CN109504652B - Method for promoting biological interaction and application - Google Patents
Method for promoting biological interaction and application Download PDFInfo
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- CN109504652B CN109504652B CN201811450846.XA CN201811450846A CN109504652B CN 109504652 B CN109504652 B CN 109504652B CN 201811450846 A CN201811450846 A CN 201811450846A CN 109504652 B CN109504652 B CN 109504652B
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
- C07H13/12—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by acids having the group -X-C(=X)-X-, or halides thereof, in which each X means nitrogen, oxygen, sulfur, selenium or tellurium, e.g. carbonic acid, carbamic acid
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
The invention provides a method for promoting the interaction of organisms and application thereof, which relate to the technical field of biochemistry, and comprise the steps of enabling the organisms needing to interact to be respectively metabolically modified with basic metabolic substances of click chemical groups capable of carrying out pairing reaction to obtain the organisms with the click chemical groups and the organisms with the click chemical groups capable of carrying out pairing reaction with the click chemical groups, and promoting the interaction of the organisms through the pairing reaction among the click chemical groups. The method can make organism tolerant better without changing its fundamental activity characteristic; meanwhile, the distance between the organisms is rapidly shortened through the bioorthogonal reaction between the organisms, the specific surface recognition of the two organisms is realized, and the interaction between the organisms is further promoted. The method provided by the invention is more direct and simple to operate; the reaction condition is mild, and the toxicity is low; meanwhile, the method has good reproducibility and is suitable for popularization and application.
Description
Technical Field
The invention relates to the technical field of biochemistry, in particular to a method for promoting interaction of organisms and application thereof.
Background
"cell recognition" is the recognition and identification of one biological cell from another, and is associated with the development of many important diseases, for example, the recognition of host cells by pathogenic bacteria cells, which are only recognized to infect and cause disease. Meanwhile, cell recognition is also related to the treatment of a plurality of serious diseases, for example, the recognition of tumor cells by immune cells in immunotherapy and the like, so that the enhancement of cell recognition plays an important role and has research significance for the treatment of a plurality of diseases.
Cell-to-cell recognition involves signaling through selective interaction of cell surface receptors or ligands with other cell surface ligands or receptors, resulting in a series of physiological and biochemical reactions. Regardless of the type of recognition system, there is a common fundamental property, either selectivity or specificity.
However, natural recognition of cells is easily interfered by complex microenvironment or other factors in the body, for example, the pressure of tumor cells to the immune system of the body, and the antigen expression, MHC-1 molecule, p-microglobulin and the like on the surface of cell membrane are gradually and selectively lost, so that the antigen presentation can be reduced in the initial stage of immunity, and the specific cytotoxic effect in the immune effect stage can be weakened. In addition, due to the existence of immune-critical mechanisms, tumor cells are gradually insensitive to cytokines secreted by immune effector cells such as interferon during the process of progression. These phenomena suggest that the immune system has a plastic effect on tumors while resisting tumors, i.e., the immune selective pressure is applied to tumor cells, so that the weakly immunogenic tumor cells can escape and grow further.
Although the current click chemical group modifies monosaccharide or choline analogue and realizes the modification of cell surface through the metabolic pathway of the substance in cells, the click chemical group is modified on the surface of tumor by the method in various documents, and the tumor targeting effect of chemical reagents such as nano diagnosis and treatment integrated reagent, molecular probe and the like is realized by the modification of pairing group. However, this technique has not been practically applied to the recognition of two kinds of cells, particularly in the treatment of immune cells. The bridging bond of the click group proposed by Seok Hyun Yun project group can realize the combination of two cells, but the length of the bridging bond can not ensure the distance between two cells to be properly recognized (as shown in fig. 1A and fig. 1B), and the method is relatively complicated. For this reason, it is important to develop a method capable of improving the recognition between organisms and further promoting the interaction between organisms.
Disclosure of Invention
It is a first object of the present invention to provide a method for facilitating biological interaction that alleviates at least one of the technical problems of the prior art.
The second purpose of the invention is to provide the application of the method for promoting the interaction of organisms in the preparation of the medicine for enhancing the immunotherapy.
The present invention provides a method of promoting biological interaction, the method comprising:
the method comprises the steps of enabling organisms needing to interact to metabolize basic metabolic substances modified with click chemical groups capable of carrying out pairing reaction respectively to obtain the organisms with the click chemical groups and the organisms with the click chemical groups capable of carrying out pairing reaction with the click chemical groups, and promoting the interaction of the organisms through the pairing reaction among the click chemical groups.
Further, the organism comprises one or more of a cell, a bacterium, or a virus;
preferably, the basal metabolic substance comprises one or more of a monosaccharide, a monosaccharide derivative, choline, a choline analogue, or an amino acid;
preferably, the monosaccharide derivative comprises mannosamine hydrochloride, glucosamine hydrochloride or galactosamine hydrochloride;
the choline analogs include dimethylethanolamine.
Further, the click chemistry group comprises an azide, an alkyne, an octyne, a diphenylcyclooctyne, or a tetrazine;
preferably, the click chemistry group comprises DBCO, DIBO, DIFO, BARAC or BCN.
