Targeted nanoscale particles, targeted cells, and methods for producing and using the same

Abstract

The present invention relates to targeted nanoscale particles, targeted cells, and methods for producing and using the same. [Solution] Here, targeted nanoscale particles composed of multiple proteins linked to each other via first binding moieties are bound to the outer surface of targeted cells, and the targeted nanoscale particles further include second binding moieties and bind to the outer surface of the target cells via the second binding moieties. In an exemplary embodiment, the targeted nanoscale particles simultaneously bind to chimeric antigen receptor T cells and leukemia cells, promoting interaction between the two types of cells and thereby facilitating recognition and killing of leukemia cells by the chimeric antigen receptor T cells. Furthermore, the protein cavities within the targeted nanoscale particles provide space for loading chemotherapeutic drugs, enabling combined drug loading and chimeric antigen receptor T cell therapy with other therapies.

Description

[Technical Field]

[0001] This application claims priority to an application filed on December 2, 2022, bearing application number CN202211539334.7 and entitled "Targeted Nanoscale Particles, Targeted Cells, and Methods for Manufacturing and Uses Thereof," the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the technical field of biological immunotherapy, and in particular to targeted nanoscale particles, targeted cells, and methods for producing and using the same. [Background technology]

[0003] Chimeric antigen receptor T cells (CAR T) have achieved remarkable results in the treatment of hematological malignancies. Currently, CAR T products targeting CD19 have been approved for sale, and several CAR T products targeting other targets are undergoing clinical trials. However, as leukemia progresses, expression of target antigens decreases. Studies have shown that more than 30% of B-lineage leukemia patients experience relapse due to decreased CD19 target expression after treatment. Downregulation of target antigen expression severely impacts the recognition and killing of leukemia cells by CAR T cells, ultimately limiting the therapeutic efficacy of CAR T. Therefore, enhancing the recognition and killing of non-target leukemia cells by CAR T is key to improving the therapeutic efficacy of CAR T.

[0004] The information in the background art is merely intended to explain the general background of the present invention and should not be taken as an admission or in any way implying that such information constitutes existing technology known to those skilled in the art. Summary of the Invention [Problem to be solved by the invention]

[0005] The inventors have found through research that the ferritin receptor CD71 can be stably and highly expressed on the surface of leukemia cells in various disease stages, and that CAR T cells show significant upregulation of CD71 expression when expanded in vitro. Based on this, the present invention develops nanoscale protein particles that promote cell-cell interactions, thereby facilitating the recognition and killing of leukemia cells by, for example, CAR T. In addition, the protein of the present invention has an internal cavity that provides space for loading chemotherapeutic drugs. Therefore, this dosage form can be loaded with drugs to achieve combined treatment with CAR T and other therapies, ultimately enhancing the therapeutic effects against various disease stages of leukemia. Specifically, the present invention includes the following: [Means for solving the problem]

[0006] In one aspect of the present invention, a T cell conjugate is provided, comprising a T cell and a ferritin cluster, wherein the T cell binds to the ferritin cluster, and the ferritin cluster is composed of ferritin.

[0007] In one embodiment, in the T cell conjugate according to the present invention, the T cells are activated T cells (typically activated with commercially available activation magnetic beads, and may also be activated with an antibody), preferably in vitro expanded T cells, also preferably novel chimeric antigen receptor T cells (CAR T cells) or T cell receptor T cells (TCR T cells).

[0008] In one embodiment, in the T cell conjugate according to the invention, the ferritin cluster binds to CD71 on the T cell via a receptor-ligand interaction.

[0009] In one embodiment, in the T cell conjugate described in the present invention, the average particle size of the ferritin clusters is 50 to 1500 nm, preferably 300 to 1500 nm, also preferably 400 to 1000 nm, and more preferably 500 to 800 nm.

[0010] In one embodiment, in the T cell conjugate described in the present invention, the ferritin cluster is formed by aggregation and cross-linking of human-derived ferritin, and preferably, heavy chain subunits account for at least 30% of the ferritin subunits exposed on the surface of the ferritin cluster.

[0011] In one embodiment, the T cell conjugate according to the invention enhances targeting of target cells expressing CD71 via the ferritin cluster.

[0012] In one embodiment, in the T cell conjugates described in the present invention, the target cells are mainly tumor cells, including solid tumor cells and hematological tumor cells, such as liver cancer, lung cancer, colon cancer, cervical cancer, prostate cancer, ovarian cancer, breast cancer, thyroid cancer, esophageal cancer, gastric cancer, leukemia, etc., preferably leukemia cells, such as B-ALL, T-ALL, AML, etc.

[0013] The present invention also provides use of the T cell binder in the manufacture of a medicament for treating or ameliorating a cancer or tumor associated with CD71 expression, such as liver cancer, lung cancer, colon cancer, cervical cancer, prostate cancer, ovarian cancer, breast cancer, thyroid cancer, esophageal cancer, gastric cancer, leukemia, etc., preferably leukemia cells, preferably leukemia.

[0014] In one aspect of the present invention, a targeted nanoscale particle is provided, the targeted nanoscale particle being configured such that multiple proteins are linked to each other via a first binding moiety, the targeted nanoscale particle further comprising a second binding moiety, and binding to the outer surface of a target cell via the second binding moiety.

[0015] In certain embodiments, the targeted nanoscale particles according to the present invention are capable of binding to the outer surface of a targeted cell.

[0016] In one embodiment, in the targeted nanoscale particle according to the present invention, the first binding moieties comprise an active group and are connected to each other via a linking arm.

[0017] In one embodiment, in the targeted nanoscale particle described in the present invention, the outer surface of the target cell contains a receptor for the protein, and the receptor can bind to the protein via the second binding moiety.

[0018] In one embodiment, in the targeted nanoscale particles described herein, the targeted cells and target cells are cells that naturally express CD71, wherein the targeted cells include at least one of T cells, NK cells, B cells, lymphocytes, cytokine-induced killer cells, innate lymphocytes, and / or leukocytes.

[0019] In one embodiment, in the targeted nanoscale particles described in the present invention, the type of T cells is not particularly limited, and preferably, the T cells include modified T cells, including CAR T cells and TCR T cells, and unmodified T cells, including tumor-infiltrating T cells, PBMC-derived T cells, or other T cells isolated from the body.

[0020] In one embodiment, the targeted nanoscale particles described in the present invention have a size of 50 nm or more and 5000 nm or less, preferably 100 nm or more and 3000 nm or less, also preferably 300 to 2000 nm, more preferably 300 to 1000 nm.

[0021] In certain embodiments, in the targeted nanoscale particles according to the present invention, the protein comprises an internal cavity.

[0022] In certain embodiments, in the targeted nanoscale particles according to the present invention, the internal cavity may or may not contain a drug.

[0023] In one embodiment, in the targeted nanoscale particles according to the present invention, the protein is at least one selected from natural ferritin, recombinant whole heavy chain ferritin, and genetically engineered ferritin.

[0024] In one aspect of the present invention, there is provided a method for producing the targeted nanoscale particles described above, the method comprising: (1) aggregating and precipitating proteins under electrolytic or non-electrolytic conditions to form protein clusters; and (2) cross-linking the protein clusters in the presence of a cross-linking agent to obtain cross-linked targeted nanoscale particles. In one embodiment, in the method for producing targeted nanoscale particles according to the present invention, the type of the crosslinker is not particularly limited as long as it can crosslink protein clusters, and examples thereof include glutaraldehyde, NHS-PEG, x -NHSDSS, disuccinimidyl suberate, paraformaldehyde and polyethyleneimine.

