Antibody-conjugated liposomes

Abstract

A liposome, particularly a nanoparticle, conjugated with an antibody. The surface of the nanoparticle contains an antibody. The number of antibodies is 5 to 60, preferably 5 to 50, more preferably 5 to 40. The nanoparticle, for example, a liposome, can exert a more effective therapeutic effect on tumors and improve multidrug resistance to tumors. Therefore, this breaks down the technical prejudice that the more antibodies there are on the liposome surface, the better the target cell binding effect, and lays the foundation for further clinical development.

Description

[Technical field]

[0001] The present disclosure is in the field of medicine, and in particular relates to antibody-conjugated liposomes. [Background technology]

[0002] There are several liposomal drugs on the domestic and international market, especially liposomal formulations containing antitumor chemotherapeutic agents such as doxorubicin, irinotecan, and paclitaxel. The in vivo distribution and circulation properties of liposomal drugs can be changed by encapsulation, while the concentration of drug accumulated in tumor tissue (passive targeting effect) can be increased through the EPR effect. The therapeutic index can be greatly improved by improving efficacy and / or reducing toxic side effects. However, due to the lack of specific interaction between conventional liposomes and tumor cells, the drug in the liposome must be released from the liposome before being taken up into the tumor tissue. The drug may not be utilized, and the efficacy of the drug cannot be fully maximized. Therefore, the application of liposomes is limited.

[0003] Therefore, there is a need in the art to develop liposomes for the effective treatment of tumors. Summary of the Invention

[0004] It is an object of the present invention to provide liposomes for the effective treatment of tumors.

[0005] In a first aspect of the present disclosure, a nanoparticle is provided, the surface of which comprises an antibody.

[0006] The number of antibodies is 5 to 60, preferably 5 to 50, and more preferably 5 to 40.

[0007] In another preferred example, the number of antibodies is 5 to 30, preferably 5 to 25, preferably 5 to 20, more preferably 5 to 15, more preferably 8 to 12, and most preferably 10.

[0008] In another preferred example, the number of antibodies is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40.

[0009] In another preferred embodiment, the outer surface of the nanoparticle comprises an antibody.

[0010] In another preferred embodiment, the antibody is conjugated to a nanomaterial.

[0011] In another preferred embodiment, the nanomaterial comprises a lipid material.

[0012] In another preferred example, the antibody comprises a HER2 antibody (human epidermal growth factor receptor 2 antibody).

[0013] In another preferred example, the HER2 antibody comprises a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a single chain Fv fragment (scFv) of a HER2 antibody.

[0014] In another preferred embodiment, the Fab fragment of the HER2 antibody comprises a heavy chain and a light chain, The heavy chain is selected from the group consisting of: (1) The amino acid sequence shown in SEQ ID NO:1 (2) A group formed by substituting, deleting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:1, (3) A group having 80%, preferably 90%, more preferably 95%, more preferably 98%, or more preferably 99% homology to the amino acid sequence shown in SEQ ID NO:1; The light chain is selected from the group: (a) the amino acid sequence shown in SEQ ID NO:2; (b) a group formed by substituting, deleting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:2; (c) A group having a homology of ≧80%, preferably ≧90%, more preferably ≧95%, more preferably ≧98%, or more preferably ≧99% to the amino acid sequence shown in SEQ ID NO:2.

[0015] In another preferred embodiment, the antibody further comprises a second antibody, wherein the two antibodies are of different types.

[0016] In another preferred example, the second antibody comprises a CD3 antibody (human cluster of differentiation 3 antibody).

[0017] In another preferred example, the CD3 antibody comprises a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a single chain Fv fragment (scFv) of the CD3 antibody.

[0018] In another preferred example, the number of the second antibodies is 0 to 60, preferably 3 to 40, preferably 3 to 36, more preferably 6 to 18, and more preferably 9.

[0019] In another preferred embodiment, the Fab fragment of the CD3 antibody comprises a heavy chain and a light chain, The heavy chain is selected from the group consisting of: (1) the amino acid sequence shown in SEQ ID NO:3; (2) A group formed by substituting, deleting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:3; (3) A group having a homology of ≥80%, preferably ≥90%, more preferably ≥95%, even more preferably ≥98%, or even more preferably ≥99% to the amino acid sequence shown in SEQ ID NO:3; The light chain is selected from the group: (a) the amino acid sequence shown in SEQ ID NO:4; (b) a group formed by substituting, deleting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:4; (c) A group having a homology of ≧80%, preferably ≧90%, more preferably ≧95%, more preferably ≧98%, or more preferably ≧99% to the amino acid sequence shown in SEQ ID NO:4.

[0020] In another preferred embodiment, the number of antibodies on the surface of the nanoparticles is calculated based on the following formula:

number

[0021] In another preferred embodiment, the nanoparticle is a liposome or a nano-sized particle.

[0022] In another preferred embodiment, the nanoparticles are drug-loaded nanoparticles.

[0023] In another preferred embodiment, the nanoparticle is a drug-loaded liposome.

[0024] In another preferred example, the nanoparticle is a drug-loaded liposome whose outer surface is modified with an antibody.

[0025] In another preferred embodiment, the nano-sized particles are drug-loaded nano-sized particles.

[0026] In another preferred example, the payload includes a drug that is encapsulated within a nanoparticle (eg, a liposome or nano-sized particle).

[0027] In another preferred embodiment, the drug includes an anti-tumor drug.

[0028] In another preferred embodiment, the anti-tumor agent is selected from the following group: anthracyclines, platinum-based agents, fluorouracil, camptothecin, taxanes, or combinations thereof.

[0029] In another preferred embodiment, the anti-tumor agent comprises an immunomodulatory agent.

[0030] In another preferred embodiment, the antitumor agent comprises doxorubicin or resiquimod.

[0031] In another preferred embodiment, the liposome comprises a lipid substance.

[0032] In another preferred embodiment, the lipid material is the lipid bilayer material of a liposome.

[0033] In another preferred example, the lipid material comprises one or more of hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-mPEG), phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, cholesterol, polyethylene glycol glycerol fatty acid esters, and polyethylene glycol glyceryl phosphatidylethanolamine.

[0034] In another preferred example, the antibody is conjugated to a lipid substance.

[0035] In another preferred example, the antibody is conjugated to DSPE-mPEG.

[0036] In another preferred example, the antibody is conjugated to the liposome via DSPE-mPEG.

[0037] In another preferred example, the DSPE-mPEG comprises DSPE-mPEG2000.

[0038] In another preferred example, the free thiol group on cysteine ​​at position 229 of the heavy chain of the HER2 antibody is conjugated with DSPE-mPEG.

[0039] In another preferred example, the conjugation method includes: DSPE-PEG reacts with maleimide to form DSPE-PEG-MAL, and then the maleimide group of DSPE-PEG-MAL reacts with the free thiol group on the cysteine ​​at position 229 of the heavy chain of the HER2 antibody, and the HER2 antibody is conjugated to the DSPE-PEG.

[0040] In another preferred example, the particle size of the nanoparticles is 60 to 250 nm.

[0041] In another preferred example, the particle size of the nanoparticles is 80 to 120 nm.

[0042] In another preferred embodiment, the nanoparticles have a PDI of less than 0.1, more preferably less than 0.09, more preferably less than 0.05.

[0043] In another preferred example, in the liposome, the weight ratio of drug to lipid substance (drug-lipid ratio) is 0.02 to 0.1, preferably 0.022 to 0.085, more preferably 0.025 to 0.05, more preferably 0.028 to 0.04, or more preferably 0.033 to 0.037.

[0044] In another preferred example, the liposome is a drug-loaded liposome, and the weight ratio of drug to lipid material (drug-lipid ratio) is 1:1-100, preferably 1:10-70, preferably 1:25-35, or preferably 1:28-32.

[0045] In another preferred example, the antibodies in the nanoparticles are modified using a post-insertion method.

[0046] In another preferred embodiment, the liposomes are prepared by the following method: (i) adding a lipid material to an organic solvent and mixing to obtain a lipid mixture; (ii) adding the lipid mixture to the aqueous phase to obtain a blank liposome solution; (iii) adding the drug solution to the empty liposome solution and incubating to obtain drug-loaded liposomes; (iv) The antibody-conjugated lipid material and the drug-loaded liposome mixture are mixed and incubated to obtain liposomes.

[0047] In another preferred embodiment, the lipid material in step (i) is selected from the following group: HSPC, Cholesterol (Chol), and DSPE-mPEG, or a combination thereof.

[0048] In another preferred example, the weight ratio of HSPCs to cholesterol is 2-4:1.

[0049] In another preferred example, the weight ratio of HSPC to DSPE-mPEG is 3 to 20:1.

[0050] In another preferred example, the weight ratio of HSPC to DSPE-mPEG is 13-17:1.

[0051] In another preferred embodiment, the organic solvent in step (ii) comprises ethanol.

[0052] In another preferred embodiment, the aqueous phase in step (ii) is water or an aqueous ammonium sulfate solution.

[0053] In another preferred example, the concentration of the aqueous ammonium sulfate solution in step (ii) is 100 to 450 mM.

[0054] In another preferred example, the concentration of the aqueous ammonium sulfate solution in step (ii) is 150 to 270 mM.

[0055] In another preferred example, the pH of the aqueous ammonium sulfate solution in step (ii) is 4.0 to 6.0.

[0056] In another preferred example, the pH of the aqueous ammonium sulfate solution in step (ii) is 4.8 to 5.2.

[0057] In another preferred embodiment, in step (ii), the lipid mixture is added to an aqueous phase, extruded with stirring, and then dialyzed to obtain an empty liposome solution.

[0058] In another preferred example, dialysis is carried out in a HEPES buffer (8-12 mM, pH 6.6-6.8).

[0059] In another preferred example, the volume ratio of the organic solvent to the aqueous phase is 1:8-12.

[0060] In another preferred embodiment, in step (iii), the weight ratio of the drug to the empty liposomes is: The (drug-lipid ratio) is 0.02 to 0.1, preferably 0.022 to 0.085, more preferably 0.025 to 0.05, more preferably 0.028 to 0.04, or more preferably 0.033 to 0.037.

[0061] In another preferred example, in step (iii), the weight ratio of the drug to the empty liposome (drug-lipid ratio) is 1:25-35, preferably 1:28-32.

[0062] In a second aspect of the present disclosure, a composition is provided, the composition comprising nanoparticles according to the first aspect of the present disclosure.

[0063] In another preferred embodiment, the composition is a pharmaceutical composition.

