Pharmaceutical preparation, its preparation method, and uses

Abstract

The present invention discloses a pharmaceutical preparation, its preparation method, and uses. The pharmaceutical preparation comprises an IL-15 reagent and platelets. The IL-15 reagent comprises IL-15, and IL-15 is natural IL-15 or its mutant, or a fusion protein with IL-15 activity. The IL-15 reagent is linked to the platelets by covalent bonds or affinity. The platelets are natural platelets or modified platelets. The molar ratio of the molar number of the IL-15 reagent to the molar number of the coupling agent is 100:1 - 1:10000, and the ratio of the mass of the IL-15 reagent to the number of platelets is 1 μg:10² - 1 μg:10¹¹. The pharmaceutical preparation of the present invention improves the effectiveness and persistence of IL-15 during in vivo use, and can significantly reduce the dosage of IL-15 when administered in vivo.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering, and in particular to a pharmaceutical preparation and a preparation method and use thereof. Background Art

[0002] Interleukins (such as IL-2, IL-6, IL-12 and IL-15) are a type of cytokines that have a significant impact on immune function. Currently, in in vitro experiments, most interleukins can achieve a certain degree of inhibitory effect on tumor cells. However, when conducting in vivo experiments, the experimental results are often poor. This is because the drugs are decomposed after entering the living body or cannot accurately target the lesions. Therefore, when conducting in vivo experiments, relatively satisfactory results have to be achieved by increasing the drug dosage and the frequency of administration.

[0003] IL-15 is one of the most promising drug candidates in the field of tumor immunotherapy, but IL-15 still has certain shortcomings. For example, the expression level of IL-15 from natural sources is low, and IL-15 generally has shortcomings such as short half-life and peripheral toxicity, which can easily cause systemic immune side effects.

[0004] By connecting IL-15 to platelets, the activity of IL-15 can be increased to a certain extent and the half-life can be prolonged. However, during the coupling process, the specific amount of drug coupling cannot be accurately quantified, which is not conducive to the clinical use of the drug obtained after coupling.

[0005] In addition, IL-15Rα, as a receptor for IL-15, is also beneficial to promote stronger activation of human immune cells when compounded with immune factors. As disclosed in the Chinese invention with publication number CN111936156A: Tumor targeting of constructs containing IL-15 or IL-15 / IL-15Rα complexes in mouse models improves the anti-tumor response in immune-competent animals transplanted with homologous tumors or T-cell and B-cell-deficient SCID mice (NK cells retained) injected with human tumor cell lines. However, there are no reports in the prior art on the delivery of IL-15 by chemical coupling or affinity on platelets, as well as related drugs and their effects. Summary of the invention

[0006] In order to overcome the above disadvantages, the object of the present invention is to provide a pharmaceutical preparation and a preparation method and use thereof. The pharmaceutical preparation of the present invention improves the targeting and sustainability of IL-15 during in vivo use by coupling interleukin IL-15 with platelets, which can significantly reduce the amount of IL-15 used during in vivo administration, and the pharmaceutical preparation of the present invention can more accurately quantify the final effective dose of IL-15, which is convenient for clinical use.

[0007] The present invention provides a pharmaceutical preparation, which comprises: an IL-15 reagent, the IL-15 reagent comprising IL-15, the IL-15 being natural IL-15 or a mutant thereof, or a fusion protein having IL-15 activity, platelets, the IL-15 reagent being linked to the platelets, the platelets being natural platelets or modified platelets, and the molar ratio of the number of moles of the IL-15 reagent to the number of moles of the coupling agent being 100:1 to 1:10000.

[0008] The ratio of the mass of the IL-15 reagent to the number of platelets is 1 μg:10 2 -1 μg:10 11 . Thus, the pharmaceutical preparation of the present invention links platelets with IL-15, enabling IL-15 to have better targeting and in vivo stability, and the pharmaceutical preparation of the present invention can accurately quantify the amount of IL-15 coupled to a unit number of platelets. Therefore, it provides convenience for the actual use of IL-15 and is easy to quantify the addition amount.

[0009] Further, the platelets and the IL-15 reagent are linked by a chemical bond, and the IL-15 reagent has a linker formed by linking with a coupling agent.

[0010] Further, the platelets and the IL-15 reagent are linked by intermolecular forces, and the intermolecular forces include ionic bonds, hydrogen bonds, van der Waals forces and hydrophobic interactions.

[0011] Furthermore, the IL-15 reagent further comprises an IL-15Rα linking reagent, the IL-15 and the IL-15Rα linking reagent are linked by intermolecular forces, the IL-15Rα linking reagent can be coupled to the platelets, the IL-15Rα linking reagent is obtained by coupling IL-15Rα and its derivatives with at least one coupling agent, and the IL-15Rα linking reagent has a linker formed by linking with a coupling agent; the IL-15Rα linking reagent expresses IL-15Rα and its derivatives on the surface of platelets through genetic engineering, and the IL-15Rα and its derivatives are natural IL-15Rα or IL-15 affinity proteins or peptides, or IL-15 receptor molecules, or IL-15 antibodies. Thus, the pharmaceutical preparation of the present invention can indirectly link IL-15 with platelets through the affinity between IL-15Rα and IL-15, with high affinity and without affecting the activity of IL-15.

[0012] Further, the coupling agent is a bifunctional coupling agent.

[0013] Further, both ends of the bifunctional coupling agent have the same or different active groups, and the active groups include: active groups capable of reacting with sulfhydryl groups; active groups capable of reacting with amino groups; active groups capable of reacting with carboxylic acids and hydroxyl groups; active groups capable of reacting with aldehydes, ketones or carboxylic acids; and active groups for click reactions. Therefore, the effective connection between platelets and IL-15 reagents can be achieved by using the bifunctional coupling agent.

[0014] Further, the bifunctional coupling agent is selected from at least one of the following coupling agents:

[0015] NHS-haloacetyl coupling agents through amino-thiol reactions;

[0016] NHS-maleimide coupling agents through amino-thiol reactions;

[0017] NHS-pyridine disulfide coupling agents through amino-thiol reactivity;

[0018] Carbodiimide or NHS ester coupling agents through carboxyl-amino reactions;

[0019] NHS esters and azide-phosphine or alkyne coupling agents through chemoselective ligation;

[0020] NHS esters and aromatic azides, phenyl azides, bisaziridines, psoralens or photoactive amino acid coupling agents through photoreactive ligation.

[0021] Further, the preparation method of the pharmaceutical preparation includes a coupling rate detection step. The coupling rate detection step is to perform mass spectrometry detection on IL-15 before and after connection with the coupling agent using a mass spectrometer, calculate the mass spectrometry intensity ratio of each detection peak of IL-15 coupled with the linker based on the ratio of the abundances of the original peaks of IL-15, and obtain the average coupling amount of all proteins according to the formula. In the formula, n represents the number of linkers, and Dn intensity represents the mass spectrometry intensity ratio of IL-15 with different numbers of connected linkers. Therefore, by detecting the coupling rate of IL-15 and the coupling agent in the present invention, the number of connections of the linker can be accurately quantified, and further, an effective connection with platelets can be ensured in the subsequent process, thereby improving the overall biological activity of the pharmaceutical preparation and effectively reducing the actual usage amount of IL-15.

