Targeting nano-scale particle, targeting cell, and preparation method and application of targeting nano-scale particle and targeting cell

Abstract

The invention discloses a targeting nanoscale particle, a targeting cell as well as a preparation method and application of the targeting nanoscale particle and the targeting cell. Wherein the outer surface of the targeting cell is combined with a targeting nano-scale particle, the targeting nano-scale particle is formed by mutually connecting a plurality of proteins through a first combination part, the targeting nano-scale particle further comprises a second combination part, and the targeting nano-scale particle is combined with the outer surface of the target cell through the second combination part. In an exemplary embodiment, the targeting nanoscale particles can promote interaction between the chimeric antigen receptor T cells and the leukemia cells by being simultaneously combined with the chimeric antigen receptor T cells and the leukemia cells, and then the chimeric antigen receptor T cells are promoted to recognize and kill the leukemia cells. In addition, a space is provided for loading of chemotherapeutic drugs by a protein internal cavity in the targeting nanoscale particles, and combined therapy of the chimeric antigen receptor T cells and other therapies is realized while the drugs are loaded.

Description

Targeted nanoparticles, targeted cells, and preparation methods and uses thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to application number CN202211539334.7, filed on December 2, 2022, entitled “Targeted nanoscale particles, targeted cells, preparation methods and uses thereof,” the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to the technical field of biological immunotherapy, and in particular to targeted nano-scale particles, targeted cells, and preparation methods and uses thereof. Background Art

[0004] Chimeric Antigen Receptor T cells (CAR T) have achieved remarkable success in the treatment of hematologic malignancies. Currently, CAR T products targeting CD19 have been approved for marketing, and several other CAR T products targeting other targets are undergoing clinical trials. However, during the progression of leukemia, target antigen expression decreases. Studies have shown that over 30% of patients with B-lineage leukemia who relapse after treatment experience decreased expression of the CD19 target. This downregulation of target antigen expression severely impacts CAR T cells' ability to recognize and kill leukemia cells, ultimately limiting the efficacy of CAR T. Therefore, promoting CAR T's ability to recognize and kill low-target leukemia cells is key to improving CAR T's efficacy.

[0005] The information in the background technology is only intended to illustrate the general background of the invention and should not be regarded as an admission or any form of suggestion that this information constitutes the prior art known to a person skilled in the art.

[0006] Summary of the Invention

[0007] Through research, the inventors found that the ferritin receptor CD71 can be stably and highly expressed on the surface of leukemia cells of various disease courses, and the expression of CD71 is greatly upregulated when CAR T cells are expanded in vitro. Based on this, the present invention has developed a method based on nanoscale protein particles to promote cell-to-cell interactions, thereby promoting, for example, CAR T to recognize and kill leukemia cells. In addition, the protein of the present invention has an internal cavity, which provides space for the loading of chemotherapy drugs. Therefore, the dosage form can also be loaded with drugs to achieve combined treatment of CAR T with other therapies, ultimately enhancing the efficacy of leukemias with various disease courses. Specifically, the present invention includes the following contents.

[0008] In one aspect of the present invention, a T cell binder 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.

[0009] In certain embodiments, according to the T cell conjugate of the present invention, the T cells are activated T cells (generally activated by commercial activation magnetic beads, and antibody activation, etc.), preferably T cells that have been expanded in vitro, and further preferably are newly made chimeric antigen receptor T cells (CAR T cells) or T cell receptor T cells (TCR T cells).

[0010] In certain embodiments, according to the T cell binder of the present invention, the ferritin cluster is bound to CD71 of the T cell through receptor-ligand interaction.

[0011] In certain embodiments, according to the T cell binder of the present invention, the average particle size of the ferritin clusters is 50-1500 nm, preferably 300-1500 nm, further preferably 400-1000 nm, and more preferably 500-800 nm.

[0012] In certain embodiments, according to the T cell binder of the present invention, the ferritin cluster is formed by aggregation and cross-linking of human ferritin, and preferably, heavy chain subunits account for at least 30% of the ferritin subunits exposed on the surface of the ferritin cluster.

[0013] In certain embodiments, according to the T cell binder of the present invention, the T cell binder enhances the targeting of target cells expressing CD71 through the ferritin cluster.

[0014] In certain embodiments, according to the T cell binder of the present invention, the target cells are mainly tumor cells, including solid tumor cells and blood 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.

[0015] The present invention also provides the use of the above-mentioned T cell binder in the preparation of a drug for treating or improving cancers or tumors associated with the expression of CD71, 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.

[0016] In one aspect of the present invention, a targeted nanoscale particle is provided, wherein the targeted nanoscale particle is composed of multiple proteins interconnected by a first binding portion, and the targeted nanoscale particle also includes a second binding portion, and is bound to the outer surface of the target cell through the second binding portion.

[0017] In certain embodiments, the targeted nanoscale particle according to the present invention is capable of binding to the outer surface of a targeted cell.

[0018] In certain embodiments, according to the targeted nanoparticles of the present invention, the first binding portions comprise active groups, and the first binding portions are connected via a connecting arm.

[0019] In certain embodiments, according to the targeted nanoparticles of the present invention, the outer surface of the target cell comprises a receptor for the protein, and the receptor can bind to the protein through the second binding portion.

[0020] In certain embodiments, according to the targeted nanoparticles of the present invention, 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, natural lymphoid cells and / or leukocytes.

[0021] In certain embodiments, according to the targeted nanoparticles of the present invention, the type of T cells is not particularly limited. Preferably, the T cells include modified and unmodified T cells, wherein the modified T cells include CAR T cells and TCR T cells, and the unmodified T cells include tumor-infiltrating T cells, PBMC-derived T cells or other T cells isolated from the body.

[0022] In certain embodiments, the targeted nanoparticles according to the present invention are 50 nm or more and 5000 nm or less, preferably 100 nm or more and 3000 nm or less, further preferably 300-2000 nm, and more preferably 300-1000 nm.

[0023] In certain embodiments, according to the targeted nanoscale particle of the present invention, the protein comprises an internal cavity.

[0024] In certain embodiments, the targeted nanoscale particle according to the present invention, wherein the internal cavity contains or does not contain a drug.

[0025] In certain embodiments, according to the targeted nanoparticles of the present invention, the protein is selected from at least one of natural ferritin, recombinant full heavy chain ferritin, and genetically engineered ferritin.

[0026] One aspect of the present invention provides a method for preparing the above-mentioned targeted nanoparticles, comprising:

[0027] (1) causing the protein to aggregate under electrolyte conditions or non-electrolyte conditions to form protein clusters; and

[0028] (2) cross-linking the protein clusters in the presence of a cross-linking agent to obtain cross-linked targeted nanoparticles.

[0029] In certain embodiments, according to the method for preparing targeted nanoparticles of the present invention, the type of the cross-linking agent is not particularly limited, as long as it can cross-link protein clusters, and examples thereof include but are not limited to: glutaraldehyde, NHS-PEG x -NHSDSS, disuccinimidyl suberate, paraformaldehyde, and polyethyleneimine.

[0030] In certain embodiments, according to the method for preparing targeted nanoparticles of the present invention, a step of allowing immunotherapeutic drugs and / or small molecule chemical drugs to enter the cavity of the protein is optionally performed before step (1).

