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-guided immunoliposomes and their applications Technical Field

[0001] This application relates to the fields of immunoliposomes, T-cell immunotherapy, and other technical fields, and particularly to a T-cell-guided immunoliposome drug. Technical Background

[0002] Liposomes are vesicle-like drug carriers with a bilayer structure, formed from phospholipids and / or cholesterol. Liposomes are artificially prepared bilayer vesicle structures formed from lipid molecules. Due to the unique structure of liposomes, lipid molecules have hydrophilic heads and hydrophobic tails, allowing hydrophilic drugs to be contained in the internal aqueous phase, while hydrophobic drugs can be embedded within the lipid bilayer. Furthermore, liposomes exhibit good biocompatibility, low toxicity, and good biosafety, thus attracting widespread attention and being applied in the field of drug delivery systems. After development and research, liposomal targeting technology has endowed liposomal drugs with unique in vivo drug distribution and metabolic behaviors compared to traditional drugs. The dosage form characteristics of liposomes allow drugs to accumulate at target sites and then specifically interact with target cells. By optimizing target selection and improving the structure and properties of targeting ligands, it is hoped that the clinical efficacy of liposomal drugs can be improved, including reducing systemic toxicity and / or increasing the therapeutic index.

[0003] The research and development of targeted liposome drug delivery systems primarily focuses on designing specific target sites based on various targets. These sites often possess local specificity and / or sufficient expression abundance. Compared to delivery vehicles or free drugs lacking specific target delivery heads, targeted liposome drugs developed based on this design philosophy exhibit higher organ-specificity. For example, increased drug concentration at the target site leads to improved pharmacokinetics. However, these targeted drug delivery systems only work on specific cell types and may not effectively eliminate tumors, potentially leading to minimal residual disease and multidrug resistance in tumors.

[0004] In recent years, with the development of tumor immunology, it has been clarified that when targeted delivery is only performed on tumor cells, the therapeutic effect is often determined by a combination of factors. These include the heterogeneity of molecular markers among different tumor cells within the same tumor from the same patient, the varying contributions of different immune cell types to the immune microenvironment within the same tumor, and interactions within the tumor itself. Therefore, researchers have broadened the design concepts for targeted delivery based on the characteristics of the tumor microenvironment. For example, drug carriers targeting 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 some extent, its design concept remains limited to targeting a single cell type. However, there are extensive interactions and influences among different cell types within a tumor, such as lymphocyte infiltration, macrophage phagocytosis, antigen presentation, and cell-cell communication based on cytokines and exosomes. These interactions constitute immune surveillance at the tumor site, maintaining a dynamic balance between tumor clearance inhibition and tumor growth promotion. Blocking or promoting cell-cell interactions has proven feasible and effective in clinical practice, for example, through antigen-specific T-cell-based vaccine therapy, antibody therapy relying on NK cell cytotoxicity, and immune checkpoint inhibitor therapy to prevent tumor immune escape. Conversely, drug delivery targeting a single cell type often only targets a specific cell and modulates its physiological processes, but cannot affect cell-cell interactions within the tumor tissue to achieve effective treatment.

[0006] In this invention, different antibodies targeting different cell types are loaded into a single liposome. This liposome, through the action of the antibodies, can bind to different cell types sequentially or simultaneously to promote target cell recognition, communication, activation, apoptosis, or clearance. Furthermore, this invention achieves targeted drug delivery to multiple cell types via a single liposome using structurally stable liposomes, thereby avoiding drug interactions that may occur with combination therapies and improving drug efficacy.

[0007] Technical Summary

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

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

[0010] In a first aspect, the present invention discloses a liposome with immune cell redirection function. This immune cell redirection liposome comprises a liposome structure on which a targeting antibody / fragment and an immune cell binding antibody / fragment are modified on the 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 effector functions.

[0011] Secondly, T-cell-guided immune liposomes 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, on the same T-cell-guided immunoliposome, the molar ratio of the targeting antibody / fragment to the immune cell-binding antibody / fragment is between 10 / 1 and 1 / 10.

[0013] Preferably, the same T-cell-guided immunoliposome surface contains approximately 1-1000 targeting antibodies / fragments and 1-1000 immune cell-binding antibodies / fragments.

[0014] In some preferred embodiments disclosed in this invention, the targeting antibody / fragment and the immune cell-binding antibody / fragment are respectively coupled to lipid molecules, and the coupled lipid-antibody complex structure 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 coupled to maleimide groups present in the lipid molecule by coupling.

[0015] Preferably, the target cells targeted by the targeting antibody / fragment mainly include: tumor cells, microorganisms, and cells infected by microorganisms. Therefore, the antigens targeted by the targeting antibody / fragment mainly originate 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.

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

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

[0018] Preferably, in this invention, the immune cells with immune effects are effector cells capable of exerting cytotoxic, apoptotic, or eliminative effects on target cells. Immune cells with immune effects include: T lymphocytes, NK cells, NKT cells, monocytes, dendritic cells, macrophages, or neutrophils.

[0019] In this invention, immune cells bind antibodies / fragments to antigens expressed by immune effector cells. Therefore, immune cell-redirected liposomes can simultaneously or sequentially bind to tumor cells, lymphocytes, or other immune cells (such as myeloid cells). The same immune cell-redirected liposome may first bind to tumor cells and then to immune cells, or vice versa.

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

[0021] In some embodiments disclosed in this invention, the cell antigen of the immune cells with an immune effect is CD3, and the immune cell binding antibody / fragment is a CD3 antibody.

[0022] Preferably, the immune cell binding antibody / fragment can also be a CD3 antibody / fragment, a PD1 antibody / fragment, a CTLA4 antibody / fragment, or 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 complexes, small molecules, cytokines, immune checkpoint inhibitors, or gene editing.