[0022] Further, before the platelets are coupled with the IL-15 reagent, it further includes a pretreatment step. The pretreatment step is to incubate the centrifuged and resuspended platelets with a pretreatment reagent, and the mixing ratio of the mass of the pretreatment reagent to the number of platelets can be 1:10 2 -1:10 11, the incubation conditions are: temperature is 4 - 40 °C, pH is 6.0 - 8.0, and incubation time is 0.5 - 48 h.

[0023] Furthermore, it further includes the step of conjugating with the IL-15 reagent after the platelet pretreatment, which is achieved by mixing and incubating the IL-15 reagent with the pretreated platelets. In this step, the ratio of the mass of the IL-15 reagent to the number of platelets is 1 - 10 μg : 10 2 -10 11 , the incubation temperature is 4 - 40 °C, the incubation pH is 6.0 - 8.0, and the incubation time is 0.25 - 48 h.

[0024] Further, the IL-15 reagent is coated inside the platelets or mixed and linked with the platelet molecules. The IL-15 reagent and the platelets are respectively mixed and linked to form a mixed linked form of the IL-15 reagent and the platelets.

[0025] The present invention also provides a pharmaceutical composition, which contains an effective amount of the aforementioned pharmaceutical preparation and a pharmaceutically acceptable carrier.

[0026] The present invention also provides the application of the aforementioned pharmaceutical preparation in the preparation of a drug for treating related diseases, and the related diseases include inflammatory diseases, HIV, autoimmune diseases, and tumors.

[0027] The present invention also provides the combined use of the aforementioned pharmaceutical preparation with a drug for cell therapy and / or a drug for treating related diseases. Brief Description of the Drawings

[0028] Figure 1 It is a flow cytometry result graph of platelet phenotype in Example 1 of the present invention;

[0029] Figure 2 It is a flow cytometry result graph of platelet activation state in Example 1 of the present invention;

[0030] Figure 3 It is a flow cytometry result graph of P-IL-15 phenotype in Example 1 of the present invention;

[0031] Figure 4 It is a flow cytometry result graph of P-IL-15 activation state in Example 1 of the present invention;

[0032] Figure 5(a) shows the conjugation efficiency of P-IL-15 in Example 2 of the present invention when the ratio of IL-15:SMCC is 1:3;

[0033] Figure 5(b) is a schematic diagram of the binding force between P-IL-15 and the receptor IL-15Rα in Example 2 of the present invention;

[0034] Figure 5(c) is a schematic diagram of the binding affinity between P-IL-15 and receptor IL-2Rβ in the second embodiment of the present invention;

[0035] Figure 5(d) is a schematic diagram of the binding affinity between P-IL-15 and receptor IL-2Rγ in the second embodiment of the present invention;

[0036] Figure 6(a) is the chromatogram of IL-15 in the second embodiment of the present invention;

[0037] Figure 6(b) is the chromatogram of SMCC-IL-15 in the second embodiment of the present invention;

[0038] Figure 7(a) is the mass spectrum of IL-15 in the second embodiment of the present invention;

[0039] Figure 7(b) is the mass spectrum of SMCC-IL-15 in the second embodiment of the present invention;

[0040] Figure 8 is the coupling ratio of IL-15 and SMCC in the second embodiment of the present invention;

[0041] Figure 9 is the standard curve of antibody mass-fluorescence value in the third embodiment of the present invention;

[0042] Figure 10 shows the effect of P-IL-15 on the expression of Granzyme B in NK92 cells in the fourth embodiment of the present invention. Among them, fluorescence Figure 1 represents the Isotpype control sample group; fluorescence Figure 2 represents the Media sample group; fluorescence Figure 3 represents the Platelet sample group; fluorescence Figure 4 represents the IL-2 sample group; fluorescence figure 5 represents the IL-15 sample group; fluorescence figure 6 represents the P-IL-15 sample group. Figure 10(a) is a schematic diagram when the fluorescence images of each sample group are shown separately, and Figure 10(b) is a schematic diagram when the fluorescence images of each sample group are shown combined;

[0043] Figure 11 is the effect of P-IL-15 on the proliferation of NK92 cells in the fourth embodiment of the present invention;

[0044] Figure 12(a) is a bar chart showing the effect of P-IL-15 prepared in the fourth embodiment of the present invention on the survival rate of NK92 cells;

[0045] Figure 12(b) is a line chart showing the effect of P-IL-15 prepared in the fourth embodiment of the present invention on the survival rate of NK92 cells;

[0046] Figure 13(a) shows the proportion of CD4 T cells in the spleen of mice in the fifth embodiment of the present invention;

[0047] Figure 13(b) shows the proportion of CD8 T cells in the spleen of mice in the fifth embodiment of the present invention;

[0048] Figure 14(a) shows the comparison of the number of lung lesions in mice in the sixth embodiment of the present invention;

[0049] Figure 14(b) is a comparison photo of the lung lesions in mice in the sixth embodiment of the present invention;

[0050] Figure 14(c) shows the comparison of the body weight changes in mice in the sixth embodiment of the present invention;

[0051] Figure 15(a) is a fluorescence image of the coupling effect of P-IL-15Rα in the seventh embodiment of the present invention when the ratio of IL-15Rα:SMCC is 1:15;

[0052] Figure 15(b) is a schematic diagram of the binding force between P-IL-15Rα and IL-15 in the seventh embodiment of the present invention;

[0053] Figure 16(a) is a fluorescence image of the coupling effect of P-IL-15Rα in the seventh embodiment of the present invention when the preservation solution is PBS and the incubation time is 19 h;

[0054] Figure 16(b) is a fluorescence image of the coupling effect of P-IL-15Rα in the seventh embodiment of the present invention when the preservation solution is serum and the incubation time is 19 h;

[0055] Figure 16(c) is a fluorescence image of the coupling effect of P-IL-15Rα in the seventh embodiment of the present invention when the preservation solution is PBS and the incubation time is 24 h;

[0056] Figure 16(d) is a fluorescence image of the coupling effect of P-IL-15Rα in the seventh embodiment of the present invention when the preservation solution is serum and the incubation time is 24 h;

[0057] Figure 17(a) is a fluorescence image of the binding effect of P-IL-15Rα:IL-15 in the eighth embodiment of the present invention when the preservation solution is PBS and the incubation time is 19 h;

[0058] Figure 17(b) is a fluorescence image of the binding effect of P-IL-15Rα:IL-15 in the eighth embodiment of the present invention when the preservation solution is serum and the incubation time is 19 h;

[0059] Figure 17(c) is a fluorescence image of the binding effect of P-IL-15Rα:IL-15 in the eighth embodiment of the present invention when the preservation solution is PBS and the incubation time is 24 h;

[0060] Figure 17(d) is a fluorescence image of the binding effect of P-IL-15Rα:IL-15 in the eighth embodiment of the present invention when the preservation solution is serum and the incubation time is 24 h. Detailed implementation manners

[0061] The following elaborates on the preferred embodiments of the present invention in conjunction with the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making the protection scope of the present invention more clearly defined.

[0062] Term Explanation:

[0063] "mP" refers to murine platelets, and "hP" refers to human platelets;

[0064] "APC-IL-15Ab" refers to IL-15 antibody stained with APC;

[0065] "P-IL-15-LD" refers to the low-dose P-IL-15 sample group;

[0066] "P-IL-15-HD" refers to the high-dose P-IL-15 sample group;

[0067] "Isotype Control" refers to isotype control.

