WO2024099265A1 - 工程化嵌合抗原受体免疫细胞及其应用 - Google Patents

工程化嵌合抗原受体免疫细胞及其应用 Download PDF

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WO2024099265A1
WO2024099265A1 PCT/CN2023/129932 CN2023129932W WO2024099265A1 WO 2024099265 A1 WO2024099265 A1 WO 2024099265A1 CN 2023129932 W CN2023129932 W CN 2023129932W WO 2024099265 A1 WO2024099265 A1 WO 2024099265A1
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seq
cells
amino acid
acid sequence
car
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PCT/CN2023/129932
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French (fr)
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姜福伟
靳胜男
王义芳
陈玲
王庆杨
王超
杨翠青
曹卓晓
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上海先博生物科技有限公司
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  • the present application relates to the field of immunology, and in particular, to immune cells expressing chimeric antigen receptors and interleukin 15 (IL15).
  • IL15 interleukin 15
  • CAR-T cells engineered T cells
  • CAR-NK cells natural killer cells
  • CARs chimeric antigen receptors
  • Autologous or allogeneic immune effector cells are activated in vitro and infused into the patient to kill tumor cells in the patient.
  • CAR-T cells and CAR-NK cells are difficult to spread and have poor persistence, especially when treating solid tumors.
  • suppressive tumor microenvironments TCE
  • TCR T cell receptor
  • co-stimulatory signaling limit cell trafficking, and inactivate CAR-T cells.
  • TCE suppressive tumor microenvironments
  • Interleukin 15 is a 14-15 kDa glycoprotein, and its amino acid sequence is shown in SEQ ID NO: 1 (NP_000576.1, NCBI database).
  • IL15 mRNA is present in a variety of tissues, including hematopoietic cells (such as monocytes, macrophages, and dendritic cells) and non-hematopoietic cells (such as keratinocytes, fibroblasts, neurons, skeletal muscle, and epithelial cells), but IL15 protein is only concentrated in specific cells.
  • IL15 can enhance T cell and NK cell immune responses, thereby improving the reliability and efficacy of cell therapy.
  • IL15 is structurally similar to interleukin 2 (IL2), and can maintain the proliferation of CD8+ memory T cells (CD8+ TM) while inhibiting IL-2-induced T cell death, thereby better maintaining long-term anti-tumor immunity.
  • IL2 interleukin 2
  • TM CD8+ memory T cells
  • mbIL15 membrane-bound IL15
  • the therapeutic efficacy of CAR-T cells is related to the ratio of T cell subsets (CD4 + or CD8 + ), differentiation (naive, memory, effector), activation (expression of activation markers) and the functional status of CAR-T cells.
  • Central memory T cells (TCM, CD62L + CD45RO + ) are T cells with long-term memory and can home to lymph nodes to receive antigen re-stimulation after the initial T cells (Naive T Cell) are activated by antigens. Under the re-stimulation of antigens, a large number of cloned effector memory T cells (TEM, CD62L - CD45RO + ) carrying allogeneic antigens can continue to be produced.
  • TEM cloned effector memory T cells
  • Stem cell-like memory T cells are an important component of memory T cells, with stem cell-like self-renewal, multi-differentiation potential and immune reconstitution characteristics.
  • Effector T cells TE, CD62L - CD45RO -
  • T cells that respond to antigen stimulation. Cells formed through proliferation and differentiation.
  • In vitro expansion of T cells is a key step in the CAR-T cell production process.
  • Recent clinical studies have shown that functional memory T cell subsets, including TSCM, TCM and other less differentiated T cell subsets, are responsible for long-term anti-tumor responses in patients. Therefore, during in vitro T cell expansion, producing a higher proportion of TSCM and TCM in T cell products is crucial for significant clinical improvements in cell therapy.
  • LAG3 (Lymphocyte-activation gene 3, LAG3 or CD223) is a lymphocyte activation gene belonging to the Ig family. It is mainly expressed in activated T cells, NK cells, B cells and plasmacytoid dendritic cells and is an immune negative regulator.
  • TIM3 T cell immunoglobulin-3, TIM3 or CD366
  • CD4 + Th1 cells CD8 + Tc1 cells
  • Th17 cells regulatory T cells
  • dendritic cells dendritic cells
  • monocytes monocytes. It is involved in immune tolerance and T cell exhaustion in chronic viral infections and cancers. LAG3 and TIM3 can be used as markers of T cell exhaustion.
  • the present application provides CAR-T cells that only express mbIL15, which achieve excellent proliferation and killing activity with exogenous proteins of smaller molecular weight.