Further, click chemical group modification is carried out on amino or hydroxyl of the basic metabolite, so as to obtain a basic metabolite-click chemical analogue;
wherein the basal metabolic substance is monosaccharide or a monosaccharide derivative;
preferably, click chemical group modification is carried out on amino or hydroxyl of the basic metabolite, and after the hydroxyl is blocked, the basic metabolite-click chemical analogue is obtained;
preferably, the hydroxyl group of the basal metabolite-click chemistry analogue is blocked by acetic anhydride;
preferably, the click chemistry group is BCN and the basal metabolite-click chemistry analogue has a structural formula as shown in formula (I):
wherein, in the formula (I), R1 is monosaccharide or monosaccharide derivative;
more preferably, the general structure of the basal metabolite-click chemistry analogue is as follows:
further preferably, the structure of the basal metabolite-click chemistry analogue is at least one of the following (ii) - (iv):
wherein, as in the above formulae (i) to (iv), R2-R5、R6-R9、R10-R13Each independently is hydrogen or acetyl.
Further, providing a bromo-intermediate of a click chemistry group, and reacting the bromo-intermediate of the click chemistry group with a basal metabolite to generate a basal metabolite-click chemistry analog;
wherein the basal metabolite is choline or a choline analogue.
Further, basal metabolic substances modified with click chemical groups capable of carrying out pairing reaction are co-cultured with the organism, and after the metabolism of the organism, the organism with the click chemical groups and the organism with the click chemical groups capable of carrying out pairing reaction with the click chemical groups are obtained.
Further, the organism requiring interaction is a cell;
preferably, the method for promoting the interaction of organisms comprises the following steps:
(a) incubating the first cell and a basic metabolite modified with a click chemical group in a cell culture solution to obtain a first cell with the click chemical group;
(b) co-incubating a second cell with a basic metabolite modified with a click chemical group capable of reacting in a pairing manner with the click chemical group in a cell culture solution to obtain a second cell having a click chemical group capable of reacting in a pairing manner with the click chemical group;
(c) mixing the first cell obtained in the step (a) and the second cell obtained in the step (b), and promoting the interaction of the first cell and the second cell through a pairing reaction between click chemical groups;
preferably, the basal metabolite modified with a click chemistry group is a BCN-modified monosaccharide analog, and the basal metabolite modified with a click chemistry group capable of pairing-reacting with the click chemistry group is a nitrogen-modified monosaccharide analog.
Further, the concentration of the basal metabolic substance modified with the click chemistry group is 5-50umol/L, preferably 10-40umol/L during co-incubation; the concentration of the basic metabolite modified with the click chemical group capable of reacting with the click chemical group in a pairing manner is 5-50umol/L, preferably 10-40 umol/L;
preferably, the co-incubation time is 48-96 hours, preferably 48-84 hours, more preferably 48-72 hours;
preferably, the first cell is mixed with the second cell in a cell number ratio of 1:0.2-5, preferably 1: 0.5-2.
Further, step (a) further comprises the step of removing impurities after co-incubation to obtain a first cell having a click chemistry group;
preferably, step (b) further comprises the step of removing impurities after co-incubation, resulting in a second cell having a click chemistry group capable of pairing reaction with said click chemistry group;
preferably, step (a) and step (b) are each independently cell-washed and/or centrifuged to remove impurities.
In addition, the invention also provides the application of the method for promoting the interaction of organisms in preparing the medicament for enhancing the immunotherapy.
The method for promoting the interaction of the organisms comprises the steps of enabling the organisms needing to interact to be respectively metabolically modified with basic metabolites of click chemical groups capable of carrying out pairing reaction to obtain the organisms with the click chemical groups and the organisms with the click chemical groups capable of carrying out pairing reaction with the click chemical groups, and promoting the interaction of the organisms through the pairing reaction among the click chemical groups. The method modifies basic metabolites by different click chemical groups capable of carrying out pairing reaction, realizes the expression of the click chemical groups on the surfaces of different organisms through the self metabolism of the organisms, and enables the tolerance of the organisms to be better on the basis of not changing the fundamental activity characteristics of the organisms; and then, the quick pairing reaction of click chemical groups is utilized, the distance between the organisms is quickly shortened through the bioorthogonal reaction between the organisms, the specific surface recognition of the two organisms is realized, and the interaction between the organisms is further promoted. In addition, because the biological system does not usually contain cycloalkynyl and azido, the whole reaction system only has the reaction between the organism containing cycloalkynyl and the organism containing azido, thereby ensuring the specificity. Compared with connection through a bridging bond reaction, the method provided by the invention is more direct, simple and convenient, saves the steps of intermediate reaction and is simple to operate; the reaction condition is mild, and the toxicity is lower; meanwhile, the method has good reproducibility and is suitable for popularization and application.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1A is a schematic representation of a prior art bridge connecting two cells;
FIG. 1B is a schematic representation of a prior art two cell type linkage connected by a secondary modified bridging bond;
FIG. 2 is a schematic representation of the pairing reaction of a cell having a click chemistry group and a cell having a click chemistry group capable of pairing reaction with the click chemistry group according to the present invention;
