, or low-molecular-weight hydrophobic or hydrophilic molecules, such as lactisole, as well as ligand-drug or ligand-radioisotope conjugates, capable of binding to the TAS1R3 receptor, or ligands functionalized with reactive groups, for use as pharmacological and/or diagnostic vehicles against tumor cells, in particular against CTCs (circulating tumor cells) and against primary, disseminated, or metastatic tumor cells.
In the present invention, “TAS1R3 receptor” is understood as a taste G protein-coupled transmembrane receptor, which is expressed in taste buds, but also in other tissues such as liver and pancreas, and in tumor cells. There are multiple references to this receptor such as; HGNC: 15661 (NCBI Vega: OTTHUMG00000003071); Entrez Gene: 83756; Ensembl: ENSG00000169962; OMIM: 605865; o UniProtKB: Q7RTX0. It can be found forming heterodimers with the TAS1R1 receptor or TAS1R2 and act as a detector of sweet taste or umami, being activated by different amino acids, glucose, etc. See
In the present invention, a ligand for the TAS1R3 receptor is understood to be molecules capable of interacting with the TAS1R3 receptor, such as mono and disaccharides, artificial sweeteners such as sucralose, cyclamate, neoesperidine, dihydrochalcone, and derivatives, sweetness inhibitors such as lactisole and derivatives, proteins such as brazzein, or fragments thereof, as well as others specifically designed by selection systems such as antibodies, fragments of antibodies, peptides, aptamers, small molecules, proteins, etc.
In the present invention, “conjugates with ligands” are chemical conjugates that incorporate a ligand and a molecule with therapeutic activity (drug, radiopharmaceutical, etc.), or for diagnostic purposes (radioisotope, chelator, gadolinium, fluorophores, etc.).
In the present invention, “ligands functionalized with reactive groups” mean ligands that have been chemically modified to present a reactive group that can subsequently be used in a chemical or biochemical reaction, or for the subsequent binding or selective interaction of other molecules.
As used herein, the term “biological sample isolated from the patient” refers to a biological tumor sample or that comprises tumor tissue removed by a routine surgical procedure for diagnostic use in routine clinical protocol. Additionally, this term is understood to encompass any tissue or biological sample isolated from blood, serum or plasma comprising tumor tissue or circulating tumor cells or substances released by the tumor.
As used herein, the term “tissue involvement” refers to the presence of tumor cells in the tissue.
As used herein, the term “determination of TAS1R3 expression” refers to primers, antibodies or any other system that can evaluate the expression of said protein or any of its expression products such as RNA.
As used herein, the term “patient” refers, preferably, to a human. However, it can refer to a mammal, including non-primate mammals (e.g., horse, dog, cat, rat, mouse, cow or pig) and primate mammals (e.g., monkey).
As used herein, the term “molecular marker/tumor biomarker” refers to a biological marker of cancer, i.e., substance (s) produced by the tumor or by stromal cells closely related to the presence thereof, and that offers information of clinical interest on the state of tissue involvement affected by tumor cells. By “molecular marker/tumor biomarker” is also meant a biological marker of cancer, that is, substance (s) produced by the tumor or by stromal cells closely related to the presence thereof, and which offers the possibility of directing therapies against said cells through the use of receptor ligands, preferably ligands in the form of conjugates.
The determination of TAS1R3 expression levels can be carried out by immunological techniques such as for example, ELISA, ELONA, immunoblotting, immunofluorescence or immunohistochemistry, flow cytometry and aptahistochemistry. Immunoblotting is based on the detection of proteins previously separated by gel electrophoresis under denaturing conditions and immobilized on a membrane, generally nitrocellulose by incubation with a specific antibody and a development system (for example, chemiluminescence). Immunofluorescence analysis requires the use of an antibody specific for the target protein for the analysis of expression. ELISA and ELONA are based on the use of antigens, antibodies or aptamers labeled with enzymes so that conjugates formed between the target antigen and the labeled antibody result in the formation of enzymatically active complexes. Since one of the components (the antigen, or the labeled antibody or aptamer) is immobilized on a support, the antigen-antibody/aptamer complexes are immobilized on the support and thus, can be detected by the addition of a substrate that it is converted by the enzyme into a product that is detectable by, for example, spectrophotometry or fluorometry.