  • the present application provides an engineered immune cell that co-expresses a chimeric antigen receptor (CAR) and all or a functional portion of membrane-bound IL15 (mbIL15).
  • CAR chimeric antigen receptor
  • mbIL15 membrane-bound IL15
  • the second aspect of the present application provides a fusion protein comprising all or a functional portion of IL15 fused to all or a portion of a transmembrane protein, wherein the transmembrane protein is selected from NKG2D, OX40, 2B4 or EpCAM.
  • the third aspect of the present application provides an isolated nucleic acid molecule, which comprises a nucleic acid molecule encoding CAR and mbIL15 co-expressed on the immune cells described in any one of the above, or comprises a nucleic acid molecule encoding the fusion protein described in any one of the above; preferably, the nucleic acid molecule is DNA or RNA.
  • the fourth aspect of the present application provides a vector comprising the above-mentioned nucleic acid molecule.
  • the fifth aspect of the present application provides a method for preparing the immune cell described in any one of the above items, which comprises introducing the nucleic acid molecule into the immune cell.
  • the sixth aspect of the present application provides a pharmaceutical composition comprising the immune cell described in any one of the above items, the fusion protein described in any one of the above items, or the nucleic acid molecule described in any one of the above items.
  • the seventh aspect of the present application provides the immune cell described in any one of the above items, the product obtained by the method, or the use of the pharmaceutical composition for preparing a drug for treating tumors in an individual.
  • the eighth aspect of the present application provides a method for treating a tumor in an individual in need thereof, comprising administering to the individual any of the above-mentioned immune cells, the product obtained by the method, or the pharmaceutical composition.
  • the ninth aspect of the present application provides the immune cell described in any one of the above items, the product obtained by the method, or the pharmaceutical composition, for use in preventing and/or treating tumors in an individual.
  • the mbIL15 armored CAR-T cells disclosed in the present application have at least one or more of the following advantages: (1) IL15 is expressed on the cell surface and is confined to the tumor microenvironment; (2) It has a higher CAR expression positive rate or cell proliferation multiple, and expression stability; (3) The proportion of TCM in the amplified T cells is higher; (4) It is highly specific for specific antigen epitopes; (5) It has excellent Tumor cell killing and inhibition capabilities, including but not limited to: showing superior tumor killing effects in long-term killing tests or at medium and low doses; (6) lower side effects, strong specificity, and greater safety.
  • Figure 1 Schematic diagram of the mbIL15-armored second-generation chimeric antigen receptor retroviral plasmid targeting human GCC.
  • Figure 3 The proportion of each subset in mbIL15 CAR-T cells on days 8 and 11 after activation.
  • D Detection results of inhibitory receptors TIM3 and LAG3 in CD8 + cell population on day 8 after activation;
  • Figure 4 Detection of mbIL15 CAR-T cell killing activity against LS174T-luc cells.
  • Figure 5 Multiple rounds of killing activity detection of mbIL15 CAR-T cells on day 11 after activation.
  • Figure 6 Schematic diagram of the mbIL15-armored second-generation chimeric antigen receptor retroviral plasmid targeting human DLL3.
  • FIG. 7 Expression of mbIL15 armored CAR on the surface of T cells on days 8 and 10 after activation.
  • Figure 8 Multiple rounds of killing activity detection of mbIL15DLL3 CAR-T cells on day 11 after activation and mbIL15DLL3 CAR-T cells frozen and revived on day 10 after activation.
  • a and B are multiple rounds of killing activity detection of mbIL15DLL3 CAR-T cells on day 11 after activation against SHP77-luc and NCI-H2171-luc cells;
  • C and D are multiple rounds of killing activity detection of mbIL15DLL3 CAR-T cells frozen and revived on day 10 after activation against SHP-77-luc and NCI-H2171-luc cells.
  • Figure 9 Detection of the anti-tumor efficacy of mbIL15 DLL3 CAR-T cells frozen and revived on the 10th day after activation on the SHP-77 cell subcutaneous tumor model in mice.
  • Figure 10 Schematic diagram of the second generation chimeric antigen receptor retroviral plasmid targeting human CD19 and armored with mbIL15.
  • compositions comprising A and B should be understood as the following technical solutions: a composition consisting of A and B, and a composition containing other components in addition to A and B, all fall within the scope of the aforementioned "a composition”.
  • immune cells refer to any cells associated with the host immune response or inflammatory response, including but not limited to T cells, NK cells, NKT cells, T cells and NK cells induced by other techniques, and other immunoregulatory cells.
