A modified chimeric antigen receptor T cell and its preparation method and use

Abstract

The present invention discloses a modified chimeric antigen receptor T cell, its preparation method, and its use. The present invention prepares cells, such as chimeric antigen receptor T cells, modified with a hydrophobic two-dimensional sheet material. Compared to simple chimeric antigen receptor T cells, these cells can bind to more leukemia cells, enhancing their recognition and killing abilities. Furthermore, the nanomaterial's high specific surface area provides space for drug loading, enabling the combination of CAR T cells with other therapies.

Description

Technical Field

[0001] The present invention relates to the field of immunotherapy, and in particular to a modified cell such as a chimeric antigen receptor T cell, and a preparation method and use thereof. Background Art

[0002] Chimeric Antigen Receptor T Cell (CAR T) therapy is an immunotherapy technology that genetically modifies T cells to achieve targeted tumor killing. Currently, CAR T therapy has achieved remarkable results in the treatment of tumors or cancers, especially leukemia, with many leukemia patients fully recovering with its help.

[0003] However, CAR T therapy still faces significant challenges. For example, clinical trials of B-lineage lymphoblastic leukemia have shown that over 30% of patients experience reduced expression of the target antigen CD19 after treatment, significantly limiting the efficacy of CAR T. Therefore, promoting CAR T's ability to recognize and kill low-target leukemia cells is key to enhancing CAR T's efficacy.

[0004] 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. Summary of the Invention

[0005] To address at least some of the technical issues in the prior art, the present invention designs cells modified with hydrophobic two-dimensional sheet materials, such as CAR T cells. In certain embodiments, compared to CAR T cells alone, these cells can bind to more leukemia cells, enhancing CAR T cell recognition and killing. Furthermore, the nanomaterial's high surface area provides space for drug loading, enabling combined CAR T therapy with other therapies. Specifically, the present invention encompasses the following.

[0006] In a first aspect, the present invention provides a modified cell comprising a cell and a hydrophobic two-dimensional sheet material located in the cell, wherein the particle size of the sheet material is 10-2000 nm.

[0007] In certain embodiments, according to the modified cell of the present invention, the hydrophobic two-dimensional sheet material comprises a nano-inorganic material, and the nano-inorganic material is selected from at least one of the group consisting of carbon elemental materials and their oxides, carbon nitride and molybdenum disulfide.

[0008] In certain embodiments, according to the modified cell of the present invention, the nano-inorganic material has a particle size of 50-900 nm.

[0009] In certain embodiments, according to the modified cell of the present invention, the nanoinorganic material has an oxidation degree of 10%-50%.

[0010] In certain embodiments, according to the modified cells of the present invention, the cells are immune cells, such as T cells, DC cells, etc., and at least a portion of the nanoinorganic material is located in the cell membrane or in the cell membrane of the cell, such as a chimeric antigen receptor T cell.

[0011] A second aspect of the present invention provides a method for preparing a modified cell, comprising the following steps:

[0012] (1) Dispersing the hydrophobic two-dimensional sheet material in water and then crushing it;

[0013] (2) isolating nanomaterials with a fixed particle size distribution, incubating them with cells, and then removing free hydrophobic two-dimensional sheet materials, wherein the particle size of the hydrophobic two-dimensional sheet material is 10-2000 nm.

[0014] In certain embodiments, according to the method for preparing modified cells of the present invention, the hydrophobic two-dimensional sheet material comprises a nano-inorganic material, and the nano-inorganic material is selected from at least one of the group consisting of carbon elemental materials and their oxides, carbon nitride and molybdenum disulfide.

[0015] In certain embodiments, according to the method for preparing modified cells of the present invention, the nano-inorganic material has a particle size of 50-900 nm.

[0016] In certain embodiments, according to the method for preparing modified cells of the present invention, the nano-inorganic material has an oxidation degree of 10%-50%.

[0017] In certain embodiments, according to the method for preparing modified cells of the present invention, in step (1), the concentration of the hydrophobic two-dimensional sheet material is 0.01-10 g / mL.

[0018] In certain embodiments, according to the method for preparing modified cells of the present invention, the mixing ratio of the hydrophobic two-dimensional sheet material and the cells is (1-20) μg: (1×10 6 -2×10 6 ) cells.

[0019] The third aspect of the present invention provides a method for increasing, promoting or enhancing intercellular forces in vitro, comprising the steps of bringing a first cell and a second cell into proximity and contact with each other, wherein the first cell is the modified cell described in the first aspect, such as a chimeric antigen receptor T cell, and the second cell is an abnormally proliferating or multiplying cell.

[0020] The fourth aspect of the present invention provides a method for enhancing cell membrane fluidity in vitro, which includes the step of contacting a hydrophobic two-dimensional sheet material with cells such as chimeric antigen receptor T cells, wherein the particle size of the hydrophobic two-dimensional sheet material is 10-2000 nm.

[0021] In certain embodiments, according to the method of the present invention, the hydrophobic two-dimensional sheet material comprises a nano-inorganic material, and the nano-inorganic material is selected from at least one of the group consisting of carbon elemental materials and their oxides, carbon nitride and molybdenum disulfide.

[0022] In certain embodiments, according to the method of the present invention, the nanomaterial has a particle size of 50-900 nm.

[0023] In certain embodiments, according to the method of the present invention, the nanomaterial has an oxidation degree of 10%-50%.