[0025] In certain embodiments, the method for producing targeted nanoscale particles according to the present invention optionally includes, prior to step (1), the step of introducing an immunotherapeutic agent and / or a small molecule chemical agent into the cavity of the protein.

[0026] In one aspect of the present invention, a targeted cell is provided, wherein the outer surface of the targeted cell is bound to a targeted nanoscale particle described in the first aspect, and a plurality of proteins are linked to each other via a first binding moiety to form the targeted nanoscale particle, and the targeted nanoscale particle further comprises a second binding moiety and binds to the outer surface of the target cell via the second binding moiety.

[0027] In one embodiment, in the targeted cells according to the present invention, the first binding moieties comprise an active group and are connected to each other via a linking arm.

[0028] In one embodiment, in the targeted cells described in the present invention, the outer surface of the target cell comprises a receptor for the protein, and the receptor can bind to the protein via the second binding moiety.

[0029] In one embodiment, in the targeted cells according to the present invention, the targeted cells and target cells are cells that naturally express CD71.

[0030] In one embodiment, the targeted cells described in the present invention are at least one selected from αβ T cells, γδ T cells, natural killer (NK) cells, innate lymphoid cells (ILCs), cytokine-induced killer (CIK) cells, cytotoxic T lymphocytes (CTLs), lymphokine-activated killer (LAK) cells, T lymphocytes, and peripheral blood mononuclear cells. Preferably, the targeted cells are at least one selected from CAR T cells, TCR T cells, and tumor-infiltrating lymphocytes, and the target cells include cells derived from a hematological tumor, a solid tumor, or a combination thereof.

[0031] In one embodiment, in targeting cells according to the present invention, the targeted nanoscale particles are 50 nm or more and 5000 nm or less, preferably 300 nm or more and 3000 nm or less. In certain embodiments, in targeted cells according to the present invention, the protein comprises an internal cavity. In certain embodiments, in the targeted cells according to the present invention, the internal cavity may or may not contain a drug. In one embodiment, in the targeted cells according to the present invention, the protein is at least one selected from natural ferritin, recombinant whole heavy chain ferritin, and genetically engineered ferritin.

[0032] In one aspect of the present invention, there is provided a method for producing targeted cells, the method comprising: (1) aggregating and precipitating ferritin under electrolyte or non-electrolyte conditions to form protein clusters; (2) cross-linking the clusters in the presence of a cross-linking agent to obtain cross-linked targeted nanoscale particles; (3) bringing the targeted nanoscale particles and cells into close contact with each other to obtain the targeted cells.

[0033] In one embodiment, the method of manufacture described herein optionally includes introducing an immunotherapeutic agent and / or a small molecule chemical agent into the cavity of the protein prior to step (1).

[0034] In one aspect of the present invention, there is provided a method for promoting mutual proximity between cells, comprising the step of promoting proximity and contact between cells using targeted nanoscale particles, wherein multiple proteins are linked to each other via first binding moieties to form the targeted nanoscale particles, and the targeted nanoscale particles further comprise second binding moieties and bind to the outer surface of the cells via the second binding moieties.

[0035] In one embodiment, the method according to the present invention is an in vitro method, and the cells include a first cell and a second cell. The first cell and the second cell may be the same cell or different cells. Preferably, the first cell and the second cell are different cells. Also preferably, the first cell has a targeting nanoparticle bound to its surface, for example, via CD71, and the second cell has CD71 on its surface.

[0036] In one embodiment, in the method described herein, the cell is at least one selected from a CAR T cell, a TCR T cell, and a tumor-infiltrating lymphocyte, or is a cancer or tumor cell, such as a leukemia cell, and the cancer or tumor comprises a hematological tumor, a solid tumor, or a combination thereof.

[0037] In one embodiment, in the method according to the present invention, the hematological tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0038] In one embodiment, in the method described herein, the solid tumor is selected from the group consisting of gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphatic cancer, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, cervical cancer, endometrial cancer, mesothelioma, pancreatic cancer, or a combination thereof.

[0039] In one aspect of the present invention, there is provided a pharmaceutical composition comprising a targeted nanoscale particle or targeted cell according to the present invention. In certain embodiments, the pharmaceutical compositions according to the present invention also include immunotherapeutic agents and / or small molecule chemical agents.

[0040] In one aspect of the present invention, there is provided the use of targeted nanoscale particles or targeted cells, or pharmaceutical compositions according to the present invention, in the manufacture of a medicament for treating cancer or tumor, wherein said cancer or tumor comprises a CD71-positive tumor or carcinoma, preferably B-lineage leukemia.

[0041] In one aspect, the present invention provides the use of a targeted nanoscale particle or targeted cell, or pharmaceutical composition according to the present invention, in combination with other drugs, including, but not limited to, diagnostic, prophylactic, and / or therapeutic agents. [Effects of the Invention]

[0042] The technical effects of the present invention include, but are not limited to: In an exemplary embodiment of the present invention, a ferritin cluster nanoformulation is prepared using an aggregation-precipitation-crosslinking method. The formulation can be modified onto the CAR T surface by binding to CD71 on the CAR T surface. It has been found that the relatively large particle size of the clusters prevents ferritin clusters from being taken up by CAR T cells, which have relatively poor endocytosis, thereby ensuring effective long-term retention of ferritin clusters on the CAR T surface. The modified CAR T cells enhance cell-cell interaction by binding the surface ferritin clusters to CD71 on the leukemia cell surface, promoting CAR T recognition of leukemia antigen targets and ultimately activating the CAR T cells. Furthermore, leukemia cells can sequester and ingest ferritin clusters from the CAR T surface through relatively strong endocytosis. This process facilitates targeted killing of leukemia cells by drugs within the ferritin cavity, ensuring the combined use of CAR T and other therapies for leukemia treatment.

[0043] In the present invention, it has been further discovered that the nanoprotein particles have an internal cavity that can be loaded with various drugs, such as arsenic trioxide, doxorubicin, triiron tetroxide, etc., thereby realizing combined therapy of ferritin with various other therapies. Furthermore, ferritin can be modified, for example, by genetic engineering, to further expand the cavity volume of ferritin or to modify the amino acid residues inside ferritin, ensuring the loading of a greater number and variety of drugs and realizing combined therapy of more therapies with CAR T. [Brief explanation of the drawings]