[0068] Example 1: Mercaptan Modification of Platelets

[0069] The platelets in this example can be platelets from natural sources or modified platelets, such as platelets with enhanced immune activity or platelets with specific response pathways and corresponding activities after genetic modification.

[0070] In some embodiments, taking platelets from natural sources as an example, in the process of mercaptan modification of platelets, the mercaptan modifier used can be Traut's reagent or other modification reagents.

[0071] Taking Traut's reagent as the mercaptan modifier as an example, the description is as follows:

[0072] Step 1.1: Take the required human platelets, centrifuge for 10 min, discard the supernatant, add 1 ml of PBS, centrifuge for 10 min, discard the supernatant, after resuspending with PBS, take 1×10 8 human platelets per tube, add freshly prepared Traut's reagent, and make up to 500 μl with PBS, and incubate at a certain temperature.

[0073] The mixing ratio of the mass (μg) of Traut's reagent to the number (pieces) of platelets in this example can be 1:10 2 -1:10 3 or 1:10 3 -1:10 4 or 1:10 4 -1:10 5 or 1:105 -1:10 6 or 1:10 6 -1:10 7 or 1:10 7 -1:10 8 or 1:10 8 -1:10 9 or 1:10 9 -1:10 10 or 1:10 10 -1:10 11 .

[0074] The incubation temperature in this example can be 4 - 10 °C, or 10 - 20 °C, or 20 - 30 °C, or 30 - 40 °C. The incubation time can be 0.5 - 5 hours, or 5 - 10 hours, or 10 - 15 hours, or 15 - 20 hours, or 20 - 24 hours, or 24 - 48 hours.

[0075] The pH value of the PBS in this example can be 6.0 - 7.0, or 7.0 - 8.0, or 8.0 - 9.0.

[0076] In some embodiments, the pH of the PBS in this example is 7.75, the incubation temperature is 20 - 30 °C, and the incubation time is 20 - 60 min.

[0077] Step 1.2: After reacting for 30 min, remove the excess Traut's reagent by centrifugation for 20 min, wash once with 1 ml of PBS, and centrifuge at 1000 g for 10 min. In this step, activated platelets are collected by centrifugation. In some embodiments, the centrifugation speed in Step 1.2 is 450 - 1250 g, and the pH of the PBS is 6.5 - 7.5. In possible embodiments, the centrifugation speed in Step 1.2 is 850 - 1000 g, and the pH of the PBS is 6.7 - 7.2.

[0078] Example 2: Preparation of Pharmaceutical Preparation (P - IL - 15)

[0079] In some embodiments, a chemical bond is formed between platelets and IL - 15. For example, the connection between platelets and IL - 15 is achieved through the action of a coupling agent.

[0080] First, IL - 15 is connected to the coupling agent to form a linker, and then it is coupled with the platelets obtained in Example 1.

[0081] The IL-15 of the present invention is natural IL-15, or its mutant, or a fusion protein having IL-15 activity.

[0082] In some embodiments, the coupling agent is selected from bifunctional coupling agents.

[0083] In some embodiments, both ends of the bifunctional coupling agent have the same or different reactive groups, and the reactive groups include: reactive groups capable of reacting with sulfhydryl groups; reactive groups capable of reacting with amino groups; reactive groups capable of reacting with carboxylic acids and hydroxyl groups; reactive groups capable of reacting with aldehydes, ketones or carboxylic acids; and reactive groups for click reactions.

[0084] In some embodiments, the coupling agent has a group capable of reacting through an amino group and reacting with -NH2, such as NHS ester, imidoester, pentafluorophenyl ester, hydroxymethylphosphine, aromatic azide; in other possible embodiments, the coupling agent has a group capable of reacting through a sulfhydryl group and reacting with -SH, such as iodoacetyl, maleimide, pyridyl disulfide, vinyl sulfone (HBVS); in other possible embodiments, the coupling agent has a group capable of reacting through a carboxyl-amino reaction and reacting with -COOH, such as carbodiimide (EDC); in other possible embodiments, the coupling agent has a group capable of reacting through an aldehyde group and reacting with -CHO group, such as hydrazide, alkoxyamine, NHS ester; in other possible embodiments, the coupling agent has a group capable of coupling with a drug and / or a platelet through a photoreaction, such as bisaziridine, aromatic azide; in other possible embodiments, the coupling agent has a group capable of reacting through a hydroxyl group and coupling with -OH, such as isocyanate coupling agent; in other possible embodiments, the coupling agent has a coupling agent capable of reacting through an azide reaction and coupling with -N3, such as alkynes, phosphines.

[0085] In some embodiments of the present invention, the heterobifunctional coupling agent is selected from at least one of the following coupling agents: NHS-haloacetyl coupling agents through amino-thiol reaction; NHS-maleimide coupling agents through amino-thiol reaction; NHS-pyridyl disulfide coupling agents through amino-thiol reactivity; carbodiimide or NHS ester coupling agents through carboxyl-amino reaction; NHS ester and azide-phosphine or alkyne coupling agents through chemoselective ligation; NHS ester and aromatic azide, phenyl azide, bisaziridine, psoralen or photoreactive amino acid coupling agents through photoreactive ligation.

[0086] In some embodiments of the present invention, the heteromorphic bifunctional coupling agent is an NHS-haloacetyl coupling agent that reacts through amino-thiol groups, including but not limited to the following coupling agents: SIA (succinimidyl iodoacetate), SBAP (succinimidyl 3-(bromoacetamidopropionate)), SIAB (N-succinimidyl (4-iodoacetyl) aminobenzoate methyl ester), Sulfo-SIAB (sulfosuccinimidyl (4-iodoacetyl) aminobenzoate methyl ester).

[0087] In some embodiments of the present invention, the heteromorphic bifunctional coupling agent is an NHS-maleimide coupling agent that reacts through amino-thiol groups, including but not limited to the following coupling agents: AMAS (N-α-maleimidoacetic acid succinimidyl ester), BMPS (N-β-maleimidopropionic acid succinimidyl ester), GMBS (N-γ-maleimidobutyric acid succinimidyl ester), Sulfo-GMBS (N-γ-maleimidobutyric acid sulfosuccinimidyl ester), MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), Sulfo-MBS (m-maleimidobenzoyl-N-hydroxy sulfosuccinimide ester), SMCC (4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester), Sulfo-SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate), EMCA (N-ε-maleimidohexanoic acid), EMCS (ε-maleimidohexanoic acid N-hydroxysuccinimidyl ester), Sulfo-EMCS (ε-maleimidohexanoic acid N-hydroxy sulfosuccinimidyl ester), SMPB (4-(p-maleimidophenyl) butyric acid succinimidyl ester), Sulfo-SMPB (sulfosuccinimidyl-4-(N-maleimidophenyl) butyrate), SMPH (6-(β-maleimidopropionylamino) hexanoic acid succinimidyl ester), LC-SMCC (4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester-(6-aminohexanoate)), Sulfo-KMUS (N-κ-maleimide-undecanoic acid sulfosuccinimidyl ester), SM(PEG)n (polyethyleneglycolated SMCC coupling agent, n is 2-24).

