A multi-cell targeted liposome

Abstract

This invention belongs to the field of liposome formulations in the preparation of tumor drugs, specifically involving the construction and application of a multi-cell targeted liposome. Because a single liposome contains multiple antibodies targeting different cells, and through molecular self-assembly, it forms a liposome with a stable structure and long-term circulation characteristics in vivo. Through the action of antibodies, it can sequentially or simultaneously bind to multiple different cells, promoting recognition, communication, cytotoxicity, apoptosis, or clearance between different cells.

Description

Technical Field

[0001] This invention belongs to the fields of pharmaceutical formulation, liposome drug carrier and antibody targeting technology, and specifically relates to a multi-cell targeted liposome drug formulation that acts simultaneously with immune effector cells and target cells. Background Technology

[0002] Liposomes are drug carriers with a bilayer vesicle structure based on phospholipids and / or cholesterol. Liposomes are artificially prepared bilayer vesicles formed from lipid molecules. Because lipid molecules have hydrophilic heads and hydrophobic tails,

[0003] Liposomes can encapsulate hydrophilic drugs in their internal aqueous phase (inner aqueous phase) and embed hydrophobic drugs within their lipid bilayer membrane. Furthermore, due to their excellent tissue compatibility, low toxicity, and biosafety, liposomes have garnered widespread attention and application in drug delivery systems. In particular, the development of liposome-targeting technology has endowed lipid drug carriers with unique in vivo behavior, enabling them to accumulate at target sites and interact specifically with target cells. It is hoped that by optimizing target selection and improving the structure and properties of targeting ligands, improvements in the clinical efficacy of liposomal drugs can be achieved, including reducing systemic toxicity and increasing the therapeutic index.

[0004] The development strategy for targeted liposome drug delivery systems involves designing various types of active targeting ligands and drug carriers targeting receptors that are highly or specifically expressed on the surface of tumor cells. Actively targeted drugs developed based on this design approach exhibit optimized lesion-site targeting capabilities compared to free drugs delivered without a targeting carrier, such as increased drug concentration at the target site and improved pharmacodynamics. However, because this type of targeted drug delivery system only acts on a specific type of tumor cell, it is not only insufficient to effectively eliminate tumors but may also induce minimal residual disease and multidrug resistance in tumors.

[0005] In recent years, thanks to improved understanding of tumor heterogeneity, the mechanisms behind the limitations of single-cell targeting have been elucidated. These include differences in tumor molecular markers among different patients, differences in molecular markers among different tumor cells within the same tumor of the same patient, and the presence of different cell types and their interactions within the same tumor. To address the characteristics of tumor heterogeneity, researchers have expanded their targeted delivery design approaches. For example, dual-target liposomes can enhance the binding affinity between the carrier and cells, promoting drug uptake by cells; drug carriers targeting tumor-associated cells can inhibit tumor growth through other mechanisms, such as acting on tumor angiogenesis, fibroblasts, and tumor stem cells.

[0006] While these targeted delivery technologies offer some therapeutic improvements, their design principles remain largely confined to the established framework of single-cell targeting. Extensive dynamic interactions exist between different cells within a tumor, including lymphocyte infiltration, macrophage phagocytosis, antigen presentation, and cytokine- and exosome-based intercellular communication. These interactions encompass both immune surveillance and clearance that inhibit tumor growth and the tumor microenvironment that promotes it. Furthermore, blocking or promoting these intercellular interactions has proven feasible and effective in clinical practice, as exemplified by antigen-specific T-cell-based vaccine therapies, antibody therapies relying on NK cell cytotoxicity, and immune checkpoint inhibitor therapies that block tumor immune escape. In contrast, single-cell-targeted drug delivery systems can only modulate physiological processes by targeting specific cells but cannot leverage intracellular interactions within tumor tissue to achieve effective treatment.

[0007] Therefore, antibodies targeting different cells are modified onto individual liposomes, which then self-assemble into liposomes with stable structures and long-term in vivo circulation characteristics. These liposomes, acting sequentially or simultaneously with the antibodies, bind to multiple different cells, thereby promoting or blocking intercellular interactions to regulate cell recognition, communication, cytotoxicity, apoptosis, or cell clearance. Furthermore, using lipid drug carriers enables targeted drug delivery to multiple cells, improving pharmacological effects while avoiding potential drug interactions associated with combination therapies. Summary of the Invention

[0008] In order to overcome the problems existing in the prior art, the purpose of this invention is to provide a multi-cell targeted liposome drug formulation that can sequentially or simultaneously bind to multiple different cells, as well as its preparation and application.

[0009] To achieve the above-mentioned objectives and other related objectives, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a multi-cell-targeting liposome, the structure of which includes a liposome, a major antibody modified on the surface of the liposome, and a helper antibody, wherein the major antibody specifically binds to target molecules on the surface of target cells, and the helper antibody specifically binds to immune effector cells.

[0011] Furthermore, the multi-cell-targeting liposomes can simultaneously or sequentially bind to immune effector cells and target cells, thereby enabling immune effector cells to recognize target cells and activate immune effects.

[0012] Furthermore, the molar ratio of the major antibody to the auxiliary antibody on each liposome is between 100 / 1 and 1 / 1.

[0013] Furthermore, each liposome has 1-1000 major antibodies and 1-1000 auxiliary antibodies on its surface.

[0014] Furthermore, the primary antibody and the auxiliary antibody are displayed on the liposome surface through covalent linkage or hydrophobic-hydrophilic interaction, respectively.

[0015] As exemplified in some embodiments of the present invention, the primary antibody and the auxiliary antibody are respectively linked to lipid molecules in the liposomes. The lipid molecules have maleimide groups. The primary antibody and the auxiliary antibody are respectively linked to the maleimide groups of the lipid molecules. The primary antibody and the auxiliary antibody are respectively linked to the lipid molecules via thioether bonds.

[0016] Furthermore, the target cells are selected from tumor cells, microorganisms, or cells infected by microorganisms. Therefore, the antigens targeted by the primary antibody are selected from microbial antigens, tumor-associated antigens, tumor cell surface-specific antigens, antigens highly expressed on the surface of tumor cells, and antigens highly expressed in tumor tissue or tumor blood vessels. In other words, the target molecules are selected from microbial antigens, tumor-associated antigens, tumor cell surface-specific antigens, antigens highly expressed on the surface of tumor cells, and antigens highly expressed in tumor tissue or tumor blood vessels.

[0017] Furthermore, the specific target molecule of the main antibody is selected from CD19, CD20, PSMA, Her2 / neu, EGFR, LGR5, or PDL1.

[0018] In some embodiments of the present invention, CD19 is listed as a specific target molecule of the major antibody. CD19 is the human CD19 antigen for human B-cell lymphoblastic leukemia or human B-cell lymphoma.

