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    • 专利摘要:
    Use of a gnp-llo complex91-99 for the treatment and prevention of cancer. The present invention relates to the use of a gnp-llo complex91-99 to provide immune responses for the treatment or prophylaxis of cancer. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2633932A1
    申请号:ES201600160
    申请日:2016-02-24
    公开日:2017-09-26
    发明作者:Carmen ÁLVAREZ DOMINGUEZ；Ricardo CALDERÓN GONZÁLEZ；Elisabet FRANDE CABANES；Eva FERRÁNDEZ FERNÁNDEZ；Sonsoles YÁÑEZ DIAZ；Soledad PENADÉS ULLATE；Marco MARRADI；Isabel GARCÍA MARTÍN
    申请人:Servicio Cantabro De Salud；Fundacion Instituto de Investigacion Marques de Valdecilla；
    IPC主号:
    专利说明:

    The present invention relates to the use of a GNP-LL091 _99 complex to provide immune responses for the treatment or prophylaxis of cancer. STATE OF THE TECHNIQUE
    The natural ability of the immune system to detect and destroy abnormal cells could prevent the formation of many types of cancer. However, some cancers manage to avoid being detected and destroyed by the immune system, due to the production of signals that reduce the ability of the immune system to detect and destroy tumor cells, or they may have modifications that make it more difficult than the system Immune recognize them and attack them.
    In this sense, immunotherapies are treatments that restore or intensify the ability of the immune system to fight cancer. In just a few years, the rapid advance of the discipline of cancer immunology has produced several new methods to treat it that increase the potency of immune responses against tumors. These therapies stimulate the activities of specific components of the immune system or counteract the signals produced by cancer cells that suppress immune responses.
    Particularly, the so-called cancer vaccines or therapeutic vaccines, are designed to treat existing cancers by strengthening the body's natural defenses to fight cancer. Vaccines for treatment can act in several ways:
    • delay or stop the growth of cancer cells;
    • cause tumor reduction;
    • prevent cancer from re-forming; or
    • Eliminate cancer cells that have not been destroyed with other forms of treatment.
    The formulation of effective cancer treatment vaccines requires detailed knowledge of how immune system cells and cancer cells interact. To be effective, cancer treatment vaccines must stimulate specific immune responses to the correct target. Immune responses must also be strong enough to break through the barriers cancer cells use to protect themselves from attacks by B cells and cytolytic T cells.
    On the other hand, the attention of gold nanoparticles (AuNPs) has recently grown, especially in terms of their applications in biomedicine, such as cancer treatment and immunization by nano-vaccines. The interest for this type of nanoparticles is given by its ability to penetrate blood vessels and tissue barriers and to be directed to a specific cell by means of specifically hoisted functional molecules (Cristina Popescu, R. et al. Current topies in medicinal chemistry, 2015, 15 (15), 1532-1542.)
    Recently, gold gluconanoparticles (Au-GNP) loaded with the peptide from Listeria monocytogenes listeriolysin O (LL091-99) have been described in order to obtain an effective vaccine against the human pathogen of Listeria monocytogenes (Listeria). Additionally, in order to improve the immunogenicity of the vaccine, a new Advax ™ adjuvant was used, a polysaccharide of natural plant origin that, when crystallized in the delta polymorphic form, becomes immunologically active (Rodríguez-Del Rio E. et al. Vaccine, 2015, 33, 1465-1473). DESCRIPTION OF THE INVENTION
    The object of the invention is to provide an effective vaccine in the treatment and / or prevention of cancer. The therapeutic vaccines of the present invention consist of gold nanoparticles comprising peptide LL091-99. These nanoparticles (GNP-LL091 .99) achieve a significant tumor regression of 90%, and prevent metastatic formation in the lungs by more than 95%.
    Several therapies were tested against murine mela noma in order to provide comparative data demonstrating the improved technical effect of the vaccine of the invention against other antitumor therapies described above.