[0088] In some embodiments of the present invention, the heteromorphic bifunctional coupling agent is an NHS-pyridine disulfide coupling agent with amino-thiol reactivity, including but not limited to: SPDP (3-(2-pyridyldithio)propionic acid N-succinimidyl ester), LC-SPDP (N-succinimidyl 6-(3-(2-pyridyldithio)propionamido)hexanoate), Sulfo-LC-SPDP (sulfosuccinimidyl 6-(3'-(2-pyridyldithio)propionamido)hexanoate), SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene), PEGn-SPDP (PEGylated long-chain SMCC coupling agent, n is 2-24).

[0089] In some embodiments of the present invention, the heteromorphic bifunctional coupling agent is a carbodiimide or NHS ester coupling agent with carboxyl-amino reactivity, including but not limited to: EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), NHS (N-hydroxysuccinimide), Sulfo-NHS (N-hydroxythiosuccinimide).

[0090] In some embodiments of the present invention, the heteromorphic bifunctional coupling agent is an NHS ester and azide-phosphine or alkyne coupling agent with chemoselective ligation, including but not limited to: azide-NHS ester, azide-PEG4-NHS, NHS-phosphine, alkyne, succinimidyl ester (3-propynyloxypropionic acid, succinimidyl ester), iodoacetamide alkyne, Click-iTAHA (L-azidohomoproline), Click-iT HPG (L-homopropargylglycine).

[0091] In some embodiments of the present invention, the heterobifunctional coupling agent is an NHS ester and an aromatic azide, phenyl azide, bisaziridine, psoralen or photoreactive amino acid coupling agent linked by a photoreactive bond, including but not limited to: ANB-NOS (N-5-azido-2-nitrobenzoic acid succinimidyl ester), Sulfo-SANPAH (sulfosuccinimidyl 6-(4'-azido-2'-nitroanilino)hexanoate), SDA (NHS-bisaziridine) (succinimidyl 4,4'-azidopentanamide), Sulfo-SDA (Sulfo-NHS-bisaziridine) (sulfosuccinimidyl 4,4'-azidopentanamide), LC-SDA (NHS-LC-bisaziridine) (succinimidyl 6-(4,4'-azidopentanamide)hexanoate), Sulfo-LC-SDA (Sulfo-NHS-LC-diazirine) (sulfosuccinimidyl 6-(4,4'-azidopentanamide)hexanoate), Sulfo-SDAD (Sulfo-NHS-SS-bisaziridine), (sulfosuccinimidyl 2-((4,4'-azidopentanamide)ethyl)-1,3'-dithiopropionate), SPB (succinimidyl-(4-(psoralen-8-yloxy))-butyrate), L-Photo-leucine, L-Photo-methionine.

[0092] In some embodiments of the present invention, the homobifunctional coupling agent is selected from at least one of the following coupling agents: an NHS ester coupling agent that reacts with platelets and a drug respectively through an amino-amino reaction; an imidate or difluoro coupling agent that reacts with platelets and a drug respectively through an amino-amino reaction; a maleimide coupling agent that reacts with platelets and a drug respectively through a thiol-thiol reaction.

[0093] In some possible embodiments of the present invention, the homobifunctional coupling agent is an NHS ester coupling agent that is coupled to platelets and drugs respectively through amino - amino reactions, including but not limited to the following coupling agents: DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), BS3 (bis(sulfosuccinimidyl) suberate), BS(PEG)n (PEGylated bis(sulfosuccinimidyl) suberate) (n is 5 - 9), DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate)), DST (diamide of tartaric acid), BSOCOES (bis(2-(succinimido - oxycarbonyloxy)ethyl) sulfone), EGS (ethylene glycol bis(N - hydroxysuccinimide butanedioate)), Sulfo - EGS (ethylene glycol bis(sulfosuccinimide butanedioate)), TSAT (Tris-(succinimidyl ester) aminotriacetic acid), BSG - d0 (bis(sulfosuccinimidyl) glutarate - d0), BS2G - d4 (bis(sulfosuccinimidyl) 2,2,4,4 - glutarate - d4), BS3 - d0 (bis(sulfosuccinimidyl) suberate - d0), BS3 - d4 (bis(sulfosuccinimidyl) 2,2,7,7 - suberate - d4), DSSO (disuccinimidyl sulfoxide), DSBU (disuccinimidyl dibutylurea).

[0094] In some other possible embodiments of the present invention, the homobifunctional coupling agent is an imidate or difluoro coupling agent that is coupled to platelets and drugs respectively through amino - amino reactions, including but not limited to the following coupling agents: DMA (dimethyl adipate), MP (dimethyl pimelimidate), DMS (dimethyl octanedioate), DTBP (Wang and Richard’s reagent), DFDNB (1,5 - difluoro - 2,4 - dinitrobenzene).

[0095] In some other possible embodiments of the present invention, the homobifunctional coupling agent is a maleimide coupling agent that is coupled to platelets and drugs respectively through thiol - thiol reactions, including but not limited to the following coupling agents: BMOE (bismaleimide ethane), BMB (1,4 - bismaleimide butane), BMH (bismaleimide hexane), BM(PEG)2 (1,8 - bismaleimide - diethylene glycol), BM(PEG)3 (1,11 - bismaleimide - triethylene glycol), DTME (dithiomaleimide ethane), TMEA ((tris(2 - maleimidoethyl)amine).

[0096] In some embodiments, taking the coupling agent Sulfo - SMCC as an example for illustration, the specific steps of this embodiment are as follows.

[0097] Step 2.1: Freshly prepare a Sulfo-SMCC solution at a certain concentration. The concentration of Sulfo-SMCC can be 0 - 1 mg / ml, or 1 - 100 mg / ml, or 100 - 1000 mg / ml. For example, the concentration of Sulfo-SMCC is 0.5 mg / ml, or 3 mg / ml, or 100 mg / ml.

[0098] Step 2.2: Preparation of the IL-15 reagent: React IL-15 with sulfo-SMCC at 4 degrees Celsius in a certain molar ratio (here, the "molar ratio" refers to the number of moles of IL-15 / the number of moles of Sulfo-SMCC).

[0099] The molar ratio in Step 2.2 can be 100:1 - 1:10, or 1:10 - 1:100, or 1:100 - 1:1000, or 1:1000 - 1:10000. For example, the molar ratio is 1:3, or 1:6, or 1:100, or 1:1000 or 2:1, or 100:2. In some embodiments, IL-15 is purchased from Beijing Yizhuang International Protein Drug Technology Co., Ltd., with the product number BY015. SMCC is purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with the product number M123456.

[0100] The reaction time of IL-15 and sulfo-SMCC can be 0.5 - 5 hours, or 5 - 10 hours, or 10 - 15 hours, or 15 - 20 hours, or 20 - 24 hours, or 24 - 48 hours.

[0101] Step 2.3: After the reaction is completed, transfer the reaction mixture obtained in Step 2.2 to a pre-centrifuged ultrafiltration tube (cut-off = 3KDa). After removing the excess Sulfo-SMCC linker by centrifugation of the mixture, filter to obtain the protein.

[0102] In some embodiments, the centrifugation conditions are 14000g, 10 min, 4 degrees Celsius, centrifuge forward 3 times and then reverse 1 time; use a 0.2μm filter during filtration.

[0103] Step 2.4: Mix SMCC-IL15 with the platelets prepared in Example 1 to prepare systems under different conditions. Incubate and slowly shake on a shaker to obtain P-IL-15.