[0025] Preferably, the targeting antibody / fragment and the 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 in this invention, the targeting antibody / fragment and the immune cell binding antibody / fragment are Fab' fragments obtained by removing and reducing the Fc fragment with an antibody. This fragment includes a light chain variable region and a heavy chain variable region.

[0027] Preferably, in some preferred embodiments disclosed in this invention, the targeting antibody / fragment is the Fab' fragment of a CD19 antibody, and the amino acid sequence of its light chain (LC) variable region (VL) 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 in this invention, the targeting antibody / fragment is the Fab' fragment of the HER2 antibody, and the amino acid sequence of its light chain (LC) variable region (VL) is as shown in SEQ ID NO.3, specifically:

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

[0031] Preferably, the targeting antibody / fragment can be an 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 an 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 in this invention, the immune cell-binding antibody / fragment is the Fab' fragment of a CD3 antibody, and the amino acid sequence of its light chain (LC) is shown in SEQ ID NO.11, specifically:

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

[0039] Preferably, the immune cell-binding antibody / fragment can be a PD-1 antibody, the amino acid sequence of which is 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 which is shown in SEQ ID NO.14, specifically:

[0041] This invention does not impose specific restrictions on the composition of T cell-guided immune liposomes, as long as they conform to the definition of liposomes. The components of liposomes can be selected from the following phospholipid combinations, including lecithin, hydrogenated soybean phosphatidylcholine, hydrogenated lecithin choline, stearic acid glycerol phosphatidylcholine, myristate glycerol phosphatidylcholine, palmitate glycerol phosphatidylcholine, stearic acid glycerol phosphatidylcholine, palmitic acid-myristate glycerol phosphatidylcholine, palmitic acid-palmitoate glycerol phosphatidylcholine, palmitic acid-oleamide glycerol phosphatidylcholine, stearic acid-linoleylcholine, dioleoylcholine, hydrogenated palmitate glycerol phosphatidylcholine, stearic acid phosphatidylcholine, myristate phosphatidylcholine, palmitic acid phosphatidylcholine, stearic acid phosphatidylcholine, palmitic acid phosphatidylcholine, myristoyl phosphatidylethanolamine, palmitic acid phosphatidylethanolamine, cerebroside phosphatidylserine, myristate phosphatidylserine, and palmitic acid phosphatidylcholine. Acylserine, lecithin glycerol, distearate glycerol phosphatidylglycerol, myristate glycerol phosphatidylglycerol, palmitate glycerol phosphatidylglycerol, linoleic acid glycerol phosphatidylglycerol, dioleoyl glycerol phosphatidylglycerol, cerebrosphingolipid, myristate sphingolipid or stearate sphingolipid, cholesterol, 1,2-dioleoyl-3-trimethylammonium propane (chloride) (DOTAP), dioleoylchloropropyltrimethylammonium chloride (DOTMA), dimethyltetradecylammonium bromide (DDAB), dimethylaminoethylaminocholesterol (DC-Chol), spanmin-5-carboxyaminoacetic acid octacosanamide (DOGS), dioleoyl succinate methyl dimethylpropylammonium chloride trifluoroacetate (DOSPA), cholesterol and DSPE-PEG-2000, or a combination of two or more of them.

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

[0043] Preferably, the T-cell-guided immunoliposomes can encapsulate drugs in the internal aqueous phase of the liposomes, or they can be free of drugs.

[0044] In some preferred embodiments disclosed in this invention, the average number of immune cell-binding antibodies / fragments in each T-cell-guided immunoliposome is 1 to 9. Preferably, the average number of anti-CD3 fragments in each T-cell-guided immunoliposome is 1 to 8. Preferably, the average number of anti-CD3 fragments in each T-cell-guided immunoliposome is 1 to 7. Preferably, the average number of anti-CD3 fragments in each T-cell-guided immunoliposome is 1 to 6. Preferably, the average number of anti-CD3 fragments in each T-cell-guided immunoliposome is 1 to 5. Preferably, the average number of anti-CD3 fragments in each T-cell-guided immunoliposome is 1 to 4. Preferably, the average number of anti-CD3 fragments in each T-cell-guided immunoliposome is 1 to 3. Preferably, the average number of anti-CD3 fragments in each T-cell-guided immunoliposome is 1 to 2.

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

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

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

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

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

[0050] Preferably, the T-cell-directed immunoliposomes can be loaded with one or more immunomodulatory chemical molecules with immunomodulatory functions. Preferably, the chemical molecules may include one or more anticancer drugs and / or immunomodulatory drugs. In some preferred embodiments disclosed in this invention, the immunomodulatory drugs encapsulated in the liposomes include resiquimod (also known as CD11301; R848; S28463; VML600) and imiquimod (trade name: MBS8, Guretolimod (DSP-0509), TransCon™, 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 one or more combinations thereof.

[0051] In addition, it includes other immunomodulatory drugs suitable for liposome encapsulation in the art. 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, it includes other pharmaceutical products suitable for liposome encapsulation in the art. 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 in this invention, the drug encapsulated in the T-cell-guided immunoliposomes includes dasatinib. Preferably, in some preferred embodiments disclosed in this invention, the drug encapsulated in the T-cell-guided immunoliposomes includes resiquimod.

[0054] Further preferably, in some preferred embodiments disclosed in this invention, the drug encapsulated in the T-cell-directed immunoliposome comprises resiquimod. The ratio of resiquimod to lipid material is 1:10 to 1:100 (by weight), and resiquimod can be encapsulated inside the T-cell-directed immunoliposome or in its phospholipid bilayer.

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

[0056] The total concentration of rethimod in the T-cell-directed immunoliposome solution ranges from 0.5 mg / mL to 1 mg / mL, and the particle size of the T-cell-directed immunoliposomes is approximately 80 nm.