    In this sense, different Listeria monocytogenes peptides have been used, among which are peptide 91-99 of listeriolysin O (LL091-99) and peptide 1-22 of glyceraldehyde-3-phosphate dehydrogenase (GAPDH1_22) , nanoparticles (gold nanoparticles synthesized with different Listeria monocytogenes peptides),
    or dendritic cells loaded with a Listeria monocytogenes LL091 _ peptide
    99) '
    In this way, it has been found that gold nanoparticles alone do not achieve any therapeutic effect on melanoma. Nor is the LL091 -99 peptide only as therapy. However, this peptide synthesized in the nanoparticles (GNPLL091-99) does achieve a significant tumor regression (Figure 1) and also prevents the formation of metastases in the lungs (Figure 2).
    On the other hand, this therapeutic effect is specific to the LL091 -99 peptide of listeriolysin O of Listería monocytogenes, because other nanoparticles synthesized by conjugation to another peptide of another virulence factor of Listería monocytogenes such as the GAPDH1-22 peptide, glyceraldehyde-3 -phosphate dehydrogenase, does not present this therapeutic activity against mela noma
    Therefore, in a first aspect, the present invention relates to the use of a nanoparticle comprising a core of gold atoms to which at least covalently bind:
    to. a first ligand comprising a peptide 91-99 of listeriolysin O from Listería monocytogenes; Y
    b. a second ligand comprising a group of carbohydrates, for the manufacture of a medicament for the treatment and / or prevention of cancer.
    In the present invention, the LL091-99 peptide is the restricted-H2-Kd epitope of listeriolysin O (LLO), and consists of amino acids 91 to 99 (H-Gly-Tyr-Lys-Asp-GlyAsn-Glu-Tyr- Ile-OH).
    In a preferred embodiment, the carbohydrates of the second ligand are selected from the list comprising N-acetylglucosamine (GlcNAc), glucose, mannose, xylose, or fructose. In a more preferred embodiment, this second ligand comprises glucose.
    In another preferred embodiment, the particles may have an average number of total ligands attached to the gold metal core of at least one peptide-like ligand and one of carbohydrate-like ligand, in total two ligands. More preferably of 20 ligands, more preferably of 50 ligands, more preferably of 60 ligands and, even more preferably, of 70 ligands (Red et al., ChemBiochem 2004, 5, 291297). In this sense, the glucose: peptide ratio can be between 8: 1 and
    10: 1 and more preferably between 9: 1.
    This glucose / peptide ratio of the nanoparticle of the invention is quantitatively determined through 1 H NMR spectra of the 500 MHz nanoparticle solutions to identify signals unambiguously belonging to the individual components of each ligand confirming that the intensity of these signals It corresponds to those provided according to the proportion of the different ligands in the original solution.
    In a preferred embodiment, the nanoparticle populations may have different densities of the ligands bound to the nucleus.
    In another preferred embodiment, the gold nanoparticles loaded with the LL091_99 peptide have a nanometric size, which allows their capture by the cells and, therefore, the presentation of the antigen on the cell surface.
    Preferably, these nanoparticles have gold nuclei with average diameters between 1.5 and 2.5 nm, and more preferably between 1.8 and 2.0 nm (Rodríguez-Del Rio E. et al. Vaccine, 2015, 33 , 1465-1473) (Figure 4). The average diameter of the gold nuclei can be measured using techniques well known in the art such as a transmission electron microscope. When considering, in addition to the gold core, the carbohydrate ligand (gluconanoparticle, GNP), the total average diameter of the particles is between 4.5 and 5.5 nm, and finally the size of the nanoparticle when incorporating the peptide ligand ( GNP-LL091 -99) increases the average diameter between 8 and 10 nm.
    Thus, in a preferred embodiment, the size of the nanoparticle of the invention, which consists of a gold gluconanoparticle loaded with the peptide LL091-99 (GNP-LL091_99). It is in the range of 12.5 to 15.5 nm.
    In another preferred embodiment, the nanoparticles of the invention are water soluble. This can be used in its purification and, importantly, it means that they can be used in solution to present the immobilized ligand on the surface of the particle. The fact that the nanoparticles are soluble has the advantage that the ligands are presented in a natural conformation. For therapeutic applications, the nanoparticles are not toxic, they are soluble and stable under physiological conditions.
