Construction, preparation and use of functionally enhanced universal car-DNT cell

Abstract

Provided are the construction, preparation and use of a functionally enhanced universal CAR-DNT cell. Specifically, provided is a chimeric antigen receptor construct. A CAR function-enhancing element and IL-10 are sequentially fused to the C-terminus of a tumor antigen-targeting CAR via a self-cleaving peptide, and the CAR construct is introduced into a DNT cell, thereby obtaining a functionally enhanced universal CAR-DNT cell, namely, an IL10-CD19-CAR-mbIL15-DNT cell. The cell specifically targets CD19, and has a stronger and more sustained cell killing activity and better safety, thus providing a new therapy for CD19-mediated diseases.

Description

Construction, preparation and application of a functionally enhanced universal CAR-DNT cell Technical Field

[0001] This invention belongs to the field of general immune cell therapy technology, specifically relating to the construction, preparation and application of a functionally enhanced general CAR-DNT cell. Background Technology

[0002] Double negative T (DNT) cells refer to CD3 cells that are normally present in human peripheral blood. + CD4 - CD8 - DNT cells are a subset of T cells, accounting for 1-10% of the total number of T cells. DNT cells express CD3 molecules and αβ- or γδ-T cell receptors (TCRs) on their surface, but do not express CD4 and CD8 molecules, nor do they bind to the CD1d tetramer loaded by αGalCer specific to invariant natural killer T (iNKT) cells. Therefore, they differ from conventional T cells, NK cells, and iNKT cells. DNT cells specifically recognize target cells in an MHC-unrestricted manner through cell surface receptors such as NKG2D, DNAM-1, and LFA-1, and kill target cells by secreting a series of cytotoxic molecules and cytokines, without inducing graft-versus-host disease (GvHD) or host resistance to graft reaction (HvGR). Developing a commercially available universal DNT cell therapy holds great potential.

[0003] CD19 is a glycoprotein on the surface of B cells, composed of 556 amino acids, and belongs to the type I transmembrane protein family. It is expressed early in B cell development and continues until differentiation into plasma cells. CD19 is a member of the immunoglobulin (Ig) superfamily and, as a component of the B cell surface signal transduction complex, participates in regulating the signal transduction process of the BCR. CD19 expression is limited to B cell lineages and is not expressed on the surface of pluripotent hematopoietic stem cells. CD19 is expressed on the surface of most B-cell lymphomas, mantle cell lymphomas, ALLs, CLLs, hairy cell leukemia, and some acute myeloid leukemia cells. In recent years, CAR-T cells targeting CD19 have also been used to treat autoimmune diseases such as systemic lupus erythematosus. Therefore, CD19 is a valuable immunotherapeutic target in the treatment of leukemia, lymphoma, and autoimmune diseases.

[0004] Interleukin-15 (IL-15) is an important cytokine with the ability to induce anti-tumor responses. The biological characterization of IL-15 is extremely complex; it exists in several functional forms: the soluble monomeric form (sIL-15), the soluble complex sIL-15 / IL-15Rα, the transtransmitted form tp-IL-15, and the transmembrane form tmb-IL-15. The main mechanism of the IL-15 signaling pathway is that after IL-15 binds to the IL-15Rα subunit on the cell surface, IL-15 remains on the cell surface and forms an immune synapse with IL-2R / IL-15Rβ-γc on nearby effector cells, activating the Jak / Stat pathway (Jak1 and Jak3, Stat3 and Stat5) to transduce signals intracellularly. The IL-15Rα chain endows IL-15 with specificity and allows it to bind to cytokines with high affinity. Studies have shown that DNT cells highly express natural cell receptors (NCRs) such as NKG2D, DNAM-1, NKp30, and TRAIL. These receptors are further upregulated in expression upon stimulation by IL-15, thereby enhancing the antitumor activity of DNT cells.

[0005] Interleukin-10 (IL-10) is a pleiotropic cytokine that can exert immunosuppressive or immunostimulatory effects in various cell types, primarily produced by monocytes and B cells. After binding to the IL-10R receptor complex, IL-10 activates the Jak / Stat pathway (JAK1, TYK2, STAT1, STAT3) to transduce signals intracellularly, exerting its biological effects. Studies have shown that in vivo injection of IL-10 can reprogram the metabolism of tumor-bearing T cells, shifting their metabolism towards oxidative phosphorylation, increasing mitochondrial respiration, and thus significantly enhancing depleted CD8+ cells. + The expansion and effector function of TIL cells. Studies have also shown that CAR T cells overexpressing IL10 improve mitochondrial health in CAR T cells via a mitochondrial pyruvate carrier-dependent pathway, increasing their metabolic shift towards oxidative phosphorylation, improving T cell exhaustion-related dysfunction, and thus enhancing their anti-tumor capabilities.

[0006] Numerous studies have been conducted in this field on the use of CAR-T cell therapy to treat CD19-related cancers; however, these therapies still have many limitations in their application. Therefore, there is still a need in the field to develop more effective and safer immunotherapy for the treatment of CD19-related diseases. Summary of the Invention

[0007] The purpose of this invention is to provide a universal CAR-DNT cell with enhanced function and higher safety, as well as its construction method, preparation method and application.

[0008] In a first aspect of the invention, a chimeric antigen receptor (CAR) construct is provided, the structure of which is shown in Formula I:

[0009] X-A1-E-A2-IL10 (I)

[0010] In the formula,

[0011] Each "-" independently represents a linking peptide or peptide bond;

[0012] X represents a CAR that targets tumor antigens;

[0013] A1 and A2 are self-shearing elements;

[0014] E is a CAR function-enhancing element selected from the following group: membrane-bound fusion proteins such as IL15, IL2, IL4, IL7, IL18, IL21, IL23, and IL36; secreted cytokines such as IL15, IL2, IL4, IL7, IL18, IL21, IL23, and IL36; and IL7R and IL21R mutants.

[0015] IL10 is interleukin-10 or its active fragment or mutant.

[0016] In another preferred embodiment, the tumor antigen is selected from the group consisting of CD19, CD20, CD22, CD33, CD5, CD7, BCMA, DLL3, claudin18.2, CD70, HER2, EGFR, PSMA, or combinations thereof.

[0017] In another preferred embodiment, X is a CAR that targets the tumor antigen CD19 and contains a single-chain variable region (scFv) of an antibody that targets CD19.

[0018] In another preferred embodiment, the structure of X is shown in equation II below.

[0019] L-scFv-H-TM-C-CD3ζ (II)

[0020] In the formula,

[0021] Each "-" independently represents a linking peptide or peptide bond;

[0022] L represents the absence of a signal peptide;

[0023] scFv is the single-chain variable region of an antibody that targets tumor antigens;

[0024] H represents the hingeless region;

[0025] TM represents a transmembrane domain;

[0026] C is a co-stimulatory signaling molecule;

[0027] CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ.

[0028] In another preferred embodiment, the scFv is a CD19-targeting scFv.

[0029] In another preferred embodiment, the amino acid sequence of the CD19-targeting scFv is shown in SEQ ID NO:2.

[0030] In another preferred embodiment, L is a signal peptide of a protein selected from the group consisting of MNDU3, CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0031] In another preferred embodiment, the L is a signal peptide derived from MNDU3.

[0032] In another preferred embodiment, H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof.

[0033] In another preferred embodiment, H is the hinge region derived from CD8.

[0034] In another preferred embodiment, the amino acid sequence of H is shown in SEQ ID NO:4.

[0035] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD8, CD28, ICOS, CD3 epsilon, CD45, CD4, CD5, CD9, CD16, MUC1-Tn, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0036] In another preferred embodiment, the TM is a transmembrane region derived from CD8.