[0044] [Figure 1] 1 shows a flow chart of an exemplary CAR-T cell leukemia treatment. [Figure 2] FIG. 1 is a transmission electron micrograph of exemplary ferritin and ferritin clusters. [Figure 3] 1 is an atomic force microscopy measurement showing the forces acting between an exemplary CAR T and leukemia cells. [Figure 4]Proliferation status of different CAR T cells after co-incubation with leukemia cells, detected using the CFSE method. [Figure 5] The positive rates of Granzyme B and interferon gamma (IFNγ) after co-incubation of different CAR T with leukemia cells were shown by flow cytometry detection. [Figure 6] Killing results when CAR T and FnC-CAR T were incubated with leukemia cells according to different ratios. [Figure 7] This shows the results of detecting changes in peripheral blood leukemia burden over time after CAR T, FnC-CAR T, and high-dose CAR T were reinjected into leukemia model mice. [Figure 8] Atomic force microscopy results showing the interaction forces between CAR T and FnC-CAR T and CD19-low expressing leukemia cells. [Figure 9] This shows the results of detecting proliferation after incubating CAR T and FnC-CAR T with CD19-low expressing leukemia cells. [Figure 10] Flow cytometry detection of activation status after co-incubation of CAR T with CD19-low expressing leukemia cells. [Figure 11] This is the result of detecting the killing of leukemia cells with low CD19 expression by CAR T. [Figure 12] This shows the results of detecting changes in peripheral blood leukemia burden over time after CAR T, FnC-CAR T, and high-dose CAR T were reinjected into CD19-low-expressing leukemia model mice. [Figure 13] Figure 1 shows that leukemia cells take over FnC from the CAR T cell surface as observed by dynamic fluorescence imaging. [Figure 14] These are the results of detecting changes in peripheral blood leukemia burden over time after CAR T with different treatment starting points, FnC-CAR T, and FnC-CAR T loaded with arsenic trioxide were reinjected into the bodies of high-burden, low-CD19 expression leukemia model mice. [Figure 15]The interaction between tumor-infiltrating lymphocytes and FnC-TILs and tumor cells, as well as the activation status of immune cells. [Figure 16] The interaction between FnC-TCR T and tumor cells and the activation status of immune cells. [Figure 17] The interaction between FnC-EGFRCAR T and tumor cells and the activation status of immune cells. [Figure 18] When CAR T cells were expanded in vitro, CD71 expression was significantly upregulated. In the figure, Rest T refers to T cells freshly isolated from human-derived PBMCs without any other manipulation, and CAR T refers to CAR T manufactured using T cells isolated from human-derived PBMCs and expanded in vitro. [Figure 19] Figure 1 shows the killing statistics of bone marrow samples from patients with clinically different leukemias by different CAR T cells. [Figure 20] This shows the results of detecting changes in peripheral blood leukemia burden over time after CAR T, FnC-CAR T, and high-dose CAR T were reinjected into T-ALL leukemia PDX model mice. [Figure 21] This shows the results of detecting changes in peripheral blood leukemia burden over time after CAR T, FnC-CAR T, and high-dose CAR T were reinjected into CD7-low-expressing T-ALL leukemia PDX model mice. [Figure 22] This shows the results of detecting changes in peripheral blood leukemia burden over time after FnC-CAR T and FnAsC-CAR T were reinjected into high-burden CD7-low-expressing T-ALL leukemia PDX model mice. DETAILED DESCRIPTION OF THE INVENTION

[0045] Various exemplary embodiments of the present invention will now be described in detail, which should not be construed as limiting the present invention, but should be understood as more detailed descriptions of specific aspects, characteristics, and embodiments of the present invention. Unless specific techniques or conditions are specified in the examples, they are carried out according to techniques or conditions described in the literature in the field (see, for example, J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Scientific Press, translated by Huang Pedang et al.) or according to the product instructions. Unless the manufacturer is specified, the reagents or equipment used are all common products available on the market.

[0046] It should be understood that the terms used herein are merely for the purpose of describing particular embodiments and are not intended to limit the present invention. Numerical ranges in the present invention should also be understood to specifically disclose the upper and lower limits of the range, as well as each intermediate value therebetween. Each smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within a stated range, is also included within the present invention. The upper and lower limits of these smaller ranges may independently be included or excluded within the range.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The present invention describes only preferred methods and materials; however, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials related to such publications. In the event of a conflict with any incorporated publication, the present specification shall control.

[0048] targeted cells The targeted cells of the present invention can have significant killing ability against tumor cells expressing CD71 on their surface or CD71-positive tumors, and can thereby be applied to the treatment or amelioration of diseases associated with CD71 expression, forming the basis for the treatment of CD71-positive tumors.

[0049] In the present invention, a "targeted cell" refers to an immune cell that exerts an immune effector function. Preferably, the immune cell is at least one selected from immune cells cultured and differentiated from pluripotent stem cells or embryonic stem cells, T lymphocytes or modified T cells, NK cells, peripheral blood mononuclear cells (PBMCs), and hematopoietic stem cells. More preferably, the immune cell is a T lymphocyte (sometimes abbreviated as T cell herein). Even more preferably, the immune cell is a modified T cell, i.e., a CAR-T cell or a TCR-T cell. In some embodiments, the T cell may be CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, CD4- / CD8-, or a combination thereof. In some embodiments, the T cell expresses a chimeric antigen receptor and produces IL-2, IFN, and / or TNF upon binding to target cells. In some embodiments, the CD8+ T cell expresses a chimeric antigen receptor and lyses antigen-specific target cells upon binding to target cells.

[0050] In the present invention, CD71 is expressed on the surface of the T cell, and the targeted nanoscale particle of the present invention can bind to it, and the targeted nanoscale particle includes a nanoscale particle or multimeric structure formed by multiple ferritins. "Bind" and "immunoreact with" or "target" are used interchangeably to refer to a non-covalent interaction that occurs between an immunoglobulin molecule and an antigen specific for the immunoglobulin.

[0051] The ferritin of the present invention may be natural ferritin, recombinant full-heavy chain ferritin, or genetically engineered ferritin, and is not particularly limited thereto. The specific sequence of the ferritin is not particularly limited, as long as it has biological activity or binding properties targeting the ferritin receptor CD71 or has improved affinity. Such other variants can be obtained using methods known in the art and are all within the scope of the present invention. Those skilled in the art can modify the amino acid sequence of a polypeptide or protein using recombinant methods and / or synthetic chemistry techniques to produce mutant polypeptides or proteins. For example, amino acid substitutions or modifications can be used to obtain ferritin with even improved affinity.

[0052] The term "modified" as used herein refers to an amino acid modification that does not significantly affect or change the binding properties of ferritin. Such modifications include amino acid substitutions, additions, and deletions. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. The ferritins of the present invention may include glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications, etc.

[0053] In the present invention, multiple proteins are linked to each other by first binding moieties to form the targeted nanoparticles, and the first binding moieties contain active groups, and are covalently linked to each other via linking arms. Examples of active groups include, but are not limited to, -COOH, -NH2, and -HS, and the linking arms include aldehyde groups and / or short-chain small molecules of polyethylene glycol derivatives. In one embodiment, the aldehyde group-containing compound includes glutaraldehyde. Preferably, the polyethylene glycol derivative is NHS-PEG. x -NHS, where the subscript x represents multiple repeating units of polyethylene glycol, and preferably x is an integer of 1 to 100.

[0054] In some embodiments, multiple ferritins can form nanoscale particles or multimeric structures, each of which has a cavity large enough to accommodate a selectable drug or small molecule chemical. The resulting targeted nanoscale particles have a particle size of 50 to 1500 nm, preferably 100 to 1500 nm, and more preferably 300 to 1500 nm, and even more preferably 300 to 800 nm, such as 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 600, 700, 800 nm, or any range therebetween. This particle size relatively weakens the ability of CAR-T cells to uptake the nanoscale particles and ensures that the nanoscale particles are attached to the surface of the CAR-T cells. More importantly, this particle size range allows target cells to capture the nanoscale particles from the CAR-T cells through endocytosis or endocytosis. The targeted nanoscale particles of the present invention can also promote interaction between cells; in an exemplary embodiment, the targeted nanoscale particles simultaneously bind chimeric antigen receptor T cells and leukemia cells, thereby promoting interaction between the two types of cells and thereby promoting recognition and killing of leukemia cells by the chimeric antigen receptor T cells.

[0055] In one embodiment, the target cells include cancer or tumor cells derived from a cancer or tumor patient, a patient suspected of having cancer or tumor, or a patient who has been treated for cancer or tumor, preferably a tumor-positive cell expressing the ferritin receptor CD71 on its cell surface. Also preferably, the target cells are leukemia cells.

[0056] In the present invention, the targeted nanoparticle further comprises a second binding moiety, and binds to the outer surface of a target cell via the second binding moiety, where the outer surface of the target cell expresses the ferritin receptor CD71, and the ferritin receptor CD71 can bind to the protein via the second binding moiety. Preferably, the second binding moiety is different from the first binding moiety.

