T cell-directed immunoliposome and use thereof

Abstract

The present invention relates to the field of uses of liposome formulations in preparation of tumor drugs, and in particular to a T cell-directed immunoliposome (TRAFsome). A targeting antibody and an immune cell engaging antibody have different valencies and are optimized separately, and a drug encapsulated in the liposome can be an immunomodulator, an anti-cancer drug or a combination thereof.

Description

T cell-directed immunoliposomes and their uses Technical Field

[0001] The present disclosure relates to the technical fields of immunoliposomes, T cell immunotherapy, and more particularly to a T cell-directed immunoliposome drug. Technical Background

[0002] Liposomes are bilayered, vesicular drug delivery systems made from phospholipids and / or cholesterol. Liposomes are artificially constructed bilayered structures formed from lipid molecules. Due to the unique structure of liposomes, the lipid molecules have a hydrophilic head and a hydrophobic tail, allowing hydrophilic drugs to be contained in the internal aqueous phase while hydrophobic drugs can be embedded within the lipid bilayer. Furthermore, liposomes exhibit excellent biocompatibility, low toxicity, and good biosafety, attracting widespread attention and application in drug delivery systems. The development and research of liposome-targeted technologies have endowed liposome-based drugs with unique in vivo drug distribution and metabolism behaviors. The liposome formulation allows drug accumulation at target sites and specific interaction with target cells. By optimizing target selection and improving the structure and properties of targeting ligands, it is expected that the clinical efficacy of liposome-based drugs will be enhanced, including reducing systemic toxicity and / or increasing the therapeutic index.

[0003] The research and development of targeted liposome drug delivery systems focuses on designing targeted sites through various targets, often with local specificity and / or sufficient expression abundance. Compared to delivery vehicles or free drugs that do not contain specific targeting heads, targeted liposome drugs developed based on this design concept have higher organ specificity for lesions. For example, drug concentration at the target site is increased, and pharmacodynamics is improved. However, this type of targeted drug delivery system only works on specific cell types and may not effectively eliminate tumors. It may also cause problems such as minimal residual disease and multidrug resistance in tumors.

[0004] In recent years, with the development of tumor immunology, it has been clarified that when only tumor cells are targeted for delivery, the therapeutic effect is often determined by a combination of multiple factors. For example, the heterogeneity of different tumor cell molecular markers within the same tumor of the same patient, or the different immune types of immune cells within the same tumor contribute differently to the immune microenvironment, or the interaction within the tumor for relief, etc. Therefore, based on the characteristics of the tumor microenvironment, researchers have broadened the design concept of targeted delivery. For example, drug carriers targeted to tumor-associated cells can inhibit tumor growth through other mechanisms, such as acting on tumor angiogenesis, T cells, myeloid cells and tumor stem cells.

[0005] While this targeted delivery technology has improved therapeutic efficacy to a certain extent, its design concept is still limited to targeting a single cell type. However, extensive interactions and interactions occur between different cell types within a tumor, such as lymphocyte infiltration, macrophage phagocytosis, antigen presentation, and intercellular communication via cytokines and exosomes. These interactions constitute immune surveillance at the tumor site, maintaining a dynamic balance between tumor clearance and tumor growth. Blocking or promoting these cell-cell interactions has been proven feasible and effective in clinical practice, such as vaccine therapies based on antigen-specific T cells, antibody therapies that rely on NK cell cytotoxicity, and immune checkpoint inhibitors to prevent tumor immune escape. In contrast, drug delivery targeting a single cell type often only targets a specific target cell and modulates its physiological processes, but fails to influence the intercellular interactions within tumor tissue to achieve effective treatment.

[0006] In the present invention, antibodies targeting different cell types are loaded into a single liposome. Through the action of the antibodies, the liposome can bind to different cell types sequentially or simultaneously, promoting recognition, communication, activation, apoptosis, or clearance of target cells. Furthermore, the present invention achieves targeted drug delivery to multiple cell types using a single liposome through its structurally stable liposomes, thereby avoiding the potential for drug interactions associated with combination therapies and improving drug efficacy.

[0007] Technical Summary

[0008] The present invention provides an immunoliposome with the function of mediating immune cell redirection, also known as a T cell redirecting immunoliposome (T-cell redirecting antibody fragment-anchored liposome, TRAFsome). The T cell redirecting immunoliposome can bind to one or more different types of cells sequentially or simultaneously. The present invention will also disclose the preparation process and application method of the T cell redirecting immunoliposome. In addition, the present invention also provides a method for treating cancer characterized by the presence of solid tumors by administering the T cell redirecting immunoliposome. In this article, the terms "cancer characterized by the presence of solid tumors" and "tumor" can be used interchangeably.

[0009] The objectives of this disclosure will be achieved through the following technical solutions:

[0010] In the first aspect of the present invention, a liposome with immune cell redirection function is disclosed. The immune cell redirection liposome comprises a liposome structure with a targeting antibody / fragment and an immune cell-binding antibody / fragment modified on the liposome surface. The targeting antibody / fragment can specifically bind to the surface of target cells, while the immune cell-binding fragment can bind to immune cells with immune effects.

[0011] Secondly, T cell-directed immunoliposomes can bind to immune effector cells and target cells simultaneously or sequentially, enabling immune effector cells to recognize target cells and activate immune effects.

[0012] Preferably, the molar ratio of the targeting antibody / fragment to the immune cell binding antibody / fragment on the same T cell-targeting immunoliposome is between 10 / 1 and 1 / 10.

[0013] Preferably, there are about 1-1000 targeting antibodies / fragments and 1-1000 immune cell binding antibodies / fragments on the surface of the same T cell-directed immunoliposome.

[0014] In some preferred embodiments disclosed herein, the targeting antibody / fragment and the immune cell-binding antibody / fragment are each conjugated to a lipid molecule, and the conjugated lipid-antibody complex further forms a liposome structure and is anchored on the liposome surface. In some cases, the targeting antibody / fragment and the immune cell-binding antibody / fragment are conjugated to maleimide groups present on the lipid molecule.

[0015] Preferably, the target cells targeted by the targeting antibodies / fragments primarily include tumor cells, microorganisms, and cells infected by microorganisms. Therefore, the antigens targeted by the targeting antibodies / fragments primarily include 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.

[0016] Preferably, the targeting antibody / fragment targets the following antigens: HER2, CD19, CD20, PSMA, Her2 / neu, EGFR, LGR5 or PDL1.

[0017] In some preferred examples disclosed in the present invention, the specific target molecule of the targeting antibody / fragment is CD19 or HER2.

[0018] Preferably, in the present invention, immune cells with immune effects are effector cells that can produce cytotoxic effects, promote apoptosis, or clear target cells. Immune cells with immune effects include: T lymphocytes, NK cells, NKT cells, monocytes, dendritic cells, macrophages, or neutrophils.

[0019] In the present invention, immune cell-binding antibodies / fragments bind to antigens expressed by immune effector cells. Thus, immune cell-redirecting liposomes can bind to tumor cells, lymphocytes, or other immune cells (e.g., myeloid cells) simultaneously or sequentially. The same immune cell-redirecting liposome may first bind to tumor cells and then to immune cells, or vice versa.

[0020] In some preferred examples disclosed in the present invention, the immune cells with immune effects are T lymphocytes.

[0021] In some embodiments disclosed herein, the cellular antigen of the immune cell with immune effector function is CD3, and the immune cell binding antibody / fragment is a CD3 antibody.

[0022] Preferably, the immune cell binding antibody / fragment may also be a CD3 antibody / fragment, a PD1 antibody / fragment, a CTLA4 antibody / fragment and a CD40L antibody / fragment.

[0023] Preferably, the T lymphocytes can be primitive T cells or T cells 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 or T cell receptor chimeric T cells.

[0024] Activation or expansion of immune cells can be achieved through antigens, antibodies, peptides, peptide-major histocompatibility complex, small molecules, cytokines, immune checkpoint inhibitors, or gene editing.

[0025] Preferably, the targeting antibody / fragment and immune cell binding antibody / fragment can be in the form of IgG, Fab' fragment, F(ab')2 fragment, Fab fragment, single-chain Fv fragment, nanobody, bispecific antibody, unibody or minibody.

[0026] Preferably, in some preferred embodiments disclosed herein, the targeting antibody / fragment and the immune cell-binding antibody / fragment are Fab' fragments obtained by removing the Fc fragment from the antibody and reducing it, which fragments include the light chain variable region and the heavy chain variable region.