[0024] In a fifth aspect, the present invention provides use of the modified cells described in the first aspect, such as immune cells, particularly T cells, such as chimeric antigen receptor T cells, in the preparation of the following medicaments, wherein the medicaments include:

[0025] (1) Drugs that improve, alleviate or treat tumors related to abnormal cell proliferation or growth;

[0026] (2) drugs that increase, promote, activate or activate the positive ratio of IFNγ and granzyme B in immune cells;

[0027] (3) drugs that increase or promote the lysis of abnormally proliferating or multiplying cells;

[0028] (4) drugs that reduce or decrease the proportion of abnormally proliferating or multiplying cells;

[0029] (5) Drugs that increase, promote, activate or activate chimeric antigen receptor T cells to recognize low-expression antigens.

[0030] The sixth aspect of the present invention provides a pharmaceutical composition for improving, alleviating or treating abnormal cell proliferation or tumors related to proliferation, wherein the pharmaceutical composition comprises the modified cells described in the first aspect.

[0031] In certain embodiments, the pharmaceutical composition according to the present invention further comprises a therapeutic agent for combined administration.

[0032] In certain embodiments, according to the pharmaceutical composition of the present invention, the therapeutic agent comprises an immune checkpoint inhibitor, an antibody therapeutic drug, a chemical drug, a miscellaneous drug, an alkylating agent, an antimetabolite, an anti-tumor antibiotic, a hormone drug, an immunomodulator or an antimicrobial agent.

[0033] The present invention discovered that hydrophobic two-dimensional sheet materials can promote cell recognition of target cells, such as CAR T cells recognizing leukemia cells. The reason for this is unclear, but it may be that the hydrophobic two-dimensional sheet material inserts into the cell membranes of CAR T and leukemia cells through van der Waals and hydrophobic forces, thereby enhancing the interaction between the cells. In addition, the nano-inorganic material of the present invention has a large specific surface area, which can efficiently load drugs, thereby achieving combined treatment of CAR T and immune checkpoint blockade. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Atomic force image of graphene oxide is shown.

[0035] Figure 2 Atomic force microscopy detection of CAR T and CAR T@GO with CD19 Normal Interaction force between PDC cells (A); CAR T@nanomaterials and CD19 Normal The interaction force between PDC cells (B). Figure 2 In B, the left column of the left figure represents CAR T and CD19 Normal The interaction force between PDC cells, the right column represents the interaction between CAR T@graphene and CD19 Normal The interaction between PDC cells; the left column in the middle figure represents the interaction between CAR T and CD19 Normal The interaction force between PDC cells, the right column represents CAR T@carbon nitride and CD19 Normal The interaction between PDC cells; the left column of the right figure represents the interaction between CAR T and CD19 Normal The interaction force between PDC cells, the right column represents CAR T@MoS2 and CD19 Normal The interaction between PDC cells.

[0036] Figure 3 Shown are the changes in CAR T cell membrane fluidity after co-incubation with GO.

[0037] Figure 4 The proliferation detection results after incubation of CAR T and CAR T@GO with leukemia cells are shown.

[0038] Figure 5The flow cytometric analysis shows the activation of interferon γ (IFNγ) and granzyme B by CAR T and CAR T@GO co-incubated with leukemia cells.

[0039] Figure 6 The figure shows the killing effect of CAR T and CAR T@GO on leukemia cells after incubation with leukemia cells at different ratios.

[0040] Figure 7 The figure shows the changes in peripheral blood leukemia burden over time after 100w dose of CAR T, 100w dose of CAR T@GO and 400w high dose of CAR T were reinfused into leukemia model mice.

[0041] Figure 8 The figure shows the changes in body temperature of leukemia model mice over time after 100W dose of CAR T, 100W dose of CAR T@GO and 400W high dose of CAR T were reinfused into the body of leukemia model mice.

[0042] Figure 9 The figure shows the changes in the survival of leukemia model mice after 100w dose of CAR T, 100w dose of CAR T@GO and 400w high dose of CAR T were reinfused into the body.

[0043] Figure 10 The interaction between CAR-T and CAR-T@GO and CD19 low-expressing leukemia cells is shown.

[0044] Figure 11 The figure shows the proliferation detection of CAR-T and CAR-T@GO after incubation with CD19 low-expressing leukemia cells.

[0045] Figure 12 The figure shows the activation flow cytometry results after CAR-T and CAR-T@GO were co-incubated with CD19 low-expressing leukemia cells.

[0046] Figure 13 This is a killing test after CAR-T and CAR-T@GO were co-incubated with CD19 low-expressing leukemia cells at different ratios.

[0047] Figure 14 The figure shows the changes in peripheral blood leukemia burden over time after 100w CAR T, 100w CAR T@GO, and 400w CAR T were reinfused into CD19 low-expressing leukemia model mice.

[0048] Figure 15 The figure shows the killing test after CAR T and CAR T@GO were co-incubated with CD19 low-expressing leukemia cells at different ratios.

[0049] Figure 16 The figure shows the changes in peripheral blood leukemia burden over time after 100w CAR T, 100w CAR T@GO and 100w CAR T@GO-i were reinfused into high-load CD19 low-expression leukemia model mice. DETAILED DESCRIPTION

[0050] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0051] 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 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 any stated value or intermediate value within a 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.

[0052] 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 related to the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail. Unless otherwise indicated, "%" is a percentage based on weight.

[0053] Modified cells

[0054] A first aspect of the present invention provides a modified cell comprising a cell and a hydrophobic two-dimensional sheet material positioned on the cell. The hydrophobic two-dimensional sheet material can be any two-dimensional sheet-like nano-inorganic material having a surface having a certain hydrophobic property. It is understood that the nano-inorganic material can be composed of carbon atoms or other materials, and can be in a non-oxidized state or an oxidized state.