When an immunological method is used, any reagent, such as antibodies or aptamers, known to bind to target proteins with high affinity to detect the amount of target proteins, can be used. However, the use of an antibody is preferred, for example polyclonal sera, hybridoma supernatants or monoclonal antibodies, fragments of antibodies, Fv, Fab, Fab′ and F(ab′)2, scFv, diabodies, triabodies, tetrabodies and humanized antibodies. On the other hand, the determination of protein expression levels can be carried out by means of immunohistochemical techniques well known in the state of the art. To carry out the determination by immunohistochemistry, and/or suitable histochemistry, the sample can be a fresh, frozen or paraffin-embedded sample and fixed using a protective agent of the formalin type. For immunohistochemical determination, the sample is stained with an antibody or aptamer specific for TAS1R3 and the frequency of cells that have been stained and the intensity of staining determined. Typically, the sample is assigned a value indicative of the expression and total and that is calculated based on the frequency of stained cells (value that varies between 0 and 4) and of the intensity in each of the stained cells (variable value between 0 and 4). Typical criteria for assigning expression values to samples have been described in detail, for example, in Handbook of Immunohistochemistry and In Situ Hybridization in Human Carcinomas, M. Hayat E d., 2004, Academic Pres s. Additionally, immunohistochemistry makes it possible to identify which type of cells present in cancerous tissue are those that present altered levels of marker expression. Preferably, the immunohistochemical detection is carried out in parallel with cell samples that serve as a positive marker and as a negative marker and, as a reference, healthy tissues of the same origin as the tumor being analyzed can be used. It is also common to use a background control.
In those cases, in which a large number of samples is to be analyzed (for example, when several samples from the same patient or samples from different patients are to be analyzed), the use of matrix formats and/or automated procedures is possible. In one embodiment, the use of tissue microarrays (tissue microarrays or TMA) that can be obtained using different techniques is possible. The samples that are part of the microarrays can be analyzed in different ways including immunohistochemistry, in situ hybridization, in situ PCR, RNA or DNA analysis, morphological inspection and combinations of any of the above. Methods for processing tissue microarrays have been described, for example, in Konenen, J. et al., (Nat. Med. 1987, 4: 844-7). Tissue microarrays are prepared from cylindrical cores of 0.6 to 2 mm in diameter from tissue samples embedded in paraffin and re-embedded in a single receptor block. In this way, tissue from multiple samples can be inserted into a single block of paraffin. The determination of the expression levels of TAS1R3 needs to be correlated with the reference values corresponding to the median value of the TAS1R3 expression levels measured in a collection of tumor tissues in biopsy samples from subjects with cancer. Said reference sample is typically obtained by combining equal amounts of samples from a population of subjects. In general, typical reference samples will be obtained from subjects who are clinically well documented and in whom the disease is well characterized by one of the usual methods (digital rectal examination, occult blood test in the stool, sigmoidoscopy, colonoscopy, biopsy), determination of tumor markers such as carcinoembryonic antigen, ultrasound, CT, nuclear magnetic resonance, positron emission tomography). In such samples, the normal (of reference) concentrations of the biomarker can be determined, for example by providing the average concentration over the reference population. Several considerations are taken into account when determining the reference concentration of the marker. Such considerations include the type of sample involved (for example tissue or CSF), the age, weight, sex, general physical condition of the patient and the like. For example, equal amounts of a group of at least 2, at least 10, at least 100 to preferably more than 1000 subjects are taken as reference group, preferably classified according to the above considerations, for example of various age categories. The collection of samples from which the reference level derives will preferably be made up of subjects suffering from the same type of cancer as the patient under study.