[0031] One aspect of the present invention provides a targeted cell, wherein the targeted nanoscale particle described in the first aspect is bound to the outer surface of the targeted cell, multiple proteins are interconnected through a first binding portion to form the targeted nanoscale particle, and the targeted nanoscale particle also includes a second binding portion, and is bound to the outer surface of the target cell through the second binding portion.

[0032] In certain embodiments, according to the targeted cell of the present invention, the first binding portion comprises an active group, and the first binding portions are connected by a connecting arm.

[0033] In certain embodiments, according to the targeted cell of the present invention, the outer surface of the target cell comprises a receptor for the protein, and the receptor can bind to the protein through the second binding portion.

[0034] In certain embodiments, according to the targeted cells of the present invention, the targeted cells and the target cells are cells that naturally express CD71.

[0035] In certain embodiments, the targeted cells according to the present invention are selected from at least one of αβT cells, γδT 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 selected from at least one of CAR T cells, TCR T cells, and tumor-infiltrating lymphocytes; and the target cells include cells from hematological tumors, solid tumors, or a combination thereof.

[0036] In certain embodiments, according to the targeted cell of the present invention, the targeted nanoscale particle is larger than 50 nm and smaller than 5000 nm, preferably larger than 300 nm and smaller than 3000 nm.

[0037] In certain embodiments, according to the targeted cell of the present invention, the protein comprises an internal cavity.

[0038] In certain embodiments, according to the targeted cell of the present invention, the internal cavity may or may not contain a drug.

[0039] In certain embodiments, according to the targeted cell of the present invention, the protein is at least one selected from natural ferritin, recombinant full heavy chain ferritin, and genetically engineered ferritin.

[0040] One aspect of the present invention provides a method for preparing targeted cells, comprising:

[0041] (1) precipitating ferritin to form protein clusters under electrolyte or non-electrolyte conditions;

[0042] (2) cross-linking the clusters in the presence of a cross-linking agent to obtain cross-linked targeted nanoparticles;

[0043] (3) Bringing the targeted nanoparticles and cells close to and in contact with each other to obtain the targeted cells.

[0044] In certain embodiments, according to the preparation method of the present invention, immunotherapeutic drugs and / or small molecule chemical drugs are optionally allowed to enter the cavity of the protein before step (1).

[0045] One aspect of the present invention provides a method for promoting the approach between cells, which includes the step of using targeted nanoparticles to promote the approach and contact between cells, wherein a plurality of proteins are interconnected through a first binding portion to form the targeted nanoparticles, and the targeted nanoparticles also include a second binding portion, and are bound to the outer surface of the cell through the second binding portion.

[0046] In certain embodiments, 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. Also preferably, the surface of the first cell has a targeting nanoparticle bound to it, for example, via CD71, and the surface of the second cell has CD71.

[0047] In certain embodiments, according to the method of the present invention, the cell is selected from at least one of a CAR T cell, a TCR T cell, and a tumor infiltrating lymphocyte, or a cancer or tumor cell, such as a leukemia cell, wherein the cancer or tumor comprises a blood tumor, a solid tumor, or a combination thereof.

[0048] In certain embodiments, according to the method of the present invention, the blood tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0049] In certain embodiments, according to the method described herein, the solid tumor is selected from the group consisting of gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymphoma, 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.

[0050] In one aspect, the present invention provides a pharmaceutical composition comprising the targeted nanoparticles or targeted cells described in the present invention.

[0051] In certain embodiments, the pharmaceutical composition according to the present invention further comprises immunotherapy drugs and / or small molecule chemical drugs.

[0052] One aspect of the present invention provides the use of the targeted nanoparticles or the targeted cells, or the pharmaceutical composition according to the present invention in the preparation of a medicament for treating cancer or tumors, wherein the cancer or tumor includes a CD71-positive tumor or cancer, preferably B-lineage leukemia.

[0053] In one aspect, the present invention provides the use of the targeted nanoparticles or targeted cells according to the present invention, or the pharmaceutical composition, in combination with other drugs. Other drugs include, but are not limited to, diagnostic agents, preventive agents, and / or therapeutic agents.

[0054] The technical effects of the present invention include but are not limited to:

[0055] In an exemplary embodiment of the present invention, a ferritin cluster nanoformulation was prepared using a coagulation-crosslinking method. It was found that the formulation can be modified on the surface of CAR T by binding to CD71 on the surface of CAR T, and due to the large particle size of the cluster, the uptake of ferritin clusters by CAR T cells with weak endocytosis ability is avoided, thereby ensuring the long-term effective retention of ferritin clusters on the surface of CAR T. The modified CAR T cells enhance the intercellular interaction force through the binding of surface ferritin clusters to CD71 on the surface of leukemia cells, promote CAR T's recognition of leukemia antigen targets, and thus promote the activation of CAR T cells. In addition, the strong endocytosis ability of leukemia cells enables them to capture and ingest ferritin clusters from the surface of CAR T. This process promotes the targeted killing of leukemia cells by drugs in the ferritin cavity, ensuring the combined treatment of leukemia with CAR T and other therapies.

[0056] The present invention further discovered that nanoparticles have internal cavities capable of loading a variety of drugs, such as arsenic trioxide, doxorubicin, and ferroferric oxide, thereby enabling combined treatment with ferritin and other therapies. Furthermore, ferritin can be genetically engineered to further expand the volume of the ferritin cavity or modify its internal amino acid residues to accommodate a greater number and variety of drugs, enabling combined treatment with CAR T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG1 shows a flow chart of an exemplary CAR-T cell therapy for leukemia.

[0058] FIG2 is a transmission electron micrograph of exemplary ferritin and ferritin clusters.

[0059] Figure 3 shows atomic force microscopy measurements showing the forces between an exemplary CAR T cell and leukemia cells.

[0060] Figure 4 shows the proliferation of different CAR T cells after co-incubation with leukemia cells using the CFSE method.

[0061] FIG5 shows the positive ratios of granzyme B and interferon γ (IFNγ) detected by flow cytometry after co-incubation of different CAR T cells with leukemia cells.

[0062] Figure 6 shows the killing results of CAR T and FnC-CAR T incubated with leukemia cells at different ratios.

[0063] FIG7 shows the detection results of the changes in peripheral blood leukemia burden over time after CAR T, FnC-CAR T, and high-dose CAR T were reinfused into leukemia model mice.

[0064] Figure 8 shows the atomic force microscopy test results, showing the interaction force between CAR T and FnC-CAR T and CD19 low-expressing leukemia cells.

[0065] Figure 9 shows the proliferation detection results of CAR T and FnC-CAR T after incubation with CD19 low-expressing leukemia cells.

[0066] Figure 10 shows the activation of CAR T cells after co-incubation with CD19 low-expressing leukemia cells by flow cytometry.

[0067] FIG11 shows the results of CAR T cell killing of leukemia cells with low CD19 expression.

[0068] Figure 12 shows the results of the detection of changes in peripheral blood leukemia burden over time after CAR T, FnC-CAR T and high-dose CAR T were reinfused into CD19 low-expressing leukemia model mice.

[0069] FIG13 shows dynamic fluorescence imaging observation of leukemia cells seizing FnC from the surface of CAR T cells.

