Construction, preparation and application of function-enhanced universal double-target BCMA / CD19-mbIL15-CAR-DNT cell

Abstract

The invention provides construction, preparation and application of a universal double-target BCMA (bovine serum albumin) / CD19-CAR-DNT (Chimeric Antigen Receptor-DNT) cell with enhanced functions. Specifically, the invention provides a chimeric antigen receptor construct of double-target BCMA / CD19, a membrane binding interleukin 15 (mbIL15) is fused at the C terminal of a bispecific CAR targeting BCMA and CD19 through a self-cleavage peptide, and the CAR construct is introduced into a DNT cell, so that a universal double-target BCMA / CD19-CAR-DNT cell with enhanced functions is obtained. The BCMA / CD19-CAR-DNT cell disclosed by the invention is targeted to BCMA and CD19 at the same time, and has stronger and more lasting cell killing activity, so that a new therapy is provided for BCMA and / or CD19 mediated diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of universal immune cell therapy, and particularly relates to construction, preparation and application of a universal double-target BCMA / CD19-mbIL15-CAR-DNT cell with enhanced function. BACKGROUND

[0002] Double negative T (DNT) cells refer to a CD3 + CD4 - CD8 - negative T cell subpopulation normally present in human peripheral blood, accounting for 1-10% of the total number of T cells. DNT cells express CD3 molecules and αβ- or γδ-T cell receptors (TCR) on the cell surface, do not express CD4 and CD8 molecules, and do not bind to CD1d tetramer loaded with αGalCer specific for invariant natural killer T (iNKT) cells, and thus are different from conventional T cells, NK cells and NKT cells. DNT cells specifically recognize target cells in an MHC-unrestricted manner through cell surface receptors such as NKG2D, LFA-1 and DNAM-1, and achieve killing and clearance of target cells by secreting a series of cytotoxic molecules and cytokines, but do not cause graft-versus-host disease (GvHD) and host-versus-graft reaction (HvGR), and thus have great potential for development of universal DNT cell therapy.

[0003] Multiple myeloma (MM) is a hematological tumor disease characterized by abnormal proliferation of plasma cells (PCs) and production of large amounts of pathological immunoglobulins. The tumor cells originate from plasma cells in the bone marrow, and have the characteristics of high incidence and high mortality. Among hematological tumors, the global incidence of multiple myeloma (MM) is second only to lymphoma, accounting for more than 10% of hematological malignancies, with an incidence of about 2-3 / 100,000 people and a mortality rate of about 1-2 / 100,000 people. The market potential for treating multiple myeloma is huge.

[0004] B-cell maturation antigen (BCMA), also known as CD269, is a member of the tumor necrosis factor receptor superfamily member 17 (TNFRS17), consisting of 184 amino acid residues, belonging to the type I transmembrane protein, and the extracellular region sequence is very short, only one sugar recognition domain of B cell surface molecules. As a cell surface protein only expressed on the B cell lineage, it binds to B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL), and plays an important regulatory role in B cell proliferation, survival, and maturation and differentiation into plasma cells. BCMA is not expressed in normal tissues and organs of the human body except mature B cells and plasma cells, and is not expressed in CD34 + hematopoietic stem cells, but is highly expressed in malignant multiple myeloma cells, and as the disease progresses, BCMA expression increases, and gene and protein expression profiling analysis confirms that BCMA is the most selectively expressed transmembrane protein on multiple myeloma cell lines. In recent years, CAR-T products targeting BCMA have also been used in the treatment of autoimmune diseases such as multiple sclerosis. Therefore, BCMA is a very valuable immunotherapy target.

[0005] CD19 is a B cell surface glycoprotein consisting of 556 amino acids, which belongs to the type I transmembrane protein. It is expressed at an early stage of B cell development and continues until it differentiates into plasma cells. CD19 is a member of the immunoglobulin (Ig) superfamily and is involved in the regulation of BCR signal transduction as one of the elements of the B cell surface signal transduction complex. The expression of CD19 is limited to the B cell lineage, and the surface of pluripotent hematopoietic stem cells does not express it. CD19 is expressed on the surface of most B cell lymphomas, mantle cell lymphoma, ALL, CLL, hairy cell leukemia, and a portion of acute myeloid leukemia cells. In addition, in 2015, an article reported by U Penn and Novartis in NEJM pointed out that in a reported case of multiple myeloma, although 99.95% of the malignant proliferating plasma cells lacked CD19 expression, the patient still achieved complete cure after CD19-CAR-T treatment. Therefore, it is believed that CD19 + tumor cells are multiple myeloma tumor stem cells. In recent years, CAR-T cells targeting CD19 have also been used in the treatment of autoimmune diseases such as systemic lupus erythematosus. Therefore, in the treatment of leukemia, lymphoma, multiple myeloma and autoimmune diseases, CD19 is a very valuable immunotherapy target.

[0006] Interleukin-15 (IL-15) is an important cytokine with the ability to induce anti-tumor response. The biological characterization of IL-15 is extremely complex, and it exists in several functional forms: soluble monomer form (sIL-15), soluble complex sIL-15 / IL-15Rα, transmembrane form tp-IL-15, and 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, IL-15 stays on the cell surface and forms an immunological synapse with IL-2R / IL-15Rβ-γc on the nearby effector cells, activating the Jak / Stat pathway (Jak1 and Jak3, Stat3 and Stat5) to transduce signals in cells. The IL-15Rα chain confers IL-15 specificity and binds to the cytokine with high affinity. Studies have shown that DNT cells highly express natural killer receptors (NCR) such as NKG2D, DNAM-1, NKp30, and TRAIL, and these natural killer receptors are further up-regulated under the stimulation of IL-15, thereby enhancing the anti-tumor activity of DNT cells.

[0007] At present, there are many studies in the field for treating BCMA and CD19 related cancers using CAR-T cell therapy, but these therapies still have many limitations in application. Therefore, there is still a need in the art to develop more effective immune cell therapies for the treatment of BCMA and CD19 related diseases. SUMMARY

[0008] The purpose of the present application is to provide a functionally enhanced universal dual-target BCMA / CD19-mIL15-CAR-DNT cell, as well as a construction method, a preparation method and an application thereof.

[0009] In a first aspect of the present application, a chimeric antigen receptor (CAR) construct is provided, and the structure of the CAR construct is shown in the following formula I or II,

[0010] X-A-E (I)

[0011] E-A-X (II)

[0012] In the formula,

[0013] Each "-" is independently a connecting peptide or a peptide bond;

[0014] X is a CAR targeting tumor antigens BCMA and CD19;

[0015] A is a self-cleavage element;

[0016] E is a membrane-bound interleukin 15 (mbIL15) fusion protein.