FIG. 3 is a schematic reaction scheme of example 1 of the present invention;
FIG. 4A is a laser scan of T cells metabolized on the surface with azide groups provided in example 1 of the present invention;
FIG. 4B is a laser scan of Raji cells with BCN groups metabolized on the surface as provided in example 1 of the present invention;
FIG. 4C is a laser scanning image of co-incubation of T cells metabolized to have azide groups on the surface and Raji cells metabolized to have BCN groups on the surface, as provided in example 1 of the present invention;
FIG. 4D is a micrograph of T cells metabolized to azide groups on the surface and Raji cells metabolized to BCN groups on the surface after co-incubation, which is provided in example 1 of the present invention;
FIG. 5A is a laser scan of T cells with no azide groups on their surface as provided in example 1 of the present invention;
FIG. 5B is a laser scan of Raji cells without BCN groups on their surface as provided in example 1 of the present invention;
FIG. 5C is a laser scan of co-incubation of T cells without azide groups on their surface and Raji cells without BCN groups on their surface as provided in example 1 of the present invention;
FIG. 5D is a micrograph of T cells without azide groups on their surface and Raji cells without BCN groups on their surface after co-incubation, which is provided in example 1 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The present invention provides a method of promoting biological interaction, the method comprising:
the method comprises the steps of enabling organisms needing to interact to metabolize basic metabolic substances modified with click chemical groups capable of carrying out pairing reaction respectively to obtain the organisms with the click chemical groups and the organisms with the click chemical groups capable of carrying out pairing reaction with the click chemical groups, and promoting the interaction of the organisms through the pairing reaction among the click chemical groups.
The key point of the technology is that the chemical synthesis of the molecules with different colors can be rapidly and reliably completed through the splicing of small units. In certain embodiments, click chemistry is embodied as a generalized cycloaddition reaction between an azide and an alkyne.
"click chemistry group" is sometimes used to refer to a first reactive functional group that is capable of participating in a click chemistry reaction with an appropriate second reactive functional group, where the second reactive functional group is also a click chemistry group. The first and second click chemistry groups, or entities (e.g., molecules) comprising such groups, are complementarily pairable. In certain embodiments, the click chemistry group is an alkyne or alkyne derivative capable of undergoing a cycloaddition reaction with complementary paired azide-bearing molecules and biomolecules; in another embodiment, the click chemistry group is an azide or azide derivative capable of undergoing a cycloaddition reaction with a complementary pair of an alkyne bearing molecule and a biomolecule.
The "organism" contains proteins and nucleic acids in its basic constituent, which are microscopic organisms that require the intake of nutrients from the outside and have a metabolic action.
The "basal metabolic substance" is a substance that can perform a chemical process related to physiology, such as digestion, absorption, transportation, and decomposition, in a living body, and the kind of the basal metabolic substance is not limited, and all substances that can be metabolized by a living body are the basal metabolic substances defined in the present invention.
The "interaction of organisms" may be, for example, but not limited to, recognition, targeting or killing between organisms.
The method for promoting the interaction of the organisms modifies basic metabolites by different click chemical groups capable of carrying out pairing reaction, realizes the expression of the click chemical groups on the surfaces of different organisms through the self metabolism of the organisms, and ensures that the tolerance of the organisms is better on the basis of not changing the fundamental activity characteristics of the organisms; and then, the quick pairing reaction of click chemical groups is utilized, the distance between the organisms is quickly shortened through the bioorthogonal reaction between the organisms, the specific surface recognition of the two organisms is realized, and the interaction between the organisms is further promoted. In addition, because the biological system does not usually contain cycloalkynyl and azido, the whole reaction system only has the reaction between the organism containing cycloalkynyl and the organism containing azido, thereby ensuring the specificity. Compared with connection through a bridging bond reaction, the method provided by the invention is more direct, simple and convenient, saves the steps of intermediate reaction and is simple to operate; the reaction condition is mild, and the toxicity is lower; meanwhile, the method has good reproducibility and is suitable for popularization and application.
In some preferred embodiments, the organism comprises one or more of a cell, a bacterium, or a virus.
Preferably, the basal metabolic substance comprises one or more of a monosaccharide, a monosaccharide derivative, choline, a choline analogue, or an amino acid. When the basic metabolic substance is monosaccharide, monosaccharide derivative, choline analogue or amino acid, metabolism of the substance in the organism is more easily performed, thereby exposing click chemistry groups on the surface of the organism.
Wherein, monosaccharide refers to sugar containing 3-6 carbon atoms in molecular structure, such as glyceraldehyde of three-carbon sugar; erythrose and threose of the four-carbon sugar family; arabinose, ribose, xylose, lyxose of five-carbon sugar; glucose, mannose, fructose, galactose, which are six-carbon sugars; choline (α -hydroxy-iv, iv-trimethylethanolamine) is a positively charged tetravalent base, a constituent of all biological membranes and a precursor of acetylcholine in cholinergic neurons; amino acids are compounds in which a hydrogen atom on a carbon atom of a carboxylic acid is substituted with an amino group, and amino acids contain two functional groups, an amino group and a carboxyl group, which are basic units constituting proteins.