    In another preferred embodiment, the nanoparticle can also comprise a label, such as a fluorescent or fluorophore group, a radionuclide, a magnetic marker, a dye, an active NMR atom or an atom that is capable of being detected using surface plasmon resonance. Between the markers
    Preferred magnetic groups include paramagnetic groups comprising Mn +, Gd + 3, Eu + 2, Cu + 2, v + 2, Co + 2, N¡ + 2, Fe + 2, Fe + 3 or lanthanides + 3. Preferred NMR active atoms include Mn + 2, Gd + 3, Eu + 2, Cu + 2, v + 2, Co + 2, Ni + 2, Fe + 2, Fe + 3 or
    lanthanides + 3.
    "Fluorophore" means any compound that can emit a light signal
    or colored after her excitement. Examples of fluorophores are, but are not limited to: fluorescein, rhodamine, coumarin, cyanine, and their derivatives, GFP, YFP YRFP, Oregon green, eosin, Texas red, naphthalene derivatives, coumarin derivatives, oxadiazole derivatives, pyrene derivatives , oxacin derivatives (such as Nile red or Nile blue), acridine derivatives, tetrapyrrole derivatives, Alexa Fluorine, DyLight Fluorine, CY5, T AMRA, JOE or biotin.
    The nanoparticles of the invention were prepared according to synthetic methodology already known in the state of the art (Rodríguez-Del Rio, E. et al. Vaccine, 2015, 33 (12), 1465-1473), according to which nuclei are used which they comprise a gold atom in which derivatized ligands with disulfide linkers that react with HAuCI4 (tetrachloric acid) are used, in the presence of reducing agent to produce the nanoparticles (Example 1).
    In a particular embodiment, a nanoparticle loaded with the 9199 peptide of listeriolysin O (LL091-99) was designed for prophylactic and / or therapeutic vaccination against melanoma.
    In a preferred embodiment, the present invention relates to the use of the nanoparticles of the invention, as described above, for the manufacture of a medicament for the treatment and / or prevention of melanoma.
    In another preferred embodiment, that medicament is a therapeutic and / prophylactic vaccine.
    In the present invention, the term "vaccination" includes an active immunization, that is, an induction of a specific immune response due to administration, e.g. ex. by subcutaneous, intradermal, intramuscular, oral or nasal routes, of small amounts of the peptide that is recognized by the vaccinated individual as foreign and is therefore immunogenic in a suitable formulation.
    In another preferred embodiment, the nanoparticles of the invention can be attached to a third type of ligand consisting of an adjuvant, or this adjuvant can be part of a pharmaceutical composition together with the nanoparticles as described above.
    In the present invention, the term "adjuvant" refers to an agent, as long as it does not possess an antigenic effect by itself, which can stimulate the immune system by increasing its response to the vaccine. For example, peptides or carbohydrates that stimulate collaborating T cells that stimulate the innate immune network. When additional adjuvants are used, the invention allows the use of a single administration vehicle for administering both the antigen and the adjuvants, or multiple antigens or adjuvants.
    In another preferred embodiment, the nanoparticles described herein can be formulated in pharmaceutical compositions useful in the treatment and / prevention of cancer, preferably melanoma.
    The administration guidelines for these pharmaceutical compositions include the enteral or parenteral routes. Parenteral administration includes administration by the following routes: intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraocular, transepithelial, intraperitoneal and topical (including dermal, ocular, rectal, nasal, inhalation and aerosol) and rectal systemic routes. More preferably, the nanoparticles of the invention or the pharmaceutical compositions comprising them are administered intravenously, cutaneously, subcutaneously, or intraperitoneally.
    In another preferred embodiment, the pharmaceutical compositions may be in the forms of solid or liquid compositions. These compositions generally comprise a vehicle of a certain type, for example, a solid vehicle such as gelatin or an adjuvant or an inert diluent, or a liquid vehicle such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. These compositions and preparations generally contain at least O, 1% by weight of the compound.