[0017] In another preferred embodiment, the CAR targeting tumor antigens BCMA and CD19 comprises an antibody single chain variable region (scFv) targeting BCMA and an antibody single chain variable region targeting CD19; wherein the amino acid sequence of the scFv targeting BCMA is set forth in SEQ ID NO: 4, and the amino acid sequence of the scFv targeting CD19 is set forth in SEQ ID NO: 2.

[0018] In another preferred embodiment, the CAR construct has the structure shown in Formula I.

[0019] In another preferred embodiment, the X has the structure shown in Formula III,

[0020] L-scFv1-scFv2-H-TM-C-CD3ζ (III)

[0021] In the formula,

[0022] Each “-” is independently a connecting peptide or a peptide bond;

[0023] L is nothing or a signal peptide;

[0024] scFv1 is an antibody single chain variable region targeting BCMA, and scFv2 is an antibody single chain variable region targeting CD19; or, scFv1 is an antibody single chain variable region targeting CD19, and scFv2 is an antibody single chain variable region targeting BCMA;

[0025] H is nothing or a hinge region;

[0026] TM is a transmembrane domain;

[0027] C is a costimulatory signaling molecule;

[0028] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

[0029] In another preferred embodiment, the amino acid sequence of the scFv targeting BCMA is set forth in SEQ ID NO: 4.

[0030] In another preferred embodiment, the amino acid sequence of the scFv targeting CD19 is set forth in SEQ ID NO: 2.

[0031] In another preferred embodiment, the 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.

[0032] In another preferred embodiment, the L is a signal peptide derived from MNDU3.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] In another preferred embodiment, the membrane-bound interleukin 15 (mbIL15) fusion protein has a structure as shown in Formula IV, L'-M-I-R (IV)

[0047] In the formula,

[0048] Each "-" is independently a connecting peptide or a peptide bond;

[0049] L' is nothing or a signal peptide;

[0050] M is interleukin 15 (IL-15);

[0051] I is a flexible linker;

[0052] R is interleukin 15 receptor alpha (IL-15Rα).

[0053] In another preferred embodiment, M comprises wild-type IL-15, or an active fragment thereof, or a mutant thereof.

[0054] In another preferred embodiment, the amino acid sequence of M is shown in SEQ ID NO: 26.

[0055] In another preferred embodiment, the IL-15Rα is a complete IL-15Rα element.

[0056] In another preferred embodiment, the IL-15Rα comprises a transmembrane region and an intracellular region.

[0057] In another preferred embodiment, the IL-15Rα comprises, in order from N-terminus to C-terminus, an extracellular region that binds to IL-15, a transmembrane region, and an intracellular region.

[0058] In another preferred embodiment, the amino acid sequence of the IL-15Rα is shown in SEQ ID NO: 28.

[0059] In another preferred embodiment, the flexible linker is a connecting peptide, preferably the amino acid of the connecting peptide is shown in SEQ ID NO: 30.

[0060] In another preferred embodiment, L' is an IgE signal peptide, and the amino acid sequence thereof is shown in SEQ ID NO: 32.

[0061] In another preferred embodiment, the membrane-bound interleukin 15 (mbIL15) fusion protein has an amino acid sequence as shown in SEQ ID NO: 24.

[0062] In another preferred embodiment, the self-cleavage element comprises T2A, P2A.

[0063] In another preferred embodiment, the self-cleaving element is P2A, and the amino acid sequence of P2A is shown as SEQ ID NO: 22.

[0064] In another preferred embodiment, the full-length amino acid sequence of the CAR construct is shown as SEQ ID NO: 33.

[0065] In a second aspect of the present application, a polynucleotide molecule is provided, wherein the nucleic acid molecule encodes the CAR construct according to the first aspect of the present application.

[0066] In a third aspect of the present application, a vector is provided, wherein the vector comprises the polynucleotide molecule according to the second aspect of the present application.

[0067] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentivirus vector, adenovirus vector, adeno-associated virus vector (AAV), retrovirus vector, transposon, or a combination thereof.

[0068] In another preferred embodiment, the vector is selected from the group consisting of plasmid, viral vector.

[0069] In another preferred embodiment, the vector is in the form of a viral particle.

[0070] In another preferred embodiment, the vector is a lentivirus vector.

[0071] In a fourth aspect of the present application, a host cell is provided, wherein the host cell comprises the vector according to the third aspect of the present application or the polynucleotide molecule according to the second aspect of the present application is integrated into the chromosome of the host cell, or the CAR construct according to the first aspect of the present application is expressed in the host cell.

[0072] In another preferred embodiment, the host cell comprises eukaryotic cells and prokaryotic cells.

[0073] In another preferred embodiment, the host cell comprises Escherichia coli and yeast.

[0074] In a fifth aspect of the present application, an engineered immune cell is provided, wherein the immune cell expresses the CAR construct according to the first aspect of the present application.

[0075] In another preferred embodiment, the cell is an isolated cell.

[0076] In another preferred embodiment, the CAR targeting tumor antigens BCMA and CD19 and a membrane-bound interleukin 15 (mbIL15) fusion protein are independently expressed on the cell membrane of the immune cell.

[0077] In another preferred embodiment, the immune cell is from a human or a non-human mammal.

[0078] In another preferred embodiment, the cell comprises a T cell, an NK cell.

[0079] In another preferred embodiment, the cell is a double negative T cell (DNT) cell.

[0080] In another preferred embodiment, the engineered immune cell can be a chimeric antigen receptor DNT cell (CAR-DNT cell), a chimeric antigen receptor T cell (CAR-T cell), or a chimeric antigen receptor NK cell (CAR-NK cell).

[0081] In a sixth aspect of the present application, there is provided a preparation comprising the CAR construct of the first aspect of the present application, the polynucleotide molecule of the second aspect of the present application, the vector of the third aspect of the present application, or the immune cell of the fifth aspect of the present application, and a pharmaceutically acceptable carrier.

[0082] In another preferred embodiment, the preparation is a liquid preparation.

[0083] In another preferred embodiment, the dosage form of the preparation is an injection.

[0084] In another preferred embodiment, the concentration of the immune cell (e.g. CAR-DNT cell) in the preparation is 1 x 10 3 -1 x 10 8 cells / ml, preferably 1 x 10 4 -1 x 10 7 cells / ml.

[0085] In another preferred embodiment, the preparation further comprises a second active ingredient against a tumor, preferably comprising a second antibody or a chemotherapeutic agent.

[0086] In a seventh aspect of the present application, there is provided the use of the CAR construct of the first aspect of the present application, the polynucleotide molecule of the second aspect of the present application, the vector of the third aspect of the present application, or the immune cell of the fifth aspect of the present application, or the preparation of the sixth aspect of the present application, for the manufacture of a medicament or preparation for the prevention and / or treatment of a disease.