Preferably, the monosaccharide derivative comprises mannosamine hydrochloride, glucosamine hydrochloride or galactosamine hydrochloride;
the choline analogs include dimethylethanolamine.
In some preferred embodiments, the click chemistry group comprises an azide, an alkyne, an octyne, a diphenylcyclooctyne, or a tetrazine.
Preferably, the click chemistry group comprises DBCO, DIBO, DIFO (cyclo-octyne difluoride), BARAC (biarylazacycloctynenone) or BCN ((1R,8S,9R) -bicyclo [6.1.0] non-4-alkynyl-9-ylmethanol).
In some preferred embodiments, the basic metabolite-click chemistry analog, i.e., the basic metabolite modified with a click chemistry group, is obtained by performing click chemistry group modification on an amino group or a hydroxyl group of the basic metabolite;
wherein the basal metabolic substance is monosaccharide or a monosaccharide derivative.
Preferably, the basic metabolite-click chemical analogue is obtained after blocking a hydroxyl group by performing click chemical group modification on an amino group or the hydroxyl group of the basic metabolite.
The hydroxyl of the basic metabolite-click chemical analogue is sealed, so that the basic metabolite-click chemical analogue can achieve a better cell entering effect, the recognition efficiency among organisms is improved, and the interaction among the organisms is further promoted.
Preferably, the hydroxyl group of the basal metabolite-click chemistry analogue is blocked by acetic anhydride.
In a specific embodiment, the click chemistry group is BCN and the basal metabolite-click chemistry analog has a structural formula as shown in formula (I):
wherein, in the formula (I), R1 is monosaccharide or monosaccharide derivative;
more preferably, the general structure of the basal metabolite-click chemistry analogue is as follows:
further preferably, the structure of the basal metabolite-click chemistry analogue is at least one of the following (ii) - (iv):
wherein, in formula (i), the monosaccharide ring (oxygen-containing six-membered ring) contains at least one-OAc substituent or- (C1-C4) alkylene-OAc substituent (directly bonded to a carbon atom on the ring), and each carbon atom is bonded to at most one of the above substituents; the remaining carbon atoms (except the carbon atom attached to the amino group) are linked to hydroxyl groups.
In the formulae (ii) to (iv), R2-R5、R6-R9、R10-R13Each independently is hydrogen or acetyl.
And the cellular compatibility is better due to the acetylated monosaccharide analogueThus, as in the above formula (II), R2-R5Wherein at least one R group is acetyl; more preferably, R2-R5Are all acetyl;
in the general formula (III), R6-R9Wherein at least one R group is acetyl; more preferably, R6-R9Are all acetyl;
in the general formula (IV), R10-R13Wherein at least one R group is acetyl; more preferably, R10-R13Are all acetyl groups.
In a specific embodiment, the monosaccharide is mannose and the mannose-based metabolite is used as a basis for the synthesis of the tetraacetylated BCN monosaccharide analog (Ac4Mann-BCN) by the following synthetic route:
in some preferred embodiments, a bromo intermediate of a click chemistry group is provided, and the bromo intermediate of the click chemistry group is reacted with a base metabolite to produce a base metabolite-click chemistry analog, i.e., a base metabolite modified with a click chemistry group;
wherein the basal metabolite is choline or a choline analogue.
In a specific embodiment, the bromo intermediate of the click chemistry group is obtained by reacting the click chemistry group with a halogen-substituted linear amine. Typical halogen substituted linear amines may be 2-bromoethylamine, 2-bromopropylamine or 2-bromobutylamine.
In some preferred embodiments, the basal metabolite modified with the click chemistry group capable of a pairing reaction is co-cultured with an organism, and after metabolism by the organism, an organism having the click chemistry group and an organism having the click chemistry group capable of a pairing reaction with the click chemistry group are obtained.
Through co-culture of the basic metabolite modified with the click chemical groups capable of carrying out pairing reaction and the organism, the click chemical groups can be metabolized on the surface of the organism, the click chemical groups capable of carrying out pairing reaction are exposed on the surfaces of different organisms, and therefore interaction of two organisms is achieved through bioorthogonal reaction between the click groups.
In some preferred embodiments, the organism in need of interaction is a cell. The model is shown in fig. 2.
Preferably, the method for promoting the interaction of organisms comprises the following steps:
(a) incubating the first cell and a basic metabolite modified with a click chemical group in a cell culture solution to obtain a first cell with the click chemical group;
(b) co-incubating a second cell with a basic metabolite modified with a click chemical group capable of reacting in a pairing manner with the click chemical group in a cell culture solution to obtain a second cell having a click chemical group capable of reacting in a pairing manner with the click chemical group;
(c) mixing the first cell obtained in step (a) and the second cell obtained in step (b), and promoting the interaction between the first cell and the second cell through a pairing reaction between click chemistry groups.