[0055] In the present invention, the hydrophobic two-dimensional sheet material is preferably located on the cell membrane, and it is also preferred that at least a portion of the hydrophobic two-dimensional sheet material is exposed to the surface of the cell membrane. The term "modified" refers to the use of a hydrophobic two-dimensional sheet material to modify cells such as chimeric antigen receptor T cells. Preferably, the hydrophobic two-dimensional sheet material is bound to the surface of the cell membrane by van der Waals forces and hydrophobic forces, or at least a portion of the hydrophobic two-dimensional sheet material is wrapped in the cell membrane, so that the hydrophobic two-dimensional sheet material and the cell membrane of the cell are "bridged". This "bridging" refers to the insertion of the hydrophobic two-dimensional sheet material into the cell membrane of the cell, and the lipid molecules on both sides of the cell membrane of the cell diffuse to the lipid-deficient area, thereby climbing on the surface of the hydrophobic two-dimensional sheet material to form a "bridging" structure.

[0056] In the present invention, the hydrophobic two-dimensional sheet material also includes any suitable modification groups, examples of which include but are not limited to: amino, hydroxyl, alkyl, alkenyl, silane, phosphate and oxide groups, so that the hydrophobic two-dimensional sheet material has improved surface properties and increased biocompatibility.

[0057] In the present invention, the hydrophobic two-dimensional sheet material is preferably a nano-inorganic material, and the nano-inorganic material is at least one selected from the group consisting of a carbon material and its oxide, carbon nitride, and molybdenum disulfide, preferably a carbon material and its oxide. In the present invention, the carbon material and its oxide include at least one of graphene, graphene oxide, and graphyne, preferably graphene oxide (or graphene oxide).

[0058] In the present invention, the hydrophobic two-dimensional sheet material has a particle diameter of 10-2000nm, preferably 10-900nm, also preferably a particle diameter of 50-900nm, further preferably 50-500nm, for example 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500nm, etc. The size of the hydrophobic two-dimensional sheet material can be adjusted as needed, and considering intercellular interactions, its particle size range can be selected to be less than the particle size range of 900nm. The thickness of the nano-inorganic material is not particularly limited, but is preferably 0.01-10nm, also preferably 0.01-5nm, more preferably 0.5-2nm, for example 0.5, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0 nm.

[0059] In the present invention, the term "particle size" should be understood broadly. Here, "particle size" refers to the longest straight-line distance through the interior of a hydrophobic two-dimensional sheet material. Particle size determination methods are known in the art and can be described using cumulative percentiles or major distribution areas based on particle size distribution measured, for example, by microscopy or scanning electron microscopy.

[0060] In the present invention, the nano-inorganic material may have a certain degree of oxidation. The term "degree of oxidation" refers to the concentration of oxygen elements (such as hydroxyl groups, carboxyl groups, epoxy groups, etc.) on its surface, and is usually expressed as the number of oxygen atoms (or oxygen content). The determination of the degree of oxidation is known in the art and can be characterized, for example, by means of adsorbed water, the amount of gaseous adsorbates (such as N2, CO2, etc.), Raman spectroscopy, etc. The inventors have found through research that nano-materials with a degree of oxidation of 10%-50%, preferably 20-40%, and more preferably 25-38%, for example, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% are beneficial for promoting intercellular interaction. Nano-inorganic materials with different degrees of oxidation can be obtained from commercially available products or can be synthesized using methods known in the art, without particular limitation.

[0061] Method for preparing modified cells

[0062] A second aspect of the present invention provides a method for preparing a modified cell, comprising the following steps:

[0063] (1) dispersing a hydrophobic two-dimensional sheet material in water and then crushing it, wherein the particle size of the hydrophobic two-dimensional sheet material is 10-2000 nm;

[0064] (2) Isolating a hydrophobic two-dimensional sheet material with a fixed particle size distribution and incubating it with cells such as chimeric antigen receptor T cells, and then removing the free hydrophobic two-dimensional sheet material.

[0065] Step (1) of the present invention is to disperse the hydrophobic two-dimensional sheet material in water and then crush it. In a preferred embodiment, the present invention is to disperse graphene oxide with a certain degree of oxidation in water and obtain a single-layer graphene oxide with a specific particle size by centrifugation. After being dispersed in water, it is preferably pre-treated by ultrasound to achieve better dispersion performance and crushing effect. The ultrasound frequency is not particularly limited and can be adjusted as needed.

[0066] In step (1) of the present invention, the concentration of the hydrophobic two-dimensional sheet material in water is 0.01-10 g / mL, preferably 0.5-5 g / mL, for example 0.5 g / mL, 0.8 g / mL, 1.0 g / mL, 2 g / mL, 3 g / mL, 4 g / mL, 5 g / mL.

[0067] When the hydrophobic two-dimensional sheet material is graphene, in order to obtain graphene oxide with different oxidation degrees, the graphene oxide can be treated with a reducing agent or an oxidizing agent, and the reducing agent or the oxidizing agent includes: concentrated nitric acid, concentrated sulfuric acid, ascorbic acid, etc.

[0068] In step (2) of the present invention, a hydrophobic two-dimensional sheet material with a specific particle size distribution is obtained by centrifugation. Centrifugation can be performed using an instrument known in the art, such as an ultracentrifuge, and the centrifugation parameters can be set to 180,000-200,000 × g. Subsequently, the separated hydrophobic two-dimensional sheet material with a specific particle size distribution is mixed and incubated with cells, such as chimeric antigen receptor T cells, and then the free hydrophobic two-dimensional sheet material is removed. Preferably, the mixing ratio of the hydrophobic two-dimensional sheet material and the chimeric antigen receptor T cells is (1-20) μg: (1×10 6 -2×10 6 ) cells. Also preferably, the mixing ratio of the hydrophobic two-dimensional sheet material and the chimeric antigen receptor T cells is (5-10) μg: (1×10 6 -2×10 6 ) cells. Further preferably, the mixing ratio of the hydrophobic two-dimensional sheet material and the chimeric antigen receptor T cells is 10 μg: (1×10 6 -2×10 6 ) cells.