  • T cell means a T lymphocyte, which belongs to a group of white blood cells called lymphocytes and participates in humoral or cell-mediated immunity. T cells can be distinguished from other lymphocyte types such as B cells and natural killer cells (NK cells) by the presence of special markers such as T cell receptors (TCR) on their cell surfaces. Other markers for identifying T cells include CD1a, CD3, CD4, CD8, and other markers known to the skilled person that may be associated with T cell status and/or function.
  • NK cells naturally killer cells
  • MHC Major Histocompatibility Complex
  • NK cells are unique in that they have the ability to recognize stressed cells regardless of whether peptides from pathogens are present on MHC molecules. Because they do not require the initial insight of pre-activation to kill the target, they are named "natural killer cells.” NK cells are large granular lymphocytes (LGL) and are known to differentiate and mature in the bone marrow (where they then enter the circulation).
  • LGL large granular lymphocytes
  • NKT cell used herein means natural killer T cell, refers to CD-ld restricted T cell expressing T cell receptor (TCR).
  • TCR T cell receptor
  • MHC major histocompatibility complex
  • NKT cells recognize lipid antigens presented by CD1d (non-classical MHC molecules).
  • CD1d non-classical MHC molecules.
  • Invariant or type I NKT cells express a very limited TCR library-typical ⁇ chain (Va24-Jal 8 in humans), which is related to the limited spectrum of ⁇ chain ( ⁇ 1 in humans).
  • the second NKT cell group is called non-classical or non-variant type II NKT cell, which shows more heterogeneous TCR ⁇ use.
  • type I NKT cells are suitable for immunotherapy.
  • Adaptive or invariant (type I) NKT cells can be identified by the expression of at least one or more of the following markers: TCR Va24Jal 8, Vbl 1, Vbl 2, Vbl 3, Vbl 4, Vbl 5, Vbl 6, Vbl 7, Vbl 8, Vbl 9, Vbl 10, Vbl 11, Vbl 12, Vbl 13, Vbl 14, Vbl 15, Vbl 16, Vbl 17, Vbl 18, Vbl 19, Vbl 20, Vbl 21, Vbl 22, Vbl 23, Vbl 24, Vbl 25, Vbl 26, Vbl 27, Vbl 28, Vbl 29, Vbl 30, Vbl 31, Vbl 32, Vbl 33, Vbl 34, Vbl 35, Vbl 36, Vbl 37, Vbl 38, Vbl 39, Vbl 40, Vbl 41, Vbl 42, Vbl 43, Vbl 44, Vbl 45, Vbl 46, Vbl
  • IL15 interleukin 15
  • IL15 refers to a cytokine that regulates T and NK cell activation and proliferation. This cytokine shares many biological activities with interleukin 2. It was found that they bind to common receptor subunits and can compete for the same receptors, and thus negatively regulate each other's activity. It was shown that the number of CD8+ memory cells is controlled by the balance between IL-15 and IL-2.
  • This cytokine induces the activation of JAK kinases and the phosphorylation and activation of transcriptional activators STAT3, STAT5 and STAT6, and can increase the expression of apoptosis inhibitors BCL2L1/BCL-x (L) through the transcriptional activation activity of STAT6, and thus prevent apoptosis.
  • a “functional portion” (“biologically active portion”) of IL-15 refers to a portion of IL-15 that retains one or more functions of full-length or mature IL- 15. Such functions include promoting immune cell survival, regulating NK cell and T cell activation and proliferation, and maintaining immune cell development from hematopoietic stem cells.
  • IL-15 is wild-type IL-15.
  • IL-15 is mammalian IL-15 (e.g., human (Homo sapiens) interleukin 15 (IL15), transcript variant 3, mRNA, NCBI reference sequence: NP_000576.1; domestic dog (canis lupus familiaris) interleukin 15, mRNA, NCBI reference sequence: NM_001197188.1; domestic cat (Felis catus) interleukin 15 (IL15), mRNA, NCBI reference sequence: NM_001009207.1).
  • human (Homo sapiens) interleukin 15 (IL15), transcript variant 3, mRNA, NCBI reference sequence: NP_000576.1; domestic dog (canis lupus familiaris) interleukin 15, mRNA, NCBI reference sequence: NM_001197188.1; domestic cat (Felis catus) interleukin 15 (IL15), mRNA, NCBI reference sequence: NM_001009207.1 e.g., human (Homo sapiens)
  • mammalian IL-15 is human IL-15.
  • transmembrane protein or “membrane protein” is a protein located at and/or within a membrane, such as a phospholipid bilayer of a biological membrane (e.g., a biological membrane such as a cell membrane).