Typical cell culture solutions may be, for example, but are not limited to, DMEM culture solution or 1640 culture solution.
In practice, the first and second cells may be incubated for a period of time before co-incubation with the basal metabolite having click chemistry groups. For example, DMEM medium containing 10% fetal bovine serum at 37 deg.C with 5% CO2Culturing under the condition of 95% humidity, changing culture solution every other day, and subculturing once every 4-6 days. Before co-incubation, cells were washed with buffer and the medium was replaced with fresh.
Preferably, the basal metabolite modified with a click chemistry group is a BCN-modified monosaccharide analog, and the basal metabolite modified with a click chemistry group capable of pairing-reacting with the click chemistry group is a nitrogen-modified monosaccharide analog.
In some preferred embodiments, the concentration of the basal metabolite modified with click chemistry groups is 5-50umol/L, such as but not limited to 5, 10, 20, 30, 40 or 50umol/L, preferably 10-40 umol/L; when the concentration of the basic metabolite having the click chemical group is within the above range, the metabolic efficiency of the organism is higher, the recognition efficiency between organisms can be improved, and the interaction between organisms is further promoted.
The concentration of the basic metabolite modified with the click chemical group capable of reacting with the click chemical group pairing is 5-50umol/L, for example, but not limited to, 5, 10, 20, 30, 40 or 50umol/L, preferably 10-40 umol/L; when the concentration of the basic metabolite having the click chemical group that reacts with the click chemical group pairing is within the above range, the metabolic efficiency of the organism is higher, the recognition efficiency between organisms can be improved, and the interaction between organisms is further promoted.
Preferably, the co-incubation time is 48-96 hours, such as but not limited to 48 hours, 60 hours, 72 hours, 84 hours or 96 hours, preferably 48-84 hours, more preferably 48-72 hours; when the co-incubation time is within the above range, the efficiency of organism metabolism is higher, the efficiency of organism-to-organism recognition can be improved, and the organism interaction can be promoted.
Preferably, the first cell is mixed with the second cell in a cell number ratio of 1:0.2-5, such as but not limited to 1:0.5, 1:1, 1:1.5, 1:2, 1:3, 1:4 or 1:5, preferably 1: 0.5-2. When the mixing ratio of the first cell and the second cell is within the above range, the metabolic efficiency of the living body is higher, the recognition efficiency between the living bodies can be improved, and the interaction between the living bodies can be promoted.
In some preferred embodiments, step (a) further comprises the step of removing impurities after co-incubation, resulting in a first cell having a click chemistry group; the buffer solution with the pH value of 7.2-7.6 is preferably used for washing, so that redundant basic metabolites with click chemical groups can be removed, and the reaction efficiency is improved; and/or centrifuging to collect the co-incubated organisms. Typical buffers may be PBS buffer or HEPES buffer.
Preferably, step (b) further comprises the step of removing impurities after co-incubation, resulting in a second cell having a click chemistry group capable of pairing reaction with said click chemistry group; the buffer solution with the pH value of 7.2-7.6 is preferably used for washing, so that redundant basic metabolites with click chemical groups can be removed, and the reaction efficiency is improved; and/or centrifuging to collect the co-incubated organisms. Typical buffers may be PBS buffer or HEPES buffer.
In addition, the invention also provides the application of the method for promoting the interaction of organisms in preparing the medicament for enhancing the immunotherapy.
By promoting the interaction of organisms, the target recognition and the immune recognition between immune cells and target cells can be promoted, for example, the target recognition and the immune recognition between T cells and cancer cells, the target recognition and the immune recognition between macrophages and bacteria, and the target recognition and the immune recognition between natural killer cells and viruses can be promoted, so that the interaction of the organisms is promoted, and the immunotherapy is enhanced.
In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments.
Example 1
(a) Metabolism of BCN mannose analogs (Ac4Mann-BCN) in B lymphoma cells (Raji cells)
(1) Activation of BCN
Dissolving 5mmol of BCN in 100mL of anhydrous dichloromethane, adding 10mmol of pyridine, stirring for several minutes, adding 5mmol of p-nitrophenyl chloroformate (pNC), and reacting at normal temperature for 30 min;
and after the reaction is finished, adding a saturated ammonium chloride solution, extracting, drying an organic phase by using anhydrous sodium sulfate, evaporating to dryness, and separating and purifying by using silica gel column chromatography to obtain white solid BCN-pNC with the yield of 70%.
(2) And (3) synthesis of BCN-Man:
dissolving 4.4mmol of mannosamine hydrochloride in 10mL of N, N-dimethylformamide, adding 10mmol of BCN-pNC and 10mmol of pyridine, and reacting at normal temperature for half an hour;
after the reaction is finished, evaporating to dryness, and separating and purifying by silica gel column chromatography to obtain white solid BCN-Man, wherein the yield is 72%;
(3) synthesis of tetraacetylated BCN-mannose analogs (Ac4Mann-BCN)
Dissolving 5mmol of BCN-Man in 20mL of N, N-Dimethylformamide (DMF), adding 10mmol of acetic anhydride and 10mmol of pyridine, fully stirring, and reacting at normal temperature overnight;
the reaction solution was concentrated and purified by silica gel column chromatography to give Ac4Mann-BCN as a white solid with a yield of 65%.