[0069] In step (2) of the present invention, the incubation time is not particularly limited, as long as the modified cells of the present invention, such as chimeric antigen receptor T cells, can be formed, and is generally 6-72 hours, preferably 6-24 hours. After the incubation is completed, the hydrophobic two-dimensional sheet material that is not connected or bridged to the cells is removed by centrifugation.

[0070] Methods for increasing, promoting or enhancing intercellular forces

[0071] Another aspect of the present invention provides a method for increasing, promoting or enhancing intercellular forces. The method described herein includes a method for increasing, promoting or enhancing intercellular forces in vitro, the method including the step of bringing a first cell and a second cell into close proximity and contact with each other, wherein the first cell is the modified cell described herein, and the second cell is an abnormally proliferating or proliferating cell.

[0072] As used herein, the term "abnormally proliferating or multiplying cells" includes cells that grow or multiply in an uncontrolled manner, examples of which include, but are not limited to, tumor cells or cancer cells, virus-infected cells, cells associated with the immune system, leukemia cells, and the like. In a preferred embodiment, the second cell of the present invention is a leukemia cell. In another preferred embodiment, the second cell of the present invention is a leukemia cell with reduced expression of a target antigen. In yet another preferred embodiment, the second cell of the present invention is a leukemia cell with low expression of the target CD19 antigen.

[0073] Without being bound by any theory, cells having any biomembrane structure and capable of being targeted (specifically recognized or bound) by CAR-T cells can be used as the second cell of the present invention.

[0074] Similar to the hydrophobic two-dimensional sheet material and modified cell bridge, the hydrophobic two-dimensional sheet material can be inserted into the cell membrane of the second cell through "bridging" after the CAR-T cell targets the second cell, and the lipid molecules on both sides of the cell membrane of the second cell diffuse to the lipid-deficient area, thereby climbing on the surface of the hydrophobic two-dimensional sheet material, increasing, promoting, and enhancing the interaction between the chimeric antigen receptor T cell and the abnormally proliferating or proliferating cells. This improvement in interaction is beneficial for treatment because it shortens the distance between the CAR T and the abnormally proliferating or proliferating cells, thereby promoting interaction between the two cells and promoting CAR T to recognize and kill the abnormally proliferating or proliferating cells.

[0075] Methods for detecting intercellular forces are known in the art. For example, atomic force microscopy can be used to detect the forces between a cell on a probe, such as a traditional CAR-T or a cell modified by the present invention (i.e., a modified CAR-T), and a second cell.

[0076] Methods to enhance cell membrane fluidity

[0077] The present invention provides a method for enhancing cell membrane fluidity, the method described herein includes a method for enhancing cell membrane fluidity in vitro, the method comprising the step of contacting a hydrophobic two-dimensional sheet material with a cell. Cell membrane fluidity is indicated by the time from cell membrane fluorescence quenching to fluorescence recovery. In certain embodiments, the inventors have found through research that the time it takes for chimeric antigen receptor T cells to recover to half of their maximum intensity is significantly reduced, indicating that the method increases the cell membrane fluidity of chimeric antigen receptor T cells, and this improvement in fluidity is beneficial for their capture and recognition of targeted cell membrane antibodies.

[0078] use

[0079] The present invention also provides the use of the modified cells in preparing the following drugs:

[0080] (1) Drugs that improve, alleviate or treat abnormal cell proliferation or tumors;

[0081] (2) drugs that increase, promote, activate or activate the positive ratio of IFNγ and granzyme B in immune cells;

[0082] (3) drugs that increase or promote the lysis of abnormally proliferating or multiplying cells;

[0083] (4) drugs that reduce or decrease the proportion of abnormally proliferating or multiplying cells;

[0084] (5) drugs that increase, promote, activate, or activate cell recognition of low-expression antigens;

[0085] (6) Drugs that increase, promote, or enhance intercellular forces;

[0086] (7) Drugs that enhance cell membrane fluidity.

[0087] The terms "improve, alleviate or treat" as used herein refer to improving a condition before or after the onset of a disease or disorder. The degree of alleviation or prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100% as measured by any standard technique compared to an untreated control group under the same conditions. In the present invention, the term "treat" refers to therapeutic treatment and preventive or prophylactic measures, the purpose of which is to prevent or slow (reduce) undesirable physiological changes or disorders, such as the progression of tumors or cancer diseases, reducing the risk of CAR T side effect cytokine release syndrome (CRS), etc. Beneficial or desired clinical results include, but are not limited to, the following, 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 alleviation (whether partial or complete). "Treatment" also means prolonged survival compared to the expected survival period when not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to having the condition or disorder or those in whom the condition or disorder is to be prevented.

[0088] In the present invention, tumors include blood tumors, solid tumors or a combination thereof. In certain embodiments, the blood tumors include but are not limited to 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.

[0089] In certain embodiments, the solid tumor includes but is not limited to gastric cancer, gastric cancer peritoneal metastasis, 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 cancer, adrenal tumor, bladder tumor, Lewis lung cancer, non-small cell lung cancer (NSCLC), brain glioma, cervical cancer, endometrial cancer, mesothelioma, pancreatic cancer or a combination thereof.