[0057] In some preferred embodiments disclosed in this invention, rethimod acts as an immunomodulator in T-cell-directed immunoliposomes to induce an appropriate immune response. This immune response includes the activation of T lymphocytes and the secretion of pro-inflammatory cytokines.

[0058] In some preferred embodiments disclosed in this invention, the T-cell-directed immunoliposomes loaded with retinoic acid are composed of the following lipid materials, the molar ratios of which are shown in Table 1:

[0059] Table 1. Lipid materials and proportions for T cell-guided immunoliposomes

[0060] In a third aspect of the invention, the preparation of the aforementioned resimilart-loaded T-cell-guided immunoliposomes and their antitumor use are provided.

[0061] Compared with traditional technologies, this disclosure has the following beneficial effects:

[0062] The T-cell-guided immunoliposomes of this invention share some similar mechanisms of action with bispecific antibodies. Furthermore, this invention further optimizes the activation of tumor-associated specific immunity by adjusting the density and relative proportion of targeting antibodies / fragments and immune cell-binding antibodies / fragments. In addition, the T-cell-guided immunoliposomes further modulate appropriate immune responses through the encapsulation of immunomodulatory drugs. Finally, compared to multispecific antibodies, the production, preparation, and quality control of T-cell-guided immunoliposomes are more feasible.

[0063] Legend and Introduction

[0064] Figure 1: Schematic diagram of different T cell redirection mechanisms, including bispecific antibody (BITE), dual-affinity redirection antibody (DART), and T cell-directed immune liposome (TRAFsome).

[0065] Figure 2A: Reduced and non-reduced SDS-PAGE images 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 antibody F(ab')2, and anti-CD19 Fab'. Figure 2C: SDS-PAGE images of liposomes bound to anti-CD3 single-chain antibody (scFv). Figure 2D: Estimation of antibody surface density on liposomes containing different amounts of Fab'.

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

[0067] Figure 4: T-cell-guided immunoliposomes loaded with rethimod mediating intercellular redirection.

[0068] Figure 5: Evaluation of the tumor cell killing effect of T cell-guided immunoliposome-mediated effector cells loaded with rethimod.

[0069] Figure 6: Antitumor activity of T-cell-guided immunoliposomes loaded with rethimod in a humanized tissue xenograft tumor model of adenoid cystic carcinoma patients.

[0070] Figure 7: Antitumor activity of resimilart-loaded T cell-guided immunoliposomes in a gastric cancer N87 tumor model in PBMC-reconstructed mice.

[0071] Figure 8: Antitumor activity of rethimod-loaded T cell-guided immunoliposomes in a humanized Balb / C mouse 4T1 tumor model with Her2 expression in CD3ε.

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

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

[0074] Figure 11: Evaluation of T-cell killing of tumor cells mediated by dasatinib-encapsulated T-cell-directed immunoliposomes. Figure 11A: Schematic diagram of the experimental procedure. Figures 11B-11C: Graphs of 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 administration. Figure 11E: Percentage of human T cells in the spleen 24 hours after the last administration. Figure 11F: Serum levels of various cytokines in mice 3 hours after the last administration.

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

[0076] Further explanation of preferred examples

[0077] Liposomes are artificially prepared vesicle structures composed of lipid molecules with a phospholipid bilayer structure. The lipid structure typically includes a hydrophilic head and a hydrophobic tail. In a polar solvent composed of water, the hydrophobic tails of the lipid molecules will move closer together due to hydrophobic interactions, while the hydrophilic heads will align closely towards the aqueous phase, thus 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 within the phospholipid bilayer.

[0078] The active targeting effect of liposomes is achieved by binding ligands to the surface of the liposome. These ligands can be antibodies, peptides, small molecules, etc. The ligands interact with the receptors on the target cells. When using antibodies or antibody fragments as ligands, liposomes exhibit better binding activity and specificity compared to small molecule and peptide ligands. Based on the self-assembly principle of lipid molecules, two or more types of ligands can also be bound to the surface of a liposome.

[0079] Based on antibody-modified liposome technology, this 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-target liposome preparations only use different ligand combinations to improve selectivity for target cells. The T-cell-guided immunoliposomes used in this invention further optimize and modulate the physiological functions of immune cells, such as recognition, binding, and activation, through different ligand ratios, promoting their interaction with target cells. Therefore, this invention provides a new mechanism for targeted liposomes.

[0080] Existing multispecific antibody complex strategies are shown in Figure 1. These include molecules similar to bispecific T-cell coupling agents (BITE) or diamidophoric redirected antibodies (DART), each containing two targeting antibodies in a 1:1 ratio. In contrast, modifying liposomes with multiple different targeting antibodies is not only more convenient but also allows for density adjustments and ratio variations of different types of targeting antibodies on the liposome surface (e.g., T-cell-directed immunoliposomes). Furthermore, T-cell-directed immunoliposomes have a larger particle size than existing antibodies and complexes such as BiTe or DART (typically around 5 nm in diameter). The particle size of T-cell-directed immunoliposomes can be up to 10 times larger. Therefore, the pharmacokinetic behavior and tissue / organ distribution of T-cell-directed immunoliposomes are superior to those of known BiTe or DART. Moreover, T-cell-directed immunoliposomes can overcome the manufacturing difficulties caused by antibody mismatch during the design and preparation of multispecific antibody complexes.

[0081] Furthermore, the T-cell-guided immunoliposomes demonstrated in this invention are also compatible with liposome drug encapsulation technology. This allows for the encapsulation of drugs within T-cell-guided immunoliposomes, enabling targeted drug delivery and release, thus improving drug selectivity and altering the release site and timing. For example, to mitigate a common adverse reaction in tumor immunotherapy—the cytokine storm—immunomodulators encapsulated in T-cell-guided immunoliposomes can achieve more precise immune regulation, thereby minimizing systemic excessive immune responses. This further enhances the efficacy of T-cell-guided immunoliposomes.