[0087] In another preferred embodiment, the disease is a BCMA and / or CD19 mediated disease.

[0088] In another preferred embodiment, the disease comprises a cancer or a tumor, an autoimmune disease.

[0089] In another preferred embodiment, the cancer or tumor comprises a solid tumor and a hematological tumor.

[0090] 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 a combination thereof.

[0091] In another preferred embodiment, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, multiple sclerosis, or a combination thereof.

[0092] In an eighth aspect of the present application, there is provided a kit for preparing the engineered immune cell of the fifth aspect of the present application, the kit comprising a container, and the polynucleotide molecule of the second aspect of the present application, or the vector of the third aspect of the present application, in the container.

[0093] In a ninth aspect of the present application, there is provided a method for preparing the engineered immune cell of the fifth aspect of the present application, the method comprising the steps of:

[0094] (a) providing an immune cell to be engineered; and

[0095] (b) transducing the polynucleotide molecule of the second aspect of the present application, or the vector of the third aspect of the present application, into the immune cell, thereby obtaining the engineered immune cell.

[0096] In another preferred embodiment, the immune cell to be engineered is a DNT cell, a T cell, or an NK cell.

[0097] In another preferred embodiment, the method further comprises the step of detecting the functionality and effectiveness of the obtained engineered immune cell.

[0098] In a tenth aspect of the present application, there is provided a method for treating a disease, the aspect comprising administering to a subject in need of treatment an effective amount of the vector of the third aspect of the present application, the immune cell of the fifth aspect of the present application, or the formulation of the sixth aspect of the present application.

[0099] In another preferred embodiment, the disease is a BCMA and / or CD19 mediated disease.

[0100] In another preferred embodiment, the disease comprises a cancer or tumor, an autoimmune disease.

[0101] In another preferred embodiment, the cancer or tumor comprises a solid tumor and a hematological tumor.

[0102] 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 a combination thereof.

[0103] In another preferred embodiment, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, multiple sclerosis, or a combination thereof.

[0104] It should be understood that, in the scope of the present application, each of the technical features described above and each of the technical features described in detail below (such as the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0105] Figure 1 Schematic diagram of the CAR structure constructed for the present application.

[0106] Figure 2 CAR-DNT cell amplification fold and viability results are shown.

[0107] Figure 3 CAR-DNT cell CAR positive rate detection results are shown.

[0108] Figure 4 CAR-DNT cell phenotype detection results are shown.

[0109] Figure 5 CAR-DNT cell cytokine release results after co-culture with tumor cells are shown.

[0110] Figure 6 CAR-DNT cell killing effect results are shown.

[0111] Figure 7 CAR-DNT cell tumor killing results in multiple rounds are shown.

[0112] Figure 8 CAR-DNT cell in vitro survival ability results are shown. DETAILED DESCRIPTION

[0113] The present inventors have conducted extensive and in-depth research and first constructed a function-enhanced universal double-target BCMA / CD19-mbIL15-CAR-DNT cell. The present application provides a double-target BCMA / CD19 chimeric antigen receptor construct, which fuses an IL-15 / IL-15R alpha complex (i.e., membrane-bound interleukin 15 (mbIL15)) at the C-terminus of the double-specificity CAR targeting BCMA and CD19 through a self-cleaving peptide, and introduces the CAR construct into DNT cells, thereby obtaining a function-enhanced universal double-target BCMA / CD19-mbIL15-CAR-DNT cell. The BCMA / CD19-mbIL15-CAR-DNT cell of the present application simultaneously targets BCMA and CD19 and has a significant killing effect on BCMA and CD19 positive cells; and the BCMA / CD19-CAR-DNT cell of the present application expresses an IL-15 / IL-15R alpha complex on the cell membrane surface, which can further enhance the tumor killing activity and persistence of DNT cells.

[0114] On this basis, the present application is completed.

[0115] Terms

[0116] In order that the disclosure can be more readily understood, certain terms are first defined. As used in this application, unless specifically stated otherwise, each of the following terms has the meaning given below. Additional definitions are set forth throughout the application.

[0117] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0118] The term "administering" refers to physically introducing the product of the present application into a subject using any of a variety of methods and delivery systems known to those in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, e.g., by injection or infusion.

[0119] The term "antibody" (Ab) shall include, but is not limited to, an immunoglobulin which specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2 and CH3. Each L chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each of VHand VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0120] It should be understood that the names of the amino acids herein are in accordance with the international one-letter code, and the corresponding three-letter abbreviations of the names of the amino acids are: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gin (Q), Glu (E), Gly (G), His (H), lie (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), Val (V).

[0121] As used herein, the terms "BCMA / CD19-mbIL15-CAR-DNT cells" and "BCMA / CD19-CAR-mbIL15-DNT cells" can be used interchangeably, and both refer to the universal dual-targeted DNT cells with enhanced function constructed by the present application, which simultaneously target BCMA and CD19, and express membrane-bound interleukin 15 (mbIL15) on the cell membrane surface.

[0122] Membrane-bound interleukin 15 (mbIL15)

[0123] The chimeric antigen receptor (CAR) construct of the present application comprises a membrane-bound interleukin 15 connected to the N-terminus or C-terminus of the CAR targeting tumor antigens BCMA and CD19 through a self-cleavage element (such as P2A or T2A). The membrane-bound interleukin 15 is a fusion protein formed by IL-15 and IL-15Rα, also known as "IL-15 / IL-15Rα complex", which comprises IL15 or its mutants or active fragments thereof, and the complete IL-15Rα comprising transmembrane region and intracellular region, and can be used to enhance the persistence of CAR modified immune cells and / or enhance the cytotoxicity of CAR modified immune cells.

[0124] The membrane-bound interleukin 15 (mbIL15) of the present application is a complex expressed on the surface of immune cells, which has the structure of IL-15 and IL-15Rα connected by a linker. In an embodiment of the present application, the amino acid sequence of IL-15 in the membrane-bound interleukin 15 (mbIL15) is shown as SEQ ID NO: 26; the amino acid sequence of IL-15Rα is shown as SEQ ID NO: 28; the amino acid sequence of the linker is shown as SEQ ID NO: 30; and the full-length amino acid sequence of the membrane-bound interleukin 15 (mbIL15) is shown as SEQ ID NO: 24.

[0125] Chimeric antigen receptor (CAR) construct

[0126] The present application provides a chimeric antigen receptor (CAR) construct comprising a bispecific CAR targeting tumor antigens BCMA and CD19 and a membrane-bound interleukin 15 (mbIL15) fusion protein.

[0127] In a preferred embodiment, the membrane-bound interleukin 15 (mbIL15) fusion protein is connected to the C-terminus of the bispecific CAR structure through a self-cleaving element, and is expressed on the membrane of the CAR modified cell after cleavage.