[0064] In another preferred embodiment, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier.

[0065] In another preferred example, the dosage form of the pharmaceutical composition is an oral formulation, an injectable formulation, or a topical formulation.

[0066] In another preferred example, the dosage form of the pharmaceutical composition is selected from the following: tablets, capsules, granules, suspensions, pills, solutions, syrups, or injections.

[0067] In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection solution.

[0068] In another preferred example, the injection solution is selected from the following: a liquid formulation, a suspension formulation, and a lyophilized powder for injection.

[0069] In another preferred example, the injection is selected from the following: intravenous injection, subcutaneous injection, and in situ injection.

[0070] In a third aspect of the present disclosure, there is provided a use of a nanoparticle according to the first aspect of the present disclosure, or a composition according to the second aspect of the present disclosure, for the preparation of a medicament for use in the prevention and / or treatment of a tumor.

[0071] In another preferred embodiment, the tumor is selected from the following group: breast cancer, gastric cancer, ovarian cancer, liver cancer, or a combination thereof.

[0072] In another preferred embodiment, the tumor comprises a receptor-expressing tumor.

[0073] In another preferred embodiment, the receptor comprises HER2.

[0074] In another preferred embodiment, the receptor is conjugated to an antibody.

[0075] In another preferred example, the conjugate comprises a specific bond.

[0076] In another preferred embodiment, the tumor comprises a HER2-overexpressing tumor.

[0077] In another preferred example, HER2 expression in tumor cells is A1, and overexpression of HER2 means that A1 / A0>1.2, preferably >1.5, compared to HER2 expression of A0 in normal cells.

[0078] In another preferred embodiment, the tumor comprises a tumor that is multi-drug resistant to an anti-tumor agent.

[0079] In a fourth aspect of the present disclosure, there is provided a method of preventing and / or treating a tumor by administering to a subject in need thereof a nanoparticle according to the first aspect of the present disclosure, or a composition according to the second aspect of the present disclosure.

[0080] In another preferred example, the subject comprises a human or non-human mammal.

[0081] It should be understood that within the scope of the present disclosure, the various technical features described above and specifically described in the following sections (e.g., embodiments, etc.) may be combined with each other to form novel or preferred technical solutions, which will not be listed in detail herein due to space limitations. [Brief description of the drawings]

[0082] [Figure 1A] FIG. 1A shows the results of non-reducing SDS-PAGE detection of the conjugation between HER2 Fab and DSPE-PEG2000-MAL. [Figure 1B] FIG. 1B shows the results of RP-HPLC detection of the conjugation between HER2 Fab and DSPE-PEG2000-MAL. [Figure 1C] FIG. 1C shows the results of non-reducing SDS-PAGE detection of the purification of aHER2-DSPE. [Figure 1D] FIG. 1D shows the results of RP-HPLC detection of the purification of aHER2-DSPE. [Diagram 2] Figure 2 shows the particle size distribution of doxorubicin liposomes (aHER2-LDX) with different numbers of antibodies inserted. Figures 2A, 2B, and 2C show the particle size distribution of aHER2-LDX with antibody numbers of 10, 20, and 40, respectively. [Diagram 3]FIG. 3 shows the post-insertion efficiency of aHER2-DSPE after insertion into LDX as detected by Sepharose 4B gel column. [Figure 4] Figure 4 shows flow cytometry detection of HER2 antigen expression on the surface of SK-Br-3, BT474, NCI-N87, MDA-MB-453, MCF-7, MDA-MB-231, MGC-803, and A2780 cells. rMFI indicates relative mean fluorescence intensity. [Figure 5A] FIG. 5A shows inhibition of tumor cell proliferation by 10-C aHER2-LDX, 20-C aHER2-LDX, 10-B aHER2-LDX, 20-B aHER2-LDX, and A-LDX in the HER2-overexpressing cell line SK-Br-3. [Figure 5B] FIG. 5B shows inhibition of tumor cell proliferation by 10-C aHER2-LDX, 20-C aHER2-LDX, 10-B aHER2-LDX, 20-B aHER2-LDX, and A-LDX in the HER2-overexpressing cell line NCI-N87. [Figure 5C] FIG. 5C shows inhibition of tumor cell proliferation by 10-C aHER2-LDX, 20-C aHER2-LDX, 10-B aHER2-LDX, 20-B aHER2-LDX, and A-LDX in the HER2-overexpressing cell line BT474. [Figure 6] Figure 6 shows the affinity activity of HER2 antibody-immunoliposomes as detected by Gator label-free bioanalyzer. The lipid concentration on the left is 0.5 mg / mL, and on the right is 1 mg / mL. [Figure 7A] Figure 7A shows the in vitro binding intensity of HER2Fab immunofluorescence liposome aHER2DiI-Lip to the HER2-overexpressing cell line SK-Br-3 at various antibody densities. aHER2DiI-Lip-3 represents fluorescently labeled aHER2-LDX conjugated to three HER2Fab antibody fragments, etc. [Figure 7B]Figure 7B shows the in vitro binding intensity of immunofluorescence liposomal aHER2DiI-Lip of HER2Fab to the HER2-negative cell line MDA-MB-231 at various antibody densities. aHER2DiI-Lip-3 represents fluorescently labeled aHER2-LDX conjugated to three HER2Fab antibody fragments, etc. [Figure 8] FIG. 8 shows the uptake and drug release of HER2 antibody-conjugated doxorubicin-loaded liposomes by the HER2-overexpressing cell line BT474. [Figure 9] FIG. 9 shows the cytotoxic effect of HER2Fab antibody-targeted doxorubicin-loaded liposomes against doxorubicin-resistant breast cancer cells MCF-7 / Adr. [Figure 10] FIG. 10 shows the pharmacological results of aHER2-LDX in a HER2-overexpressing BT474 breast cancer xenograft model in Balb / C-Nude mice. [Figure 11] FIG. 11 shows the pharmacological results of aHER2-LDX in a HER2-overexpressing NCI-N87 gastric cancer xenograft model in Balb / C-Nude mice. [Figure 12] FIG. 12 shows the pharmacological results of aHER2-LDX in a xenograft model of HER2-low-expressing MCF-7 breast cancer in Balb / C-Nude mice. [Figure 13A] FIG. 13A shows the pharmacokinetics of aHER2-LDX with respect to the agent doxorubicin in BT474 tumor-bearing mice. [Figure 13B] FIG. 13B shows the pharmacokinetics of aHER2-LDX relative to HER2Fab in BT474 tumor-bearing mice. [Figure 14A] FIG. 14A shows in vitro activation of CD25 expression on the surface of Jurkat cells by CD3×HER2 dual-targeted liposomes at different densities. [Figure 14B] FIG. 14B shows in vitro activation of CD69 expression on the surface of Jurkat cells by CD3×HER2 dual-targeted liposomes at different densities. [Figure 15]Figure 15 shows the cytotoxicity of pre-activated effector cells (PBMCs) mediated by CD3 x HER2 dual-targeted liposomes against SK-Br-3 target cells at different densities, as detected by LDH assay 48 hours after drug exposure. [Figure 16] Figure 16 shows the expression of CD25 and CD69 on the T cell surface mediated by CD3 x HER2 dual targeted liposomes in co-cultures of target cells SK-Br-3 and pre-activated effector cells (PBMC) at different densities. Detected by flow cytometry 48 hours after drug exposure. (A) CD25 expression in CD4 positive cells. (B) CD69 expression in CD4 positive cells. (C) CD25 expression in CD8 positive cells. (D) CD69 expression in CD8 positive cells. [Figure 17] FIG. 17 shows the pharmacological study results of CD3×HER2 dual-targeted liposomes and CD3×HER2 dual-targeted R848-loaded liposomes in a humanized model of HER2-overexpressing NCI-N87 gastric cancer cells in NCG-B2M-KO severely immunodeficient mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0083] Through extensive and detailed research, the present inventors have developed nanoparticles, such as liposomes, whose surface contains antibodies. Research has shown that a liposome surface containing 10-40 liposomes can exert a more effective therapeutic effect on tumors and reduce the multidrug resistance of tumors. The present invention has been completed based on this fact.

[0084] term As used herein, the term "pharmaceutical composition of the present disclosure" refers to a pharmaceutical composition comprising an antitumor drug and a liposome, the composition having a drug-lipid ratio in the range of 0.01 to 0.15, preferably 0.02 to 0.1.

[0085] As used herein, human epidermal growth factor receptor 2, abbreviated as HER2, aHER2, or [sic]HER2, is also referred to as Neu or HER2 / neu.

[0086] As used herein, representative names for individual cell lines are provided in Table A below: [Table 1]

[0087] antibody In a preferred example of the disclosure, the antibodies described herein include HER2 antibodies (human epidermal growth factor receptor 2 antibodies).

[0088] The basic unit structure of a complete antibody consists of four peptide chains, including two heavy chains and two light chains linked by disulfide bonds. The shape of an antibody resembles the letter Y, and the hinge region of the Y structure is flexible. Each peptide chain has a constant region (highly conserved in all antibodies) and a variable region (specific to a particular antibody). The light chain variable region is named VL, and the light chain constant region is named CL. Similarly, the variable and constant regions of the heavy chain are named VH and CH, respectively. Carbohydrates are typically attached to the CH2 region of the heavy chain. The Fc region contains only the constant region (CH) of the heavy chain, while the antigen-binding Fab region (Fab) contains the variable regions of the heavy and light chains, as well as the constant regions connected to the variable regions of both the heavy and light chains.

[0089] In preferred examples of the present disclosure, the HER2 antibody comprises a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a single chain Fv fragment (scFv) of a HER2 antibody.

[0090] Typically, the Fab fragment of the HER2 antibody contains a heavy chain and a light chain, The heavy chain is selected from the group consisting of: (1) The amino acid sequence shown in SEQ ID NO:1 (2) A group formed by substituting, deleting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:1, (3) a group having a homology of ≥80%, preferably ≥90%, more preferably ≥95%, more preferably ≥98%, or more preferably ≥99% to the amino acid sequence shown in SEQ ID NO:1; The light chain is selected from the group: (a) the amino acid sequence shown in SEQ ID NO:2; (b) a group formed by substituting, deleting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:2; (c) A group having a homology of ≧80%, preferably ≧90%, more preferably ≧95%, more preferably ≧98%, or more preferably ≧99% to the amino acid sequence shown in SEQ ID NO:2.

[0091] Nanoparticles The present disclosure provides a nanoparticle, the surface of which comprises an antibody.