[0027] Preferably, in some preferred embodiments disclosed herein, the targeting antibody / fragment is a Fab' fragment of a CD19 antibody, and the amino acid sequence of the light chain (LC) variable region (VL) thereof is as shown in SEQ ID NO. 1, specifically:

[0028] Preferably, the amino acid sequence of the heavy chain (HC) variable region (VH) of the Fab' fragment of the CD19 antibody is as shown in SEQ ID NO.2, specifically:

[0029] Preferably, in some preferred embodiments disclosed herein, the targeting antibody / fragment is a Fab' fragment of a HER2 antibody, and the amino acid sequence of the light chain (LC) variable region (VL) thereof is as shown in SEQ ID NO. 3, specifically:

[0030] Preferably, the amino acid sequence of the heavy chain (HC) of the Fab' fragment of the HER2 antibody is as shown in SEQ ID NO.4, specifically:

[0031] Preferably, the targeting antibody / fragment can be a scFv fragment of an EGFR antibody, and the scFv VH sequence of the EGFR antibody is shown in SEQ ID NO.5, specifically:

[0032] Preferably, the targeting antibody / fragment can be a scFv fragment of an EGFR antibody, and the scFv VL sequence of the EGFR antibody is shown in SEQ ID NO.6, specifically:

[0033] Preferably, the targeting antibody / fragment can be a PSMA antibody, and the VH sequence of the PSMA antibody is shown in SEQ ID NO.7, specifically:

[0034] Preferably, the targeting antibody / fragment can be a PSMA antibody, and the VL sequence of the PSMA antibody is shown in SEQ ID NO. 8, specifically:

[0035] Preferably, the targeting antibody / fragment can be a PD-L1 antibody, and the heavy chain sequence of the PD-L1 antibody is shown in SEQ ID NO.9, specifically:

[0036] Preferably, the targeting antibody / fragment can be a PD-L1 antibody, and the light chain sequence of the PD-L1 antibody is shown in SEQ ID NO.10, specifically:

[0037] Preferably, in some preferred embodiments disclosed herein, the immune cell-binding antibody / fragment is a Fab' fragment of a CD3 antibody, and the amino acid sequence of its light chain (LC) is as shown in SEQ ID NO. 11, specifically:

[0038] Preferably, the amino acid sequence of the heavy chain (HC) of the Fab' fragment of the CD3 antibody is as shown in SEQ ID NO. 12, specifically:

[0039] Preferably, the immune cell binding antibody / fragment may be a PD-1 antibody, the amino acid sequence of its VH being as shown in SEQ ID NO. 13, specifically:

[0040] Preferably, the immune cell binding antibody / fragment can be a PD-1 antibody, the amino acid sequence of its VL is shown in SEQ ID NO. 14, specifically:

[0041] The present invention has no particular limitation on the composition of the T cell-directed immunoliposome, as long as it meets the definition of a liposome. The components of the liposomes can be selected from the following phospholipid combinations, including lecithin, hydrogenated soybean phosphatidylcholine, hydrogenated egg phosphatidylcholine, stearic acid glyceryl phosphatidylcholine, myristic acid glyceryl phosphatidylcholine, palmitic acid glyceryl phosphatidylcholine, stearic acid glyceryl phosphatidylcholine, palmitic acid-myristic acid glyceryl phosphatidylcholine, palmitic acid-stearic acid glyceryl phosphatidylcholine, stearic acid-palmitic acid glyceryl phosphatidylcholine, palmitic acid-oleamide glyceryl phosphatidylcholine, stearic acid-linoleoylcholine, dioleoylcholine, hydrogenated palmitic acid glyceryl phosphatidylcholine, stearic acid phosphatidic acid, myristic acid phosphatidic acid, palmitic acid phosphatidic acid, stearic acid phosphatidic acid, palmitic acid phosphatidic acid, myristyl phosphatidylethanolamine, palmitic acid phosphatidylethanolamine, brain phosphatidylserine, myristic acid phosphatidylserine, palmitic acid phosphatidyl Serine, egg phosphatidylglycerol, distearate glycerylphosphatidylglycerol, myristate glycerylphosphatidylglycerol, palmitate glycerylphosphatidylglycerol, linoleate glycerylphosphatidylglycerol, dioleate glycerylphosphatidylglycerol, cerebrolysin, myristate sphingolipid or stearate sphingolipid, cholesterol, 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), dioleoylchloropropyltrimethylammonium chloride (DOTMA), dimethylcosylammonium bromide (DDAB), dimethylaminoethylaminocholesterol (DC-Chol), spanmin-5-carboxyaminoacetic acid octacosylamide (DOGS), dioleate succinylaminomethyl dimethylpropylammonium chloride trifluoroacetate (DOSPA), cholesterol and DSPE-PEG-2000, or a combination of two or more.

[0042] Preferably, in some preferred embodiments disclosed herein, the components of the T cell-directed immunoliposome include phosphatidylcholine, cholesterol, a targeting antibody / fragment coupled to a lipid molecule, and an immune cell-binding antibody / fragment coupled to a lipid molecule.

[0043] Preferably, the T cell-targeting immunoliposomes may encapsulate drugs in the internal aqueous phase of the liposomes, or may not contain drugs.

[0044] In some preferred examples disclosed herein, the average number of immune cell binding antibodies / fragments per T cell-targeted immunoliposome is 1 to 9. Preferably, the average number of anti-CD3 fragments per T cell-targeted immunoliposome is 1 to 8. Preferably, the average number of anti-CD3 fragments per T cell-targeted immunoliposome is 1 to 7. Preferably, the average number of anti-CD3 fragments per T cell-targeted immunoliposome is 1 to 6. Preferably, the average number of anti-CD3 fragments per T cell-targeted immunoliposome is 1 to 5. Preferably, the average number of anti-CD3 fragments per T cell-targeted immunoliposome is 1 to 4. Preferably, the average number of anti-CD3 fragments per T cell-targeted immunoliposome is 1 to 3. Preferably, the average number of anti-CD3 fragments per T cell-targeted immunoliposome is 1 to 2.

[0045] Further preferably, the average number of immune cell binding antibodies / fragments per T cell-targeting immunoliposome is 2 to 9. Further preferably, the average number of anti-CD3 fragments per T cell-targeting immunoliposome is 2 to 6. Further preferably, the average number of anti-CD3 fragments per T cell-targeting immunoliposome is 3 to 9. Further preferably, the average number of anti-CD3 fragments per T cell-targeting immunoliposome is 3 to 8. Further preferably, the average number of anti-CD3 fragments per T cell-targeting immunoliposome is 3 to 6.

[0046] More preferably, the average number of immune cell binding antibodies / fragments per T cell-targeting immunoliposome is 4 to 9. More preferably, the average number of anti-CD3 fragments per T cell-targeting immunoliposome is 4 to 8. More preferably, the average number of anti-CD3 fragments per T cell-targeting immunoliposome is 5 to 7. More preferably, the average number of anti-CD3 fragments per T cell-targeting immunoliposome is 6.

[0047] In some preferred examples disclosed in the present invention, the average number of targeting antibodies / fragments (targeting HER2) per T cell-directed immunoliposome is 3 to 20. Preferably, the average number of targeting antibodies / fragments (targeting HER2) per T cell-directed immunoliposome is 3 to 18. Preferably, the average number of targeting antibodies / fragments (targeting HER2) per T cell-directed immunoliposome is 3 to 15. Preferably, the average number of targeting antibodies / fragments (targeting HER2) per T cell-directed immunoliposome is 3 to 12. Preferably, the average number of targeting antibodies / fragments (targeting HER2) per T cell-directed immunoliposome is 3 to 10.

[0048] Preferably, the average number of targeting antibodies / fragments (targeting HER2) per T cell-directed immunoliposome is 5 to 20. Preferably, the average number of targeting antibodies / fragments (targeting HER2) per T cell-directed immunoliposome is 6 to 20. Preferably, the average number of targeting antibodies / fragments (targeting HER2) per T cell-directed immunoliposome is 7 to 20. Preferably, the average number of targeting antibodies / fragments (targeting HER2) per T cell-directed immunoliposome is 8 to 20. Preferably, the average number of targeting antibodies / fragments (targeting HER2) per T cell-directed immunoliposome is 10 to 20.