[0037] In another preferred embodiment, the amino acid sequence of the TM is shown in SEQ ID NO:6.

[0038] In another preferred embodiment, C is a co-stimulatory signaling molecule selected from the group consisting of: 4-1BB (CD137), ICOS, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or combinations thereof.

[0039] In another preferred embodiment, C is a co-stimulatory signaling molecule derived from 4-1BB.

[0040] In another preferred embodiment, the amino acid sequence of C is shown in SEQ ID NO:8.

[0041] In another preferred embodiment, the amino acid sequence of CD3ζ is shown in SEQ ID NO:10.

[0042] In another preferred embodiment, the CAR function-enhancing element is a membrane-bound IL15 fusion protein (mbIL15), the structure of which is shown in Formula III.

[0043] L'-MIR (III)

[0044] In the formula,

[0045] Each "-" independently represents a linking peptide or peptide bond;

[0046] L' represents either no signal peptide or a signal peptide;

[0047] M represents interleukin-15 (IL-15);

[0048] I represents a flexible joint;

[0049] R stands for interleukin-15 receptor α (IL-15Rα).

[0050] In another preferred embodiment, M includes wild-type IL-15, or its active fragment, or a mutant thereof.

[0051] In another preferred embodiment, the amino acid sequence of M is shown in SEQ ID NO:16.

[0052] In another preferred embodiment, the IL-15Rα is a complete IL-15Rα element.

[0053] In another preferred embodiment, the IL-15Rα comprises a transmembrane region and an intracellular region.

[0054] In another preferred embodiment, the IL-15Rα comprises, from the N-terminus to the C-terminus, an extracellular region, a transmembrane region, and an intracellular region that bind to IL-15.

[0055] In another preferred embodiment, the amino acid sequence of the IL-15Rα is shown in SEQ ID NO:18.

[0056] In another preferred embodiment, the flexible linker is a linker peptide, preferably, the amino acid composition of the linker peptide is as shown in SEQ ID NO:20.

[0057] In another preferred embodiment, L' is an IgE signal peptide with the amino acid sequence shown in SEQ ID NO:22.

[0058] In another preferred embodiment, the amino acid sequence of the membrane-bound IL15 fusion protein (mbIL15) is shown in SEQ ID NO:14.

[0059] In another preferred embodiment, the self-shearing element includes T2A and P2A.

[0060] In another preferred embodiment, the self-cleaving element is P2A, whose amino acid sequence is shown in SEQ ID NO:12.

[0061] In another preferred embodiment, the amino acid sequence of the IL10 is shown in SEQ ID NO:24.

[0062] In another preferred embodiment, the full-length amino acid sequence of the CAR construct is shown in SEQ ID NO:25.

[0063] In a second aspect of the invention, a polynucleotide molecule is provided, wherein the nucleic acid molecule encodes a CAR construct as described in the first aspect of the invention.

[0064] In a third aspect of the invention, a carrier is provided, the carrier containing a polynucleotide molecule as described in the second aspect of the invention.

[0065] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors (AAV), retroviral vectors, transposons, or combinations thereof.

[0066] In another preferred embodiment, the vector is selected from the group consisting of plasmids and viral vectors.

[0067] In another preferred embodiment, the carrier is in the form of viral particles.

[0068] In another preferred embodiment, the vector is a lentiviral vector.

[0069] In a fourth aspect of the invention, a host cell is provided, said host cell containing a vector or chromosome as described in the third aspect of the invention, or expressing an exogenous polynucleotide molecule as described in the second aspect of the invention, or expressing a CAR construct as described in the first aspect of the invention.

[0070] In another preferred embodiment, the host cell includes eukaryotic cells and prokaryotic cells.

[0071] In another preferred embodiment, the host cells include Escherichia coli and yeast.

[0072] In a fifth aspect of the invention, an engineered immune cell is provided, said immune cell expressing the CAR construct as described in the first aspect of the invention.

[0073] In another preferred embodiment, the cells are isolated cells.

[0074] In another preferred embodiment, the CAR targeting the tumor antigen and the CAR enhancing element are independently expressed on the cell membrane of the immune cell, and the immune cell secretes interleukin-10 or its active fragment or mutant.

[0075] In another preferred embodiment, a CD19-targeting CAR and membrane-bound IL15 fusion protein (mbIL15) is independently expressed on the cell membrane of the immune cell, and the immune cell secretes interleukin-10 or its active fragment or mutant.

[0076] In another preferred embodiment, the immune cells are derived from human or non-human mammals.

[0077] In another preferred embodiment, the cells include T cells and NK cells.

[0078] In another preferred embodiment, the cells are double-negative T cells (DNT cells).

[0079] In another preferred embodiment, the engineered immune cells may be chimeric antigen receptor DNT cells (CAR-DNT cells), chimeric antigen receptor T cells (CAR-T cells), or chimeric antigen receptor NK cells (CAR-NK cells).

[0080] In a sixth aspect of the invention, a formulation is provided comprising the CAR construct of the first aspect of the invention, the polynucleotide molecule of the second aspect of the invention, the carrier of the third aspect of the invention, or the engineered immune cell of the fifth aspect of the invention, and a pharmaceutically acceptable carrier.

[0081] In another preferred embodiment, the formulation is a liquid formulation.

[0082] In another preferred embodiment, the dosage form of the preparation is an injection.

[0083] In another preferred embodiment, the concentration of the immune cells (e.g., CAR-DNT cells) in the formulation is 1 × 10⁻⁶. 3 -1×10 8 Cells / ml, preferably 1×10⁻⁶ 4 -1×10 7 Cells / ml

[0084] In another preferred embodiment, the formulation further comprises a second active ingredient for antitumor activity, preferably including a second antibody or chemotherapeutic agent.

[0085] In a seventh aspect of the invention, there is provided the use of the CAR construct of the first aspect of the invention, the polynucleotide molecule of the second aspect of the invention, the carrier of the third aspect of the invention, or the engineered immune cell of the fifth aspect of the invention, or the formulation of the sixth aspect of the invention, for the preparation of a medicament or formulation for the prevention and / or treatment of a disease.

[0086] In another preferred embodiment, the disease is a CD19-mediated disease.

[0087] In another preferred embodiment, the disease includes: cancer or tumor, autoimmune disease.

[0088] In another preferred embodiment, the cancer or tumor is selected from the group consisting of multiple myeloma (MM), lymphoma, leukemia, B-cell lymphoma, mantle cell lymphoma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, or combinations thereof.

[0089] In another preferred embodiment, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, multiple sclerosis, or a combination thereof.

[0090] In an eighth aspect of the invention, a kit is provided for preparing engineered immune cells as described in the fifth aspect of the invention, the kit comprising a container and a polynucleotide molecule as described in the second aspect of the invention or a carrier as described in the third aspect of the invention located within the container.

[0091] In a ninth aspect of the present invention, a method for preparing engineered immune cells as described in the fifth aspect of the present invention is provided, the method comprising the following steps:

[0092] (a) Providing immune cells to be modified; and

[0093] (b) The polynucleotide molecule described in the second aspect of the present invention or the vector described in the third aspect of the present invention is introduced into the immune cells to obtain the engineered immune cells.

[0094] In another preferred embodiment, the immune cells to be modified are DNT cells, T cells, or NK cells.

[0095] In another preferred embodiment, the method further includes a step of testing the function and effectiveness of the obtained engineered immune cells.

[0096] In a tenth aspect of the invention, a method for treating a disease is provided, the method comprising administering to a subject requiring treatment an effective amount of the carrier described in the third aspect of the invention, the engineered immune cells described in the fifth aspect of the invention, or the preparation described in the sixth aspect of the invention.

[0097] In another preferred embodiment, the disease is a CD19-mediated disease.