[0070] Figure 14 shows the detection results of the changes in peripheral blood leukemia load over time after CAR T, FnC-CAR T with different treatment starting points and FnC-CAR T loaded with arsenic trioxide were re-infused into high-load CD19 low-expression leukemia model mice.

[0071] Figure 15 shows the interaction between tumor-infiltrating lymphocytes and FnC-TIL and tumor cells, as well as the activation of immune cells.

[0072] Figure 16 shows the interaction between FnC-TCR T and tumor cells and the activation of immune cells.

[0073] Figure 17 is FnC- EGFR The interaction between CAR T and tumor cells and the activation of immune cells.

[0074] Figure 18 shows that the expression of CD71 is significantly upregulated when CAR T cells are expanded in vitro. In the figure, Rest T refers to T cells newly isolated from human PBMCs without any other manipulation; CAR T refers to CAR T prepared using T cells isolated from human PBMCs and expanded in vitro.

[0075] FIG19 shows the statistical results of the killing effect of different CAR T cells on bone marrow samples from different clinical leukemia patients.

[0076] FIG20 shows the detection results of the changes in peripheral blood leukemia burden over time after CAR T, FnC-CAR T, and high-dose CAR T were reinfused into T-ALL leukemia PDX model mice.

[0077] Figure 21 shows the detection results of the changes in peripheral blood leukemia burden over time after CAR T, FnC-CAR T and high-dose CAR T were re-infused into CD7 low-expressing T-ALL leukemia PDX model mice.

[0078] Figure 22 shows the detection results of the changes in peripheral blood leukemia load over time after FnC-CAR T and FnAsC-CAR T were re-infused into high-load CD7 low-expressing T-ALL leukemia PDX model mice. DETAILED DESCRIPTION

[0079] Now, various exemplary embodiments of the present invention are described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and embodiments of the present invention. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this area (e.g., with reference to "Molecular Cloning Experiment Guide" by J. Sambrook et al., translated by Huang Peitang et al., 3rd edition, Science Press) or the product instructions are used. Reagents or instruments used without indicating the manufacturer are conventional products that can be obtained commercially.

[0080] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value within any stated value or stated range and any other stated value or intermediate value within the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0081] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0082] Targeted cells

[0083] The targeted cells of the present invention have significant killing ability against tumor cells expressing CD71 on their surface or CD71-positive tumors, and can thus be used to treat or improve diseases related to CD71 expression, laying the foundation for the treatment of CD71-positive tumors.

[0084] In the present invention, "targeted cells" are immune cells that can perform immune effector functions. Preferably, the immune cells are selected from at least one of: immune cells differentiated by culturing 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 cells are T lymphocytes (sometimes referred to herein as T cells). Further preferably, the immune cells are modified T cells, i.e., CAR-T cells or TCR-T cells. In some embodiments, T cells can be CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, CD4- / CD8-, or a combination thereof. In some embodiments, T cells produce IL-2, IFN, and / or TNF when expressing a chimeric antigen receptor and binding to a target cell. In some embodiments, CD8+T cells lyse antigen-specific target cells when expressing a chimeric antigen receptor and binding to a target cell.

[0085] In the present invention, CD71 is expressed on the surface of the T cells, and the targeted nanoparticles of the present invention are capable of binding thereto, wherein the targeted nanoparticles comprise nanoparticles or multimeric structures formed by multiple ferritins. "Binding" and "immunoreactive with" or "targeting" are used interchangeably to refer to the non-covalent interaction between an immunoglobulin molecule and an antigen specific for the immunoglobulin.

[0086] The ferritin of the present invention may be natural ferritin, or may be recombinant full heavy chain ferritin or genetically engineered ferritin, without particular limitation. The specific sequence of ferritin is not particularly limited, as long as it has biological activity or binding properties targeting the ferritin receptor CD71 or has improved affinity for ferritin. Such other variants can be obtained using methods known in the art and are all included in 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 for producing variant polypeptides or proteins. For example, amino acid substitution or modification can be used to obtain ferritin with further improved affinity.

[0087] As used herein, the term "modified" refers to amino acid modifications that do not significantly affect or alter the binding characteristics 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 ferritin 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, among others.

[0088] In the present invention, multiple proteins are interconnected through a first binding portion to form the targeted nanoparticles, the first binding portion comprises an active group, and the first binding portions are covalently connected through a connecting arm. Examples of active groups include but are not limited to -COOH, -NH2 and -HS, and the connecting arm comprises an aldehyde group and / or a short chain small molecule of a polyethylene glycol derivative. In certain embodiments, the aldehyde-containing compound comprises glutaraldehyde. Preferably, the polyethylene glycol derivative is NHS-PEG. x -NHS, wherein the subscript x represents multiple repeating units of polyethylene glycol. Also preferably, x is an integer of 1-100.

[0089] In certain embodiments, a plurality of ferritins can form nanoparticles or polymer structures, and the ferritins have a cavity sufficient to accommodate an optional drug or small molecule chemical drug, and the particle size of the targeted nanoparticles formed is 50-1500 nm, preferably 100 nm or more and 1500 nm or less, further preferably 300-1500 nm, and further preferably 300-800 nm, such as 320, 340, 360, 380, 400, 420, 440, 460, 480, 500 nm, 600 nm, 700 nm, 800 nm or any numerical range therebetween. This particle size makes the uptake of nanoparticles by CAR-T cells weak, ensuring that the nanoparticles are modified on the surface of CAR-T cells. More importantly, this particle size range enables target cells to capture nanoparticles from CAR-T cells through their endocytosis or endocytosis. In addition, the targeted nanoparticles of the present invention can promote related interactions between cells. In an exemplary embodiment, the targeted nanoparticles can promote the interaction between the two cells by simultaneously binding to chimeric antigen receptor T cells and leukemia cells, thereby promoting the recognition and killing of leukemia cells by chimeric antigen receptor T cells.

[0090] In certain embodiments, the target cells include cancer or tumor cells from a cancer or tumor patient, a patient suspected of having cancer or tumor, or a cancer or tumor patient undergoing treatment, preferably a positive tumor expressing the ferritin receptor CD71 on the cell surface. Also preferably, the target cells are leukemia cells.

[0091] In the present invention, the targeted nanoparticle further includes a second binding portion, and binds to the outer surface of the target cell via the second binding portion. The target cell expresses the ferritin receptor CD71 on its outer surface, and the ferritin receptor CD71 is capable of binding to the protein via the second binding portion. Preferably, the second binding portion is different from the first binding portion.

[0092] Preparation method

[0093] The present invention further provides a method for preparing targeted cells, which comprises steps (1)-(3). Wherein, in step (1), the protein is precipitated under electrolyte conditions or non-electrolyte conditions to form protein clusters, and the electrolyte condition refers to the addition of an electrolyte solution to the ferritin solution. The electrolyte solution is not particularly limited, and in certain embodiments, a saturated ammonium sulfate solution is used. The non-electrolyte condition refers to the addition of a non-electrolyte solution to the ferritin solution, and the non-electrolyte solution is not particularly limited, and in certain embodiments, the non-electrolyte solution is ethanol. The purpose of step (1) is to precipitate ferritin to form ferritin clusters, and therefore, the electrolyte and non-electrolyte related reagents can be used as long as they can destroy the stability of the protein solution. It will be understood by those skilled in the art that, for example, ethanol and saturated ammonium sulfate solution can be used simultaneously or separately to form ferritin clusters.