[0092] The number of antibodies is 5 to 60, preferably 5 to 50, and more preferably 5 to 40.

[0093] In a preferred example of the present disclosure, the number of antibodies is 5 to 30, preferably 5 to 25, preferably 5 to 20, more preferably 5 to 15, more preferably 8 to 12, and most preferably 10.

[0094] Typically the number of antibodies is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40.

[0095] In a preferred example of the present disclosure, the outer surface of the nanoparticle comprises an antibody.

[0096] In a preferred example of the present disclosure, the antibody is conjugated to a nanomaterial.

[0097] In a preferred embodiment of the present disclosure, the nanomaterial comprises a lipid material.

[0098] In a preferred example of the present disclosure, the nanoparticles are liposomes or nano-sized particles.

[0099] In another preferred embodiment, the nanoparticles are drug-loaded nanoparticles.

[0100] In another preferred embodiment, the nanoparticle is a drug-loaded liposome.

[0101] In another preferred embodiment, the nano-sized particles are drug-loaded nano-sized particles.

[0102] In a preferred example of the present disclosure, the payload includes a drug that is encapsulated within a nanoparticle (eg, a liposome or nano-sized particle).

[0103] In a preferred embodiment of the present disclosure, the drug comprises an anti-tumor drug.

[0104] Typically, the anti-tumor agent is selected from the following group: anthracyclines, platinum-based agents, fluorouracil, camptothecins, taxanes, or combinations thereof.

[0105] Typically, the anti-tumor agent includes doxorubicin.

[0106] In a preferred embodiment of the present disclosure, the liposome comprises a lipid material.

[0107] In a preferred embodiment of the present disclosure, the lipid material is a lipid bilayer material of a liposome.

[0108] Typically, the lipid material comprises one or more of HSPC, DSPE-mPEG2000, phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, cholesterol, polyethylene glycol glycerol fatty acid esters, and polyethylene glycol glyceryl phosphatidylethanolamine.

[0109] In a preferred example of the present disclosure, the antibody is conjugated to a lipid substance.

[0110] Typically, the antibody is conjugated to DSPE-PEG.

[0111] Typically, the free thiol group on cysteine ​​at position 229 of the heavy chain of the HER2 antibody is conjugated to DSPE-mPEG.

[0112] In a preferred example of the present disclosure, the conjugation method includes: DSPE-PEG reacts with maleimide to form DSPE-PEG-MAL, and then the maleimide group of DSPE-PEG-MAL reacts with the free thiol group on the cysteine ​​at position 229 of the heavy chain of the HER2 antibody, and the HER2 antibody is conjugated to the DSPE-PEG.

[0113] In another preferred example, the particle size of the nanoparticles is 60 to 250 nm.

[0114] In another preferred example, the particle size of the nanoparticles is 80 to 100 nm.

[0115] In a preferred example of the present disclosure, in the liposome, the weight ratio of drug to lipid substance (drug-lipid ratio) is 0.02 to 0.1, preferably 0.022 to 0.085, more preferably 0.025 to 0.05, more preferably 0.028 to 0.04, or more preferably 0.033 to 0.037.

[0116] In a preferred embodiment of the present disclosure, the weight ratio of drug to lipid substance (drug:lipid ratio) in the liposome is 1:25-35, preferably 1:28-32.

[0117] In a preferred example of the present disclosure, the liposomes are made by the following method: (i) adding a lipid material to an organic solvent and mixing to obtain a lipid mixture; (ii) adding the lipid mixture to the aqueous phase to obtain a blank liposome solution; (iii) adding the drug solution to the empty liposome solution and incubating to obtain drug-loaded liposomes; (iv) The antibody-conjugated lipid material and the drug-loaded liposome mixture are mixed and incubated to obtain liposomes.

[0118] In another preferred embodiment, the lipid material in step (i) is selected from the following group: HSPC, Chol, and DSPE-mPEG2000, or a combination thereof.

[0119] In another preferred example, the weight ratio of HSPCs to cholesterol is 2-4:1.

[0120] In another preferred example, the weight ratio of HSPC to DSPE-mPEG2000 is 3 to 20:1.

[0121] In another preferred example, the weight ratio of HSPC to DSPE-mPEG2000 is 15 to 17:1.

[0122] In another preferred embodiment, the organic solvent in step (ii) comprises ethanol.

[0123] In another preferred embodiment, the aqueous phase in step (ii) is water or an aqueous ammonium sulfate solution.

[0124] In another preferred example, the concentration of the aqueous ammonium sulfate solution in step (ii) is 100 to 450 mM.

[0125] In another preferred example, the concentration of the aqueous ammonium sulfate solution in step (ii) is 230 to 270 mM.

[0126] In another preferred example, the pH of the aqueous ammonium sulfate solution in step (ii) is 4.0 to 6.0.

[0127] In another preferred example, the pH of the aqueous ammonium sulfate solution in step (ii) is 4.8 to 5.2.

[0128] In another preferred embodiment, in step (ii), the lipid mixture is added to an aqueous phase, extruded with stirring, and then dialyzed to obtain an empty liposome solution.

[0129] In another preferred example, dialysis is carried out in a HEPES buffer (8-12 mM, pH 6.6-6.8).

[0130] In another preferred example, the volume ratio of the organic solvent to the aqueous phase is 1:8-12.

[0131] In another preferred example, in step (iii), the weight ratio of the drug to the empty liposomes (drug-lipid ratio) is 0.02 to 0.1, preferably 0.022 to 0.085, more preferably 0.025 to 0.05, more preferably 0.028 to 0.04, or more preferably 0.033 to 0.037.

[0132] In another preferred example, in step (iii), the weight ratio of the drug to the empty liposome (drug-lipid ratio) is 1:25-35, preferably 1:28-32.

[0133] composition The present disclosure further provides a composition, the composition comprising nanoparticles according to the present disclosure.

[0134] In a preferred embodiment of the present disclosure, the composition is a pharmaceutical composition.

[0135] In a preferred embodiment of the present disclosure, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" ingredient is a substance suitable for use in humans and / or mammals without excessive adverse side effects (e.g., toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. Such pharma- ceutically acceptable carriers include, but are not limited to, saline, buffer, glucose, water, and combinations thereof. In general, a pharmaceutical formulation must be compatible with the mode of administration. The dosage form of the pharmaceutical composition of the present disclosure is selected from the following: tablets, capsules, granules, suspensions, pills, solutions, syrups, or injections.

[0136] In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection solution.

[0137] In another preferred embodiment, the injection solution is selected from an injection solution, a suspension, and a lyophilized powder for injection.

[0138] In another preferred example, the injection is selected from the following: intravenous injection, subcutaneous injection, and in situ injection.

[0139] The preparation is effected in the conventional manner using saline or aqueous solutions containing glucose or other adjuvants.The pharmaceutical compositions are preferably prepared under sterile conditions.

[0140] The effective amount of the active ingredient of the present disclosure may vary depending on the mode of administration and the severity of the disease being treated. The selection of a preferred effective amount can be determined by a person skilled in the art based on various factors (e.g., clinical trials). Such factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, the severity of the disease of the patient being treated, the patient's body weight, the patient's immune status, the route of administration, and the like. In general, satisfactory results can be obtained when the active ingredient of the present disclosure is administered at a dose of about 0.00001 mg to 50 mg / kg of animal body weight (preferably 0.0001 mg to 10 mg / kg of animal body weight) per day. For example, several individual doses may be given daily, or the dose may be proportionally reduced, as required by the exigencies of the treatment situation.

[0141] Typically, when the pharmaceutical composition of the present disclosure is administered orally, the average daily dose is 10-500 mg, preferably 20-300 mg, more preferably 50-250 mg for a 60 kg human subject. The daily dose may be taken in one, two or more administrations.

[0142] Pharmaceutically acceptable carriers according to the present disclosure include (but are not limited to) water, saline, nanogel, or combinations thereof. The choice of carrier should be compatible with the mode of administration, which is known to those skilled in the art.

[0143] In a preferred embodiment of the present disclosure, the pharmaceutical composition is in the form of an oral formulation, an injectable formulation, or a topical formulation.

[0144] In a preferred example of the present disclosure, the dosage form of the pharmaceutical composition is selected from the following: tablets, capsules, granules, suspensions, pills, solutions, syrups, or injections.

[0145] In a preferred embodiment of the present disclosure, the dosage form of the pharmaceutical composition is an injection solution.

[0146] In a preferred example of the present disclosure, the injection solution is selected from the following: a liquid formulation, a suspension formulation, and a lyophilized powder for injection.

[0147] In a preferred example of the present disclosure, the mode of administration of the injection is selected from the following: intravenous injection, subcutaneous injection, and in situ injection.

[0148] tumor The tumor according to the present disclosure is preferably selected from the following group: breast cancer, gastric cancer, ovarian cancer, liver cancer, or a combination thereof.

[0149] In a preferred example of the present disclosure, the tumor comprises a receptor-expressing tumor.

[0150] In another preferred embodiment, the receptor comprises HER2.

[0151] In another preferred embodiment, the receptor is conjugated to an antibody.

[0152] In another preferred example, the conjugate comprises a specific bond.

[0153] In a preferred example of the present disclosure, the tumor comprises a HER2-overexpressing tumor.

[0154] In a preferred example of the present disclosure, HER2 expression in tumor cells is A1, and overexpression of HER2 means that A1 / A0>1.2, preferably >1.5, compared to HER2 expression of A0 in normal cells.

[0155] In a preferred embodiment of the present disclosure, the tumor comprises a tumor that is multi-resistant to anti-tumor drugs.

[0156] Uses and Methods The present disclosure further provides the use of a nanoparticle or a composition according to the present disclosure for the preparation of a medicament for use in the prevention and / or treatment of a tumor.

[0157] The present disclosure further provides a method for preventing and / or treating tumors by administering to a subject in need thereof a nanoparticle or a composition according to the present disclosure.

[0158] In a preferred embodiment of the present disclosure, the subject includes a human or non-human mammal.

[0159] Key advantages of the present disclosure include: In the present disclosure, nanoparticles, such as liposomes, are developed. When the surface of nanoparticles, such as liposomes, contains 10-40 liposomes, it can exert a more efficient therapeutic effect on tumors and reduce the multidrug resistance of tumors. This breaks the commonly accepted technical prejudice that the presence of many antibodies on the liposome surface improves the binding with target cells and lays the foundation for further clinical development.