[0057] Manufacturing method The present invention further provides a method for producing targeted cells, comprising steps (1) to (3). In step (1), protein clusters are formed by aggregating and precipitating proteins under electrolyte or non-electrolyte conditions. The electrolyte conditions refer to adding an electrolyte solution to a ferritin solution. The electrolyte solution is not particularly limited, and in one embodiment, a saturated ammonium sulfate solution is used. The non-electrolyte conditions refer to adding a non-electrolyte solution to a ferritin solution. The non-electrolyte solution is not particularly limited, and in one embodiment, the non-electrolyte solution is ethanol. Since the purpose of step (1) is to aggregate and precipitate ferritin to form ferritin clusters, the electrolyte and non-electrolyte-related reagents may be any reagents capable of impairing the stability of the protein solution. Those skilled in the art will understand that, for example, ethanol and saturated ammonium sulfate solution can be used simultaneously or separately to form ferritin clusters.

[0058] In step (2), the clusters are crosslinked in the presence of a crosslinking agent to obtain crosslinked targeted nanoscale particles. Preferably, the crosslinking agent is glutaraldehyde and NHS-PEG. x It is understood that those skilled in the art can use other cross-linking agents to cross-link multiple ferritins to form aggregates or clusters having a certain particle size, and such cross-linking agents further include disuccinimidyl suberate, paraformaldehyde, and polyethyleneimine. After step (2), a washing step is further included to remove excess reactants.

[0059] In step (3), the targeting nanoscale particles and cells are brought into close contact with each other, thereby obtaining targeted cells in which ferritin nanoclusters are modified on the surface of CAR-T cells by binding to CD71. Preferably, the bringing of the targeting nanoscale particles and cells into close contact with each other is performed under ex vivo conditions.

[0060] Those skilled in the art will understand that the present invention may further include a step of introducing an immunotherapeutic agent and / or a small molecule chemical agent into the protein cavity prior to step (1) to achieve combined administration or combined treatment, and therefore, the step of loading another drug is also within the scope of the present invention. The specific step of loading a drug is not particularly limited, and different drug loading methods can be used depending on the type of drug. For example, the drug may be loaded before forming clusters, or by dissociation and self-assembly, or directly via the tunnels of ferritin.

[0061] Method for promoting cell proximity or binding The present invention further provides a method for promoting cell proximity or cell binding, comprising the step of promoting cell-cell proximity and contact using targeted nanoscale particles. Preferably, the bringing of the targeted nanoscale particles and the cells into close proximity and contact with each other is performed under ex vivo conditions. It should be noted that the cells herein include a first cell and a second cell, and the first cell is at least one selected from CAR T cells, TCR-T cells, and tumor-infiltrating lymphocytes. The second cell is selected from cancer or tumor cells. In one embodiment, the targeted nanoscale particles bind to CD71 on the surface of at least one of the cells selected from CAR T cells, TCR-T cells, and tumor-infiltrating lymphocytes to form a conjugate, and the conjugate binds to CD71 on the surface of the cancer or tumor cells via the targeted nanoscale particles. During this process, the inventors found that CD71 expression was significantly upregulated during the in vitro construction and expansion of CAR-T cells (see Figure 18), and that co-incubating and binding targeting nanoparticles with the CAR-T significantly enhanced the intercellular interaction, promoting the recognition of cancer or tumor antigen targets by CAR-T cells and thereby activating CAR-T cells. Therefore, the modified CAR-T cells of the present invention can effectively solve the problem that downregulation of target antigen expression seriously affects the recognition and killing of leukemia cells by CAR T cells, and thus can significantly promote the recognition and killing of non-target leukemia cells by CAR T cells.

[0062] Pharmaceutical Composition The present invention also provides pharmaceutical compositions comprising the targeted cells described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Pharmaceutical compositions can be prepared in the form of a lyophilized preparation or an aqueous solution by mixing an active agent having a desired purity with an optional pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are non-toxic to recipients at the dosage and concentration used, and may include at least one of buffers, antioxidants, preservatives, isotonicity agents, stabilizers, and surfactants. In addition, pharmaceutical compositions must be sterilized so that they can be used for in vivo administration. Pharmaceutical compositions can be sterilized by filtration through a sterile filtration membrane. In some embodiments, the pharmaceutical composition may contain at least one additive selected from the group consisting of cytotoxic agents, chemotherapeutic agents, cytokines, immunosuppressants, growth inhibitors, and active agents required for the specific indication to be treated. The specific amount of additives added may be adjusted according to actual needs.

[0063] Purpose The present invention also provides the use of reagents comprising the targeted cells or pharmaceutical compositions described herein in the manufacture of a medicament for treating or ameliorating cancer. Preferably, the treatment or amelioration of cancer or tumor refers to the ability to stimulate or enhance immune function in cancer or tumor patients. Preferably, the cancer or tumor refers to a cancer associated with CD71 expression.

[0064] In the present invention, "cancer or tumor associated with CD71 expression" refers to a disease caused directly or indirectly by cells expressing CD71, and is preferably a cancer or tumor in which CD71 is highly or overexpressed. Preferably, the cancer or tumor includes a hematological cancer or tumor, a solid tumor, or a combination thereof.

[0065] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or blood-derived) cancers include leukemia, including acute leukemia (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myelocytic leukemia, and myeloblastic, promyelocytic, granulo-monocytic, monocytic, and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myelocytic leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and aggressive forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelohyperplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0066] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma mesothelioma, lymphoid malignancies, pancreatic cancer, and ovarian cancer.

[0067] Therefore, the present invention also provides a method for treating and / or preventing cancer, comprising administering a therapeutically effective amount of targeted cells or pharmaceutical compositions to a subject in need thereof. As used herein, the terms "subject" and "patient" are used interchangeably to refer to any animal that may require treatment with the targeted cells or pharmaceutical compositions described herein. Thus, subjects and patients include, but are not limited to, primates (including humans), dogs, cats, mice, and other mammalian subjects. Preferably, the subject is a human.

[0068] In the present invention, the term "treatment" refers to therapeutic treatment and prophylactic or preventative therapeutic measures aimed at preventing or alleviating (reducing) the progression of undesired physiological changes or disorders, such as hematological immune disorders. Beneficial or desired clinical results include detectable and undetectable results, including, but not limited to, amelioration of symptoms, reduction in disease severity, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, and improvement or palliation and alleviation (either partial or total) of the disease state. "Treatment" also means prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those suffering from a disease or disorder, as well as those susceptible to a disease or disorder, or those in need of prevention of the disease or disorder.

[0069] As used herein, the term "effective amount" refers to an amount of a drug or agent that elicits the biological or pharmacological response of a tissue, system, animal, or human that is desired, for example, by a researcher or clinician. The term "therapeutically effective amount" also refers to an amount that results in improved treatment, cure, prevention, or reduction of a disease, disorder, or side effect, or reduces the rate of progression of a disease or condition, compared to a corresponding subject not receiving that amount. The term also includes within its scope an amount that effectively enhances normal physiological function. Generally, the effective amount herein will vary depending on various factors, such as the given drug or compound, pharmaceutical formulation, route of administration, type of disease or disorder, and subject to be treated, but can still be routinely determined by one of ordinary skill in the art. The effective amount of the targeted cells or pharmaceutical compositions of the present invention can be readily determined by one of ordinary skill in the art using conventional methods known in the art.

[0070] Administration of the targeted cells or pharmaceutical compositions of the present invention can be by any convenient method, including nebulization, injection, swallowing, infusion, implantation, or transplantation. The targeted cells or pharmaceutical compositions described herein can be administered to a patient by subcutaneous, intradermal, intratumoral, intranodal, intraspinal, intramuscular, intravenous (iv) injection, or intraperitoneal administration. In one embodiment, the T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention are preferably administered by iv injection. Compositions containing T cells can be injected directly into a tumor, lymph node, or site of infection.