[0128] In a preferred embodiment, the bispecific CAR targeting tumor antigens BCMA and CD19 is composed of a signal peptide, an antibody single chain variable region targeting BCMA, an antibody single chain variable region targeting CD19, a CD28 hinge region, a CD28 transmembrane region, a CD28 co-stimulatory domain, and a CD3ζ in series.

[0129] Specifically, the chimeric antigen receptor (CAR) of the present application comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain comprises a target-specific binding element (also referred to as an antigen binding domain). The intracellular domain comprises a co-stimulatory signaling region and a zeta chain portion. The co-stimulatory signaling region refers to a portion of the intracellular domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules required for the efficient response of lymphocytes to antigen, other than antigen receptors or their ligands. In a preferred embodiment, the CAR of the present application comprises a co-stimulatory signaling molecule derived from CD28.

[0130] A linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term "linker" generally refers to any oligo- or polypeptide that serves to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of the polypeptide chain. The linker can comprise 0-300 amino acids, preferably 2 to 100 amino acids, and most preferably 3 to 50 amino acids.

[0131] In a preferred embodiment of the application, the extracellular domain of the CAR provided herein comprises an antigen binding domain targeting BCMA and CD19. The CAR of the application, when expressed in an immune effector cell, is capable of antigen recognition based on antigen binding specificity. When it binds to its cognate antigen, it affects tumor cells, causing them to not grow, be caused to die, or otherwise be affected, and leading to a reduction or elimination of the tumor burden of the patient. The antigen binding domain is preferably fused to an intracellular domain from one or more of a costimulatory molecule and a zeta chain.

[0132] As used herein, "antigen binding domain" "single chain antibody fragment" each refers to a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a single Fv fragment having antigen binding activity. An Fv antibody contains the variable region of the heavy chain, the variable region of the light chain, but no constant region, and has the smallest antibody fragment with all antigen binding sites. Generally, the Fv antibody further comprises a polypeptide linker between the VHand VLdomains, and is capable of forming a structure required for antigen binding. The antigen binding domain is usually a scFv (single-chain variable fragment). The size of the scFv is generally 1 / 6 of a complete antibody. The single chain antibody is preferably an amino acid chain sequence encoded by one nucleotide chain. As a preferred mode of the present application, the scFv comprises an antibody single chain variable region specifically recognizing BCMA and CD19, preferably a humanized single chain antibody.

[0133] For the hinge region and the transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, the 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 a different surface membrane protein, thereby minimizing interactions with other members of the receptor complex.

[0134] Vectors

[0135] The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by directly isolating the gene from cells and tissues comprising the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0136] The present application also provides vectors in which the expression cassettes of the present application are inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, as they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have advantages over vectors derived from onco-retroviruses such as murine leukemia viruses, as they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of being less immunogenic.

[0137] Briefly, the expression cassettes or nucleic acid sequences of the present application are typically operably linked to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of expression of the desired nucleic acid sequences.

[0138] The expression constructs of the present application can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, e.g., U.S. Patent Nos. 5,399,346; 5,580,859; 5,589,466, incorporated herein by reference in their entireties. In another embodiment, the present application provides a gene therapy vector.

[0139] The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Particular vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0140] Further, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and described in, e.g., 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, herpes viruses, and lentiviruses. Typically, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0141] A number of viral-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. A number of adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0142] Additional promoter elements, such as enhancers, can modulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the initiation site, although it has recently been shown that many promoters also contain functional elements downstream of the initiation site. The spacing between promoter elements is often flexible, such that a promoter functions when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by as much as 50 bp before activity begins to decline. Depending on the promoter, individual elements can appear to act cooperatively or independently to initiate transcription.

[0143] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation factor- 1 alpha (EF-1 alpha). However, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus immediate early promoter, the Rous Sarcoma Virus promoter, and human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the present application should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present application. The use of inducible promoters provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0144] To assess expression of a CAR polypeptide or portion thereof, the expression vector introduced into a cell can also comprise either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vector. In other aspects, a selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0145] Reporter genes are used to identify potentially transfected cells and to assess functionality of regulatory sequences. Generally, a reporter gene is one that is not present or expressed by the recipient organism or tissue and which encodes a polypeptide whose expression is clearly indicated by some readily detectable property, such as enzyme activity. After the DNA has been introduced into the recipient cells, expression of the reporter gene is determined at an appropriate time. Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted 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 obtained commercially. Generally, a construct having a minimum of 5 flanking regions that shows the highest level of reporter gene expression is identified as the promoter. Such a promoter region can be linked to a reporter gene and used to assess the ability of an agent to modulate promoter-driven transcription.

[0146] Methods of introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell, by any of a number of methods in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0147] Physical methods of introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods of producing cells that include vectors and / or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). The preferred method of introducing a polynucleotide into a host cell is calcium phosphate transfection.

[0148] Biological methods of introducing a polynucleotide of interest into a host cell 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, e.g., human, cells. Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus, and adeno-associated virus, among others. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0149] Chemical means of introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, and beads; and lipid- based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., a artificial membrane vesicle).

[0150] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. It is contemplated that a lipid formulation is used to introduce a nucleic acid into a host cell (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with a lipid can be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution comprising a lipid, mixed with a lipid, associated with a lipid, contained in a lipid as a suspension, contained in or complexed with a micelle, or otherwise associated with a lipid. The lipids, lipid / DNA, or lipid / expression vector associated with the composition are not limited to any particular structure in solution. For example, they can exist in a bilayer structure, as micelles, or have a "collapsed" structure. They can also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. A lipid is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include fat droplets, which naturally occur in the cytoplasm and comprise long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes, among such compounds.

[0151] In a preferred embodiment of the application, the vector is a lentiviral vector.

[0152] Formulations

[0153] The present application also provides a formulation comprising the CAR construct of the first aspect of the application, the polynucleotide molecule of the second aspect of the application, the vector of the third aspect of the application, or the immune cell of the fifth aspect of the application, and a pharmaceutically acceptable carrier.

[0154] In particular, the present application provides a formulation comprising the CAR-DNT cells of the present application, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the concentration of the CAR-DNT cells in the formulation is 1 x 10 3 1 x 10 8 cells / ml, more preferably 1 x 10 4 1 x 10 7 cells / ml.

[0155] In one embodiment, the formulation can include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextranes, mannitol; proteins; polypeptides 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 application are preferably formulated for intravenous administration.

[0156] Therapeutic applications

[0157] The present application also provides the use of the vector of the third aspect of the present application, the immune cell of the fifth aspect of the present application, or the formulation of the sixth aspect of the present application for treating a disease mediated by BCMA and / or CD19.