    For intravenous, cutaneous or subcutaneous injection, or injection at the site of the ailment, the active substance will be in the form of an aqueous solution acceptable parenterally that does not contain pyrogens and has the appropriate pH, isotonicity and stability. Those skilled in the art are well trained to prepare suitable solutions using, for example, solutions of the compounds or a derivative thereof, e.g. eg, in physiological saline solution, a dispersion prepared with glycerol, liquid polyethylene glycol or oils.
    In addition to the nanoparticles of the invention, the pharmaceutical compositions may comprise one or more of an excipient, carrier, buffer, stabilizer, isotonic agent, preservative or antioxidant, or other pharmaceutically acceptable materials well known to those skilled in the art. These materials must be non-toxic and not interfere with the effectiveness of the active substance. The precise nature of the vehicle or other material may depend on the route of administration.
    Liquid pharmaceutical compositions are typically formulated to have a pH between about 3.0 and 9.0; more preferably between about 4.5 and 8.5 and, even more preferably, between about 5.0 and 8.0. The pH of a composition can be maintained by using a buffer such as acetate, citrate, phosphate, succinate, Tris or histidine, typically using in the range of about 1 mM to 50 mM. The pH of the compositions can be adjusted on the other hand using physiologically acceptable acids or bases.
    Generally, preservatives are included in pharmaceutical compositions to retard microbial growth, extending the life of the compositions and allowing multiple use of the presentation. Examples of preservatives include phenol, metacresol, benzyl alcohol, para-hydroxybenzoic acid and its esters, methylparaben, propylparaben, benzalkonium chloride and benzetronium chloride. Typically, preservatives are used in the range of about 0.1 to 1.0% (w / v).
    Preferably, the pharmaceutical compositions are administered to an individual in a prophylactically effective amount or a therapeutically effective amount (as the case may be, prevention or treatment), this being sufficient to show a benefit to the individual.
    In the sense used in this description, the term "therapeutically effective amount" refers to that amount of the component of the pharmaceutical composition that when administered to a mammal, preferably a human, is sufficient to produce prevention and / or treatment. , as defined below, of a disease or pathological condition of interest in the mammal, preferably a human. The therapeutically effective amount will vary, for example, according to the age, body weight, general state of health, sex and diet of the patient; the mode and time of administration; the rate of excretion, the combination of drugs; the severity of the particular disorder or pathological condition; and the subject undergoing therapy, but can be determined by a specialist in the art according to his own knowledge.
    The term "excipient" refers to a substance that aids the absorption of any of the components of the product of the invention, stabilizes said components or aids in the preparation of the pharmaceutical composition in the sense of giving it consistency or providing flavors that do so. nicer. Thus, the excipients could have the function of keeping the components together such as starches, sugars or cellulose, sweetening function, dye function, drug protection function such as to isolate it from air and / or moisture, function filling a tablet, capsule or any other form of presentation such as dibasic calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph. Therefore, the term "excipient" is defined as that matter which, included in the galenic forms, is added to the active principles or to their associations to enable their preparation and stability, modify their organoleptic properties or determine the physicochemical properties of the Pharmaceutical composition and its bioavailability. The "pharmaceutically acceptable" excipient must allow the activity of the compounds of the pharmaceutical composition, that is, to be compatible with said components. Examples of excipients are binders, fillers, disintegrators, lubricants, coatings, sweeteners, flavorings and colorizers. More specific non-limiting examples of acceptable excipients are starches, sugars, xylitol, sorbitol, calcium phosphate, steroid fats, talc, silica or glycerin among others.
    The "galenic form or pharmaceutical form" is the provision to which the active ingredients and excipients are adapted to constitute a medicament. It is defined by the combination of the way in which the pharmaceutical composition is presented by the manufacturer and the way in which it is administered.
    A "vehicle" or carrier is preferably an inert substance. The function of the vehicle is to facilitate the incorporation of other compounds, allow a better dosage and administration or give consistency and form to the pharmaceutical composition. Therefore, the carrier is a substance that is used to dilute any of the components of the pharmaceutical composition of the present invention to a given volume or weight; or that even without diluting said components it is capable of allowing a better dosage and administration or giving consistency and form to the medicine. It's vehicle is pharmaceutically acceptable. When the form of presentation is liquid, the pharmaceutically acceptable carrier is the diluent.