[0160] The present disclosure will be further described below with specific embodiments.It should be understood that these embodiments are provided only for the purpose of illustrating the present disclosure, and are not intended to limit the scope of the present disclosure.In the following embodiments, the experimental method without specific conditions is typically carried out under conventional conditions or as recommended by the manufacturer.Unless otherwise stated, percentage percentage and weight fraction are weight percentage and weight part.

[0161] Embodiment The heavy chain amino acid sequence of the Fab fragment of the HER2 antibody (aHER2 Fab) is shown in SEQ ID NO: 1, in which the cysteine ​​(C) at position 229 of the heavy chain contains a free thiol group connected to a linker (DSPE-PEG-MAL), and the light chain amino acid sequence is shown in SEQ ID NO: 2. SEQ ID NO:1 (N-terminus to C-terminus): EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCAA SEQ ID NO:2 (N-terminus to C-terminus): DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0162] The heavy chain amino acid sequence of the Fab fragment of the CD3 antibody (aCD3 Fab) is shown in SEQ ID NO: 3, in which C at position 231 of the heavy chain contains a free thiol group connected to a linker (DSPE-PEG-MAL), and the light chain amino acid sequence is shown in SEQ ID NO: 4. SEQ ID NO:3 (N-terminus to C-terminus): EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVTTYADSVKGRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCAA SEQ ID NO:4 (N-terminus to C-terminus): DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0163] 1. Preparation of liposomes encapsulating doxorubicin (LDX) 900mg HSPC, 300mg cholesterol, and 60mg DSPE-mPEG2000 were added to 2mL absolute ethanol and heated to 60℃ in a water bath. Then a magnetic stirrer was added to stir until completely dissolved, and a clear liquid mixture was obtained, which was the lipid mixture. At the same time, 3.304g ammonium sulfate was placed in a glass bottle, 100mL ultrapure water was added, stirred until dissolved, and then the pH was adjusted to 5.0 to obtain an aqueous solution of ammonium sulfate with a concentration of 250mM. This solution was prepared immediately before use. The dissolved lipid mixture was injected into 19mL of aqueous ammonium sulfate solution using a syringe, then stirred and extruded six times to perform dialysis in HEPES buffer (10mM, pH6.8) using a 10kD dialysis membrane to obtain empty liposomes with an external aqueous phase of HEPES solution.

[0164] According to the concentrations of drug and empty liposomes listed in Table 1, 10 mg / mL doxorubicin stock solution was added to the corresponding doxorubicin stock solution and the above empty liposome suspension and incubated with stirring at 60° C. for 30 min. After drug loading was completed, a 100 kD dialysis membrane was used to remove free drug to produce formulations A, B, C and D of doxorubicin-loaded liposomes, denoted as A-LDX, B-LDX, C-LDX and D-LDX.

[0165] A Nano-S90 nanoparticle size analyzer was used for detection. For the various formulations, the particle size of doxorubicin-loaded liposomes was about 85 nm, with a polydispersity index (PDI) of less than 0.1. By electron microscopy, doxorubicin-loaded liposome formulations A, B, C, and D all had a round shape and uniform size distribution. [Table 2]

[0166] 2. Preparation of immunoliposomes modified with HER2 Fab antibody fragments (aHER2-LDX) 2.1 Preparation of HER2 Fab antibody fragment-liposome molecule (aHER2-DSPE) At position 229 of the heavy chain, C of the HER2 Fab antibody contains a free thiol group, which is exposed by reduction at a specific site. The free thiol group chemically reacts with the maleimide group of the lipid molecule DSPE-PEG-MAL to generate the HER2 Fab antibody fragment lipid molecule (represented as: aHER2-DSPE). The specific procedure is as follows: First, the concentration of HER2 Fab antibody was determined by ultraviolet spectrophotometry or BCA method. Then, reduction was performed using β-mercaptoethylamine (β-MEA) reducing agent. HER2 Fab antibody and β-MEA were mixed in a molar ratio of 10:1, and HER2 Fab antibody was reduced at room temperature for about 45-90 minutes. After reduction, desalting was performed using a Zeb desalting column or an AKTA instrument to remove the reducing agent from the solution. DSPE-PEG-MAL and DSPE-mPEG2000 were mixed in a molar ratio of 1:1, dissolved in 30 mM HEPES solution or PBS solution (containing 2 mM EDTA), and the pH was adjusted to 6.5-6.8 to obtain a mixed micelle solution of DSPE-PEG2-MAL and DSPE-mPEG2000. Then, HER2 Fab antibody and DSPE-PEG-MAL were added separately in a molar ratio of 1:1. The reaction was carried out at 4°C for 2 hours to obtain aHER2-DSPE micelles. Then, L-cysteine ​​was added to a final concentration of 1 mM to block any unreacted DSPE-PEG-MAL, and the mixture was incubated at 4°C for less than 16 hours. The reaction of HER2 Fab antibody with DSPE-PEG-MAL was not completed, so the resulting mixture consisted of HER2 Fab antibody, DSPE-PEG-MAL, DSPE-mPEG2000, and conjugated aHER2-DSPE. Therefore, purification was then carried out using gel columns and ion exchange columns to obtain aHER2-DSPE single micelles.

[0167] The conjugate aHER2-DSPE was characterized by non-reducing SDS-PAGE and RP-HPLC.

[0168] The results in Figure 1A from non-reducing SDS-PAGE gel analysis indicate that a 1:1 molar ratio is the preferred reaction ratio for HER2 Fab antibody and DSPE-PEG-MAL. The results in Figure 1B are detected by RP-HPLC and show that the conjugation efficiency is greater than 40% when the molar ratio of HER2 Fab antibody to DSPE-PEG-MAL is 1:1. In Figures 1C and 1D, the purified aHER2-DSPE is characterized using non-reducing SDS-PAGE and RP-HPLC methods and shows a purity of greater than 95%.

[0169] 2. Preparation of doxorubicin-loaded liposomes modified with HER2 Fab antibody fragments (aHER2-LDX) To accurately create docetaxel-loaded liposomes with specific numbers of HER2 Fab antibodies, a computational method is provided as detailed below: calculating on the basis that the surface of one liposome is conjugated with X antibody molecules; Known parameters: (1) the average relative molecular weight of the protein, expressed as MW; (2) lipid mass, represented as Y; (3) the average relative molecular weight of lipids, expressed as L (calculated based on the lipid ratios); (4) On average, each liposome contains N lipid molecules (calculated based on the average liposome size); (5) Avogadro steady state NA The amount of lipid is Y, and to create each liposome with X antibody molecules on its surface, the required amount of antibody, denoted as P, is: Calculation formula:

number

[0170] Figures 2A-2C and Table 2 list the average particle size and PDI of aHER2-LDX with 10, 20 and 40 antibody numbers, respectively. The average particle size of all three is about 80-100 nm, and the PDI is less than 0.1. Moreover, the average particle size is independent of the number of antibodies immobilized on the liposome surface. [Table 3]

[0171] Furthermore, the present disclosure provides a method for detecting the post-insertion efficiency of aHER2-DSPE after insertion into liposomes, the specific procedure is as follows: Sepharose 4B packing was loaded into an 8 mm x 105 mm gel column, and the column was equilibrated with PBS buffer. 120 μL of liposomes post-inserted with aHER2-LDX were loaded onto the top of the column, and the eluent was collected by gravity. Free aHER2-DSPE and aHER2-LDX liposomes could be completely separated. Quantitative analysis of aHER2-DSPE was performed using RP-HPLC to determine the amount of free aHER2-DSPE by M free aHER2-DSPE on aHER2-LDX liposome is expressed as M post-insertion The post-insertion efficiency E post-insertion =M post-insertion / (M free +M post-insertion )×100%.

[0172] Experimental results show that the post-insertion efficiency of aHER2-DSPE after insertion into LDX is greater than 95% (shown in FIG. 3), suggesting the superiority of the post-insertion method provided in this disclosure for generating antibody-modified drug-loaded liposomes.

[0173] 3. HER2 expression in different tumor cell lines The expression of HER2 antigen on the cell surface of SK-Br-3, BT474, NCI-N87, MDA-MB-453, MCF-7, MDA-MB-231, MGC-803, and A2780 was detected using flow cytometry. The results are shown in Figure 4 and Table 3. [Table 4]

[0174] As can be seen from the above experimental data, SK-Br-3, NCI-N87, and BT474 are HER2-overexpressing cell lines, MDA-MB-453 is a cell line that moderately expresses HER2, and MCF-7, A2780, MGC-803, and MDA-MB-231 are HER2 low-expressing / negative cell lines.

[0175] 4. Formulation screening of immunoliposomes modified with HER2 antibody (aHER2-LDX) Using the feedback system control (FSC) described in the paper “Optimization of drug combinations using Feedback System Control. Nature Protocols. 2016;11:302-315”, a total of 10 parameters, including the number of aHER2 Fab antibodies on the surface of aHER2-LDX (0, 10, 20, 40, 80), LDX formulations (A, B, C, D), and empty liposomes (E), were evaluated to evaluate the cytotoxicity (IC) of drugs against HER2-overexpressing and HER2-low expressing cells. 50 ) was examined in the evaluation.

[0176] 4.1. Creation of aHER2-LDX with various drug combinations By adjusting the amounts of aHER2-DSPE and doxorubicin-loaded liposomes added in the above-mentioned aHER2-LDX preparation process, aHER2-LDX with 0, 10, 20, 40 and 80 aHER2 Fab antibody numbers on the surface were prepared, and designated as LDX formulations A, B, C and D, respectively, and empty liposome E. A total of 25 combinations were prepared by combining pairs of these 10 parameters as follows: A-LDX, B-LDX, C-LDX, D-LDX, empty liposome E, 10-D aHER2-LDX, 10-C aHER2-LDX, 10-B aHER2-LDX, 10-A aHER2-LDX, 20-D aHER2-LDX, 20-C aHER2-LDX, 20-B aHER2-LDX, 20-A aHER2-LDX, 40-D aHER2-LDX, 40-C aHER2-LDX, 40-B aHER2-LDX, 40-A aHER2-LDX, 80-D aHER2-LDX, 80-C aHER2-LDX, 80-B aHER2-LDX, 80-A aHER2-LDX, 10-E aHER2-LDX, 20-E aHER2-LDX, 40-E aHER2-LDX, 80-E aHER2-LDX (Note: the number before the hyphen represents the number of HER2 Fab antibodies. The letters after the hyphen represent the various LDX formulations. See Table 1 above for formulation information).