[0049] Further preferably, the average number of targeting antibodies / fragments (targeting HER2) per T cell-directed immunoliposome is 8 to 15. Preferably, the average number of targeting antibodies / fragments (targeting HER2) per T cell-directed immunoliposome is 9 to 12. Preferably, the average number of targeting antibodies / fragments (targeting HER2) per T cell-directed immunoliposome is 9 to 11. Preferably, the average number of targeting antibodies / fragments (targeting HER2) per T cell-directed immunoliposome is 10.

[0050] Preferably, the T cell-directed immunoliposomes can be loaded with one or more immunomodulatory chemical molecules having immunomodulatory functions. Preferably, the chemical molecules can include one or more anticancer drugs and / or immunomodulatory drugs. In some preferred embodiments disclosed herein, the immunomodulatory drugs encapsulated in the liposomes include Resiquimod (also known as CD11301; R848; S28463; VML600), Imiquimod (trade name: ), MBS8, Guretolimod (DSP-0509), TransConTM, GSK1795091, GSK2245035, VTX-2337, eritoran (E5564), SD-101, Selgantolimod, RO7119929, CpG1018, Vidutolimod (CMP-001), SHR2150, MGN1703, CYT003-QbG10, CPG-7909, Tilsotolimod, JNJ-64794964 (AL-034 / TQ-A3334), BNT411, APR003, BDB001, or a combination of one or more of them.

[0051] In addition, other immunomodulatory drugs suitable for liposome encapsulation in the art are also included. For example, see U.S. Patents 8,202,974, 9,050,376, 9,211,320, 9,248,170, 9,295,732, 9,458,184, 10,076,535, 10,259,793 and 11,026,964, and U.S. Patent Application Publications 2018 / 0360974, 2018 / 0085388, 2020 / 0316211, 2021 / 0059953, 2021 / 0371440 and 2022 / 0192997.

[0052] In addition, other drugs suitable for liposome encapsulation in the art are also included. For example, see U.S. Patents 8,597,654, 8,895,717, 9,326,953, 9,351,997, 9,789,193, 9,993,551, 10,376,519, 10,426,753, 10,618,905, 10,653,774, 10,654,831 and 11,096,900, and U.S. Patent Application Publications 2005 / 240131, 2020 / 0001110, 2020 / 0283406, 2021 / 0077397, 2022 / 0160729 and 2021 / 0275453.

[0053] Preferably, in some preferred embodiments disclosed herein, the drug encapsulated by the T cell-directed immunoliposomes comprises dasatinib. Preferably, in some preferred embodiments disclosed herein, the drug encapsulated by the T cell-directed immunoliposomes comprises resiquimod.

[0054] Further preferably, in some preferred embodiments disclosed herein, the drug encapsulated in the T cell-targeting immunoliposomes comprises resiquimod. The ratio of resiquimod to lipid material is 1:10 to 1:100 (weight ratio), and resiquimod can be encapsulated within the interior of the T cell-targeting immunoliposomes or in the phospholipid bilayer thereof.

[0055] Preferably, the ratio of resiquimod to lipid material is 1:15 (weight ratio). Preferably, the ratio of resiquimod to lipid material is 1:25 (weight ratio). Preferably, the ratio of resiquimod to lipid material is 1:50 (weight ratio).

[0056] The total concentration of resiquimod in the T cell-directed immunoliposome solution is between 0.5 mg / mL and 1 mg / mL, and the particle size of the T cell-directed immunoliposome is about 80 nm.

[0057] In some preferred embodiments disclosed herein, resiquimod acts as an immunomodulator in T cell-targeted immunoliposomes to induce an appropriate immune response, including activation of T lymphocytes and secretion of pro-inflammatory cytokines.

[0058] In some preferred embodiments disclosed in the present invention, the T cell-targeting immunoliposomes encapsulating resiquimod are composed of the following lipid materials, and the molar ratios thereof are shown in Table 1:

[0059] Table 1 Lipid materials and proportions of T cell-targeted immunoliposomes

[0060] In the third aspect of the present invention, the preparation of the aforementioned T cell-directed immunoliposomes encapsulating resiquimod and their anti-tumor uses are provided.

[0061] Compared with the traditional technology, the present invention has the following advantages:

[0062] The T cell-targeting immunoliposomes of the present invention share a similar mechanism of action as bispecific antibodies. Furthermore, the present invention further optimizes activation of tumor-associated specific immunity by adjusting the density and relative ratio of the targeting antibody / fragment and the immune cell-binding antibody / fragment. Furthermore, T cell-targeting immunoliposomes can further modulate appropriate immune responses by encapsulating immunomodulatory drugs. Finally, compared to multispecific antibodies, the production, preparation, and quality control of T cell-targeting immunoliposomes are more feasible.

[0063] Legend and Introduction

[0064] Figure 1: Schematic diagram of different T cell redirection mechanisms, including bispecific antibodies (BITEs), dual-affinity redirecting antibodies (DARTs), and T cell-directed immunoliposomes (TRAFsomes).

[0065] Figure 2A: Reducing and non-reducing SDS-PAGE of liposomes bound to full-length CD3 antibody, anti-CD3 F(ab')2, and anti-CD3 Fab'. Figure 2B: SDS-PAGE images of liposomes bound to full-length CD19 antibody, anti-CD19 F(ab')2, and anti-CD19 Fab'. Figure 2C: SDS-PAGE images of liposomes bound to anti-CD3 single-chain antibody (scFv) and anti-CD3 single-chain antibody (scFv). Figure 2D: Estimated surface density of antibodies on liposomes containing varying amounts of Fab'.

[0066] Figure 3A: Schematic diagram of anti-Her2 conjugated lipid (TL01). Figure 3B: Schematic diagram of anti-CD3 conjugated lipid (TL02).

[0067] FIG4 : T cell-directed immunoliposomes loaded with resiquimod mediate cell-to-cell redirection.

[0068] FIG5 : Evaluation of the tumor cell killing effector cell-mediated by T cell-directed immunoliposomes loaded with resiquimod.

[0069] Figure 6: Antitumor activity of resiquimod-loaded T cell-directed immunoliposomes in adenoid cystic carcinoma patient-derived human tissue xenograft tumor model

[0070] Figure 7: Antitumor activity of resiquimod-encapsulated T cell-directed immunoliposomes in the gastric cancer N87 tumor model in PBMC-reconstituted mice

[0071] Figure 8: Antitumor activity of resiquimod-encapsulated T cell-directed immunoliposomes in a CD3ε humanized Balb / C mouse model bearing Her2-expressing 4T1 tumors

[0072] Figure 9: Evaluation of T cell-mediated tumor cell killing by dasatinib-encapsulated T cell-directed immunoliposomes

[0073] Figure 10A: T cell activation mediated by T cell targeting immunoliposomes with different anti-CD3 lipid conjugations on a single liposome. Figure 10B: T cell lysis of CD19+ Raji cells and wild-type K562 cells by T cell targeting immunoliposomes with different anti-CD3 lipid conjugations on a single liposome.

[0074] Figure 11: Evaluation of T cell-mediated tumor cell killing by dasatinib-entrapped T cell-targeting immunoliposomes. Figure 11A: Schematic diagram of the experimental flow. Figures 11B-11C: Graphs showing mean (Figure 11B) and individual (Figure 11C) tumor growth curves within different groups. Figure 11D: Percentage of human T cells in the spleen of mice 24 hours after the last dose. Figure 11E: Percentage of human T cells in the spleen 24 hours after the last dose. Figure 11F: Levels of various cytokines in the serum of mice 3 hours after the last dose.

[0075] Figure 12: Activation of γδ T lymphocytes, IL-2-stimulated LAK cells, and T lymphocytes expanded with aCD3 / aCD28 magnetic beads by T cell-directed immunoliposomes. Figure 12A: Lysis of Raji cells after 5 hours of coculture with PBMCs (left), γδ T cells (center left), IL-2-stimulated PBMCs (center right), or cells expanded with aCD3 / aCD28 at various effector to target (E:T) ratios. Figure 12B: Lysis of Raji cells at a fixed E:T ratio of 10:1 using human γδ T cells at various effector to target (E:T) ratios and a 5 μg / ml lipid dose (left), human γδ T cells at various E:T ratios and a 50 μg / ml lipid dose (center right), or human γδ T cells at various E:T ratios and a 0.5 μg / ml lipid dose (right).