    In each case the form of presentation of the medicament will be adapted to the type of administration used, therefore, the composition of the present invention can be presented in the form of solutions or any other form of clinically permitted administration and in a therapeutically effective amount. The pharmaceutical composition of the invention can be formulated in solid, semi-solid, liquid or gaseous forms, such as tablet, capsule, powder, granule, ointment, solution, suppository, injectable, inhalant, gel, syrup, nebulizer, microsphere or aerosol, preferably in the form of a tablet, capsule, powder, granule, solution, suppository or syrup.
    The above mentioned compositions may be prepared using conventional methods, such as those described in the Pharmacopoeias of different countries and in other reference texts.
    In a preferred embodiment, the pharmaceutical composition of the invention may comprise another active substance. In addition to the requirement of therapeutic efficacy, where said pharmaceutical composition may require the use of other therapeutic agents, there may be additional fundamental reasons that compel or strongly recommend the use of a combination of a compound of the invention and another therapeutic agent. The term "active principle" is any matter, whatever its origin, human, animal, plant, chemical or other, to which an appropriate activity is attributed to constitute a medicine.
    In another preferred embodiment, the nanoparticles of the invention, or the pharmaceutical compositions that comprise them, can be used together with other medicaments in combination therapies. The other drugs may be part of the same composition or of a different composition, for administration at the same time or at different times.
    In a preferred embodiment, the compositions of the invention are preferably administered to patients at a dose of between 0.25 mg and 5.0 mg of active ingredient per kg of body weight and day, and more preferably, between 0.5 mg and 2.5 mg / kg / day.
    Additionally, the described nanoparticles of the invention can be used alone or in combination with current therapies for the treatment of cancer, as an example: surgery, radiation, chemotherapy. Similarly, the GNP-LL091 _99 described prophylactically administered lead to a rapid innate immune response against the tumor, with a subsequent development of a specific antigen adaptive immune response, emphasizing its use in prophylactic or therapeutic vaccines against cancer.
    The term "cancer", as used in the present description, refers to the neoplastic disease in which the cells, of abnormal morphology, have an uncontrolled growth reaching to generate a tumor. Examples of cancer include, but are not limited to, liver or hepatocarcinoma, prostate, lung, pancreatic, colon, breast, gynecological cancers, such as ovarian, uterus, cervix, vagina or vulva, skin cancer, such as melanoma, esophageal cancer, gastric cancer, bladder cancer, urinary tract cancer, thyroid cancer, kidney cancer, brain cancer, sarcoma, lymphoma or leukemia.
    The term "melanoma", as used in the present invention, refers to any tumor resulting from the proliferation of melanocytes that appear predominantly in the skin but also in the eye or intestine and includes, without limitation, melanomas, metastatic melanomas, melanocarcinomas, melanoepitheliomas, melanosarcomas, mela noma in situ, superficial mela noma that extends superficially, modular melanoma, malignant lentigo noma mela, acral lentiginous noma mela, invasive melanoma, familial atypical mole and melanoma syndrome. Preferably cutaneous noma mela, noma in situ mela, superficially extending noma mela, inasic noma mela and metastatic noma mela.
    Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention. BRIEF DESCRIPTION OF THE FIGURES
    FIG. 1. Comparative effect of different therapies against murine melanoma after tumor establishment for 7 days subcutaneously in the left ventral area and subsequent inoculation into the tumor with different therapies for 5 days (1A). After these treatments, the mice were sacrificed and the tumors recovered, their size being measured with a caliber (1 B).
    FIG. 2. Metastasis in lung of the murine mela noma. After treatment with different therapies, the mice were sacrificed and the lungs were removed, which are washed with PBS. Tumor nodules were counted under a magnifying glass that are seen as black spots in the lungs.
    FIG. 3. General scheme of the GNP-LL091-99 synthesized (3A). Chemical structure of the nanoparticle with the gold core and the two ligands, the ~ -D-glucose and the LL091-99 (3B) peptide _
    FIG. 4. Image of electron transmission microscopy (100,000 x magnification) (4A). Histogram of the nanometric size of the gold core of GNP-LL091_99 (4B).
