Plasmid Combinations and Their Application in the Preparation of Modified Immune Cells

Abstract

The present disclosure provides a method using a 4-plasmid system to produce modified immune effector cells. The method comprises forming a lentivirus using four plasmids in 293T cells, extracting and obtaining the lentivirus, and then transfecting immune effector cells using the lentivirus to express a chimeric antigen receptor. The present disclosure also provides for the use of immune effector cells obtained using the described method and compositions containing immune effector cells.

Description

Technology Field

[0001] The present disclosure relates to the field of biopharmaceuticals. More specifically, the present disclosure relates to a method of transfecting 293T cells using a 4-plasmid system, comprising forming a lentivirus by using four plasmids in 293T cells, extracting and obtaining the lentivirus, and then transfecting T cells with the lentivirus to express a chimeric antigen receptor targeting CD19. The present disclosure further relates to immune effector cells obtained using the present method, a composition comprising immune effector cells, and the use thereof. Background Technology

[0002] Current clinical treatments for acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), and B-cell lymphoma primarily involve chemotherapy, stem cell transplantation, and biological therapies. While these treatments can achieve specific efficacy, relapsed or refractory leukemia remains a major, difficult-to-resolve problem. Cellular immunotherapy of tumors has emerged as a key topic in recent research as a novel treatment strategy. CD19 is widely expressed on the surface of almost all B-cell tumor cells, whereas it is rarely expressed on other parenchymal cells and hematopoietic stem cells.

[0003] Tumor cells can generate immune escape by downregulating the expression of molecules involved in various pathways, such as T cell recognition and antigen response, or by reducing immunogenicity, thereby preventing the organism's immune system from effectively eliminating the tumor. Research has revealed that chimeric antigen receptor T cells (CAR-T cells) can recognize antigens on the surface of tumor cells and specifically kill them, and thus are useful for the treatment of tumors.

[0004] Lentiviruses produced using currently available plasmid packaging systems in conventional technology have low concentrations of viable viruses and low transfection titers. To achieve the ideal effect of T cell transfection, it is necessary to add much higher doses of lentivirus, along with disadvantages such as higher costs, excessive residue, and poor safety performance. Despite the fact that 4-plasmid packaging systems have been used in conventional technology to replace 3-plasmid packaging systems, the appropriate ratio in which plasmids can be combined to provide higher transfection titers while ensuring superior safety performance has not yet been determined. Therefore, screening the appropriate ratio of four plasmids for packaging lentiviral vectors to produce modified immune effector cells remains an urgent need.

[0005] Summary of the Invention

[0006] To overcome the disadvantages of the prior art, the present disclosure provides a 4-plasmid packaging system comprising a target plasmid and three helper plasmids, wherein the target plasmid is free of non-essential components for virus packaging and thereby effectively minimizes potential safety risks. By using the four plasmids in specific proportions, the present disclosure can effectively increase the transfection titer of the obtained lentivirus and provide CAR-T cells exhibiting superior clinical therapeutic efficacy and higher safety performance obtained by transfecting T cells with a lentivirus vector loaded with a specific CAR.

[0007] The present disclosure provides a chimeric antigen receptor comprising the amino acid sequence shown in SEQ ID NO. 1. The present disclosure further provides a nucleic acid encoding the chimeric antigen receptor, a vector comprising the nucleic acid, the chimeric antigen receptor, an immune effector cell comprising the nucleic acid molecule and / or the vector, a method for producing the immune effector cell, a composition comprising the immune effector cell, and a use of the chimeric antigen receptor.

[0008] A chimeric antigen receptor according to the present disclosure comprises at least one of the following advantageous effects:

[0009] (1) Stable expression on the surface of immune cells (e.g., T cells) at high expression levels;

[0010] (2) Powerful ability to kill CD19-positive target cells;

[0011] (3) The ability to promote immune cells to secrete cytokines, for example, the ability of T cells to secrete cytokines (INF-γ or IL-6) by at least 1, 2, or 3 times;

[0012] (4) It is non-hemolytic and does not easily induce hemolysis or agglutination of red blood cells;

[0013] (5) No vascular irritation, and no local or systemic abnormalities after administration;

[0014] (6) There is no risk of carcinogenicity In vivo or In vitro It is non-tumor-genic;

[0015] (7) Ability to extend the survival time of cancer patients;

[0016] (8) Ability to effectively improve the severity of cancer (e.g., acute lymphoblastic leukemia in adults, acute lymphoblastic leukemia in children and / or non-Hodgkin lymphoma); and

[0017] (9) Ability to exhibit higher safety performance with a lower risk of causing side effects (e.g., cytokine release syndrome or CAR-T-cell-associated encephalopathy syndrome).

[0018] In one embodiment, the present disclosure provides a plasmid combination, wherein the plasmid combination comprises plasmid Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev; and plasmid Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev are present in a ratio of 2-6:1-1.5:1-3:1-1.5.

[0019] In a specific embodiment, plasmid Seq1 in the plasmid combination comprises a nucleic acid molecule encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO. 1.

[0020] In a specific embodiment, plasmid Seq1 in the plasmid combination comprises a nucleic acid molecule encoding a chimeric antigen receptor, wherein the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO. 2.

[0021] In certain embodiments, plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev in the plasmid combination are present in a ratio of 11.8:3.53:6.33:2.3, 13.8:3.48:5.31:2.54 or 14:4.67:4.67:4.67.

[0022] In one embodiment, the present disclosure provides a method for preparing a lentivirus vector, the method comprising the step of introducing a plasmid combination into a cell. In certain embodiments, the step of introducing refers to the step of transfecting.

[0023] In one embodiment, the present disclosure provides a method for obtaining a lentivirus vector by transfecting a cell with a 4-plasmid packaging system, wherein the plasmid packaging system comprises plasmid Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev, and plasmid Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev are present in a ratio of 2-6:1-1.5:1-3:1-1.5.

[0024] In a specific embodiment, plasmid Seq1 may express a chimeric antigen receptor comprising the amino acid sequence shown in SEQ ID NO. 1.

[0025] In a specific embodiment, plasmid Seq1 comprises an isolated nucleic acid molecule encoding a chimeric antigen receptor, wherein the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO. 2.

[0026] In certain embodiments, plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev are present in a ratio of 11.8:3.53:6.33:2.3 or 13.8:3.48:5.31:2.54.

[0027] In a specific embodiment, the cell is 293T. In a specific embodiment, the cell is 293T / 17.

[0028] In one embodiment, the present disclosure provides a chimeric antigen receptor comprising the amino acid sequence shown in SEQ ID NO. 1.

[0029] In another aspect, the present disclosure further provides an isolated nucleic acid molecule encoding the chimeric antigen receptor described herein.

[0030] In another aspect, the present disclosure further provides an isolated nucleic acid molecule encoding a chimeric antigen receptor, wherein the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO. 2.

[0031] In another aspect, the present disclosure further provides a plasmid comprising a nucleic acid molecule described herein.

[0032] In another aspect, the present disclosure further provides a method for producing a modified immune effector cell, comprising the step of producing and obtaining a lentivirus vector according to a method for producing a lentivirus.

[0033] In certain embodiments, the method further comprises the step of introducing a lentivirus vector into an immune effector cell. In certain embodiments, the step of introducing refers to the step of transfecting.

[0034] In certain embodiments, immune effector cells are selected from a group consisting of T lymphocytes and natural killer cells.

[0035] In another aspect, the present disclosure further provides a modified immune effector cell comprising a modified immune effector cell produced and obtained by a method for producing a modified immune effector cell.

[0036] In certain embodiments, immune effector cells are selected from a group consisting of T lymphocytes and natural killer cells.

[0037] In certain embodiments, immune effector cells may express the chimeric antigen receptor described herein.

[0038] In another aspect, the present disclosure further provides a composition comprising modified immune effector cells obtained by the method described herein.

[0039] In certain embodiments, immune effector cells are selected from a group consisting of T lymphocytes and natural killer cells.

[0040] In certain embodiments, the chimeric antigen receptor described herein is expressed on the surface of an immune effector cell.

[0041] In another aspect, the present disclosure further provides for the use of immune effector cells in the manufacture of immune effector cells produced by the above method, and / or in compositions and / or medicines comprising immune effector cells, chimeric antigen receptors, nucleic acid molecules, and vectors, wherein the medicines are useful for the treatment of diseases or disorders associated with CD19 expression.

[0042] In another aspect, the present disclosure further provides a method for treating a disease or disorder associated with CD19 expression, comprising applying a composition and / or immune effector cells.

[0043] In another aspect, the present disclosure further provides a composition and / or immune effector cells for use in the treatment of a disease or disorder associated with CD19 expression.

[0044] In certain embodiments, the disease or disorder associated with CD19 expression includes a non-solid tumor.

[0045] In certain embodiments, non-solid tumors include leukemia and / or lymphoma.

[0046] In certain embodiments, diseases or disorders associated with CD19 expression include acute lymphoblastic leukemia and / or B-cell lymphoma.

[0047] In certain embodiments, acute lymphoblastic leukemia includes acute lymphoblastic leukemia in adults and / or acute lymphoblastic leukemia in children.

[0048] In a specific embodiment, the medicine for treating acute lymphoblastic leukemia is 0.25 × 10 8 Up to 0.5×10 8 It is administered as a dose of CAR-positive T cells.

[0049] In certain embodiments, B-cell lymphoma includes non-Hodgkin lymphoma.

[0050] In a specific embodiment, the medicine for treating non-Hodgkin lymphoma is 1×10 8Up to 2×10 8 It is administered as a dose of CAR-positive T cells.

[0051] Those skilled in the art will readily recognize other aspects and advantages of the present disclosure from the following detailed description. In the following detailed description, only exemplary embodiments of the present disclosure are illustrated and described. As will be recognized by those skilled in the art, the content of the present disclosure allows those skilled in the art to make modifications to the specific embodiments disclosed herein without departing from the spirit and scope of the invention of the present disclosure. Accordingly, the drawings and examples in the description of the present disclosure are merely illustrative rather than restrictive. Brief explanation of the drawing

[0052] Specific features related to the present disclosure are set forth in the appended claims. The features and advantages of the disclosure related to the present specification can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows: Figure 1a shows the transfection titer after transfection with a 4-plasmid system. Figure 1b shows the physical titer after transfection with a 4-plasmid system. Figure 2a shows the detection results of CAR molecules expressed on the surface of CNCT19 cells. Figure 2b shows the detection results of CAR molecules expressed on the surface of CNCT19 cells. Figure 3 shows the retention rate of tumor cells under different co-culture conditions. Figure 4 shows the death of target cells (CHO-CD19) by CNCT19 cells, which is monitored in real time by a real-time cell analysis (RTCA) dual-purpose (DP) system. Figure 5a shows the change in INF-γ concentration in the supernatant under different co-culture conditions. Figure 5b shows the change in IL-6 concentration in the supernatant under different co-culture conditions. Figure 6a shows the observation results of each test tube before shaking for 3 hours. Figure 6b shows the observation results of each test tube after shaking for 3 hours. Figure 7a shows a micrograph of the locally injected site after administration of CAR-T cells (HE staining, 10× objective lens). Figure 7b shows a micrograph of the locally injected site after administration of the sodium chloride injection solution (HE staining, 10× objective lens). Figure 8 shows the formation of soft agar colonies 3 weeks after cell inoculation in each group. Figure 9 shows the survival curve of NCG mice with Nalm-6 xenograft tumors treated with different cells. Figure 10 shows the distribution of tissue after a single administration of CNCT19 cells. Fig. 11 shows animals with tumors and animals without tumors. In vivo This is a comparison of distributed CNCT19 cells. Figure 12 shows changes in CNCT19 cells distributed in various tissues of animals after a single administration. Specific details for implementing the invention

[0053] The following specific embodiments illustrate specific embodiments of the disclosure. Those familiar with the art can easily understand the other advantages and benefits of the disclosure from the contents disclosed herein.