[0094] In step (2), the clusters are cross-linked in the presence of a cross-linking agent to obtain cross-linked targeted nanoparticles. Preferably, the cross-linking agent includes glutaraldehyde and NHS-PEG. x -NHS. It will be appreciated that those skilled in the art may also use other cross-linking agents to enable cross-linking of multiple ferritins to form aggregates or clusters of a certain particle size. Such cross-linking agents also include disuccinimidyl suberate, paraformaldehyde, and polyethyleneimine. After step (2), a washing step is further included to remove excess reactants.

[0095] In step (3), the targeted nanoparticles and cells are brought into close proximity and contact with each other, so that the ferritin nanoclusters can be modified on the surface of the CAR-T by binding to CD71 to obtain the targeted cells. Preferably, the targeted nanoparticles and cells are brought into close proximity and contact with each other under in vitro conditions.

[0096] It will be appreciated by those skilled in the art that, in order to achieve combined administration or combined therapy, a step of allowing immunotherapy drugs and / or small molecule chemical drugs to enter the cavity of the protein is further included before step (1). Therefore, the additional drug loading step is also within the scope of protection of the present invention. The specific drug loading steps are not particularly limited, and different drug loading methods can be used depending on the type of drug. For example, the drug can be loaded before cluster formation, or loaded by dissociation self-assembly, or directly loaded through the pores of ferritin.

[0097] Methods for promoting cell proximity or binding

[0098] The present invention further provides a method for promoting cell proximity or binding, which includes the step of using targeted nano-scale particles to promote cells to approach and contact each other. Preferably, the targeted nano-scale particles and cells are brought into proximity and contact with each other under in vitro conditions. It should be noted that the cells here include a first cell and a second cell, wherein the first cell is selected from at least one of CAR T cells, TCR-T cells, and tumor-infiltrating lymphocytes. The second cell is selected from cancer or tumor cells. In certain embodiments, the targeted nano-scale particles bind to CD71 on the cell surface of at least one of CAR T cells, TCR-T cells, and tumor-infiltrating lymphocytes to form a conjugate, and the conjugate is formed by binding of the targeted nano-scale particles to CD71 on the surface of cancer or tumor cells. In this process, the inventors found that the expression of CD71 was significantly upregulated during the in vitro construction and amplification of CAR-T (see Figure 18). By co-incubating and binding the targeted nano-scale particles with the CAR T, the intercellular interaction force was greatly enhanced, which promoted the recognition of cancer or tumor antigen targets by CAR-T cells, and then promoted the activation of CAR-T cells. Therefore, the modified CAR-T cells of the present invention can effectively solve the problem that the downregulation of target antigen expression seriously affects the recognition and killing of leukemia cells by CAR T cells, and thus the modified CAR-T cells of the present invention can significantly promote the recognition and killing of low-target leukemia cells by CAR T.

[0099] Pharmaceutical composition

[0100] The present invention also provides a pharmaceutical composition comprising the targeted cells described herein.

[0101] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0102] Pharmaceutical compositions can be prepared by mixing an active agent of the desired purity with an optional pharmaceutically acceptable carrier in the form of a lyophilized formulation or an aqueous solution. The pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration used and may include at least one of a buffer, an antioxidant, a preservative, an isotonic agent, a stabilizer, and a surfactant. In addition, in order for the pharmaceutical compositions to be useful for in vivo administration, they must be sterile. Pharmaceutical compositions can be sterilized by filtration through a sterile filtration membrane.

[0103] In some embodiments, the pharmaceutical composition may contain at least one additive selected from the group consisting of a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, a growth inhibitor, and an active pharmaceutical ingredient required for the specific indication to be treated. The specific amount of the additive may be adjusted according to actual needs.

[0104] use

[0105] The present invention also provides use of an agent in preparing a drug for treating or ameliorating cancer, wherein the agent comprises the targeted cell or pharmaceutical composition described herein.

[0106] Preferably, the treatment or improvement of cancer or tumor refers to the ability to stimulate or enhance the immune function of cancer or tumor patients.

[0107] Preferably, the cancer or tumor is a cancer associated with CD71 expression.

[0108] In the present invention, "cancer or tumor associated with CD71 expression" refers to a disease directly or indirectly caused by the expression of CD71 on cells, preferably a cancer or tumor with high or overexpression of CD71. Preferably, the cancer or tumor includes a hematological cancer or tumor, a solid tumor, or a combination thereof.

[0109] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelo-monocytic, monocytic, and erythroleukemias), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0110] Solid tumors are abnormal masses of tissue that do not typically contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcomas, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, and ovarian cancer.

[0111] The present invention therefore also provides a method for treating / preventing cancer, comprising administering a therapeutically effective amount of a targeted cell or pharmaceutical composition to a subject in need thereof. As used herein, the terms "subject" and "patient" are used interchangeably herein to refer to any animal that may be in need of treatment with the targeted cell or pharmaceutical composition described herein. Subjects and patients therefore include, but are not limited to, primates (including humans), canines, felines, mice, and other mammalian subjects. Preferably, the subject is human.

[0112] In the present invention, the term "treatment" refers to therapeutic treatment and preventive or prophylactic measures, the purpose of which is to prevent or slow down (reduce) the progression of undesirable physiological changes or disorders, such as immune diseases of the blood system. Beneficial or desired clinical results include, but are not limited to, the following results, whether detectable or undetectable, including relief of symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete). "Treatment" also refers to a prolonged survival period compared to the expected survival period when not receiving treatment. Those in need of treatment include those who already have a disease or disorder, as well as those who are prone to a disease or disorder, or those who need to prevent the disease or disorder.

[0113] As used herein, the term "effective amount" refers to the amount of a drug or pharmaceutical agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human being, such as that sought by a researcher or clinician. In addition, the term "therapeutically effective amount" refers to an amount that causes improved treatment, cure, prevention, or alleviation of a disease, condition, or side effect, or an amount that reduces the rate of progression of a disease or condition, compared to a corresponding subject that has not received the amount. The term also includes within its scope an amount that effectively enhances normal physiological function. Generally, the effective amount herein varies according to various factors, such as a given drug or compound, pharmaceutical formulation, route of administration, type of disease or condition, subject being treated, and the like, but can still be routinely determined by those skilled in the art. The effective amount of the targeted cell or pharmaceutical composition of the present invention can be easily determined by those skilled in the art by conventional methods known in the art.

[0114] The administration of the targeted cells or pharmaceutical compositions of the present invention can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The targeted cells or pharmaceutical compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous (iv) injection or intraperitoneally. In certain embodiments, the T cell compositions of the present invention are administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention are preferably administered by iv injection. The compositions comprising the T cells can be injected directly into the tumor, lymph node or infection site.

[0115] Combination therapy

[0116] The present invention also provides the use of the targeted cells or pharmaceutical compositions described herein 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 drugs and / or small molecule chemical drugs, examples of which include, but are not limited to, chemotherapeutic drugs, radioisotopes, cytokines, nucleic acids, and anti-tumor or anti-inflammatory drugs. These drugs can be loaded into the ferritin internal cavity of the targeted nanoparticles of the present invention, or administered before, after, or simultaneously with the administration of the targeted cells with or without the drug-loaded targeted nanoparticles.