[0019] Furthermore, the immune effector cells are effector cells capable of exerting cytotoxic, pro-apoptotic, or clearance effects on target cells. These immune effector cells are selected from T lymphocytes, NK cells, NKT cells, macrophages, or neutrophils. Therefore, the antigens targeted by the helper antibody are selected from antigens expressed by tumor cells, lymphocytes, or myeloid cells. Thus, the multi-cell-targeting liposome can bind to tumor cells, lymphocytes, or myeloid cells simultaneously or sequentially. That is, the same liposome may bind to tumor cells first and then lymphocytes, or vice versa.

[0020] In some embodiments of the present invention, the immune effector cells are listed as T lymphocytes.

[0021] Some embodiments of the present invention cite CD3 as the effector antigen of the immune effector cells and CD3 antibody as the helper antibody.

[0022] Furthermore, the auxiliary antibody is selected from CD3 antibody and fragment, PD1 antibody and fragment, CTLA4 antibody and fragment, and CD40L antibody and fragment.

[0023] The T lymphocytes can be unactivated T cells or T cells that have been activated or expanded in vitro, such as antigen-specific T lymphocytes, tumor-infiltrating lymphocytes, cytotoxic T cells, helper T cells, lymphokine-activated killer cells (LAK cells), γδ T cells, chimeric antigen receptor T cells, and T cell receptor chimeric T cells.

[0024] The T lymphocytes are either unactivated T cells or T cells that have been activated or expanded in vitro.

[0025] The activation or amplification can be achieved through antigens, antibodies, peptides, peptide-major histocompatibility complexes, small molecules, cytokines, immune checkpoint inhibitors, or gene editing.

[0026] Furthermore, the primary antibody and the auxiliary antibody are selected from IgG, Fab' fragment, F(ab')2 fragment, Fab fragment, single-chain Fv fragment, minibody, nanobody, unibody, or diabody.

[0027] Some embodiments of the present invention exemplify that the antibody is a Fab' fragment of an antibody, obtained by removing the Fc fragment from the antibody and then reducing it. It includes a light chain variable region and a heavy chain variable region.

[0028] Some embodiments of the present invention exemplify the Fab' fragment of a CD19 antibody as the main antibody, the amino acid sequence of which is shown in SEQ ID NO.1, specifically:

[0029] DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIK.

[0030] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2, specifically:

[0031] EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS.

[0032] In addition, the primary antibody may be a Her2 antibody, the LC sequence of which is shown in SEQ ID NO.3, specifically:

[0033] MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPP TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0034] The HC sequence of the Her2 antibody is shown in SEQ ID NO.4, specifically as follows:

[0035] MEFGLSWVFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV.

[0036] The primary antibody may also be an scFv fragment of an EGFR antibody, the VH sequence of which is shown in SEQ ID NO.5, specifically:

[0037] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA.

[0038] The VL sequence of the EGFR antibody is shown in SEQ ID NO.6, specifically as follows:

[0039] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK.

[0040] The primary antibody may also be a PSMA antibody, the VH sequence of which is shown in SEQ ID NO.7, specifically:

[0041] QVQLVESGGGLVKPGESLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVAIISDGGYYYTYYSDIIKGRFTISSRDNAKNSLYLQMNSLKAEDTAVYYCARGFPLLRHGAMDYWGQGT LVTVSS.

[0042] The VL sequence of the PSMA antibody is shown in SEQ ID NO.8, specifically as follows:

[0043] DIQMTQSPSSSLSASVGDRVTITCKASQNVDTNVAWYQQKPGQAPKSLIYSASYRYSDVPSRFSGSASGTDFTLTISSVQSEDFATYYCQQYDSYPYTFGGGTKLEIK.

[0044] The primary antibody may also be a PDL1 antibody, the heavy chain sequence of which is shown in SEQ ID NO.9, specifically:

[0045] EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYIMMWVRQA PGKGLEWVSS 50

[0046] IYPSGGITFY ADTVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARIK 100

[0047] LGTVTTVDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK 150

[0048] DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT 200

[0049] YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP 250

[0050] KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 300

[0051] STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ 350

[0052] VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV 400

[0053] LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450.

[0054] The light chain sequence of the PDL1 antibody is shown in SEQ ID NO.10, specifically as follows:

[0055] QSALTQPASV SGSPGQSITI SCTGTSSDVG GYNYVSWYQQ HPGKAPKLMI 50

[0056] YDVSNRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYYC SSYTSSSTRV 100

[0057] FGTGTKVTVL GQPKANPTVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV 150

[0058] AWKADGSPVK AGVETTKPSK QSNNKYAASS YLSLTPEQWK SHRSYSCQVT 200

[0059] HEGSTVEKTV APTECS.

[0060] In some embodiments of the present invention, the auxiliary antibody is exemplified as a Fab' fragment of a CD3 antibody, the VH sequence of which is shown in SEQ ID NO.11, specifically:

[0061] DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS.

[0062] The VL sequence of the CD3 antibody is shown in SEQ ID NO.12, specifically as follows:

[0063] DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK.

[0064] Alternatively, the VH sequence of the CD3 antibody is as shown in SEQ ID NO.13, specifically:

[0065] EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS.

[0066] The VL sequence of the CD3 antibody is shown in SEQ ID NO.14, specifically as follows:

[0067] QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLA.

[0068] The auxiliary antibody may also be a PD-1 antibody, the VH sequence of which is shown in SEQ ID NO.15, specifically:

[0069] QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS.

[0070] The VL sequence of the PD-1 antibody is shown in SEQ ID NO.16, specifically as follows:

[0071] LEMAEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK.

[0072] This invention does not impose any specific restrictions on the composition of the liposomes; any liposome is acceptable. The components of the liposomes may be selected from lecithin, hydrogenated soybean phosphatidylcholine, hydrogenated lecithin phosphatidylcholine, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, 1-myristoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-stearoyl phosphatidylcholine, 1-stearoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-... -Oleoylphosphatidylcholine, 1-stearoyl-2-linoleoylphosphatidylcholine, dioleoylphosphatidylcholine, hydrogenated dipalmitoylphosphatidylcholine, distearylphosphatidylcholine, dimyristoylphosphatidyl, dimyristoylphosphatidyl, dimyristoylphosphatidyl, dimyristoylphosphatidyl, dimyristoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, cerebrophosphatidylserine, dimyristoylphosphatidyl Acylserine, dipalmitoylphosphatidylserine, methionine, dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearylphosphatidylglycerol, dioleoylphosphatidylglycerol, sphingomyelin, dipalmitoylsphingomyelin or distearylsphingomyelin, cholesterol, dioleoyloxypropyltrimethylammonium chloride (DOTAP), dioleoylchloropropyltrimethylammonium chloride (DOTMA), bis(octadecyl)dimethylammonium bromide (DDAB), dimethylaminoethylaminopropionyl-cholesterol (DC-Chol), spermine-5-carboxyaminoacetic acid octadecylamide (DOGS), dioleoylsuccinylglycerol choline ester (DOSC), dioleoylchlorosuccinylamine carboxyamide ethyl dimethylpropyltrifluoroacetate ammonium (DOSPA), or any one or a combination of two or more thereof.