The reaction scheme is shown in FIG. 3.
(4) 2 x 10 to5Each Raji cell was cultured in 24-well plates in DMEM medium containing 10% fetal bovine serum at 37 ℃ with 5% CO2And incubating overnight under the culture condition of 95% humidity, adding an Ac4Mann-BCN dimethyl sulfoxide solution with a certain concentration into a culture solution of Raji cells to enable the final concentration to be 25umol/L, and incubating the mixed solution in an incubator for 72 hours to enable the surface of the Raji cells to fully metabolize BCN groups. After the co-incubation is finished, the mixed solution is collected and centrifuged, cells are washed twice by PBS, centrifuged and collected, and Raji cells (BCN-Raji cells) with surface modified BCN groups are obtained.
(b) Metabolism of azide-modified mannose analogs (AC4ManAz) in immune cells (T cells)
2 x 10 to5The individual T cells were cultured in 24-well plates in DMEM medium containing 10% fetal bovine serum at 37 ℃ with 5% CO2And incubated overnight under a culture condition of 95% humidity, a solution of AC4ManAz dimethyl sulfoxide at a certain concentration was added to the culture solution of T cells to a final concentration of 25umol/L, and the mixture was incubated in an incubator for 72 hours. The azide (N3) group was metabolized well on the T cell surface. After the end of the co-incubation, the mixture was collected, centrifuged, and the cells were washed twice with PBS, centrifuged, and collected to obtain T cells (N3-T cells) with surface-modified azide groups.
(c) Interaction of two cells
BCN-Raji cells and N3-T cells were mixed in a certain ratio (1:1), and the interaction between the two cells mediated by bioorthogonal reaction was analyzed by confocal laser analysis after co-incubation of modified T cells (N3-T cells) and modified Raji cells (BCN-Raji cells), as shown in FIGS. 4A-4D and FIGS. 5A-5D. Raji cells and T cells which do not carry BCN and N3 groups are used as a control, and the result shows that N3-T cells can be rapidly gathered around the BCN-Raji cells, the combination speed is increased by more than 8 times compared with a control group, and the killing rate of the cells on the BCN-Raji cells is increased by more than 3 times.
Example 2
(a) Metabolism of DBCO glucose analogs in liver cancer cells (HepG2 cells)
(1) Synthesis of DBCO-glucose (DBCO-Glc):
dissolving 4.4mmol of mannosamine hydrochloride in 10mL of N, N-Dimethylformamide (DMF), adding 20mmol of DBCO-COOH, 20mmol of NHS and 20mmol of EDC & HCl, and reacting at normal temperature overnight; after the reaction is finished, evaporating to dryness, and separating and purifying by silica gel column chromatography to obtain white solid DBCO-Glc with the yield of 78%.
(2) Synthesis of tetraacetylated DBCO-glucose (Ac4DBCO-Glc)
5mmol of DBCO-Glc was dissolved in 20mL of N, N-Dimethylformamide (DMF), and 15mmol of acetic anhydride and 15mmol of pyridine were added thereto, followed by stirring thoroughly and reacting at room temperature overnight. The reaction solution was concentrated, and purified by silica gel column chromatography to obtain Ac4DBCO-Glc as a white solid with a yield of 70%.
(3) Will be 1 × 105Individual HepG2 cells were cultured in 24-well plates in DMEM medium containing 10% fetal bovine serum at 37 ℃ with 5% CO2And incubating overnight under the culture condition of 95% humidity, adding a DBCO glucose analogue dimethylformamide solution with a certain concentration into a culture solution of HepG2 cells to enable the final concentration to be 5umol/L, and incubating the mixed solution in an incubator for 96 hours to enable the surface of the HepG2 cells to sufficiently metabolize DBCO groups. After the co-incubation was completed, the mixture was collected, centrifuged, and the cells were washed twice with PBS, centrifuged, and collected to obtain HepG2 cells (DBCO-HepG2 cells) surface-modified with DBCO groups.
(b) Metabolism of azide-modified glucose analogs in immune cells (T cells)
Will 4 generate105The individual T cells were cultured in 24-well plates in DMEM medium containing 10% fetal bovine serum at 37 ℃ with 5% CO2And incubated overnight under a culture condition of 95% humidity, a solution of azide-modified glucose analog dimethylformamide at a certain concentration was added to the culture solution of T cells to a final concentration of 50umol/L, and the mixture was incubated in an incubator for 48 hours. The azide (N3) group was metabolized well on the T cell surface. After the end of the co-incubation, the mixture was collected, centrifuged, and the cells were washed twice with PBS, centrifuged, and collected to obtain T cells (N3-T cells) with surface-modified azide groups.