    FIG. 5. Comparative effect of different therapies in neonates against the murine mela noma after the establishment of the tumor for 5 days subcutaneously to 9-day-old female mice of the Balb / c strain and subsequent inoculation into the tumor with different therapies for 5 days . After these treatments, the mice were sacrificed and the tumors recovered, their size measured with a caliber. EXAMPLES
    The invention will now be illustrated by tests carried out by the inventors, which demonstrates the effectiveness of the product of the invention.
    Example 1. Preparation of gold nanoparticles synthesized with LL0lU peptides: ~ (GNP-LL091-99) or GAPDH1-22 (GNP-GAPDH1_221
    Peptides LL091-99 and GAPDH1_22 with a C-terminal cysteamide, LL091_ 99C (O) NHCH2CH2SH or GAPDH1_22C (O) NHCH2CH2SH (1 mg, 0.85 IJmol, 95% purity) are commercially purchased from GenScript and together with 5- (mercapto) pentyl-a-Dglucopyranoside GlcC5SH (2.1 mg, 7.4 IJmol) is dissolved in deuterated water (750 IJL). The 1H NMR analysis of the mixture shows a Glc: LL091_99 -9: 1 ratio (eg 90% of Glc with respect to 10% of LL091-99 or GAPDH1_d. These prepared solutions of the ligands (0.011 M, 4 equivalences) are added to an aqueous solution of HAuCI4 (100 IJL, 0.025 M, 1 equivalence) followed by an aqueous solution of NaBH4 (67.5 IJL, 1 M, 27 equivalents) in four portions under rapid stirring (Ufoaming '). The dark dispersion is Stir for 2h and filter using 3 KDa MWCO membranes by filtration centrifugation.The black colloid is recovered with water and lyophilized (0.482 mg) The residue is redispersed in a minimum volume of water, loaded with a SnakeSkin dialysis membrane (Pierce, 10KDa MWCO) and dialyzed against 3 L of water with gentle agitation and recharged with fresh dialyzed water every 8 hours for a total of 72. After lyophilization, 0.456 mg of GNP-LL091-99 is obtained (Figure 3) or GNP-GAPOH1_22.
    The glucose / peptide ratio in the GNP is quantitatively determined by NMR (qNMR) on a Bruker AVANCE 500 MHz spectrometer: 0.456 mg of GNP-LL091-99 and GNPGAPOH1-22 are dispersed in 020 99.9% (200 ... IL). 80 ¡..IL of these solutions are added to 40¡IL of 0.05% of sodium salt solution of 3- (trimethylsilyl) propionic acid2,2,3,3-d4 (TSP-d4) at 020 and 60 ¡..IL of 020. The 1H-NMR analysis of the mixture allows the calculation of the amount of peptide in GNP-GAPOH1_22 and GNP-LL091-99: 8.9¡ ..Ig (LL091_99) / O, 182 mg (nanoparticle) UVNis (Beckman Coulter OU 800 spectrometer, H20, 0.1 mg / mL). A typical surface plasmon band is observed at 520 nm. TEM (JEOL JEM-2100F running at 200 kV): a single drop (5¡IL) of the aqueous dispersion (ca. 0.1 mg mL-1 in MilliQ water) of the GNPs is placed on a copper grid covered with an ultrafine carbon film (Electron Microscopy Sciences). The rack is allowed to air dry for 12 hours at room temperature.
    Example 2. Comparative study of tumor size reduction between the therapy of the invention and known antitumor therapies
    For the study of tumor size reduction using different therapies and their comparison with that of the invention, 1 x 106 B16F10 murine melanoma cells were inoculated subcutaneously in the left ventral flank of female mice of 8-12 weeks of age of strain C57BU6.