Once this median or threshold value has been established, the level of this marker expressed in tumor tissues of patients with this median value can be compared, and in this way, be assigned to the “increased” or “decreased” level of expression. Due to the variability between subjects (for example, aspects related to age, race, etc.) it is very difficult (if not practically impossible) to establish absolute reference values of TAS1R3 expression. Thus, in a particular embodiment, the reference values for “increased” or “decreased” expression of TAS1R3 expression are determined by calculating the percentiles by conventional means involving testing the TAS1R3 expression levels in one or more isolated samples in subjects for whom the disease is well documented by one of the methods mentioned above. The “reduced” levels of APTX can then be assigned, preferably, to samples where the TAS1R3 expression levels are equal to or less than the 50th percentile in the normal population, including, for example, expression levels equal to or lower than the 60th percentile in the normal population, equal to or less than the 70th percentile in the normal population, equal to or less than the 80th percentile in the normal population, equal to or lower than the 90th percentile in the normal population, and equal to or lower than the 95th percentile in the normal population. The “increased” levels of TAS1R3 can then be preferably assigned to samples where the TAS1R3 expression levels are equal to or exceed the 50th percentile in the normal population, including, for example, expression levels equal to or in excess of the 60th percentile in the normal population, equal to or in excess of the 70th percentile in the normal population, equal to or in excess of the 80th percentile in the normal population, equal to or in excess of the 90th percentile in the normal population, and equal to or in excess of the 95th percentile in the normal population. The term “increased expression levels of TAS1R3,” as used herein, refers to levels of TAS1R3 higher than those appearing in a reference sample. In particular, a sample can be considered to have increased levels of TAS1R3 expression when the expression levels are, with respect to the reference sample, at least 1.1 times, 1.5 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or even more with respect to the sample isolated from the patient.
The authors of the present invention have identified in CTCs (circulating tumor cells) (see example 1) and in tumors, a new receptor, TAS1R3 (a receptor of taste), and have determined that the expression of said receptor varies depending on the presence of glucose, and is related to the presence of primary, disseminated or metastatic tumor cells.
In this sense, as can be seen in
These data demonstrate the diagnostic potential of this biomarker, given that
The present invention, therefore, relates to a new prognostic/diagnostic tumor biomarker capable of detecting the presence of primary, disseminated or metastatic tumor cells in a biological sample such as a tissue or a sample of blood, serum or plasma. Also, the invention relates to the detection of TAS1R3 expression levels in a biological sample such as a tissue or a serum or plasma blood sample, to diagnose the involvement or presence in said tissue or biological sample of tumor cells, whether primary, disseminated or metastatic. Said information would also have a prognostic value since the identification of metastatic tumor cells or the comparison of the level and/or concentration of the TAS1R3 receptor in a biological sample such as a tissue or a sample of blood serum or plasma with the level and/or concentration of the TAS1R3 receptor in a biological sample from a healthy individual, or from a part of a healthy organ or tissue or biological sample without tumor involvement, or from a reference value, would provide useful information in an individual's prognosis such as information about the overall survival (OS) of a patient suffering from cancer. In this sense, the determination of the expression of TAS1R3 and the identification of the cell type that expresses it, will allow predicting the involvement of the tissue affected by tumor cells. Additionally, the expression levels of the TAS1R3 receptor will allow predicting the possible involvement implied by metastatic tumor cells, thus providing a clear prognostic value.
Therefore, a first aspect of the present invention refers to a method for the prediction or in vitro diagnosis of tumor involvement or presence (neoplastic or metastatic involvement or presence) in a patient, characterized by comprising the following steps:
Additionally, in a second aspect, the present invention refers to a method for the in vitro prognosis of the clinical course of a patient suffering from a tumor involvement or presence (neoplastic or metastatic involvement or presence), characterized by comprising the following steps:
Negative clinical evolution of the patient is understood as, preferably, an estimated overall survival of less than 5 years, preferably less than 3 years.