[0177] 4.2. Cytotoxicity experiments to evaluate the cytotoxic effect of aHER2-LDX The cytotoxic effect of HER2-LDX on tumor cells was evaluated using Promega's CellTiter-Glo® Luminescent Cell Viability Assay kit. The specific procedure is as follows: various tumor cells were digested with trypsin one day in advance, and then seeded into 96-well clear-bottom black plates at a cell density of 5,000-10,000 cells per well in 100 μL of culture medium. After the cells were fully attached to the well surface, drugs were added according to a concentration series of 0, 0.25, 0.5, 1, 2, 5, 10, 20, 50, 100, and 150 μg / mL, and for each concentration, a total volume of culture medium and three replicate wells were prepared. The plates were then incubated at 37°C in an incubator for 24 hours. After 24 hours, the drug-containing culture medium was removed, and fresh complete culture medium was added to continue the incubation for 48 hours. CellTiter-Glo® buffer was prepared in advance and equilibrated to room temperature. Meanwhile, the CellTiter-Glo® lyophilized substrate was also equilibrated to room temperature. The CellTiter-Glo® buffer was transferred to the CellTiter-Glo® substrate, and the solution was mixed by shaking or inversion to make it uniform. The CellTiter-Glo® substrate was completely dissolved within 1 minute. The 96-well cell culture plate was then removed from the cell culture incubator and equilibrated to room temperature for approximately 30 minutes. Control wells (no cells) containing culture medium only were prepared to obtain background luminescence. Then, 100 μL of CellTiter-Glo® reagent was added to each well, the 96-well cell culture plate was placed on a horizontal shaker, and the contents were mixed for 2 minutes to promote cell lysis. The plate was then left at room temperature for 10 minutes until the fluorescent signal stabilized. After the signal stabilized, the luminescence values ​​were recorded using a TECAN Spark 10M microplate reader.

[0178] Statistical analysis was performed on the recorded luminescence signal values ​​using Graph Prism 8.0 analysis software to determine the IC for each drug combination. 50The values ​​were calculated. The results are shown in Tables 4-1, 4-2, and 4-3. Figures 5A, 5B, and 5C show the inhibition of tumor cell growth by 10-C aHER2-LDX, 20-C aHER2-LDX, 10-B aHER2-LDX, 20-B aHER2-LDX, and Formulation A-LDX in the HER2-overexpressing cell lines SK-Br-3, NCI-N87, and BT474, respectively.

[0179] These data clearly show that: HER2 Fab antibody itself has minimal cytotoxicity. The cytotoxic effect can be enhanced by lowering the drug lipid concentration ratio. The drug toxicity to cells can be further enhanced by surface modification of LDX with HER2 Fab antibody fragments. Taken together, the above results indicate that 10-40 antibody numbers are the preferred combination, and furthermore, 10-C aHER2-LDX showed the best results for the drug combination. Experiments were performed on cell lines with high, medium, and low HER2 expression. The results showed that aHER2-LDX exhibited strong cytotoxicity across the entire HER2 expression spectrum, as detailed in Table 4-4. [Table 5] [Table 6] [Table 7] [Table 8]

[0180] 5. Investigation of the binding activity of liposomes with different antibody numbers The binding activity of immunoliposomes with different antibody numbers and probes with different HER2 antigen levels was analyzed and observed under in vitro conditions using a Gator label-free bioanalyzer. The specific procedures are as follows: (1) The samples and buffer to be analyzed were added to the corresponding 96-well plates, and the probes were placed in the corresponding wells. (2) The temperature of the plate was set to the current temperature of the shaker, and the speed of shaker B was set to 1,000 rpm, whereby the new probes were pre-wetted in K buffer for 3 minutes. (3) Test procedure settings: 1) Criterion 1: K buffer, 120 seconds; 2) Loading: Human Her2 / ErbB2(23-450) protein, Fc Tag, 240 seconds; 3) Criterion 2: K buffer, 120 seconds; 4) Binding: Approximately 200 seconds for the sample solution to be analyzed (until the binding curve is stable); 5) Dissociation: Approximately 200 seconds for K buffer (until the dissociation curve is stable); 6) Regeneration: RE buffer, 5 seconds, Q buffer, 5 seconds, one cycle for each group for a total of three cycles.

[0181] The binding-dissociation curves were fitted and analyzed using Gator analysis software. The results are shown in Figure 6. From Figure 6, it can be observed that the number of bound liposomes increases with increasing antigen levels. However, the binding kinetics did not show a strong correlation with lipid concentration and antigen levels. By increasing the number of antibodies on the liposome surface, the binding rate can be accelerated, but the overall binding capacity is not significantly affected.

[0182] 6. Investigating the binding ability of aHER2-Lip conjugated to HER2 antibodies with different numbers of Fab fragments to tumor cells DiI fluorochrome-labeled liposomes were prepared (denoted as DiI-Lip) and surface-modified with 3, 6, 13, 25, 50, 100, and 200 aHER2 Fab antibody fragments (denoted as aHER2-DiI-Lip-x, where x represents the number of antibody fragments modified). Flow cytometry was used to analyze binding to both the HER2-overexpressing cell line SK-Br-3 and the HER2-low / negative cell line MDA-MB-231.

[0183] The prepared aHER2-DiI-Lip liposomes were adjusted to a lipid concentration of 0.5 mg / mL. Then, 10 μL of the sample was diluted with 2.5 × 10 5 SK-Br-3 cells, and 2.5 × 10 5 The cells were mixed with 100 MDA-MB-231 cells separately and then incubated in the dark at 4 °C for 30 min. After incubation, the cells were washed 2-3 times with 1 mL of PBS buffer, and finally resuspended in 200 μL of PBS buffer and analyzed by flow cytometer.

[0184] The results are shown in Figures 7A and 7B. The results show that: when there are only three aHER2 Fab antibodies on the liposome surface, aHER2-DiI-Lip exhibits significant binding activity with SK-Br-3 cells. When the number of aHER2 Fab antibodies reaches about 13-25, the fluorescence intensity of aHER2-DiI-Lip binding to SK-Br-3 cells tends to saturate. When the number of aHER2-DiI-Lip surface antibodies is less than 100, there is basically no binding activity with the low expression / negative cell line MDA-MB-231. However, when the number of antibodies exceeds 100, binding occurs, but with weak binding strength.

[0185] 7. Investigating cellular uptake of aHER2-LDX The aHER2 Fab antibody fragment was conjugated to doxorubicin liposomes and added to the HER2-overexpressing cell line BT474 to detect the doxorubicin drug content in the cells.

[0186] BT474 cells were digested with trypsin one day prior to cell division and diluted to 5 × 10 5Cells / well were seeded in a 6-well plate. According to the amount of doxorubicin, C-LDX and 10-C aHER2-LDX were added at two doses of 10 μg and 50 μg, respectively, and blank control wells were set up. After adding the drugs, the cells were incubated at 4 °C for 4 h. Next, the cells were gently washed 2-3 times with PBS buffer, then the plate was scraped and centrifuged, after which the supernatant was removed. The cells were lysed by three freeze-thaw cycles at -80 °C. Proteins were precipitated by adding 100 μL of acetonitrile / methanol (v / v, 1:1), followed by centrifugation at 10,000 rpm for 10 min. The supernatant was then collected and filtered through a 0.22 μm organic membrane, and the doxorubicin content was analyzed by HPLC.

[0187] As seen in Figure 8, only the doxorubicin-loaded liposomes conjugated to the HER2 Fab antibody fragment were able to efficiently deliver the drug into the cells, suggesting that aHER2-LDX could specifically target HER2-overexpressing tumor cells and mediate endocytosis.

[0188] 8. Investigating aHER2-LDX in the treatment of chemotherapy-resistant tumors Addition of doxorubicin-loaded liposomes conjugated to a HER2 antibody to doxorubicin-resistant cells has been shown to be able to reverse tumor cell resistance to chemotherapy drugs.

[0189] To investigate the performance of HER2 Fab antibody targeted doxorubicin-loaded liposomes in doxorubicin-resistant tumor cell lines, MCF-7 / doxorubicin-resistant breast cancer was selected for the study. The cytotoxic effect of HER2 / neu antibody targeted doxorubicin-loaded liposomes on tumor cells was evaluated using Promega's CellTiter-Glo® Luminescent Cell Viability Assay kit. A parallel comparison against non-targeted formulations C and A doxorubicin-loaded liposomes was also performed using trastuzumab as a control. Cytotoxicity curves were fitted using Graph Prism 8.0 analysis software to obtain individual IC 50 The values ​​were calculated and the results are shown in Figure 9.

[0190] The results of the cytotoxicity study showed that the IC of HER2 Fab antibody-targeted doxorubicin-loaded liposomes, doxorubicin-loaded liposomes of formulations C and A, and trastuzumab in MCF-7 / Adr were significantly higher than that of the control. 50 The values ​​are 13.70 μg / mL, 46.41 μg / mL, and 101.30 μg / mL, respectively, which again indicates that the HER2 Fab antibody-targeted doxorubicin-loaded liposomes exhibit strong cytotoxicity against doxorubicin-resistant cell lines. Therefore, the liposomes may be effective in clinical practice in treating patients with doxorubicin-resistant tumors.

[0191] 9. In vivo pharmacology testing of aHER2-LDX conjugated to various amounts of HER2 antibody in animals 9.1.Establishment of nude mouse xenograft model of BT474 breast cancer Balb / C-Nude Construction of BT474 breast cancer Balb / C-Nude nude mouse xenograft model: SPF grade female Balb / C-Nude nude mice, weighing about 18g, aged 4-5 weeks, were purchased and acclimatized in the animal breeding room for about one week. After the BT474 cells grew to logarithmic growth phase, they were digested with trypsin, centrifuged, and then collected. The cells were then washed 2-3 times with PBS buffer, resuspended in PBS buffer, and counted. The cell density was then adjusted to 1×10^8 cells / mL. The Matrigel matrix gel was removed from the -20℃ freezer, pre-thawed on ice, and mixed with the Matrigel matrix gel and cell suspension at a volume ratio of 1:1. 5×10 6 ~1×10 7 Based on the amount of cells, a 1 mL syringe was used to aspirate the corresponding volume of cell suspension and inoculated it subcutaneously into the right axillary region of nude mice, and pressure was applied to the needle puncture site for 30 seconds after inoculation to prevent leakage of the cell solution. After inoculation, a noticeable subcutaneous papule was observed. The inoculated mice were observed regularly to monitor tumor growth.