[0073] As illustrated in some embodiments of the present invention, the liposomes are composed of phosphatidylcholine, cholesterol, lipids linking the major antibody, and lipid molecules linking the auxiliary antibody.

[0074] Furthermore, the liposomes can be blank liposomes or drug-loaded liposomes.

[0075] A second aspect of the present invention provides a method for preparing the aforementioned multi-cell targeted liposomes, which employs a post-insertion method and includes the following steps:

[0076] (1) The major antibody and the auxiliary antibody are respectively linked to lipid molecules to obtain major antibody-lipid molecules and auxiliary antibody-lipid molecules;

[0077] (2) The obtained main antibody-lipid molecules and auxiliary antibody-lipid molecules are mixed with the constructed liposomes and incubated to obtain a mixed solution;

[0078] (3) The resulting mixed solution is dialyzed or ultrafiltered to remove unloaded antibodies and antibody-lipid complexes, thereby obtaining multi-cell targeted liposomes.

[0079] Preferably, the molar ratio of the main antibody to the lipid molecule is in the range of (0.1-5):1000.

[0080] Preferably, the molar ratio of the auxiliary antibody to the lipid molecule is in the range of (0.1-5):1000.

[0081] A third aspect of the present invention provides the application of the aforementioned multi-cell-targeting liposomes in the preparation of immunotherapeutic drugs and antitumor drugs.

[0082] A fourth aspect of the present invention discloses a method for treating tumors, comprising the steps of: administering the aforementioned multi-cell-targeting liposomes to a tumor patient. The method may be in vitro. The method may be for non-therapeutic purposes. The tumor may be a CD19-positive tumor on its cell surface.

[0083] Compared with the prior art, the present invention has the following beneficial effects:

[0084] The multi-cell targeted liposomes of the present invention have a similar mechanism of action to multi-target antibodies, but further propose to optimize the effector cell activation effect against different target cells and target molecules by adjusting the density and relative ratio of the main antibody and the auxiliary antibody; furthermore, the effect of the drug loaded in the liposome on the target cells and effector cells is further enhanced; finally, the production, preparation and quality control of multi-cell liposomes are more feasible than those of multi-target antibodies. Attached Figure Description

[0085] Figure 1A Reduced and non-reduced SDS-PAGE images of the full-length CD3 antibody, purified anti-CD3 antibody F(ab')2 fragment, and anti-CD3 antibody Fab' fragment-polymer-DSPE ligation product, stained with Coomassie brilliant blue.

[0086] Figure 1B SDS-PAGE images of CD19 whole antibody, purified anti-CD19 antibody F(ab')2 fragment, and anti-CD19 antibody Fab' fragment-polymer-DSPE conjugate, stained with Coomassie brilliant blue.

[0087] Figure 1C SDS-PAGE images of anti-CD3 single-chain antibody (scFv) fragment and anti-CD3 single-chain antibody (scFv) fragment-polymer-DSPE conjugate, stained with Coomassie brilliant blue.

[0088] Figure 2 : Particle size distribution of multi-cell targeted, single-target, and non-targeted liposomes.

[0089] Figure 3A Jurkat specifically binds to target cells of CD3 and CD19 multi-cell and single-cell liposomes.

[0090] Figure 3B :Target cell Raji-specific binding of CD3 and CD19 multi-cell targeted and single-target liposomes.

[0091] Figure 4 : It enables intercellular binding of CD3+ and CD19+ target cells to multi-cell targeted, single-target, and non-targeted liposomes.

[0092] Figure 5A The cytotoxic effect of pre-activated effector cells mediated by multi-cell targeted liposomes on target cells Raji was investigated. The cytotoxicity was detected by LDH assay 24 hours after drug administration. The effector cells were T cells that had been activated and expanded for 3 days after CD3 and CD28 antibody administration.

[0093] Figure 5B The cytotoxic effect of multicellular targeted liposome-mediated pre-activated effector cells on target cells Raji was investigated. The cytotoxicity was detected 24 hours after drug administration using the LDH method. The effector cells were LAK cells that had been activated and expanded by human IL-2.

[0094] Figure 6A The study aimed to investigate the cytotoxic effect of pre-activated effector cells mediated by multi-cell targeted liposomes on target cells Raji. The effector cells were human γδT cells expanded with zoledronic acid. The cytotoxicity was detected 24 hours after drug administration using the LDH method, and the cytotoxicity was assessed at different effector cell / target cell ratios.

[0095] Figure 6B This study investigated the cytotoxic effect of pre-activated effector cells mediated by multi-cell targeted liposomes on target Raji cells. The effector cells were human γδT cells expanded with zoledronic acid. The cytotoxicity was detected 24 hours after drug administration using the LDH method, and the study presented the cytotoxic effects of different doses and targeted liposomes.

[0096] Figure 6CThis study investigated the cytotoxic effect of pre-activated effector cells mediated by multi-cell targeted liposomes on target Raji cells. The effector cells were human γδT cells expanded with zoledronic acid. The cytotoxicity was detected 24 hours after drug administration using the LDH method, and the study presented the cytotoxic effects of different doses and targeted liposomes.

[0097] Figure 7A The study aimed to investigate the cytotoxic effect of unstimulated effector cells on target cells Raji mediated by multicellular targeted liposomes. The effector cells were unactivated human lymphocytes. The cytotoxicity, i.e., the target cell killing status, was detected 5 hours after drug administration using PI staining.

[0098] Figure 7B The study aimed to investigate the cytotoxic effect of unstimulated effector cells on target cells Raji mediated by multicellular targeted liposomes. The effector cells were unactivated human lymphocytes. The cytotoxicity, i.e., the target cell killing status, was detected 24 hours after drug administration using PI staining.

[0099] Figure 8A : This study investigated the killing effect of unstimulated lymphocytes on Raji cells mediated by multicellular targeting liposomes, with unactivated human lymphocytes as the effector cells; cell killing at different effector / target cell ratios (PI staining method).

[0100] Figure 8B This study demonstrates the killing effect of unstimulated lymphocytes on Raji cells mediated by multicellular targeted liposomes, with unactivated human lymphocytes as the effector cells; it also illustrates the cell killing mediated by liposomes of different doses and targeting methods (LDH method).

[0101] Figure 8C This study demonstrates the killing effect of unstimulated lymphocytes on Raji cells mediated by multicellular targeted liposomes, with unactivated human lymphocytes as the effector cells; it also illustrates the cell killing mediated by liposomes of different doses and targeting methods (LDH method).