[0054] The disclosure is further explained below: According to the present disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Additionally, related terms and experimental procedures used herein regarding protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology are all terms and procedures widely used in the field. Meanwhile, to better understand the present disclosure, definitions and descriptions of related terms are provided below.

[0055] As used herein, the term "Chimeric Antigen Receptor (CAR)" generally refers to a fused protein comprising an extracellular domain capable of binding to an antigen and at least one intracellular domain. The CAR is a core component of a chimeric antigen receptor T cell (CAR-T) and may include a targeting moiety (e.g., a moiety that binds to a tumor-associated antigen (TAA)), a hinge region, a transmembrane region, and an intracellular domain. In this disclosure, the CAR may be combined with an intracellular domain for T cell receptor activation based on the antigen specificity of an antibody. T cells expressing the CAR can specifically recognize and eliminate malignant cells expressing a target antigen.

[0056] As used herein, the term “isolated” generally refers to that which is obtained from a natural state by artificial means. If a specific “isolated” substance or component appears in nature, it may imply that the natural environment in which it is located has been altered, or that the substance was isolated from the natural environment, or both. For example, some non-isolated polynucleotides or polypeptides occur naturally in living animals, and the same polynucleotides or polypeptides isolated from this natural state and in high purity are referred to as isolated polynucleotides or polypeptides. The term “isolated” does not exclude the possibility of mixing with artificial or synthetic materials, nor does it exclude the presence of other impurities that do not impair the activity of the substance.

[0057] As used herein, the term “immune effector cell” generally refers to a cell that participates in an immune response, such as promoting an immune effector response. In this disclosure, immune effector cells may be selected from the group consisting of T lymphocytes and natural killer cells.

[0058] As used herein, the term “specifically binds and / or specifically recognizes” generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody (or CAR structural fragment), which can determine the presence of the target when a heterogeneous cellular population of the molecule (including biomolecules) is present. For example, an antibody (or CAR structural fragment) that specifically binds to a target (which may be an epitope) is an antibody (or CAR structural fragment) that binds to the target in a more convenient manner with higher affinity and binding strength and / or for a longer period compared to binding to other targets.

[0059] As used herein, the term “isolated nucleic acid molecule” generally refers to a nucleotide, deoxyribonucleotide, or ribonucleotide of any length in its isolated form, or an analogue isolated from its natural environment or artificially synthesized.

[0060] As used herein, the terms “plasmid” or “vector” generally refer to a tool for delivering nucleic acids into which a polynucleotide encoding a specific protein may be inserted, thereby enabling the expression of the protein. A vector may be transformed, transduced, or transfected into a host cell so that the genetic material element it carries may be expressed in the host cell. For example, vectors include plasmids: phagemids; cosmids; artificial chromosomes (e.g., yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC)); phages such as λ phage or M13 phage; and animal viruses, etc. Types of animal viruses as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomatous vacuolar viruses (e.g., SV40). Vectors may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain replication origins. Vectors may also contain components that aid in entry into the cell, such as viral particles, liposomes, or protein coats, but are not limited to these materials.

[0061] As used herein, the term “composition” generally refers to a composition suitable for administration to a patient. For example, a composition according to the present disclosure may comprise the immune effector cells described herein. Furthermore, the composition may also comprise one or more suitable formulations of a (pharmaceutically effective) carrier, stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier, and / or preservative. Acceptable components of the composition are non-toxic to the receptor at any dose and concentration used. The compositions of the present disclosure include, but are not limited to, liquid, and freeze-dried compositions.

[0062] As used herein, the term “CD19” refers to a cluster of differentiated (CD) 19 proteins, which is a cluster of epitopes that can typically be detected in leukemia precursor cells. Amino acid and nucleic acid sequences of human and Muirline CD19 are available in public databases (e.g., GenBank, UniProt, and Swiss-Prot). For example, the amino acid sequence of human CD19 is accessible under UniProt / Swiss-Prot accession number P15391, and the nucleotide sequence encoding human CD19 is accessible under accession number NM_001178098. According to the present disclosure, “CD19” may include proteins having mutations (e.g., point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type CD19).

[0063] As used in this specification, the term “object” generally refers to a human or non-human animal including, but not limited to, cats, dogs, horses, pigs, cattle, sheep, rabbits, mice, rats, or monkeys.

[0064] As used herein, the term “lentivirus vector” generally refers to a vector comprising one or more nucleic acid sequences derived from at least a portion of a lentivirus genome. A lentivirus vector may comprise one or more non-coding sequences of proteins from a lentivirus.

[0065] The term “target plasmid” as used herein may include, for example, a heterogeneous nucleic acid sequence to be delivered into a cell (e.g., a nucleic acid sequence encoding CAR), and may additionally include, for example, one or more lentivirus genes or parts thereof.

[0066] The "helper plasmid" as used herein may contain one or more genes encoding a lentivirus protein or a part thereof. For example, it may include a gene encoding a lentivirus capsid protein, and as another example, a gene encoding an env protein or a part thereof. A virus may be produced by transfecting a host cell with a target plasmid and one or more helper plasmids, which may be used to infect target cells (e.g., T cells) to express one or more transplant genes (e.g., a gene encoding CAR) contained in a heterologous nucleic acid sequence in the target cells.

[0067] As used in this specification, the term "comprising" generally refers to the inclusion of an explicitly specified feature but without excluding other elements.

[0068] As used herein, the term “about” generally refers to a change within a range greater or less than the specified value, such as a change within a range greater or less than 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.

[0069] 4-Plasmid System

[0070] In one embodiment, the present disclosure provides a plasmid combination, wherein the plasmid combination comprises plasmids Seq1, PMD2.G, pMDLg-pRRE, and pRSV-Rev.

[0071] A 4-plasmid system is used to transfect a lentivirus according to the method described in the description thereof in this disclosure. The 4-plasmid system may include a target plasmid (Seq1) and three helper plasmids (PMD2.G, pMDLg-pRRE and pRSV-Rev).

[0072] According to the present disclosure, the target plasmid may be Seq1. Seq 1 carries the gene sequence of interest. In the vector plasmid, the gene of interest is inserted into the genome of a target cell (293 cell) by a lentiviral vector to achieve stable expression of the CAR. The transfer plasmid has the amino acid sequence shown in SEQ ID NO. 1 and the nucleic acid sequence shown in SEQ ID NO. 2.

[0073] Helper plasmid 1 may be PMD2.G, which is used to provide the VSV-G gene encoding the vesicular stomatitis virus glycoprotein G. The VSVG-envelope-like lentivirus vector expands the vector's target cell affinity range and increases the stability of the lentivirus, thereby allowing the lentivirus vector to be concentrated by high-speed centrifugation to obtain higher titers.

[0074] Helper plasmid 2 is used to provide the Rev protein-binding site and may be pMDLg-pRRE containing Gag and Pol genes. The Gag gene encodes major structural proteins of the viral particle, such as nucleocapsid proteins, membrane proteins, and capsid proteins. The Pol gene encodes viral replication-associated enzymes, such as proteases, reverse transcriptases, and integrases. It plays an important role in viral assembly.

[0075] Helper plasmid 3 can be pRSV-Rev, which is used to regulate the expression levels of Gag and Pol genes and to provide the Rev gene to guide the replication process of single-stranded DNA, and can regulate splicing / RNA transfer. It plays an important role in viral assembly.

[0076] In certain embodiments, plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev are 2-6: 1-1.5: 1-3: 1-1.5, e.g. 2-5: 1-1.5: 1-3: 1-1.5, 2-4: 1-1.5: 1-3: 1-1.5, 2-3: 1-1.5: 1-3: 1-1.5, 3-6: 1-1.5: 1-3: 1-1.5, 3-5: 1-1.5: 1-3: 1-1.5, 3-4: 1-1.5: 1-3: 1-1.5, 4-6: 1-1.5: 1-3: 1-1.5, 4-5: 1-1.5: 1-3: It may be present in plasmid combinations in a ratio of 1-1.5, or 5-6: 1-1.5: 1-3: 1-1.5. In certain embodiments, plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev are 1-1.5: 1-3: 1-1.5, e.g. 2.1: 1-1.5: 1-3: 1-1.5, 2.2: 1-1.5: 1-3: 1-1.5, 2.3: 1-1.5: 1-3: 1-1.5, 2.4: 1-1.5: 1-3: 1-1.5, 2.5: 1-1.5: 1-3: 1-1.5, 2.6: 1-1.5: 1-3: 1-1.5, 2.7: 1-1.5: 1-3: 1-1.5, 2.8: 1-1.5: 1-3: 1-1.5, 2.9: 1-1.5: 1-3: 1-1.5, 3.0: 1-1.5: 1-3: 1-1.5, 3.1: 1-1.5: 1-3: 1-1.5, 3.2: 1-1.5: 1-3: 1-1.5, 3.3: 1-1.5: 1-3: 1-1.5, 3.4: 1-1.5: 1-3: 1-1.5, 3.5: 1-1.5: 1-3: 1-1.5, 3.6: 1-1.5: 1-3: 1-1.5, 3.7: 1-1.5: 1-3: 1-1.5, 3.8: 1-1.5: 1-3: 1-1.5, 3.9: 1-1.5: 1-3: 1-1.5, 4.0: 1-1.5: 1-3: 1-1.5, 4.1: 1-1.5: 1-3: 1-1.5, 4.2: 1-1.5: 1-3: 1-1.5, 4.3: 1-1.5: 1-3: 1-1.5, 4.4: 1-1.5: 1-3: 1-1.5, 4.5: 1-1.5: 1-3: 1-1.5, 4.6: 1-1.5: 1-3: 1-1.5, 4.7: 1-1.5: 1-3: 1-1.5, 4.8: 1-1.5: 1-3: 1-1.5, 4.9: 1-1.5: 1-3: 1-1.5, 5.0: 1-1.5: 1-3: 1-1.5, 5.1: 1-1.5: 1-3: 1-1.5, 5.2: 1-1.5: 1-3: 1-1.5, 5.3: 1-1.5: 1-3: 1-1.5, 5.4: 1-1.5: 1-3: 1-1.5, 5.5: 1-1.5: 1-3: 1-1.5, It may exist in the ratio of 5.6: 1-1.5: 1-3: 1-1.5, 5.7: 1-1.5: 1-3:1-1.5, 5.8: 1-1.5: 1-3: 1-1.5, 5.9: 1-1.5: 1-3: 1-1.5, or 6.0: 1-1.5: 1-3: 1-1.5.

[0077] In certain embodiments, plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev may be present in the plasmid combination in a ratio of 2-6: 1-1.5: 1-3: 1-1.5, e.g. 2-6: 1-1.3: 1-3: 1-1.5, 2-6: 1.2-1.5: 1-3: 1-1.5, or 2-6: 1.1-1.4: 1-3: 1-1.5. In certain embodiments, plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev may be present in a ratio of 2-6: 1-1.5: 1-3: 1-1.5, e.g., 2-6: 1.1: 1-3: 1-1.5, 2-6: 1.2: 1-3: 1-1.5, 2-6: 1.3: 1-3: 1-1.5, 2-6: 1.4: 1-3: 1-1.5, or 2-6: 1.5: 1-3: 1-1.5.