[0082] The T-cell-guided immunoliposomes described in this invention contain targeting antibodies / fragments, immune cell-binding antibodies / fragments, and are encapsulated with immunomodulators. Therefore, they can promote the binding and recognition of immune cells with target cells and regulate the immune function and effects of immune cells. T-cell-guided immunoliposomes possess a unique in vivo mechanism of action and offer significant advantages over existing technologies in terms of production, preparation, and activity.

[0083] This invention relates to T-cell-guided immunoliposomes as pharmaceuticals for immunotherapy and antitumor therapy, applied to the treatment of tumors. As understood in the art, "treatment" is a method of obtaining beneficial or desired results, including clinical outcomes. These results may include one or more of the following aspects: alleviating or improving one or more cancer symptoms, reducing the severity of disease or disorder, stabilizing the state of disease or disorder, delaying or slowing the progression of disease or disorder, remission of disease or disorder, and killing at least one cancer cell targeted by the disclosed composition or inducing an immune response against cancer cells.

[0084] In some preferred embodiments disclosed in this invention, 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, pancreas, stomach, and esophageal cancers. Preferably, HER2 / neu+ tumors are breast cancers. 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 can be a HER2-low expression or HER2-negative tumor.

[0085] Preferably, in some preferred embodiments disclosed in this invention, the drug encapsulated in the T-cell-guided immunoliposomes includes dasatinib. Preferably, in some preferred embodiments disclosed in this invention, the drug encapsulated in the T-cell-guided immunoliposomes includes resiquimod.

[0086] In a further preferred embodiment disclosed in this invention, the drug encapsulated in the T-cell-guided immunoliposome comprises rectimote. The ratio of rectimote to lipid material is 1:10 to 1:100 (by weight), and rectimote can be encapsulated within the T-cell-guided immunoliposome or in its phospholipid bilayer.

[0087] The total concentration of retimoid in the T-cell-guided immunoliposome solution ranged from 0.5 mg / mL to 1 mg / mL, and the encapsulation efficiency of retimoid was greater than 90%.

[0088] In some preferred embodiments disclosed in this invention, the T-cell-directed immunoliposomes loaded with rethimod are composed of the following lipid materials, the molar ratios of which are shown in Table 2. TL01 is a Her2-binding antibody-conjugated lipid, and TL02 is a CD3-binding antibody-conjugated lipid.

[0089] Table 2. Lipid materials and proportions for T cell-guided immunoliposomes

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

[0091] This invention introduces two different antibody-conjugated lipids onto a single liposome during liposome preparation and further encapsulates small molecules with immunomodulatory functions. The composition of the different lipids and the content of the encapsulated immunomodulatory molecules can be adjusted according to clinical needs.

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

[0093] T-cell directed immunoliposomes can be further encapsulated with drugs. T-cell directed immunoliposomes encapsulated with rasimodil, which has immunomodulatory functions, have a significant redirection effect that mediates the killing of tumor cells by immune cells.

[0094] 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 following specific embodiments; it should also be understood that the terminology used in the following embodiments is only for describing specific embodiments and does not limit the scope of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally based on conventional conditions or conditions recommended by the manufacturer.

[0095] When numerical values ​​are given in the embodiments, it should be understood that both endpoints of each numerical range and either of them can be chosen, unless otherwise stated. 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. Apart from the specific methods, apparatus, and materials used in the embodiments, this invention can be implemented using any prior art methods, apparatus, and materials similar to or equivalent to those mentioned in the embodiments, based on the understanding of one of ordinary skill in the art and the record of this invention.

[0096] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques from 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 more information, please 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 regular 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 (edited by PM Wassarman and AP Wolfe), Academic Press, San Diego, 1999; and *METHODS IN MOLECULAR BIOLOGY*, Vol. 119, Chromatin Protocols (edited by PB Becker), Humana Press, Totowa, 1999, etc.

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

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

[0099] 1. Preparation of F(ab')2 fragments of anti-CD19 and anti-CD3 antibodies. The Fc fragment of IgG was cleaved by protease digestion to prepare the F(ab')2 fragment. Human IgG or mouse IgG was digested with pepsin, and mouse IgG1 was digested with fig protease. The specific steps are as follows: 500 μl of antibody was centrifuged and then passed through a desalting column; the flow solution was added to the protease solution and mixed at 37°C; the mixture was incubated with pepsin for 4 hours or fig protease for 24 hours, centrifuged at 5000g for 1 minute to collect the flow solution, the enzyme was immobilized by washing with protein A binding buffer, and the flow solutions were collected and combined to obtain the enzyme digestion product.

[0100] The enzyme digestion products were incubated in a protein A column for 10 minutes at an insulated temperature. The flow rate was collected by centrifugation, and the protein A column was washed with protein A binding buffer, with the flow rate collected again. The flow rates were combined to obtain a purified enzyme digestion product purified by the protein A column, with the Fc fragment and large IgG fragment removed. The enzyme digestion products were dialyzed through a 50 kDa dialysis bag using PBS solution (pH = 7.0) as the dialysis medium. The dialysis sequence was 2 hours, 2 hours, and 16 hours to obtain purified antibody F(ab')2 fragments with the Fc fragment removed.