[0076] Further explanation of preferred examples

[0077] Liposomes are artificially prepared vesicles composed of lipid molecules with a phospholipid bilayer structure. Lipid structures often consist of a hydrophilic head and a hydrophobic tail. In a polar solvent composed of water, the hydrophobic tails of the lipid molecules are drawn together due to hydrophobic interactions, while the hydrophilic heads are closely aligned toward the aqueous phase, forming a bilayer membrane structure. Based on this structure, hydrophilic drugs can be encapsulated in the internal aqueous phase of the liposome, while hydrophobic drugs can be embedded in the phospholipid bilayer.

[0078] The active targeting effect of liposomes is achieved by binding ligands to the liposome surface. These ligands can be antibodies, peptides, small molecules, etc. Ligands interact with target cell receptors. When antibodies or antibody fragments are used as ligands, liposomes exhibit greater binding activity and specificity than small molecule and peptide ligands. Due to the self-assembly principle of lipid molecules, two or more types of ligands can be bound to the surface of a single liposome.

[0079] Based on antibody-modified liposome technology, the present invention achieves a novel and unique killing mechanism and effect against target cells by cleverly combining targeting antibodies / fragments and immune cell-binding antibodies / fragments. Currently known multi-targeted liposome preparations only use different ligand combinations to improve selectivity for target cells. The T cell-directed immunoliposomes used in the present invention also optimize and regulate the physiological functions of immune cells such as recognition, binding and activation through different ligand ratios, promoting their interaction with target cells. Therefore, the present invention provides a new mechanism of targeted liposomes.

[0080] The existing multi-specific antibody complex strategy is shown in Figure 1. They include molecules similar to bispecific T cell coupling agents (BITEs) or dual affinity redirecting antibodies (DARTs), which contain two targeting antibodies in a ratio of 1:1. In contrast, modifying a variety of different targeting antibodies on liposomes is not only more convenient, but also allows the density and ratio of different types of targeting antibodies to be adjusted on the liposome surface (such as T cell-directed immunoliposomes). In addition, T cell-directed immunoliposomes have a larger particle size than existing antibodies and complexes such as BiTe or DART (usually about 5 nanometers in diameter). The particle size of T cell-directed immunoliposomes may be 10 times larger than that of BiTe or DART. Therefore, the pharmacokinetic behavior and tissue / organ distribution of T cell-directed immunoliposomes are more advantageous than those of known BiTe or DART. In addition, T cell-directed immunoliposomes can also overcome the production difficulties caused by antibody mismatches in the design and preparation of multi-specific antibody complexes.

[0081] In addition, the T cell-directed immunoliposomes presented in the present invention are also compatible with liposome drug encapsulation technology. The drug can be encapsulated in the T cell-directed immunoliposome to achieve targeted delivery and release of the drug, that is, to improve the selectivity of the drug and change the release site and timing of the drug. For example, to slow down the common adverse reactions in tumor immunotherapy, namely the cytokine storm produced in the patient's body, the immunomodulators encapsulated by the T cell-directed immunoliposomes can achieve more precise immunoregulation, thereby minimizing systemic excessive immune responses. This further improves the efficacy of the T cell-directed immunoliposomes.

[0082] The T cell-targeted immunoliposomes described in this invention contain targeting antibodies / fragments, immune cell-binding antibodies / fragments, and an encapsulated immunomodulator. They can promote the binding and recognition of immune cells with target cells and modulate the immune function and effects of immune cells. T cell-targeted immunoliposomes have a unique mechanism of action in vivo and offer significant advantages over existing technologies in terms of production, preparation, and activity.

[0083] The present invention relates to T cell-targeted immunoliposomes as agents for immunotherapy and anti-tumor therapy, for use in the treatment of tumors. As understood in the art, "treatment" is an approach to achieving a beneficial or desired result, including a clinical result. These results may include one or more of the following: alleviation or amelioration of one or more cancer symptoms, reduction in the severity of the disease or disorder, stabilization of the disease or disorder, delay or slowing of the progression of the disease or disorder, remission of the disease or disorder, and killing of at least one cancer cell targeted by the disclosed compositions or inducing an immune response against the cancer cell.

[0084] In some preferred embodiments disclosed herein, the tumor is a HER2 / neu-positive (Her2 / neu+) tumor. HER2 / neu+ tumor cells have more HER2 / neu than normal cells. Preferably, HER2 / neu-positive (Her2 / neu+) tumors include breast, ovarian, bladder, pancreatic, gastric, and esophageal cancers. Preferably, the HER2 / neu+ tumor is breast cancer. HER2 / neu is also commonly referred to as Erb-b2 receptor tyrosine kinase 2 (ERBB2), c-erbB-2, and human epidermal growth factor (EGF) receptor 2 (HER2). Preferably, the tumor may be a HER2-low-expressing or HER2-negative tumor.

[0085] Preferably, in some preferred embodiments disclosed herein, the drug encapsulated by the T cell-directed immunoliposomes comprises dasatinib. Preferably, in some preferred embodiments disclosed herein, the drug encapsulated by the T cell-directed immunoliposomes comprises resiquimod.

[0086] Further preferably, in some preferred embodiments disclosed herein, the drug encapsulated in the T cell-targeting immunoliposomes comprises resiquimod. The ratio of resiquimod to lipid material is 1:10 to 1:100 (weight ratio), and resiquimod can be encapsulated within the interior of the T cell-targeting immunoliposomes or within the phospholipid bilayer thereof.

[0087] The total concentration of resiquimod in the T cell-directed immunoliposome solution is between 0.5 mg / mL and 1 mg / mL, and the encapsulation efficiency of resiquimod is greater than 90%.

[0088] In some preferred embodiments disclosed herein, the T cell-targeting immunoliposomes encapsulating resiquimod are composed of the following lipid materials, the molar ratios of which are shown in Table 2. Among them, TL01 is a Her2-binding antibody-conjugated lipid, and TL02 is a CD3-binding antibody-conjugated lipid.

[0089] Table 2 Lipid materials and proportions of T cell-directed immunoliposomes

[0090] In some preferred embodiments disclosed in the present invention, the technical solution of the present invention has the following advantages:

[0091] The present invention introduces two different antibody-coupled lipids into the same liposome during liposome preparation, and further encapsulates small molecules with immunomodulatory functions. The composition of different lipids and the content of encapsulated immunomodulatory molecules can be adjusted according to clinical needs.

[0092] The Her2-binding antibody-conjugated lipid TL01 was used to bind to Her2 on tumor cells, while the CD3-binding antibody-conjugated lipid TL02 was used to bind to CD3 on immune cells. Fluorescently labeled T cell-directed immunoliposomes showed strong binding activity when co-incubated with Her2-positive tumor cells and peripheral mononuclear cells (PBMCs) and mediated immune cell killing of Her2-expressing tumor cells.

[0093] T cell-directed immunoliposomes can further encapsulate drugs. T cell-directed immunoliposomes encapsulating resiquimod, which has immunomodulatory function, have a significant redirection effect in mediating immune cell killing of tumor cells.

[0094] Before further describing the specific embodiments of the present invention, it should be understood that the scope of the present invention is not limited to the following specific embodiments. It should also be understood that the terms used in the following examples are intended only to describe the specific embodiments and do not limit the scope of the present invention. The test methods described in the following examples, where specific conditions are not specified, are generally based on conventional conditions or manufacturer recommended conditions.

[0095] When numerical values ​​are given in the examples, it should be understood that both endpoints of each numerical range and any range between them can be selected unless otherwise indicated. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by professionals. In addition to the specific methods, equipment, and materials used in the examples, any methods, equipment, and materials in the prior art that are similar or equivalent to those described in the examples can be used to implement the present invention, according to the knowledge of professionals, the level of technology in the art, and the record of the present invention.

[0096] Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein utilize conventional techniques in the fields of molecular biology, biochemistry, chromosome structure and analysis, analytical chemistry, cell culture, and recombinant DNA technology. These techniques have been described in detail in previous literature. For further information, see Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed., Cold Spring Harbor Laboratory Press, 1989, and 3rd ed., 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987, and its periodic updates; the METHODS IN ENZYMOLOGY series published by Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE AND FUNCTION, 3rd ed., Academic Press, San Diego, 1998; METHOD IN ENZYMOLOGY, vol. 304, Chromatin (P. M. Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, vol. 119, Chromatin Protocols (P. P. Becker, ed.), Humana Press, Totowa, 1999, etc.

[0097] Unless otherwise specified, all experimental materials used in the following examples were purchased from regular reagent / consumables suppliers.