Additionally, a third aspect the present invention refers to a method for the in vitro monitoring of the clinical evolution of a patient suffering from a tumor involvement or presence (neoplastic or metastatic involvement or presence), characterized by comprising the following steps:
Additionally, a fourth aspect the present invention refers to a method for monitoring the response to in vitro treatment of a patient suffering from a tumor involvement or presence (neoplastic or metastatic involvement or presence), characterized by comprising the following steps:
As already stated in the “definitions” section, as used herein, the term “determination of the expression of TAS1R3” refers to antibodies or any other system that can evaluate the expression of said protein, such as aptamers. In order to obtain antibodies or aptamers useful in the determination of TAS1R3 expression levels, a sequence analysis of the human TAS1R3 protein was carried out. From these data, it was possible to predict that the sequences most suitable from the immunological point of view (present in the N-terminal, extracellular region) would be the following:
Based on these data, the peptide was synthesized (Acetyl-LSQQLRMKGDYVLGGC-Amide) using the heterologous expression system Escherichia coli. The resulting protein was purified to homogeneity with a final purity of 90-95%, 2 mg conjugated to KLH, 3 mg conjugated to BSA. The design of the genomic construct was carried out based on the literature (Maitrepierre et al., 2012). Likewise, a study of storage buffer formulation was carried out to preserve the integrity of the protein (restricting the use of detergents and polysaccharides in the final formula). The batch obtained was aliquoted in labeled sterile vials and stored at −80° C. until use. After several immunizations of the mice with 4 plus 2 doses of 50 μg of peptide, the bleeds were titrated in order to evaluate the increase of the response against the peptide. The results show that the mice generated some response against the peptide, although with very low values that discourage the achievement of production.
Therefore, in a preferred embodiment of the first to fourth aspect of the invention, the determination of the expression of TAS1R3 in the cells of a biological sample such as a tissue or a sample of blood serum or plasma, is carried out with antibodies or fragments thereof or aptamers capable of binding to SEQ ID NO 1 or SEQ ID NO 2.
In a preferred embodiment of the first to fourth aspect of the invention, the present invention is characterized in that the negative predictive value of said method is greater than 80%, more preferably greater than 90%, 95%, 96%, 97%, 98% or 99%. That is, when the expression of TAS1R3 is not detected in the cells in the biological sample of the patient, it means that said sample will not be affected by the tumor.
As used herein, the term “negative predictive value” refers to the percentage of cases that having the negative test results in not having the disease (tissue involvement affected by tumor cells).
In another preferred embodiment, the present invention is characterized in that the patient suffers from a solid tumor, preferably suffers from a disease selected from among the following group: breast cancer, melanoma, uveal melanoma, pancreatic cancer, lung cancer, prostate cancer, stomach cancer, head and neck cancer, sarcoma, glioblastoma, neuroblastoma, cancer of the colon and rectum, cancer of the head and neck, kidney and bladder cancer, and hepatocarcinoma.
In another preferred embodiment, the present invention is characterized in that the cell type identification technique defined in step b) is selected from among the following group: immunohistochemistry, immunofluorescence, aptahistochemistry, and flow cytometry. Preferably, the technique for cell type identification defined in step b) is immunohistochemistry, and more preferably, immunohistochemistry comprises the following steps:
In another preferred embodiment, the present invention is characterized in that the sample already obtained from a tissue defined in step a) is selected from among the following group: fixed tissue sample, fresh tissue sample, and frozen tissue or blood sample, serum or plasma. Preferably, it is a fixed tissue sample or a sample of blood, serum or plasma.
In another preferred embodiment, the present invention is characterized in that the patient is a mammal. Preferably, the mammal is selected from the following group: human, primate and non-primate. More preferably, the patient is a human.
The present invention also relates, in a fifth aspect of the invention, to TAS1R3 for use as a tumor biomarker, preferably in the method described above.
The present invention also relates to a kit for use in the method described above.
The embodiment of the invention can be carried out in fresh tissue, fixed or frozen by flow cytometry, immunohistochemistry, immunofluorescence or any other technique that allows the identification of the cell type known by a person skilled in the art.
On the other hand, and due to the enormous potential of this receptor for the selective direction of molecules, such as nanoparticles, drugs, or radioisotopes, and taking into account that we know of the existence of molecules that interact with said receptor, and are not endogenous, and, therefore, competition phenomena do not occur; we have selected several ligands of the receptor, in particular several sweeteners, to validate the hypothesis that it is possible to design therapies against it. We have also functionalized nanostructures with said ligands, to direct them against tumors that express TAS1R3, such as primary tumors or disseminated tumor cells such as CTCs (circulating tumor cells) and metastatic cells. We have also observed that some ligands, such as lactisole, or nanostructures functionalized with it, can give rise to therapeutic effects, in terms of tumor toxicity and proliferation.
In order to carry out said functionalization, the ligands described in Table 2 were acquired, and either they were joined by covalent binding to a hydrophobic moiety (e.g., LACT-C16), or by incubation on preformed nanoemulsions (ex. BRA). The sequences of the peptides used and the physicochemical properties of the resulting nanoemulsions are described below (Table 2).