[0158] The disease associated with high expression of BCMA and / or CD19 includes cancer or tumor, autoimmune disease. Among them, the cancer or tumor includes hematological tumor and solid tumor; in particular, the cancer or tumor includes (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), hairy cell leukemia, etc. The autoimmune disease includes (but not limited to) systemic lupus erythematosus, multiple sclerosis, etc.

[0159] The CAR-modified immune cells of the present application can be administered alone or as a pharmaceutical composition in combination with diluents and / or with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present application can include a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextranes, mannitol; proteins; polypeptides 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 application are preferably formulated for intravenous administration.

[0160] The pharmaceutical compositions of the present application can be administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease - although appropriate dosages can be determined by clinical trials.

[0161] When referring to an "immunologically effective amount", "anti-tumor effective amount", "tumor-inhibiting effective amount", or "therapeutic amount", the precise amount of the composition of the present application to be administered will be determined by a physician, considering such factors as the age, weight, tumor size, extent of infection or metastasis, and individual condition of the patient (subject). It can be generally stated that: the pharmaceutical composition comprising the T cells described herein can be administered at a dose of 10 4 to 10 9 cells / kg body weight, preferably 10 5 to 10 6 cells / kg body weight (including all integer values within those ranges). The T cell composition can also be administered multiple times at these doses. The cells can be administered by using infusion techniques well known in the art of immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by monitoring the patient's disease signs and adjusting the treatment accordingly, by those skilled in the medical arts.

[0162] The administration of the subject composition can be performed in any convenient manner, including by spray, injection, ingestion, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection or intraperitoneally. In one embodiment, the DNT cell composition of the present application is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the DNT cell composition of the present application is preferably administered by i.v. injection. The DNT cell composition of the present application can be injected directly into a tumor, lymph node or site of infection.

[0163] In certain embodiments of the application, the cells activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels are administered to a patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) any number of relevant therapeutic modalities, including but not limited to treatment with agents such as anti-viral therapies, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab treatment for MS patients or efalizumab treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the DNT cells of the application can be used in conjunction with chemotherapy, radiation, immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the application are administered to a patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) bone marrow transplantation, with chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), and cyclophosphamide. For example, in one embodiment, a subject can undergo standard therapy of high dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, following transplantation, the subject receives infusion of expanded immune cells of the application. In an additional embodiment, the expanded cells are administered prior to or following surgery.

[0164] The dosage of the above treatments administered to a patient will vary with the precise attributes of the condition being treated and the recipient of the treatment. Dosage ratios for human administration can be implemented in accordance with accepted practices in the art. Generally, 1 x 10 6 to 1 x 10 10 modified DNT cells of the application are administered to a patient by, for example, intravenous infusion.

[0165] The main advantages of the present application are:

[0166] (1) The BCMA and CD19 dual-targeting universal DNT cells (BCMA / CD19-CAR-mbIL15-DNT) constructed in the present application can simultaneously target BCMA and CD19 and exhibit significantly effective cytotoxicity against BCMA-positive cells and CD19-positive cells, and thus can be used for the treatment of BCMA and / or CD19-mediated diseases.

[0167] (2) The BCMA and CD19 dual-targeting universal DNT cells constructed in the present application have stronger and more persistent cell-killing activity, and thus can achieve better therapeutic effects.

[0168] (3) In the process of preparing the universal BCMA / CD19-CAR-mbIL15-DNT cell, the CAR lentivirus infection condition is optimized, which greatly improves the efficiency of CAR lentivirus infection of DNT cells.

[0169] (4) The universal BCMA / CD19-CAR-mbIL15-DNT cell prepared by the application is derived from immune cells donated by healthy donors, can be produced on a large scale, saves cost, and greatly reduces the economic burden of patients in clinical application.

[0170] The application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not to limit the scope of the application. The experimental methods in the following examples are not specified, and the conditions are generally according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0171] Example 1: Construction of CAR lentivirus vector expression

[0172] The expression of CAR lentivirus vector is entrusted to Nanjing Kingsriver Biotechnology Co., Ltd. for DNA synthesis, plasmid cloning and sequencing verification, and the CAR structure is as shown in Figure 1 The CD19-CAR structure is composed of humanized anti-CD19 scFv, CD8 hinge, CD8 transmembrane region, 4-1BB costimulatory domain and CD3ζ intracellular activation domain; the BCMA-CAR structure is composed of murine anti-BCMA scFv, CD8 hinge, CD8 transmembrane region, 4-1BB costimulatory domain and CD3ζ intracellular activation domain; the BCMA / CD19-CAR structure is composed of murine anti-BCMA scFv and humanized anti-CD19 scFv in series, CD28 hinge, CD28 transmembrane region, CD28 costimulatory domain and CD3ζ intracellular activation domain; and the BCMA / CD19-CAR-mbIL15 structure is composed of murine anti-BCMA scFv and humanized anti-CD19 scFv in series, CD28 hinge, CD28 transmembrane region, CD28 costimulatory domain, CD3ζ intracellular activation domain and membrane-bound mbIL15.

[0173] The scFv sequence of anti-BCMA is derived from murine antibody C11D5.3, and the scFv sequence of anti-CD19 is derived from humanized murine antibody FMC63.

[0174] The nucleotide sequences and amino acid sequences of each element are shown in Table 1.

[0175] Table 1: CAR structural element nucleotide sequences and amino acid sequences

[0176]

[0177]

[0178]

[0179]

[0180] Example 2: CAR lentivirus packaging and titer detection

[0181] 1. Plate T225 of logarithmic growth of 293T cells, after 48 hours of culture, the confluence density reaches 80%~90% for transfection.

[0182] 2. Prepare a sterile 50ml centrifuge tube, first add 1.8ml Opti-MEM transfection medium, then add lentivirus packaging plasmid, the specific amount is shown in Table 2, vortex mix, incubate at room temperature for 5min;

[0183] Table 2: Lentivirus packaging plasmid information

[0184] Plasmid name CAR lentivirus plasmid pMDL g / pRRE pRSV-Rev pMD2.0G Mass (ug) 79.68 25.8 28.0 13.0

[0185] 3. Take 36μl PEI pro and add it to a 15ml centrifuge tube containing 1.8ml Opti-MEM transfection medium, mix well, and incubate at room temperature for 5min;

[0186] 4. Add the Opti-MEM transfection medium containing PEI pro to the Opti-MEM transfection medium containing plasmid DNA, mix well, and incubate at room temperature for 20min, then add Opti-MEM transfection medium to 25ml, mix well;

[0187] 5. Discard the culture medium of 293T cells in T225 culture bottle, add 25ml transfection complex to T225 culture bottle;

[0188] 6. After 6h of transfection, replace the transfection reagent with 50ml DMEM+10%FBS complete medium;

[0189] 7. Continue to culture for 48h, harvest the virus, centrifuge to remove floating dead 293T cells, then filter, concentrate, aliquot, store at-80℃ and determine the titer of the virus-containing culture medium.