(c) Interaction of two cells
DBCO-HepG2 cells and N3-T cells are mixed according to a certain ratio (1:0.2), the interaction between the two cells is detected through a laser confocal analysis experiment provided in example 1, and the interaction is detected by taking HepG2 cells and T cells which do not carry DBCO and N3 groups as a control, so that the N3-T cells can be rapidly gathered around the DBCO-HepG2 cells, the combination speed is increased by more than 7 times compared with that of the control group, and the killing rate of the DBCO-HepG2 cells is increased by more than 2 times.
Example 3
(a) Metabolism of BCN-choline analogues in cervical cancer cells (HeLa cells)
Will be 4X 105Individual HeLa cells were cultured in 24-well plates in 1640 medium containing 10% fetal bovine serum at 37 ℃ with 5% CO2And incubating overnight under the culture condition of 95% humidity, adding a BCN-choline analogue dimethyl sulfoxide solution with a certain concentration into a culture solution of the HeLa cells to enable the final concentration to be 50umol/L, and incubating the mixed solution in an incubator for 48 hours to enable the surface of the HeLa cells to fully metabolize BCN groups. After the co-incubation was completed, the mixture was collected, centrifuged, and the cells were washed twice with HEPES, centrifuged, and collected to obtain HeLa cells (BCN-HeLa cells) with BCN groups modified on the surface.
(b) Metabolism of azide-modified choline analogs in immune cells (T cells)
Will be 1 × 105The individual T cells were cultured in 24-well plates in 1640 medium containing 10% fetal bovine serum at 37 ℃ with 5% CO2And incubating overnight under the culture condition of 95% humidity, adding a certain concentration of azide-modified choline analogue dimethyl sulfoxide solution into a culture solution of the T cells to enable the final concentration to be 5umol/L, and incubating the mixed solution in an incubator for 48 hours. The azide (N3) group was metabolized well on the T cell surface. After the end of the co-incubation, the mixture was collected, centrifuged, and the cells were washed twice with HEPES, centrifuged, and collected to obtain T cells (N3-T cells) with surface-modified azide groups.
(c) Interaction of two cells
BCN-HeLa cells and N3-T cells are mixed according to a certain ratio (1:5), the interaction between the two cells is detected through a laser confocal analysis experiment provided in example 1, and the HeLa cells and T cells which do not carry BCN and N3 groups are used as a control, so that N3-T cells can be rapidly gathered around the BCN-HeLa cells, the combination speed is increased by more than 8 times compared with that of the control group, and the killing rate of the BCN-HeLa cells is increased by more than 2 times.
Example 4
(a) Metabolism of BCN mannose analogues (Ac4Mann-BCN) in (Raji cells) (1) Ac4See example 1 for the synthetic procedure of Mann-BCN.
(2) 2 x 10 to5Each Raji cell was cultured in 24-well plates in DMEM medium containing 10% fetal bovine serum at 37 ℃ with 5% CO2And incubating overnight under the culture condition of 95% humidity, adding an Ac4Mann-BCN dimethyl sulfoxide solution with a certain concentration into a culture solution of Raji cells to enable the final concentration to be 25umol/L, and incubating the mixed solution in an incubator for 72 hours to enable the surface of the Raji cells to fully metabolize BCN groups. After the co-incubation is finished, the mixed solution is collected and centrifuged, cells are washed twice by PBS, centrifuged and collected, and Raji cells (BCN-Raji cells) with surface modified BCN groups are obtained.
(b) Azide-modified mannose Analogs (AC)4Manaz) metabolism in anaerobic bacterium YB1
2 x 10 to5YB1 is cultured in a strain test tube, AC4ManAz dimethyl sulfoxide solution with certain concentration is added into culture solution of YB1 to make the final concentration be 50umol/L, and the mixed solution is incubated for 72 hours in an incubator. Make itThe YB1 surface was fully metabolized to azide (N3) groups. After the co-incubation is finished, the mixed solution is collected and centrifuged, cells are washed twice by PBS, centrifuged and collected to obtain T cells (N) with surface modified azide groups3-YB1)。
(c) Bacterial-cellular interactions
Mixing BCN-Raji cells and N3-YB1 cells according to a certain ratio (1:1), and analyzing modified anaerobic bacteria (N) by laser confocal analysis3-YB1) and modified Raji cells (BCN-Raji cells) due to bioorthogonal reaction mediated bacterial-cell interaction after co-incubation. Compared with untreated bacteria and tumor cells, the N3-YB1 bacteria can be rapidly gathered around BCN-Raji tumor cells to enhance the targeting effect of the BCN-Raji tumor cells on tumors.
Example 5
(a) Metabolism of BCN mannose analogues (Ac4Mann-BCN) in (Raji cells)
(1) See example 1 for the synthetic procedure of Ac4 Mann-BCN.
(2) 2 x 10 to5A549 cells were cultured in 24-well plates in DMEM medium containing 10% fetal bovine serum at 37 ℃ with 5% CO2And incubating overnight under the culture condition of 95% humidity, adding an Ac4Mann-BCN dimethyl sulfoxide solution with a certain concentration into a culture solution of the A549 cells to ensure that the final concentration is 25umol/L, and incubating the mixed solution in an incubator for 72 hours to ensure that BCN groups are fully metabolized on the surface of the A549 cells. And after the co-incubation is finished, collecting the mixed solution, centrifuging, washing the cells twice by using PBS, centrifuging, and collecting the cells to obtain the A549 cells (BCN-A549 cells) with the surface modified BCN groups.