[0071] Combination treatment The present invention also provides the use of the targeted cells or pharmaceutical compositions described in the present invention in combination with other drugs. Preferably, the other drugs include detection agents, prophylactic agents, and / or therapeutic agents. Also preferably, the other drugs are immunotherapeutic agents and / or small molecule chemical agents, examples of which include, but are not limited to, chemotherapeutic agents, radioisotopes, cytokines, nucleic acids, antitumor or anti-inflammatory drugs. The drugs can be loaded into the ferritin internal cavity of the targeted nanoscale particles of the present invention, or can be administered before, after, or simultaneously with the administration of the targeted cells of the targeted nanoscale particles, with or without a drug loaded. [Example]

[0072] 1.Material Whole heavy chain recombinant ferritin (obtained by recombinant bacterial expression), PBS buffer, deionized water, absolute ethanol, ammonium sulfate, glutaraldehyde, NHS-PEG X-NHS (Sigma), polylysine (Sigma), Cell-Tak adhesive (Corning), CFSE dye (Sigma), flow cytometry intracellular staining kit (Becton, Dickinson and Company), human-derived CD3 flow cytometry antibody (Biolegend), human-derived IFNγ flow cytometry antibody (Biolegend), human-derived granzyme B flow cytometry antibody (Biolegend), human-derived CD19 flow cytometry antibody (Biolegend), red blood cell lysate (Solarbio), LDH release detection kit (Solarbio). 2. Method The production of targeted cells of the present invention comprises the following steps. (1) a step of aggregating and precipitating proteins under electrolyte or non-electrolyte conditions to form protein clusters; Non-electrolyte conditions: Ferritin was dissolved in PBS buffer (pH 7.2-7.4) to a final concentration of 10 mg / mL. Then, absolute ethanol was added dropwise to the solution under magnetic stirring at 1000 rpm at room temperature until the solution became visibly turbid. Electrolyte conditions: Ferritin was dissolved in PBS buffer (pH 7.2-7.4) to a final concentration of 10 mg / mL. Saturated ammonium sulfate solution was then added dropwise to the solution under magnetic stirring at 1000 rpm at room temperature until visible turbidity appeared. (2) The clusters were crosslinked in the presence of a crosslinker to obtain crosslinked targeted nanoscale particles. The crosslinking step involved adding a 25% glutaraldehyde solution directly to the turbid solution obtained in step (1) to a final concentration of 0.25%-1%, or adding NHS-PEG-NHS to a final concentration of approximately 1%. The mixture was then allowed to react for 1.5 hours at room temperature while stirring at 1000 rpm. After the reaction was complete, PBS buffer was added to the mixture at room temperature while stirring at 1000 rpm, diluting the mixture to twice its original volume. Finally, the mixture was centrifuged at 10,000 g for 10 minutes, the supernatant was discarded, and the precipitate was washed three times with PBS buffer to obtain the ferritin cluster nanoformulation. The clusters were then stored in PBS buffer. (3) contacting the targeted nanoparticles and cells in close proximity to each other to obtain the targeted cells. 6 According to the ratio of CAR T, CAR T cells and ferritin cluster nanoformulations were co-incubated for 1 h and then centrifuged to wash away excess ferritin clusters. 0.5 mg of ferritin clusters was based on protein mass. 3. Detection of acting force The forces acting between CAR T and FnC-CAR T and leukemia cells were detected using atomic force microscopy. First, a 6 cm diameter Petri dish was coated with polylysine by incubation, and then leukemia cells were cultured in the Petri dish and adsorbed to the bottom of the Petri dish using polylysine. Next, CAR T or FnC-CAR T was attached to the probe of an atomic force microscope using Cell-Tak adhesive, and then the force acting between the CAR T or FnC-CAR T on the probe and the leukemia cells was detected using an atomic force microscope. 4. Detection of proliferation after incubating CAR T and FnC-CAR T with leukemia cells The CFSE method was used to detect the proliferation status after co-incubation of CAR T with leukemia cells. CFSE dye was used to stain CAR T, and then the partially stained CAR T was co-incubated with FnC to construct CFSE-stained FnC-CAR T. Next, the CFSE-stained CAR T and FnC-CAR T were co-incubated with leukemia cells, respectively. Finally, after 0 h, 24 h, and 72 h of co-incubation, respectively, the CFSE fluorescent signal of CAR T or FnC-CAR T was detected using flow cytometry to detect both proliferation states (where CAR T or FnC-CAR T was labeled with human-derived CD3 antibody). 5. Activation of CAR T and FnC-CAR T by co-incubation with leukemia cells Flow cytometry was used to detect the activation of CAR T and FnC-CAR T co-incubated with leukemia cells. To characterize the degree of activation, IFNγ and granzyme B antibodies were used to detect the positive ratios of IFNγ and granzyme B after co-incubation of CAR T and FnC-CAR T with leukemia cells according to the intracellular staining protocol provided by Becton, Dickinson and Company, where CAR T or FnC-CAR T was labeled with a human-derived CD3 antibody. 6. Killing of leukemia cells incubated with CAR T and FnC-CAR T at different ratios Leukemia cell killing by CAR T was characterized by detecting the release of LDH in the supernatant. CAR T or FnC-CAR T was mixed with leukemia cells at different ratios and then cultured in a 96-well plate. After 24 hours, the cell mixture was removed and centrifuged, and the supernatant was collected. LDH release in the supernatant was detected using an LDH release detection kit (Solarbio). The rate of leukemia cell lysis was characterized by comparison with the positive control group. 7. Changes in peripheral blood leukemia burden over time After reinjection of CAR T, FnC-CAR T, and high-dose CAR T into leukemia model mice, the time course of changes in peripheral blood leukemia burden was detected. First, leukemia modeling: Leukemia cells isolated from the bone marrow of clinical leukemia patients were used at 5 × 10 6 The cells were then intravenously reinjected into severely immunodeficient mice (NTG mice, Speifuku Co., Ltd.) at the appropriate cell dose. Approximately 7 days after reinjection, retro-orbital blood samples were taken and the proportion of human leukemia cells in the white blood cells of the mouse blood samples was detected by flow cytometry. A proportion of 1% or higher was considered to indicate successful leukemia modeling. Treatment regimen: After successful modeling of the leukemia model, the model mice were randomly divided into groups (8 or more per group), and then treated with PBS buffer (200 μL / mouse), CAR T (1 × 10 6 / fish), FnC-CAR T(1×10 6 / mouse), high dose CAR T (5×10 6The mice were intravenously reinjected with 100 mg of 1000 mg of human leukemia cells (100 mg / animal). To assess the changes in the leukemia burden in the mice after treatment, the proportion of human leukemia cells in the peripheral blood of the mice was measured every 5 days.