[0190] Lentivirus titer determination: 293T cells were plated into 12-well plates the day before virus titer determination, 1 ml of 293T cells at a concentration of 1E5 / ml was added to each well; 1 ul, 0.1 ul, and 0.01 ul of concentrated lentivirus was added to the 293T cells. 48 hours later, lentivirus titer was determined by FACS. Virus titer = CAR positive cell number x 1E5 / volume of virus added x 1000 (TU / ml). The results of lentivirus titer determination of different CAR structures are shown in Table 3.

[0191] Table 3: CAR lentivirus titer

[0192]

[0193] Example 3: Preparation and detection of CAR-DNT cells

[0194] 3.1 DNT cell activation

[0195] 1. 12-well plates were coated with 1.5 ug / ml CD3 antibody at 4°C overnight;

[0196] 2. The water bath was turned on and set to 37°C. The cryopreserved DNT cells (CD3 + CD4 - CD8 - ) were quickly placed in the water bath and shaken rapidly to completely thaw the cell solution within 2 min;

[0197] 3. One 50 ml centrifuge tube was taken and 20 ml of the corresponding medium was added. The thawed DNT cells were added to the centrifuge tube, which was centrifuged at 500 x g for 5 min. The supernatant was discarded and the cells were washed 2-3 times;

[0198] 4. The cells were resuspended in X-VIVO + 10 ug / ml gentamicin + 10% ICSR + 250 IU / ml IL2. The DNT cells were adjusted to 2.5 x 10 6 cells / ml, and 300 ul / well of the cell suspension was added to the 24-well plates coated with CD3 antibody and washed with PBS.

[0199] 3.2 CAR lentivirus infection of DNT cells

[0200] 1. The amount of virus to be used was calculated according to the CAR lentivirus titer and MOI = 2. One control well (without virus) was set up, and the corresponding amount of CAR lentivirus was added to each well;

[0201] 2. According to the final volume of the virus supernatant added, 1% DMSO was added to each well to promote the efficiency of lentivirus infection of DNT cells;

[0202] 3. On day 2, day 3 and day 4, 0.3 ml, 0.4 ml and 1 ml of liquid medium were added respectively, and the culture was continued to day 5, and the cells were transferred to a T25 culture flask for culture, and the subsequent culture density was maintained at about 1 x 10 6

[0203] 4. On day 9, day 12 and day 14, samples were taken respectively, counted by K2 counting instrument, and the proliferation fold and viability of CAR-DNT cells were calculated.

[0204] The proliferation fold and viability of CAR-DNT cells are shown in Figure 2 Table 5. The proliferation fold of BCMA / CD19-CAR-mbIL15-DNT, BCMA / CD19-CAR-DNT, BCMA-CAR-DNT and CD19-CAR-DNT cells on day 14 was 949.0, 802.0, 697.5 and 332.5 respectively, and the viability was 88.9%, 90.1%, 78.6% and 78.9% respectively. The proliferation fold and viability of double-target BCMA / CD19-CAR-mbIL15-DNT and BCMA / CD19-CAR-DNT cells were significantly better than those of the other two groups of single-target CAR-DNT cells.

[0205] 3.3 Detection of CAR-DNT phenotype and positive rate

[0206] 1. Take 500 μL CAR-DNT cells into a 1.5 ml EP tube, add 1 ml of 1 x PBS, and centrifuge at 500 x g for 5 minutes;

[0207] 2. Discard the supernatant, resuspend with 100 μL of 1 x PBS, add antibodies to each tube respectively, and incubate in a 4°C refrigerator for 30 minutes;

[0208] 3. Add 1 ml of 1 x PBS to wash the cells, centrifuge at 500 x g for 5 minutes, discard the supernatant, resuspend with 200 μL of 1 x PBS, and detect on a flow cytometer;

[0209] The results of CAR-DNT cell positive rate detection in this example are shown in Figure 3 ​As shown, the CAR positive rate of BCMA-CAR-DNT cells was 80.5%, the positive rate of CD19-CAR-DNT cells was 83.5%, the CAR positive rate of BCMA / CD19-CAR-DNT cells was 70.0%, the CAR positive rate of BCMA / CD19-CAR-mbIL15-DNT cells was 49.5%, and the CAR+mbIL15 positive rate was 30.0%. CAR molecules were successfully expressed on the membranes of DNT cells with different CAR structures, and double-target BCMA / CD19-CAR-mbIL15-DNT cells not only successfully expressed double-target CAR molecules, but also successfully expressed mbIL15 molecules.

[0210] The results of phenotype detection of DNT cells with different CAR structures in this embodiment are shown in the following table: Figure 4 As shown, the proportion of CD3+ T cells in BCMA / CD19-CAR-mbIL15-DNT cells was 96.8%, the proportion of CD3 + CD4 - The proportion of DNT cells with CD8 - was 98.1%, the proportion of CD45RA + CD62L + The proportion of TSCM was 8.9%, the proportion of CD45RA - CD62L + The proportion of TCM was 58.3%; the proportion of CD3 + T cells in BCMA / CD19-CAR-DNT was 97.2%, the proportion of CD3 + CD4 - CD8 - The proportion of DNT cells was 99.2%, the proportion of CD45RA + CD62L + The proportion of TSCM was 14.7%, the proportion of CD45RA - CD62L + The proportion of TCM was 46.4%, the proportion of CD3 + T cells in BCMA-CAR-DNT was 91.6%, the proportion of CD3 + CD4 - CD8 - The proportion of DNT cells was 91.1%, the proportion of CD45RA + CD62L + The proportion of TSCM was 7.6%, the proportion of CD45RA - CD62L + The proportion of TCM was 21.6%; the proportion of CD3 + T cells in CD19-CAR-DNT was 93.6%, the proportion of CD3 + CD4 - CD8- DNT cells were 93.9%, CD45RA + CD62L + TSCM proportion was 17.4%, CD45RA - CD62L + TCM proportion was 56.1%.

[0211] The results showed that the proportion of CD3 + T cells in BCMA / CD19-CAR-mbIL15-DNT, BCMA / CD19-CAR-DNT, BCMA-CAR-DNT and CD19-CAR-DNT cells was more than 90%, among which the proportion of CD3 + CD4 - CD8 - The proportion of DNT cells was more than 90%, with no significant difference. Except that the proportion of Tscm+Tcm cells in BCMA-CAR-DNT cells was less than 50%, the proportion of Tscm+Tcm cells in other CAR-DNT cells was more than 50%.