  • a biological membrane e.g., a biological membrane such as a cell membrane.
  • Membrane proteins enable membranes to carry out their unique activities. The complement of proteins attached to the membrane varies depending on the cell type and subcellular localization. Some proteins are only bound to the membrane surface, while other proteins have one or more regions embedded in the membrane and/or domains on one or both sides of the membrane. Protein domains on the surface of the extracellular membrane are generally involved in cell-cell signaling or interactions.
  • transmembrane domain The region within the membrane is referred to herein as a "transmembrane domain", particularly those that form channels and pores to move molecules across the membrane.
  • a "transmembrane domain” is a three-dimensional protein structure that is thermodynamically stable in a membrane, such as a membrane of a vesicle (e.g., a cell). Examples of transmembrane domains include a single alpha helix, a stable complex of several transmembrane alpha helices, a transmembrane beta barrel, the beta helix of gramicidin A, or any other structure.
  • a transmembrane helix is typically about 20 amino acids long.
  • membrane proteins are divided into two major categories, integral (intrinsic) and peripheral (extrinsic), based on the nature of membrane-protein interactions. Most biological membranes contain both types of membrane proteins.
  • Integral membrane proteins have one or more fragments embedded in the phospholipid bilayer. Integral membrane proteins include transmembrane proteins and lipid anchored proteins. Most integral proteins contain residues with hydrophobic side chains that interact with the fatty acyl groups of membrane phospholipids, thereby anchoring the protein to the membrane. Most integral proteins span the entire phospholipid bilayer. These transmembrane proteins include one or more transmembrane domains and 4 to several hundred residues long domains extending into the aqueous medium on each side of the bilayer.
  • the transmembrane domain is one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) alpha helices and/or beta strands ( ⁇ strand).
  • Transmembrane alpha helical domains are usually embedded in the membrane by hydrophobic interactions with lipids inside the bilayer and may also be embedded in the membrane by ionic interactions with the polar head groups of phospholipids (e.g., glycophorin).
  • the structure of the beta strand is usually in the form of a transmembrane barrel structure (e.g., porin).
  • Some integral proteins are anchored to one of the leaflets of the membrane by covalently bound fatty acids.
  • lipid-anchored cytoplasmic proteins e.g., myristate or palmitate
  • a fatty acyl group is attached to the N-terminal glycine residue via an amide bond.
  • Peripheral membrane proteins or exogenous proteins do not interact with the hydrophobic core of the phospholipid bilayer. Instead, they are usually bound to the membrane indirectly through interactions with integral membrane proteins or directly through interactions with lipid polar head groups.
  • Peripheral proteins localized to the cytoplasmic face of the plasma membrane include the cytoskeletal proteins spectrin and actin in erythrocytes and the enzyme protein kinase C. This enzyme shuttles between the cytosol and the cytoplasmic face of the plasma membrane and plays a role in signal transduction.
  • Other peripheral proteins are localized to the outer (outside the lipid membrane) surface of the plasma membrane.
  • transmembrane protein includes receptor, ligand, immunoglobulin, glycophorin or its combination.
  • the specific example of transmembrane protein includes CD8 ⁇ , CD4, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD28, CD137, Fc ⁇ RI ⁇ , T cell receptor (TCR, such as TCR ⁇ and/or TCR ⁇ ), nicotinic acetylcholine receptor, GABA receptor, NKG2D, OX40, 2B4, EpCAM or its combination.
  • TCR T cell receptor
  • TCR TCR, such as TCR ⁇ and/or TCR ⁇
  • nicotinic acetylcholine receptor GABA receptor
  • NKG2D such as TCR ⁇ and/or TCR ⁇
  • EpCAM EpCAM
  • the specific example of immunoglobulin includes IgG, IgA, IgM, IgE, IgD or its combination.
  • glycophorin includes glycophorin A, glycophorin D or its combination.
  • chimeric antigen receptor herein refers to an artificial cell surface receptor that is modified to be expressed on immune effector cells and specifically binds to an antigen, comprising at least (1) an extracellular antigen binding domain, such as a variable heavy chain or light chain of an antibody, (2) a transmembrane domain that anchors CAR into immune effector cells, and (3) an intracellular signaling domain.
  • CAR can redirect T cells and other immune effector cells to selected targets, such as cancer cells, using the extracellular antigen binding domain in a non-MHC restricted manner.
  • signal peptide herein refers to a fragment in a protein or polypeptide that is used to guide the protein or polypeptide into the secretory pathway, transfer to the cell membrane and/or cell surface.