[0073] 2. Experimental results 2.1 Ferritin and ferritin clusters Figure 1 shows an overall schematic diagram of the treatment of leukemia using the modified CAR-T cells of the present invention, and transmission electron microscope images of the produced ferritin and ferritin clusters are shown in Figure 2. According to the method of the present invention, we have successfully constructed ferritin clusters with particle sizes of approximately 300 to 500 nm. 2.2 Detection of acting force The force between CAR T and leukemia cells measured by atomic force microscopy was significantly increased after FnC modification (from approximately 2 nN to approximately 43 nN), as can be seen in Figure 3. 2.3 Detection of proliferation status after co-incubation of CAR T with leukemia cells using CFSE assay As can be seen in Figure 4, after FnC modification, there is a significant increase in proliferation after co-incubation of CAR T with leukemia cells. 2.4 Detection of CAR T and FnC-CAR T co-incubation activation with leukemia cells by flow cytometry Figure 5 shows the positive rates of Granzyme B and interferon gamma (IFNγ) after co-incubation of CAR T with leukemia cells, as detected by flow cytometry, to characterize CAR T activation. As can be seen from Figure 5, after FnC modification, the activation rate after co-incubation of CAR T with leukemia cells significantly increased. 2.5 Killing of CAR T and FnC-CAR T incubated with leukemia cells according to different ratios Figure 6 shows the leukemia cell killing by CAR T by detecting the release of LDH from the supernatant, with the vertical axis representing the killing rate. As can be seen from Figure 6, the leukemia cell killing ability of CAR T after FnC modification was significantly increased, and the killing rate could still reach over 80% at a ratio of 1:4. 2.6 Changes in peripheral blood leukemia burden over time After reinjection of CAR T, FnC-CAR T, and high-dose CAR T into leukemia model mice, we monitored the time course of peripheral blood leukemia burden. As shown in Figure 7, peripheral blood samples were taken from the model mice at different time points, and the percentage of leukemia cells in the peripheral blood was measured to characterize the therapeutic effect. As can be seen from Figure 7, FnC-modified CAR T completely inhibited leukemia progression, achieving a therapeutic effect nearly equivalent to that of high-dose CAR T. [Example]

[0074] 1. Interaction between CAR T and FnC-CAR T and CD19-low expressing leukemia cells The present invention further detected the interaction between CAR T and CD19 low-expressing leukemia cells, and the interaction between CAR T and FnC-CAR T and CD19 low-expressing leukemia cells detected by atomic force microscopy is shown in Figure 8. 2. Detection of proliferation after incubation of CAR T and FnC-CAR T with CD19-low expressing leukemia cells Similar to the CFSE method described above, the present invention detected the proliferation status after co-incubation of CAR T with leukemia cells with low CD19 expression. The results are shown in Figure 9, demonstrating that FnC modification promotes the activity of CAR T and leukemia cells, thereby promoting proliferation. 3. Flow cytometry of activation after co-incubation of CAR T and FnC-CAR T with CD19-low expressing leukemia cells Similar to the activation flow cytometry diagram described above, the present invention characterized the activation status after co-incubation of CAR T with CD19 low-expressing leukemia cells, and the results are shown in Figure 10. 4. Detection of killing after co-incubation of CAR T and FnC-CAR T with CD19-low expressing leukemia cells according to different ratios Similar to the LDH release detection killing figures above, the present invention characterized the killing of CD19 low-expressing leukemia cells by CAR T, and the results demonstrated enhanced killing of CD19 low-expressing leukemia cells by CAR T after FnC modification, as shown in Figure 11. 5. Changes in peripheral blood leukemia burden over time after reinfusion of CAR T, FnC-CAR T, and high-dose CAR T into CD19-low expressing leukemia model mice Similar to the in vivo therapeutic effect diagrams described above, the present invention detected the time course of peripheral blood leukemia burden after reinfusion of CAR T, FnC-CAR T, and high-dose CAR T into an animal model of CD19 low-expressing leukemia, and the results are shown in Figure 12. [Example]

[0075] Cell labeling: In this experiment, CAR T cells were labeled with red fluorescent Celltracker dye, leukemia cells were labeled with CFSE, and ferritin cluster nanoformulations were labeled with Cy5-se dye. The excitation wavelengths for the three were 561 nm, 488 nm, and 633 nm, respectively.

[0076] Specific experimental steps: According to the above-mentioned methods, the above fluorescently labeled CAR T cells and fluorescently labeled ferritin cluster nanoformulations were prepared into FnC-CAR T. Then, the FnC-CAR T was mixed with the above fluorescently labeled leukemia cells in a 96-well plate, and the capture of leukemia cells by FnC on the surface of FnC-CAR T was monitored in real time using a high-content imaging analysis system (Operetta CLS).

[0077] The present invention used a high-content imaging analysis system to observe the situation in which leukemia cells plunder FnC from the surface of CAR T when FnC-CAR T was co-incubated with leukemia cells. The results are shown in Figure 13. A high-content dynamic fluorescence imaging system was used to observe the leukemia cells plundering FnC from the surface of CAR T cells. As can be seen from the figure, leukemia cells can plunder FnC from the surface of CAR T, while CAR T does not ingest FnC. [Example]

[0078] The present invention detected changes in peripheral blood leukemia burden over time after reinjection of CAR T, FnC-CAR T, and FnC-CAR T loaded with arsenic trioxide into the bodies of high-burden CD19 low-expressing leukemia model mice.

[0079] The main steps of this experiment are as follows: Construction of arsenic-loaded FnC-CAR T: First, arsenic trioxide, a chemotherapy drug, was loaded onto ferritin to induce Fn As and then Fn As Fn As Finally, Fn was constructed according to the method described above. As C and CAR T, Fn As It was built on C-CAR T. Evaluation of in vivo therapeutic efficacy: A CD19-low expressing leukemia model was established using CD19-low expressing leukemia cells according to the method described above. Treatment was initiated when the proportion of human-derived leukemia cells in the mouse peripheral blood leukocytes reached 10% or higher. PBS (200 μL / mouse), CAR T (1 × 10 6 / fish), FnC-CAR T(1×10 6 / fish), Fn As C-CAR T(1×10 6 Mice were treated with intravenous reinfusion of 1000 mg / animal of 1000 mg ...

[0080] The results are shown in Figure 14, which is the same as the in vivo therapeutic effect diagram for the aforementioned CD19-low expression leukemia model, except that the starting point of treatment was a peripheral blood leukemia burden of 10% (the starting point for the two aforementioned models was 1%). [Example]

[0081] The T cells used in this experiment were clinical melanoma infiltrating lymphocytes (Tumor Infiltrating Lymphocytes, TILs). The specific steps are as follows: Production of FnC-TIL: In this experiment, TILs were isolated from tumor samples from clinical melanoma patients, centrifuged and washed, and then activated and expanded in vitro using magnetic beads stimulated with human IL-2 and CD3 & CD28. The resulting TILs were then used to produce FnC-TILs using FnC and TILs according to the above-mentioned method for producing FnC-CAR T.

[0082] Detection of interaction forces between TILs and tumor cells: In this experiment, similar to the previous experiment, melanoma cell suspensions were cultured on 6 cm Petri dishes coated with polylysine, and then TILs or FnC-TILs were attached to the probe of an atomic force microscope using Cell-Tak adhesive, and the interaction forces between the two were detected using an atomic force microscope.

[0083] In vitro activation of TILs: TILs and FnC-TILs were mixed with the above melanoma sample cell suspension and cultured in a 96-well plate. After 72 h, the IFNγ and granzyme B positive rates of TILs and FnC-TILs were detected using flow cytometry according to the previously described method to characterize their activation status.

[0084] The present invention further prepared FnC-TILs based on tumor-infiltrating lymphocytes and studied their interaction with tumor cells and immune cell activation. The results are shown in Figure 15, where the left graph shows the statistics of interaction between tumor-infiltrating lymphocytes and FnC-TILs and tumor cells measured by atomic force detection. The right graph shows the double-positive ratio of granzyme B (GrB) and interferon-γ (IFNγ) after co-incubation of TILs and FnC-TILs with tumor cells, characterizing TIL activation. The TILs in this graph are melanoma versus solid tumors. [Example]

[0085] The present invention further produced FnC-TCR T based on TCR T cells, and investigated the interaction between it and tumor cells and the activation status of immune cells. The T cells used in this experiment were engineered TCR T cells. The specific experiment is as follows.