[0102] Figure 9A This study investigated the use of multi-cell-targeting liposomes to mediate the killing of unstimulated lymphocytes. Effector cells were unactivated lymphocytes, and target cells were Raji cells. Cytotoxicity was assessed 5 hours after drug administration using the LDH method. Notably, the primary antibody in the multi-cell-targeting liposomes was no longer the Fab'2 fragment of the anti-CD3 antibody UCHT1 clone, but instead a different clone of anti-CD3 IgG monoclonal antibody (2C11).

[0103] Figure 9BThe study aimed to investigate the target cell killing effect of multicellular liposome-mediated non-stimulated lymphocytes, with effector cells being unactivated human lymphocytes and target cells being CD19+ human B lymphocytic leukemia SUP-B15 cells; cytotoxicity was detected 24 hours after drug administration using the LDH method.

[0104] Figure 9C This study investigated the target cell killing effect of multi-cell-targeting liposomes mediated by unstimulated lymphocytes. The effector cells were unactivated CD4+ T lymphocytes, and the target cells were LGR5+ human non-small cell lung cancer A549 cells. Cytotoxicity was assessed 5 hours after drug administration using the LDH assay. The auxiliary antibody for the multi-cell-targeting liposomes was an LGR5 antibody.

[0105] Figure 10 This study describes the secretion of IFN-γ by unstimulated lymphocytes in the killing of Raji cells by multi-cell targeted liposomes. The effector cells are unactivated human lymphocytes. Among them, PxP refers to the non-targeted liposome group, HxP refers to the CD19 single-targeted liposome group, UxP refers to the CD3 single-targeted liposome group, and UxH refers to the CD19 and CD3 multi-cell targeted liposome group.

[0106] Figure 11 The study investigated the effect of different concentrations of EGTA on the killing of Raji cells by unstimulated lymphocytes mediated by multicellular targeted liposomes. The effector cells were unactivated human lymphocytes.

[0107] Figure 12 The study describes the target cell killing effect mediated by multi-cell targeted liposomes on unstimulated lymphocytes. The effector cells were unactivated human lymphocytes, and the target cells were CD19+ Raji cells. The cytotoxicity was detected by the LDH method 24 hours after drug administration. UxH-lipo refers to the multi-cell targeted blank liposome group, and UxH-DAS-lipo refers to the multi-cell targeted dasatinib liposome group. Detailed Implementation

[0108] Liposomes are artificially prepared bilayer vesicles formed by lipid molecules. The structure of a lipid molecule typically consists of three parts: a hydrophilic head, a hydrophobic tail, and a connecting portion. In an aqueous medium, due to hydrophobic interactions, the hydrophobic tails of the lipid molecules approach each other, while the hydrophilic heads align facing the aqueous phase, forming a bilayer. Based on this structure, hydrophilic drugs can be encapsulated within the aqueous phase of the liposome (the inner aqueous phase), or hydrophobic drugs can be embedded within the lipid bilayer membrane.

[0109] The targeting effect of liposomes is achieved through surface modification with ligands. Ligands can be antibodies, peptides, small molecules, etc., and interact with specific receptors on target cells. Modifying the liposome surface with antibodies and their fragments exhibits better binding activity and specificity compared to small molecule and peptide ligands. Applying the principle of lipid molecule self-assembly, modifying the surface of a liposome with two or more different ligands also shows good feasibility.

[0110] This invention, based on antibody-modified liposome technology, achieves a novel and unique liposome-mediated killing mechanism and effect against tumor cells through a clever combination of major antibodies targeting target cells and helper antibodies targeting effector cells. Currently known multi-target liposome formulations utilize different ligand combinations to simultaneously bind different antigens, thereby improving the efficiency and selectivity of targeted delivery to specific cells. The multi-cell-targeting liposomes proposed in this invention regulate physiological functions such as recognition, binding, and activation between different cells by promoting or blocking interactions between various cells. Therefore, this presents a completely new direction for ligand-targeted liposome technology.

[0111] On the other hand, constructing multi-cell-targeting liposomes on the surface of lipid drug carriers is not only much simpler than constructing two different targeting antibody sequences on the same antibody structure, but also allows for optimization of anti-tumor efficacy by adjusting the density and ratio of the primary and auxiliary antibodies on the liposome surface. Furthermore, this method of constructing and preparing multi-cell-targeting liposomes avoids problems such as antibody mismatch, overcoming shortcomings in production and efficacy. In particular, liposomes possess unique pharmacokinetic and tissue distribution characteristics in vivo, which can significantly improve the short in vivo half-life limitation of current multi-targeting antibodies.

[0112] Furthermore, the multi-cell-targeting liposomes obtained in this invention can be combined with liposome drug delivery technology, which is more convenient, diverse, and controllable compared to existing clinical combination therapies using multi-targeting antibodies and other drugs. By encapsulating drugs in liposomes, targeted delivery and release of drugs can be achieved, i.e., improving drug selectivity and altering the release site and timing. For example, adverse reactions to tumor immunotherapy include cytokine storms, so patients are often required to take dexamethasone beforehand to suppress this adverse reaction. Another example is that some immunotherapies require simultaneous systemic administration of cytokine drugs such as IL-2 to achieve the desired immune activation. Liposomes, as drug delivery carriers, can achieve targeted drug delivery by encapsulating drugs and utilizing primary and secondary antibodies.

[0113] Therefore, this invention, by preparing multi-cell-targeting liposomes containing major antibodies against target cells and auxiliary antibodies against effector cells, can induce immune cells to bind to and recognize tumor cells, thereby activating the effector function of immune cells. These multi-cell-targeting liposomes possess a unique in vivo mechanism of action and represent a substantial advancement in both production and in vivo efficacy compared to clinically used multi-targeting antibodies. Furthermore, drug-loaded liposomes can be used to prepare multi-cell-targeting formulations, achieving better therapeutic effects.

[0114] As illustrated in some embodiments of the present invention, the technical solution of the present invention has the following advantages:

[0115] (1) This invention starts from the preparation of multi-cell targeted liposomes, and modifies two different specific targeting antibodies or antibody fragments on the same liposome. The liposome preparation and surface targeting modification technology is mature, simple to design, easy to produce and purify on a large scale, and the targeting group can be adjusted according to clinical needs, which is conducive to the development and design of various multi-cell targeted combinations with different specificities according to diseases;

[0116] (2) The two antibody fragment-lipid conjugates of the present invention that specifically bind to CD19 and CD3 antigens, when incubated with multi-cell targeted and single-target liposomes prepared by fluorescently labeled liposomes and CD19-positive and CD3-positive cells respectively, all showed strong binding activity and this binding was specific.

[0117] (3) The multi-cell targeted liposomes of the present invention that specifically bind to CD19 and CD3 antigens were co-incubated with CD19-positive and CD3-positive cells with different fluorescent labels. The two fluorescent double-positive signals were detected by flow cytometry, which showed that the multi-cell targeted liposomes could effectively mediate intercellular binding, that is, enable immune effector cells to recognize target cells.