    After 7 days post-inoculation, 50¡Ig / ml of the following therapies were inoculated into the tumors for 5 days: LL091-99 (listeriolysin O peptide only from Listeria monocytogenes, LL091-99), GNP (empty gluconanoparticles ), GNP-LL091-99 (gluconanoparticles synthesized with the listeriolysin O peptide of Listería monocytogenes, LL091-99), GNP-GAPOH1_22 (nanoparticles synthesized with the glyceraldehyde-3-phosphate peptide dehydrogenase of Listería monocytogenes), GAPOH1_22
    or no particle (NT). Also included, as a control, were a vector previously used in therapy, dendritic cells loaded with the LL091-99 peptide (1 x106 OC-LL091-99).
    After this treatment, the mice were sacrificed and the tumors recovered, their size measured with a caliber. The results are shown in Figure 1.
    Conclusions:
    • 10 nm gold nanoparticles by themselves do not achieve any therapeutic effect on melanoma, as previously described
    (ChemBioChem 2004, 5, 291-297);
    • The LL091-99 peptide alone did not achieve any therapeutic effect on melanoma;
    • The LL091 -99 peptide synthesized in the nanoparticles, GNP-LL091_99, does achieve a significant tumor regression of 90% (Figure 1);
    • Other nanoparticles synthesized by conjugation to another peptide of another virulence factor of Listeria monocytogenes such as the GAPDH1_22 peptide, glyceraldehyde-3-phosphate dehydrogenase does not exhibit this therapeutic activity against melanoma.
    Example 3. Comparative study of metastasis inhibition between the therapy of the invention and known antitumor therapies
    For the study of lung metastases in murine melanoma after the use of different therapies and their comparison with that of the invention, 1 x 106 B16F10 murine melanoma cells were inoculated subcutaneously as in Figure 1. After 7 days post- inoculation, they were inoculated in the tumors 50) .Jg / ml of the following therapeutic particles for 5 days: GNP (empty gluconanoparticles), GNP-LL091 _ 99 (gluconanoparticles synthesized with the listeriolysin O peptide of Listeria monocytogenes, LL091-99 ), GNP-GAPDH1_22 (nanoparticles synthesized with the Listeria monocytogenes glyceraldehyde-3-phosphate dehydrogenase peptide, GAPDH1-22)
    or no particle (NT). Also included as a control were the vector previously used in therapy consisting of dendritic cells loaded with the peptide LL091-99 (1 x106 DC-LL091-99).
    After this treatment, the mice were sacrificed and the lungs were removed, which were washed with PBS. Subsequently, the tumor nodules were counted under a magnifying glass that are observed as black spots in the lungs.
    Figure 2 shows that LL091 _99 peptide synthesized in the nanoparticles, GNP-LL091-99, prevents metastatic formation in the lungs by more than 95%.
    Example 4.-Comparative study of tumor size reduction in neonates between the invention therapy and known anti-tumor therapies.
    For the study of tumor size reduction in neonates using different therapies and their comparison with that of the invention, 5 x 105 B16F10 murine melanoma cells were inoculated subcutaneously in the intra-scapular area of female mice of 9 days old of the strain Balb / c.
    After 5 days post-inoculation, 50 ~ g / ml of the following therapies were inoculated into the tumors for 5 days: LL091-99 (Listeria monocytogenes listeriolysin O peptide, LL091-99), GNP (empty gluconanoparticles), GNP-LL091 _99 (gluconanoparticles synthesized with the listeriolysin O peptide of Listeria monocytogenes, LL091-99), GNP-GAPDH1_22 (nanoparticles synthesized with the glyceraldehyde-3-phosphate dehydrogenase peptide of Listeria monocytogenes2, GAPDH1-2). Also included, as a control, were a vector previously used in therapy, dendritic cells loaded with the peptide LL091-99 (1 x105 DC-LL091-99).
    After this treatment, the mice were sacrificed and the tumors recovered, their size measured with a caliber. The results are shown in Figure 5.
    Conclusions:
    • The 10 nm gold nanoparticles by themselves do not achieve any therapeutic effect on melanoma, nor in neonates of another mouse strain such as Balb / c,
    • The LL091 -99 peptide alone did not achieve any therapeutic effect on melanoma;
    • The LL091 -99 peptide synthesized in the nanoparticles, GNP-LL091_99, does achieve a significant tumor regression in infants of 95% (Figure 5);
    • Other nanoparticles synthesized by conjugation to another peptide of another virulence factor of Listeria monocytogenes such as the GAPDH1-22 peptide, glyceraldehyde-3-phosphate dehydrogenase do not exhibit this therapeutic activity in neonates against melanoma.