Once the previous functionalization was carried out, we observed a high internalization capacity of those functionalized nanoemulsions with ligands against TAS1R3 in cells that express the receptor (tumor cells). For example, in the case of nanoemulsions functionalized with lactisole, the DiD signal encapsulated in the nanoemulsions was greater than with respect to those other non-functionalized nanoemulsions. In addition, we observed that when tumor cells expressed the target receptor with less intensity (in this case TAS1R3 in SW620 cells cultured in medium with higher glucose content (low expression, example 2)), the intensity of the signal decreased (example 4).
The present invention demonstrates, therefore, that the functionalization of nanosystems with ligands against TAS1R3, expressed in the cell membrane of tumor cells, in particular in the membrane of disseminated and metastatic tumor cells, makes it a unit with a strong therapeutic potential. In particular, said nanosystems can be used as vehicles for the development of combination therapies. Additionally, said potential to functionalize structures other than nanoemulsions, such as quantum dots, are described in Example 5 of the present invention. This discovery can be extrapolated to any type of nanosystem, such as nanoparticles, micelles, or liposomes, i.e., ligands against this receptor can serve as selective vehicles that serve to functionalize any type of nanosystem. Furthermore, this discovery can be extrapolated to any type of system, such as any ligand-drug conjugate and/or ligand-radioisotope comprising ligands against this receptor and can serve as selective intracellular vehicles that functionalize said drug or radioisotope.
Therefore, the present invention demonstrates that once any type of nanostructure, liposome, drug or radioisotope with ligands has been functionalized against the receptor of the present invention, a vehiculation of said structure is observed towards those cells that express the receptor, in particular a vehiculation towards primary, disseminated or metastatic tumor cells.
Thus, a sixth aspect of the invention relates to the use of the TAS1R3 receptor as a molecular marker/tumor biomarker to direct therapies against tumor cells through the use of receptor ligands, preferably ligands in the form of conjugates or immunotoxins. The immunotoxins are conjugates of a specific target agent, typically an antibody or fragment thereof directed to the tumor, with a cytotoxic agent, such as a toxin moiety. The target agent is designed to direct the toxin to the cells that carry the target antigen, and kill such cells.
The present invention, therefore, in a seventh aspect of the invention, provides conjugates of:
In a preferred embodiment of the seventh aspect of the invention, the target agent are antibodies or fragments thereof, or peptides, or aptamers capable of binding to SEQ ID NO 1 or SEQ ID NO 2.
An eighth aspect of the invention relates to the conjugate of the seventh aspect of the invention, for use in therapy or in vivo diagnosis. Preferably, for use in the in vivo treatment or diagnosis of cancer, more preferably, for use in the treatment or in vivo diagnosis of a solid tumor, preferably a tumor selected from among the following group: breast cancer, melanoma, uveal melanoma, pancreatic cancer, lung cancer, prostate cancer, stomach cancer, head and neck cancer, sarcoma, glioblastoma, neuroblastoma, colon and rectal cancer, head and neck cancer, kidney and bladder cancer, and hepatocarcinoma, or disseminated or metastatic cells.
Throughout the description and claims the word “comprises” and its variants do not intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practical implementation of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.