[0118] (4) The multi-cell targeted liposomes of the present invention that specifically bind CD19 and CD3 antigens are co-incubated with immune effector cells and tumor cells, and exhibit a strong killing ability of immune effector cells against tumor cells, and this killing is mediated by the perforin-granzyme pathway.

[0119] (5) The multi-cell targeted liposomes of the present invention, which specifically bind to CD19 and CD3 antigens, were co-incubated with immune effector cells and tumor cells. ELISA detection showed that the multi-cell targeted liposomes could effectively stimulate the secretion of IFN-γ cytokines by immune effector cells.

[0120] (6) The multi-cell targeted liposomes of the present invention, which specifically bind to CD19 and CD3 antigens, demonstrate the ability to regulate cell interactions by adjusting the density of surface antibodies, as detected by LDH.

[0121] (7) The CD19 and CD3 antigen-specific binding multi-cell targeted liposomes of the present invention not only possess the advantages of multi-target antibodies, but also allow for the preparation of multi-cell targeted drug delivery systems using drug-loaded liposomes. Using two antibody fragments specifically binding to CD19 and CD3 antigens—lipid conjugates—and drug-loaded liposomes carrying the immunomodulatory drug dasatinib as raw materials, the prepared drug-loaded multi-cell targeted liposomes exhibit effective inhibition of immune effector cell-mediated cell killing under high drug loading. Drug-loaded multi-cell targeted liposomes can serve as targeted drug carriers, developing into targeted drug delivery systems for diseases, achieving better therapeutic effects.

[0122] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0123] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0124] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature; see Sambrook et al., *MOLECULAR CLONING: A LABORATORY MANUAL*, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., *CURRENT PROTOCOLS IN MOLECULAR BIOLOGY*, John Wiley & Sons, New York, 1987 and periodic updates; these series *METHODS IN ENZYMOLOGY*, Academic Press, San Diego; Wolffe, *CHROMATINSTRUCTURE AND FUNCTION*, Third edition, Academic Press, San Diego, 1998; *METHODS IN ENZYMOLOGY*, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and *METHODS IN MOLECULAR*. BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0125] Unless otherwise specified, all experimental materials used in the following examples were purchased from regular biochemical reagent stores.

[0126] Experimental reagents and cells

[0127] Lecithin (EPC), hydrogenated soybean lecithin (HSPC), and distearate phosphatidylethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG2000-mal) were purchased from NOF Corporation (Tokyo, Japan); cholesterol (Chol) and distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) were purchased from Avanti Polar Lipids Corporation (AL, US).

[0128] DiI was purchased from Sigma and CFSE was purchased from Invitrogen.

[0129] RPMI-1640 medium, fetal bovine serum, and penicillin-streptomycin dual antibiotic reagent (100×) were purchased from Gibico.

[0130] Raji and Jurkat human lymphoma cells were purchased from the Institute of Cell Biology, Chinese Academy of Sciences.

[0131] Example 1: Preparation of multi-cell targeted liposomes

[0132] 1. Preparation of anti-CD19 and anti-CD3 antibody F(ab')2 fragment

[0133] The Fc fragment of IgG was excised to prepare the F(ab')2 fragment by immobilized pepsin or ficin. Human and mouse IgG were digested with pepsin, while mouse IgG1 was digested with ficin. The specific steps were as follows: 500 μL of antibody was centrifuged and passed through a desalting column; the flow-through buffer was added to the immobilized enzyme and mixed at 37°C; after incubation with pepsin for 4 h or ficin for 24 h, the flow-through buffer was collected by centrifugation at 5,000 g for 1 min; the immobilized enzyme was washed with Protein A Binding Buffer, and the flow-through buffer was collected. The combined flow-through buffers yielded the digested product.

[0134] The digestion products were incubated in the protein A column for 10 min at a constant temperature. The flow-through was then collected by centrifugation. The Protein A column was washed with Protein A Binding Buffer, and the flow-through was collected again. The combined flow-throughs yielded the digestion products purified by the Protein A column, with IgG and Fc fragments removed. The digestion products were then dialyzed through a 50 kDa dialysis bag in PBS solution (pH 7.0) at 2 h, 2 h, and 16 h intervals to obtain the purified antibody F(ab')2 fragment with Fc fragment removed.

[0135] Using existing technology, it was confirmed that the obtained anti-CD19 antibody F(ab')2 fragment was obtained by removing the Fc fragment from the full-length anti-CD19 antibody.

[0136] Confirmed using existing technology, the obtained anti-CD3 antibody F(ab')2 fragment is derived from the full-length anti-CD3 antibody after removing the Fc fragment. It includes a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region is shown in SEQ ID NO.1, specifically:

[0137] DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIK.

[0138] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2, specifically:

[0139] EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS.

[0140] 2. Preparation of Fab' fragment-polymer-lipid conjugates of anti-CD19 and anti-CD3 antibodies

[0141] Using β-mercaptoethylamine as a reducing agent, the antibody F(ab')2 fragment was reduced to Fab' and chemically linked to the maleimide group of DSPE-PEG2000-Mal. The specific steps were as follows: The antibody F(ab')2 fragment (1-10 mg / ml) was mixed with 50 mM EDTA-containing β-mercaptoethylamine, and reacted at 37°C with shaking for 90 min under N2 protection. Afterwards, the mixture was centrifuged through a desalting column, and the flow-through was collected. The reduced product Fab' of the antibody F(ab')2 fragment without β-mercaptoethylamine was obtained. Then, 600 μM of DSPE-PEG2000-Mal micelles in 30 mM HEPES solution was added to make the molar ratio of Fab' to DSPE-PEG2000-Mal 1:1, and the reaction was carried out at 10°C with shaking for 16 h under N2 protection. The antibody Fab' fragment-polymer-lipid conjugate DSPE-PEG2000-Mal-Fab' micelles was obtained. The obtained ligation products were examined for ligation using SDS-PAGE. The results are as follows: Figure 1A and Figure 1B As shown.

[0142] 3. Preparation of Fab' fragment-polymer-lipid conjugate of anti-CD3 antibody

[0143] Using tricarboxyethylphosphine (TCEP) as a reducing agent, the antibody scFv fragment and its polymer were reduced to scFv containing free thiol groups and chemically linked to the maleimide group of DSPE-PEG2000-Mal. The specific steps were as follows: 800 μL of antibody scFv fragment (1-10 mg / mL) was mixed with a HEPES solution containing 1.2 Mm TCEP, making the molar ratio of scFv to TCEP 1:5. Immediately afterwards, 600 μM of DSPE-PEG2000-Mal micelles in a 30 mM HEPES solution were added, and the reaction was carried out at 10 °C with shaking under N2 protection for 16 h. The antibody scFv fragment-polymer-lipid conjugate DSPE-PEG2000-Mal-scFv micelles was obtained. The ligation status of the obtained conjugate product was examined by SDS-PAGE. The results are as follows: Figure 1C As shown.