[0078] In certain embodiments, plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev are 2-6: 1-1.5: 1-3: 1-1.5, e.g. 2-6: 1-1.5: 1-2.5: 1-1.5, 2-6: 1-1.5: 1-2.0: 1-1.5, 2-6: 1-1.5: 1-1.5: 1-1.5, 2-6: 1-1.5: 1.5-3: 1-1.5, 2-6: 1-1.5: 1.5-2.5: 1-1.5, 2-6: 1-1.5: 1.5-2: 1-1.5, 2-6: 1-1.5: 2-3: 1-1.5, 2-6: It may exist in plasmid combinations in a ratio of 1-1.5: 2-2.5: 1-1.5, or 2-6: 1-1.5: 2.5-3: 1-1.5. In certain embodiments, plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev are 2-6: 1-1.5: 1-3: 1-1.5, e.g. 2-6: 1-1.5: 1.1: 1-1.5, 2-6: 1-1.5: 1.2: 1-1.5, 2-6: 1-1.5: 1.3: 1-1.5, 2-6: 1-1.5: 1.4: 1-1.5, 2-6: 1-1.5: 1.5: 1-1.5, 2-6: 1-1.5: 1.6: 1-1.5, 2-6: 1-1.5: 1.7: 1-1.5, 2-6: 1-1.5: 1.8: 1-1.5, 2-6: 1-1.5: 1.9: 1-1.5, 2-6: 1-1.5: 2: 1-1.5, 2-6: 1-1.5: 2.1: 1-1.5, 2-6: 1-1.5: 2.2: 1-1.5, 2-6: 1-1.5: 2.3: 1-1.5, 2-6: 1-1.5: 2.4: 1-1.5, 2-6: 1-1.5: 2.5: 1-1.5, 2-6: 1-1.5: 2.6: 1-1.5, 2-6: 1-1.5: 2.7: 1-1.5, 2-6: 1-1.5: 2.8: 1-1.5, It may exist in a ratio of 2-6: 1-1.5: 2.9: 1-1.5, or 2-6: 1-1.5: 3: 1-1.5.

[0079] In certain embodiments, plasmids Seq1, PMD2.G, pMDLg-pRRE, and pRSV-Rev may be present in the plasmid combination in a ratio of 11.8: 3.53: 6.33: 2.3, 13.8: 3.48: 5.31: 2.54, or 14: 4.67: 4.67: 4.67. In certain embodiments, plasmids Seq1, PMD2.G, pMDLg-pRRE, and pRSV-Rev may be present in a ratio of 11.8: 3.53: 6.33: 2.3. In certain embodiments, plasmids Seq1, PMD2.G, pMDLg-pRRE, and pRSV-Rev may be present in a ratio of 13.8: 3.48: 5.31: 2.54. In certain embodiments, plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev may be present in a ratio of 14: 4.67: 4.67: 4.67.

[0080] Chimeric antigen receptor, nucleic acid, vector, immune effector cell, and composition

[0081] The plasmid Seq1 of the present disclosure may express a chimeric antigen receptor comprising the amino acid sequence shown in SEQ ID NO. 1. The present disclosure further provides a chimeric antigen receptor comprising an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence shown in SEQ ID NO. 1.

[0082] In certain embodiments, the chimeric antigen receptor described herein may specifically bind to and / or recognize a tumor antigen. For example, the chimeric antigen receptor described herein may specifically bind to and / or recognize a CD19 antigen.

[0083] In certain embodiments, the chimeric antigen receptor described herein may stimulate immune effector cells to secrete cytokines. Immune effector cells may be selected from the group consisting of T lymphocytes and natural killer cells. Cytokines may be selected from the group consisting of IFN-γ and IL-6. Immune effector cells may be mammalian immune effector cells. T lymphocytes may be mammalian T lymphocytes, and natural killer cells may also be mammalian natural killer cells. T lymphocytes may be human T lymphocytes, and natural killer cells may also be human natural killer cells.

[0084] In certain embodiments, the chimeric antigen receptor described herein is non-hemolytic and does not stimulate blood vessels.

[0085] In certain embodiments, the chimeric antigen receptor described herein is In vitro It is non-tumor-genic.

[0086] In certain embodiments, the chimeric antigen receptor described herein is In vivo It is non-tumor-genic.

[0087] In certain embodiments, the chimeric antigen receptor described herein can effectively treat a tumor. The tumor may be a CD19-positive tumor. For example, the chimeric antigen receptor described herein can effectively extend the survival time of a patient with a CD19-positive tumor. For example, the chimeric antigen receptor described herein can effectively extend the survival time of a patient with a non-solid tumor. For example, the chimeric antigen receptor described herein can effectively extend the survival time of a patient with a lymphoma and / or leukemia. As another example, the chimeric antigen receptor described herein can effectively extend the survival time of an adult patient with acute lymphoblastic leukemia. As yet another example, the chimeric antigen receptor described herein can effectively extend the survival time of a pediatric patient with acute lymphoblastic leukemia. As yet another example, the chimeric antigen receptor described herein can effectively extend the survival time of a patient with B-cell lymphoma (e.g., non-Hodgkin lymphoma).

[0088] In certain embodiments, the chimeric antigen receptor described herein can effectively treat acute lymphoblastic leukemia in adults.

[0089] In certain embodiments, the chimeric antigen receptor described herein can effectively treat acute lymphoblastic leukemia in children.

[0090] In certain embodiments, the chimeric antigen receptor described herein can effectively treat non-Hodgkin lymphoma.

[0091] The plasmid Seq1 of the present disclosure comprises an isolated nucleic acid molecule encoding the chimeric antigen receptor described herein.

[0092] The plasmid Seq1 of the present disclosure comprises an isolated nucleic acid molecule encoding a chimeric antigen receptor, wherein the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO. 2.

[0093] The plasmid Seq1 of the present disclosure comprises an isolated nucleic acid molecule encoding a chimeric antigen receptor, wherein the nucleic acid molecule comprises a nucleic acid sequence similar to the sequence shown in SEQ ID NO. 2 and is a nucleic acid molecule encoding a chimeric antigen receptor.

[0094] In a specific embodiment, a nucleic acid sequence similar to the sequence shown in SEQ ID NO. 2 refers to a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO. 2.

[0095] In certain embodiments, a nucleic acid sequence similar to the sequence shown in SEQ ID NO. 2 means that the nucleic acid molecule may encode a chimeric antigen receptor that is different from the nucleic acid sequence shown in SEQ ID NO. 2 due to a wobble (degeneracy) of the base at position 3 of the nucleic acid codon.

[0096] The present disclosure comprises variants of genes and proteins having one or more biological activities (e.g., variants of the amino acid sequence shown in SEQ ID NO. 1, or variants of the nucleic acid sequence shown in SEQ ID NO. 2 as described herein). Such variants of proteins or polypeptides comprise proteins or polypeptides that have been or may be modified using recombinant DNA technology to have altered or additional properties; for example, variants confer enhanced stability in plasma or increased activity for the protein. Variants may differ from the reference sequence and may differ, for example, from naturally occurring polynucleotides, proteins, or peptides. At the nucleotide sequence level, naturally occurring variant genes and non-naturally occurring variant genes will have at least about 50%, more typically at least about 70%, and much more typically at least about 80% identity (90% or higher identity) with respect to the reference gene. At the amino acid sequence level, naturally occurring variant proteins and non-natural variant proteins will typically have at least about 70%, more typically at least about 80%, and much more typically at least about 90% or more identity with the reference protein, while allowing for higher percentage non-identity regions in non-conserved regions (e.g., percentage identity is less than 70%, e.g., less than 60%, less than 50%, or even less than 40%). In other embodiments, the sequence has at least 60%, 70%, 75% or more identity with the reference sequence (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity). Modification procedures for introducing nucleotides and amino acids into polynucleotides, proteins, or polypeptides are known to those skilled in the art (e.g., see Sambrook et al. (1989)).

[0097] The terms “identity,” “homology,” and their grammatical variations as used herein generally mean that two or more entities are identical when their sequences are “aligned.” Thus, for example, when two polypeptides have the same sequence, they have the same amino acid sequence at least within a reference region or part. If two polynucleotides have the same sequence, they have the same polynucleotide sequence at least within a reference region or part. Identity may be identity of a defined region (region or domain) of a sequence. The “region” or “domain” of identity refers to the same part of two or more reference entities. Thus, if two protein or nucleic acid sequences are identical in one or more sequence regions or regions, they have identity in that region. An “aligned” sequence refers to a polynucleotide or protein (amino acid) sequence that, compared to a reference sequence, often contains supplementary or additional bases or amino acids (gaps). The degree of identity (homology) between two sequences can be determined using computer programs and mathematical algorithms. These algorithms that calculate percent sequence identity (homology) generally calculate sequence gaps and mismatches in the regions or areas being compared. For example, the BLAST (e.g., BLAST 2.0) search algorithm (see, e.g., Altschul et al., J.Mol. Biol. 215: 403(1990), publicly available at NCBI) provides the following exemplary search parameters: mismatch -2, gap opening 5, gap extension 2.

[0098] According to the present disclosure, a nucleic acid molecule may be a nucleotide of any length, a deoxyribonucleotide or a ribonucleotide in its isolated form, or an analog isolated from its natural environment or artificially synthesized, as long as it can encode the chimeric antigen receptor described herein.

[0099] In another aspect, the present disclosure provides a vector comprising a nucleic acid molecule described herein.

[0100] According to the present disclosure, a vector may be transformed, transduced, or transfected into a host cell so that the genetic material element it carries can be expressed in the host cell. For example, the vector includes plasmids; phages; cosmids; artificial chromosomes (e.g., yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC)); phages such as λ phage or M13 phage; and animal viruses, etc. Types of animal viruses as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papilloma vacuolar viruses (e.g., SV40). As another example, the vector may contain various elements that control expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. Additionally, the vector may include a replication origin. Furthermore, the vector may contain components that aid in entry into the cell, such as viral particles, liposomes, or protein coats, but is not limited to these materials.

[0101] In one embodiment, the present disclosure provides a method for preparing a lentivirus vector, the method comprising introducing a plasmid combination into a cell.

[0102] In another aspect, the present disclosure provides an immune effector cell comprising a chimeric antigen receptor, a nucleic acid molecule, and / or a vector as described herein.

[0103] According to the present disclosure, immune effector cells may be selected from the group consisting of T lymphocytes and natural killer cells. In certain embodiments, the immune effector cells may be human immune effector cells. For example, the immune effector cells may be human T lymphocytes. In another example, the immune effector cells may be human natural killer cells.

[0104] According to the present disclosure, the chimeric antigen receptor described in this specification is expressed on the surface of immune effector cells.

[0105] In certain embodiments, the immune effector cells described herein can effectively kill tumor cells. The tumor cells may be CD19-positive cells. For example, the immune effector cells described herein can significantly reduce the retention rate of CD19-positive human leukemia cell line Nalm-6 cells.

[0106] In certain embodiments, the immune effector cells described herein can effectively promote cytokine secretion when in contact with CD19-positive cells. The cytokines may be selected from the group consisting of IFN-γ and IL-6. For example, co-culturing the immune effector cells described herein with the CD19-positive human leukemia cell line Nalm-6 cells results in a significant increase in the secretion of IFN-γ and IL-6 cytokines.