[0192] 9.2. Dosage and Efficacy Assessment Tumor size: 100-200mm 3 When the mice reached 100 mg / kg / day, they were randomly divided into six groups, with at least six mice in each group, including a control group, a formulation A doxorubicin-loaded liposome group, a formulation C doxorubicin-loaded liposome group, and HER2 Fab antibody-targeted formulation C doxorubicin-loaded liposome groups with 10, 20, and 40 antibody numbers (i.e., 10-C aHER2-LDX group, 20-C aHER2-LDX group, and 40-C aHER2-LDX group).

[0193] After grouping, the control group received empty liposomes via the tail vein. Nude mice in the Formulation A doxorubicin-loaded liposome group were administered Formulation A doxorubicin-loaded liposomes at a dose of 2 mg / kg via the tail vein according to body weight. Nude mice in the Formulation C doxorubicin-loaded liposome group were also administered Formulation C doxorubicin-loaded liposomes at a dose of 2 mg / kg via the tail vein according to body weight. Nude mice in the 10-C aHER2-LDX group, nude mice in the 20-C aHER2-LDX group, and nude mice in the 40-C aHER2-LDX group were administered HER2 Fab antibody-targeted Formulation C doxorubicin liposomes at a dose of 2 mg / kg via the tail vein according to body weight. The drug was administered once a week for a total of five administrations. The weight of the mice and the tumor growth curves were recorded every other day. After administration, the mice were euthanized (the control group was euthanized on day 18 due to tumor volume exceeding the limit).

[0194] The inhibitory effect of the test substances on breast cancer BT474 mouse xenografts and their complete cure ability were mainly tested.

[0195] Measurement of tumor volume and weight of tumor-bearing mice: Measurements were performed every other day using a caliper. Tumor volume was calculated as V=0.5 a×b 2 The calculation was performed as follows: a and b represent the length and width diameters of the tumor, respectively.

[0196] Tumor growth inhibition rate TGI (%) = [1-(Di-D0) / (Ci-C0] x 100, where Di-D0>0. D0 is the average tumor volume at the time of the first administration in the treatment group (drug). D i is the mean tumor volume at a particular measurement time after the start of dosing in the treatment group. C0 is the mean tumor volume at the time of the first dose in the control group (control). C i is the mean tumor volume at a particular measurement time after the start of dosing in the control group.

[0197] The results of the in vivo pharmacological study in BT474 breast cancer Balb / C-Nude nude mouse xenografts are shown in FIG. 10. The results show that the tumor growth inhibition rates (TGI%) of formulation A doxorubicin-loaded liposomes, formulation C doxorubicin-loaded liposomes, and 10-C aHER2-LDX, 20-C aHER2-LDX, and 40-C aHER2-LDX on day 18 after dosing were 50.55%, 60.12%, 69.66%, 61.98%, and 62.94%, respectively. This suggests that the HER2 Fab antibody-targeted formulation C doxorubicin-loaded liposomes have a stronger tumor growth inhibition ability than formulation C doxorubicin-loaded liposomes and formulation A doxorubicin-loaded liposomes. However, the tumor inhibition effect did not increase further even with the increase in the number of surface antibodies of the doxorubicin-loaded liposomes, and these results are consistent with the results of the cytotoxicity experiment. These experimental results clearly demonstrate that: 1) doxorubicin-loaded liposomes with low lipid ratio not only exert stronger cytotoxic effects on tumor cells in vitro, but also exhibit superior tumor growth inhibition in vivo. 2) In addition, the HER2 Fab antibody-targeted formulation C doxorubicin-loaded liposomes further enhanced the ability to inhibit tumor growth. These suggest that surface modification with antibodies on doxorubicin-loaded liposomes may enhance targeting and improve the efficacy of tumor inhibition.

[0198] 10. Investigating the in vivo dose-dependent efficacy of aHER2-LDX in animals 10.1. Construction of NCI-N87 breast cancer Balb / C-Nude nude mouse xenograft model The NCI-N87 gastric cancer cell line with high cell surface HER2 expression was selected to establish a xenograft model in Balb / C-Nude nude mice.

[0199] We purchased 4-5 week-old female Balb / C-Nude nude mice, weighing about 18g, SPF grade, and allowed them to acclimate in the animal breeding room for about a week. After the NCI-N87 cells grew to the logarithmic growth phase, they were digested with trypsin, centrifuged, and then collected. The cells were then washed 2-3 times with PBS buffer, resuspended in PBS buffer, and counted. The cell density was then adjusted to 2×10^8 cells / mL. The Matrigel matrix gel was removed from the -20°C freezer and pre-thawed on ice, and the Matrigel matrix gel and cell suspension were mixed in a 1:1 volume ratio. Based on the amount of 1*10^7 cells, a 1mL syringe was used to aspirate the corresponding volume of cell suspension and inoculated it subcutaneously into the right axillary region of the nude mice, and pressure was applied to the needle puncture site for 30 seconds after inoculation to prevent leakage of the cell solution. After inoculation, a noticeable subcutaneous papule was observed. Inoculated mice were observed regularly to monitor tumor growth.

[0200] 10.2. Dosage and Efficacy Assessment Tumor size: 100-200mm 3 When the mice reached 100 mg / kg, they were randomly divided into seven groups, with at least six mice in each group: control, trastuzumab, formulation A doxorubicin-loaded liposome, and HER2 Fab antibody-targeted formulation C doxorubicin-loaded liposome (10-C aHER2-LDX) at doses of 0.5, 2, 5, and 10 mg / kg.

[0201] After grouping, the control group was given empty liposomes via the tail vein. Nude mice in the formulation A doxorubicin-loaded liposome group were administered doxorubicin-loaded liposomes at a drug-lipid ratio of 0.17 and a dose of 2 mg / kg via the tail vein according to body weight. Nude mice in the 10-C aHER2-LDX group were similarly administered HER2 Fab antibody-targeted formulation C doxorubicin-loaded liposomes at doses of 0.5, 2, 5 and 10 mg / kg via the tail vein according to body weight. At the same time, the trastuzumab group was set as the control group, and was administered 5 mg / kg once a week for a total of five administrations. The body weight and tumor growth curves of the mice were recorded every other day, and the mice were euthanized after the end of administration.

[0202] The inhibitory effects and complete cure ability of the test substances on NCI-N87 gastric cancer xenograft model in mice were mainly tested. (1) Measurement of tumor volume: Tumor volume was measured every other day using a caliper. Tumor volume was calculated as V=0.5 a×b 2 The calculation was performed as follows: a and b represent the length and width diameters of the tumor, respectively. (2) Tumor growth inhibition rate TGI (%) = [1-(D i -D0) / (C i -C0]×100. In the formula, D i -D0>0. D0 is the average tumor volume at the time of the first administration in the treatment group (drug). D i is the mean tumor volume at a particular measurement time after the start of dosing in the treatment group. C0 is the mean tumor volume at the time of the first dose in the control group (control). C i is the mean tumor volume at a particular measurement time after the start of dosing in the control group.

[0203] The results of in vivo pharmacological testing on NCI-N87 gastric cancer Balb / C-Nude nude mouse xenografts are shown in Figure 11. The tumor inhibition rates of formulation A doxorubicin-loaded liposomes, trastuzumab, and 10-C aHER2-LDX at doses of 0.5, 2, 5, and 10 mg / kg were 77.27%, 14.49%, 28.41%, 81.71%, 88.75%, and 96.33%, respectively. The results indicate that doxorubicin-loaded liposomes conjugated to 10 HER2 Fab antibodies have excellent tumor growth inhibition effects in vivo on HER2-overexpressing NCI-N87 gastric cancer tumor models, and exhibit a dose-dependent relationship.

[0204] 11.Investigation of in vivo pharmacology of aHER2-LDX in animal models of HER2 low-expressing tumors 11.1.Establishment of nude mouse xenograft model of MCF-7 breast cancer Balb / C-Nude The MCF-7 breast cancer cell line with low cell surface HER2 expression was selected to establish a xenograft model in Balb / C-Nude nude mice.

[0205] The method of constructing the tumor model was the same as in 10.1. Construction of nude mouse xenograft model of NCI-N87 breast cancer Balb / C-Nude

[0206] 11.2. Dosage and Efficacy Assessment Tumor size: 100-200mm 3 When the mice reached 100 mg / kg, they were randomly divided into six groups, with at least six mice in each group: control, trastuzumab, formulation A doxorubicin-loaded liposome, and HER2 Fab antibody-targeted formulation C doxorubicin-loaded liposome (10-C aHER2-LDX) at doses of 2, 5, and 10 mg / kg.

[0207] After grouping, the control group received empty liposomes via the tail vein. Nude mice in the formulation A doxorubicin-loaded liposome group were administered doxorubicin-loaded liposomes at a dose of 2 mg / kg with a drug-lipid ratio of 0.17 via the tail vein according to body weight. Nude mice in the 10-C aHER2-LDX group were similarly administered HER2 Fab antibody-targeted formulation C doxorubicin-loaded liposomes at doses of 2, 5 and 10 mg / kg via the tail vein according to body weight. At the same time, the trastuzumab group was set as the control group, and was administered 5 mg / kg once a week for a total of five administrations. The body weight and tumor growth curves of the mice were recorded every other day, and the mice were euthanized after the end of administration.

[0208] The inhibitory effects of the test substances on breast cancer MCF-7 mouse xenografts and their complete cure potential were mainly tested.

[0209] The method for evaluating efficacy was the same as in 10.2.

[0210] The results of in vivo pharmacological testing of MCF-7 breast cancer Balb / C-Nude nude mouse xenografts are shown in Figure 12. The tumor inhibition rates of trastuzumab, formulation A doxorubicin-loaded liposomes, and 10-C aHER2-LDX at doses of 2, 5, and 10 mg / kg are 2.91%, 62.16%, 69.67%, 92.18%, and 97.38%, respectively. The results also show that doxorubicin-loaded liposomes conjugated to 10 HER2 Fab antibodies have excellent tumor growth inhibition effects in vivo on HER2-low-expressing MCF-7 breast cancer tumor models, and exhibit a dose-dependent relationship. The results suggest that doxorubicin-loaded liposomes modified with HER2 Fab antibodies have the same therapeutic effect on HER2-low-expressing tumors as on HER2-overexpressing tumors, suggesting that they may be widely applicable.

[0211] 12. Investigating the in vivo pharmacokinetic properties of aHER2-LDX in animals The in vivo pharmacokinetic properties of doxorubicin-loaded liposomes with various antibody numbers were investigated in a tumor-bearing mouse model.