[0086] Construction of FnC-TCR T: FnC and TCR T were constructed into FnC-TCR T according to the construction method of FnC-CAR T described above.

[0087] Interaction force between TCR T and tumor cells: Human melanoma cell lines were cultured in 6 cm Petri dishes. After 24 h, TCR T or FnC-TCR T were attached to an atomic force microscope probe using Cell-Tak adhesive, and then the interaction force between TCR T or FnC-TCR T and melanoma cells was detected using an atomic force microscope.

[0088] In vitro activation verification: TCR T or FnC-TCR T were cultured with melanoma cells in a 96-well plate. After 72 h, the IFNγ and granzyme B positive rates of TCR T and FnC-TCR T were detected using flow cytometry according to the previously described method to characterize their activation status.

[0089] The results are shown in Figure 16. The left figure shows the force statistics between TCR T and FnC-TCR T and tumor cells measured by atomic force detection. The right figure shows the double positive ratio of granzyme B (GrB) and interferon gamma (IFNγ) after co-incubation of TCR T and FnC-TCR T with tumor cells to characterize TCR T activation. This figure demonstrates that the enhancing effect of the FnC strategy is also effective against TCR T. [Example]

[0090] The present invention provides EGFR CAR T ( EGFR FnC-based CAR T cells EGFR CAR T was further produced to study the interaction between it and tumor cells and the activation status of immune cells.

[0091] The T cells used in this experiment were human-derived EGFR-specific CAR T ( EGFR CAR T). The specific experimental steps are as follows:

[0092] FnC- EGFR CAR T production: FnC and EGFR CAR T, FnC- EGFR It was constructed in CAR T.

[0093] EGFR Detection of interaction between CAR T and tumor cells: Human liver cancer cell lines were cultured in 6 cm Petri dishes. After 24 h, they were transfected using Cell-Tak adhesive. EGFR CAR T or FnC- EGFR The CAR T was attached to the probe of an atomic force microscope, and then the atomic force microscope was used to EGFR CAR T or FnC- EGFR The interaction between CAR T and liver cancer cells was detected.

[0094] In vitro activation validation: EGFR CAR T or FnC- EGFRCAR T cells were cultured with human liver cancer cells in a 96-well plate. After 72 hours, the cells were analyzed by flow cytometry according to the method described above. EGFR CAR T and FnC- EGFR The positive rates of IFNγ and granzyme B of CAR T were detected to characterize their activation status.

[0095] The results are shown in Figure 17, where the left figure shows the EGFR CAR T( EGFR CAR T) and FnC- EGFR Statistics of the interaction between CAR T and tumor cells. EGFR To characterize CAR T activation, EGFR CAR T and FnC- EGFR The double-positive rate of granzyme B (GrB) and interferon gamma (IFNγ) after co-incubation of CAR T with tumor cells. This figure demonstrates that the enhancing effect of the FnC strategy is also effective for other types of CAR T. [Example]

[0096] This example shows the statistical results of killing bone marrow samples from clinically different leukemia patients by different CAR T cells, and the experimental steps are as follows:

[0097] FnC-CAR T and Fn As Construction of C-CAR T: The CAR T cells used in this experiment were CD7-CAR T, CD33-CAR T, and CD19-CAR T for T-ALL, AML, and B-ALL, respectively. The three types of cells were then used to construct the corresponding FnC-CAR T and Fn As C-CAR T was constructed.

[0098] Kill test: CAR T, FnC-CAR T and Fn AsC-CAR T cells were mixed with bone marrow samples from leukemia patients at a 1:1 ratio and then cultured in a 96-well plate. After 24 hours, the cell mixture was removed and centrifuged. The supernatant was collected and LDH release was detected using an LDH release detection kit (Solarbio). The rate of leukemia cell lysis was characterized by comparison with a positive control group.

[0099] The results are shown in Figure 19. In Figure 19, the CAR T cells for the bone marrow sample of the T-ALL patient are CD7-CAR T cells, the CAR T cells for the bone marrow sample of the AML patient are CD33-CAR T cells, and the CAR T cells for the bone marrow sample of the B-ALL patient are CD19-CAR T cells. The results showed that the targeted cells of the present invention can target and effectively kill cancer cells in patients with different types of leukemia. [Example]

[0100] This example demonstrates the time course of changes in peripheral blood leukemia burden after reinjection of CAR T, FnC-CAR T, and high-dose CAR T into T-ALL leukemia PDX model mice. First, a leukemia T-ALL PDX model was constructed. Leukemia cells isolated from the bone marrow of clinical T-ALL leukemia patients were injected into the PDX model at 5 × 10 6 The cells were then intravenously reinjected into severely immunodeficient mice (NTG mice, Speifuku Co., Ltd.) at the appropriate cell dose. Approximately 7 days after reinjection, retro-orbital blood samples were taken and the proportion of human leukemia cells in the white blood cells of the mouse blood samples was determined by flow cytometry. A proportion of 1% or higher was considered to indicate successful modeling of T-ALL PDX.

[0101] Treatment regimen: After successful modeling of the T-ALL PDX model, the model mice were randomly divided into groups (8 or more per group) and then treated with PBS buffer (200 μL / mouse), CAR T (1 × 10 6 / fish), FnC-CAR T(1×10 6 / mouse), high dose CAR T (5×106 The mice were intravenously reinjected with 100 mg of 1000 mg of human leukemia cells (100 mg / animal). To assess the changes in the leukemia burden in the mice after treatment, the proportion of human leukemia cells in the peripheral blood of the mice was measured every 5 days.

[0102] The results are shown in Figure 20. In Figure 20, the CAR T cells used were CD7-CAR T cells. The results showed that the targeted cells of the present invention can significantly reduce the peripheral blood leukemia burden in T-ALL leukemia PDX model mice. [Example]

[0103] This example shows the results of detecting changes in peripheral blood leukemia burden over time after reinjection of CAR T, FnC-CAR T, and high-dose CAR T into CD7-low-expressing T-ALL leukemia PDX model mice. Low The purpose of this study was to establish a T-ALL PDX model. Leukemia cells isolated from the bone marrow of clinical T-ALL leukemia patients were downregulated with a CD7 knockdown virus to create a CD7-low PDX model (CD7 Low ) leukemia cells were constructed. Then, 5 × 10 6 CD7-low expressing T-ALL cells were intravenously reinjected into severely immunodeficient mice (NTG mice, Speifuku Co., Ltd.) according to the cell / mouse dose. Approximately 7 days after reinjection, orbital blood samples were taken from the mice, and the percentage of human leukemia cells in the leukocytes of the blood samples was detected by flow cytometry. If the percentage was 1% or higher, the cells were considered to be CD7-low expressing T-ALL cells. Low We considered the modeling of T-ALL PDX to be successful.

[0104] Treatment regimen: CD7 Low After successful modeling of the T-ALL PDX model, the model mice were randomly divided into groups (8 or more per group) and then injected with PBS buffer (200 μL / mouse), CAR T (1 × 10 6 / fish), FnC-CAR T(1×10 6 / mouse), high dose CAR T (5×10 6The mice were intravenously reinjected with 100 mg of 1000 mg of human leukemia cells (100 mg / animal). To assess the changes in the leukemia burden in the mice after treatment, the proportion of human leukemia cells in the peripheral blood of the mice was measured every 5 days.