[0098] In another preferred embodiment, the disease includes: cancer or tumor, autoimmune disease, or nervous system disease.

[0099] In another preferred embodiment, the cancer or tumor is selected from the group consisting of multiple myeloma (MM), lymphoma, leukemia, B-cell lymphoma, mantle cell lymphoma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, or combinations thereof.

[0100] In another preferred embodiment, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, multiple sclerosis, or a combination thereof.

[0101] In another preferred embodiment, the objects include humans and non-human mammals.

[0102] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0103] Figure 1 is a schematic diagram of the CAR structure constructed in this invention.

[0104] Figure 2 shows the fold expansion and viability of CAR-DNT cells.

[0105] Figure 3 shows the CAR positivity rate and mbIL15 expression detection results of CAR-DNT cells.

[0106] Figure 4 shows the CAR-DNT cell phenotype detection results.

[0107] Figure 5 shows the IL-10 secretion results of CAR-DNT cells.

[0108] Figure 6 shows the cytokine release results after co-culturing CAR-DNT cells with tumor cells.

[0109] Figure 7 shows the results of CAR-DNT cell killing activity.

[0110] Figure 8 shows the results of multiple rounds of tumor killing by CAR-DNT cells.

[0111] Figure 9 shows the results of CAR-DNT cell survival in vitro.

[0112] Figure 10 shows the in vivo efficacy and safety evaluation results of CAR-DNT cells. Detailed Implementation

[0113] Through extensive and in-depth research, and through numerous screenings and verifications, the inventors have, for the first time, constructed a universal CAR-DNT cell with enhanced function and improved safety. This invention provides a chimeric antigen receptor construct targeting tumor antigens (e.g., CD19). An IL-15 / IL-15Rα complex (i.e., membrane-bound interleukin-15 (mbIL15)) and IL-10 are sequentially fused to the C-terminus of a CD19-targeting CAR via a self-cleaving peptide. This CAR construct is then introduced into DNT cells, thereby obtaining a universal CAR-DNT cell with enhanced function and improved safety, namely the IL10-CD19-CAR-mbIL15-DNT cell of this invention. The IL10-CD19-CAR-mbIL15-DNT cells of the present invention have a significant killing effect on CD19-positive cells and express the IL-15 / IL-15Rα complex on the cell membrane surface, while secreting IL-10, which can further enhance the tumor-killing activity and persistence of DNT cells, and the two show a synergistic effect. In addition, thanks to the structure of the CAR construct of the present invention, the IL10-CD19-CAR-mbIL15-DNT cells secrete a lower level of IL-10 and release a relatively low level of cytokines when the target cells are activated, thereby improving the safety during administration.

[0114] Based on this, the present invention was completed.

[0115] the term

[0116] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0117] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0118] The term “giving” means the physical introduction of the product of the present invention into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0119] The term "antibody" (Ab) should include, but is not limited to, immunoglobulins that specifically bind to antigens and comprise at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or their antigen-binding portions. Each H chain contains a heavy chain variable region (abbreviated VH) and a heavy chain constant region. The heavy chain constant region contains three constant domains CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated VL) and a light chain constant region. The light chain constant region contains one constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0120] It should be understood that the amino acid names in this article adopt the internationally accepted single-letter identifiers, and the corresponding three-letter abbreviations of the amino acid names are: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), Val (V).

[0121] CAR Function Enhancement Components

[0122] The CAR constructs of the present invention contain CAR function-enhancing elements that can be expressed on the surface of CAR-modified immune cells, thereby enhancing the function of CAR-modified immune cells. Available CAR function-enhancing elements include (but are not limited to): membrane-bound fusion proteins such as IL15, IL2, IL4, IL7, IL18, IL21, IL23, and IL36; secreted cytokines such as IL15, IL2, IL4, IL7, IL18, IL21, IL23, and IL36; and IL7R and IL21R mutants.

[0123] In one embodiment of the invention, the chimeric antigen receptor (CAR) construct of the present invention comprises membrane-bound interleukin 15 linked to the C-terminus of a CAR targeting the tumor antigen CD19 via a self-cleaving element (e.g., P2A or T2A). The membrane-bound interleukin 15 (or membrane-bound IL15) is a fusion protein formed from IL-15 and IL-15Rα, or referred to as the “IL-15 / IL-15Rα complex,” comprising IL-15 or a mutant thereof or an active fragment thereof, and intact IL-15Rα comprising transmembrane and intracellular regions, which can be used to enhance the persistence and / or cytotoxicity of CAR-modified immune cells.

[0124] The membrane-bound interleukin 15 (mbIL15) of the present invention is a complex expressed on the surface of immune cells, the structure of which is IL-15 and IL-15Rα linked by a linker. In one embodiment of the present invention, the amino acid sequence of IL-15 in the membrane-bound interleukin 15 (mbIL15) is shown in SEQ ID NO:16; the amino acid sequence of IL-15Rα is shown in SEQ ID NO:18; the amino acid sequence of the linker is shown in SEQ ID NO:20; and the full-length amino acid sequence of the membrane-bound interleukin 15 (mbIL15) is shown in SEQ ID NO:14.

[0125] Chimeric antigen receptor (CAR) constructs

[0126] The present invention provides a chimeric antigen receptor (CAR) construct comprising a CAR targeting a tumor antigen, a CAR function-enhancing element, and IL-10.

[0127] In one specific embodiment of the present invention, a CAR construct targeting CD19 is provided, comprising a CAR targeting CD19, a membrane-bound interleukin-15 (mbIL15) fusion protein, and IL-10.

[0128] In a preferred embodiment, the membrane-bound interleukin-15 (mbIL15) fusion protein and IL-10 are sequentially linked to the C-terminus of the CAR structure via a self-cleaving element, and after cleavage, mbIL15 is expressed on the membrane of the CAR-modified cell, while the cell secretes IL-10.

[0129] In a preferred embodiment, the CD19-targeting CAR is composed of a signal peptide, a single-chain variable region of an antibody targeting CD19, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB co-stimulatory domain, and a CD3ζ domain linked together.

[0130] Specifically, the chimeric antigen receptor (CAR) of the present invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen-binding domain). The intracellular domain includes a co-stimulatory signaling region and a ζ-chain portion. The co-stimulatory signaling region refers to a portion of the intracellular domain containing a co-stimulatory molecule. The co-stimulatory molecule is a cell surface molecule required for an effective lymphocyte response to an antigen, rather than an antigen receptor or its ligands. In a preferred embodiment, the CAR of the present invention comprises a co-stimulatory signaling molecule derived from 4-1BB.

[0131] A linker may be incorporated between the extracellular and transmembrane domains of the CAR, or between the cytoplasmic and transmembrane domains of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular or cytoplasmic domain of the polypeptide chain. Linkers may comprise 0-300 amino acids, preferably 2 to 100 amino acids, and most preferably 3 to 50 amino acids.

[0132] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen-binding domain targeting CD19. When expressed in immune effector cells, the CAR of the present invention is capable of antigen recognition based on antigen-binding specificity. When it binds to its associated antigen, it affects tumor cells, causing them to stop growing, be induced to die, or otherwise be affected, resulting in a reduction or elimination of the patient's tumor burden. The antigen-binding domain is preferably fused with an intracellular domain derived from one or more of the co-stimulatory molecule and the ζ chain.

[0133] As used herein, "antigen-binding domain" and "single-chain antibody fragment" refer to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments with antigen-binding activity. Fv antibodies contain variable regions of the antibody heavy chain and light chain, but no constant region, and are the smallest antibody fragments possessing all antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding. The antigen-binding domain is typically scFv (single-chain variable fragment). The size of an scFv is generally 1 / 6 that of a complete antibody. Single-chain antibodies are preferably a single amino acid chain sequence encoded by a single nucleotide chain. As a preferred embodiment of the invention, the scFv contains a single-chain variable region that specifically recognizes BCMA and CD19, preferably a humanized single-chain antibody.