[0117] Example 1

[0118] 1. Materials

[0119] Full heavy chain recombinant ferritin (expressed by engineered bacteria), PBS buffer, deionized water, anhydrous ethanol, ammonium sulfate, glutaraldehyde, NHS-PEG X -NHS (Sigma), poly-lysine (Sigma), Cell-Tak adhesive (Corning), CFSE dye (Sigma), flow cytometry intracellular staining kit (Becton, Dickinson and Company), human CD3 flow cytometry antibody (Biolegend), human IFNγ flow cytometry antibody (Biolegend), human granzyme B flow cytometry antibody (Biolegend), human CD19 flow cytometry antibody (Biolegend), red blood cell lysate (Solarbio), LDH release detection kit (Solarbio).

[0120] 2. Methods

[0121] The preparation of the targeted cells of the present invention comprises the following steps:

[0122] (1) Proteins are aggregated to form protein clusters under electrolyte or non-electrolyte conditions;

[0123] Non-electrolyte conditions: Dissolve ferritin in PBS buffer (pH 7.2-7.4) to a final concentration of 10 mg / mL. Add anhydrous ethanol dropwise to the solution at room temperature with magnetic stirring at 1000 rpm until the solution becomes visibly turbid.

[0124] Electrolyte Conditions: Dissolve ferritin in PBS buffer (pH 7.2-7.4) to a final concentration of 10 mg / mL. Add saturated ammonium sulfate solution dropwise to the solution at room temperature with magnetic stirring at 1000 rpm until the solution becomes visibly turbid.

[0125] (2) The clusters are cross-linked in the presence of a cross-linking agent to obtain cross-linked targeted nanoparticles. The cross-linking in this step is to directly add 25% glutaraldehyde solution to the turbid solution in step (1) to a final concentration of 0.25%-1%; or add NHS-PEG-NHS to a final concentration of about 1%. Then react for 1.5 hours at room temperature and 1000 rpm. After the reaction is completed, PBS buffer is added to the system at room temperature and 1000 rpm to dilute the system to twice the original volume. Finally, the system is centrifuged at 10000g for 10 minutes, the supernatant is discarded, and the precipitate is washed 3 times with PBS buffer to obtain a ferritin cluster nanoparticle form. The cluster is stored in PBS buffer.

[0126] (3) The targeted nanoparticles and cells are brought into contact with each other to obtain the targeted cells: CAR T cells and ferritin cluster nanoparticles are mixed at a rate of 0.5 mg ferritin clusters / 10 6 Incubate with CAR T cells for 1 hour, then centrifuge to remove excess ferritin clusters. The protein mass is used as the standard for 0.5 mg of ferritin clusters.

[0127] 3. Force detection

[0128] Atomic force microscopy was used to detect the interaction forces between CAR T and FnC-CAR T and leukemia cells.

[0129] First, a 6cm diameter culture dish was coated with poly-lysine, and then leukemia cells were cultured in the dish. Poly-lysine was used to adsorb the leukemia cells to the bottom of the dish. Next, Cell-Tak adhesive was used to adhere the CAR T or FnC-CAR T to the probe of an atomic force microscope. The atomic force microscope was then used to measure the interaction force between the CAR T or FnC-CAR T on the probe and the leukemia cells.

[0130] 4. Proliferation detection after incubation of CAR T and FnC-CAR T with leukemia cells

[0131] The CFSE method was used to detect the proliferation of CAR T cells after co-incubation with leukemia cells.

[0132] CAR T was stained with CFSE dye, and then some of the stained CAR T were co-incubated with FnC to construct CFSE-stained FnC-CAR T. CFSE-stained CAR T and FnC-CAR T were then co-incubated with leukemia cells, respectively. Finally, flow cytometry was used to detect the CFSE fluorescence signal of CAR T or FnC-CAR T at 0 h, 24 h, and 72 h after co-incubation to detect the proliferation status of the two (where CAR T or FnC-CAR T was labeled with a human CD3 antibody).

[0133] 5. CAR T and FnC-CAR T co-incubated with leukemia cells for activation

[0134] Flow cytometry was used to detect the activation of CAR T and FnC-CAR T by co-incubation with leukemia cells.

[0135] According to the intracellular staining protocol provided by Becton, Dickinson and Company, IFNγ and granzyme B antibodies were used to detect the positive ratio of IFNγ to granzyme B after CAR T and FnC-CAR T were co-incubated with leukemia cells to characterize the degree of activation (where CAR T or FnC-CAR T was labeled with human CD3 antibody).

[0136] 6. Killing effect of CAR T and FnC-CAR T incubated with leukemia cells at different ratios

[0137] The killing of leukemia cells by CAR T was characterized by detecting the release of LDH in the supernatant.

[0138] CAR T or FnC-CAR T cells were co-cultured with leukemia cells at varying ratios and then cultured in 96-well plates. After 24 hours, the cell mixture was removed and centrifuged. The supernatant was collected and LDH release was measured using an LDH release assay kit (Solarbio). The leukemia cell lysis rate was determined by comparison with the positive control.

[0139] 7. Changes in peripheral blood leukemia burden over time

[0140] After CAR T, FnC-CAR T, and high-dose CAR T were reinfused into leukemia model mice, the changes in peripheral blood leukemia burden over time were detected.

[0141] First, leukemia modeling: leukemia cells isolated from the bone marrow of clinical leukemia patients were cultured at a rate of 5×10 6The cells were intravenously infused into severely immunodeficient mice (NTG mice, Spefox) at a dose of 100 cells / mouse. Approximately 7 days after the infusion, orbital blood samples were collected and flow cytometry was used to measure the proportion of human leukemia cells in the white blood cells of the blood samples. A leukemia model was considered successful when the proportion was no less than 1%.

[0142] Treatment plan: After the leukemia model was successfully established, the model mice were randomly divided into groups (no less than 8 mice in each group), and PBS buffer (200 μL / mouse), CAR T (1×10 6 / ), FnC-CAR T (1×10 6 / ), high-dose CAR T (5×10 6 The drug was then intravenously infused back into the model mice. Every five days after infusion, the proportion of human leukemia cells in the mice's peripheral blood leukocytes was measured to assess changes in the leukemia burden in the model mice after treatment.

[0143] 2. Experimental results

[0144] 2.1 Ferritin and ferritin clusters

[0145] Figure 1 shows an overall schematic diagram of the modified CAR-T cells of the present invention for treating leukemia. The prepared ferritin and ferritin clusters are shown in transmission electron microscopy in Figure 2. Ferritin clusters were successfully constructed according to the method of the present invention, with a particle size of approximately 300-500 nm.

[0146] 2.2 Force detection

[0147] The interaction force between CAR T and leukemia cells was measured by atomic force microscopy. As shown in Figure 3, the interaction force between CAR T and leukemia cells increased significantly after FnC modification (from about 2nN to about 43nN).

[0148] 2.3 Detection of proliferation of CAR T cells after co-incubation with leukemia cells using CFSE assay

[0149] As shown in Figure 4, after FnC modification, the proliferation of CAR T cells increased significantly after co-incubation with leukemia cells.