[0104] The ratio of the mass of SMCC-IL-15 (μg) to the number of platelets can be 1:10 2 -1:10 3 and can also be 1:10 3 -1:104 , or it can be 1:10 4 -1:10 5 , or it can be 1:10 5 -1:10 6 , or it can be 1:10 6 -1:10 7 , or it can be 1:10 7 -1:10 8 , or it can be 1:10 8 -1:10 9 , or it can be 1:10 9 -1:10 10 , or it can also be 1:10 10 -1:10 11 . For example, it can be 4:1×10 8 , or 1:1×10 9 or 3:1×10 10 .

[0105] The incubation temperature in Step 2.4 can be 4 - 10 degrees Celsius, or it can be 10 - 20 degrees Celsius, or it can be 20 - 30 degrees Celsius or 30 - 40 degrees Celsius.

[0106] The incubation time in Step 2.4 can be 0.25 - 1 hour, or it can be 1 - 2 hours, or it can be 2 - 4 hours, or it can be 4 - 16 hours or 16 - 48 hours.

[0107] In some embodiments, the incubation temperature is room temperature and the incubation time is 1 hour.

[0108] In some embodiments, the correspondence between the number of platelets of the pharmaceutical preparation and the mass of IL - 15 is 1.0×10 8 platelets corresponding to 0.1 - 1.0 μg of IL - 15.

[0109] Step 2.5: Centrifuge the reaction mixture obtained in Step 2.4 at room temperature for 20 minutes to remove unbound IL - 15, and resuspend it with 1 ml of PBS. Count the finally conjugated IL - 15 platelets and calculate the platelet yield.

[0110] The pH value of the PBS in this step can be 6.0 - 7.0, or it can be 7.0 - 8.0 or 8.0 - 9.0.

[0111] In some embodiments, the centrifugation speed in Step 2.5 is 1000g and the pH of the PBS is 6.7 - 7.5.

[0112] Step 2.6: After collecting the sample, according to the platelet yield, take 1.5×10 6After quantifying the platelets to 100 μl, add 1 μl of APC-IL-15Ab, incubate in the dark at room temperature for 30 min, add 1 ml of PBS, centrifuge for 10 min. After removing the supernatant, resuspend with 100 μl of PBS.

[0113] Step 2.7: Take 5 μl of the P-IL-15 prepared in Step 2.4 for counting, take 1×10 6 of P-IL-15 and platelets not conjugated with IL-15, add 0.45 μg of IL-15Rα, 0.65 μg of IL-2Rβ and 0.35 μg of IL-2Rγ, make up to 100 μl with PBS, and incubate at room temperature for 30 min.

[0114] Step 2.8: Centrifuge for 10 min, remove the supernatant, resuspend with 100 μl of PBS, add 1 μl of the receptor dye PE-IgG, and incubate for 20 min. The stain for the receptor dye can be APC, FC, PE, or ECD.

[0115] Step 2.9: Centrifuge for 10 min, remove the supernatant, resuspend with 100 μl of PBS, and perform flow cytometry analysis. The results are as Figures 1-4 shown.

[0116] Step 2.10: Verify whether the phenotype and activation function of the P-IL-15 prepared in Step 2.4 are consistent with those of platelets:

[0117] The P-IL-15 prepared in Step 2.4 is counted according to the number of platelets being 2×10 7 and CaCl2 solution and thrombin are added, followed by incubation. The concentration of CaCl2 in this step is 1 - 10 mM, or 10 - 50 mM, or 50 - 100 mM, and the incubation temperature is 4 - 10, or 10 - 20, or 20 - 40 degrees Celsius.

[0118] In some embodiments, the specific activation reaction conditions are 20 μl of CaCl2 (100 mM), 4 μl of thrombin (100 U / ml), prepare a 100 μl system, incubate at 37 degrees Celsius for 15 min.

[0119] Take the activated and non-activated P-IL-15 and Platelet, prepare a 100 μl system according to 2×10 6 platelets, add 1 μl of the corresponding antibodies (CD9, CD41, CD61, CD62p, and CD154 antibodies), incubate in the dark for 20 min, add 1 ml of PBS, centrifuge at 2000 g for 10 min, after resuspending with 100 μl of PBS, perform analysis on the machine. The results are as Figures 1-4As shown, the results indicate that the phenotypes of platelets before and after coupling with IL-15 are basically the same, indicating that the coupling process does not have a significant impact on the immune properties of platelets.

[0120] The phenotypes and activation states of platelets are respectively as Figure 1 and Figure 2 shown, and the phenotypes and activation states of P-IL-15 are respectively as Figure 3 and Figure 4 shown.

[0121] Here, taking the molar ratio of IL-15 reagent in the preparation in step 2.2 as 1:3 as an example for the experiment, the flow cytometry detection results of platelets coupled with IL-15 are shown in Figure 5(a). In the figure, the fluorescence map of the lighter gray area represents the peak emergence of the substance bound to IL-15. It can be seen that the coupling efficiency of P-IL-15 of the present invention is relatively high, reaching more than 95%. This indicates that IL-15 obtained according to the method and preparation conditions of the present invention can be effectively linked to platelets.

[0122] It can be seen from Figures 5(b)-(d) that P-IL-15 prepared by the present invention can bind to the IL-15Rα receptor and retains the receptor binding ability; P-IL-15 prepared by the present invention can bind to the IL-2Rβ receptor and retains the receptor binding ability; P-IL-15 prepared by the present invention cannot bind to the single IL-2Rγ receptor.

[0123] In step 2.3, there is also a step of determining whether IL-15 is successfully coupled with the coupling agent Sulfo-SMCC. By comparing the chromatograms of IL-15 before and after coupling with Sulfo-SMCC by chromatography, the results are shown in Figures 6(a) and 6(b) respectively. It can be seen from the figures that compared with the chromatogram of IL-15, there is an additional side peak near the peak emergence position of the original IL-15 in the chromatogram of IL-15-SMCC. This indicates that IL-15 of the present invention is successfully coupled with the coupling agent Sulfo-SMCC.

[0124] In addition, the present invention also uses mass spectrometry to perform mass spectrometry detection on IL-15 modified by Sulfo-SMCC to determine the number and distribution of linkers (connectors) connected to IL-15. Mass spectrometry comparative analysis is performed on IL-15 and SMCC-

[0125] IL-15 respectively, and the results are as Figure 7(a) and 7(b)As shown in the figure. By comparing the mass spectrometry spectrum of SMCC-IL-15 with that of IL-15, the peak with the same elution position as the original IL-15 was defined as L0, which represents that the IL-15 molecule eluting at this position was not successfully conjugated with SMCC. Subsequently, the peaks successively away from L0 were named L1, L2 in sequence, and their molecular weights increased successively.

[0126] As Figure 8 shown, in the IL-15-SMCC sample, based on the proportion of the abundance of the original peak of pure IL-15, the mass spectrometry intensity ratios of IL-15 conjugated with different numbers of linkers were calculated, and the possible ratios of IL-15 conjugated with different numbers of linkers were obtained. The results are shown in Table 1. Among them, the ratio of L0 was calculated by the ratio of its mass spectrometry intensity value 748076.28 to the total mass spectrometry intensity value (748076.28 + 402882.45 + 85458.16). Considering the interference of side peaks eluting after the main peak, such as sugars, comprehensively, the various ratios shown in Table 1 were obtained.