[0212] Example 4: Cytokine release detection of CAR-DNT cells

[0213] 1. Preparation of effector cells: Take non-transduced DNT cells and CAR-DNT cells and place them in 15 ml centrifuge tubes, respectively, and add 5 ml of 1x PBS, centrifuge at 500xg for 5 min, and wash twice; resuspend the cells with X-VIVO+5% FBS basic medium, count with K2, and adjust the density of CAR-DNT cells to 8x10 5 cells / ml, with an effector target ratio of 1:1, for standby.

[0214] 2. Preparation of target cells: Take the non-target cells (CHO-luciferase) and target cells (CHO-CD19-luciferase, CHO-BCMA-luciferase, CHO-BCMA-CD19-luciferase, H929-CD19-luciferase, Raj i-luciferase) in logarithmic growth phase; centrifuge at 500xg for 3 min in a 15 ml centrifuge tube, discard the supernatant, and wash twice; resuspend the cells with X-VIVO+5% FBS basic medium, count with a cell counter and detect the cell viability, and finally dilute the cell density to 8x10 5 cells / ml concentration, for standby.

[0215] 3. Effector-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 at a ratio of 1:1 and added to a U-shaped 96-well plate. The plate was placed in a 37°C, 5% CO2 incubator for 20-24 hours. The supernatant of the co-culture was collected in a 1.5 ml EP tube and stored for later use.

[0216] 4. Cytokine detection: According to the requirements of the CBA detection kit instructions, the required cytokines and sample numbers were determined. The capture beads were prepared, and the cytokine capture bead bottle to be detected was taken out and mixed well. Each bead was taken out (the number of samples to be detected + the number of negative controls) x 10 μl / 6 of capture beads, and each bead was mixed well by vortexing. N 1.5 ml EP tubes (N = sample number + control number) were taken, 10 μl of mixed beads were added to each tube (sample, negative control), 10 μl of the corresponding test reagent (sample, control) was added to each tube, 10 μl of PE Detection Reagent was added to each tube, and the mixture was mixed well. After incubation at room temperature for 3 hours in the dark, 500 μl of wash buffer was added to each sample, mixed well, and centrifuged at 500 x g for 5 minutes. The supernatant was discarded, and the sample was resuspended with 200 μl of wash buffer and centrifuged at 500 x g for 5 minutes. The supernatant was discarded, and the sample was resuspended with 150 μl of wash buffer. The sample was detected by flow cytometry.

[0217] The results of this example are shown in Table 1. Figure 5As shown, after co-culture of BCMA / CD19-CAR-mbIL15-DNT, BCMA / CD19-CAR-DNT with target cells (CHO-CD19-luciferase, CHO-BCMA-luciferase, CHO-BCMA-CD19-luciferase, H929-CD19-luciferase, Raji-luciferase), there is a large amount of TNFα, IFNγ inflammatory cytokine release, and after co-culture with non-target cells (CHO-luciferase), there is no TNFα, IFNγ inflammatory cytokine release; after co-culture of BCMA-CAR-DNT with target cells (CHO-BCMA-luciferase, CHO-BCMA-CD19-luciferase, H929-CD19-luciferase, Raji-luciferase), there is a large amount of TNFα, IFNγ inflammatory cytokine release, and after co-culture with non-target cells (CHO-luciferase, CHO-CD19-luciferase), there is no TNFα, IFNγ inflammatory cytokine release; after co-culture of CD19-CAR-DNT with target cells (CHO-CD19-luciferase, CHO-BCMA-CD19-luciferase, H929-CD19-luciferase, Raji-luciferase), there is a large amount of TNFα, IFNγ inflammatory cytokine release, and after co-culture with non-target cells (CHO-luciferase, CHO-BCMA-luciferase), there is no TNFα, IFNγ inflammatory cytokine release.

[0218] The cytokine release results show that CAR-DNT cells with different CAR structures have high specific targeting and inflammatory cytokine secretion ability.

[0219] Example 5: Detection of CAR-DNT cell killing ability

[0220] The firefly luciferase reporter gene activity chemiluminescence method was used to detect the CAR-DNT cell killing ability, and the specific experimental steps were as follows:

[0221] 1. Effect cell preparation: take the non-transduced DNT cells and CAR-DNT cells, respectively, and place them in 15 ml centrifuge tubes, add 5 ml of 1×PBS, centrifuge at 500×g for 5 min, and wash twice; resuspend the cells with X-VIVO+5% FBS basic medium, count with K2, and adjust the density of CAR-DNT cells to 8×10 5 cells / ml, 4×105 2 x 10 5 2 x 10

[0222] 2. Target cell preparation: Take the non-target cells (CHO-luciferase) and target cells (CHO-CD19-luciferase, CHO-BCMA-luciferase, CHO-BCMA-CD19-luciferase, H929-CD19-luciferase, Raj i-luciferase) in the logarithmic growth phase in a 15 ml centrifuge tube, centrifuge at 500 x g for 3 minutes, discard the supernatant, and wash twice; resuspend the cells with X-VIVO + 5% FBS basic medium, count with a cell counter and detect the cell viability, and finally dilute the cell density to a concentration of 2 x 10 5

[0223] 3. Chemiluminescence detection: mix 50 μL target cells and 50 μL CAR-DNT cells into a white flat-bottom 96-well plate, incubate at 37°C, 5% CO2 for 24 hours; add 100 uL GMOne-Step TM Luciferase Reporter Gene Detection Kit to detect the substrate, mix well, and stand for 5 minutes. Detect on a Spectra MaxL microplate reader (wavelength 570 nm).

[0224] 4. Calculate the killing rate according to the RLU value: killing rate (%) = 1- (target cells + effector cells) fluorescence value / target cell fluorescence value x 100%

[0225] The results of this example are shown in Figure 6 BCMA / CD19-CAR-mbIL15-DNT, BCMA / CD19-CAR-DNT, BCMA-CAR-DNT, and CD19-CAR-DNT do not have killing effect on non-target cells CHO-luciferase cells that do not express CD19 and BCMA target antigens, indicating that all CAR-DNT cells have target point-dependent tumor cell killing effect and do not produce non-specific killing effect;

[0226] ​The double-target BCMA / CD19-CAR-mbIL15-DNT cells and the BCMA / CD19-CAR-DNT cells can not only kill target cells expressing CD19 antigens, but also kill target cells expressing BCMA antigens. The BCMA-CAR-DNT cells cannot kill target cells expressing only CD19 antigens, and the CD19-CAR-DNT cells cannot kill target cells expressing only BCMA antigens, indicating that the double-target CAR-DNT cells have more broad-spectrum double-target anti-tumor activity than the single-target CAR-DNT cells.