[0098] Example 1. Preparation of T cell-directed immunoliposomes containing anti-CD19-coupled lipids and anti-CD3-coupled lipids

[0099] 1. Prepare F(ab')2 fragments of anti-CD19 and anti-CD3 antibodies. Prepare F(ab')2 fragments by removing the Fc fragment of IgG through protease digestion. Human IgG or mouse IgG was digested with pepsin, while mouse IgG1 was digested with ficin. The specific steps are as follows: centrifuge 500 μl of antibody and then pass it through a desalting column; add the flow-through to the protease solution and mix at 37°C; incubate with pepsin for 4 hours or ficin for 24 hours, centrifuge at 5000 g for 1 minute, collect the flow-through, wash the immobilized enzyme with Protein A binding buffer, collect the flow-through, and pool the flow-throughs to obtain the enzymatic digestion products.

[0100] The enzymatic digestion product was mixed at a constant temperature on a Protein A column for 10 minutes, and the flow-through was collected by centrifugation. The Protein A column was washed with Protein A binding buffer and the flow-through was collected. The flow-throughs were combined to obtain a Protein A column-purified enzymatic digestion product from which the Fc fragment and large IgG fragments had been removed. The enzymatic digestion product was dialyzed against a 50KD dialysis bag using a PBS solution (pH = 7.0). The dialysis sequence was 2 hours, 2 hours, and 16 hours to obtain a purified antibody F(ab')2 fragment from which the Fc fragment had been removed.

[0101] The obtained anti-CD3 antibody F(ab')2 fragment was confirmed to be the full-length anti-CD3 antibody with the Fc fragment removed. The amino acid sequence of the light chain variable region is shown in SEQ.ID.NO.1, specifically:

[0102] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 2, specifically:

[0103] 2. Preparation of anti-CD19 and anti-CD3 conjugated lipids. Antibody F(ab')2 fragments were reduced to Fab' fragments using β-mercaptoethylamine as a reducing agent. The Fab' fragments were then chemically conjugated to the maleimide groups of DSPE-PEG2000-Mal. The specific steps were as follows: The F(ab')2 fragment (1-10 mg / ml) was mixed with β-mercaptoethylamine containing 50 mM EDTA and reacted at 37°C with shaking for 90 minutes under nitrogen. The mixture was centrifuged, passed through a desalting column, and the flow-through was collected. The β-mercaptoethylamine was removed from the mixture to yield Fab', the reduced product of the antibody F(ab')2 fragment. A 30 mM HEPES solution containing 600 μM DSPE-PEG2000-Mal micelles was then added to achieve a 1:1 molar ratio of Fab' to DSPE-PEG2000-Mal. The reaction was then incubated at 10°C with shaking for 16 hours under nitrogen. The results of SDS-PAGE monitoring of this process are shown in Figures 2A and 2B.

[0104] 3. Preparation of anti-CD3 scFv-bound lipids. Using tricarboxyethylphosphine (TCEP) as a reducing agent, an antibody scFv fragment containing a free sulfhydryl group was obtained, which was then chemically coupled to the maleimide group of DSPE-PEG2000-Mal. The specific steps were as follows: 800 μl of the scFv fragment (1-10 mg / ml) was mixed with a HEPES solution containing 1.2 mM TCEP, resulting in a molar ratio of scFv to TCEP of 1:5. A 30 mM HEPES solution containing 600 μM DSPE-PEG2000-Mal micelles was then immediately added. The mixture was shaken at 10°C under nitrogen protection for 16 hours. The SDS-PAGE results of this process are shown in Figure 2C.

[0105] 4. Preparation of T cell-directed immunoliposomes containing anti-CD19-conjugated lipids and anti-CD3-conjugated lipids. Liposomes made from HSPC, cholesterol, and DSPE-PEG-2000 were co-incubated with antibody-conjugated liposomes to prepare T cell-directed immunoliposomes. The prepared T cell-directed immunoliposomes were washed five times by 300 kD ultrafiltration. The particle size of the liposomes was approximately 80-90 nm, and the PDI was approximately 0.1, as measured by dynamic light scattering. The average number of Fab's per liposome was calculated by dividing the molar concentration of incorporated Fab' by the molar concentration of the liposomes (assuming a molecular weight of 64 million daltons for large unilamellar vesicles (LUVs)). Based on these copy numbers, the average density of Fab' on the liposome surface was estimated (Figure 2D).

[0106] Example 2 Preparation of T cell-directed immunoliposomes containing anti-Her2-coupled lipids and anti-CD3-coupled lipids (TL01 and TL02)

[0107] A CHO cell line was established to express anti-HER2 Fab. The sequence of the anti-HER2 Fab fragment is as follows:

[0108] Heavy-Chain (SEQ ID NO.4):

[0109] Light-Chain (SEQ ID NO.3):

[0110] In addition, a stable CHO cell line was established to express the anti-CD3 Fab fragment. The sequence of this fragment is shown below:

[0111] Heavy chain (SEQ ID NO.12):

[0112] Light chain (SEQ ID NO.11):

[0113] Subsequently, the expressed and purified anti-HER2 Fab and anti-CD3 Fab were chemically linked to the maleimide group of DSG-PEG2000-Mal. The products were named TL01 (anti-Her2 conjugated lipid) and TL02 (anti-CD3 conjugated lipid), respectively. The structures of TL01 and TL02 are shown in Figures 3A and 3B.

[0114] Prepare T cell-directed immunoliposomes containing TL01 and TL02 using the following ingredients:

[0115] Table 3 Weighing of T cell-directed immunoliposomes

[0116] The steps for preparing T cell-directed immunoliposomes containing TL01 and TL02 are as follows:

[0117] (1) Weigh 42.9 g of hydrogenated soybean phosphatidylcholine (HSPC, molecular weight 783.8), 2.86 g of stearic acid phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and 14.3 g of cholesterol. Dissolve them in 150 ml of ethanol and mix in a 65°C water bath to obtain an ethanol lipid mixture.

[0118] (2) The ethanol mixture obtained in step (1) was added to 2000 ml of ammonium sulfate buffer solution (pH 5.0, composed of 250 mM ammonium sulfate and water) and incubated in a 65° C. water bath for 30 minutes to obtain liposome vesicles.

[0119] (3) The liposome vesicles obtained in step (2) are extruded through a polycarbonate membrane to finally obtain liposomes with an average particle size of about 75 nm in the aqueous phase.

[0120] (4) The liposomes obtained in step (3) are dialyzed through a dialysis membrane with a pore size of 10,000 Daltons. The dialyzate is a 10 mM HEPES and 0.9% (w / w) NaCl solution at a pH between 6 and 8. The resulting liposomes have a small unilamellar liposome structure with a phospholipid bilayer structure, and there are pH and ion concentration gradients in the inner and outer aqueous phases of the liposomes.

[0121] (5) 120.0 g of TL01 solution (solution concentration: 3.4 mg / ml) and 59.5 g of TL02 solution (solution concentration: 4.0 mg / ml) were added to the blank liposome solution prepared in step (4). The drug was loaded at 60°C for 30 min. After drug loading, the suspension was diafiltration to obtain purified T cell-targeted immunoliposomes. The particle size distribution of the T cell-targeted immunoliposomes was approximately 80-90 nm, and the PDI was approximately 0.1.

[0122] Example 3 Preparation of T cell-targeted immunoliposomes encapsulating resiquimod

[0123] TRAFsomes containing targeting antibodies and immune cell-binding antibodies can also encapsulate the immunomodulator resiquimod, which can increase the efficacy of resiquimod while reducing drug toxicity. The steps for encapsulating the drug into pre-prepared TRAFsomes are as follows: 2000 resiquimod is dissolved in 100mM sodium acetate buffer at pH 5.5. The concentration of the resiquimod solution is 2mg / ml. The resiquimod solution is added to the pre-formed T cell-directed immunoliposomes and incubated for 30 minutes. The encapsulation efficiency (EE) of resiquimod is between 85% and 100%.