[0134] For the hinge region and transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one implementation, a transmembrane domain naturally associated with one of the domains in the CAR is used. In some examples, the transmembrane domain can be selected, or modified by amino acid substitution, to avoid binding such a domain to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0135] carrier

[0136] The nucleic acid sequence encoding the desired molecule can be obtained using recombination methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a vector known to contain the gene, or by directly isolating the gene from cells and tissues containing the gene using standard techniques. Optionally, the gene of interest can be synthesized.

[0137] This invention also provides vectors in which the expression cassette of this invention is inserted. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses, such as murine leukemia viruses, because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

[0138] In short, the expression cassette or nucleic acid sequence of this invention is typically operatively linked to a promoter and incorporated into an expression vector. This vector is suitable for replication and integration into eukaryotic cells. A typical cloning vector contains transcription and translation terminators, an initial sequence, and a promoter that can be used to regulate the expression of the desired nucleic acid sequence.

[0139] The expression constructs of the present invention can also be used with standard gene delivery protocols for nucleic acid immunotherapy and gene therapy. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0140] This nucleic acid can be cloned into many types of vectors. For example, it can be cloned into vectors including, but not limited to, plasmids, phage particles, phage derivatives, animal viruses, and granules. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0141] Furthermore, the expression vector can be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and has been described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, a suitable vector contains at least one origin of replication functioning in an organism, a promoter sequence, a convenient restriction enzyme site, and one or more optional markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0142] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0143] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. These are typically located in a 30–110 bp region upstream of the start site, although recent studies have shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible to maintain promoter function when an element is inverted or moved relative to another. In the thymidine kinase (TK) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, individual elements can function cooperatively or independently to initiate transcription.

[0144] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the early promoter of simian virus 40 (SV40), mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ's sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Furthermore, the invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered as part of the invention. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operatively linked to the inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0145] To assess the expression of CAR peptides or portions thereof, the expression vector introduced into cells may also contain either or both of an optional marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a population of cells seeking transfection or infection via a viral vector. Alternatively, the optional marker may be carried on a separate DNA segment and used in co-transfection procedures. Both the optional marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful optional markers include, for example, antibiotic resistance genes such as neo.

[0146] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Typically, a reporter gene is a gene that is either absent from or expressed by the recipient organism or tissue, and that encodes a polypeptide whose expression is clearly indicated by readily detectable properties such as enzyme activity. After DNA has been introduced into the recipient cells, reporter gene expression is measured at an appropriate time. Suitable reporter genes may include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well-known and can be prepared using known techniques or are commercially available. Typically, a construct with at least five flanking regions exhibiting the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be ligated into reporter genes and used to evaluate the ability of reagents to regulate promoter-driven transcription.

[0147] Methods for introducing genes into cells and expressing genes into cells are known in the art. Within the scope of expression vectors, the vector can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0148] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, and so on. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Calcium phosphate transfection is a preferred method for introducing polynucleotides into host cells.

[0149] Biological approaches to introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0150] Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, and beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in both in vitro and in vivo is the liposome (e.g., an artificial membrane capsule).

[0151] In the case of using a non-viral delivery system, an exemplary delivery tool is a liposome. Consider using a lipid formulation to introduce nucleic acid into host cells (in vitro, ex vivo, or in vivo). Alternatively, the nucleic acid may be associated with a lipid. Lipid-associated nucleic acid can be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with both the liposome and the oligonucleotide, trapped within the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, conjugated with lipids, contained in lipids as a suspension, contained in or complexed with micelles, or otherwise associated with lipids. The lipids, lipid / DNA, or lipid / expression vector associated with the composition are not limited to any specific structure in solution. For example, they may be present in a bilayer structure, as micelles, or have a “collapsed” structure. They may also be simply dispersed in solution, possibly forming aggregates of varying sizes or shapes. Lipids are fatty substances and can be naturally occurring or synthetic lipids. For example, lipids include fat droplets, which occur naturally in the cytoplasm and in compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols and aldehydes.

[0152] In a preferred embodiment of the present invention, the carrier is a lentiviral carrier.

[0153] preparation

[0154] The present invention also provides formulations containing the CAR constructs described in the first aspect of the present invention, the polynucleotide molecules described in the second aspect of the present invention, the vectors described in the third aspect of the present invention, or the immune cells described in the fifth aspect of the present invention, and pharmaceutically acceptable vectors.

[0155] Specifically, the present invention provides CAR-DNT cells containing the present invention, and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injectable formulation. Preferably, the concentration of the CAR-DNT cells in the formulation is 1 × 10⁻⁶. 3 -1×10 8 Cells / ml, more optimal 1×10 4 -1×10 7 Cells / ml

[0156] In one embodiment, the formulation may include buffer solutions such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The formulations of the present invention are preferably formulated for intravenous administration.

[0157] Therapeutic applications

[0158] The present invention also provides the use of the vector described in the third aspect of the present invention, the immune cells described in the fifth aspect of the present invention, or the preparations described in the sixth aspect of the present invention for the treatment of CD19-mediated (or CD19-related) diseases.

[0159] The diseases associated with high CD19 expression include cancer or tumors and autoimmune diseases. Specifically, the cancers or tumors include hematologic malignancies; including (but not limited to) multiple myeloma (MM), lymphoma, leukemia, B-cell lymphoma, mantle cell lymphoma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and hairy cell leukemia. The autoimmune diseases include (but are not limited to) systemic lupus erythematosus and multiple sclerosis.

[0160] The CAR-modified immune cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as IL-2, IL-17, or other cytokines or cell populations. In short, the pharmaceutical compositions of the present invention may include target cell populations as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0161] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease—although the appropriate dosage can be determined by clinical trials.

[0162] When referring to "immunologically effective amount," "antitumor effective amount," "tumor-suppressive effective amount," or "therapeutic amount," the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and disease condition. It can generally be indicated that a pharmaceutical composition including T cells described herein can be administered in doses of 10... 4 Up to 10 9 A dose of cells / kg body weight, preferably 10. 5 Up to 10 6 The T-cell composition can be administered at a dose of cells per kg of body weight (including all integer values ​​within those ranges). These doses can also be administered multiple times. The cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a specific patient can be readily determined by a physician skilled in the medical field by monitoring the patient's disease signs and thus adjusting the treatment accordingly.

[0163] The application of the target composition can be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously (iv), or intraperitoneally. In one embodiment, the DNT cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the DNT cell composition of the present invention is preferably administered by intravenous injection. The DNT cell composition of the present invention can be directly injected into tumors, lymph nodes, or sites of infection.

[0164] In some embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination with any number of relevant treatment modalities (e.g., before, simultaneously with, or after), including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or nastatinumab treatment for MS patients or erfaizumab treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the DNT cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell composition of the invention is administered to a patient in combination with bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiotherapy (XRT), or cyclophosphamide (e.g., before, simultaneously with, or after). For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives an injection of the expanded immune cells of the present invention after transplantation. In an additional embodiment, the expanded cells are administered before or after surgery.

[0165] The dosage of the above treatments administered to patients will vary depending on the precise nature of the condition being treated and the recipient of the treatment. The dosage ratios administered to individuals can be implemented according to accepted practices in the field. Typically, 1 × 10⁻⁶ ppm can be administered per treatment or per course of treatment. 6 One to 1×10 10 The modified DNT cells of this invention are administered to a patient, for example, via intravenous infusion.