  • the "transmembrane (TM) region" of a chimeric antigen receptor refers to a polypeptide structure that enables the chimeric antigen receptor to be expressed on the surface of an immune cell (e.g., a lymphocyte, NK cell, or NKT cell) and guides the immune cell to respond to a target cell.
  • the transmembrane domain may be natural or synthetic, and may also be derived from any membrane-bound protein or transmembrane protein. When the chimeric antigen receptor binds to the target antigen, the transmembrane domain is capable of signal transduction.
  • the "hinge region" of a chimeric antigen receptor generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane region and the antigen binding region.
  • the hinge region is used to provide greater flexibility and accessibility to the antigen binding region.
  • the hinge region may be derived in whole or in part from natural molecules, such as in whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from an antibody constant region.
  • the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence.
  • intracellular signaling domain refers to a portion of a protein that transduces effector function signals and directs cells to perform a specified function.
  • the intracellular signaling domain is responsible for the primary signaling within the cell after the antigen binding domain binds to the antigen, resulting in the activation of immune cells and immune responses.
  • the intracellular signaling domain is responsible for activating at least one of the normal effector functions of the immune cells in which the CAR is expressed.
  • Exemplary intracellular signaling domains include CD3 ⁇ .
  • a "co-stimulatory signal binding domain” includes a co-stimulatory signal binding domain in an antigen presenting cell (e.g., aAPC, dendritic cell, B
  • an antigen presenting cell e.g., aAPC, dendritic cell, B
  • the cognate co-stimulatory molecule on the T cell such as a T cell, specifically binds to a cognate co-stimulatory molecule on the T cell, thereby providing a signal that mediates T cell responses, including but not limited to proliferation, activation, differentiation, etc., in addition to the primary signal provided by, for example, the TCR/CD3 complex binding to the MHC molecule loaded with the peptide.
  • the co-stimulatory signal binding domain may include but is not limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, agonists or antibodies that bind to Toll ligand receptors and ligands that specifically bind to B7-H3.
  • CD7 CD80
  • B7-2 CD86
  • PD-L1, PD-L2, 4-1BBL OX40L
  • IX40L inducible co-stimulatory ligand
  • IAM intercellular adhesion molecule
  • CD30L CD40, CD70, CD83, HLA-G, MICA, MICB
  • HVEM lymphotoxi
  • Co-stimulatory signal binding domains also include, among others, antibodies that specifically bind to co-stimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.
  • antibodies that specifically bind to co-stimulatory molecules present on T cells such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.
  • LFA-1 lymphocyte function-associated antigen-1
  • GUI2c refers to mammalian guanylate cyclase C (GUCY2C), preferably human GUCY2C protein.
  • the term “GUCY2C” can be used interchangeably with "STAR", “GUC2C", “GCC” or “ST receptor”.
  • the nucleotide sequence of human GUCY2C is disclosed as GenBank Accession No. NM_004963, and the amino acid sequence of human GUCY2C is disclosed as GenBank Accession No. NP_004954.
  • Naturally occurring allelic variants have an amino acid sequence that is at least 95%, 97% or 99% identical to the protein described in GenBank Accession No. NP.Sub.-004954.
  • GUCY2C protein is a transmembrane cell surface receptor protein that plays an important role in maintaining intestinal fluid, electrolyte homeostasis and cell proliferation.
  • the "DLL3 (Delta-Like Ligand 3)" used in this article is a single-pass transmembrane protein attached to the cell surface and is a member of the Notch ligand family.
  • the human DLL3 gene is located on chromosome 19q13, and its open reading frame is approximately 1800bp long.
  • the human DLL3 protein consists of 619 amino acids, and the complete structure includes 1 DSL domain, 1 intracellular domain, and 6 epidermal growth factor-like domains.
  • the DSL gene sequence at the N-terminus of the extracellular domain is highly conserved in the ligand family and is a functional domain required for binding to the Notch receptor.
  • the DLL3 intracellular domain is short and its function is still unclear.
  • DLL3 is highly expressed in SCLC and other neuroendocrine tumors, but rarely expressed in normal tissues. Activation of DLL3 can play a role in promoting or suppressing cancer. DLL3 is widely expressed in human cancers, including small cell lung cancer, glioma, pancreatic cancer, melanoma, breast cancer, pituitary tumors, endometriomas, acute myeloid leukemia, liver cancer, bladder cancer, colon cancer, prostate cancer, kidney cancer and esophageal cancer.