[0124] Example 4 Evaluation of T cell-directed immunoliposome-mediated T cell redirection to tumor cells

[0125] The present invention uses a mixed lymphocyte reaction system to evaluate the T cell redirection effect mediated by T cell-directed immunoliposomes. In this experiment, HER2-positive N87 cells were used as target cells and CD3-positive Jurkat cells were used as model T cells. A total of 1×10 5 The total number of N87 cells was 1 × 10 5 Jurkat cells. The cell mixture was placed in a 37°C incubator and incubated overnight (or for 16 hours). The cells were then collected, placed in a flow cytometer tube, and centrifuged at 500×g for 5 minutes. Jurkat cells were stained with antibodies (APC anti-CD3 antibody, catalog number 300311, PE / Cyanine7 anti-human CD25 antibody, catalog number 356108, PE anti-human CD69 antibody, catalog number 310906). After washing the cells twice, flow cytometry was used to assess the binding between Jurkat-N87 cells (CD3-APC and CFSE-FITC fluorescence double positive). The results are shown in Figure 4.

[0126] Example 5 Evaluation of T cell-directed immunoliposomes in mediating T cell killing of tumor cells

[0127] To evaluate the effector cell function mediated by T cell-directed immunoliposomes, we used a mixed lymphocyte reaction assay. Target cells (HER2-expressing cells: N87, SK-Br-3, and BT474 cell lines) and effector cells (freshly isolated PBMCs from different donors) were used to investigate the cytotoxic activity of immune effector cells induced by resiquimod-encapsulated T cell-directed immunoliposomes.

[0128] Specifically, effector and target cells were resuspended in phenol red-free complete medium at a cell concentration of 2 × 10 6 cells / ml and 5×10 5 cells / ml. 100 μl of effector cells and 100 μl of target cells were added to a 96-well plate and the cells were thoroughly mixed. At this point, the ratio of target cells to effector cells was 1:5. 10 μl of T cell-directed immunoliposomes loaded with resiquimod at different concentrations were added to each well and mixed thoroughly. The 96-well plate was placed in a cell culture incubator and incubated for 24 hours. 45 minutes before the end of the incubation, 10 μl of lysis buffer was added to the volume correction control wells and the target cell maximum lysis wells. After the incubation, the 96-well plate was centrifuged at 250 g for 4 minutes. After centrifugation, 50 μl of the supernatant was transferred to a new 96-well plate, 50 μl of LDH substrate was added to the new plate, and the cells were incubated at 22°C for 15 minutes. After the incubation, the absorbance at a wavelength of 490 nm was measured using a microplate reader. The results are shown in Figure 5.

[0129] These experimental data clearly demonstrate that the resiquimod-encapsulated T cell-targeted immunoliposomes used in the present invention can effectively mediate the killing of target cells by effector cells. Effector cells can include unstimulated lymphocytes, in vitro expanded lymphocytes, in vitro activated lymphocytes, and in vitro differentiated lymphocytes. The cytotoxic effect relies on cell lysis and is dose-dependent.

[0130] Example 6 Antitumor Activity of Resiquimod-Encapsulated T Cell-Directed Immunoliposomes in a Human Tissue Xenograft Tumor Model of Adenoid Cystic Carcinoma Patients

[0131] In this study, a humanized tissue xenograft tumor model of adenoid cystic carcinoma was established using the NCG animal model. Peripheral mononuclear cells were humanized in mice bearing humanized tissue xenograft tumors. The antitumor activity of resiquimod-encapsulated T cell-directed immunoliposomes was evaluated using PBS as a control and resiquimod-encapsulated T cell-directed immunoliposomes as the test drug.

[0132] About 21 days after immune reconstitution with human peripheral mononuclear cells (hu-PBMCs), 12 mice were subcutaneously injected with human tissue xenogeneic cells from patients with adenoid cystic carcinoma and randomly divided into two groups, named HF5050 group and control group. For the HF5050 group, mice were injected with resiquimod-encapsulated T cell redirecting immune lipids every three days starting on the 7th day after tumor implantation, while the control group received PBS treatment. The HF5050 group with resiquimod-encapsulated T cell redirecting immune lipids was administered with a resiquimod dose of 0.28 mpK, and the treatment lasted for 3 weeks. Individual tumor growth curves were collected and their anti-tumor growth effects were evaluated. The individual tumor growth curves are plotted in Figures 6B and 6C and summarized in Figure 6A (mean ± SEM, n = 6). Statistical analysis was performed by comparing the HF5050 group and the control group using a t-test.

[0133] Encouragingly, after the eighth dose, the HF5050 group showed effective anti-tumor growth effect with a TGI of 44%.

[0134] Example 7 Antitumor activity of resiquimod-encapsulated T cell-directed immunoliposomes in the gastric cancer N87 tumor model of PBMC-reconstituted mice

[0135] In addition, the tumor growth inhibition test of gastric cancer N87 by T cell-directed immunoliposomes loaded with resiquimod was also tested. NOD / ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22B2mem21Cd4 / Gpt gene knockout mice were used as model mice for this experiment. The mice were subcutaneously inoculated with N87 cells and then injected with human peripheral mononuclear cells (hu-PBMC) through the tail vein for 14 days for immune reconstitution. Then 12 mice were randomly selected from them and divided into two groups, named HF5050 group and control group. For the HF5050 group, mice were injected with immune-regulating resiquimod-loaded T cell-directed immunoliposomes every three days starting from the 14th day after tumor implantation, while the control group received PBS treatment. The HF5050 group, which included T cell-targeted immunoliposomes encapsulated with resiquimod, was administered with a dose of 0.08 mpK for 3 weeks. Individual tumor growth curves were collected and evaluated for anti-tumor growth. Individual tumor growth curves are plotted in Figures 7B and 7C and summarized in Figure 7A (mean ± SEM, n = 6). Statistical analysis was performed by comparing the HF5050 group with the control group using a t-test.

[0136] Example 8 Antitumor Activity of Resiquimod-Encapsulated T Cell-Directed Immunoliposomes in a CD3ε Humanized Balb / C Mouse Model Bearing Her2-Expressing 4T1 Tumors

[0137] The antitumor activity of resiquimod-encapsulated T cell-targeted immunoliposomes was also tested in a homograft tumor model. This study used CD3ε-humanized Balb / C mice as a model. A tumor model harboring a 4T1 cell line engineered to express humanized Her2 was established in this animal model. Twelve mice were subcutaneously injected with 4T1 cells expressing humanized Her2. After tumors established, the mice were divided into two groups, designated HF5050 and control groups. Mice in the HF5050 group received resiquimod-encapsulated T cell-targeted immunoliposomes every three days starting on day 7 after tumor implantation, while the control group received PBS. The HF5050 group, which received resiquimod-encapsulated T cell-targeted immunoliposomes, was administered at a dose of 0.08 mpk for three weeks. Individual tumor growth curves were collected and evaluated for their antitumor efficacy. Individual tumor growth curves are plotted in Figures 8A and 8B and summarized in Figure 8C (mean ± SEM, n = 6). Statistical analysis was performed by comparing the HF5050 group with the control group using t-test.

[0138] Example 9 Evaluation of T cell-mediated killing of tumor cells by dasatinib-encapsulated T cell-directed immunoliposomes

[0139] First, dasatinib-encapsulated T cell-targeted immunoliposomes were prepared and evaluated for their ability to activate effector cells. 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 a 96-well plate and plated to mix thoroughly by pipetting. At this point, the target cell to effector cell ratio was 1:10. 10 μl of drug-encapsulated liposomes containing different dasatinib concentrations but the same lipid concentration was added to each well and mixed thoroughly by pipetting. The 96-well plate was placed in a cell culture incubator and incubated for 24 hours. Forty-five minutes before the end of the incubation period, 10 μl of lysis buffer was added to the volume correction control wells and the target cell lysis wells, respectively. After the incubation period, the 96-well plate was centrifuged at 250 g for 4 minutes. After centrifugation, 50 μl of the supernatant was transferred to a new 96-well plate, and 50 μl of LDH substrate was added to the new plate. The plate was incubated at 22°C for 15 minutes. After incubation, the absorbance was measured at a wavelength of 490 nm using a microplate reader.

[0140] The results shown in Figure 9 indicate that the dasatinib-carrying TRAFsome is effective in directing various effector cell functions toward target cells.

[0141] Example 10 Evaluation of T cell targeting immunoliposomes loaded with different numbers (titers) of CD3

[0142] First, liposomes (i.e., LUVs) with an average diameter of 80-90 nm were prepared. Different anti-CD3 conjugated lipids (TL02) were loaded onto different T cell-directed immunoliposomes. The specific number (titer) is shown in Table 4. The number of copies of anti-CD3 conjugated lipids on each liposome can be estimated by the aforementioned method (Li et al., Pharm. Res. 38(9): 1593-1600(2021)). In short, since the diameter of each liposome is distributed in 80 nm and it is a single bilayer structure, it is estimated that the therapeutic weight of each liposome is approximately 64 million Daltons. The molar ratio of anti-CD3 conjugated lipid to liposome is the average valence of anti-CD3 on each liposome, which can also be estimated according to the aforementioned method (Xie et al., Mabs 14(1): 2115205(2022)), the content of which has been incorporated by reference.