[0101] Technical confirmation revealed that the obtained anti-CD3 antibody F(ab')2 fragment is a 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 coupled lipids. The antibody F(ab')2 fragment was reduced to the Fab' fragment using β-mercaptoethylamine as a reducing agent. Then, the Fab' fragment was chemically coupled to the maleimide group of DSPE-PEG2000-Mal. The specific steps are as follows: The F(ab')2 fragment (1-10 mg / ml) was mixed with β-mercaptoethylamine containing 50 mM EDTA and reacted with shaking at 37°C for 90 minutes under nitrogen protection; centrifuged, passed through a desalting column, and the flow-through was collected. β-mercaptoethylamine was removed to obtain Fab', the reduced product of the antibody F(ab')2 fragment. Then, a 30 mM HEPES solution containing 600 μM DSPE-PEG2000-Mal micelles was added, making the molar ratio of Fab' to DSPE-PEG2000-Mal 1:1, and reacted with shaking at 10°C for 16 hours under nitrogen protection. The SDS-PAGE monitoring results for this process are shown in Figures 2A and 2B.

[0104] 3. Preparation of anti-CD3 scFv binding lipids. Tricarboxyethylphosphine (TCEP) was used as a reducing agent to obtain antibody scFv fragments containing free thiol groups, which were then chemically coupled to the maleimide group of DSPE-PEG2000-Mal. The specific steps are as follows: 800 μl of the 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, a 30 mM HEPES solution containing 600 μM DSPE-PEG2000-Mal micelles was added, and the reaction was carried out under nitrogen protection at 10 °C with shaking 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-coupled lipids and anti-CD3-coupled lipids. Liposomes prepared from HSPC, cholesterol, and DSPE-PEG-2000 were co-incubated with antibody-bound liposomes to prepare T-cell-directed immunoliposomes. The prepared T-cell-directed immunoliposomes were washed five times by 300 kDa ultrafiltration. The liposome particle size was approximately 80-90 nm, and the PDI was approximately 0.1, as measured by dynamic light scattering. The average number of Fab' copies on each liposome was calculated by dividing the molar concentration of incorporated Fab' by the molar concentration of the liposome (assuming a molecular weight of 64 million Daltons for large monolayer vesicles (LUVs)). Based on these copy numbers, the average Fab' density on the liposome surface was estimated (Figure 2D).

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

[0107] A CHO cell line was established to express anti-HER2Fab. The sequence of the anti-HER2Fab 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-HER2Fab and anti-CD3Fab were chemically linked to the maleamide group of DSG-PEG2000-Mal, respectively. The products were named TL01 (anti-Her2-coupled lipid) and TL02 (anti-CD3-coupled lipid), respectively. The structures of TL01 and TL02 are shown in Figures 3A and 3B.

[0114] T cell-guided immunoliposomes containing TL01 and TL02 were prepared using the following components:

[0115] Table 3. Weighing of T cell-guided immunoliposomes

[0116] The steps for preparing T cell-guided 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 stearate 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) Add the ethanol mixture obtained in step (1) to 2000 mL of ammonium sulfate buffer solution (pH 5.0, consisting of 250 mM ammonium sulfate and water) and incubate in a 65°C water bath for 30 minutes to obtain liposome vesicles.

[0119] (3) The liposome vesicles obtained in step (2) are squeezed through a polycarbonate membrane, and finally liposomes with an average particle size of about 75 nanometers are obtained 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 dialysate is a solution of 10 mM HEPES and 0.9% (w / w) NaCl with a pH value between 6 and 8. The resulting liposomes are small single-chambered liposomes with a phospholipid bilayer structure, and there are pH and ion concentration gradients in the aqueous phases inside and outside the liposomes.

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

[0122] Example 3: Preparation of T-cell-guided immunoliposomes loaded with resimilarin

[0123] TRAFsomes containing both targeted binding antibodies and immune cell binding antibodies can also encapsulate the immunomodulatory agent rectimote, increasing its efficacy while reducing toxicity. The encapsulation process for the drug into pre-prepared TRAFsomes is as follows: 2000 g of rectimote is dissolved in 100 mM pH 5.5 sodium acetate buffer. The concentration of the rectimote solution is 2 mg / ml. The rectimote solution is then added to pre-prepared T-cell-directed immunoliposomes and incubated for 30 minutes. The encapsulation efficiency (EE) of rectimote is between 85% and 100%.

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

[0125] This invention evaluates the T-cell redirection effect mediated by T-cell-directed immunoliposomes using a mixed lymphocyte reaction system. 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 × 10⁶ cells were used in each reaction / plate. 5 The number of N87 cells and the total number of cells was 1×10. 5 Jurkat cells were collected. The cell mixture was incubated overnight (or 16 hours) at 37°C. Cells were then collected and centrifuged at 500×g for 5 minutes in flow cytometry tubes. 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, the binding between Jurkat-N87 cells was assessed by flow cytometry (double positive for CD3-APC and CFSE-FITC fluorescence). The results are shown in Figure 4.

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

[0127] To evaluate T-cell-directed immunoliposome-mediated effector cell function, we employed 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 rethimod-loaded T-cell-directed immunoliposome-induced immune effector cells.

[0128] Specifically, effector cells and target cells were resuspended in phenol red-free complete culture medium at cell concentrations of 2 × 10⁻⁶. 6 cells / ml and 5×10 5 Cells / ml. Add 100 μl of effector cells and 100 μl of target cells to a 96-well plate and mix thoroughly. At this point, the ratio of target cells to effector cells is 1:5. Add 10 μl of T-cell-directing immunoliposomes loaded with different concentrations of resimilartodextrin to each well and mix thoroughly. Place the 96-well plate in a cell culture incubator and incubate for 24 hours. 45 minutes before the end of incubation, add 10 μl of lysis buffer to the volume correction control well and the target cell maximum lysis well. After incubation, centrifuge the 96-well plate at 250 g for 4 minutes. After centrifugation, transfer 50 μl of supernatant to a new 96-well plate, add 50 μl of LDH substrate to the new plate, and incubate at 22 °C for 15 minutes. After incubation, measure the absorbance at 490 nm using a microplate reader. The results are shown in Figure 5.