  • CD19 refers to the Cluster of Differentiation 19 protein (CD19), which is an antigenic determinant detectable on leukemic precursor cells. CD19 is expressed on most B-lineage cancers, such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma.
  • antigen-binding fragment is used in the broadest sense and refers to a fragment that specifically binds to an antigen.
  • antigen-binding molecules include, but are not limited to, antibodies or antibody mimetics.
  • Antibody mimetics refers to organic compounds or binding domains that can specifically bind to an antigen but are unrelated to the antibody structure, and exemplarily, antibody mimetics include, but are not limited to, affibodies, affitins, affilins, designed ankyrin repeat proteins (DARPins), nucleic acid aptamers, or Kunitz-type domain peptides.
  • DARPins ankyrin repeat proteins
  • antibody is used in the broadest sense and refers to a polypeptide or combination of polypeptides that contains sufficient sequence from the variable region of the immunoglobulin heavy chain and/or sufficient sequence from the variable region of the immunoglobulin light chain, so as to be able to specifically bind to an antigen.
  • Antibodies herein encompass various forms and various structures, as long as they exhibit the desired antigen binding activity.
  • Antibodies herein include alternative protein scaffolds or artificial scaffolds with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which contain mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds containing, for example, biocompatible polymers.
  • Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art that can be used to transplant CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.
  • antibody includes complete antibodies and any antigen-binding fragments (i.e., “antigen-binding portions”) or single chains thereof.
  • Antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2, and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • the VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs, which are 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 can interact with antigens.
  • the constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • immunoglobulins Due to the different amino acid compositions and arrangement sequences of the constant regions of the heavy chains of immunoglobulins, their antigenicity is also different. Based on this, the "immunoglobulins" herein can be divided into five categories, or isotypes of immunoglobulins, namely, IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are ⁇ chains, ⁇ chains, ⁇ chains, ⁇ chains, and ⁇ chains, respectively.
  • the same class of Ig can be divided into different subclasses according to the differences in the amino acid composition of its hinge region and the number and position of the disulfide bonds of the heavy chain, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2.
  • Light chains are divided into ⁇ chains or ⁇ chains by the differences in the constant regions.
  • Each of the five classes of Ig can have ⁇ chains or ⁇ chains.
  • antibody in this article also includes antibodies that do not contain light chains, for example, heavy-chain antibodies (HCAbs) produced by camelids such as dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanicoes (Lama guanicoe) and alpacas (Vicugna pacos), and immunoglobulin new antigen receptors (Ig new antigen receptors, IgNARs) found in cartilaginous fish such as sharks.
  • HCAbs heavy-chain antibodies
  • camelids such as dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanicoes (Lama guanicoe) and alpacas (Vicugna pacos)
  • the term “heavy chain antibody” refers to an antibody lacking the light chain of a conventional antibody.
  • the term specifically includes, but is not limited to, a homodimeric antibody comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.
  • the term “nanoantibody” refers to the natural heavy chain antibody lacking light chain that exists in camels. Cloning its variable region can obtain a single domain antibody consisting of only the heavy chain variable region, also called VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.
  • VHH Very domain of heavy chain of heavy chain antibody
  • nucleic acid and single domain antibody used in this article have the same meaning and can be used interchangeably. They refer to cloning the variable region of a heavy chain antibody to construct a single domain antibody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, a heavy chain antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.
  • CH1 light chain and heavy chain constant region 1
  • antibodies herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g. sharks).
  • Antigen binding fragment and “antibody fragment” used herein are used interchangeably herein, and they do not have the entire structure of a complete antibody, but only contain a partial or partial variant of a complete antibody, and the partial or partial variant has the ability to bind to an antigen.
  • Antigen binding fragment or “antibody fragment” herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, scFv, diabody, and single domain antibody.
  • variable region herein refers to the region of an antibody heavy chain or light chain that is involved in enabling the antibody to bind to an antigen
  • variable domains of the heavy and light chains of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007).
  • a single VH or VL domain may be sufficient to confer antigen binding specificity.
  • CDR complementarity determining region
  • HVR hypervariable region
  • VH heavy chain variable region
  • VL light chain variable region
  • HCDR the heavy chain variable region
  • LCDR the light chain variable region
  • framework region or “FR region” are interchangeable and refer to those amino acid residues other than CDR in the heavy chain variable region or light chain variable region of an antibody.
  • a typical antibody variable region consists of 4 FR regions and 3 CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • CDR herein can be annotated and defined by methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system or the IMGT numbering system, and the tool websites used include but are not limited to the AbRSA website (http://cao.labshare.cn/AbRSA/cdrs.php), the abYsis website (www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi) and the IMGT website (http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results).