[0090] In applications of improving, alleviating or treating abnormal cell proliferation or proliferation-related tumors, specific antibodies are detected by staining or the proliferation status of CAR-T or tumors is identified by flow cytometry.

[0091] In applications of increasing, promoting, activating, or activating the positive ratio of IFNγ and granzyme B in immune cells, examples of the immune cells include natural killer cells (NK cells) and T cells, including but not limited to CD8+ T cells and CD4+ T cell subsets (such as Th1 cells). The positive ratio of IFNγ and granzyme B is detected by IFNγ and granzyme B antibodies to characterize the degree of activation of CAR-T.

[0092] In the application of increasing or promoting the lysis of abnormally proliferating or proliferating cells, the rate of lysis of abnormally proliferating or proliferating cells can be characterized by detecting the release of LDH in the supernatant using an LDH release detection kit.

[0093] Herein, abnormally proliferating or proliferating cells also include those abnormally proliferating or proliferating cells with low expression of target antigens. The present invention enhances intercellular interactions and increases cell membrane fluidity through modified chimeric antigen receptor T cells, thereby promoting CAR T's recognition of abnormally proliferating or proliferating cells with low expression of target antigens.

[0094] It is understood that the application of the present invention also includes the combined administration with other therapeutic agents to improve, alleviate or treat abnormal cell proliferation or proliferation-related tumors or diseases. Examples of the therapeutic agents include, but are not limited to, immune checkpoint inhibitors, antibody therapeutics, chemical drugs, miscellaneous drugs, alkylating agents, antimetabolites, antitumor antibiotics, hormone drugs, immunomodulators or antimicrobial agents. The therapeutic agent can be loaded on the surface or inside the nanomaterial of the present invention, or it can be connected to the surface or inside the nanomaterial of the present invention through a surfactant group, thereby achieving simultaneous administration with the modified cell CAR-T. Other dosing regimens can also be selected, such as sequentially administering the therapeutic agent before or after the administration of the modified CAR-T of the present invention. In a preferred embodiment, the high specific surface area of ​​the nanomaterial in the modified cell of the present invention, such as CAR-T, is utilized to load PD-1 / PD-L1 or CTLA-4, thereby achieving combined therapy with immune checkpoint blockade.

[0095] Pharmaceutical composition

[0096] Another aspect of the present invention provides a pharmaceutical composition for improving, alleviating or treating abnormal cell proliferation or proliferation-related tumors, which comprises modified cells and optionally a pharmaceutically acceptable excipient, carrier or diluent.

[0097] As used herein, the term "pharmaceutically acceptable excipient, carrier, or diluent" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, vehicle, solvent, or encapsulating material, that participates in the transport or delivery of a pharmaceutical agent from one organ or part of the body to another. Each carrier is "acceptable" if it is compatible with the other ingredients of the formulation and is not harmful to the patient. Some examples of pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives and analogs such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl lauryl ester; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; and other nontoxic and compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate, magnesium stearate and polyoxyethylene-polypropylene oxide copolymers, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0098] The term "subject" as used herein refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, and the like, that is about to receive a specific treatment. Generally, "subject" and "patient" are used interchangeably herein to refer to a human subject.

[0099] The dosage of the modified chimeric antigen receptor T cells of the present invention and the pharmaceutical compositions comprising the same depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality and individual response of the patient or animal, the route of administration, the number of administrations, and the purpose of treatment. Therefore, the therapeutic dose of the present invention can vary widely. In general, the dosage of the modified chimeric antigen receptor T cells and the pharmaceutical compositions comprising the same in the present invention is well known to those skilled in the art. The dosage can be administered in a single dosage form or divided into several, for example, two, three or four dosage forms. The dosage administered is within the expected range of the clinician or laboratory personnel, for example, the dosage can be appropriately adjusted through effectiveness and safety testing to obtain the optimal dosage.

[0100] The total dose required for each treatment can be divided into multiple doses or administered as a single dose. The modified chimeric antigen receptor T cells of the present invention and the pharmaceutical composition comprising the same can be administered alone or in combination with other therapeutic drugs or symptomatic drugs and treatment regimens and the dosage can be adjusted.

[0101] There is no particular limitation on the administration of the modified cells of the present invention, such as chimeric antigen receptor T cells, or pharmaceutical compositions comprising the same. Representative administration methods include, but are not limited to, oral administration, parenteral injection, intramuscular injection, intravenous injection, intravenous drip, enema, spray, topical application, or intraperitoneal injection.

[0102] Modified cells such as chimeric antigen receptor T cells for parenteral injection, pharmaceutical compositions containing the same may include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0103] Example 1

[0104] In this example, chimeric antigen receptor T cells modified with graphene oxide, graphyne, carbon nitride, and molybdenum disulfide as exemplary hydrophobic two-dimensional sheet materials were prepared as follows.

[0105] 1. Experimental Materials and Methods

[0106] 1. Materials

[0107] Graphene oxide, graphyne, carbon nitride, molybdenum disulfide, poly-lysine (Sigma), ascorbic acid (Solarbio), Cell-Tak adhesive (Corning), CFSE dye (Sigma), DiO cell membrane green fluorescent probe (Beyotime), 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), human CD20 flow cytometry antibody (Biolegend), red blood cell lysate (Solarbio), LDH release detection kit (Solarbio).

[0108] 2. Methods

[0109] 2.1 The preparation of graphene oxide with different particle sizes and oxidation degrees includes the following steps:

[0110] 1 g of graphene oxide powder was dissolved in 10 mL of deionized water and ultrasonicated in an ice bath at 30% power for 2 h. The monolayer graphene oxide with a particle size of 100 nm was then separated using an ultracentrifuge (speed 180,000-200,000 × g). In addition, the monolayer graphene oxide with a particle size of 1000 nm was separated (speed 60,000-80,000 × g).