[0144] 4. Preparation of anti-CD19 and anti-CD3 single-target and multi-cell targeted liposomes

[0145] Single-target and multi-cell-targeting liposomes were prepared by a post-interpolation method. For multi-cell-targeting liposomes, the prepared anti-CD19 and anti-CD3 Fab' fragment-polymer-lipid conjugate DSPE-PEG2000-Mal-Fab' or scFv fragment-polymer-lipid conjugate was mixed with liposomes at a molar ratio of (0.1-5):(0.1-5):1000 and reacted at 37°C under N2 protection with shaking for 24 h. Then, the liposomes were washed five times by 300KD ultrafiltration to remove unbound antibody Fab' fragments, thus obtaining anti-CD19 and anti-CD3 multi-cell-targeting liposomes. For anti-CD3 single-target liposomes, the anti-CD19 Fab' fragment-polymer-lipid linker DSPE-PEG2000-Mal-Fab' was replaced with DSPE-mPEG2000 in equal molar amounts; for anti-CD19 single-target liposomes, the anti-CD3 Fab' fragment-polymer-lipid linker DSPE-PEG2000-Mal-Fab' was replaced with DSPE-mPEG2000 in equal molar amounts. Results are as follows: Figure 2 As shown in Table 1, the particle size of the prepared targeted liposomes is around 80-90 nm, and the PDI is approximately 0.1.

[0146] Table 1

[0147]

[0148] Example 2: Evaluation of the in vitro specific binding of multi-cell targeted liposomes

[0149] Using CD19-positive Raji cells and CD3-positive Jurkat cells as examples, we investigated the in vitro specific binding of multicellular targeting liposomes.

[0150] Jurkat,Clone E6-1 human T-cell lymphoma cell line and Raji human B-cell lymphoma cell line were purchased from the Cell Bank of the Chinese Academy of Sciences. The cell lines were cultured in complete medium containing 1% plc of penicillin-dextrose antibody and 10% FBS in RPMI-1640 (GIBCO).

[0151] The in vitro specific binding capacity of multi-cell-targeting liposomes was investigated by flow cytometry analysis of CD3-positive Jurkat cells, CD19-positive Raji cells, and human peripheral blood lymphocytes. (1×10⁻⁶) 6 Raji or Jurkat cells were resuspended in 400 μL of complete culture medium and added to 48-well plates. 1 μg of single- or multi-cell-targeting DiI-labeled liposomes was added to each well. Cells were incubated at 37°C for 2 h. Cells were then transferred to flow cytometry tubes, centrifuged at 500 × g for 5 min, and the supernatant was discarded. Cells were washed twice with PBS. Cells were then resuspended in 400 μL of PBS and analyzed using a Becton Dickinson FACS LSRII flow cytometer. Results are as follows: Figure 3A and Figure 3B As shown.

[0152] Example 3: Evaluation of Multi-cell Targeting Liposome-Mediated Intercellular Binding

[0153] Using CD19-positive Raji cells and CD3-positive Jurkat cells as examples, it was demonstrated that multi-cell targeted liposomes can mediate cell-cell binding between different target cells.

[0154] Jurkat cells were stained with CFSE, and Raji cells were stained with DiI. 1.25 × 10⁻⁶ 5 Raji-DiI and 1.25×10 5 Jurkat-CFSE cells were resuspended and mixed in 400 μL of phenol red-free complete medium and added to 48-well plates. 1 μg of single- or multi-cell-targeting liposomes was added to each well. The plates were incubated at 37°C for 30 min. Flow cytometry was used to analyze CFSE-positive Jurkat cells, DiI-positive Raji cells, and CFSE / DiI double-positive cell clusters to investigate the target cell binding mediated by multi-cell-targeting liposomes. Results are as follows: Figure 4 As shown.

[0155] Example 4: Evaluation of the killing effect of multi-cell targeted liposome-mediated effector cells on tumor cells.

[0156] The cell culture medium was formulated as follows: 5% FBS, 1% penicillin antibiotics, and 100 IU / ml hIL-2 were added to phenol red-free RPMI-1640 medium.

[0157] 1. LDH method for evaluating tumor cell killing effect

[0158] Through Promega's CytoTox A non-radioactive cytotoxicity assay kit was used to detect the killing effect of multi-cell targeted liposome-mediated effector cells on tumor cells.

[0159] Effector cells (unstimulated human lymphocytes, CD3 / CD28 antibody-amplified and activated human T cells, γδT cells, and LAK cells) and target cells (Raji cells, SUP-B15 cells, and A549 cells) were resuspended in phenol red-free complete medium at concentrations of 1*10^6 cells / ml and 1.11*10^5 cells / ml, respectively. 100 μL of effector cells and 90 μL of target cells were added to 96-well plates and mixed thoroughly by pipetting, resulting in a target cell to effector cell ratio of 1:10. 10 μL of different concentrations of targeted liposomes were added to each well and mixed thoroughly by pipetting. The 96-well plates were incubated in a cell culture incubator for 24 hours. 45 minutes before the end of incubation, 10 μL of lysis buffer was added to the volume correction control wells and the wells with the largest target cell lysis. After incubation, centrifuge the 96-well plate at 250g for 4 min. After centrifugation, transfer 50 μL of the supernatant to a new 96-well plate, add 50 μL of LDH substrate to the new plate, and incubate at 22°C for 15 min. After incubation, measure the absorbance at 490 nm using a microplate reader.

[0160] The results are as follows Figure 5A , Figure 5B , Figure 6A , Figure 6B , Figure 6C , Figure 8B , Figure 8C As shown in Figure 9.

[0161] These experimental data fully demonstrate that the multi-cell targeted liposomes of the present invention can effectively mediate the effective killing of target cells by effector cells. The effector cells may include unstimulated lymphocytes, in vitro expanded lymphocytes, in vitro activated lymphocytes, and in vitro induced differentiated lymphocytes. Moreover, this cell-killing effect depends on cell lysis and is dose-dependent.

[0162] 2. Evaluation of tumor cell killing effect using PI staining method

[0163] Effector cells (unstimulated human lymphocytes, CD3 / CD28 antibody-amplified and activated human T cells, γδT cells, and LAK cells) and CFSE-labeled target cells (Raji cells) were resuspended in phenol red-free complete medium at concentrations of 1*10^6 cells / ml and 1.11*10^5 cells / ml, respectively. 100 μL of effector cells and 90 μL of target cells were added to 96-well plates and mixed by pipetting, resulting in a target cell to effector cell ratio of 1:1, 1:5, and 1:10. 10 μL of different concentrations of targeted liposomes were added to each well and mixed by pipetting. The 96-well plates were incubated in a cell culture incubator for 24 hours. After incubation, the cell culture was collected into a flow cytometry tube, 5 μL of 1 mg / ml PI was added, and the cells were incubated for 15 minutes. Then, 200 μL of flow cytometry buffer was added and the cells were loaded.