[0143] Table 4 Density of anti-CD3 on the liposome surface and molar ratio of anti-CD3 to liposome when single T cell reprogrammed immunoliposomes are loaded with different anti-CD3 conjugated lipids

[0144] Since anti-CD3 conjugated lipids can freely diffuse along the liposome surface, their interaction with target cells may be multivalent. In fact, the radius of the TCR complex on naive T cells is about 1-3 nm, and on activated T cells it is about 35-70 nm (Lillemeier et al., Nat. Immunol. 11(1):90-96 (2010). Therefore, when CD3 is coupled to magnetic beads, although the freedom of movement of CD3 is reduced by being fixed on the magnetic beads, it has better activation efficiency. Another study found that when CD3 is coupled to microbeads smaller than 50 nm, it cannot activate naive T cells, but it has better activity against T cells that have experienced antigen stimulation. This may be because there are more TCR complexes on the surface of activated T cells. (Lo et al., J. Immunol. 191(10):5107-5114 (2013)

[0145] As shown in Figure 10A, increasing the amount of anti-CD3 Fab in the T cell-directed immunoliposomes resulted in an increase in the number of T cells expressing CD25 and CD69. T cell-directed immunoliposomes with anti-CD3 conjugated lipids at 9 or 18 valencies converted approximately 20% of CD8+ T cells to CD25+ and approximately 40% of CD8+ T cells to CD69+. Increasing the CD3 valency to 35 or 70 further increased the percentages of CD25+ and CD69+ CD8+ T cells. T cell-directed immunoliposomes containing both anti-CD3 conjugated lipids and targeting antibodies enhanced T cell recruitment in the presence of target cells. TRAFsomes with anti-CD3 valencies of 9 or 18 converted approximately 40% of CD8+ T cells to CD25+ and approximately 60% of CD8+ T cells to CD69+. Increasing the CD3 valency to 35 or 70 further increased the percentages of CD25+ and CD69+ CD8+ T cells. These data suggest that when T cell-directed immunoliposomes contain both targeting antibodies and immune cell-binding antibodies, less anti-CD3-conjugated lipid may be required to achieve the desired effect.

[0146] Since liposomes containing only anti-CD3 conjugated lipids can induce some degree of T cell activation when there are more than 18 copies per liposome, it is possible that, while not wishing to be bound by theory, limiting the amount of anti-CD3 conjugated lipids in each T cell-targeting immunoliposome will minimize nonspecific T cell activation. As shown in FIG10B , T cell-targeting immunoliposomes containing only 9 anti-CD3 Fabs induce significant lysis of Raji cells but minimal lysis of K562 cells. On the other hand, anti-CD3 liposomes with a higher number of anti-CD3 Fabs induce cytotoxicity against both cell types independent of CD19 expression.

[0147] Example 11 Antitumor activity of dasatinib-loaded T cell-directed immunoliposomes in a B cell lymphoma tumor model in PBMC-reconstituted mice

[0148] The antitumor activity of dasatinib-loaded T cell-directed immunoliposomes was tested in a Raji+-PBMC co-transplantation model using NOD-PrkdcscidIL2rgtm1 mice (Figure 11A). NOD-PrkdcscidIL2rgtm1 immunodeficient mice were subcutaneously injected with Raji cells (1×10 6 ) and PBMC (1×10 7) mixed with Matrigel. As a control, mice with only Raji cells but no human PBMCs were also injected. Starting 7 days after tumor transplantation, 9×35 TRAFsomes and 70×35 TRAFsomes were injected every 3 days, where 9×35 and 70×35 respectively indicate that each T cell-directed immunoliposome was loaded with 9 anti-CD3 coupled lipids and 70 anti-CD3 coupled lipids. Both dasatinib-encapsulated T cell-directed immunoliposomes were given three doses. Based on in vitro data, the dose response of dasatinib-encapsulated T cell-directed immunoliposomes with 9 or 70 anti-CD3 coupled lipids was compared (Table 5). CD19+ Raji cells were transplanted into Matrigel together with PBMCs. Due to the reconstitution of the immune system, these Raji+-PBMC co-transplanted tumors generally grow slower than tumors with only Raji cells but no PBMCs. Implanted tumors were allowed to reach approximately 200 mm before treatment. 3 . Individual tumor growth curves are plotted in Figure 11C and summarized in Figure 11B (mean ± SEM, n = 5). Statistical analysis was performed by comparison with a blank vehicle control group. Encouragingly, a low dose (65 μg lipid per injection) of 9×35 dasatinib-encapsulated T cell-directed immunoliposomes effectively shrank established tumors. A higher dose of 9×35 dasatinib-encapsulated T cell-directed immunoliposomes also resulted in a reduction in tumor volume. Treatment with 70×35 dasatinib-encapsulated T cell-directed immunoliposomes at a low dose of 63 μg lipid per injection resulted in a tumor reduction similar to that of 9×35 dasatinib-encapsulated T cell-directed immunoliposomes. Surprisingly, treatment with 70×35 dasatinib-encapsulated T cell-directed immunoliposomes at a low dose of 80 μg lipid per injection resulted in a significant reduction in tumor volume after just 3 injections.

[0149] Table 5 Dose response experiment design with different numbers of anti-CD3 conjugated lipids inserted into the liposome surface

[0150] After the final injection, mice were sacrificed and the remaining human CD3+ T cells in their spleens were analyzed (Figure 11D). In the high-dose and medium-dose groups of 70×35 dasatinib-encapsulated T cell-directed immunoliposomes, there were almost no remaining human T cells. In the low-dose 70×35 dasatinib-encapsulated T cell-directed immunoliposomes and the medium-dose and low-dose 9×35 dasatinib-encapsulated T cell-directed immunoliposomes, the number of human T cells was almost equivalent to that in the blank control group. However, the high-dose 9×35 dasatinib-encapsulated T cell-directed immunoliposomes group was slightly different, with fewer human T cells in the spleen but slower tumor growth.

[0151] Blood samples collected on day 26 (24 hours before the last injection) and day 27 (3 hours after the last injection) were also analyzed. The percentage of circulating human CD45+CD3+ cells in total leukocytes is shown in Figure 11E, and the levels of human inflammatory cytokines in plasma are shown in Figure 11F (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, by two-way ANOVA with Tukey's multiple comparison test). It is worth noting that the number of human T cells and the levels of various inflammatory cytokines in plasma in the high-dose group of 9×35 dasatinib-encapsulated T cell-directed immunoliposomes reached a considerable level.

[0152] Although not limited by theory, treatment of mice with TRAFsomes having 70 anti-CD3-coupled lipids may result in T cell exhaustion or tolerance. It has been shown that multivalent CD3 ligands without IL-2 auxiliary stimulation can lead to T cell anergy, which may lead to failure of T cell immunotherapy (Duré and Macian, Mol. Immunol. 46 (5): 999-1006 (2009); Lechler et al., Immunology 103 (3): 262-269 (2001)). Composite T cell activating factors comprising multiple anti-CD3 copies may be particularly noteworthy (Zhukovsky et al., Curr. Opin. Immunol. 40: 24-35 (2016); Vafa and Trinklein, Front. Oncol. 10 (446): 1-7 (2020)). The data disclosed herein show that this problem can be alleviated by reducing anti-CD3 titers and / or adding immunomodulators within liposomes. This allows for the engagement of a broader T cell repertoire, including γδ T cells, which may be beneficial due to the potential for disruption of pre-existing tumor antigen evasion mechanisms.

[0153] Example 12 Using T cell-directed immunoliposomes to encapsulate different drugs

[0154] T cell directed immunoliposomes can be loaded with one or more immunomodulatory chemical molecules with immunomodulatory function. Preferably, the chemical molecules can include one or more anticancer drugs and / or immunomodulatory drugs. In some preferred embodiments disclosed in the present invention, the immunomodulatory drugs encapsulated in the liposomes include Resiquimod (also known as CD11301; R848; S28463; VML600), Imiquimod (trade name: ), MBS8, Guretolimod (DSP-0509), TransConTM, GSK1795091, GSK2245035, VTX-2337, eritoran (E5564), SD-101, Selgantolimod, RO7119929, CpG1018, Vidutolimod (CMP-001), SHR2150, MGN1703, CYT003-QbG10, CPG-7909, Tilsotolimod, JNJ-64794964 (AL-034 / TQ-A3334), BNT411, APR003, BDB001, or a combination of one or more of them.