[0111] The oxidation degree of graphene oxide without any treatment is about 27%. In order to expand the range of oxidation degree, graphene oxide with an oxidation degree of about 20% was obtained by antigenic acid reduction. Specifically, 3 mg of graphene oxide and 15 mg of ascorbic acid were dissolved in 10 ml of deionized water. After stirring at room temperature for 24 hours, graphene oxide with particle sizes of 100 nm and 1000 nm was separated using an ultracentrifuge.

[0112] In addition to graphene oxides with oxidation degrees of 27% and 20%, graphene oxide with an oxidation degree of 40% and a large particle size of 1000 nm or more was purchased from a commercial product, and graphene oxides with sizes of 100 nm and 1000 nm were obtained by the above-mentioned ultrasonic crushing and centrifugation methods.

[0113] The materials used in this example are specifically shown below.

[0114]

[0115] 2.2 CAR-T@GO preparation includes the following steps:

[0116] The ratio of the mass of graphene oxide sheets to the number of CAR-T cells is 5-10 μg: (1×10 6 -2×10 6 ) cells, CAR-T cells were co-incubated with graphene oxide for 24 h, and then the excess graphene oxide was removed by centrifugation at 500×g to obtain CAR-T@GO.

[0117] 2.3 Preparation of CAR-T@nanomaterials

[0118] Graphene, carbon nitride, and molybdenum disulfide were used to prepare CAR-T@nanomaterials according to the above steps 2.1-2.2.

[0119] 3. Atomic force testing of graphene oxide

[0120] Use atomic force microscopy to detect the particle size of graphene oxide: first, take 10μl of 1mg / mL graphene oxide solution and dissolve it in 1mL of anhydrous ethanol. After mixing it evenly, drop it on the mica sheet of atomic force microscope. After the anhydrous ethanol evaporates, use atomic force microscope for detection.

[0121] 4. Force detection

[0122] Atomic force microscopy was used to detect the interaction forces between CAR-T, CAR-T@nanomaterials, CAR-T@GO and leukemia cells.

[0123] First, a 6-cm-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 CAR-T or CAR-T@nanomaterials or CAR-T@GO 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 CAR-T@nanomaterials or CAR-T@GO on the probe and the leukemia cells.

[0124] 5. Cell membrane fluidity detection

[0125] The fluorescence recovery after photobleaching method was used to detect that co-incubation with GO enhanced the fluidity of the CAR-T cell membrane, thereby enhancing the capture of leukemia cell surface antigens by CAR on the surface of CAR-T cells.

[0126] Take 1w cells of CAR-T@GO, add 2μl of cell membrane green fluorescent probe DiO dissolved in DMSO, stain in a 37℃ incubator for 30min, then add PBS and centrifuge to wash the cells twice, and then add the cell suspension to a confocal dish. Place the dish under a rotating disk confocal microscope and observe with a 100x oil lens. After selecting the area on the cell membrane that needs to be quenched, use a laser to quench the fluorescence of the cell membrane under the 488nm channel. At the same time, collect images from the start of quenching to the period when the fluorescence returns to equilibrium. Fit the fluorescence curve using the analysis curve in the software to analyze and obtain T 1 / 2 , that is, the time it takes for fluorescence to recover to half of its maximum intensity. T 1 / 2 The smaller the value, the faster the membrane fluidity.

[0127] 6. Proliferation detection after incubation of CAR-T and CAR-T@GO with leukemia cells

[0128] The CFSE method was used to detect the proliferation of CAR-T cells after co-incubation with leukemia cells.

[0129] CAR-T cells were stained with CFSE dye, and then partially incubated with stained CAR-T@GO to construct CFSE-stained CAR-T@GO. CFSE-stained CAR-T cells and CAR-T@GO cells were then incubated with leukemia cells. Finally, flow cytometry was used to detect the CFSE fluorescence signal of CAR-T cells or CAR-T@GO cells at 0, 24, and 72 hours after incubation to monitor their proliferation (where CAR-T cells or CAR-T@GO cells were labeled with a human CD3 antibody).

[0130] 7. CAR-T and CAR-T@GO co-incubated with leukemia cells for activation

[0131] Flow cytometry detection of activation of CAR-T and CAR-T@GO after co-incubation with leukemia cells.

[0132] 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 CAR-T@GO were co-incubated with leukemia cells to characterize the degree of activation (where CAR-T or CAR-T@GO was labeled with human CD3 antibody).

[0133] 8. Killing effect of CAR-T and CAR-T@GO incubated with leukemia cells at different ratios

[0134] The killing of leukemia cells by CAR-T was characterized by detecting the release of LDH in the supernatant.

[0135] CAR-T or CAR-T@GO was co-mixed 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 the LDH release assay kit (Solarbio) was used to measure LDH release in the supernatant. The leukemia cell lysis rate was then compared with the positive control group.

[0136] 9. Changes in peripheral blood leukemia burden over time

[0137] After 100w CAR-T, 100w CAR-T@GO, and 400w CAR-T were reinfused into leukemia model mice, the changes in peripheral blood leukemia burden over time were detected.

[0138] First, leukemia modeling was performed: leukemia cells isolated from the bone marrow of clinical leukemia patients were divided into 5×10 6 The cells were intravenously infused into severely immunodeficient mice (NTG mice, Spav). Approximately 7 days after the infusion, blood samples were collected from the mouse orbitals. The proportion of human leukemia cells in the white blood cells of the mouse blood samples was measured by flow cytometry. A leukemia model was considered successful when the proportion was no less than 1%.