The analysis of the gene expression of the TAS1R3 receptor in CTCs (circulating tumor cells) of patients with metastatic lung cancer was performed from peripheral blood samples (patients=10, controls n=4), using magnetic particles for their isolation (EpCAM-based CELLection™ Epithelial Enrich Dynabeads® kit), following the diagram shown in
A genomic analysis of differential expression was also carried out. As depicted in
Next, we proceeded to validate the expression of the candidate genes in an independent cohort of patients and controls. Using quantitative PCR, it was confirmed that the expression levels of this receptor were significantly higher in the patients (
To obtain information about the prognostic value of the marker, a Kaplan-Meier survival analysis was performed using the SPSS statistical package (
Regarding the evaluation of expression in cell cultures, several types of cell lines were used, obtained from ATCC (American Type Culture Collection) (
Regarding the immunofluorescence studies shown in
For the qualitative study of proteins, Western Blot was performed from proteins isolated from the SW620 cell line, and the results are shown in
The expression of TAS1R3 in the colon line SW620 varies depending on the glucose content in the culture medium (high levels of glucose give rise to a lower expression, in relation to that observed in
Cell proliferation studies were carried out in SW620 cells,
The presence of lactisole in the culture medium, a ligand of the TAS1R3 receptor, causes an increase in the expression of the receptor under study (
To determine whether after cell incubation with the ligand cell senescence is induced, 500,000 cells of the SW620 line were cultured in 6-well plates and medium with low glucose concentration. In several wells, ligand (3.5 mg/mL) was added. After 72 hours, the wells were washed and fixed (2% formaldehyde, 0.2% glutaraldehyde in PBS) for 15 minutes at room temperature. The solution was eliminated giving three washes with 1×PBS, and then the staining solution was applied, calculated for one milliliter of solution: 200 μL citric acid/phosphate buffer, pH 6.0 (dibasic sodium phosphate (Na2HPO4) 0.2 M+0.1 M citric acid), 30 μL 5 M NaCl, 50 μL potassium ferricyanide (K3[Fe(CN)6]) 100 mM, 50 μL potassium ferrocyanide (K4[Fe(CN)6]) 100 mM, 2 μL magnesium chloride (MgCl2) 1 M, 50 μL solution X-gal (20 mg/mL of 5-bromo-4-chloro-3-indolyl β D-galactopyranoside in dimethylformaldehyde) and 618 μL distilled water. It was incubated in the oven at 37° C. overnight, the wells were washed with distilled water in motion twice, to then apply PBS 1× and observe the possible senescent cells under the Vert microscope. Al, Zeiss®. Blue staining is observed, corresponding to cells in senescence.
For the test of colony formation in media with the free ligand, the same process was carried out but in this case, the cells were seeded in 1000 ml/L of glucose (low glucose concentration) and 3.5 mg/mL of ligand was added to each of the 24 wells. The results are shown in
This finding is extremely interesting, since it could present this ligand with a dual function, for the active direction of nanoparticles and as a drug.
The functionalization of the nanoemulsions with different ligands against TAS1R3 took place as described below. Nanoemulsions were obtained spontaneously after adding 100 μL of ethanol, containing oleic acid and sphingomyelin (5 mg and 500 μg respectively), together with 500 μg of lactisole covalently linked to a C16-C18 chain, on 1 mL of Milli-Q water (MilliporeMilli-Q System®) under gentle magnetic stirring. High-performance liquid chromatography (HPLC) was used to confirm the inclusion of the ligand in the nanoemulsions using an HPLC system (1260 Infinity II, Agilent) equipped with a G7111A pump, a G7129A autosampler and a G7114A UV-Vis detector, with an InfinityLab Poroshell 120EC-C18 column (Agilent, 4.6×00 mm, 4 μm pore size).
To study the effect of functionalization on the ability of nanoemulsions to interact with cells that express the receptor of interest, flow cytometry and confocal microscopy techniques were used. Specifically, SW620 cells were used, and confocal microscopy was used. For these experiments, 80,000 cells were seeded per well in 8-well micro-chambers (PLC30108, SPL LifeSciences). After 24 hours, the cells were incubated with nanoemulsions prepared from oleic acid and a sphingomyelin derivative labeled with nitrobenzoxadiazole (NBD), which in turn encapsulated DiR (OLM; O:SM1:0.1), and those same nanoemulsions functionalized with lactisole (OLM-L; O:SM:Lact 1:0.1:0.1), at a final concentration in the nanoemulsion well of 0.12 mg/mL. After 4 hours of incubation at 37° C., the cells were washed with 1×PBS twice, and then fixed with 4% paraformaldehyde for 15 minutes, after which the wells were washed twice with PBS 1×, and then Cell nuclei were stained