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

[0130] Example 6: Antitumor activity of T-cell-guided immunoliposomes loaded with retinoic acid in a humanized tissue xenograft tumor model of adenoid cystic carcinoma patients.

[0131] In this experiment, an NCG animal model was used to establish a humanized tissue xenograft tumor model from adenoid cystic carcinoma patients, and peripheral mononuclear cells were reconstructed in mice with the established humanized tissue xenograft tumor. Resimod-loaded T-cell-directed immunoliposomes were used as the test drug, with PBS as the control, to evaluate the antitumor activity of resimod-loaded T-cell-directed immunoliposomes.

[0132] Approximately 21 days after immune reconstitution with human peripheral mononuclear cells (hu-PBMCs), 12 mice were subcutaneously injected with xenografted human tissue cells from adenoid cystic carcinoma patients and randomly divided into two groups: the HF5050 group and the control group. In the HF5050 group, mice received retsimod-loaded T-cell redirected immunolipids every three days starting on day 7 post-tumor implantation, while the control group received PBS treatment. The HF5050 group, treated with retsimod-loaded T-cell redirected immunolipids, received retsimod at a dose of 0.28 mpK for 3 weeks. Individual tumor growth curves were collected, and the anti-tumor growth effect was evaluated. Individual tumor growth curves are plotted in Figures 6B and 6C, and summarized in Figure 6A (mean ± SEM, n = 6). Statistical analysis was performed using a t-test to compare the HF5050 group and the control group.

[0133] Encouragingly, after the 8th dose, the HF5050 group showed effective anti-tumor growth effect with a TGI of 44%.

[0134] Example 7: Antitumor activity of T cell-guided immunoliposomes loaded with rethimod in a gastric cancer N87 tumor model in PBMC-reconstructed mice.

[0135] In addition, the tumor growth inhibition of gastric cancer N87 was tested using T-cell-directed immunoliposomes loaded with retsimolet. NOD / ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22B2mem21Cd4 / Gpt gene knockout mice were used as the model mice for this experiment. Mice were subcutaneously inoculated with N87 cells, followed by immune reconstitution 14 days after intravenous injection of human peripheral mononuclear cells (hu-PBMCs). Twelve mice were then randomly selected and divided into two groups: the HF5050 group and the control group. In the HF5050 group, mice received T-cell-directed immunoliposomes loaded with retsimolet every three days starting on day 14 post-tumor implantation, while the control group received PBS treatment. The HF5050 group, which received retsimolet-encapsulated T-cell-directed immunoliposomes, was treated with retsimolet at a dose of 0.08 mpK for 3 weeks. Individual tumor growth curves were collected, and the anti-tumor growth effect was evaluated. 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 resimilar T-cell guided immunoliposomes loaded with retsimole in a humanized Balb / C mouse 4T1 tumor model loaded with Her2 expression and CD3ε.

[0137] The antitumor activity of retsimolet-encapsulated T-cell-guided immunoliposomes was also tested in an allogeneic tumor model. In this study, humanized CD3ε Balb / C mice were used as a model, and a tumor model was constructed in this animal model using engineered 4T1 cell lines expressing humanized Her2. A total of 12 mice were injected subcutaneously with 4T1 cell lines expressing humanized Her2. After tumor growth, they were divided into two groups, designated HF5050 and a control group. Mice in the HF5050 group received retsimolet-encapsulated T-cell-guided immunoliposomes every three days starting on day 7 after tumor implantation, while the control group received PBS treatment. The HF5050 group received retsimolet at a dose of 0.08 mpK for 3 weeks, and individual tumor growth curves were collected to evaluate its antitumor growth effect. 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 a t-test.

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

[0139] First, dasatinib-encapsulated T-cell-directed immunoliposomes were prepared, and their activation effect on effector cells was evaluated. Effector cells (unstimulated lymphocytes) and target cells (Raji cells) were resuspended in phenol red-free intact 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 each well of a 96-well plate, and the cells were spread evenly by pipetting. At this point, the ratio of target cells to effector cells was 1:10. 10 μl of drug-encapsulated liposomes with different dasatinib concentrations but the same lipid concentration were added to each well and mixed by pipetting. The 96-well plates were placed in a cell culture incubator and incubated for 24 hours. 45 minutes before the end of incubation, 10 μl of lysis buffer was added to the volume-corrected control wells and the targeted cell lysis wells. After incubation, the 96-well plates were 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 490 nm using a microplate reader.

[0140] The results shown in Figure 9 demonstrate that TRAFsome carrying dasatinib is effective in guiding the function of various effector cells toward target cells.

[0141] Example 10 Evaluation of loading different numbers (titers) of CD3 onto T cell-guided immunoliposomes

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

[0143] Table 4. Density of anti-CD3 on the liposome surface and its molar ratio to the liposome when loading different anti-CD3-coupled lipids onto single T cell-redefined immunoliposomes.

[0144] Since anti-CD3-coupled lipids can diffuse freely along the surface of liposomes, 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, while it is about 35-70 nm on activated T cells (Lillemeier et al., Nat. Immunol. 11(1):90-96 (2010). Therefore, when CD3 is coupled to magnetic beads, although the degree of freedom of movement of CD3 is reduced by fixing it 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 on T cells that have experienced antigen stimulation. This may be because there is more TCR complex 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 number of anti-CD3 Fab' in T cell-directed immunoliposomes leads to an increase in the number of T cells expressing CD25 and CD69. T cell-directed immunoliposomes with anti-CD3 conjugated lipids of 9 or 18 valences convert approximately 20% of CD8+ T cells to CD25+ and approximately 40% to CD69+. Increasing the CD3 valence to 35 or 70 further increases the percentage of CD25+ and CD69+CD8+ T cells. T cell-directed immunoliposomes containing both anti-CD3 conjugated lipids and targeting antibodies enhance T cell enhancement in the presence of target cells. TRAFsomes with anti-CD3 valences of 9 or 18 convert approximately 40% of CD8+ T cells to CD25+ and approximately 60% to CD69+. Increasing the CD3 valence to 35 or 70 further increases the percentage of CD25+ and CD69+CD8+ T cells. These data suggest that when T-cell-guided immunoliposomes contain both targeting antibodies and immune cell-binding antibodies, a smaller amount of anti-CD3 conjugated lipids may be required to achieve the desired effect.