[0107] In certain embodiments, the immune effector cells described herein are non-hemolytic and do not stimulate blood vessels. For example, In vitro In hemolysis tests, the immune effector cells described herein will not induce hemolysis and blood agglutination. As another example, the immune effector cells described herein do not stimulate blood vessels.

[0108] In certain embodiments, the immune effector cells described herein are In vitro It is non-tumor-genic.

[0109] In certain embodiments, the immune effector cells described herein are In vivo It is non-tumor-genic.

[0110] In certain embodiments, the immune effector cells described herein can effectively treat a tumor. The tumor may be a CD19-positive tumor. For example, the immune effector cells described herein can effectively extend the survival time of a patient with a CD19-positive tumor. As another example, the immune effector cells described herein can effectively extend the survival time of an adult patient with acute lymphoblastic leukemia. As yet another example, the immune effector cells described herein can effectively extend the survival time of a pediatric patient with acute lymphoblastic leukemia. As yet another example, the immune effector cells described herein can effectively extend the survival time of a patient with B-cell lymphoma (e.g., non-Hodgkin lymphoma).

[0111] In certain embodiments, the immune effector cells described herein can effectively treat acute lymphoblastic leukemia in adults.

[0112] In certain embodiments, the immune effector cells described herein can effectively treat acute lymphoblastic leukemia in children.

[0113] In certain embodiments, the immune effector cells described herein can effectively treat B-cell lymphoma (e.g., non-Hodgkin lymphoma).

[0114] In another aspect, the present disclosure provides a composition comprising immune effector cells as described herein.

[0115] According to the present disclosure, the composition may also comprise one or more suitable formulations of a (pharmaceutically effective) carrier, stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier, and / or preservative. The acceptable components of the composition are non-toxic to the receptor at any dose and concentration used. The compositions of the present disclosure include, but are not limited to, liquids and frozen or lyophilized compositions.

[0116] In certain embodiments, the composition may be for parenteral, transdermal, lumenal, arterial, intrathecal and / or nasal administration, or direct injection into tissue. For example, the composition may be administered to a patient or subject via infusion or injection. In other embodiments, administration of the composition may be performed by different means, e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, local, or intradermal administration. In other embodiments, the composition may be administered without interruption. Uninterrupted (or continuous) administration may be realized by a small pump system worn by the patient to measure the inflow of the therapeutic agent into the patient's body, as described in WO2015 / 036583.

[0117] According to the present disclosure, the dosage regimen of the composition may be a dose of a rapid infusion agent, multiple divided doses administered over time, or the dose may be reduced or increased in proportion to the severity and urgency of the treatment situation. In certain embodiments, the treatment regimen may be administered once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. In certain embodiments, the administration regimen comprises intravenous administration, and the dose is 0.1 × 10⁻⁶ 8 Up to 3×10 8 CAR-positive T cells, e.g., 0.15 × 10⁶ 8 Up to 2×10 8 CAR-positive T cells, 0.5×10 8 Up to 2×10 8CAR-positive T cells, 1×10 8 Up to 2×10 8 CAR-positive T cells, 0.2×10 8 Up to 2×10 8 CAR-positive T cells, 0.2×10 8 Up to 1×10 8 CAR-positive T cells, 0.25×10⁶ 8 Up to 1×10 8 CAR-positive T cells, 0.25×10⁶ 8 Up to 0.5×10 8 CAR-positive T cells, or 0.5×10⁶ 8 CAR-positive T cells, or 2×10⁶ 8 It can be administered in the range of CAR-positive T cells.

[0118] The dosage for administration may vary for different indications. In certain embodiments, immune effector cells for the treatment of adult patients with relapsed and refractory acute lymphoblastic leukemia are 0.25 × 10⁶ 8 Up to 0.5×10 8 CAR-positive T cells, or 0.5×10⁶ 8 CAR-positive T cells, e.g., 0.3 × 10⁶ 8 Up to 0.5×10 8 , 0.4×10 8 Up to 0.5×10 8 , 0.25×10 8 Up to 0.4×10 8 , 0.3×10 8 Up to 0.4×10 8 , or 0.4×10 8 Up to 0.5×10 8 It can be administered at a dose of CAR-positive T cells. In certain embodiments, immune effector cells for the treatment of adult patients with relapsed and refractory acute lymphoblastic leukemia are 0.25 × 10⁶ 8 , 0.26×10 8 , 0.27×10 8 , 0.28×10 8 , 0.29×10 8 , 0.3×108 , 0.31×10 8 , 0.32×10 8 , 0.33×10 8 , 0.34×10 8 , 0.35×10 8 , 0.36×10 8 , 0.37×10 8 , 0.38×10 8 , 0.39×10 8 , 0.4×10 8 , 0.41×10 8 , 0.42×10 8 , 0.43×10 8 , 0.44×10 8 , 0.45×10 8 , 0.46×10 8 , 0.47×10 8 , 0.48×10 8 , 0.49×10 8 or 0.5×10 8 It can be administered in doses of CAR-positive T cells.

[0119] In a specific embodiment, immune effector cells for the treatment of pediatric patients with relapsed and refractory acute lymphoblastic leukemia are 0.25 × 10⁶ 8 Up to 0.5×10 8 CAR-positive T cells, or 0.5×10⁶ 8 CAR-positive T cells, e.g., 0.3 × 10⁶ 8 Up to 0.5×10 8 , 0.4×10 8 Up to 0.5×10 8 , 0.25×10 8 Up to 0.4×10 8 , 0.3×10 8 Up to 0.4×10 8 , or 0.4×10 8 Up to 0.5×10 8 It can be administered at a dose of CAR-positive T cells. In some embodiments, immune effector cells for the treatment of pediatric patients with relapsed and refractory acute lymphoblastic leukemia are 0.25 × 10⁶ 8 , 0.26×108 , 0.27×10 8 , 0.28×10 8 , 0.29×10 8 , 0.3×10 8 , 0.31×10 8 , 0.32×10 8 , 0.33×10 8 , 0.34×10 8 , 0.35×10 8 , 0.36×10 8 , 0.37×10 8 , 0.38×10 8 , 0.39×10 8 , 0.4×10 8 , 0.41×10 8 , 0.42×10 8 , 0.43×10 8 , 0.44×10 8 , 0.45×10 8 , 0.46×10 8 , 0.47×10 8 , 0.48×10 8 , 0.49×10 8 or 0.5×10 8 It can be administered in doses of CAR-positive T cells.

[0120] In another embodiment, immune effector cells for the treatment of patients with relapsed and refractory non-Hodgkin lymphoma are 1×10 8 Up to 2×10 8 CAR-positive T cells, or 2×10⁶ 8 CAR-positive T cells, e.g., 1×10⁶ 8 Up to 1.8×10 8 , 1×10 8 Up to 1.5×10 8 , 1×10 8 Up to 1.3×10 8 , 1.3×10 8 Up to 2×10 8 , 1.3×10 8 Up to 1.5×10 8 , 1.5×10 8 Up to 2×10 8 , 1.5×108 Up to 1.8×10 8 or 1.8×10 8 Up to 2×10 8 It can be administered at a dose of CAR-positive T cells. In another embodiment, immune effector cells for the treatment of patients with relapsed and refractory Hodgkin lymphoma are 1 × 10⁶ 8 , 1.1×10 8 , 1.2×10 8 , 1.3×10 8 , 1.4×10 8 , 1.5×10 8 , 1.6×10 8 , 1.7×10 8 , 1.8×10 8 , 1.9×10 8 or 2.0×10 8 It can be administered in doses of CAR-positive T cells.

[0121] Manufacturing method and use

[0122] In another aspect, the present disclosure further provides a method for producing an immune effector cell, comprising the step of transducing a plasmid or vector described herein into an immune effector cell.

[0123] In certain embodiments, immune effector cells are selected from a group consisting of T lymphocytes and natural killer cells.

[0124] In another aspect, the present disclosure further provides for the use of chimeric antigen receptors, nucleic acid molecules, vectors and / or immune effector cells in the manufacture of a medicine, wherein the medicine is useful for the treatment of a disease or disorder associated with CD19 expression. The dose of the medicine for administration may refer to the dose defined for immune effector cells as mentioned above.

[0125] In another aspect, the present disclosure further provides a method for treating a disease or disorder associated with CD19 expression, comprising applying the chimeric antigen receptor, nucleic acid molecule, vector, and / or immune effector cell described herein to a subject in need of treatment. According to the present disclosure, administration of the composition may be performed in different ways, e.g., intravenously, intratumorally, intraperitoneally, subcutaneously, intramuscularly, locally, or intradermally.

[0126] In another aspect, the chimeric antigen receptor, nucleic acid molecule, vector, and / or immune effector cell described herein are useful for the treatment of diseases or disorders associated with CD19 expression.

[0127] According to the present disclosure, the medicine may include a T-cell immunotherapy agent.

[0128] According to the present disclosure, diseases or disorders associated with CD19 expression may include non-solid tumors.

[0129] According to the present disclosure, diseases or disorders associated with CD19 expression may include leukemia and / or lymphoma.

[0130] In certain embodiments, the disease or disorder associated with CD19 expression may include acute lymphoblastic leukemia (ALL), e.g., adult acute lymphoblastic leukemia (ALL) and / or pediatric acute lymphoblastic leukemia (ALL).

[0131] In another embodiment, the disease or disorder associated with CD19 expression may include adult chronic lymphoblastic leukemia (CLL). In another embodiment, the disease or disorder associated with CD19 expression may include B-cell lymphoma. For example, B-cell lymphoma may include non-Hodgkin lymphoma.

[0132] According to the present disclosure, the subject may include a human or non-human animal. For example, the subject may include, but is not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0133] Without being bound by any theory, the following examples are described only to illustrate the chimeric antigen receptors, immune effector cells, methods of preparation, and uses of the present disclosure and are not intended to limit the scope thereof. The examples do not include detailed descriptions of traditional methods, such as methods used to construct vectors and plasmids, methods for inserting genes encoding proteins into these vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and are disclosed in many publications, including Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.

[0134] All patents, applications, and references cited in this disclosure are incorporated herein by reference in their entirety to the extent that each document is cited individually by reference. If any material incorporated by reference contradicts or is inconsistent with this Detailed Description, the Detailed Description replaces any such material.

[0135] Examples

[0136] Example 1. Construction and preparation of a lentivirus vector

[0137] A fragment containing the CAR structure described in the present disclosure (its amino acid and base sequences are denoted as SEQ ID NO. 1 and SEQ ID NO. 2, respectively) was artificially synthesized and constructed into a modified empty vector (Manufacturer: SBI Corporation, Catalog No.: CD500-CD800). A lentiviral vector was obtained using three helper plasmids for packaging, and the obtained lentiviral vector was named the expression vector CNCT19. The specific steps are described as follows.

[0138] 1.1 Construction of Plasmid Vectors

[0139] In the present disclosure, a 4-plasmid system containing a target plasmid (Seq1), PMD2.G, pMDLg-pRRE, and pRSV-Rev was used.

[0140] First, the target plasmid Seq1 was constructed. An empty vector (Manufacturer: SBI System BioScience, Designation: PCDH-EF1-MCS-T2A-copGFP, Catalog No.: CD526A-1) was cleaved with two restriction enzymes, Nhe I (GCTAGC) and Not I (GCGGCCGC), ligated to a CAR gene fragment encoding the amino acid sequence shown in SEQ ID NO. 1 containing Nhe I and Not I enzyme-digested sequences at both ends, and the GFP sequence was removed to obtain Vector 1. Vector 1 was cleaved with two restriction enzymes, Not I (GCGGCCGC) and Sal I (GTCGAC), and the T2A sequence was removed. The sticky ends were blunted with Krenau cleavage enzyme, and Solution I ligase was added for ligation to obtain Vector 2. PCR was performed to amplify the sequence of vector ② excluding the ampicillin resistance (AmpR) gene, amplify the synthetic kanamycin resistance (KanR) gene, and then the two fragments were ligated with a recombinant enzyme to obtain target plasmid Seq1 containing the nucleotide sequence shown in Sequence No. 2.