with Hoechst 33342 (Thermo Fisher®). The mounting medium (Mowiol, Calbiochem) was applied and the samples were observed under the confocal microscope (Laser Microscope Confocal Leica SP8g). A similar experiment was carried out comparing in this case the internalization of the functionalized nanoemulsions with lactisole (O:SM:Lact 1:0.1:0.1), and labeled with DiD, after incubation in SW620 cells with cultured medium with high or low concentration of glucose, and therefore with different levels of expression of the receptor, as seen in example 2. It was found that indeed the intensity of fluorescence due to nanoemulsions decreased in the case of incubation on SW620 cells with lower expression of TAS1R3 receptor for lactisole, as can be seen in
O:SM:Lact 1:0.1:0.1 (NE-F), loaded with etoposide at 1% by weight, with respect to the other components. 10,000 cells were seeded per well in a 96-well plate. After 24 h of culture, 20 μL of the test formulation was applied to 110 μL of culture medium, in order of increasing concentration, establishing as controls a positive one, adding the vehicle in which the nanosystem is dissolved (water in the largest part of the cases), and a negative one, or total death, where a dilution of Triton 100× at 6% was applied. After 48 hours, the culture medium of the plate was aspirated, washing with 1×PBS, then applying the MTT reagent at a concentration of 5 mg/mL in 1×PBS, after dilution 1:10 in DMEM medium without supplementation, and filtered with a 0.22 μm filter. 110 μL was applied per well. After 4 hours in the incubator, the medium was removed from the plate, and a volume of 110 μL of 1×DMSO (dimethylsulfoxid, 99.7%, AcrosOrganics) was added to dissolve the formazan crystals originated by the mitochondrial enzymes. Protecting the light plate was incubated 15 minutes at 37° C., to then measure the absorbance at 570 nanometers in the spectrophotometer (Multiskan EX, ThermoLabsystems®) and obtain the EC50 values of each formulation, using the GraphPad Prism 5 program. For the encapsulation of an antitumor drug, etoposide was selected, and 13.75 μl of a solution of 40 mg/mL (550 μg of drug) was added to the organic phase. All nanoemulsions were isolated by centrifugation for 30 minutes at 14000×g 15° C. (Microcentrifuge 5415R, rotor F452411 Eppendorf®), in order to eliminate all that was not part of the nanoemulsions. As shown in
Quantum dots functionalized with an antibody against the human protein TAS1R3. The antibody against TAS1R3 (Ref sc50353, SCBT) was conjugated to quantum dots using the SiteClick™ Qdot® 655 Antibody Labeling Kit (Ref S10453, ThermoFisher) following the supplier's instructions. The concentration in the final preparation was determined by measuring the optical density at the specified wavelength and then using the formula A=ccL, where A is the absorbance, c is the molar extinction coefficient, c is the molar concentration, and L is the length of the path. We checked the size distribution of the quantum dots, observing the presence of a majority population of approx. 400 nm.
We performed an interaction test of functionalized quantum dots with lung tumor cells A549 in suspension and adherence. Incubation was carried out at 4° C. and 37° C. for 4 hours. After the incubation time, the culture medium was removed and washed with 1×PBS. The cells incubated in suspension were seeded a posteriori to allow their adherence and perform immunofluorescence studies of the TAS1R3 receptor. After 24 hours, the culture medium was removed by aspiration and washed with PBS 1×, to proceed to fix the cells with 4% paraformaldehyde, for 15 minutes at room temperature. Then, the incubation of the anti-human primary antibody TAS1R3 (rabbit) (Ref sc50353, SCBT), in 0.2% BSA, dissolved in 1×PBS in the 1:1000 dilution, incubating the plate for one hour at room temperature was carried out. The primary antibody was then washed, and the anti-rabbit secondary antibody (111-545-144 Jackson Immunoresearch) was applied, dilution 1:500 in BSA 0.2% PBS1×, which is labeled with the fluorophore (Alexa Fluor 488). At the same time, Hoechst 33342 (ThermoFisher®) (dilution 1:1000) was added and incubated for one hour, protecting the fluorophore from possible light degradation. Once the incubation time had passed, it was washed again with PBS1× three times under stirring, to then remove the walls of the micro-chamber, apply the Mowiol mounting medium (Calbiochem) and place the coverslip on the sample. It was left overnight to dry at room temperature protected from the dark, and the next day it was observed under a confocal microscope (Leica SP8® Confocal Laser Microscope.
The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.
These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 18382013.3 | Jan 2018 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/050981 | 1/15/2019 | WO | 00 |