[0146] Since liposomes containing only anti-CD3-coupled lipids, with more than 18 copies per liposome, induce some degree of T cell activation, it is not theoretically improbable that limiting the number of anti-CD3-coupled lipids per T cell-directed immunoliposome will minimize non-specific T cell activation. As shown in Figure 10B, T cell-directed immunoliposomes containing only 9 anti-CD3s induced significant Raji cell lysis, but little lysis of K562 cells. On the other hand, anti-CD3 liposomes with more anti-CD3Fab' induced cytotoxicity in both cell types independently of CD19 expression.

[0147] Example 11: Antitumor activity of dasatinib-encapsulated T-cell-guided immunoliposomes in a PBMC-reconstructed mouse model of B-cell lymphoma.

[0148] The antitumor activity of dasatinib-encapsulated 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). 6 ) and PBMC (1×10 7Raji cells were mixed with Matrigel. As a control, mice containing only Raji cells and no human PBMCs were also injected. Starting 7 days post-tumor transplantation, 9×35 TRAFsomes and 70×35 TRAFsomes were injected every 3 days, where 9×35 and 70×35 represent 9 and 70 anti-CD3-coupled lipids respectively loaded on each T-cell-directed immunoliposome. Both dasatinib-loaded T-cell-directed immunoliposomes were administered in three doses. The dose-response of dasatinib-loaded T-cell-directed immunoliposomes with 9 or 70 anti-CD3-coupled lipids was compared based on in vitro data (Table 5). CD19+ Raji cells were co-transplanted with PBMCs into Matrigel. Due to immune system remodeling, these Raji+-PBMC co-transplanted tumors generally grew slower than tumors containing only Raji cells and no PBMCs. Prior to treatment, implanted tumors were allowed to reach approximately 200 mm. 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 control group. Encouragingly, low-dose (65 μg lipid per injection) of 9×35 dasatinib-loaded T-cell-directed immunoliposomes effectively shrunk established tumors. Higher doses of 9×35 dasatinib-loaded T-cell-directed immunoliposomes also resulted in tumor volume reduction. Treatment with a low dose of 63 μg lipid per injection of 70×35 dasatinib-loaded T-cell-directed immunoliposomes resulted in tumor reduction similar to that of the 9×35 dasatinib-loaded T-cell-directed immunoliposomes. Surprisingly, treatment with a low dose of 80 μg lipid per injection of 70×35 dasatinib-loaded T-cell-directed immunoliposomes resulted in significant tumor volume reduction after only 3 injections.

[0149] Table 5. Dose-response experimental design for different numbers of anti-CD3-coupled lipids inserted into the liposome surface.

[0150] After the final injection, the mice were sacrificed, and the remaining human CD3+ T cells in their spleens were analyzed (Figure 11D). In the high- and medium-dose groups of 70×35 dasatinib-loaded T-cell-guided immunoliposomes, almost no human T cells remained. In the low-dose groups of 70×35 dasatinib-loaded T-cell-guided immunoliposomes and the medium- and low-dose groups of 9×35 dasatinib-loaded T-cell-guided immunoliposomes, the number of human T cells was almost equivalent to that in the blank control group. However, the high-dose group of 9×35 dasatinib-loaded T-cell-guided immunoliposomes showed a slight difference, 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 and Tukey's multiple comparison test). Notably, the high-dose group of 9×35 dasatinib-encapsulated T-cell-directed immunoliposomes showed considerably high levels of human T cells and plasma levels of various inflammatory cytokines.

[0152] Although not bound by theory, treatment of mice with TRAFsome containing 70 anti-CD3-coupled lipids may lead to T-cell exhaustion or tolerance. It has been shown that multivalent CD3 ligands without IL-2 co-stimulation result in T-cell unresponsiveness, potentially leading 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)). Complex T-cell activators containing multiple anti-CD3 copies may be of particular interest (Zhukovsky et al., Curr. Opin. Immunol. 40:24-35 (2016); Vafa and Trinklein, Front. Oncol. 10(446):1-7 (2020)). The data disclosed in this paper suggest that this problem can be mitigated by reducing anti-CD3 titers and / or by incorporating immunomodulators in the liposome. This allows them to participate in a broader pool of T cells, including γδT cells, which could be beneficial because there may be a pre-existing mechanism that disrupts tumor antigen evasion.

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

[0154] T-cell-guided immunoliposomes can be loaded with one or more immunomodulatory chemical molecules with immunomodulatory functions. Preferably, the chemical molecules may include one or more anticancer drugs and / or immunomodulatory drugs. In some preferred embodiments disclosed in this invention, the immunomodulatory drugs encapsulated in liposomes include resiquimod (also known as CD11301; R848; S28463; VML600) and imiquimod (trade name: MBS8, Guretolimod (DSP-0509), TransCon™, 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 one or more combinations thereof.