[0212] The method for constructing the BT474 breast cancer Balb / C-Nude nude mouse xenograft model is as described in embodiment 9.1. The subcutaneous tumor volume is 400-600 mm 3 When this was reached, groups were assigned and medication was administered.

[0213] Dosing: The first group of mice was administered 10-C aHER2-LDX via tail vein according to body weight, the drug dose was 5mg / kg. The second group of mice was administered 10-C aHER2-LDX via tail vein according to body weight, the drug dose was 5mg / kg. Blood collection: Blood was collected in EDTA anticoagulant tubes at 0.5, 1, 2, 4, 6, 8, 12, 24, 48, and 72 hours after dosing. It was centrifuged at 4°C, 3,000 rpm for 30 minutes on ice. The upper layer of plasma was carefully pipetted into a new labeled centrifuge tube and subsequently tested or stored in a -80°C freezer.

[0214] Detection of doxorubicin drug content in plasma: 40 μL of plasma sample was accurately measured and placed into a centrifuge tube, and 160 μL of acetonitrile-methanol (1:1, v / v) was added according to the plasma to organic reagent ratio of 1:4. The sample was vortexed for 1-2 min to completely precipitate the proteins in the plasma, and centrifuged at 1,000 × g rotation speed at 4 °C for 10 min. The supernatant was carefully pipetted into a new centrifuge tube, while taking care not to suck up the protein layer, and centrifuged at 1,000 × g rotation speed at 4 °C for 10 min. The sample was filtered using a 0.22 μm organic filter head, and then 50 uL was placed into a sample vial for HPLC detection. Conditions for high performance liquid chromatography (referring to the Chinese Pharmacopoeia): An Athena C18 column (120 Å, 4.6150 mm, 5 μm) with octadecylsilane bonded to silica gel as the packing material was used. Sodium dodecyl sulfate solution (1.44 g of sodium dodecyl sulfate and 0.68 mL of phosphoric acid were added and dissolved with 500 mL of water)-acetonitrile-methanol (500:500:60) was used as the mobile phase. The detection wavelength was 254 nm, the detection flow rate was 1.0 mL / min, and the sample volume was 10 μL.

[0215] Detection of HER2 Fab antibody content in plasma: The content of HER2 Fab antibody in plasma was detected by ELISA. HER2 antigen was coated on a 96-well microplate at 4°C one day before. 350μL of washing solution was added to each well and washed three times, and 300μL of blocking solution was added to each well and [blocked] at 37°C for 1 hour. Then 350μL of washing solution was added to each well and washed three times. Then, reference standard, sample, or control was added to the corresponding well at 100μL / well, and the reaction well was sealed with a plate sealer. Then, incubation was performed with shaking at 37°C for 1 hour, and then 350μL of washing solution was added to each well and washed three times. Then, 100μL of chromogenic substrate was added to each well and incubated at room temperature in the dark for 3-15 minutes. Finally, 100μL of stop solution was added to each well, mixed well, and the OD450 value was measured immediately.

[0216] Analysis: The plasma concentration results of doxorubicin-loaded liposomes with different amounts of HER2 Fab antibody in BT474 tumor-bearing mice are shown in Figure 13A. The change in HER2 Fab antibody concentration in blood samples is shown in Figure 13B. The change in drug concentration and HER2 Fab antibody in blood can be used to show the process of drug release from HER2 Fab antibody-targeted liposomes. It can be seen that liposomes with different antibody densities have different in vivo release processes. Liposomes with low antibody density have slow drug release, while HER2 Fab release is fast.

[0217] 13. Formulation screening of immunoliposomes modified with CD3×HER2 dual targeting antibody (CD3×HER2 Lip) 13.1. Preparation of immunoliposomes decorated with CD3×HER2 dual targeting antibody (CD3×HER2 Lip) Empty liposomes were prepared with reference to embodiment 1. 900mg HSPC, 300mg cholesterol, and 60mg DSPE-mPEG2000 were added to 2mL absolute ethanol and heated to 60℃ in a water bath. Then, a magnetic stirrer was added to stir until completely dissolved, and a transparent liquid mixture was obtained, which was used as the lipid mixture. At the same time, 3.304g ammonium sulfate was placed in a glass bottle, 100mL ultrapure water was added, and stirred until dissolved, and then pH was adjusted to 5.0 to obtain an aqueous solution of ammonium sulfate with a concentration of 250mM. This solution was prepared immediately before use. The dissolved lipid mixture was injected into 19mL of aqueous ammonium sulfate solution using a syringe, then stirred and extruded six times, and dialysis was performed using a 10kD dialysis membrane in HEPES buffer (10mM, pH 6.8) to obtain empty liposomes with an external aqueous phase of HEPES solution.

[0218] At position 231 of the heavy chain, C of the CD3 Fab antibody contains a free thiol group, which is exposed by reduction at a specific site. The free thiol group chemically reacts with the maleimide group of the lipid molecule DSPE-PEG-MAL to generate the CD3 Fab antibody fragment lipid molecule (represented as: aCD3-DSPE). For specific procedures, see the preparation of aHER2-DSPE in embodiment 2.1.

[0219] To generate liposomes with different combinations of CD3 and HER2 antibodies on the surface at various densities, the amount of aHER2-DSPE and aCD3-DSPE added was adjusted and incubated with empty liposomes to generate liposomes decorated with CD3 x HER2 dual targeting antibodies at different densities (represented as CD3 x HER2 Lip axb, where "a" represents the number of CD3 Fab antibodies and "b" represents the number of HER2 Fab antibodies, i.e., CD3 x HER2 Lip 9x5, CD3 x HER2 Lip 9x10, CD3 x HER2 Lip 9x20, CD3 x HER2 Lip 9x40, CD3 x HER2 Lip 9x80, and CD3 x HER2 Lip 3x10, CD3 x HER2 Lip 6x10, CD3 x HER2 Lip 18x10, and CD3 x HER2 Lip Single targeted liposomes using HER2 and CD3 antibodies were also generated separately, resulting in HER2 Lip-5, HER2 Lip-10, HER2 Lip-20, HER2 Lip-40, HER2 Lip-80, and CD3 Lip-3, CD3 Lip-6, CD3 Lip-9, CD3 Lip-18, and CD3 Lip-36.

[0220] 13.2. In vivo evaluation of immunoliposomes decorated with CD3×HER2 dual targeting antibody (CD3×HER2 Lip) The ability of CD3 x HER2 dual-targeted liposomes to activate Jurkat cells in vitro was investigated using CD3 positive Jurkat [sic: Jurkat] cells as representative.

[0221] Jurkat, clonal E6-1 human T-lymphoblastic leukemia cell line was obtained from the School of Pharmacy, Shanghai Jiao Tong University. The cell line was grown in RPMI-1640 complete culture medium containing 1% penicillin-streptomycin dual antibiotics and 10% fetal bovine serum.

[0222] The expression of CD25 and CD69 on the surface of CD3 positive Jurkat cells was analyzed by flow cytometry to investigate the in vitro activation of T cells with CD3 x HER2 dual targeting antibody liposomes and to select the optimal antibody density. Jurkat cells were cultured at 1 × 10 per well with 200 μL of culture medium. 5 Jurkat cells were resuspended in complete culture medium and added to a 96-well plate. 0.01 μg, 0.1 μg, 1 μg, 5 μg, and 10 μg of CD3 x HER2 dual-targeted liposomes, CD3 single-targeted liposomes, or HER2 single-targeted liposomes were added to each well. Incubation was continued for 48 hours in a 37°C incubator. The 96-well cell culture plate was then removed and centrifuged at 400g for 5 minutes, and the supernatant was discarded. Then, 100 μL of PBS buffer was added to resuspend the cells, and anti-human CD25 PE-Cy7 and anti-huamn [sic: anti-human] CD69 PE fluorescent antibodies were added. The cells were incubated at room temperature for 15 minutes, and then washed twice with PBS buffer. × Finally, the cells were resuspended in 100 μL of PBS buffer, loaded, and tested on a Novocyte flow cytometer. The results are shown in Figures 14A and 14B.

[0223] The ability of CD3×HER2 dual-targeted liposomes to activate T cells in vitro and mediate target cell killing by effector cells in vitro was investigated using CD3 / CD28 antibody-stimulated expanded T cells as representatives.

[0224] The cytotoxic effect of CD3×HER2 dual-targeting antibody liposomes in mediating target cell killing by effector cells in vitro was evaluated using Promega's CytoTox96® Non-Radioactive Cytotoxicity Assay kit. At the same time, the expression of CD25 and CD69 on the surface of T cells was analyzed by flow cytometry to investigate the in vitro activation of T cells by CD3×HER2 dual-targeting liposomes and to select the optimal antibody density. Effector cells (CD3 / CD28 antibody stimulated and expanded T cells) and target cells SK-Br-3 were cultured at 1.11×10 cells / mL, respectively, in RPMI-1640 complete culture medium without phenol red. 6 cells / mL and 1.0 x 10 5 The cells were resuspended at a cell concentration of 10 ... Then, 50 μL of substrate reaction mixture was added to the new plate, the plate was covered with aluminum foil or an opaque box to protect it from light, and incubated at room temperature for 30 minutes. Then, 50 μL of stop solution was added, and the absorbance value was measured at 490 nm using a microplate reader. The results are shown in FIG. 15.

[0225] The cells in the lower layer after removing the supernatant as described above were washed twice with PBS buffer, and anti-human CD4 APC-Cy7, anti-human CD8 FITC, anti-human CD25 PE-Cy7 and anti-huamn [sic: anti-human] CD69 PE fluorescent antibodies were added and incubated at room temperature for 15 minutes, after which the cells were washed twice with PBS buffer. Finally, the cells were resuspended in 100 μL of PBS buffer, loaded and tested on a Novocyte flow cytometer. The results are shown in Figures 16A and 16B.

[0226] Summarizing the results of Figures 14, 15, and 16 above, it can be concluded that the CD3×HER2 dual-targeted liposomes had significant activation effects on both CD25 and CD69 within 48 hours, and significant cell-killing effects within 48 hours. When the number of CD3 antibodies on the liposome surface was 9, the expression of CD25 / CD69 molecules decreased with the increase in the number of HER2 antibodies. When the number of HER2 antibodies on the liposome surface was 10, the expression of CD25 / CD69 molecules gradually increased with the increase in the number of CD3 antibodies. This suggests that the activation effect was more significant from this point on.