[0105] The results are shown in Figure 21. In Figure 21, the CAR T cells used were CD7-CAR T cells. The results showed that the targeted cells of the present invention can significantly reduce the peripheral blood leukemia burden in CD7-low-expressing T-ALL leukemia PDX model mice. [Example]

[0106] This example shows the results of detecting changes in peripheral blood leukemia burden over time after FnC-CAR T and FnAsC-CAR T were reinjected into the bodies of high-load CD7-low-expressing T-ALL leukemia PDX model mice. First, we investigated the leukemia-high-load CD7 Low T-ALL PDX model was constructed and CD7 Low 5 × 10 T-ALL cells 6 The cells were then intravenously reinjected into severely immunodeficient mice (NTG mice, Speifuku Co., Ltd.) according to the cell / mouse dose. At different time points after reinjection, orbital blood samples were taken from the mice. The percentage of human leukemia cells in the leukocytes of the blood samples was detected by flow cytometry. If the percentage was 10% or higher, the cells were classified as highly loaded CD7 Low We considered the modeling of T-ALL PDX to be successful.

[0107] Treatment regimen: CD7-heavy Low After successful modeling of the T-ALL PDX model, the model mice were randomly divided into groups (8 or more per group) and then injected with PBS buffer (200 μL / mouse), FnC-CAR T (1 × 10 6 / fish), Fn As C-CAR T(1×10 6 The mice were intravenously reinjected with 100 mg of 1000 mg of human leukemia cells (100 mg / animal). To assess the changes in the leukemia burden in the mice after treatment, the proportion of human leukemia cells in the peripheral blood of the mice was measured every 5 days.

[0108] The results are shown in Figure 22. In Figure 22, the CAR T cells used were CD7-CAR T cells. The results showed that the targeted cells of the present invention can significantly reduce the peripheral blood leukemia burden in high-burden CD7-low-expressing T-ALL leukemia PDX model mice.

[0109] While the present invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or modifications may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the invention. The scope of the claims should be accorded the broadest interpretation so as to cover all modifications and equivalent structures and functions.

Claims

1. A T cell complex comprising a T cell and a ferritin cluster, wherein the T cell binds to the ferritin cluster, and the ferritin cluster is composed of ferritin.

2. the T cells are activated T cells; Preferably, the T cells are in vitro expanded. The T cell conjugate of claim 1.

3. The T cell conjugate of claim 1, wherein the ferritin cluster binds to CD71 on the T cell through a receptor-ligand interaction.

4. The T cell conjugate according to claim 1, wherein the average particle size of the ferritin clusters is 50 to 1500 nm, preferably 300 to 1500 nm, also preferably 400 to 1000 nm, more preferably 500 to 800 nm.

5. The ferritin cluster is formed by aggregation and cross-linking of human-derived ferritin, Preferably, heavy chain subunits account for at least 30% of the ferritin subunits exposed on the surface of the ferritin cluster. The T cell conjugate of claim 1.

6. The T cell conjugate of claim 1 , wherein the T cell conjugate enhances targeting to target cells expressing CD71 via the ferritin cluster.

7. the target cells include tumor cells; Preferably, the target cells include solid tumor cells and / or hematological tumor cells, such as cells from liver cancer, lung cancer, colon cancer, cervical cancer, prostate cancer, ovarian cancer, breast cancer, thyroid cancer, esophageal cancer, gastric cancer, leukemia, preferably leukemia cells, such as B-ALL, T-ALL, AML, etc. The T cell conjugate of claim 6.

8. A pharmaceutical composition comprising the T cell binding substance according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

9. 10. A method for treating or ameliorating a cancer or tumor associated with CD71 expression, comprising administering to a subject in need thereof a T cell conjugate according to any one of claims 1 to 7, Preferably, the cancer or tumor is, for example, liver cancer, lung cancer, colon cancer, cervical cancer, prostate cancer, ovarian cancer, breast cancer, thyroid cancer, esophageal cancer, gastric cancer, or leukemia; method.

10. A targeted nanoscale particle, characterized in that the targeted nanoscale particle is configured such that multiple proteins are linked to each other via first binding moieties, the targeted nanoscale particle further comprises a second binding moiety, and binds to the outer surface of a target cell via the second binding moiety.

11. 11. The targeted nanoscale particle of claim 10, wherein the targeted nanoscale particle is capable of binding to the outer surface of a targeted cell.

12. 12. The targeted nanoscale particle of claim 11, wherein the first binding moieties comprise an active group and are connected to each other via a linking arm.

13. 13. The targeted nanoscale particle of claim 12, wherein the outer surface of the target cell contains a receptor for the protein, and the receptor is capable of binding to the protein via the second binding moiety.

14. 12. The targeted nanoscale particle of claim 11, wherein the target cells and target cells are cells that naturally express CD71, and the target cells include at least one of T cells, NK cells, B cells, lymphocytes, cytokine-induced killer cells, innate lymphocytes, and / or leukocytes.

15. 15. The targeted nanoscale particle of claim 14, wherein the T cells include modified T cells, including CAR T cells and TCR T cells, and unmodified T cells, including tumor-infiltrating T cells, PBMC-derived T cells, or other T cells isolated from within the body.

16. 11. The targeted nanoscale particle of claim 10, wherein the targeted nanoscale particle is greater than or equal to 50 nm and less than or equal to 5000 nm.

17. 11. The targeted nanoscale particle of claim 10, wherein the protein comprises an internal cavity.

18. 18. The targeted nanoscale particle of claim 17, wherein the internal cavity may or may not contain a drug.

19. The targeted nanoscale particle according to any one of claims 10 to 18, wherein the protein is at least one selected from natural ferritin, recombinant full-heavy chain ferritin, and genetically engineered ferritin.

20. 20. A method for producing targeted nanoscale particles according to any one of claims 19, comprising: (1) a step of aggregating and precipitating proteins under electrolytic or non-electrolytic conditions to form protein clusters; and (2) cross-linking the protein clusters in the presence of a cross-linking agent to obtain cross-linked targeted nanoscale particles; Manufacturing method.

21. 21. The method for producing targeted nanoscale particles of claim 20, optionally including, before step (1), the step of introducing immunotherapeutic agents and / or small molecule chemical agents into the protein cavity.

22. A targeted cell, characterized in that the outer surface of the targeted cell is bound to a targeted nanoscale particle according to any one of claims 10 to 19.

23. 23. The method of claim 22 for producing targeted cells, comprising: 1) a step of aggregating and precipitating proteins under electrolytic or non-electrolytic conditions to form protein clusters; 2) cross-linking the protein clusters in the presence of a cross-linking agent to obtain cross-linked targeted nanoscale particles; and 3) contacting the targeted nanoscale particles and cells in close proximity to each other to obtain the targeted cells; Manufacturing method.

24. 24. The method of claim 23, further comprising the step of introducing an immunotherapeutic agent and / or a small molecule chemical agent into the cavity of the protein prior to step 1).

25. A method for promoting mutual proximity between cells, comprising the step of promoting proximity and contact between cells using a T cell binding substance described in any one of claims 1 to 7 or a targeted nanoscale particle described in any one of claims 10 to 19, wherein a plurality of proteins are linked to each other via first binding moieties to form the targeted nanoscale particle, and the targeted nanoscale particle further comprises a second binding moiety and binds to the outer surface of a cell via the second binding moiety.

26. the cells have receptors on their surface that bind to the targeted nanoscale particles or have the targeted nanoscale particles bound to them; Preferably, the cells are at least one selected from CAR T cells, TCR T cells, tumor-infiltrating lymphocytes, cancer cells, and tumor cells.

26. The method of claim 25.

27. A pharmaceutical composition comprising a targeted nanoscale particle according to any one of claims 10 to 19 or a targeted cell according to claim 22.

28. Use of a T cell binding product according to any one of claims 1 to 7, or a targeted nanoscale particle according to any one of claims 10 to 19, or a targeted cell according to claim 22, in the manufacture of a medicament for treating or ameliorating cancer or tumors.

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