[0155] In addition, it includes other immunomodulatory drugs suitable for liposome encapsulation in the art. 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), and compretastatin. A-4 phosphate (CA4P), curcumin, doxorubicin (Dox) (Doxil, Caelyx, Lipo-Dox, Myocet, Zolsketil), mitomycin C lipid prodrug (MLP) (Promitil), alendronate (Ald), cytoarabinolipan lipid complex (DepoCyt), cytoarabinolipan lipid complex and doxorubicin (Vyxeos), doxorubicin (DNR, DaunoXome), all-trans retinoic acid (ATRA), mitoxantrone (MXT, PLM60), paclitaxel (PCX, EndoTAG), excinotecan (Onivyde / Nal-IRI), vincristine (Marqibo), mirtamiviride (Mepact), cis-bis-neodecanoate-trans-bis-neodecanoate trans-R,R-1,2-diaminocyclohexane platinum II (L-NDDP, Aroplatin) TM Cisplatin (LiPlaCis, LipoplatinTM, SPI-077), or one or more of these.

[0157] In addition, it includes other pharmaceutical products suitable for liposome encapsulation in the art. 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 in this invention, the drug encapsulated in T-cell guided immunoliposomes includes dasatinib. In some preferred embodiments disclosed in this invention, the drug encapsulated in T-cell guided immunoliposomes includes resiquimod.

[0159] T-cell-directed immunoliposomes loaded with dasatinib demonstrated their ability to enhance CTL activity in CD8+ T lymphocytes. Dasatinib is a broad-spectrum kinase inhibitor targeting multiple Src family kinases, but it has also been found to be involved in T-cell effector function. The effects of T-cell-directed immunoliposomes without and loaded with dasatinib on activated CD4+ and CD8+ T cells were compared, with T cells simultaneously stimulated against both anti-CD3 Fab' and anti-CD28 Fab' cells serving as positive controls. At doses of 1–10 nM, dasatinib significantly improved CD8+ T-cell activation, with limited effect on CD4+ T cells. Plates coated with equimolar concentrations of CD3 monoclonal antibody (UCHT1) plus soluble anti-CD28 monoclonal antibody (2 μg / ml) served as positive controls. Data are presented as mean ± standard error (n = 3). T cell-directed immunoliposomes (MTLs) and dasatinib-loaded T cell-directed immunoliposomes were compared in the presence of CD19+ Raji cells and wild-type K562 cells (CD19-negative). While nonspecific T cell activation induced by liposomes (lipid dose of 5 μg / ml) was almost as high in K562 cells as in Raji cells, the addition of dasatinib-loaded MTLs significantly reduced nonspecific activation. Lipid concentrations were fixed at 5 μg / ml, and dasatinib concentrations were fixed at 5 nM. Meanwhile, CTL activity in Raji cells was not affected in MTLs with dasatinib loadings of 1–10 nM. Dasatinib exhibited inhibitory effects on T cells at concentrations of 10 nM and above.

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

[0161] T cell-directed immunoliposomes were found to stimulate other T cell subsets, including γδ T cells, IL-2-stimulated LAK cells, or T cells expanded with anti-CD3 / anti-CD28 antibodies (aCD3 / aCD28). These pretreated T cells were considered more reactive compared to freshly isolated PBMCs. Indeed, treatment with T cell-directed immunoliposomes resulted in significant cytolytic activity of γδ T cells within 5 hours of culture (44.3% at E:T = 20:1) (Figs. 12A–12B). Data in Fig. 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, T cell-directed immunoliposomes triggered higher CTL activity in LAK cells prestimulated with IL-2, especially at a higher E:T ratio, compared to mPEG liposomes and anti-CD3 liposomes (Fig. 12A, middle right). T cells expanded using aCD3 / aCD28 also showed similar activity, but the improvement from T cell-directed immunoliposomes was smaller compared to liposomes containing only anti-CD3 Fab' (Fig. 12A, right). T cell-directed immunoliposomes required a longer time to activate resting T cells from PBMCs compared to pre-stimulated T cell subsets. Without being theoretically constrained, the variation in activation time may be due to a lack of TCR clustering in resting T cells, suggesting that T cell-directed immunoliposomes may facilitate TCR dissociation through simultaneous binding with cancer cells.

[0162] The above description consists only of some preferred embodiments of this disclosure and is not intended to limit this disclosure in any form or substance. It should be noted that those skilled in the art can make improvements and additions without departing from the methods disclosed herein, and such improvements and additions will also be protected by this disclosure. Equivalent changes, modifications, and evolutions can be made by those skilled in the art using the disclosed technical content without departing from the spirit and scope of this disclosure; these equivalent changes are considered equivalent embodiments of this disclosure. Furthermore, any changes, modifications, and evolutions to any equivalent embodiment can be made by those skilled in the art without departing from the spirit and scope of this disclosure; these equivalent changes are considered equivalent embodiments of this disclosure.

Claims

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

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

3. The T-cell-directed immunoliposome according to claim 2, wherein the average valence of the targeting antibody or its binding fragment on each liposome is 10. The valence can be calculated by dividing the molar concentration of the contained antibody or fragment by the number of moles of liposomes.

4. The T-cell-directed immunoliposome according to claim 2, wherein the average valence of the immune cell-binding antibody or fragment thereof on each liposome is 6.

5. The T-cell-guided immunoliposome according to claim 2, wherein the liposome contains an antitumor drug, an immunomodulatory drug, or a combination of an antitumor drug and an immunomodulatory drug.

6. The T-cell directed immune liposome according to claim 2, wherein the immunomodulatory drug comprises Resiquimod, imiquimod, Selgantolimod, Vidutolimod, or a combination of two or more thereof.

7. The T-cell-guided immune liposome according to claim 2, wherein the immunomodulatory drug is resimiodide.

8. The T-cell-directed immune liposome according to claims 1-7, wherein the components of the liposome comprise phosphatidylcholine, cholesterol, lipids linked to the targeting antibody or its target-binding fragment, and lipids linked to the immune cell-binding antibody or its fragment.

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