  • CDR herein includes overlaps and subsets of amino acid residues defined in different ways.
  • conservative amino acid generally refers to amino acids belonging to the same class or having similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity).
  • amino acids within each of the following groups are conservative amino acid residues of each other, and the replacement of the amino acid residues within the group is a replacement of conservative amino acids:
  • identity herein can be calculated in the following manner: to determine the percentage of "identity" of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal comparison or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at this position.
  • the percent identity between the two sequences will vary depending on the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • nucleic acid herein includes any compound and/or substance comprising a polymer of nucleotides.
  • Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group.
  • cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U) a sugar (i.e., deoxyribose or ribose) and a phosphate group.
  • nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules.
  • Nucleic acid molecules can be linear or cyclic.
  • nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms.
  • nucleic acid molecules as described herein may contain naturally occurring or non-naturally occurring nucleotides.
  • nucleic acid molecules also encompass DNA and RNA molecules that are suitable as vectors for directly expressing the constructs of the present application in vitro and/or in vivo, for example in a host or patient.
  • DNA e.g., cDNA
  • RNA e.g., mRNA
  • mRNA may be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule so that the mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038/nm.4356 or EP 2 101 823 B1).
  • the DNA or RNA described herein can be used to introduce nucleic acid into cells, for example, by encapsulating nucleic acid with materials such as liposomes such as liposome polymers (LPR), liposome complexes (LP), nanoparticles (NP) or lipid nanoparticles (LNP) to introduce the DNA or RNA into cells.
  • LPR liposome polymers
  • LP liposome complexes
  • NP nanoparticles
  • LNP lipid nanoparticles
  • the RNA molecule can be linear or circular, for example, using LNP to deliver the linear or circular RNA molecule to the inside of a cell or subject, and using the subject's expression system to express the corresponding fusion protein or CAR to produce immune cells expressing CAR in vivo.
  • isolated nucleic acid herein refers to a nucleic acid molecule that has been separated from the components of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in the following cells, which typically contain the nucleic acid molecule, but the nucleic acid molecule is present outside the chromosome or at a chromosomal position different from its natural chromosomal position.
  • vector refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors.”
  • pharmaceutical composition refers to a preparation which is formulated to allow the biological activity of the active ingredient contained therein to be exerted.
  • the pharmaceutical composition is in an effective form and does not contain additional components that are unacceptably toxic to a subject to which the pharmaceutical composition is administered.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions in the treated subject, such as the progression of cancer.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete), whether detectable or undetectable.
  • Subjects in need of treatment include those who already have a condition or disease, as well as those who are susceptible to a condition or disease or those for whom a condition or disease is to be prevented.
  • slow down, alleviate, weaken, alleviate, and relieve their meanings also include elimination, disappearance, non-occurrence, and the like.
  • subject refers to an animal, and in a specific aspect refers to a mammal.
  • mammals include primates, canines, felines, rodents, and the like. Specific examples include humans, dogs, cats, horses, cattle, sheep, goats, rabbits, guinea pigs, rats, and mice.
  • subject in need refers to an individual in need of treatment or prevention as determined by a researcher, veterinarian, physician, or other clinician. In one embodiment, the individual in need is a mammal, such as a human.
  • the mbIL15 armored CAR-T cells disclosed in the present application have at least one or more of the following advantages: (1) IL15 is expressed on the cell surface and is confined to the tumor microenvironment; (2) It has a higher CAR expression positivity rate or cell proliferation multiple, and expression stability; (3) The proportion of TCM in the amplified T cells is higher; (4) It has a high specificity for specific antigen epitopes; (5) It has excellent tumor cell killing and inhibition capabilities, including but not limited to: showing better tumor killing effects in long-term killing tests or at medium and low doses; (6) It has lower side effects, strong specificity, and is safer.
  • the present application provides an engineered immune cell that co-expresses a chimeric antigen receptor (CAR) and all or a functional portion of membrane-bound IL15 (mbIL15).
  • CAR chimeric antigen receptor
  • mbIL15 membrane-bound IL15
  • all or a functional portion of the IL15 is fused to all or a portion of a transmembrane protein selected from NKG2D, OX40, 2B4, or EpCAM.
  • the NKG2D comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 15 or 16.
  • the OX40 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 17.
  • the 2B4 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 18.
  • the EpCAM comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 19 or 20.
  • all or a functional portion of the IL15 is fused to all or a portion of the transmembrane protein via a linker.
  • the linker is CD8 ⁇ , and the preferred linker comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 14.