(b) Modification of lentiviral latin-viruses surface by azide-modified mannose analogues (AC4ManAz)
Will be 5X 105HEK293T cells were cultured in 6-well plates, incubated overnight in DMEM medium containing 10% fetal bovine serum at 37 deg.C under 5% CO2 and 95% humidity, and AC was added at a given concentration4Adding ManAz dimethyl sulfoxide solution into culture solution of HEK293T cell to make its final concentration at 25umol/L, incubating the mixture in incubator for 24 hr to make HEK293T cell surface fully metabolize azide (N3) group. After the end of the co-incubation, the mixture was collected, centrifuged, and the cells were washed twice with PBS, centrifuged, and collected to obtain HEK293T cells (N3-HEK293T cells) with N3 groups modified on the surface. Lentiviral latin-viruses were then transfected with the viral package transfection calcium phosphate transfection kit (Promega). After 6-8 hours, the mixed culture medium was added with the same concentration of AC4ManAz and cultured for another 48 hours. Containing N3The supernatant of the-latin-virus was obtained by centrifugation at 25000 rpm for 2 h.
(c) Viral and cellular interactions
Contacting BCN-A549 cells with N3Mixing the latin-virus engineering viruses according to a certain ratio (1:1), and analyzing the modified engineering viruses (N) by laser confocal analysis3Latin-viruses) and modified Raji cells (BCN-A549 cells) mediated by bioorthogonal reactions. Relative to untreated viruses and cells, N3The latin-virus bacteria can rapidly gather around the BCN-A549 tumor cells to enhance the infection capacity of the cells.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (6)
1. Use of a method for promoting biological interaction in the manufacture of a medicament for enhancing immunotherapy, the method comprising:
respectively metabolically modifying basic metabolites of click chemical groups capable of carrying out pairing reaction by organisms needing to interact to obtain organisms with click chemical groups and organisms with click chemical groups capable of carrying out pairing reaction with the click chemical groups, and promoting the interaction of the organisms through the pairing reaction among the click chemical groups;
the organism comprises one or more of a cell, a bacterium, or a virus;
the basal metabolic substance is monosaccharide or a monosaccharide derivative;
the monosaccharide derivative comprises mannosamine hydrochloride;
blocking hydroxyl of the basic metabolite-click chemical analogue by acetic anhydride to obtain the basic metabolite-click chemical analogue;
the click chemistry group is BCN, and the structure of the basal metabolite-click chemistry analogue is at least one of the following (ii) - (iv):
wherein, as in the above formulae (ii) to (iv), R2-R5、R6-R9、R10-R13Each independently is hydrogen or acetyl.
2. The use according to claim 1, wherein a bromo intermediate of a click chemistry group is provided, and the bromo intermediate of the click chemistry group is reacted with a basal metabolite to produce a basal metabolite-click chemistry analogue;
wherein the basal metabolite is choline or a choline analogue.
3. The use according to claim 1, characterized in that the basal metabolic substance modified with a click chemistry group capable of a pairing reaction is co-cultured with an organism, and after metabolism by the organism, an organism having a click chemistry group and an organism having a click chemistry group capable of a pairing reaction with the click chemistry group are obtained.
4. The use according to any one of claims 1 to 3, wherein the organism in need of interaction is a cell;
the method for promoting the interaction of organisms comprises the following steps:
(a) incubating the first cell and a basic metabolite modified with a click chemical group in a cell culture solution to obtain a first cell with the click chemical group;
(b) co-incubating a second cell with a basic metabolite modified with a click chemical group capable of reacting in a pairing manner with the click chemical group in a cell culture solution to obtain a second cell having a click chemical group capable of reacting in a pairing manner with the click chemical group;
(c) mixing the first cell obtained in the step (a) and the second cell obtained in the step (b), and promoting the interaction of the first cell and the second cell through a pairing reaction between click chemical groups;
the basal metabolite modified with the click chemistry group is a BCN-modified monosaccharide analog, and the basal metabolite modified with the click chemistry group capable of reacting with the click chemistry group in a pairing manner is a nitrogen-modified monosaccharide analog.
5. The use according to claim 4, wherein the concentration of the basal metabolite modified with click chemistry groups is 5-50umol/L upon co-incubation; the concentration of the basic metabolite modified with the click chemical group capable of reacting with the click chemical group in a pairing manner is 5-50 umol/L;
the co-incubation time is 48-96 hours;
the number ratio of the first cell to the second cell is 1: 0.2-5.
6. The use of claim 4, wherein step (a) further comprises the step of removing impurities after co-incubation to obtain a first cell having a click chemistry group;
step (b) further comprises the step of removing impurities after co-incubation to obtain a second cell having a click chemistry group capable of pairing reaction with the click chemistry group;
step (a) and step (b) are each independently cell-washed and/or centrifuged to remove impurities.
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