    权利要求:
    Claims (9)
    [1]
    1. Use of a GNP-LL091 .99 complex comprising a core of gold atoms to which at least covalently bind:
    to. a ligand consisting of a peptide 91-99 of listeriolysin O from Listeria monocytogenes; Y
    b. a second ligand consisting of a group of carbohydrates, for the manufacture of a medicament for the treatment and / or prevention of
    Cancer.
    [2]
    2. Use according to claim 1 wherein the GNP-LL091 .99 complex comprises glucose ligands.
    [3]
    3. Use according to claim 2 wherein the GNP-LL091 .99 complex comprises ~ -D-glucose ligands.
    [4]
    Four. Use according to any of the preceding claims, wherein the medicament is a therapeutic and / or prophylactic vaccine.
    [5]
    5. Use according to any of the preceding claims, wherein the cancer is melanoma.
    [6]
    6. Use according to claims 1 to 5, wherein the GNP-LL091 .99 complex is administered in a dose of between 0.25 and 5.0 mg / kg / day.
    [7]
    7. Use according to claim 6, wherein the GNP-LL091-99 complex is administered in a dose comprised between 0.5 and 2.5 mg / kg / day.
    [8]
    8. Use according to any of the preceding claims, wherein the GNP-LL091 -99 complex is in a form suitable for administration by a route selected from intravenous, cutaneous, subcutaneous, nasal, intramuscular, intraocular, transepithelial, intraperitoneal, topical, and systemic rectal pathways.
    [9]
    9. Use according to claim 8, wherein the route of administration is selected from intravenous, cutaneous, subcutaneous, or intraperitoneal.
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    h
    ~ X: peptides, fluorides,
    ~, ~ r // ~ / M ~~, ~
     ~ SU, {/ 1. /
    :; :::! d ~ f: .-- ~
     ~! f ~
    ~
    B
    LL091-99 Peptide
    Do you
    COOH _ .. - NH COOH
     O ~; '- INH
    O o
     (JO h
    ~ -D-glucose (Glc)
    '"OH
     qN) -N ~
    Ha - = --- <-_ O OH s
     HO ~ O ~ s
    OH
    FIG. 3
    TO
    B
    1 00
    IIC '¡j'
    to:
    "AND
    or i
    or
    C
     FIG. 4 
    THERAPIES IN NEO NATOS
    GNP-LLOg1 • GNP LL0GNP-GAPDHDC-LL0
    99 91 • 99 1 • 22 91 • 99
    FIG. 5
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    同族专利:
    公开号 | 公开日
    US20190046627A1|2019-02-14|
    WO2017144762A1|2017-08-31|
    ES2633932B1|2018-07-30|
    EP3421045A1|2019-01-02|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2004014957A1|2002-08-12|2004-02-19|The Council Of The Queensland Institute Of Medical Research|Novel immunogenic lipopeptides comprising t-helper and cytotoxic t lymphocyte epitopes|
    WO2006099448A2|2005-03-14|2006-09-21|University Of Iowa Research Foundation|Accelerated cd8+ t-cell memory after dendritic cell vaccination|
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    ES201600160A|ES2633932B1|2016-02-24|2016-02-24|Use of a GNP-LLO91-99 complex for the treatment and prevention of cancer.|ES201600160A| ES2633932B1|2016-02-24|2016-02-24|Use of a GNP-LLO91-99 complex for the treatment and prevention of cancer.|
    EP17715958.9A| EP3421045A1|2016-02-24|2017-02-23|Use of a gnp-llo91-99 complex for the treatment and prevention of cancer|
    PCT/ES2017/070103| WO2017144762A1|2016-02-24|2017-02-23|Use of a gnp-llo91-99 complex for the treatment and prevention of cancer|
    US16/079,477| US20190046627A1|2016-02-24|2017-02-23|Use of a gnp-llo91-99 complex for the treatment and prevention of cancer|
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