[0127] Table 1 Mass spectrometry intensity ratios of IL-15 conjugated with different numbers of linkers

[0128] L0 L1 L2 ％ 58.83 34.01 7.16

[0129] According to the formula, the average conjugation amount LAR of all proteins was obtained as 0.48. In the formula, n represents the number of linkers, and Dn intensity represents the mass spectrometry intensity ratio of IL-15 conjugated with different numbers of linkers. In the figure, LAR0, LAR1, and LAR2 represent the numbers of SMCC conjugated on the IL-15 molecule are 0, 1, and 2 respectively, which are used to characterize the number of SMCC linkers that can be modified on each IL-15 molecule. Only the IL-15 conjugated with SMCC can be linked to platelets.

[0130] The conjugation ratio (LAR) of IL-15 and SMCC was calculated and the result is as Figure 8 shown. The ordinate represents the mass spectrometry intensity (intensity) of IL-15 with different conjugation numbers in all IL-15. This shows that about 48% of IL-15 was modified with the SMCC group and can be conjugated with platelets.

[0131] In other possible embodiments, IL-15 and platelets can also be linked by using platelets to encapsulate IL-15. For example, IL-15 can be directly encapsulated within the membranous outer shell formed by platelets. Platelets can also be pulverized and then linked with IL-15, or IL-15 can be modified and then linked with platelets.

[0132] Example 3: Detection of P-IL-15 Protein Loading

[0133] The protein loading of P-IL-15 prepared by setting the molar ratio of IL-15:SMCC in Example 2 to 1:3 was detected, and the content of the conjugated drug IL-15 was detected. Here, the sample group with a molar ratio of IL-15:SMCC of 1:3 was selected because it has sufficient biological activity and relatively low overall usage cost.

[0134] The specific experimental steps are as follows:

[0135] Step 3.1: Take P-IL-15 in an EP tube, add IL-15 fluorescent antibody (FITC anti-human IL-15 Antibody), pipette and mix well, and incubate in the dark at room temperature for 20 min;

[0136] The addition amount of P-IL-15 is counted by the number of platelets, which can be 1×10 3 -1×10 5 pieces, or can be 1×10 5 -1×10 7 pieces, or 1×10 7 -1×10 9 pieces. The concentration of IL-15 fluorescent antibody can be 0 - 100 μg / ml, or 100 - 1000 μg / ml, or 1 - 100 mg / ml.

[0137] In some embodiments, the addition amount of IL-15 is 1×10 6 pieces, or 2×10 7 pieces, or 1×10 6 pieces. The addition amount of IL-15 fluorescent antibody is 2 μl, and the concentration is 10 μg / ml.

[0138] Step 3.2: After incubation, centrifuge for 20 min, pay attention to the direction of the centrifuge tube. After centrifugation, carefully aspirate and discard the supernatant, add 1 ml of PBS respectively for resuspension and washing, and centrifuge again for 20 min.

[0139] Step 3.3: Prepare IL-15 fluorescent antibody standards with different concentrations. Take 0.4 μl of FITC anti-human IL-15 Antibody standard, dissolve it in 200 μl of PBS solution, and mix well; then take 100 μl of the solution mixed in the previous well and dissolve it in 100 μl of PBS solution, and mix well. Dilute step by step in turn to prepare standard solutions with different concentrations.

[0140] Step 3.4: Add the standard solution and the sample to be tested prepared in Step 3.3 into the detection wells of an opaque black microplate.

[0141] Step 3.5: Use a microplate reader to detect the fluorescence values of the standard and the sample to be tested. The excitation wavelength is 495 nm, and the detection wavelength is 550 nm. The detection results are shown in Table 2 and Figure 9 as follows. In the experiment, two groups of samples were used for parallel experiments.

[0142] Table 2 Corresponding results of protein content and fluorescence value of samples in Example 3

[0143]

[0144] Thus, it can be calculated that when using the P-IL-15 sample prepared in Example 2 and the platelet count is 1.0×10 8 cells, the mass of IL-15 loaded on P-IL-15 is 0.2933 μg. Thus, it can be shown that IL-15 has been successfully conjugated with platelets on the P-IL-15 prepared in Example 2 of the present invention, and the IL-15 conjugated on the P-IL-15 sample can be accurately quantified through this standard curve.

[0145] Example 4: Effect of P-IL-15 on the activation of NK92 cells in vitro (promoting Granzyme B expression)

[0146] Granzyme is a group of homologous serine proteases. Currently, five types of human granzyme families have been discovered, namely Granzyme A, Granzyme B, Granzyme H, Granzyme K, and Granzyme M.

[0147] Granzyme is an important effector molecule for NK cells and CTL cells (cytotoxic T cells) to kill tumor cells, and it causes apoptosis of target cells, playing an important role in the body's antiviral and anti-tumor processes. Granzyme mainly exists in the cytoplasm of activated lymphocytes such as NK cells and CTL cells, and together with perforin, it is the main effector molecule for NK and CTL cells to exert cytotoxic effects.

[0148] Therefore, by analyzing the expression of Granzyme B in NK92 cells by P-IL-15 in vitro, the activation effect of the obtained P-IL-15 on NK92 cells can be studied.

[0149] The experimental steps are as follows:

[0150] Step 4.1: Take NK92 cells, centrifuge at 1000 g for 5 min, wash once with PBS, centrifuge again, and resuspend with NK92MI medium.

[0151] Step 4.2: Inoculate cells at a density of 1×10 5 / ml / well, and then add 10 ng / ml IL-2, 40 ng / ml IL-15, and 2×10 6 platelets / ml of hP-IL-15 (activated and non-activated). Activation conditions: 2×10 7 hP-IL-15, 2 μl of 100 U / ml thrombin, 1 μl of 1 M CaCl2, for 30 min at 37 °C, and the system volume is 100 μl. In this step, the added amounts of IL-2 and IL-15 are both experimentally proven to be the amounts most conducive to the growth of NK92 cells. The added amount of IL-15 in the hP-IL-15 corresponding to 2×10 6 platelets / ml is approximately 6 ng / ml (here, calculated based on the protein loading of P-IL-15 in Example 3, and experimentally calculated that the protein amount of IL-15 loaded on 1×10 8 platelets is approximately 0.3 μg. Therefore, when the number of platelets is 2×10 6 , correspondingly, the amount of IL-15 is 6 ng).

[0152] Step 4.3: Perform cell counting on 10 μl of the culture medium on Day 1, Day 2, and Day 3.

[0153] Step 4.4: On Day 3, centrifuge to collect the cells and discard the supernatant.

[0154] Step 4.5: Add 100 μl of fixation / permeabilization solution, vortex, and incubate at 4 °C for 20 min.

[0155] Step 4.6: Add 1 ml of PBS, centrifuge at 1000 g for 10 min, and discard the supernatant.

[0156] Step 4.7: Resuspend with 100 μl of PBS, add 1.5 μl of APC anti-Granzyme B, incubate for 30 min, add 1 ml of PBS, centrifuge at 1000 g for 10 min, discard the supernatant, resuspend with 100 μl of PBS, and then perform on-machine detection.

[0157] The results are as shown in Figure 10(a) and 10(b) . It can be seen that although the amount of IL-15 conjugated in P-IL-15 is significantly lower than the amount of IL-15 used alone, P-IL-15 still has an obvious effect of promoting the expression of Granzyme B in NK92 cells, and its promoting effect is not inferior to that of using IL-15 alone. Thus, it can be shown that the potency of IL-15 can be significantly improved by conjugating it with platelets.