[0150] 2.4 Flow cytometry detection of activation of CAR T and FnC-CAR T by co-incubation with leukemia cells

[0151] Figure 5 shows the flow cytometry analysis of the positive ratios of granzyme B and interferon gamma (IFNγ) after co-incubation of CAR T cells with leukemia cells, which characterizes the activation of CAR T. As shown in Figure 5, after FnC modification, the activation ratio of CAR T cells after co-incubation with leukemia cells increased significantly.

[0152] 2.5 Killing effect of CAR T and FnC-CAR T incubated with leukemia cells at different ratios

[0153] Figure 6 shows the leukemia cell killing ability of CAR T cells as measured by supernatant LDH release, with the ordinate representing the killing ratio. As shown in Figure 6, the FnC-modified CAR T cells showed a significant increase in their ability to kill leukemia cells, still achieving a killing rate exceeding 80% at a 1:4 ratio.

[0154] 2.6 Changes in peripheral blood leukemia burden over time

[0155] After CAR T cells, FnC-CAR T cells, and high-dose CAR T cells were infused back into leukemia model mice, the leukemia burden in peripheral blood was measured over time. As shown in Figure 7, peripheral blood samples were collected from model mice at different time points, and the proportion of leukemia cells in the peripheral blood was measured to indicate the therapeutic effect. As shown in Figure 7, the FnC-modified CAR T cells were able to completely inhibit the progression of leukemia, achieving a therapeutic effect nearly equivalent to that of high-dose CAR T cells.

[0156] Example 2

[0157] 1. Interaction between CAR T and FnC-CAR T and CD19-low-expressing leukemia cells

[0158] The present invention further detected the interaction force between CAR T and CD19 low-expressing leukemia cells. The interaction force between CAR T and FnC-CAR T and CD19 low-expressing leukemia cells detected by atomic force microscopy is shown in Figure 8.

[0159] 2. Proliferation detection after incubation of CAR T and FnC-CAR T with CD19 low-expressing leukemia cells

[0160] Similar to the aforementioned CFSE method, the present invention detected the proliferation of CAR T cells after co-incubation with CD19 low-expressing leukemia cells. The results are shown in Figure 9, demonstrating that FnC modification promoted the interaction between CAR T cells and leukemia cells and thus promoted proliferation.

[0161] 3. CAR T and FnC-CAR T were co-incubated with CD19 low-expressing leukemia cells and activated by flow cytometry

[0162] Similar to the aforementioned activation flow cytometry graph, the present invention characterized the activation of CAR T cells after co-incubation with CD19 low-expressing leukemia cells, and the results are shown in FIG10 .

[0163] 4. Killing detection after CAR T and FnC-CAR T were co-incubated with CD19 low-expressing leukemia cells at different ratios

[0164] Similar to the aforementioned LDH release killing detection graph, the present invention characterized the killing of leukemia cells with low CD19 expression by CAR T, as shown in Figure 11. The results demonstrated that FnC modification enhanced the killing of leukemia cells with low CD19 expression by CAR T.

[0165] 5. Changes of peripheral blood leukemia burden over time after CAR T, FnC-CAR T, and high-dose CAR T were infused into CD19 low-expression leukemia model mice

[0166] Similar to the aforementioned in vivo therapeutic effect graph, the present invention tested the changes in peripheral blood leukemia burden over time after reinfusion of CAR T, FnC-CAR T, and high-dose CAR T into an animal model with low CD19 expression. The results are shown in Figure 12.

[0167] Example 3

[0168] Cell Labeling: In this experiment, CAR T cells were labeled with the red fluorescent Celltracker dye, leukemia cells were labeled with CFSE, and ferritin cluster nanoformulations were labeled with Cy5-se dye. The excitation wavelengths for these three markers were 561 nm, 488 nm, and 633 nm, respectively.

[0169] Specific experimental steps: According to the aforementioned method, the fluorescently labeled CAR T cells and the fluorescently labeled ferritin cluster nanoformulation were prepared into FnC-CAR T. FnC-CAR T was then mixed with the fluorescently labeled leukemia cells in a 96-well plate, and the capture of FnC on the surface of FnC-CAR T by leukemia cells was observed in real time using a high-content imaging analysis system (Operetta CLS).

[0170] The present invention used a high-content imaging analysis system to observe the seizure of FnC from the CAR T cell surface by leukemia cells when FnC-CAR T cells were co-incubated with leukemia cells. The results are shown in Figure 13. High-content dynamic fluorescence imaging was used to observe the seizure of FnC from the CAR T cell surface by leukemia cells. As can be seen from the figure, leukemia cells were able to seizure FnC from the CAR T cell surface, while CAR T cells did not take up FnC.

[0171] Example 4

[0172] The present invention detects the changes in peripheral blood leukemia load over time after CAR T, FnC-CAR T, and FnC-CAR T loaded with arsenic trioxide were reinfused into high-load CD19 low-expression leukemia model mice.

[0173] The main steps of this experiment are as follows.

[0174] Construction of arsenic-loaded FnC-CAR T: First, the chemotherapy drug arsenic trioxide was loaded into ferritin to construct Fn As , and then follow the above method to set Fn As Composition Fn As C. Finally, according to the above method, Fn As C and CAR T construct into Fn As C-CAR T.

[0175] In vivo evaluation of therapeutic efficacy: CD19 low-expressing leukemia models were constructed using CD19 low-expressing leukemia cells according to the aforementioned method. Treatment was initiated when the proportion of human leukemia cells in the peripheral blood leukocytes of mice was no less than 10%. PBS (200 μL / mouse), CAR T (1×10 6 / ), FnC-CAR T (1×10 6 / pcs)、Fn As C-CAR T (1×10 6 The drug was then intravenously infused into the mice for treatment. Finally, the proportion of human leukemia cells in the mice's peripheral blood leukocytes was monitored according to the aforementioned method to detect changes in the leukemia burden in the mice.

[0176] The results are shown in FIG14 , which are the same as the in vivo therapeutic effect diagram of the aforementioned CD19 low expression leukemia model, but the starting point of treatment is 10% of the peripheral blood leukemia load (the starting point of the aforementioned two models is 1%).

[0177] Example 5

[0178] The T cells used in this experiment are clinical melanoma infiltrating lymphocytes (TIL). The specific steps are as follows:

[0179] Preparation of FnC-TIL: In this experiment, TILs were isolated from clinical melanoma patient tumor samples. After centrifugation and washing, human IL-2 and CD3 & CD28 stimulating magnetic beads were used to activate and expand TILs in vitro. The resulting TILs were prepared using FnC and TILs according to the previously described FnC-CAR T preparation method.

[0180] Detection of the interaction force between TIL and tumor cells: This experiment is similar to the previous one. The melanoma cell suspension was cultured in a 6 cm culture dish coated with poly-lysine. Then, Cell-Tak adhesive was used to adhere TIL or FnC-TIL to the atomic force microscope probe, and the interaction force between the two was detected using atomic force microscopy.

[0181] In vitro TIL activation validation: TILs and FnC-TILs were mixed with the aforementioned melanoma sample cell suspension and cultured in 96-well plates. After 72 hours, the IFNγ and granzyme B positivity ratios of TILs and FnC-TILs were measured using flow cytometry as previously described to characterize their activation.