[0166] The main advantages of this invention are:

[0167] (1) The universal DNT cells (IL10-CD19-CAR-mbIL15-DNT) that target CD19 constructed in this invention can specifically target CD19 and exhibit significant and effective cytotoxicity against CD19-positive cells, and therefore can be used for the treatment of CD19-mediated diseases.

[0168] (2) The IL10-CD19-CAR-mbIL15-DNT cells constructed in this invention have stronger and more persistent cell killing activity, thus achieving better therapeutic effects.

[0169] (3) The IL10-CD19-CAR-mbIL15-DNT cells constructed in this invention express the IL-15 / IL-15Rα complex on the cell membrane surface and secrete IL-10, which can further enhance the tumor killing activity and persistence of DNT cells, and the two show a synergistic effect.

[0170] (4) The IL10-CD19-CAR-mbIL15-DNT cells constructed in this invention secrete a low level of IL-10 and release a relatively low level of cytokines when the target cells are activated, thereby improving the safety of the administration process.

[0171] (5) The IL10-CD19-CAR-mbIL15-DNT cells prepared by this invention use DNT cells derived from immune cells donated by healthy donors, which can be produced on a large scale, saving costs and greatly reducing the economic burden on patients in clinical applications.

[0172] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0173] Example 1: Construction of a CAR-expressing lentiviral vector

[0174] The CAR lentiviral vectors were synthesized, plasmids cloned, and sequenced by Nanjing GenScript Biotech Co., Ltd. The CAR structures are shown in Figure 1. The CD19-CAR structure consists of humanized anti-CD19 scFv, a CD8 hinge, a CD8 transmembrane region, a 4-1BB co-stimulatory domain, and a CD3ζ intracellular activation domain. The IL10-CD19-CAR structure consists of humanized anti-CD19 scFv, a CD8 hinge, a CD8 transmembrane region, a 4-1BB co-stimulatory domain, a CD3ζ intracellular activation domain, and human IL-10. The CD19-CAR-mbIL15 structure consists of humanized anti-CD19 scFv, a CD8 hinge, a CD8 transmembrane region, a 4-1BB co-stimulatory domain, a CD3ζ intracellular activation domain, and membrane-bound mbIL-15. The IL10-CD19-CAR-IL15 structure is a humanized anti-CD19 scFv... The IL10-CD19-CAR-mbIL15 structure is composed of scFv, CD8 hinge, CD8 transmembrane region, 4-1BB co-stimulatory domain, CD3ζ intracellular activation domain, human IL-10, and human IL-15.

[0175] The scFv sequences of anti-CD19 are all derived from the humanized mouse antibody FMC63.

[0176] The nucleotide and amino acid sequences of each element are shown in Table 1.

[0177] Table 1: Nucleotide and amino acid sequences of CAR structural elements

[0178] Example 2: CAR Lentiviral Packaging and Titer Detection

[0179] The CAR lentiviral vector constructed in Example 1 was packaged into lentiviruses, and the experimental steps are as follows:

[0180] (1) Take 293T cells in the logarithmic growth phase, plate them in T225 culture flasks, and after culturing for 48 hours, when the confluence density reaches 80% to 90%, they can be transfected and used.

[0181] (2) Prepare a sterile 50ml centrifuge tube, add 1.8ml of Opti-MEM transfection medium, then add lentivirus packaging plasmid. The specific dosage is shown in Table 2. Vortex mix and incubate at room temperature for 5min.

[0182] Table 2: Information on Lentiviral Packaging Plasmids

[0183] (3) Take 36 μl of PEI pro and add it to a 15 ml centrifuge tube containing 1.8 ml of Opti-MEM transfection medium, mix well, and incubate at room temperature for 5 min;

[0184] (4) Add all of the Opti-MEM transfection medium containing PEI pro to the Opti-MEM transfection medium containing plasmid DNA, mix well, incubate at room temperature for 20 min, and when the DNA-PEI complex is formed, add Opti-MEM transfection medium to 25 ml and mix well.

[0185] (5) Discard the culture medium of 293T cells in the T225 culture flask and add 25 ml of transfection complex to the T225 culture flask;

[0186] (6) Six hours after transfection, the transfection reagent was replaced with 50 ml of DMEM + 10% FBS complete medium;

[0187] (7) Continue culturing for 48 hours, harvest the virus, centrifuge to remove floating and dead 293T cells, then filter the virus-containing culture medium, concentrate, dispense, freeze at -80℃ and determine the titer.

[0188] In this embodiment, the titers of lentiviruses with different CAR structures were determined as follows: One day before the titer determination, 293T cells were seeded into 12-well plates, and 1 ml of 293T cells at a concentration of 1E5 / ml was added to each well. 1 μl, 0.1 μl, and 0.01 μl of concentrated lentivirus were then added to the 293T cells. Lentiviral titers were detected by FACS after 48 hours. The viral titer was calculated as: viral titer = number of CAR-positive cells × 1E5 / volume of added virus × 1000 (TU / ml). The titer results for lentiviruses with different CAR structures are shown in Table 3.

[0189] Table 3: CAR Lentiviral Titer

[0190] Example 3: Preparation and Detection of CAR-DNT Cells

[0191] 3.1 DNT cell activation

[0192] (1) Coat a 12-well plate with 5ug / ml CD3 antibody and incubate overnight at 4°C;

[0193] (2) Turn on the water bath, set the temperature to 37°C, and remove the frozen DNT cells (CD3+). + CD4 - CD8 - Quickly place the mixture into a water bath and shake it rapidly to completely dissolve the cell solution within 2 minutes.

[0194] (3) Take a 50ml centrifuge tube, add 20ml of the corresponding culture medium, add the thawed DNT cells to the centrifuge tube, centrifuge at 500×g for 5min, discard the supernatant, and wash 2-3 times.

[0195] (4) Resuspend cells with X-VIVO + 10ug / ml gentamicin + 10% ICSR + 250IU / ml IL2, and adjust DNT cells to 2.5×10⁻⁶. 6 Add 300 μL / well of cell suspension to a 24-well plate coated with CD3 antibody and rinse with PBS.

[0196] 3.2 CAR Lentiviral Infection of DNT Cells

[0197] (1) Calculate the amount of virus to be used based on the titer of each CAR lentivirus and MOI=2 determined in Example 2, set up a control well (without virus), and add CAR lentivirus to each well.

[0198] (2) Add 1% DMSO of sensitizing agent to each well according to the final volume of virus supernatant after addition to promote the lentiviral infection efficiency of DNT cells;

[0199] (3) On days 2, 3, and 4, 0.3 ml, 0.4 ml, and 1 ml of culture medium were added, respectively. On day 5, the cells were transferred to T25 culture flasks for further culture, maintaining a density of approximately 1 × 10⁻⁶ cells / year. 6 pcs / ml;

[0200] (4) Samples were taken on Day 9, Day 12 and Day 14 of culture, and counted using a K2 counter to calculate the fold increase and viability of CAR-DNT cells.

[0201] The fold expansion and viability of CAR-DNT cells with different CD19-CAR structures in this embodiment are shown in Figure 2 and Table 4. The fold expansion of CD19-CAR-DNT, IL10-CD19-CAR-DNT, CD19-CAR-mbIL15-DNT, IL10-CD19-CAR-IL15-DNT, and IL10-CD19-CAR-mbIL15-DNT cells on day 14 were 646.6, 501.5, 560.3, 614.4, and 612.7, respectively; the cell viability was 86%, 76%, 86%, 82%, and 87%, respectively. There was no significant difference in the fold expansion of CAR-DNT cells with different CD19-CAR structures, but the viability of IL10-CD19-CAR-DNT cells on day 14 was slightly lower than that of the other four groups of CAR-DNT cells.