[0164] The results are as follows Figure 7A , Figure 7B , Figure 8A , Figure 9B As shown.

[0165] These experimental data fully demonstrate that the multi-cell-targeting liposomes of the present invention can effectively mediate the killing of target cells by effector cells. The effector cells can include unstimulated lymphocytes, in vitro expanded lymphocytes, in vitro activated lymphocytes, and in vitro induced differentiated lymphocytes. Furthermore, this cell-killing effect depends on membrane lysis and exhibits a dose-dependent relationship. In addition, the multi-targeting antibody can effectively mediate the killing of effector cells on different target cells expressing the same antigen. Figure 9A CD19+SUP-B15 cells).

[0166] The results of changing different antibody molecules or target cells are as follows Figure 9A , Figure 9B , Figure 9C As shown. Among them, Figure 9A The main antibody was replaced with different clones (2C11) and different antibody forms (IgG) of anti-CD3 antibody; Figure 9B The multi-cell targeted liposomes used have the same primary and auxiliary antibodies, but the target cells were changed to CD19+ human leukemia SUP-B15 cells. Figure 9C The auxiliary antibody was replaced with a different targeting molecule (RSPO-1 protein) that targets different antigens (LGR), and the target cells were replaced with non-small cell lung cancer A549 cells (solid tumor model).

[0167] These experimental data fully demonstrate that the primary and secondary antibodies of the multi-cell targeted liposomes of this invention can be targeting molecules of any form and structure that target any antigen, and the target cells and effector cells can be any cells that can be bound by the primary and secondary antibodies. Because the multi-cell targeted liposomes mediate the interaction between immune effector cells and target cells based on targeting, we can effectively mediate the effect of effector cells on target cells by changing any targeting molecule (primary and secondary antibody) according to different disease types.

[0168] Example 5: Evaluation of the cell activation effect of multi-cell targeted liposome stimulation

[0169] Effector cells (unstimulated human lymphocytes) and target cells (Raji cells) were resuspended in phenol red-free complete medium at concentrations of 1*10^6 cells / ml and 1.11*10^5 cells / ml, respectively. 100 μL of effector cells and 90 μL of target cells were added to 96-well plates and mixed thoroughly by pipetting, resulting in a target cell to effector cell ratio of 10:1. 10 μL of different concentrations of targeted liposomes were added to each well and mixed thoroughly by pipetting. The 96-well plates were incubated in a cell culture incubator for 24 hours. After incubation, the 96-well plates were centrifuged at 250 g for 4 min, and 50 μL of the supernatant was transferred to a fresh 96-well plate. IFN-γ in the supernatant was detected by ELISA. The results are shown below. Figure 10 As shown, this invention's multi-cell targeted liposomes effectively mediate the killing of target cells by unstimulated lymphocytes, in vitro expanded lymphocytes, in vitro activated lymphocytes, and in vitro induced differentiated lymphocytes, while simultaneously promoting the activation of effector cells, promoting the production and release of cytokines, and the produced cytokines have anti-tumor activity in a dose-dependent manner.

[0170] Example 6: Study on the Mechanism of Action of Multi-Cell Targeted Liposomes

[0171] Effector cells (unstimulated human lymphocytes) and target cells (Raji cells) were resuspended in phenol red-free complete medium at concentrations of 1.11 × 10⁶ cells / ml and 1.11 × 10⁵ cells / ml, respectively. 90 μL of effector cells and 90 μL of target cells were added to each well of a 96-well plate and seeded by pipetting, maintaining a target cell to effector cell ratio of 10:1. 10 μL of multi-cell targeted liposomes were added to each well and mixed by pipetting. 10 μL of EGTA / MgCl₂ was added to each well, with final concentrations of 0, 0.1, 1, and 10 mM, and mixed by pipetting. The 96-well plates were incubated in a cell culture incubator for 24 hours. 45 minutes before the end of incubation, 10 μL of lysis buffer was added to the volume correction control wells and the wells with the largest target cell lysis. After incubation, centrifuge the 96-well plate at 250g for 4 min. After centrifugation, transfer 50 μL of the supernatant to a new 96-well plate, add 50 μL of LDH substrate to the new plate, and incubate at 22°C for 15 min. After incubation, measure the absorbance at 490 nm using a microplate reader.

[0172] The results are as follows Figure 11 As shown, this fully illustrates that the multi-cell targeted liposome-mediated killing effect of effector cells on target cells in this invention is based on the calcium ion-dependent granzyme / perforin pathway of effector cells.

[0173] Example 7: Evaluation of Multi-cell Targeted Liposome-based Drug Delivery

[0174] Multi-cell targeted liposomes loaded with drugs were prepared by a post-interpolation method. For the multi-cell targeted drug-loaded liposomes, the prepared anti-CD19 and anti-CD3 Fab' fragment-polymer-lipid conjugate DSPE-PEG2000-Mal-Fab' was mixed with dasatinib drug-loaded liposomes at a molar ratio of 0.1-5:0.1-5:1000, and reacted at 37°C under N2 protection with constant temperature shaking for 24 h. Then, the liposomes were washed 5 times by 300KD ultrafiltration to remove unbound antibody Fab' fragments, thus obtaining multi-cell targeted dasatinib liposomes.

[0175] Effector cells (unstimulated lymphocytes) and target cells (Raji cells) were resuspended in phenol red-free complete medium at concentrations of 1*10^6 cells / ml and 1.11*10^5 cells / ml, respectively. 100 μL of effector cells and 90 μL of target cells were added to 96-well plates and seeded by pipetting, maintaining a target cell to effector cell ratio of 1:10. 10 μL of multi-cell targeted drug-loaded liposomes with different dasatinib concentrations but the same lipid concentration were added to each well and mixed by pipetting. The 96-well plates were incubated in a cell culture incubator for 24 hours. 45 minutes before the end of incubation, 10 μL of lysis buffer was added to the volume correction control wells and the wells with the largest target cell lysis. After incubation, the 96-well plates were centrifuged at 250 g for 4 min. 50 μL of supernatant was transferred to a new 96-well plate, and 50 μL of LDH substrate was added to the new plate. The plates were incubated at 22°C for 15 min. After incubation, absorbance was measured at 490 nm using a microplate reader.

[0176] The results are as follows Figure 12 As shown, this fully demonstrates that by linking the main antibody and the auxiliary antibody to the drug-loaded liposome, the drug-loaded effect can be fully utilized based on the multi-cell targeting mechanism.