[0182] The present invention further prepared FnC-TIL based on tumor-infiltrating lymphocytes and studied the interaction between them and tumor cells and the activation of immune cells. The results are shown in Figure 1 5, where the left figure shows the statistics of the interaction between tumor-infiltrating lymphocytes and FnC-TIL and tumor cells using atomic force microscope. The right figure shows the double-positive ratio of granzyme B (GrB) and interferon gamma (IFNγ) after co-incubation of TIL and FnC-TIL with tumor cells, which indicates the activation of TIL. The TIL in this figure targets melanoma as a solid tumor.

[0183] Example 6

[0184] The present invention further prepared FnC-TCR T based on TCR T cells and studied its interaction with tumor cells and the activation of immune cells.

[0185] The T cells used in this experiment are engineered TCR T cells. The specific experiment is as follows.

[0186] Construction of FnC-TCR T: FnC and TCR T were constructed into FnC-TCR T according to the aforementioned FnC-CAR T construction method.

[0187] Interaction forces between TCR T cells and tumor cells: Human melanoma cell lines were cultured in 6 cm culture dishes. After 24 hours, TCR T cells or FnC-TCR T cells were attached to an atomic force microscope probe using Cell-Tak adhesive. The interaction forces between TCR T cells or FnC-TCR T cells and melanoma cells were then measured using atomic force microscopy.

[0188] In vitro activation validation: TCR T cells or FnC-TCR T cells were co-cultured with melanoma cells in 96-well plates. After 72 hours, the IFNγ and granzyme B positive rates of TCR T cells and FnC-TCR T cells were measured by flow cytometry as described above to characterize their activation.

[0189] The results are shown in Figure 16 . The left figure shows the atomic force analysis of the interaction forces between TCR T cells and FnC-TCR T cells and tumor cells. The right figure shows the proportion of granzyme B (GrB) and interferon gamma (IFNγ) double positive cells after co-incubation of TCR T cells and FnC-TCR T cells with tumor cells, which indicates TCR T activation. This figure demonstrates that the FnC-enhanced strategy is also effective for TCR T cells.

[0190] Example 7

[0191] The present invention further prepares a CAR T-based EGFR FnC-CAR T cells EGFR CAR T and studied its interaction with tumor cells and the activation of immune cells.

[0192] The T cells used in this experiment were human EGFR-specific CAR T ( EGFR CAR T). The specific experimental steps are as follows.

[0193] FnC- EGFR Preparation of CAR T: According to the above-mentioned FnC-CAR T construction method, FnC and EGFR CAR T is constructed into FnC- EGFR CAR T.

[0194] EGFR Detection of the interaction between CAR T and tumor cells: Human liver cancer cell lines were cultured in 6 cm culture dishes. After 24 hours, Cell-Tak adhesive was used to EGFR CAR T or FnC- EGFR CAR T cells are attached to an atomic force microscope probe and then examined using an atomic force microscope. EGFR CAR T or FnC- EGFR The interaction between CAR T and liver cancer cells.

[0195] In vitro activation verification: EGFR CAR T or FnC- EGFR CAR T cells were co-cultured with human hepatocellular carcinoma cells in 96-well plates. After 72 hours, flow cytometry was used to detect the cells. EGFR CAR T and FnC- EGFR The IFNγ and granzyme B positive ratios of CAR T cells were used to characterize their activation status.

[0196] The results are shown in Figure 17, where the left figure shows the atomic force test of EGFR CAR T ( EGFR CAR T) and FnC- EGFR Statistics of the interaction force between CAR T and tumor cells. EGFR CAR T and FnC- EGFR The double positive ratio of granzyme B (GrB) and interferon gamma (IFNγ) after CAR T cells were co-incubated with tumor cells was used to characterize the EGFR Activation of CAR T. This figure demonstrates that the enhancement effect of the FnC strategy is also effective for other types of CAR T.

[0197] Example 8

[0198] This example shows the statistical results of different CAR T cells killing bone marrow samples from different clinical leukemia patients. The experimental steps are as follows:

[0199] FnC-CAR T and Fn As Construction of C-CAR T: The CAR T cells used in this experiment are CD7-CAR T, CD33-CAR T and CD19-CAR T, which target T-ALL, AML and B-ALL respectively. Then, the corresponding FnC-CAR T and Fn As C-CAR T.

[0200] Killing detection: CAR T, FnC-CAR T and Fn As C-CAR T cells were co-mixed with bone marrow samples from leukemia patients at a 1:1 ratio and then cultured in 96-well plates. After 24 hours, the cell mixture was removed and centrifuged. The supernatant was collected and assayed for LDH release using an LDH release assay kit (Solarbio). Comparison with the positive control group indicated the lysis rate of leukemia cells.

[0201] The results are shown in Figure 19. In Figure 19, the CAR T cells targeting bone marrow samples from T-ALL patients are CD7-CAR T cells; the CAR T cells targeting bone marrow samples from AML patients are CD33-CAR T cells; and the CAR T cells targeting bone marrow samples from B-ALL patients are CD19-CAR T cells. The results show that the targeted cells of the present invention can target cancer cells from patients with different types of leukemia and effectively kill cancer cells from patients with different types of leukemia.

[0202] Example 9

[0203] This example shows the results of the time-dependent changes in peripheral blood leukemia burden after CAR T, FnC-CAR T, and high-dose CAR T were infused 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 cultured at a rate of 5×10 6 The cells were intravenously infused into severely immunodeficient mice (NTG mice, Spefox) at a dose of 100 cells / mouse. Approximately 7 days after the infusion, orbital blood was sampled and flow cytometry was used to measure the proportion of human leukemia cells in the white blood cells of the blood samples. A T-ALL PDX model was considered successful when the proportion was no less than 1%.

[0204] Treatment plan: After the T-ALL PDX model was successfully established, the model mice were randomly divided into groups (no less than 8 mice in each group), and PBS buffer (200 μL / mouse), CAR T (1×10 6 / ), FnC-CAR T (1×10 6 / ), high-dose CAR T (5×10 6 The drug was then intravenously infused back into the model mice. Every five days after infusion, the proportion of human leukemia cells in the mice's peripheral blood leukocytes was measured to assess changes in the leukemia burden in the model mice after treatment.

[0205] 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.

[0206] Example 10

[0207] This example shows the results of the time-dependent changes in peripheral blood leukemia burden after CAR T, FnC-CAR T, and high-dose CAR T were infused into T-ALL leukemia PDX model mice with low CD7 expression. Low T-ALL PDX model: Leukemia cells isolated from the bone marrow of clinical T-ALL leukemia patients were constructed by downregulating CD7 expression using CD7 knockdown virus. Low ) leukemia cells. Then press 5×10 6 The CD7 low-expressing T-ALL cells were intravenously infused into severely immunodeficient mice (NTG mice, Spav). About 7 days after the infusion, blood samples were collected from the mouse orbits and the proportion of human leukemia cells in the white blood cells of the mouse blood samples was detected by flow cytometry. When the proportion was not less than 1%, it was considered CD7 Low T-ALL PDX model was successfully established.