[0202] Table 4: Fold rate and viability of CAR-DNT cells

[0203] 3.3 Detection of CAR-DNT cell phenotype and positive rate

[0204] (1) Take 500 μL of CAR-DNT cells into a 1.5 ml EP tube, add 1 ml of 1×PBS and mix, then centrifuge at 500×g for 5 minutes;

[0205] (2) Discard the supernatant, resuspend in 100 μL 1×PBS, add anti-CD19 scFv, CD3, CD4, CD56, CD45RA, and CD62L antibodies to each tube respectively, and incubate at 4°C for 30 minutes.

[0206] (3) Add 1 ml of 1×PBS to wash the cells, centrifuge at 500×g for 5 minutes, discard the supernatant, add 200 μL of 1×PBS to resuspend, and perform flow cytometry analysis.

[0207] The CAR-DNT cell positivity rate results in this embodiment are shown in Figure 3. The CAR positivity rate of CD19-CAR-DNT cells was 83.8%; the CAR positivity rate of IL10-CD19-CAR-DNT cells was 88.1%; the CAR positivity rate of CD19-CAR-mbIL15-DNT cells was 73.4%, with a CAR+mbIL15 positivity rate of 52.5%; the CAR positivity rate of IL10-CD19-CAR-IL15-DNT cells was 62.2%; the CAR positivity rate of IL10-CD19-CAR-mbIL15-DNT cells was 30.2%, with a CAR+mbIL15 positivity rate of 16.2%. It can be seen that CAR molecules were successfully expressed on the cell membranes of DNT cells with different CD19-CAR structures. The surfaces of CD19-CAR-mbIL15-DNT cells and IL10-CD19-CAR-mbIL15-DNT cells not only successfully expressed CD19-CAR molecules but also successfully expressed mbIL15 molecules.

[0208] The phenotypic detection results of CAR-DNT cells with different structures in this embodiment are shown in Figure 4. In the CD19-CAR-DNT cells, the proportion of CD3+ T cells was 99.3%. + CD4 - CD8 - The percentage of DNT cells was 95.2%, and the CD45RA content was [missing information]. + CD62L + The TSCM ratio is 11.8%, CD45RA - CD62L + The TCM ratio was 27.7%; CD3 in IL10-CD19-CAR-DNT + The proportion of T cells was 97.8%, CD3 + CD4- CD8 - The percentage of DNT cells was 92.0%, and the CD45RA content was [missing information]. + CD62L + The TSCM ratio is 8.5%, CD45RA - CD62L + The TCM ratio is 11.8%; CD19-CAR-mbIL15-DNT contains CD3 + The proportion of T cells was 98.9%, CD3 + CD4 - CD8 - The percentage of DNT cells was 98.0%, and the CD45RA content was [missing information]. + CD62L + The TSCM ratio is 7.8%, CD45RA - CD62L + The TCM ratio is 31.0%; CD3 in IL10-CD19-CAR-IL15-DNT + The proportion of T cells was 99.1%, CD3 + CD4 - CD8 - The percentage of DNT cells was 97.3%, and the CD45RA content was [missing information]. + CD62L + The TSCM ratio is 13.2%, CD45RA - CD62L + The TCM ratio was 37.2%; CD3 in IL10-CD19-CAR-mbIL15-DNT + The proportion of T cells was 99.1%, CD3 + CD4 - CD8 - The DNT cells were 97.9%, CD45RA + CD62L + The TSCM ratio is 11.9%, CD45RA - CD62L + The TCM ratio is 45.9%.

[0209] The above results indicate that CD19-CAR-DNT, IL10-CD19-CAR-DNT, CD19-CAR-mbIL15-DNT, IL10-CD19-CAR-IL15-DNT, and IL10-CD19-CAR-mbIL15-DNT cells contain CD3+. + The proportion of T cells was above 95%, including DNT cells (CD3+). + CD4 - CD8 -)The proportions of all T cells were above 90%, with no significant difference. In IL10-CD19-CAR-mbIL15-DNT cells, Tscm+Tcm cells accounted for more than 55%, far exceeding that of CAR-DNT cells with other structures. This indicates that IL10-CD19-CAR-mbIL15-DNT cells have a higher proportion of stem cell-like T cells and better cell persistence at the time of administration.

[0210] Example 4: Detection of IL10 secreted by CAR-DNT cells

[0211] To compare whether CAR-DNT cells with different CD19-CAR structures could successfully secrete IL10 and their sustained secretory potential, cells cultured in vitro for 24 hours (cell density 8×10⁶) were collected. 5 IL10 was detected in the supernatant of CD19-CAR-DNT, IL10-CD19-CAR-DNT, CD19-CAR-mbIL15-DNT, IL10-CD19-CAR-IL15-DNT, and IL10-CD19-CAR-mbIL15-DNT cells cultured for 12 days, according to the cytokine detection method in Example 5.

[0212] The results are shown in Figure 5: After 24 hours of in vitro culture, the concentrations of IL10 secreted by IL10-CD19-CAR-DNT, IL10-CD19-CAR-IL15-DNT, and IL10-CD19-CAR-mbIL15-DNT cells were 9765.0 pg / ml, 1458 pg / ml, and 189 pg / ml, respectively, while CD19-CAR-DNT and CD19-CAR-mbIL15-DNT cells did not secrete IL10; after 12 days of in vitro culture, The concentration of IL10 secreted by IL10-CD19-CAR-DNT cells was 45864.2 pg / ml, the concentration of IL10 secreted by IL10-CD19-CAR-IL15-DNT cells was 20214.0 pg / ml, and the concentration of IL10 secreted by IL10-CD19-CAR-mbIL15-DNT cells was 942.4 pg / ml. However, CD19-CAR-DNT and CD19-CAR-mbIL15-DNT cells did not secrete IL10.

[0213] It is evident that IL10-CD19-CAR-DNT, IL10-CD19-CAR-IL15-DNT, and L10-CD19-CAR-mbIL15-DNT cells can all continuously secrete IL10. Among them, L10-CD19-CAR-mbIL15-DNT cells secrete a lower concentration of IL10, which effectively reduces the safety risks caused by excessive IL10 secretion.

[0214] Example 5: Detection of CAR-DNT Cytokine Release

[0215] (1) Effector cell preparation: Untransduced DNT cells and CAR-DNT cells were placed in 15ml centrifuge tubes, 5ml of 1×PBS was added to each tube, centrifuged at 500×g for 5min, and washed twice; the cells were resuspended in X-VIVO + 5% FBS basal medium, counted using K2, and the CAR-DNT cell density was adjusted to 8×10⁶ cells using DNT cells. 5 The concentration is 1 unit / ml, with a target-to-effect ratio of 1:1, for later use.

[0216] (2) Target cell preparation: Non-target cells (K562) and target cells (Raji-luciferase, Nalm6-luciferase) in the logarithmic growth phase were collected; centrifuged at 500×g for 3 minutes in 15ml centrifuge tubes, the supernatant was discarded, and the cells were washed twice; the cells were resuspended in X-VIVO + 5% FBS basal medium, and cell counts and viability were detected using a cell counter. Finally, the cell density was diluted to 8×10⁶. 5 Concentration per ml, for later use.

[0217] (3) Effector and target cell co-culture: The treated effector cells were co-cultured with target cells and non-target cells respectively. 100 μL of target cells and 100 μL of CAR-DNT cells were mixed 1:1 and added to a U-shaped 96-well plate. The plate was placed in a 37℃, 5% CO2 incubator and co-cultured for 20-24 hours. The supernatant of the co-culture was collected into a 1.5 ml EP tube for later use.