[0158] The proliferation effect of P-IL-15 on NK92 cells is as Figure 11 shown. It can be seen that P-IL-15 has the effect of promoting the proliferation of NK92, while Platelet has no such effect, indicating that the IL-15 conjugated with Platelet prepared in the present invention still has activity.

[0159] The effect of P-IL-15 on the activity of NK92 cells is shown in Figures 12(a) and 12(b). It can be seen from the figures that under the premise of reducing the effective drug (IL-15), the P-IL-15 prepared by the method of the present invention can still keep NK92 cells with relatively high activity.

[0160] Example 5: Effect of P-IL-15 on the phenotype of immune cells

[0161] In this example, the changes in the immune phenotype of mouse spleen T cells were detected by P-IL-15. The specific experimental steps are as follows:

[0162] Step 5.1: By means of tail vein injection, drugs were administered on Day1 and Day8 respectively, and the administration volume was 100 μl. Among them, the administration doses were as follows: low-dose group of P-IL-15: 2×10 8 red blood cells; high-dose group of P-IL-15: 6×10 8 red blood cells; MP group: 6×10 8 red blood cells; IL-15 group: According to 2 μg, with the administration volume of 100 μl, drugs were administered on Day1-7 and Day9-14 respectively.

[0163] After two weeks of drug administration, the mice were sacrificed by cervical dislocation, the spleens were dissected and placed in PBS.

[0164] Step 5.2: Transfer to a 70-mesh cell sieve, grind with a syringe piston, and add PBS for rinsing and flushing.

[0165] Step 5.3: Centrifuge the obtained grinding solution at 1000 g for 5 min, add 1 ml of red blood cell lysate after removing the supernatant, react at room temperature for 5 min, centrifuge at 1000 g for 5 min, and remove the supernatant.

[0166] Step 5.4: Take 1×10 6 cells for staining, add 2.7 μl of APC anti-CD3, FITC anti-CD4, PE anti-CD8 mix, incubate for 20 min, centrifuge, and load for detection.

[0167] The results are shown in Figures 13(a) and 13(b). From the figures, it can be seen that the proportion of CD4 T cells in the spleen of the IL-15 and P-IL-15 groups decreased slightly, while the CD8 T cells increased. Among them, IL-15 and low-dose P-IL-15 were more obvious. This indicates that the P-IL-15 prepared by the method of the present invention promoted the proliferation of CD8 T cells in vivo and changed the immune state, indicating that the P-IL-15 prepared by the present invention has sufficient ability to promote the activation of immune cells.

[0168] Example 6: Therapeutic effect of P-IL-15 on B16F10 metastatic tumors

[0169] In this example, the therapeutic effect of P-IL-15 on B16F10 metastatic tumors was evaluated. The specific experimental steps are as follows:

[0170] Step 6.1: On Day 0, mice were injected with 1.5×10 5 B16F10 for modeling and administration. On Day 7, administration was performed again, including MP (6×10 8 ), IL-15 (2 μg), P-IL15-LD (2×10 8 ), and P-IL-15-HD (6×10 8 ). Among them, IL-15 was continuously administered for 10 days. On Day 17, the mice were dissected to obtain the growth of lung lesions and the proliferation of spleen T cells.

[0171] Step 6.2: The mice were sacrificed by cervical dislocation, and the intact lung organs were dissected and placed in PBS for washing.

[0172] Step 6.3: Observe and count the number of lesions.

[0173] The experimental results are as Figures 14(a)-14(b) shown. From the figure, it can be seen that from the perspective of the number of lesions, the low-dose group has a better effect than the high-dose group. It is speculated that the pro-tumor metastasis effect of Platelet in the high-dose group is superior to the anti-tumor effect of conjugated IL-15; the overall dosage of IL-15 in the P-IL15 sample group is significantly lower than that in the simple IL-15 sample group. However, its effect on reducing the number of lesions is obvious, which also indicates that the pharmaceutical preparation P-IL15 prepared by the present invention has high biological activity.

[0174] As can be seen from Figure 14(c), there is no significant change in the body weight difference of each group of mice, indicating that the high dose of 6×10 8 P-IL-15 has no strong toxicity to mice.

[0175] Example 7: Preparation of P-IL-15Rα

[0176] In this embodiment, P-IL-15Rα is prepared by connecting IL-15Rα and its derivatives linked with a linker to platelets.

[0177] In some embodiments, IL-15Rα is the naturally occurring IL-15 receptor IL-15Rα, or can be other fragments or fusion proteins capable of binding to IL-15, such as the IL-15Rα sushi domain or IL-15Rα / IgG1 Fc.

[0178] The platelets in this embodiment are also the platelets obtained after mercapto modification using the method of Example 1. The process of coupling IL-15Rα with a coupling agent can also be prepared with reference to Steps 2.1-2.3 in Example 2. In some embodiments, IL-15Rα is purchased from Suzhou Novoprotein Scientific Inc., with the product number CK34. The molar ratio of IL-15Rα to the coupling agent is 100:1 - 1:10, or can be 1:10 - 1:100, or can be 1:100 - 1:1000, or can also be 1:1000 - 1:10000. For example, the molar ratio is 1:5, or 1:15, or 1:100, or 1:1000 or 2:1, or 100:2.

[0179] In some embodiments, the coupling agent is Sulfo-SMCC (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number M123456), with a concentration of 2 mg / ml, an addition amount of 1 μg, the molar ratio of IL-15Rα to the coupling agent is 1:15, the addition amount of IL-15Rα is 50 μg, and the reaction system is 500 μl. The reaction time can be 0.5 - 1 h, 1 - 3 h, 3 - 5 h, and the reaction temperature can be 0 - 5 °C, 5 - 8 °C, 8 - 12 °C, 12 - 16 °C, 16 - 20 °C. For example, the reaction can be carried out for 2 h, and the reaction temperature can be 4 °C.

[0180] The steps of coupling platelets with a drug reagent (i.e., the IL-15Rα after coupling in this embodiment) refer to Steps 2.4 - 2.5. When coupling IL-15Rα with platelets, the concentration of platelets can be 1×10 6 -1×10 7 、1×10 7 -1×10 8 or 1×10 8 -1×10 9 cells / ml. For example, it can be 1×10 8 cells / ml.

[0181] Subsequently, the reaction mixture was subjected to two centrifugation treatments (the centrifugation conditions for the first time can be 500 - 5000 g, the temperature is 20 - 50 degrees Celsius, and in some embodiments, the centrifugation conditions for the first time are 500 g, 1000 g, 2000 g, 2500 g, 3000 g, 4000 g or 5000 g). After centrifugation for 20 - 60 min. For example, after centrifugation at 1000 g for 20 min, it was resuspended after the second centrifugation. In some embodiments, when resuspending, PBS buffer was used, and the pH can be 6.0 - 8.0. Specifically, it can be 6.5 - 6.8, or 6.8 - 7.0, or 7.0 - 7.2, or 7.2 - 7.4 or 7.4 - 7.8. For example, the above-mentioned platelets and IL-15Rα were coupled using PBS buffer with a pH of 7.2. The amount of PBS used can be 200 - 300 μl, or 300 - 500 μl, or 500 - 700 μl, or 700 - 1000 μl. In some embodiments, the amount of PBS buffer used is 600 μl.