[0177] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention. sequence list <110> Shanghai Jiao Tong University <120> A multi-cell targeted liposome <130> 171715 <160> 16 <170> SIPOSequenceListing 1.0 <210> 1 <211> 107 <212> PRT <213> Artificial Sequence <400> 1 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 2 <211> 122 <212> PRT <213> Artificial Sequence <400> 2 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met 35 40 45 Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Leu Thr Val Phe Ser 115 120 <210> 3 <211> 236 <212> PRT <213> Artificial Sequence <400> 3 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100 105 110 His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 4 <211> 237 <212> PRT <213> Artificial Sequence <400> 4 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile 35 40 45 Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 85 90 95 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 145 150 155 160 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 225 230 235 <210> 5 <211> 119 <212> PRT <213> Artificial Sequence <400> 5 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60 Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115 <210> 6 <211> 107 <212> PRT <213> Artificial Sequence <400> 6 Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 7 <211> 121 <212> PRT <213> Artificial Sequence <400> 7 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ile Ile Ser Asp Gly Gly Tyr Tyr Thr Tyr Tyr Ser Asp Ile Ile 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Phe Pro Leu Leu Arg His Gly Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 8 <211> 107 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Asp Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Ala Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ser 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 9 <211> 450 <212> PRT <213> Artificial Sequence <400> 9 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 10 <211> 216 <212> PRT <213> Artificial Sequence <400> 10 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215 <210> 11 <211> 119 <212> PRT <213> Artificial Sequence <400> 11 Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 12 <211> 106 <212> PRT <213> Artificial Sequence <400> 12 Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 13 <211> 125 <212> PRT <213> Artificial Sequence <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 14 <211> 110 <212> PRT <213> Artificial Sequence <400> 14 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly 20 25 30 Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn 85 90 95 Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ala 100 105 110 <210> 15 <211> 113 <212> PRT <213> Artificial Sequence <400> 15 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <210> 16 <211> 111 <212> PRT <213> Artificial Sequence <400> 16 Leu Glu Met Ala Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 1 5 10 15 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 20 25 30 Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser 85 90 95 Asn Trp Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110

Claims

1. A multilamellar targeting liposome, characterized in that, The structure of the multivesicular body-targeting liposome is composed of a liposome, a primary antibody modified on the surface of the liposome, and a secondary antibody modified on the surface of the liposome, wherein the primary antibody specifically binds to a target molecule on the surface of a target cell, and the secondary antibody specifically binds to an immune effector cell; The multivesicular body-targeting liposome can simultaneously or sequentially bind to the immune effector cell and the target cell, so as to realize the recognition of the target cell by the immune effector cell and the activation of the immune effector. The liposome is a bilayer vesicle formed by lipid molecules, and the components of the liposome are composed of phosphatidylcholine, cholesterol, a lipid connected to the primary antibody, and a lipid connected to the secondary antibody. The primary antibody and the secondary antibody are modified on the surface of the liposome through a reaction of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide. The multivesicular body-targeting liposome is prepared by a method comprising the following steps: (1) connecting the primary antibody and the secondary antibody to lipid molecules, respectively, to obtain a primary antibody-lipid molecule and a secondary antibody-lipid molecule; (2) mixing and incubating the obtained primary antibody-lipid molecule and secondary antibody-lipid molecule with a liposome that has been constructed to obtain a mixed solution; (3) removing the unloaded antibodies and antibody-lipid complexes from the obtained mixed solution by dialysis or ultrafiltration to obtain the multivesicular body-targeting liposome. The particle size of the multivesicular body-targeting liposome is 80-90 nm, and the PDI is about 0.

1. The molar ratio of the primary antibody to the secondary antibody on each liposome is between 100 / 1 and 1 / 1.

2. The multilamellar liposome of claim 1, wherein, There are 1-1000 primary antibodies and 1-1000 secondary antibodies on the surface of each liposome.

3. The multilamellar liposome of claim 1, wherein, The primary antibody and the secondary antibody are displayed on the surface of the liposome through covalent connection or hydrophobic-hydrophilic interaction.

4. The multilamellar liposome of claim 1, wherein, The antigen targeted by the primary antibody is selected from microbial antigens, tumor-associated antigens, tumor cell surface-specific antigens, tumor cell surface-highly-expressed antigens, and antigens highly expressed in tumor tissues or tumor blood vessels.

5. The multilamellar liposome of claim 1, wherein, The antigen targeted by the secondary antibody is selected from antigens expressed by tumor cells, lymphocytes, or myeloid cells.

6. The multilamellar liposome of claim 1, wherein, The multivesicular body-targeting liposome can simultaneously or sequentially bind to tumor cells, lymphocytes, or myeloid cells.

7. The multilamellar liposome of claim 1, wherein, The multivesicular body-targeting liposome targets and binds to T lymphocytes, NK cells, NKT cells, macrophages, or neutrophils.

8. The multilamellar liposome of claim 1, wherein, The multivesicular body-targeting liposome targets and binds to unactivated T cells or in-vitro activated and expanded T cells.

9. The multilamellar liposome of claim 8, wherein, The T lymphocytes are selected from antigen-specific T lymphocytes, tumor-infiltrating lymphocytes, cytotoxic T cells, helper T cells, lymphocyte-activated killer cells, γδ T cells, chimeric antigen receptor T cells, and T cell receptor chimeric T cells.

10. The multilamellar liposome of claim 1, wherein, The forms of the primary antibody and the secondary antibody are selected from IgG, Fab' fragments, F(ab')2 fragments, Fab fragments, single-chain Fv fragments, single-domain antibodies, and double-domain antibodies.

11. The multilamellar liposome of claim 1, wherein, The antigen targeted by the primary antibody is selected from CD19, CD20, PSMA, Her2 / neu, EGFR, or LGR5.

12. The multilamellar liposome of claim 1, wherein, The helper antibody is selected from one or more of a CD3 antibody or an antigen-binding fragment thereof, a PD1 antibody or an antigen-binding fragment thereof, a CTLA4 antibody or an antigen-binding fragment thereof, and a CD40L antibody or an antigen-binding fragment thereof.

13. The multilamellar liposome of claim 1, wherein, The primary antibody is an antibody against CD19, and the helper antibody is a CD3 antibody.

14. The method for preparing the multi-cell targeting liposome according to any one of claims 1-13, wherein the method comprises the following steps: (1) linking the primary antibody and the helper antibody to lipid molecules respectively to obtain a primary antibody-lipid molecule and a helper antibody-lipid molecule; (2) mixing and incubating the obtained primary antibody-lipid molecule and helper antibody-lipid molecule with the liposome that has been constructed to obtain a mixed solution; (3) removing the unloaded antibodies and antibody-lipid complexes from the obtained mixed solution by dialysis or ultrafiltration to obtain the multi-cell targeting liposome.

15. The use of the multi-cell targeting liposome according to any one of claims 1-13 in the preparation of an immunotherapeutic drug or an anti-tumor drug. ​

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