[0208] Treatment plan: CD7 Low After the T-ALL PDX model was successfully established, the model mice were randomly divided into groups (no less than 8 mice in each group), and PBS buffer (200 μL / mouse), CAR T (1×10 6 / ), FnC-CAR T (1×10 6 / ), high-dose CAR T (5×10 6 The drug was then intravenously infused back into the model mice. Every five days after infusion, the proportion of human leukemia cells in the mice's peripheral blood leukocytes was measured to assess changes in the leukemia burden in the model mice after treatment.

[0209] 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.

[0210] Example 11

[0211] This example shows the results of the detection of the changes in peripheral blood leukemia load over time after FnC-CAR T and FnAsC-CAR T were infused into T-ALL leukemia PDX model mice with high load and low expression of CD7. Low T-ALL PDX model: CD7 Low T-ALL cells were collected at 5×10 6 The cells were intravenously infused into severely immunodeficient mice (NTG mice, Spefox). Orbital blood samples were collected at different time points after the infusion, and the proportion of human leukemia cells in the white blood cells of the mouse blood samples was detected by flow cytometry. When the proportion was not less than 10%, it was considered to be a high load of CD7. Low T-ALL PDX model was successfully established.

[0212] Treatment plan: high load CD7 Low After the T-ALL PDX model was successfully established, the model mice were randomly divided into groups (no less than 8 mice in each group), and PBS buffer (200 μL / mouse), FnC-CAR T (1×10 6 / pcs)、Fn As C-CAR T (1×10 6 The drug was then intravenously infused back into the model mice. Every five days after infusion, the proportion of human leukemia cells in the mice's peripheral blood leukocytes was measured to assess changes in the leukemia burden in the model mice after treatment.

[0213] 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-load, low-CD7-expressing T-ALL leukemia PDX model mice.

[0214] Although 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 modifications and variations may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims is to be given the broadest interpretation so as to encompass all such modifications and equivalent structures and functions.

Claims

1. A T cell binder, characterized in that It comprises T cells and ferritin clusters, wherein the T cells bind to the ferritin clusters, which are composed of ferritin.

2. The T cell binding substance according to claim 1, wherein The T cells are activated T cells; T cells expanded in vitro are preferred.

3. The T cell binding substance according to claim 1, wherein The ferritin clusters bind to CD71 of the T cells through receptor-ligand interactions.

4. The T cell binding substance according to claim 1, wherein The average particle size of the ferritin clusters is 50-1500 nanometers, preferably 300-1500 nanometers, further preferably 400-1000 nanometers, and more preferably 500-800 nanometers.

5. The T cell binding substance according to claim 1, wherein The ferritin clusters are formed by aggregation and cross-linking of human ferritin; Preferably, heavy chain subunits account for at least 30% of the surface-exposed ferritin subunits of the ferritin clusters.

6. The T cell binding substance according to claim 1, wherein The T cell binder enhances targeting to target cells expressing CD71 through the ferritin clusters.

7. The T cell binding substance according to claim 6, wherein The target cells include tumor cells; Preferably, the target cells include solid tumor cells and / or blood 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.

8. A pharmaceutical composition, characterized in that The invention comprises the T cell binder according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

9. A method for treating or ameliorating cancer or tumor associated with expression of CD71, characterized in that: The method comprises the steps of administering the T cell binder according to any one of claims 1 to 7 to a subject in need thereof; 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.

10. A targeted nanoparticle, characterized in that: The targeted nano-scale particle is composed of a plurality of proteins interconnected by a first binding portion. The targeted nano-scale particle also includes a second binding portion, and is bound to the outer surface of the target cell through the second binding portion.

11. The targeted nanoparticle according to claim 10, characterized in that The targeted nanoparticles are capable of binding to the outer surface of targeted cells.

12. The targeted nanoparticle according to claim 11, characterized in that The first binding parts include active groups, and the first binding parts are connected by a connecting arm.

13. The targeted nanoparticle according to claim 12, characterized in that The outer surface of the target cell contains a receptor for the protein, and the receptor can bind to the protein through the second binding portion.

14. The targeted nanoparticle according to claim 11, characterized in that 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, natural lymphoid cells and / or leukocytes.

15. The targeted nanoparticle according to claim 14, characterized in that The T cells include modified and unmodified T cells, wherein the modified T cells include CAR T cells and TCR T cells, and the unmodified T cells include tumor-infiltrating T cells, PBMC-derived T cells or other T cells isolated from the body.

16. The targeted nanoparticle according to claim 10, characterized in that The targeted nano-scale particles are larger than 50 nm and smaller than 5000 nm.

17. The targeted nanoparticle according to claim 10, characterized in that The protein comprises an internal cavity.

18. The targeted nanoparticle according to claim 17, characterized in that The interior cavity may or may not contain a drug.

19. The targeted nanoparticle according to any one of claims 10 to 18, characterized in that The protein is selected from at least one of natural ferritin, recombinant full heavy chain ferritin and genetically engineered ferritin.

20. The method for preparing targeted nanoparticles according to any one of claim 19, characterized in that: include: (1) Proteins are aggregated to form protein clusters under electrolyte or non-electrolyte conditions; and (2) cross-linking the protein clusters in the presence of a cross-linking agent to obtain cross-linked targeted nanoparticles.

21. The method for preparing targeted nanoparticles according to claim 20, characterized in that: Optionally, before step (1), a step is performed to allow immunotherapy drugs and / or small molecule chemical drugs to enter the cavity of the protein.

22. A targeted cell, characterized in that The targeted nanoparticles according to any one of claims 10 to 19 are bound to the outer surface of the targeted cells.

23. The method for preparing targeted cells according to claim 22, characterized in that: include: 1) The protein is aggregated to form protein clusters under electrolyte or non-electrolyte conditions; 2) cross-linking the protein clusters in the presence of a cross-linking agent to obtain cross-linked targeted nanoparticles; and 3) bringing the targeted nanoparticles and cells into close proximity and contact with each other to obtain the targeted cells.

24. The preparation method according to claim 23, characterized in that The step of allowing immunotherapy drugs and / or small molecule chemical drugs to enter the cavity of the protein before step 1).

25. A method for promoting the proximity of cells, characterized in that: The method comprises the steps of using the T cell binder according to any one of claims 1 to 7 or the targeted nanoscale particle according to any one of claims 10 to 19 to promote cells to approach and contact each other, wherein a plurality of proteins are interconnected through a first binding portion to form the targeted nanoscale particle, and the targeted nanoscale particle further comprises a second binding portion, and is bound to the outer surface of the cell through the second binding portion.

26. The method according to claim 25, characterized in that The surface of the cell has a receptor that binds to the targeted nano-scale particle or is bound to the targeted nano-scale particle; Preferably, the cell is selected from at least one of a CAR T cell, a TCR T cell, a tumor infiltrating lymphocyte, a cancer cell or a tumor cell.

27. A pharmaceutical composition, characterized in that Comprising the targeted nanoparticles according to any one of claims 10 to 19, or the targeted cells according to claim 22.

28. Use of the T cell binder according to any one of claims 1 to 7, or the targeted nanoparticle according to any one of claims 10 to 19, or the targeted cell according to claim 22 in the preparation of a medicament for treating or ameliorating cancer or tumor.

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