[0182] The coupling efficiency of P-IL-15Rα was detected, and the results are shown in Figure 15(a). In the figure, the peak on the right represents the fluorescence spectrum of IL-15Rα coupled to the P-IL-15Rα prepared in this example. It can be seen from the figure that the coupling rate of IL-15Rα to P-IL-15Rα in this example is close to 100%.

[0183] In addition, the coupling stability of P-IL-15Rα was also detected in this example. The specific steps are as follows:

[0184] After the P-IL-15Rα sample was resuspended, a part of the sample was taken out, quantified with serum and PBS respectively, incubated overnight at room temperature, and taken out at 19 h and 24 h respectively. After incubation with PE anti-Fc and APC anti-IL-15, it was centrifuged, the supernatant was discarded, PBS was added, and it was detected by machine.

[0185] In some embodiments, after quantification with serum and PBS to 200 - 500 μl respectively, for example, it can be quantified to 300 μl and then incubated overnight at room temperature. At 19 h and 24 h, 80 μl were taken respectively, 0.5 μl of PE anti-Fc and 1 μl of APC anti-IL-15 were added, and incubation treatment was carried out. The incubation time can be 30 - 240 min. The centrifugation conditions after incubation can be 1000 - 5000 g, and the time is 5 - 60 min. For example, after incubation for 50 min, it was centrifuged at 3000 g for 10 min, the supernatant was discarded, and an appropriate amount of PBS, such as 100 μl of PBS, was added for detection by machine. The results are respectively as Figures 16(a)-16(d)As shown. The results indicate that the P-IL-15Rα prepared in this example has good stability at 19 h and 24 h, and it has relatively good biological stability.

[0186] Example 8: Preparation of a pharmaceutical preparation (P-IL-15Rα:IL-15)

[0187] What differentiates it from Example 2 is that IL-15 and platelets can also be connected through intermolecular interactions, such as through ionic bonds, hydrogen bonds, van der Waals forces, and hydrophobic interactions.

[0188] In some embodiments, the connection between IL-15 and platelets is indirectly achieved by utilizing the binding force between IL-15Rα and its derivatives and IL-15. The specific steps are as follows:

[0189] Step 8.1: After resuspending the P-IL-15Rα sample prepared in Example 7, add IL-15 and incubate. In some embodiments, the addition amount of IL-15 can be 1 - 100 μg. A portion of the resuspended sample can be taken out and IL-15 can be added for incubation. For example, a portion of the sample resuspended in 600 μl of PBS buffer as described above can be taken and IL-15 can be added. It can be 200 - 500 μl, or 240 μl, 300 μl, 360 μl, 420 μl; the addition amount of IL-15 is 1 - 50 μg. In some embodiments, 380 μl of the above resuspended sample is taken and 10 μg of IL-15 is added for incubation for 1 h.

[0190] Step 8.2: Centrifuge the sample obtained in Step 8.1 twice more. Among them, the conditions for the first centrifugation can be 500 g - 3000 g, and the centrifugation time can be 10 - 60 min. In some embodiments, the centrifugation conditions are 500 g, 30 min; or 1000 g, 20 min; or 2000 g, 20 min. Then it is resuspended in PBS buffer solution and centrifuged again.

[0191] Step 8.3: After resuspending the sample (mP-IL-15Rα) obtained in Step 8.2, add an antibody dye for IL-15 and incubate at room temperature. The antibody dye for IL-15 can be any dye such as APC, PE, CY5, PI, ECD, etc. The addition volume of the dye to the sample can be 1:100 - 1:20. In some embodiments, after resuspending the sample obtained in Step 8.2 to 100 μl, 2 μl of the antibody dye is added and incubated for 30 min.

[0192] Step 8.4: After centrifuging the sample obtained in Step 8.3, resuspend it and then perform flow cytometry detection. In some embodiments, the centrifugation conditions in this step are 1000g for 10 min; after centrifugation, add 100 μl of PBS to resuspend and perform flow cytometry detection. The results are shown in Fig. 15(b). This shows that the P-IL-15Rα obtained in this example has a strong binding affinity with IL-15.

[0193] In addition, this example also detects the binding stability of the pharmaceutical preparation (P-IL-15Rα:IL-15). The specific steps are as follows: Quantify the mixture of P-IL-15Rα:IL15 with serum and PBS respectively, incubate overnight, take samples at 19 h and 24 h respectively, add PE anti-Fc and APC anti-IL-15, centrifuge after incubation, discard the supernatant, add PBS, and perform detection on the machine. The addition ratio of P-IL-15Rα to IL15 in the mixture of P-IL-15Rα:IL15 can refer to the addition ratio in Step 8.1 of this example, or other addition ratios can be used as needed.

[0194] In some embodiments, the incubation conditions here can be the same as those for the incubation of P-IL-15Rα with PE anti-Fc and APC anti-IL-15 above. The results of the machine detection are as Figures 17(a)-17(d) shown. The results show that partial dissociation of the P-IL-15Rα:IL-15 of the present invention occurs after 19 h and 24 h, but it has a better preservation effect in serum, indicating that the affinity is affected by the preservation solution, while the preservation solution has no obvious effect on the binding stability of P-IL-15Rα, which also shows that the covalent bond binding of IL-15Rα in the P-IL-15Rα:IL-15 prepared by the present invention to platelets is not affected by the preservation solution.

[0195] In other possible embodiments, the pharmaceutical preparation (P-IL-15 or P-IL-15Rα) obtained in the present invention can also be used in combination with other drugs for anti-tumor treatment. For example, the P-IL-15 or P-IL-15Rα:IL-15 of the present invention can be used in cell therapy methods and combined with immunocyte therapy drugs, CIK cell therapy drugs, NK cell therapy drugs, TIL cell therapy drugs, CAR-T therapy drugs, CAR-NK drugs, etc. to exert anti-tumor-related treatment. The P-IL-15 or P-IL-15Rα:IL-15 of the present invention can also be used in conventional radiotherapy and chemotherapy treatment methods as drugs for treating related diseases such as tumors.

[0196] In other possible embodiments, the P-IL-15Rα:IL-15 prepared in Example VIII can also be used to prepare drugs for treating autoimmune diseases and inflammation, because P-IL-15Rα:IL-15 has the potential to activate Tregs.

[0197] The above embodiments are only for illustrating the technical concept and characteristics of the present invention, and the purpose is to enable those who are familiar with this technology to understand the content of the present invention and implement it. It is not intended to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A pharmaceutical preparation, characterized in that, Comprising: An IL-15 reagent, the IL-15 reagent comprising IL-15, and the IL-15 being natural IL-15, Platelets, the platelets being natural platelets or modified platelets, Further comprising an IL-15Rα linking reagent, the IL-15 being linked to the IL-15Rα linking reagent through intermolecular forces, The IL-15Rα linking reagent being capable of coupling with the platelets, the IL-15Rα linking reagent being obtained by coupling IL-15Rα with at least one coupling agent, and the IL-15Rα linking reagent having a linker formed by linking with the coupling agent, The IL-15Rα being a naturally-derived IL-15 receptor molecule, The coupling agent being sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate for amino-thiol reaction.

2. A drug delivery system, characterized in that, Containing an effective amount of the pharmaceutical preparation as described in claim 1 and a pharmaceutically acceptable carrier.

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