[0218] (4) Cytokine detection: According to the instructions of the CBA detection kit, determine the cytokines to be detected and the number of samples. Prepare capture beads, take out the capture bead bottle containing the cytokines to be detected, mix vigorously, and take out (number of samples to be detected + number of negative controls) × 10 μl / 6 of each type of bead. Mix each type of bead by turbo oscillation. Take N 1.5 ml EP tubes (N = number of samples + number of controls) and add 10 μl of mixed beads to each tube (sample and negative control). Add 10 μl of the corresponding test reagent (sample and control) to each tube. Add 10 μl of PE Detection Reagent to each tube, mix thoroughly, and incubate at room temperature in the dark for 3 hours. After incubation, add 500 μl of wash buffer to each sample, mix thoroughly, centrifuge at 500×g for 5 minutes, and discard the supernatant. Resuspend with 200 μl of wash buffer, centrifuge at 500×g for 5 minutes, wash, discard the supernatant, and wash with 150 μl of wash buffer. Samples were resuspended in buffer and then analyzed by flow cytometry.

[0219] As shown in Figure 6, co-culturing CD19-CAR-DNT, IL10-CD19-CAR-DNT, CD19-CAR-DNT-mbIL15, IL10-CD19-CAR-DNT-IL15, and IL10-CD19-CAR-DNT-mbIL15 cells with target cells (Raji-luciferase and Nalm6-luciferase) resulted in the release of large amounts of inflammatory cytokines TNFα and IFNγ, and CD19-CAR-DNT-mbIL15 cells also secreted a small amount of IL2. The cytokine release results indicate that CAR-DNT cells with different CD19-CAR structures all possess highly specific targeting capabilities and can be activated by tumor target cells expressing CD19 antigen, releasing inflammatory cytokines. Among them, CD19-CAR-DNT-mbIL15 cells released relatively low levels of inflammatory cytokines, effectively mitigating the potential cytokine storm during administration and thus improving administration safety.

[0220] Example 6: Detection of CAR-DNT cell killing ability

[0221] The cell-killing ability of CAR-DNT was detected using a firefly luciferase reporter gene activity chemiluminescence assay. The specific experimental steps are as follows:

[0222] (1) Effector cell preparation: Untransduced DNT cells and CAR-DNT cells were placed in 15ml centrifuge tubes, 5ml of 1×PBS was added to each tube, centrifuged at 500×g for 5min, and washed twice; the cells were resuspended in X-VIVO + 5% FBS basal medium, counted using K2, and the CAR-DNT cell density was adjusted to 8×10⁶ cells using DNT cells. 5 cells / ml, 4×10 5 cells / ml, 2×10 5 Quantity / ml, corresponding to target-efficacy ratios of 4:1, 2:1, and 1:1, for later use.

[0223] (2) Target cell preparation: Target cells (Raji-luciferase, Nalm6-luciferase) in the logarithmic growth phase were placed in 15ml centrifuge tubes, centrifuged at 500×g for 3 minutes, the supernatant was discarded, and the cells were washed twice. The cells were resuspended in X-VIVO + 5% FBS basal medium, and cell counts and viability were detected using a cell counter. Finally, the cell density was diluted to 2×10⁻⁶. 5 concentration per ml.

[0224] (3) Chemiluminescence detection: 50 μL of target cells and 50 μL of CAR-DNT cells were mixed and added to a white flat-bottomed 96-well plate, and incubated at 37°C and 5% CO2 for 48 hours; 100 μL of GMOne-Step was added to each well. TM The substrate for the luciferase reporter gene assay kit was mixed and allowed to stand for 5 minutes. The sample was then analyzed using a Spectra MaxL microplate reader (wavelength 570 nm).

[0225] (4) Calculate the kill rate based on the RLU value. Kill rate (%) = 1 - (target cell + effector cell) fluorescence value / target cell fluorescence value × 100%

[0226] The results, shown in Figure 7, indicate that CD19-CAR-DNT, IL10-CD19-CAR-DNT, CD19-CAR-DNT-mbIL15, IL10-CD19-CAR-DNT-IL15, and IL10-CD19-CAR-DNT-mbIL15 cells all exhibited dose-dependent killing of tumor target cells. At an effector-to-target ratio of 1:1, the percentages of Raji-luciferae tumor target cells killed by CD19-CAR-DNT, IL10-CD19-CAR-DNT, CD19-CAR-DNT-mbIL15, IL10-CD19-CAR-DNT-IL15, and IL10-CD19-CAR-DNT-mbIL15 cells were 67.2%, 91.1%, 73.2%, 83.5%, and 83.7%, respectively.

[0227] Example 7: Detection of multiple rounds of tumor stimulation in CAR-DNT cells

[0228] (1) Effector cell preparation: Untransduced DNT cells and CAR-DNT cells were placed in 15ml centrifuge tubes, 5ml of 1×PBS was added to each tube, centrifuged at 500×g for 5min, and washed twice; the cells were resuspended in X-VIVO + 5% FBS medium, counted using K2, and the CAR-DNT cell density was adjusted to 2×10⁻⁶ cells / mL. 5 Quantity / ml, for later use.

[0229] (2) Target cell preparation: Take target cells (Raji-luciferase, Nalm6-luciferase) in the logarithmic growth phase into a 15ml centrifuge tube, centrifuge at 500×g for 3 minutes, discard the supernatant, and wash twice; resuspend the cells in X-VIVO + 5% FBS basal medium, count them using K2, and adjust the cell density to 2×10⁻⁶. 5 Concentration per ml, for later use;

[0230] (3) Effector and target cell co-culture: The treated effector cells and target cells were added to a 12-well plate at a 1:1 effector-target ratio. 500 μl of target cells and 500 μl of CAR-DNT cells were added to each well. The cells were cultured in a 37°C, 5% CO2 incubator.

[0231] (4) Multiple antigen stimulation: Co-culture cells for 4-5 days, harvest the co-incubated mixed cells, centrifuge at 500×g for 5 minutes, and resuspend the cells in 1ml of X-VIVO + 5% FBS medium; take 100ul of the cell stock solution from step one and add GMOne-Step TM Use luciferase reporter gene assay reagent to detect its fluorescence value; take 500 μL of the above cells and add 500 μL of 2×10⁻⁶ cells. 5 Tumor cells were cultured at a density of 100 cells / ml for 4–5 days for two rounds of tumor killing. The operation methods for the third and fourth rounds of tumor cell killing were the same as those for the second round. The fluorescence value of the surviving tumor cells in each round was counted.

[0232] The results are shown in Figure 8 and Table 5-6. After the third round of tumor killing, the fluorescence value of the residual tumor cells in the IL10-CD19-CAR-DNT-mbIL15 cell group, which contains the IL10 and mbIL5 genes in the CD19-CAR structure, was much lower than that in the CD19-CAR-DNT, IL10-CD19-CAR-DNT, CD19-CAR-DNT-mbIL15, and IL10-CD19-CAR-DNT-IL15 cell groups. This suggests that the IL10-CD19-CAR-DNT-mbIL15 cells have a stronger and more sustained ability to kill tumor (target) cells than other CAR-DNT cell groups.

[0233] Table 5: Residual fluorescence values ​​of Raji-Luciferase after multiple rounds of killing by CAR-DNT cells (mean ± SD)

[0234] Table 6: Residual fluorescence values ​​of NALM6-Luciferase after multiple rounds of killing by CAR-DNT cells (mean ± SD)

[0235] Example 8: Detection of the ability of CAR-DNT cells to survive in vitro without cytokines

[0236] (1) Take untransduced DNT cells and CAR-DNT cells and place them in 15ml centrifuge tubes respectively, add 5ml of 1×PBS to each tube, and centrifuge at 500×g for 5min.