[0227] The BCMA / CD19-CAR-mbIL15-DNT cells, the BCMA / CD19-CAR-DNT cells, the BCMA-CAR-DNT cells and the CD19-CAR-DNT cells all have dose-dependent killing of tumor target cells. When the effector-to-target ratio is 2:1 and 1:1, the double-target BCMA / CD19-CAR-mbIL15-DNT cells and the BCMA / CD19-CAR-DNT cells have stronger targeted killing activity on tumor cells than the single-target BCMA-CAR-DNT cells and the CD19-CAR-DNT cells.

[0228] Example 6: Detection of multiple rounds of tumor stimulation of CAR-DNT cells

[0229] 1. Preparation of effector cells: Take the non-transduced DNT cells and the CAR-DNT cells into 15 ml centrifuge tubes, respectively, and add 5 ml of 1x PBS, centrifuge at 500xg for 5 min, and wash twice; resuspend the cells with X-VIVO + 5% FBS medium, count with K2, and adjust the density of the CAR-DNT cells to 2x10 5 cells / ml for standby.

[0230] 2. Preparation of target cells: Take the logarithmic growth of target cells (Raji-luciferase, H929-CD19-luciferase) into 15 ml centrifuge tubes, centrifuge at 500xg for 3 min, discard the supernatant, and wash twice; resuspend the cells with X-VIVO + 5% FBS medium, count with K2, and adjust the cell density to 2x10 5 cells / ml concentration for standby.

[0231] 3. Co-culture of effector and target cells: Add the treated effector cells and target cells into a 12-well plate at an effector-to-target ratio of 1:1, add 500 μl of target cells and 500 μl of CAR-DNT cells to each well, and incubate the cells in a 37°C, 5% CO2 incubator.

[0232] 4. Multiple antigen stimulation: Co-culture cells for 4–5 days, harvest co-incubated cells, centrifuge at 500×g for 5 minutes, and resuspend cells in 1 ml of X-VIVO + 5% FBS medium; take 100 μl 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.

[0233] The results of this embodiment are as follows: Figure 7 As shown, after the third round of tumor killing, the fluorescence value of the residual tumor cells in the dual-target BCMA / CD19-CAR-DNT-mbIL15 cell group was much lower than that in the BCMA / CD19-CAR-DNT, CD19-CAR-DNT and BCMA-CAR-DNT cell groups, indicating that BCMA / CD19-CAR-DNT-mbIL15 has a stronger and more durable ability to target and kill tumors than other CAR-DNT cell groups.

[0234] Example 7: Detection of the ability of CAR-DNT cells to survive in vitro without cytokines

[0235] 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;

[0236] 2. Resuspend the cells in 5 ml of 1×PBS, centrifuge at 500×g for 5 min, discard the supernatant, and wash twice;

[0237] 3. Resuspend cells in X-VIVO + 10% ICSR medium (without cytokines), perform K2 counting, and adjust cell density to 5 × 10⁶ cells / year. 5 Cells / ml, cultured in 5ml T25 culture flasks.

[0238] 4. Sampling and counting of cell count and viability every 7 days.

[0239] The results of this embodiment are as follows: Figure 8 As shown, CAR-DNT cells cultured without IL2 factor were compared to their sustained viability in vitro. After in vitro culture to day 14, the number of dual-target BCMA / CD19-CAR-DNT-mbIL15 cells expanded and the cell viability were much higher than those of BCMA / CD19-CAR-DNT, BCMA-CAR-DNT, and CD19-CAR-DNT cells.

[0240] Example 8: In vivo efficacy and safety test of CAR-DNT cells

[0241] Raji-luciferase cells were injected into NSG mice (5x10 5 On the second day after inoculation, the in vivo expansion of tumor cells was observed by live imaging technology. According to the live imaging data, the NSG mice were divided into 5 groups, and 5x10 5 BCMA / CD19-CAR-mbIL15-DNT, BCMA / CD19-CAR-DNT, BCMA-CAR-DNT, CD19-CAR-DNT, and DNT cells were injected into the tail vein of each group of mice, respectively. The in vivo tumor proliferation of mice was observed by live imaging every 7 days, and the analysis was performed according to the change of fluorescence intensity and the change of mouse body weight.

[0242] All the documents mentioned in the present application are incorporated herein by reference as if each document were individually incorporated by reference. In addition, it should be understood that various changes and modifications to the present application can be made by those skilled in the art after studying the above teaching of the present application and that such changes and modifications are also within the scope of 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 or II below. XAE(I) EAX(II) In the formula, Each "-" independently represents a linking peptide or peptide bond; X is a CAR that targets the tumor antigens BCMA and CD19; A is a self-shearing element; E is a membrane-bound interleukin-15 (mbIL15) fusion protein.

2. The CAR construct as described in claim 1, characterized in that, The CAR targeting tumor antigens BCMA and CD19 comprises a single-chain variable region (scFv) of an antibody targeting BCMA and a single-chain variable region of an antibody targeting CD19; wherein, the amino acid sequence of the scFv targeting BCMA is shown in SEQ ID NO:4, and the amino acid sequence of the scFv targeting CD19 is shown in SEQ ID NO:

2.

3. The CAR construct as described in claim 1, characterized in that, The structure of X is shown in Equation III below. L-scFv1-scFv2-H-TM-C-CD3ζ(III) In the formula, Each "-" independently represents a linking peptide or peptide bond; L represents either no signal peptide or a signal peptide; scFv1 is the single-chain variable region of an antibody targeting BCMA, and scFv2 is the single-chain variable region of an antibody targeting CD19; or, scFv1 is the single-chain variable region of an antibody targeting CD19, and scFv2 is the single-chain variable region of an antibody targeting BCMA. 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.

4. The CAR construct as claimed in claim 1, characterized in that, The structure of the membrane-bound interleukin-15 (mbIL15) fusion protein is shown in Formula IV. L'-MIR(IV) 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. A polynucleotide molecule, characterized in that, The nucleic acid molecule encodes the CAR construct as described in any one of claims 1-4.

6. A carrier, characterized in that, The carrier contains the polynucleotide molecule as described in claim 5.

7. An engineered immune cell, characterized in that, The immune cells express the CAR construct as described in any one of claims 1-4.

8. A formulation comprising a CAR construct as described in any one of claims 1-4, a polynucleotide molecule as described in claim 5, a vector as described in claim 6, or an immune cell as described in claim 7, and a pharmaceutically acceptable vector.

9. Use of a CAR construct as described in any one of claims 1-4, a polynucleotide molecule as described in claim 5, a carrier as described in claim 6, an immune cell as described in claim 7, or a formulation as described in claim 8, for the preparation of a medicament or formulation for the prevention and / or treatment of a disease.

10. A method for preparing engineered immune cells according to the fifth aspect of the present invention, the method comprising the following steps: (a) Provide immune cells to be modified; and (b) Transfecting the polynucleotide molecule as described in claim 5 or the vector as described in claim 6 into the immune cells to obtain the engineered immune cells.

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