[0227] Overall, it can be seen that in CD3×HER2 dual-targeting liposomes, a higher number of HER2 antibodies does not necessarily lead to better results. Optimal activation of T cells is achieved with an antibody number of 5-10. Considering the cytotoxic effect, the ideal number of HER2 antibodies is 10. The higher the density of CD3 antibodies, the more effective the T cell activation, but a plateau is reached when the CD3 antibody number reaches 9 or 18. On the other hand, a higher activation of T cell immunity does not necessarily lead to better results in vivo.

[0228] 14. Preparation of immunoliposomes decorated with CD3×HER2 dual targeting antibody (CD3×HER2 R848-Lip) 285.7mg HSPC, 95mg cholesterol, and 19mg DSPE-mPEG2000 were added to 1mL absolute ethanol and heated to 60℃ in a water bath. Then a magnetic stirrer was added to stir until completely dissolved, resulting in a clear liquid mixture. At the same time, 6.607mg ammonium sulfate was placed in a glass bottle, 100mL ultrapure water was added, stirred until dissolved, and then the pH was adjusted to 5.0 to obtain an aqueous solution of ammonium sulfate with a concentration of 500mM. This solution was prepared immediately before use. The dissolved lipid mixture was injected into 9mL of aqueous ammonium sulfate solution using a syringe, stirred while hydrating at 60℃ for 45 minutes, and then extruded six times with an extruder, and dialysis was performed using a 10kD dialysis membrane in a dialysis solution (10mM HEPES, 0.9% NaCl, pH5.5) to obtain empty liposomes.

[0229] Further, 6.25 mL of 1 mg / mL R848 solution was added to 3.75 mL of empty liposomes with a lipid concentration of 40 mg / mL to obtain a total volume of 10 mL. The mixture was stirred at room temperature for 30 min to load the drug. After that, a 100 kD dialysis bag was used to remove the free drug to obtain the prepared R848 liposomes. The particle size is 80-120 nm, and the PDI is less than 0.1.

[0230] For the preparation of aHER2-DSPE in embodiment 2.1 and aCD3-DSPE in embodiment 13.1, aHER2-DSPE and aCD3-DSPE were added to the above R848 liposomes in a 60°C water bath and incubated for 10-30 minutes. The ratio of 10 HER2 antibodies to 6 CD3 antibodies was on the surface of each liposome. Ultrafiltration and dialysis were then performed.

[0231] 15. In vivo pharmacological evaluation of immunoliposomes decorated with CD3×HER2 dual targeting antibodies (CD3×HER2 Lip) and R848 liposomes (CD3×HER2R848-Lip) 15.1. Construction of humanized NCG-B2M-KO mouse xenograft NCI-N87 breast cancer model The NCI-N87 gastric cancer cell line with high cell surface HER2 expression was selected to construct a humanized model of NCG-B2M-KO severely immunodeficient mice.

[0232] We purchased 4-5 week-old female Balb / C-Nude nude mice, weighing about 18g, SPF grade, and allowed them to acclimate in the animal breeding room for about a week. After the NCI-N87 cells grew to the logarithmic growth phase, they were digested with trypsin, centrifuged, and then collected. The cells were then washed 2-3 times with PBS buffer, resuspended in PBS buffer, and counted. The cell density was then adjusted to 2×10^8 cells / mL. The Matrigel matrix gel was removed from the -20°C freezer and pre-thawed on ice, and the Matrigel matrix gel and cell suspension were mixed in a 1:1 volume ratio. Based on the amount of 1*10^7 cells, a 1mL syringe was used to aspirate the corresponding volume of cell suspension and inoculated it subcutaneously into the right axillary region of the nude mice, and pressure was applied to the needle puncture site for 30 seconds after inoculation to prevent leakage of the cell solution. After inoculation, a noticeable subcutaneous papule was observed. The inoculated mice were observed periodically to monitor tumor growth. At the same time, each mouse was intravenously injected with 1*10^7 PBMC cells from healthy volunteers via the tail vein.

[0233] 15.2. Dose and Efficacy Assessment Tumor size: 100-200mm 3 When the total number of mice reached 100, and after the mice were successfully humanized, the mice were randomly divided into seven groups, each of which had at least six mice, including an empty group (inoculated with tumor cells only), a control group (inoculated with tumor cells + PBMC cells), an R848 liposome group, a CD3×HER2 dual-targeting liposome group at doses of 0.1, 1 and 2 mg / kg, and a CD3×HER2 dual-targeting R848 liposome group at a dose of 0.1 mg / kg.

[0234] After grouping, the empty and control groups were intravenously injected with PBS solution via the tail vein. Nude mice in the CD3×HER2 dual-targeting liposome group were administered 0.1, 1, and 2 mg / kg of CD3×HER2 dual-targeting liposome via the tail vein according to body weight. Nude mice in the CD3×HER2 dual-targeting R848 liposome group were similarly administered 0.1 mg / kg of CD3×HER2 dual-targeting R848 liposome via the tail vein according to body weight. Administration was performed twice a week for a total of seven times. The body weight and tumor growth curves of the mice were recorded every other day, and the mice were euthanized after the end of administration.

[0235] The inhibitory effect of test substance on NCI-N87 gastric cancer xenograft model in mice and complete cure ability are mainly tested.For measuring tumor volume and calculating the inhibition rate of tumor growth, see embodiment 10.2.

[0236] The in vivo pharmacodynamics results of humanized NCG-B2M-KO mouse xenograft NCI-N87 breast cancer model are shown in Figure 17. CD3×HER2 dual-targeted liposomes had a significant inhibitory effect on tumor growth, and the antitumor effect could be further improved when the immunomodulatory drug R848 was encapsulated in the liposomes.

[0237] All documents mentioned in this disclosure are incorporated by reference in this application to the same extent as if each portion of the document was individually incorporated by reference. Also, after reading the above description of the contents of this disclosure, a person skilled in the art will understand that various changes or modifications can be made to the present disclosure, and such equivalents are also within the same scope as defined by the claims appended to this application.

Claims

1. A nanoparticle, wherein the nanoparticle is a drug-loaded liposome whose outer surface is modified with an antibody containing a first antibody, the liposome contains a lipid substance, the drug is encapsulated within the liposome, and the drug contains an anti-tumor drug, the number of the first antibodies is 5 to 40, the first antibody contains a HER2 antibody (human epidermal growth factor receptor 2 antibody) or a Fab fragment, Fab' fragment, F(ab')2 fragment, or single-chain Fv fragment (scFv) of a HER2 antibody, a nanoparticle.

2. The nanoparticle according to claim 1, wherein the number of the first antibodies is 5 to 15, more preferably 8 to 12, and most preferably 10.

3. The nanoparticle according to claim 1, wherein the Fab fragment of the HER2 antibody contains the following heavy chain and light chain: The heavy chain is selected from the following; (1) A heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1, (2) A heavy chain formed by substitution, deletion, or addition of one or more amino acid residues to the amino acid sequence shown in SEQ ID NO: 1, (3) A heavy chain having 80%, preferably 90%, more preferably 95%, more preferably 98%, or more preferably 99% homology with the amino acid sequence shown in SEQ ID NO: 1, The light chain is selected from the following; (a) A light chain consisting of the amino acid sequence shown in SEQ ID NO: 2, (b) A light chain formed by substitution, deletion, or addition of one or more amino acid residues to the amino acid sequence shown in SEQ ID NO: 2, (c) A light chain having ≧80%, preferably ≧90%, more preferably ≧95%, more preferably ≧98%, or more preferably ≧99% homology with the amino acid sequence shown in SEQ ID NO:

2.

4. The nanoparticle according to claim 1, wherein the antibody further contains a second antibody, the two antibodies are different types of antibodies, and optionally, the number of the second antibodies is 6 to 18, preferably 9.

5. The nanoparticle according to claim 4, wherein the second antibody contains a CD3 antibody (human differentiation antigen group 3 antibody) or a Fab fragment, Fab' fragment, F(ab')2 fragment, or single-chain Fv fragment (scFv) of a CD3 antibody.

6. The nanoparticle according to claim 5, wherein the Fab fragment of the CD3 antibody contains the following heavy chain and light chain: The heavy chain is selected from the following; (1) A heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 3, (2) a heavy chain formed by substitution, deletion, or addition of one or more amino acid residues to the amino acid sequence shown in SEQ ID NO: 3, and (3) a heavy chain having a homology of ≧80%, preferably ≧90%, more preferably ≧95%, even more preferably ≧98%, or even more preferably ≧99% with the amino acid sequence shown in SEQ ID NO: 3, The light chain is selected from the following; (a) a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4, (b) a light chain formed by substitution, deletion, or addition of one or more amino acid residues to the amino acid sequence shown in SEQ ID NO: 4, and (c) a light chain having a homology of ≧80%, preferably ≧90%, more preferably ≧95%, more preferably ≧98%, or more preferably ≧99% with the amino acid sequence shown in SEQ ID NO:

4. **Claim 7** The nanoparticle according to claim 1, wherein the anti-tumor agent is selected from the group consisting of anthracycline, platinum-based agent, fluorouracil, camptothecin, taxane, or a combination thereof. **Claim 8** The anti-tumor agent contains an immunomodulatory agent; or The nanoparticle according to claim 1, wherein the anti-tumor agent is selected from doxorubicin or resiquimod. **Claim 9** The nanoparticle according to claim 1, wherein the weight ratio of the agent to the lipid substance (agent-lipid ratio) is 1:25 to 35, preferably 1:28 to 32. **Claim 10** The lipid substance contains one or more of hydrogenated soy phosphatidylcholine (HSPC), distearoyl phosphatidylethanolamine-polyethylene glycol (DSPE-mPEG), phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, cholesterol, polyethylene glycol glycerol fatty acid ester, and polyethylene glycol glyceryl phosphatidylethanolamine, Preferably, the lipid substance is selected from HSPC, cholesterol (Cho), and DSPE-mPEG or a combination thereof, the nanoparticle according to claim 1. **Claim 11** A pharmaceutical composition comprising the nanoparticle according to claim 1 and a pharmaceutically acceptable carrier. **Claim 12** The pharmaceutical composition according to claim 11, for use in a method of preventing and / or treating HER2-expressing tumors in a subject in need thereof. Use of the pharmaceutical composition according to claim 12, wherein the tumor comprises a HER2-overexpressing tumor, a tumor moderately expressing HER2 or a HER2-low-expressing tumor. Use of the pharmaceutical composition according to claim 12, wherein the tumor comprises a doxorubicin-resistant tumor. Use of the pharmaceutical composition according to claim 12, wherein the tumor is selected from breast cancer, gastric cancer, ovarian cancer, liver cancer, or a combination thereof.

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