[0141] Subsequently, three helper plasmids were manufactured, where helper plasmid 1 was PMD2.G (purchased from Biovector with product number Biovector 12259); helper plasmid 2 was pMDLg-pRRE (purchased from Biovector Corporation with product number Biovector 012251); and helper plasmid 3 was pRSV-Rev (purchased from Biovector Corporation with product number Biovector 012253).

[0142] Four plasmids, Seq1, PMD2.G, pMDLg-pRRE, and pRSV-Rev, were used for packaging in the following four different plasmid ratios:

[0143] The ratio 1 of Seq1: PMD2.G: pMDLg-pRRE: pRSV-Rev was 11.8:3.53:6.33:2.3;

[0144] The ratio 2 of Seq1: PMD2.G: pMDLg-pRRE: pRSV-Rev was 13.8:3.48:5.31:2.54;

[0145] The ratio 3 of Seq1: PMD2.G: pMDLg-pRRE: pRSV-Rev was 14:4.67:4.67:4.67;

[0146] The ratio 4 of Seq1: PMD2.G: pMDLg-pRRE: pRSV-Rev was 2:1:1:1 and;

[0147] The ratio 5 of Seq1: PMD2.G: pMDLg-pRRE: pRSV-Rev was 7:3:5:5;

[0148] The ratio 6 of Seq1: PMD2.G: pMDLg-pRRE: pRSV-Rev was 9:3:4:6; and

[0149] In the comparative example, the ratio of Seq1:PsPAX2:PMD2.G was 14:10.5:3.5.

[0150] 1.2 Preparation of Lentivirus Vectors

[0151] (1) Cell recovery: Cryopreserved 293T / 17 cells were recovered and the recovered cells were cultured at 37℃ in 5% CO2;

[0152] (2) Subculture of cells: The recovered cells were subcultured in a T175 culture flask until the cell growth density reached 85-95% of the bottom of the flask;

[0153] (3) Cell pretreatment: Cells 5×10 6 Cells / dishes were added to 10 cm culture dishes and incubated at 37°C with 5% CO2;

[0154] (4) Preparation of packaging system: 2 ml of DMEM was taken, the target plasmid and 3 helper plasmids were added in specific ratios to form a 4-plasmid system, 28 μl of PEI was added, the mixture was thoroughly mixed, and left to stand at room temperature for 15 minutes;

[0155] (5) Plasmid transfection: Cells were pretreated and cultured until the cell growth density reached 60-90% of the bottom of the dish, and the prepared solution of the packaging system was transferred to a culture dish for transfection; transfection was performed at 37°C with 5% CO2;

[0156] (6) Collection of viruses: 16-18 hours after transfection, the medium was replaced with an equal volume of DMEM containing 10% FBS and incubation was continued for 24 hours to collect the virus liquid for the first time, then the medium was replaced and an equal volume of DMEM containing 10% FBS was added and incubation was performed for another 24 hours to collect the virus liquid for the second time, where incubation was performed at 37°C with 5% CO2;

[0157] (7) Purification of virus liquid: The lentivirus vector liquid was centrifuged at 3000 rpm at 19°C for 15 minutes, the precipitate was discarded, and the supernatant was obtained;

[0158] (8) Virus concentration: The supernatant was filtered through a high-speed centrifuge tube with a 0.45 μm filter and centrifuged at a speed of 50,000 g at 4°C for 90 minutes. The supernatant was discarded, and the precipitate was lysed in lymphocyte culture medium and stored in a low-temperature refrigerator at -80°C for later use. A lentivirus vector packaged by a 4-plasmid system was obtained and named the CNCT19 lentivirus vector.

[0159] 1.3 Determination of Physical Tit and Transfection Tit

[0160] 210 μl / tube of concentrated virus solution was taken, physical titer was determined using ELISA, and transfection titer was detected using a flow cytometer. Physical titer represents the number of lentivirus particles. Transfection titer represents the number of active lentivirus particles.

[0161] The test results are shown in Tables 1 and 2, and Figures 1a and 1b. The results in Table 1, Figure 1a, and Figure 1b show that the configuration of the lentivirus packaging system resulted in nearly identical packaging efficiency in the quality ratios of the four plasmids (Seq1: PMD2.G: pMDLg-pRRE: pRSV-Rev) of 11.8: 3.53: 6.33: 2.3, 13.8: 3.48: 5.31: 2.54, and 14: 4.67: 4.67: 4.67, and all induced higher transfection titers than the comparative example. Table 2 shows that the quality ratios of the four plasmids (Seq1: PMD2.G: pMDLg-pRRE: pRSV-Rev) of 7:3:5:5 and 9:3:4:6 induced significantly higher transfection titers than those of ratio 4 and the comparative example, reaching transfection titers that were more than twice as high as those resulting from ratio 4 and the comparative example. The results show that the transfection titers resulting from ratios 1, 2, and 3 were higher than those resulting from ratios 4, 5, and 6 and the comparative example, where the transfection titers resulting from ratios 1, 2, and 3 were more than twice as high as those resulting from ratio 4 and the comparative example.

[0162] [Table 1]: Transfection titers and physical titers resulting from ratios 1, 2, and 3

[0163]

[0164] [Table 2]: Transfection titers and physical titers resulting from ratios 4, 5, 6, and 7 and the ratios of the comparative example

[0165]

[0166] Example 2. Preparation of T cells infected with lentivirus

[0167] Infection experiments were performed according to conventional methods known to those skilled in the art. The stages of infection are briefly described as follows:

[0168] 1. Classification of T cells

[0169] Peripheral blood mononuclear cells (PBMCs) were isolated from the subject's plasma exchange cells, and then T cells were separated from the PBMC cells.

[0170] 2. T cell activation

[0171] Isolated T cells were cultured in complete lymphocyte culture medium (Xvivo15 medium + 5% FBS + 100 IU / ml IL-2 or Xvivo15 medium + 5% FBS + 20 ng / ml IL-21 + 10 ng / ml IL-7) to a final concentration of (1–2) × 10⁶ 6 The cells were resuspended in ml, and 5 to 10 μl of CD3 / CD28 stimulating magnetic beads were added. The mixture was thoroughly mixed and placed in an incubator for culture for at least 24 hours under culture conditions of 37°C + 5% CO2.

[0172] 3. Infection of T cells with lentivirus

[0173] Activated cultured T cells were extracted and polybrene was added at a final concentration of 8 μg / ml and mixed thoroughly. The CNCT19 lentivirus vector obtained in Example 1 was slowly added at an MOI of 2. After mixing thoroughly, the mixture was placed in a centrifuge and centrifuged at 1500 rpm for 1.5 hours. It was then placed in an incubator for culture for at least 24 hours under culture conditions of 37°C + 5% CO2.

[0174] 4. Extended culture of infected T cells

[0175] Infected cells are extracted and cell density is monitored (0.5~1)×10 for use in subsequent examples. 6 The cells were maintained at a cell / ml level. The obtained infected T cells were named CNCT19 cells (i.e., immune effector cells as described herein).

[0176] Example 3. Detection of CAR molecule expression on the surface of CNCT19 cells

[0177] The steps of the experiment are as follows:

[0178] (1) Centrifuge the CNCT19 cell suspension at 300g for 5 minutes, discard the supernatant, and the viable cell density is (0.5~1)×10 7 The sieve fluid was added to resuspend the cells until the cell / ml level was reached.

[0179] (2) For each sample, two flow cytometry tubes were taken and labeled as Tube 1 and Tube 2; Tube 1 was a blank control and no antibody was added to it, and Tube 2 was labeled with Alexa Fluor ® 10 μL of a 10-fold dilution of 647-goat anti-mouse IgG F(ab')2 antibody (Manufacturer: Jackson, Catalog No.: 115-605-072) was added.

[0180] (3) 100 μl of cell suspension was added to each tube, and the reaction was carried out in a dark room at room temperature for 15 to 20 minutes.

[0181] (4) 2 ml of cleaning sheath fluid was added to each tube and centrifuged at 300 g for 5 minutes.

[0182] (5) Discard the supernatant and add 20 μl of FITC-CD3 (manufacturer: Tongsheng Shidai, catalog number: Z6410047-100T) to tube 2 and incubate at room temperature in a dark room for 15 to 20 minutes.

[0183] (6) 2 ml of cleaning sheath fluid was added to each tube and centrifuged at 300 g for 5 minutes, then the supernatant was discarded, and 2 ml of sheath fluid was added to each cleaning tube and centrifuged at 300 g for 5 minutes.

[0184] (7) Discard the supernatant and add 300 μl of sieve fluid to each tube to resuspend it, then detect it by flow cytometry.

[0185] The results are presented in Figures 2a and 2b. As can be seen, Figure 2a shows the blank control tube; its right-top CD3+CAR+ quadrant represents the cell-free population. Figure 2b shows the experimental detection tube. CART cells were labeled with IgG F(ab')2 and CD3 antibodies, and a CAR expression rate of 68.6% was CD3 by flow cytometry. + It can be clearly detected in the CAR+ quadrant. The results indicate that the CAR molecule according to the present disclosure is well expressed on the surface of CNCT19 cells.

[0186] Example 4. In vitro Detection of the apoptotic effect of CNCT19 cells on target cells

[0187] The experimental steps of this example are as follows:

[0188] 1) CD19-positive human leukemia cell line Nalm-6 (purchased from Shanghai Enzyme Research Bioscience Co., Ltd., catalog number: CH179) and CD19-negative human leukemia cell line KG-1a (purchased from Shanghai Enzyme Research Bioscience Co., Ltd., catalog number: CC-Y1305) were selected as tumor cells (i.e., target cells), respectively. CD19 CAR-T cells (i.e., CNCT19 cells obtained in Example 2) and untransfected T cells (indicated by NTD) were selected as effector cells, respectively.

[0189] 2) The aforementioned target cells and effector cells were mixed at effector / target ratios of 1:2 and 2:1, respectively, and seeded into 24-well plates. The total number of co-cultured cells in each well was approximately 1 × 10⁶. 6 The plates were set to have 3 / well, and 3 parallel wells were set for each condition. Each well was refilled with 1 ml of culture solution, the plates were placed in an incubator at 37°C and 5% CO2 for culture, and the time was recorded.

[0190] 3) After 24 hours of co-culture, cell suspensions were collected from each well, transferred to 1.5 mL EP tubes, and labeled. Additionally, after centrifugation, the supernatant from each sample tube was aspirated into a new 1.5 mL EP tube and cryopreserved at -20°C for subsequent cytokine detection (see Example 6 for details).

[0191] 4) For labeling according to the type of tumor cell, the corresponding detection amount of antibody was added to the mixed cells in each well, and the procedure was performed according to the antibody instructions. Nalm-6 cells were labeled using the PE-CD10 antibody, and KG-1a cells were labeled using the Percp-cy5.5-CD45 antibody.

[0192] 5) Changes in the proportion of different target tumor cells in each sample were detected using flow cytometry.