[0155] In addition, other immunomodulatory drugs suitable for liposome encapsulation in the art are also included. For example, see U.S. Patents 8,202,974, 9,050,376, 9,211,320, 9,248,170, 9,295,732, 9,458,184, 10,076,535, 10,259,793 and 11,026,964, and U.S. Patent Application Publications 2018 / 0360974, 2018 / 0085388, 2020 / 0316211, 2021 / 0059953, 2021 / 0371440 and 2022 / 0192997.

[0156] In addition, the encapsulated anticancer drugs include cyclophosphamide (Cytoxan; Cytoxan Lyophilized), axitinib (Inlyta), bevacizumab (Avastin), cabozantinib (Cometriq), 5,6-dimethylxanthenone-4-acetic acid (DMXAA), combretastatin A-4phosphate (CA4P), curcumin, doxorubicin (Dox) (Doxil, Caelyx, Lipo-Dox, Myocet, Zolsketil), mitomycin C lipid prodrug (MLP) (Promitil), alendronic acid (Ald), cellular arabinan lipid complex (DepoCyt), cellular arabinan lipid complex and doxorubicin (Vyxeos), doxorubicin (DNR, DaunoXome), all-trans retinoic acid (ATRA), mitoxantrone (MXT, PLM60), paclitaxel (PCX, EndoTAG), exinotecan (Onivyde / Nal-IRI), vincristine (Marqibo), mifamotide (Mepact), cis-bis-neodecanoyl-trans-bis-neodecanoyl-R,R-1,2-diaminocyclohexane platinum II (L-NDDP, Aroplatin TM ), Cisplatin (LiPlaCis, LipoplatinTM, SPI-077), or a combination of one or more thereof.

[0157] In addition, other drugs suitable for liposome encapsulation in the art are also included. For example, see U.S. Patents 8,597,654, 8,895,717, 9,326,953, 9,351,997, 9,789,193, 9,993,551, 10,376,519, 10,426,753, 10,618,905, 10,653,774, 10,654,831 and 11,096,900, and U.S. Patent Application Publications 2005 / 240131, 2020 / 0001110, 2020 / 0283406, 2021 / 0077397, 2022 / 0160729 and 2021 / 0275453.

[0158] In some preferred embodiments disclosed herein, the drug encapsulated by the T cell-directed immunoliposomes comprises dasatinib. In some preferred embodiments disclosed herein, the drug encapsulated by the T cell-directed immunoliposomes comprises resiquimod.

[0159] T cell-directed immunoliposomes encapsulating dasatinib have been shown to enhance CTL activity of CD8+ T lymphocytes. Dasatinib is a broad-spectrum kinase inhibitor targeting multiple Src family kinases, but it has also been implicated in T cell effector function. The effects of unloaded and loaded T cell-directed immunoliposomes on activated CD4+ and CD8+ T cells were compared, with T cells stimulated with both anti-CD3 Fab' and anti-CD28 Fab' as positive controls. At doses of 1-10 nM, dasatinib significantly improved CD8+ T cell activation, with limited effects on CD4+ T cells. Plates coated with equimolar concentrations of CD3 monoclonal antibody (UCHT1) plus soluble anti-CD28 monoclonal antibody (2 μg / ml) were used as positive controls. Data are presented as means ± standard errors (n = 3). The researchers compared T cell activation in the presence of CD19+ Raji cells and wild-type K562 cells (CD19-negative) using T cell-directed immunoliposomes and dasatinib-loaded T cell-directed immunoliposomes. Although nonspecific T cell activation induced by liposomes (lipid dose of 5 μg / ml) was almost as high with K562 cells as with Raji cells, the addition of dasatinib-loaded MTLs significantly reduced nonspecific activation. The lipid concentration was fixed at 5 μg / ml and the dasatinib concentration was fixed at 5 nM. At the same time, CTL activity against Raji cells was unaffected in MTLs loaded with 1-10 nM dasatinib. Dasatinib had an inhibitory effect on T cells at concentrations of 10 nM and above.

[0160] Example 13 Activation of γδT lymphocytes, IL-2-stimulated LAK cells, and aCD3 / aCD28 magnetic bead-expanded T lymphocytes by T cell-directed immunoliposomes

[0161] T cell-directed immunoliposomes were found to stimulate other T cell subsets, including γδT cells, IL-2-stimulated LAK cells, or T cells amplified by anti-CD3 / anti-CD28 antibodies (aCD3 / aCD28). These pretreated T cells were found to be more reactive compared to freshly isolated PBMCs. In fact, treatment with T cell-directed immunoliposomes resulted in significant cytolytic activity of γδT cells within 5 hours of culture (44.3% at E:T=20:1) (Figures 12A-12B). The data in Figure 12B are mean ± standard error (n=3). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, compared by Student's t-test. Similarly, compared with mPEG liposomes and anti-CD3 liposomes, T cell-directed immunoliposomes triggered higher CTL activity of LAK cells pre-stimulated by IL-2, especially at higher E:T ratios (Figure 12A, middle right). T cells expanded with aCD3 / aCD28 also showed similar activity, but the improvement with T cell-directed immunoliposomes was smaller compared to liposomes containing only anti-CD3 Fab' (Figure 12A, right). T cell-directed immunoliposomes took longer to activate resting T cells from PBMCs relative to pre-stimulated T cell subsets. Without being bound by theory, the change in activation time may be due to the lack of TCR clustering in resting T cells and suggests that T cell-directed immunoliposomes may dissociate TCRs due to simultaneous binding to cancer cells.

[0162] The above are only some preferred embodiments of the present disclosure, and are not limitations of any form or substance to the present disclosure. It should be noted that for those familiar with the field, improvements and supplements can be made without departing from the method of the present disclosure, and these improvements and supplements will also be protected by the present disclosure. By using the technical content disclosed above, equivalent changes, modifications and evolutions can be made by those familiar with the technical field without departing from the spirit and scope of the present disclosure, and these equivalent changes are regarded as equivalent embodiments of the present disclosure. At the same time, any changes, modifications and evolutions to any equivalent embodiments can be made by technical personnel familiar with the above technical content without departing from the spirit and scope of the present disclosure, and these equivalent changes are regarded as equivalent embodiments of the present disclosure.

Claims

1. A T cell-directed immunoliposome, comprising a liposome, a targeting antibody or a target binding fragment thereof, and an immune cell binding antibody or a target binding fragment thereof modified on the surface of the liposome, wherein the targeting antibody or the target binding fragment specifically binds to a ligand on the surface of a target cell, and the immune cell binding antibody or fragment acts on an immune effector cell.

2. The T cell-directed immunoliposome according to claim 1, wherein the targeting antibody or target binding fragment thereof is an anti-Her2 / neu antibody or target binding fragment thereof, and the immune cell binding antibody or fragment thereof is an anti-CD3 antibody or binding fragment thereof.

3. The T cell-directed immunoliposome according to claim 2, wherein the average valency of the targeting antibody or its binding fragment on each liposome is 10. The valency can be calculated by dividing the molar concentration of the contained antibody or fragment by the molar number of liposomes. 4 . The T cell-directed immunoliposome according to claim 2 , wherein the average valency of the immune cell-binding antibody or fragment thereof on each liposome is 6. 5 . The T cell-directed immunoliposome according to claim 2 , wherein the liposome contains an anti-tumor drug, an immunomodulatory drug, or a combination of an anti-tumor drug and an immunomodulatory drug.

6. The T cell-directed immunoliposome of claim 2, wherein the immunomodulator drug comprises Resiquimod, Imiquimod, Selgantolimod, Vidutolimod, or a combination of two or more thereof.

7. The T cell-directed immunoliposome according to claim 2, wherein the immunomodulatory drug is resiquimod.

8. The T cell-directed immunoliposome according to claims 1-7, wherein the components of the liposome comprise phosphatidylcholine, cholesterol, a lipid to which the targeting antibody or its target binding fragment is attached, and a lipid to which the immune cell binding antibody or its fragment is attached.

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