[0139] 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 / ), CAR-T@GO (1×10 6 / ), CAR-T (4×10 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.

[0140] At the same time, the body temperature and survival rate of the mice were monitored every two days.

[0141] 2. Results

[0142] Figure 1 The graphene oxide atomic force image is shown, specifically showing that graphene oxide with different particle sizes and oxidation degrees is obtained by the preparation method of the present invention.

[0143] Figure 2 Atomic force microscopy detection of CAR T and CAR-T modified with graphene (CAR T@GO), CAR-T modified with other hydrophobic two-dimensional sheet materials (CAR T@ nanomaterials) and CD19 NormalThe interaction force between PDC cells and the results showed that the interaction force between CAR T and leukemia was significantly increased after modification with hydrophobic two-dimensional sheet materials.

[0144] Figure 3 The results show that the changes in the membrane fluidity of CAR T cells after co-incubation with GO. The results show that after GO is inserted into the CAR T cell membrane, the membrane fluidity is significantly increased, and the T 1 / 2 The time was reduced from 7.58s to 3.84s. The fluidity of the cell membrane is related to the capture of surface antibodies, which further indicates that CAR T@GO can recognize antigens on the surface of leukemia cells more quickly.

[0145] Figure 4 The figure shows the proliferation test results of CAR T and CAR T@GO after incubation with leukemia cells. The proliferation of CAR T after co-incubation with leukemia cells was detected by flow cytometry. The results showed that after GO modification, the proliferation of CAR T after co-incubation with leukemia cells was significantly increased.

[0146] Figure 5 The results show that the activation of interferon gamma (IFNγ) and granzyme B by CAR T and CAR T@GO co-incubated with leukemia cells was detected by flow cytometry. The results showed that compared with the positive ratio of interferon gamma (IFNγ) and granzyme B after CAR T and leukemia cells co-incubated, the activation ratio of CAR T after GO modification and co-incubation with leukemia cells increased significantly, indicating the activation of CAR T.

[0147] Figure 6 The figure shows the killing effect of CAR T and CAR T@GO on leukemia cells after incubation with leukemia cells at different ratios. The killing effect of CAR T on leukemia cells is characterized by measuring LDH release in the supernatant. The horizontal axis is CAR T:PDC, and the vertical axis is the killing rate. The results show that GO-modified CAR T has a significantly increased killing ability on leukemia cells.

[0148] Figure 7 Figure 3 shows the time-dependent changes in peripheral blood leukemia burden after infusion of 100w CAR T, 100w CAR T@GO, and a high-dose 400w CAR T into leukemia model mice. Peripheral blood samples were collected from the leukemia model mice every five days to measure the proportion of leukemia cells in the peripheral blood, demonstrating the therapeutic effect. The results demonstrated that the 100w CAR T@GO group completely inhibited leukemia progression, achieving a therapeutic effect nearly equivalent to that of the high-dose CAR T group (400w CAR T).

[0149] Figure 8Figure 3 shows the changes in body temperature over time after 100W of CAR T, 100W of CAR T@GO, and a high-dose of 400W of CAR T were infused back into leukemia model mice. Temperature monitoring of the leukemia model mice every two days revealed elevated body temperatures in the 400W CAR T group, but gradually returned to normal in the 100W CAR T@GO group, demonstrating the risk of CRS in the high-dose group.

[0150] Figure 9 The figure shows the changes in the survival of leukemia model mice after the infusion of 100w CAR T, 100w CAR T@GO, and 400w high-dose CAR T. Daily monitoring of the survival of leukemia model mice showed that although the high-dose CAR T (400w CAR T group) was able to completely inhibit the progression of leukemia, the mice began to gradually die after 22 days. In contrast, the mice in the 100w CAR T@GO group remained healthy throughout the 80-day observation period, demonstrating that the 100w CAR T@GO group has a better safety profile than the 400w CAR T group.

[0151] Example 2

[0152] 1. Interaction between CAR-T and CAR-T@GO and CD19-low-expressing leukemia cells

[0153] The present invention further detected the interaction force between CAR-T and CAR-T@GO and low-expressing leukemia cells. Atomic force microscopy was used to detect the interaction force between CAR-T and CAR-T@GO and low-expressing CD19 leukemia cells. Figure 10 As shown in the figure, the results showed that compared with CAR T, CAR T@GO significantly increased its interaction with CD19 low-expressing leukemia cells.

[0154] 2. Proliferation detection of CAR-T and CAR-T@GO after incubation with CD19 low-expressing leukemia cells

[0155] Similar to the above CFSE method, the present invention detected the proliferation of CAR-T and CAR-T@GO low-expressing leukemia cells after co-incubation. The results are as follows: Figure 11 As shown, it was further verified that FnC modification promoted the interaction between CAR T and leukemia cells and thus promoted proliferation.

[0156] 3. CAR-T and CAR-T@GO were co-incubated with CD19 low-expressing leukemia cells and activated by flow cytometry

[0157] Similar to the activation flow cytometry graph above, the present invention characterized the activation of CAR T cells after co-incubation with CD19 low-expressing leukemia cells. Figure 12 shown.

[0158] 4. Killing detection after CAR-T and CAR-T@GO were co-incubated with CD19 low-expressing leukemia cells at different ratios

[0159] The same as the above-mentioned LDH release detection killing figure, the present invention characterizes the killing of leukemia cells with low CD19 expression by CAR T, as shown in FIG. Figure 13 As shown, the results demonstrated that GO modification enhanced the killing of CAR T cells against leukemia cells with low CD19 expression.