[0193] The results are shown in Figure 3. As can be seen, compared to co-culture with untransfected T cells (i.e., NTD), co-culture of CD19-positive tumor cells Nalm-6 and CAR-T cells (i.e., CNCT19 cells) induced a significantly reduced retention rate of Nalm-6; nevertheless, there was no significant difference in the retention rate of CD19-negative tumor cells KG-1a after co-culture with various effector cells. Specifically, when the effector / target ratio was 1:2, co-culture of Nalm-6 cells and CNCT19 cells induced a retention rate of (2.8±1.3)% of target cells, which is higher than that resulting from the co-incubation of untransfected T cells with Nalm-6 cells ((12.1±1.2)%). PIt was significantly lower than <0.01). Similarly, when the effector / target ratio was 2:1, co-incubation of Nalm-6 cells and CNCT19 cells induced a target cell retention rate of (1.1±0.1)%, which was higher than that resulting from the co-incubation of T cells not transfected with Nalm-6 cells ((7.3±1.2)%). P It was significantly lower than <0.01. Furthermore, the apoptotic effect of CNCT19 cells on CD19-positive tumor cells was enhanced with increasing effector / target ratio.

[0194] Example 5. Real-time monitoring of the apoptotic function of CNCT19 cells

[0195] The experimental steps are as follows:

[0196] (1) The target cell CHO-CD19 was removed, the culture solution in the culture flask was aspirated and discarded, the culture flask was washed once with physiological saline solution, 1 ml of trypsin solution containing EDTA was added, and incubation was performed in a 37°C incubator for 2 to 6 minutes before isolation was stopped. CHO cells were purchased from Shanghai Enzyme Research Bioscience Co., Ltd. under catalog number CC-Y2110; the molecular sequence of CD19 cells was derived from NCBI, the molecular sequence of CD19 was constructed in CHO cells using molecular biology methods, and the target cell CHO-CD19 cell line was obtained through screening.

[0197] (2) Add an appropriate amount of culture medium to the culture flask to allow the target cells to form a cell suspension, and homogenize the cells by pipetting. After counting the concentration of the cell suspension with a counting plate, the cell suspension was set to the required cell concentration of 1×10⁻⁶ in the experiment. 5 It was formulated into cells / ml.

[0198] (3) 50 μl of culture medium was added to the wells of E-Plate 16 of the RTCA DP system. E-Plate 16 was placed on the RTCA station. The RTCA system would automatically scan to check whether the contact was good ("Connection OK" was displayed on the "Message" page) ("Scan Plate"). It started detecting the baseline (background) to check if the selected wells were in a normal contact state.

[0199] (4) Remove E-Plate 16 and add 100 μl of well-mixed target cell suspension 1 × 10⁶ per well 4 Cells were added to the wells. E-Plate 16 was placed on a super clean bench at room temperature for 30 minutes and then placed on the RTCA station in the incubator. After the system automatically scanned ("Scan Plate"), Step 2 was initiated to dynamically detect the cell proliferation curve in real time.

[0200] (5) E-Plate 16 was removed and a target cell suspension and a CNCT19 cell suspension were added to some wells, and a target cell suspension and a non-transfected T cell (i.e., NTD) suspension (as a control) were added to other wells, where the effector / target ratio (i.e., ratio of effector cells to target cells, i.e., CNCT19 cells: target cells; and NTD: target cells) was 1:1 and the volume of the target cell suspension was 50 μl. The E-Plate 16 detection plate was placed on an RTCA DP detection platform for 60 hours of real-time monitoring to observe the effect of CNCT19 cells on target cells.

[0201] The results are illustrated in Figure 4. In the figure, Line 1 represents the curve of the NTD control group, and Line 2 represents the curve of the CNCT19 treatment group. By comparing Line 1 and Line 2, it can be seen that the proliferation of tumor cells (i.e., target cells) was inhibited by CNCT19 cells over time. Specifically, after co-incubation with untransfected T cells, target cells did not show a significant change in growth trend (Line 1). In contrast, after co-incubation with CNCT19 cells, target cells showed a significantly reduced growth trend and even began to show a decrease in their number (Line 2). This indicates that CNCT19 has a potent ability to kill target cells.

[0202] Example 6. Detection of secreted cytokines after co-culture of target cells and CNCT19 cells

[0203] The experimental steps of this example are as follows:

[0204] 1) The supernatant sample (i.e., the sample obtained in Step 3) from the culture mixed in each well of Example 4 was removed from a -20°C refrigerator and thawed at room temperature;

[0205] 2) Each sample was processed according to the instructions using the LEGENDplex™ kit (Manufacturer: Biolegned, Catalog No.: 740013); and

[0206] 3) Different levels of cytokines in each sample were detected by flow cytometry.

[0207] The results are illustrated in Figures 5a and 5b. Compared to untransfected T cells (i.e., NTD), co-culture of Nalm-6 cells (CD19+) and CAR-T cells (i.e., CNCT19 cells) resulted in significantly increased secretion of the cytokines IFN-γ and IL-6 by CAR-T cells. Specifically, after co-culture with target cells at an effector / target ratio of 2:1 for 24 hours, CNCT19 cells were stimulated by the target cells to secrete INF-γ at a level of (6186.37±861.13) pg / ml, which is higher than the amount of INF-γ secreted by untransfected T cells (2096.85±228.16 pg / ml, P It was significantly higher than <0.05; the amount of IL-6 secreted by CNCT19 cells (32.22±1.46 pg / ml) was significantly higher than the amount secreted by untransformed T cells (12.23±4.37 pg / ml, P It was significantly higher than <0.05.

[0208] Example 7. Detection of hemolysis and stimulation of CNCT19 cells

[0209] 7.1. In vitro Hemolysis test of CNCT19 cells

[0210] The experimental steps of this example are as follows:

[0211] 1) A total of 7 glass test tubes were used and numbered from 1 to 7, and 2.5 mL of 2% rabbit red blood cell suspension (collected from New Zealand rabbits and produced by the National Institute of Food and Drug Administration of China with quality certification number 11400500032425) was added to each tube.

[0212] 2) 1×10 7Different volumes (0.5 to 0.1 mL) of CNCT19 cells at cell / mL concentrations (based on the total number of T cells) were added to tubes 1 to 5, which already contained different volumes (2.0 to 2.4 mL) of sodium chloride infusion. Meanwhile, 2.5 mL of sodium chloride infusion (negative control) and 2.5 mL of sterile water for infusion (positive control) were added to tubes 6 and 7, respectively.

[0213] 3) Each test tube had a total volume of 5.0 mL. The test tubes were placed in a 37°C ± 0.5°C incubator for 3 hours. After placing the test tubes in the incubator, it was observed whether red blood cells lysed or agglutinated at 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, and 3 hours.

[0214] The results are illustrated in Figures 6a and 6b. Figure 6a shows the observation results of each test tube before shaking for 3 hours, and Figure 6b shows the observation results of each test tube after shaking for 3 hours. In the negative control tube (No. 6), it was observed that the supernatant was colorless and transparent, red blood cells settled to the bottom of the tube, and were evenly dispersed after shaking; it was determined that there was no hemolysis or agglutination. In the positive control tube (No. 7), the solution was clear and reddish, there were no separated layers, and there were no red blood cell residues at the bottom of the tube; it was determined that there was complete hemolysis. As a result of observing each test tube with different doses of CNCT19 cells for 3 hours, it was observed that in each test tube, the supernatant was colorless and transparent, and red blood cells settled to the bottom of the tube and were evenly dispersed after shaking; it was determined that there was no hemolysis or agglutination.

[0215] 7.2. Test of Vascular Stimulation Upon Intravenous Infusion of CNCT19 Cells

[0216] The experimental steps of this example are as follows:

[0217] 1) Six New Zealand rabbits (3 males and 3 females) that had passed quarantine and had no abnormalities at the injection site were selected. An auto-control method was used: cryopreserved CAR-T cells (i.e., CNCT19 cells) and a negative control (sodium chloride injection) were intravenously injected into the right and left ears of each animal, respectively.

[0218] 2) CAR-T cells injected through the right vein are 1×10 7 The concentration was cells / mL, and 1×10 7 The dose was in cells / kg (based on the total number of T cells). Infection with sodium chloride infusion via the left bilateral vein was used as a negative control. The dose administered intravenously via both the left and right bilateral veins was 1 mL / kg.

[0219] 3) After intravenous administration, the animals were examined in their entire bodies, the injection site was examined, and a pathological examination was performed.

[0220] The results are illustrated in Figs. 7a and 7b. Fig. 7a shows a micrograph of the injection site (HE staining, 10× objective lens) after administration of CAR-T cells, and Fig. 7b shows a micrograph of the injection site (HE staining, 10× objective lens) after administration of sodium chloride. It can be seen that no systemic or local symptoms or pathological abnormalities were found in the animals compared to the negative control side after intravenous injection of cryopreserved CAR-T cells.

[0221] Example 8. In vitro Test of the carcinogenicity of CNCT19 cells

[0222] The experimental steps of this example are as follows:

[0223] 1) CAR-T cells (i.e., CNCT19 cells) from two donors were incubated in soft agar medium. The two donors were Donor 1 (healthy human donor T cells, lot number: TC20180613015) and Donor 2 (healthy human donor T cells, lot number: TC20180613016), respectively. Additionally, human embryonic lung fibroblast cell line MRC-5 was set as a negative control, and human cervical cancer cell line Hela was set as a positive control.

[0224] 2) 6-well culture plates were used for the experiment. The number of cells in each well was approximately 1 × 10⁶ 3 Three parallel wells were set up for each group. Culture plates were placed in a CO2 incubator for culture and observed for three weeks.

[0225] 3) Every week, remove the culture plates, observe under a microscope for the formation of clones, and take photographs. The experiment was stopped until clear clone formation was observed in the positive control group.

[0226] The results are shown in Figure 8. Positive control cells (Hela) formed clones in the culture medium, and it can be seen that the size of the clones increased significantly over time. On day 23 of observation, CAR-T cells from another donor and negative control cells (MRC-5) did not show clonal growth, and they all died. In vitro It did not feature immortalized proliferation.

[0227] Example 9. In vivo Experiment on the tumorigenicity of CNCT19 cells

[0228] The experimental steps of this example are as follows:

[0229] 1) Cryopreserved CAR-T cells (i.e., CNCT19 cells) and corresponding untransfected T cells (i.e., NTD) from two donors were inoculated subcutaneously into BALB / c nude mice, respectively, where the two donors were Donor 2 (healthy human donor T cells, lot number: TC20180613016) and Donor 3 (healthy human donor T cells, lot number: TC20180808019).

[0230] 2) Inoculation with CAR-T cells is 1×10 7 The procedure was conducted using cells / mouse (based on the total number of T cells). The negative control group consisted of 1 × 10⁶ mice. 7 They were inoculated with human embryonic lung fibroblast line MRC-5 cells, and the positive control group was 1×10⁶ per mouse. 6 The human cervical cancer cell line Hela cells were inoculated.

[0231] 3) Continuous observation was performed for 16 weeks to detect changes in body weight, the occurrence of nodules, and whether the nodules were induced into neoplastic nodules. The results were compared with those of negative and positive cell groups.

[0232] 4) A total anatomy was performed after observation. The weight of each organ was measured and the organ count was calculated. Histopathological examinations were performed on the inoculation site and tissues or organs with suspected symptoms.