[0237] (2) Resuspend the cells in 5 ml of 1×PBS, centrifuge at 500×g for 5 min, discard the supernatant, and wash twice;

[0238] (3) Resuspend cells in X-VIVO + 10% ICSR medium (without cytokines), perform K2 counting, and adjust cell density to 5 × 10⁻⁶ cells / year. 5 Individuals / ml, cultured in 5ml T25 culture flasks;

[0239] (4) Sample and count the number and viability of cells every 7 days.

[0240] The results are shown in Figure 9. The in vitro expansion and survival of CAR-DNT cells with different CD19-CAR structures were studied using an IL2-free in vitro culture system. After in vitro culture to day 21, the expansion fold and survival rate of IL10-CD19-CAR-DNT-mbIL15 cells were significantly better than those of CD19-CAR-DNT and DNT cells.

[0241] Example 9: In vivo efficacy and safety testing of CAR-DNT cells

[0242] Raji-luciferase cells were injected into NOG mice (5 × 10⁻⁶) via tail vein. 5 / mouse). On the third day after inoculation, the proliferation of tumor cells in vivo was observed using in vivo imaging technology. NOG mice were randomly divided into 4 groups according to the tumor fluorescence intensity. After grouping, they were injected via tail vein with PBS (control), CD19-CAR-DNT cells, IL10-CD19-CAR-DNT cells, and IL10-CD19-CAR-DNT-mbIL15 cells (5×10⁻⁶ cells), respectively. 6 ( / mouse) The proliferation of tumor cells in mice was detected by in vivo imaging, and the antitumor activity and safety were evaluated based on changes in fluorescence intensity and changes in mouse weight.

[0243] The results of this embodiment are shown in Figure 10A. In vivo imaging data on day 22 after CAR-DNT cell infusion showed that mice in the CD19-CAR-DNT, IL10-CD19-CAR-DNT, and IL10-CD19-CAR-mbIL15-DNT cell infusion groups all exhibited effective anti-tumor effects in vivo. Although the mice in the IL10-CD19-CAR-DNT cell group had a strong anti-tumor effect (Figure 10B), they began to show adverse symptoms such as weight loss and abnormal activity on day 15 after cell infusion (Figure 10C), which may be related to the high concentration of IL10 released by the IL10-CD19-CAR-DNT cells.

[0244] To compare the differences in in vivo antitumor activity between CD19-CAR-DNT cells and IL10-CD19-CAR-mbIL15-DNT cells, mice in both groups were re-infused with 1×10⁻⁶ cells on day 22. 6 Raji-luciferase tumor cells were used in a high-challenge experiment. Imaging results on day 31 showed that all five mice in the CD19-CAR-DNT group experienced tumor recurrence, with an average fluorescence value of 2.6 × 10⁻⁶. 6 P / S, while all mice in the IL10-CD19-CAR-mbIL15-DNT group had no tumor recurrence, no tumor fluorescence was detected (Figure 10 A and B), and the mice had normal weight and activity (Figure 10 C).

[0245] To further challenge the sustained antitumor effect of IL10-CD19-CAR-mbIL15-DNT cells in vivo, mice were infused with 1×10⁻⁶ cells for the third time on day 31. 6 Imaging results on day 39 showed that although the tumors in mice had recurred, the tumor burden remained very low, with an average fluorescence value of only 3.7 × 10⁻⁶. 6 P / S (Figure 10, A and B), and the mouse's weight and activity were normal (Figure 10, C).

[0246] In summary, under the experimental conditions, the enhanced IL10-CD19-CAR-mbIL15-DNT cells exhibited a more sustained antitumor effect than CD19-CAR-DNT cells, without significant toxic side effects. Furthermore, it was demonstrated that adding the mbIL5 gene to the IL10-CD19-CAR structure significantly reduced IL10 toxicity and maintained the cells' sustained antitumor activity.

[0247] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A chimeric antigen receptor (CAR) construct, characterized in that, The structure of the CAR construct is shown in Equation I below: X-A1-E-A2-IL10 (I) In the formula, Each "-" independently represents a linking peptide or peptide bond; X represents a CAR that targets tumor antigens; A1 and A2 are self-shearing elements; E is a CAR function-enhancing element selected from the following group: membrane-bound IL15, IL2, IL4, IL7, IL18, IL21, IL23, and IL36 fusion proteins, secreted IL15, IL2, IL4, IL7, IL18, IL21, IL23, and IL36 cytokines, as well as IL7R mutants and IL21R mutants; IL10 is interleukin-10 or its active fragment or mutant.

2. The CAR construct as described in claim 1, characterized in that, The structure of X is shown in equation II below: L-scFv-H-TM-C-CD3ζ (II) In the formula, Each "-" independently represents a linking peptide or peptide bond; L represents the absence of a signal peptide; scFv is the single-chain variable region of an antibody that targets tumor antigens; H represents the hingeless region; TM represents a transmembrane domain; C is a co-stimulatory signaling molecule; CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ.

3. The CAR construct as described in claim 2, characterized in that, The scFv is a CD19-targeting scFv; preferably, the amino acid sequence of the CD19-targeting scFv is shown in SEQ ID NO:

2.

4. The CAR construct as claimed in claim 1, characterized in that, The CAR functional enhancement element is a membrane-bound IL15 fusion protein (mbIL15), the structure of which is shown in Formula III, L'-MIR (III). In the formula, Each "-" independently represents a linking peptide or peptide bond; L' represents either no signal peptide or a signal peptide; M represents interleukin-15 (IL-15); I represents a flexible joint; R stands for interleukin-15 receptor α (IL-15Rα).

5. The CAR construct as described in claim 4, characterized in that, The amino acid sequence of the membrane-bound IL15 fusion protein (mbIL15) is shown in SEQ ID NO:

14.

6. The CAR construct as claimed in claim 1, characterized in that, The amino acid sequence of IL10 is shown in SEQ ID NO:

24.

7. The CAR construct as claimed in claim 1, characterized in that, The full-length amino acid sequence of the CAR construct is shown in SEQ ID NO:

25.

8. A polynucleotide molecule, characterized in that, The nucleic acid molecule encodes the CAR construct as described in any one of claims 1-7.

9. A carrier, characterized in that, The carrier contains the polynucleotide molecule as described in claim 8.

10. An engineered immune cell, characterized in that, The immune cells express the CAR construct as described in any one of claims 1-7.

11. The engineered immune cells as described in claim 10, characterized in that, The engineered immune cells mentioned are chimeric antigen receptor DNT cells (CAR-DNT cells), chimeric antigen receptor T cells (CAR-T cells), or chimeric antigen receptor NK cells (CAR-NK cells).

12. A formulation, characterized in that, The formulation contains the CAR construct as described in any one of claims 1-7, the polynucleotide molecule as described in claim 8, the vector as described in claim 9, or the engineered immune cell as described in claim 10, and a pharmaceutically acceptable vector.

13. Use of a CAR construct as described in any one of claims 1-7, a polynucleotide molecule as described in claim 8, a vector as described in claim 9, an engineered immune cell as described in claim 10, or a formulation as described in claim 12, for the preparation of a medicament or formulation for the prevention and / or treatment of a disease.

14. A kit for preparing engineered immune cells as claimed in claim 10, the kit comprising a container and a polynucleotide molecule as claimed in claim 8 or a carrier as claimed in claim 9 located within the container.

15. A method for preparing engineered immune cells as described in claim 10, the method comprising the following steps: (a) Provide immune cells to be modified; and (b) Transfecting the polynucleotide molecule as described in claim 8 or the vector as described in claim 9 into the immune cells to obtain the engineered immune cells.

16. A method of treating a disease, the method comprising administering to a subject requiring treatment an effective amount of the carrier as claimed in claim 9, the engineered immune cells as claimed in claim 10, or the formulation as claimed in claim 12.

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