[0160] 5. Changes in peripheral blood leukemia burden over time after 1 million CAR T, 1 million CAR T@GO, and 4 million CAR T were infused back into CD19 low-expressing leukemia model mice

[0161] Similar to the above-mentioned in vivo therapeutic effect graph, the present invention tested the changes in peripheral blood leukemia load over time after reinfusion of 100w CAR T, 100w CAR T@GO, and 400w CAR T into the animal model of CD19 low expression leukemia. The results are as follows: Figure 14 shown.

[0162] Example 3

[0163] The present invention detects the changes in peripheral blood leukemia load over time after CAR T, CAR T@GO, and CAR T@GO-i loaded with small molecule inhibitors were reinfused into high-load CD19 low-expression leukemia model mice. PD-1 / PD-L1 is used as an example of the small molecule inhibitor of the present invention for illustration.

[0164] 1. The main steps of this experiment are as follows:

[0165] Construction of loading PD-1 / PD-L1 small molecule inhibitor (BMS202): 10 μL of 5 mg / mL BMS202 was added to a 1 mg / mL aqueous solution of graphene oxide (the graphene oxide had an oxidation degree of 27% and a particle size of 100 nm). After stirring at room temperature for 24 hours, the graphene oxide was separated using an ultracentrifuge (speed 180,000-200,000 × g).

[0166] 2. In vitro effect evaluation

[0167] CAR-T, CAR-T@GO, and CAR-T@GO-i were co-incubated with leukemia cells with low CD19 expression at different ratios and then tested for killing. Similar to the aforementioned LDH release killing test, the present invention characterized the killing of leukemia cells with high load and low CD19 expression, such as Figure 15 As shown in the figure, the results show that CAR-T@GO-i greatly improves the killing of leukemia cells with high load and low CD19 expression.

[0168] 3. In vivo evaluation of therapeutic effects

[0169] The CD19 low-expression leukemia model was established using CD19 low-expression leukemia cells according to the aforementioned method. When the proportion of human leukemia cells in the peripheral blood leukocytes of mice was not less than 10%, treatment was started. PBS (200 μL / mouse), CAR T (1×10 6 / pcs), CAR T@GO(1×10 6 / pcs), CAR T@GO-i(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.

[0170] The results are as follows Figure 16 As shown, it is the same as the in vivo treatment effect diagram of the aforementioned CD19 low expression leukemia model, but the treatment starting point is 10% of the peripheral blood leukemia load (the starting point of the aforementioned two models is 1%).

[0171] 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 method for increasing, promoting or enhancing intercellular forces, characterized in that: The method includes the steps of bringing a first cell and a second cell into proximity and contact with each other, wherein the first cell is a modified CAR T cell, the second cell is an abnormally proliferating or proliferating cell, and the modified CAR T cell includes a CAR T cell and a hydrophobic two-dimensional sheet material located on the CAR T cell, and the particle size of the sheet material is 10-2000 nm.

2. The method for increasing, promoting or enhancing intercellular forces according to claim 1, characterized in that: The hydrophobic two-dimensional sheet material includes nano-inorganic material.

3. The method for increasing, promoting or enhancing intercellular forces according to claim 2, characterized in that: The nano inorganic material is at least one selected from the group consisting of carbon elemental materials and their oxides, carbon nitride and molybdenum disulfide.

4. The method for increasing, promoting or enhancing intercellular forces according to claim 3, characterized in that: At least a portion of the nano-inorganic material is located on or within the cell membrane of the CAR T cell.

5. The method for increasing, promoting or enhancing intercellular interaction according to claim 1, characterized in that: The modified CAR T cells are prepared by the following method: (1) dispersing the hydrophobic two-dimensional sheet material in water and then crushing it; (2) Isolating a hydrophobic two-dimensional sheet material with a fixed particle size distribution, incubating it with CAR T cells, and then removing the free hydrophobic two-dimensional sheet material.

6. The method for increasing, promoting or enhancing intercellular forces according to claim 5, characterized in that: In step (1), the concentration of the hydrophobic two-dimensional sheet material is 0.01-10 g / mL.

7. The method for increasing, promoting or enhancing intercellular forces according to claim 5, characterized in that: The mixing ratio of the hydrophobic two-dimensional sheet material and CAR T cells is (1-20) μg: (1×10 6 -2×10 6 ) cells.

8. The method for increasing, promoting or enhancing intercellular forces according to claim 1, characterized in that: The modified CAR T cells are used to prepare the following drugs: (1) Drugs that improve, alleviate or treat tumors associated with abnormal cell proliferation or growth in subjects; (2) drugs that increase, promote, activate or activate the positive ratio of IFNγ and granzyme B in immune cells; (3) drugs that increase or promote the lysis of abnormally proliferating or multiplying cells in a subject; (4) drugs that reduce or decrease the proportion of abnormally proliferating or multiplying cells in a subject; (5) Drugs that increase, promote, activate or activate chimeric antigen receptor T cells to recognize low-expression antigens.

9. The method for increasing, promoting or enhancing intercellular interaction according to claim 1, characterized in that: The modified CAR T cells are used to prepare a pharmaceutical composition.

10. The method for increasing, promoting or enhancing intercellular forces according to claim 9, characterized in that: The pharmaceutical composition further includes a therapeutic agent for co-administration.

11. The method for increasing, promoting or enhancing intercellular interaction according to claim 10, characterized in that: The therapeutic agents include immune checkpoint inhibitors, antibody therapeutic drugs, chemical drugs, miscellaneous drugs, alkylating agents, antimetabolites, antitumor antibiotics, hormone drugs, immunomodulators or antimicrobial agents.

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