[0233] The result is that CAR-T cells In vivoIt indicates that it does not induce tumorigenesis. In the negative control group (inoculated with MRC-5 cells), fluid nodules disappeared in all animals by day 9 after inoculation. In the positive control group (inoculated with HeLa cells), subcutaneous nodules gradually increased in all animals. Pathological examination indicated that these nodules were caused by the growth of tumor tissue with a 100% tumor-forming rate. In summary, this experiment was valid. In the groups inoculated with CAR-T cells (derived from two donors) and untransformed T cells, all fluid nodules disappeared by day 5 after inoculation, and no nodules re-formed prior to euthanasia on day 114. Pathological examination indicated that no tumors formed at the inoculation site or metastatic sites.

[0234] Example 10. Test of the toxicity of a single intravenous injection of CNCT19 cells in NCG mice with Nalm-6 xenograft tumors

[0235] The experimental steps of this example are as follows:

[0236] 1) 6-8 week old NCG mice were used, half of whom were male. 3 days prior to administration, a Nalm-6 cell suspension of 2.5 × 10⁶ 6 It was injected into the tail vein at a concentration of cells / mL and a dose of 10mL / kg.

[0237] 2) Selected animals were randomly divided into four sex-balanced groups (i.e., Groups 2 through 5) according to their body weight. Groups 2 through 5 were the vehicle control, T cell control, low-dose CAR-T cell group, and high-dose CAR-T cell group, respectively, with each group comprising 40 animals (20 males and 20 females). Both the tumor-free control group (i.e., Group 1) and the vehicle control group (i.e., Group 2) were provided with the vehicle control (i.e., physiological saline containing 4% (W / V) human albumin). The T cell control group (i.e., Group 3) was provided with 1 × 10⁶ untransfected T cells (i.e., NTDs). 9They were provided at a dose of cells / kg (based on the total number of T cells, the same applies hereinafter). The low-dose CAR-T cell group (Group 4) received 1×10 8 They were inoculated at a dose of cells / kg, and the high-dose CAR-T cell group (Group 5) had 1×10 9 Inoculated at a dose of cells / kg.

[0238] 3) The dosage for each animal was 25 mL / kg, and administration was performed by a single intravenous injection at a rate of about 1 mL / min.

[0239] 4) After administration, continuous observation was performed for 4 hours on the day of administration. During the study, general clinical observation was conducted once in the morning and once in the afternoon every day. Detailed clinical observation and measurement of body weight and food intake were performed once a week. During the study, body temperature, clinicopathological markers (blood cell count and blood biochemical markers), and immunological markers (T lymphocyte subsets, cytokines, and C-reactive protein) were detected.

[0240] 5) In groups 1-5, 10 animals / sex / groups were euthanized on day 2, and 5 animals / sex / groups were euthanized on day 15. Their organ weights were measured, and comprehensive anatomical observations were performed. Histopathological examinations were conducted on the major organs of the animals in groups 1 and 5.

[0241] The results of this experiment show that the maximum allowable dose of CAR-T cells (i.e., CNCT19 cells) for a single administration is 1×10 9It indicates that the cell count exceeds 1kg. During the study, none of the animals in the low-dose CAR-T cell group or the high-dose CAR-T cell group died or did not die. No abnormal responses were observed in general or detailed clinical observations. No distinct abnormal changes were observed in body weight, food intake, body temperature, C-reactive protein, or blood biochemical indicators. Histopathological examinations revealed no distinct abnormalities in the low-dose CAR-T cell group or the high-dose CAR-T cell group compared to the T-cell control group.

[0242] Example 11. Treatment of animals with CNCT19 cells

[0243] 11.1. Therapeutic Effects of CNCT19 Cells on NCG Mice with Nalm-6 Xenograft Tumors

[0244] The experimental steps of this example are as follows:

[0245] 1) 6-8 week old female NCG mice were used. 5×10⁶ ligations were inserted into the tail vein 3 days prior to administration. 5 Nalm-6 cells were injected, and here, 2.5 × 10⁶ Nalm-6 cells were placed in physiological saline. 6 Cells were lysed at a concentration of cells / ml, and 200μl of cell resuspended solution was injected into each mouse.

[0246] 2) Each experimental group was injected with the corresponding CAR-T cells (i.e., CNCT19 cells), untransformed T cells (i.e., NTD), or cell preservation solution (i.e., physiological saline containing 4% (W / V) human albumin). A total of five groups were divided as follows: the low-dose CAR-T group (5 × 10⁶ CNCT19 cells based on the total number of T cells). 6 Injection into cells / mice, same applies below), CAR-T medium-dose group (CNCT19 cells 1×10⁶ 7 (injected into cells / mice), CAR-T high-dose group (2×10⁶ CNCT19 cells) 7 Injected into cells / mice), T cell control group (2×10 non-transfected T cells) 7Cell / injection into mouse) and vehicle control (injection of 200 μl of cell preservation solution into mouse). The injection volume per mouse was 200 μl.

[0247] 3) Body weight was measured and general clinical observation was performed twice a week. The survival of the mice was recorded and survival curves were plotted.

[0248] The results are illustrated in Figure 9. As can be seen, CNCT19 cells had a significant effect on extending survival time at all doses, and these effects were clearly dose-dependent. Specifically, the median survival time for the saline control group was 24 days, for the NTD control group 23 days, and for the low-dose CNCT19 group 40 days, whereas all experimental animals in the medium-dose and high-dose CNCT19 groups survived until the end of the observation period. Compared to the saline group and the NTD control group, all dose groups of CNCT19 were able to extend the survival time of animals with leukemia by more than 16 days.

[0249] 11.2. Distribution of CNCT19 cells in animals

[0250] The experimental steps of this example are as follows:

[0251] 1) 6-8 week old NCG mice were used, half of which were male. A Nalm-6 xenograft tumor model was established by the method of Example 11.1.

[0252] 2) Both animals with tumors and animals without tumors were injected with a single tail vein of CAR-T cells (i.e., CNCT19 cells) at a rate of 5×10⁶ 6 Received at a dose of cells / mouse (based on the total number of T cells).

[0253] 3) Animals in the tumor group were euthanized as planned at 24 hours (i.e., D2), 72 hours (i.e., D4), 168 hours (i.e., D8), 336 hours (i.e., D15), 504 hours (i.e., D22), and 672 hours (i.e., D29), respectively, after administration. Animals in the tumor-free group were euthanized as planned at 24 hours (i.e., D2), 168 hours (i.e., D8), and 336 hours (i.e., D15), respectively, after administration. Animal whole blood (EDTA anticoagulant), brain, spinal cord (cervical segments), skeletal muscle, gonads (ovaries, testes, and epididymis), bladder, stomach, small intestine, mesenteric lymph nodes, bone marrow, liver, kidney, spleen, heart, lungs, and other tissues or body fluids were collected in sequence.

[0254] 4) The content of chimeric antigen receptors (i.e., CARs) in blood and various tissue samples was determined using a validated Q-PCR method.

[0255] The results were 5×10 mice with tumors and mice without tumors 6 This indicates that after administration by a single intravenous injection into cells / mice, CNCT19 cells are distributed mainly in whole blood and tissues with major blood flows such as the lungs, liver, heart, and spleen (see Fig. 10). Among these, the heart and whole blood had the largest distribution, and the area under the curve (copy number of nucleic acid molecules encoding CAR in gDNA-hours, AUC) was approximately 150,000 hours*copy number / μg. The lungs and spinal cord followed, with an AUC of approximately 40,000 to 60,000 hours*copy number / μg. For the spleen, liver, and other tissues, the AUC was less than approximately 20,000 hours*copy number / μg.

[0256] The results also indicate that the content of CNCT19 cells in mouse tissues with tumors is slightly higher than in mouse tissues without tumors (see Fig. 11). The concentrations of CNCT19 cells in the whole blood, heart, and spinal cord of mice with tumors were about 3, 6, and 5 times higher, respectively, than those in mice without tumors 24 hours after administration.

[0257] Subsequently, the drug content in each tissue gradually decreased and dropped below the methodological detection limit after 2 weeks of administration. As the disease progressed, CNCT19 cells in mice proliferated reactively due to increased CD19 antigen stimulation (see Fig. 12), and the concentration of CNCT19 cells in the brain, lungs, liver, whole blood, and spinal cord increased to over 1200 copies / μg DNA and even reached 3500 copies / μg DNA.

[0258] Example 12. Treatment of acute lymphoblastic leukemia with CNCT19 cells

[0259] 12.1. Clinical Use of CNCT19 Cells

[0260] The clinical application process is shown in Table 3, and specific experimental steps for some stages are described below.

[0261] [Table 3]. Process of Clinical Application of CNCT19 Cells

[0262]

[0263] 1. Pretreatment for Lymph Node Dissection

[0264] Lymph node dissection pretreatment is performed on Day 5 before the CNCT19 cell suspension is reinjected as planned.

[0265] The preprocessing plan is as follows:

[0266] Fludarabine is administered at 30 mg / m² once daily for 2-4 consecutive days; and

[0267] Cyclophosphamide is administered once a day at 500 mg / m² for 2 consecutive days.

[0268] The use of both drugs for pretreatment chemotherapy must begin on the same day.

[0269] 2. Re-infusion of CNCT19 cell suspension

[0270] (1) After the cells are successfully prepared, they are frozen and preserved under conditions of <-100℃, transported to a hospital under the same temperature conditions for use, and retrieved according to the experimental procedure guide before injection.

[0271] (2) The method for re-injecting cells is as follows. Cells must be retrieved according to the experimental procedure guide, and the re-injection of the cell suspension must be completed within 30 minutes of retrieval. The cell suspension is injected into the subject via a single infusion through a vein using a transfusion device. If there is more than one bag of cell suspension, it may be injected continuously without a time interval between re-injections of each bag. The subject must be closely monitored during cell re-injection. In the event of a serious adverse event, the infusion must be stopped, and appropriate treatment must be performed according to the specific adverse event. If no serious adverse event occurs, follow-up measures may be taken according to the visit workflow.

[0272] (3) The subject needs to be closely monitored for 24 hours after cell re-injection. If a serious adverse event occurs, appropriate treatment should be performed according to the specific adverse event. If no serious adverse event occurs, follow-up measures can be taken according to the visit workflow.

[0273] (4) After cell re-injection, the subject needs to remain hospitalized for observation for 14 days or for a period determined by the researcher's comprehensive assessment of the subject's condition.

[0274] 3. Management of CNCT19 Cell Suspension

[0275] To strictly manage and utilize the CNCT19 cell suspension, a rigorous system has been established for its management by specially designated personnel. Specific personnel are assigned to transport the research cell suspension to hospital departments, and the task of receiving the suspension and establishing a registration system is assigned to these specific individuals.

[0276] After injection, the packaging of the cell suspension is retrieved by drug management personnel and stored / discarded.

[0277] 4. Clinical Efficacy and Safety Results of CNCT19 Cell Suspension in the Treatment of Relapsed or Refractory Acute Lymphoblastic Leukemia

[0278] This study began in September 2016 and continued until October 2020. During the exploratory and Phase 1 clinical trials, 63 patients (23 adult patients and 40 pediatric patients) with relapsed or refractory acute lymphoblastic leukemia (ALL) were treated with CNCT. The irradiation dose administered for adult ALL was 0.25 × 10⁻⁶ 8 Up to 0.5×10 8 It was a range of CAR-positive T cells.

[0279] (a) Data on clinical efficacy are presented in Tables 4 and 5. Table 4 shows the recovery of 63 patients, including adults and children. Table 5 shows the recovery of 23 adult patients. Among the 63 patients with relapsed or refractory acute lymphoblastic leukemia, it was found that the majority (93.7%) of patients achieved complete remission and 88.9% of patients were MRD-negative after injection and re-infusion of the CNCT19 cell suspension. The results demonstrate that injection and re-infusion of the CNCT19 cell suspension can effectively treat adult and pediatric patients with relapsed or refractory acute lymphoblastic leukemia.

[0280] [Table 4]: Clinical efficacy of CNCT19 cell suspension for the treatment of acute lymphoblastic leukemia in adults and children

[0281]

[0282] [Table 5]: Clinical efficacy of CNCT19 cell suspension for the treatment of acute lymphoblastic leukemia in adults

[0283]

[0284] Note: In Tables 4 and 5

[0285] ORR represents the overall remission rate;

[0286] A negative MRD (Minimum Residual Disease) means that no tumor cells were detected (by the most sensitive method);

[0287] CR indicates complete remission; and

[0288] Cri indicates morphological complete remission with incomplete recovery of blood cell counts.

[0289] b) Data from the preliminary safety results are presented in Tables 6 and 7. Table 6 presents safety results for 63 patients, including adult and pediatric patients, and Table 7 presents safety results for 23 adult patients. Among the 63 ALL patients, the incidence rates of Grade 3 or higher CRES and encephalopathy were 19% and 20.6%, respectively. Comparisons between age groups revealed that the incidence of severe CRES in the adult group (39.1%) was higher than in the pediatric group (7.5%), while the incidence of severe CRES in the adult group (17.4%) was slightly lower than or close to that in the pediatric group (22.6%). This may be attributed to the fact that in the initial study, CNCT19 was administered based on body weight, and higher doses were given to adult patients with higher body weight. For example, the dose given to 46 subjects in the initial study was 0.71 × 10⁶ 6 Up to 4.08×10 6 The range was in the range of / kg, which is 0.16×10 8 -2.36×10 8 It corresponds to CAR-positive T cells. As initial dose-search gradually elucidated the characteristics of the CNCT19 product, safer dose ranges were selected in subsequent clinical studies. For example, 0.2 × 10⁶ 8 Up to 1.1×10 8 Dose range of CAR-positive T cells (median: 0.5×10⁶ 8Among the 17 patients who received ), the incidence of grade ≥ 3 CRS and CRES dropped to 5.9% (1 / 17).

[0290] It can be seen that CNCT19 possesses excellent safety performance, as the injection of the CNCT19 cell suspension has a low likelihood of causing serious CRS or CRES adverse effects and its overall safety is controllable. The excellent safety performance of CNCT19 improves product quality and reduces clinical risk.

[0291] [Table 6]. Safety results of CNCT19 cell suspension in adults and children with acute lymphoblastic leukemia.

[0292]

[0293] [Table 7]. Safety results of CNCT19 cell suspension in adults with acute lymphoblastic leukemia.

[0294]

[0295] Note: In Tables 6 and 7,

[0296] CRS indicates cytokine release syndrome; and

[0297] CRES indicates CAR-T-cell-associated encephalopathy syndrome.

[0298] Example 13. Treatment of relapsed or refractory non-Hodgkin lymphoma with CNCT19 cells

[0299] 13.1. Clinical Use of CNCT19 Cells

[0300] The clinical application process is shown in Table 8, and specific experimental steps for some stages are described below.

[0301] [Table 8]. Process of Clinical Application of CNCT19 Cells

[0302]

[0303] 1. Pretreatment for Lymph Node Dissection

[0304] Lymph node dissection pretreatment is performed on Day 5 before the CNCT19 cell suspension is reinjected as planned.

[0305] The preprocessing plan is as follows:

[0306] Fludarabine is administered at 30 mg / m² once daily for 2-4 consecutive days; and

[0307] Cyclophosphamide is administered once a day at 500 mg / m² for 2 consecutive days.

[0308] The use of both drugs for pretreatment chemotherapy must begin on the same day.

[0309] 2. Re-infusion of CNCT19 cell suspension

[0310] (1) After the cells are successfully prepared, they are frozen and preserved under conditions of <-100℃, transported to a hospital under the same temperature conditions for use, and retrieved according to the experimental procedure guide before injection.

[0311] (2) The method for re-injecting cells is as follows. Cells must be retrieved according to the experimental procedure guide, and the re-injection of the cell suspension must be completed within 30 minutes of retrieval. The cell suspension is injected into the subject via a single infusion through a vein using a transfusion device. If there is more than one bag of cell suspension, it may be injected continuously without a time interval between re-injections of each bag. The subject must be closely monitored during cell re-injection. In the event of a serious adverse event, the infusion must be stopped, and appropriate treatment must be performed according to the specific adverse event. If no serious adverse event occurs, follow-up measures may be taken according to the visit workflow.

[0312] (3) The subject needs to be closely monitored for 24 hours after cell re-injection. If a serious adverse event occurs, appropriate treatment should be performed according to the specific adverse event. If no serious adverse event occurs, follow-up measures can be taken according to the visit workflow.

[0313] (4) After cell re-injection, the subject needs to remain hospitalized for observation for 14 days or for a period determined by the researcher's comprehensive assessment of the subject's condition.

[0314] 3. Management of CNCT19 Cell Suspension

[0315] To strictly manage and utilize the CNCT19 cell suspension, a rigorous system has been established for its management by specially designated personnel. Specific personnel are assigned to transport the research cell suspension to hospital departments, and the task of receiving the suspension and establishing a registration system is assigned to these specific individuals.

[0316] After injection, the packaging of the cell suspension is retrieved by drug management personnel and stored / discarded.

[0317] 4. Clinical Efficacy and Safety Results of CNCT19 Cell Suspension in the Treatment of Relapsed or Refractory Non-Hodgkin Lymphoma

[0318] This study began in September 2016 and continued until October 2020. During the exploratory and Phase 1 clinical trials, 50 patients with relapsed or refractory non-Hodgkin lymphoma (NHL) were treated with CNCT19 cell suspension. The irradiation dose administered for NHL (adults only) was 1 × 10⁶ 8 Up to 2×10 8 It was a range of CAR-positive T cells.

[0319] (a) Data on clinical efficacy are shown in Table 9. Table 9 shows the recovery of 50 patients. Among 50 patients with relapsed or refractory non-Hodgkin lymphoma, it can be seen that approximately 80% of patients were in complete or partial remission after injection and re-infusion of the CNCT19 cell suspension, where the complete remission rate was 54% (27 cases) and the partial remission rate was 24% (12 cases). This demonstrates that the CNCT19 cell suspension can effectively treat patients with relapsed or refractory non-Hodgkin lymphoma.

[0320] [Table 9]: Clinical Efficacy of CNCT19 Cell Suspension for the Treatment of Non-Hodgkin Lymphoma

[0321]

[0322] Note: In Table 9

[0323] ORR represents the overall remission rate;

[0324] CR indicates complete remission; and

[0325] PR indicates partial remission.

[0326] b) Data on preliminary safety results are shown in Table 10. Table 10 presents safety results for 50 patients. It can be seen that the infusion of the CNCT19 cell suspension has a low probability of causing serious CRS or CRES adverse events and a probability of causing grade ≥ 3 adverse events of less than 10%, where the probability of causing grade ≥ 3 CRS is 0% and the probability of causing grade ≥ 3 CRES is 6%. Therefore, CNCT19 has excellent safety performance.

[0327] [Table 10]. Safety results of CNCT19 cell suspension for non-Hodgkin lymphoma

[0328]

[0329] Note: In Table 10,

[0330] CRS indicates cytokine release syndrome; and

[0331] CRES indicates CAR-T-cell-associated encephalopathy syndrome.

[0332] The foregoing detailed description is provided for illustrative and illustrative purposes only, but is not intended to limit the scope of the appended claims. Various modifications to the embodiments listed in the disclosure are obvious to those skilled in the art and are maintained within the scope of the appended claims and their equivalents.

Claims

Claim 1 As a plasmid composition, the plasmid composition comprises plasmid Seq1, PMD2.G, pMDLg-pRRE, and pRSV-Rev; and plasmid Seq1, PMD2.G, pMDLg-pRRE, and pRSV-Rev have a weight ratio of 3-6: 1-1.5: 1-3: 1-1.

5. A plasmid composition existing in proportion, wherein the plasmid Seq1 comprises a nucleic acid molecule encoding a chimeric antigen receptor, and the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO:

1. Claim 2 A plasmid composition comprising, wherein the plasmid composition comprises plasmid Seq1, PMD2.G, pMDLg-pRRE, and pRSV-Rev; wherein plasmid Seq1, PMD2.G, pMDLg-pRRE, and pRSV-Rev are present in a weight ratio of 11.8: 3.53: 6.33: 2.3, 13.8: 3.48: 5.31: 2.54, or 14: 4.67: 4.67: 4.67, and wherein the plasmid Seq1 comprises a nucleic acid molecule encoding a chimeric antigen receptor, and the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO:

1. Claim 3 A plasmid composition according to claim 1 or 2, wherein plasmid Seq1 comprises a nucleic acid molecule encoding a chimeric antigen receptor, and the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO.

2. Claim 4 A method for manufacturing a lentivirus vector, wherein the method comprises introducing a plasmid composition according to claim 1 or 2 into a cell, and the cell is 293T. Claim 5 A method for producing modified and isolated immune effector cells, comprising producing a lentivirus vector according to the method of claim 4, wherein the immune effector cells are selected from the group consisting of T lymphocytes and natural killer cells. Claim 6 A method according to claim 5, comprising introducing a lentivirus vector into an immune effector cell. Claim 7 Modified immune effector cells produced by the method according to paragraph 5 or 6. Claim 8 In paragraph 7, the immune effector cell is a modified immune effector cell capable of expressing a chimeric antigen receptor. Claim 9 A composition comprising modified immune effector cells according to claim 7, wherein the composition is useful for the treatment of non-solid tumors. Claim 10 In claim 9, the composition wherein the non-solid tumor comprises leukemia and / or lymphoma. Claim 11 A composition in which, in Clause 10, the leukemia is acute lymphoblastic leukemia and the lymphoma is B-cell lymphoma. Claim 12 A composition according to claim 11, wherein the acute lymphoblastic leukemia comprises acute lymphoblastic leukemia of an adult and / or acute lymphoblastic leukemia of a child. Claim 13 In item 10, the composition is 0.25×10 8 Pieces up to 0.5×10 8 A composition administered as a single dose of CAR-positive T cells. Claim 14 In claim 10, the composition comprises B-cell lymphoma, including non-Hodgkin lymphoma. Claim 15 In paragraph 14, the composition is 1×10 8 Pieces up to 2×10 8 A composition administered as a single dose of CAR-positive T cells. Claim 16 In paragraph 8, immune effector cells for use in the treatment of non-solid tumors. Claim 17 In paragraph 16, non-solid tumors include immune effector cells, such as leukemia and / or lymphoma. Claim 18 In paragraph 17, the leukemia is acute lymphoblastic leukemia, and said acute lymphoblastic leukemia includes acute lymphoblastic leukemia in adults and / or acute lymphoblastic leukemia in children, immune effector cells. Claim 19 In paragraph 18, 0.25×10 8 Pieces up to 0.5×10 8 Immune effector cells administered as a single dose of CAR-positive T cells. Claim 20 In paragraph 17, leukemia is an immune effector cell that is a B-cell lymphoma. Claim 21 In paragraph 20, B-cell lymphoma includes immune effector cells, such as non-Hodgkin lymphoma. Claim 22 In paragraph 21, 1×10 8 Pieces up to 2×10 8 Immune effector cells administered as a single dose of CAR-positive T cells. Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete

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