  • all or a functional portion of the IL15 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 47.
  • the mbIL15 comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 27-32 and 48.
  • the CAR comprises at least an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain.
  • the extracellular antigen-binding domain comprises a Nanobody (VHH) or an antigen-binding fragment thereof targeting GCC, wherein the Nanobody or the antigen-binding fragment thereof comprises CDR1, CDR2 and CDR3.
  • VHH Nanobody
  • the Nanobody or the antigen-binding fragment thereof comprises CDR1, CDR2 and CDR3.
  • the CDR1, CDR2 and CDR3 are respectively selected from the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 2.
  • the CDR1, CDR2 and CDR3 contain the amino acid sequences shown in SEQ ID NO: 3-5, respectively.
  • the Nanobody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 2, or comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations compared to the amino acid sequence shown in SEQ ID NO: 2.
  • the extracellular antigen binding domain comprises an antibody or an antigen-binding fragment thereof targeting DLL3, wherein the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3.
  • the HCDR1, HCDR2 and HCDR3 are respectively selected from HCDR1, HCDR2 and HCDR3 contained in VH shown in SEQ ID NO: 36; and the LCDR1, LCDR2, LCDR3 are respectively selected from LCDR1, LCDR2 and LCDR3 contained in VL shown in SEQ ID NO: 37.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 respectively have the amino acid sequences shown in SEQ ID NO: 39-44.
  • the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 36 and SEQ ID NO: 37, or comprises an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 36 and 37, or an amino acid sequence that has at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations compared to the amino acid sequence shown in SEQ ID NO: 36 and 37.
  • the extracellular antigen binding domain comprises an antibody or an antigen binding fragment thereof targeting CD19, wherein the antibody or the antigen binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3.
  • the HCDR1, HCDR2 and HCDR3 are respectively selected from HCDR1, HCDR2 and HCDR3 contained in VH shown in SEQ ID NO: 55; and the LCDR1, LCDR2 and LCDR3 are respectively selected from LCDR1, LCDR2 and LCDR3 contained in VL shown in SEQ ID NO: 56.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 respectively have the amino acid sequences shown in SEQ ID NO: 58-63.
  • the antibody or antigen-binding fragment thereof comprises the amino acids shown in SEQ ID NO: 55 and SEQ ID NO: 56.
  • the mutation is selected from insertion, deletion and/or substitution, and the substitution is preferably a conservative amino acid substitution.
  • the immune cells are selected from T cells, NK cells, NKT cells, or T cells and NK cells induced by other techniques.
  • a second aspect of the present application provides a fusion protein comprising all or a functional portion of IL15 fused to all or a portion of a transmembrane protein, wherein the transmembrane protein is selected from NKG2D, OX40, 2B4 or EpCAM.
  • the fusion protein further comprises a CAR, preferably the above-mentioned CAR.
  • the NKG2D comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 15 or 16.
  • the OX40 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 17.
  • the 2B4 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 18.
  • the EpCAM comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 19 or 20.
  • all or a functional portion of the IL15 is fused to all or a portion of the transmembrane protein via a linker.
  • the linker is CD8 ⁇ , and the preferred linker comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 14.
  • all or a functional part of the IL15 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 47.
  • the fusion protein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 27-32 and 48.
  • the third aspect of the present application provides an isolated nucleic acid molecule, which comprises a nucleic acid molecule encoding CAR and mbIL15 co-expressed on the immune cells described in any one of the above, or comprises a nucleic acid molecule encoding the fusion protein described in any one of the above; preferably, the nucleic acid molecule is DNA or RNA.
  • the fourth aspect of the present application provides a vector comprising the above nucleic acid molecule.
  • the vector is a viral vector.
  • the fifth aspect of the present application provides a method for preparing the immune cell described in any one of the above items, which comprises introducing the nucleic acid molecule into the immune cell.
  • the sixth aspect of the present application provides a pharmaceutical composition comprising the immune cell described in any one of the above items, the fusion protein described in any one of the above items, or the nucleic acid molecule described in any one of the above items.
  • the seventh aspect of the present application provides the use of any of the above-mentioned immune cells, the product obtained by the method, or the pharmaceutical composition for preparing a drug for treating a tumor in an individual.
  • the tumor is selected from a blood tumor or a solid tumor, and optionally, the blood tumor is selected from a B-cell lymphoma, a tumor expressing CD19, etc.; optionally, the solid tumor Selected from non-small cell lung cancer, gastric cancer, colon adenocarcinoma, colon cancer, small cell lung cancer, tumors expressing GCC or DLL3, etc.
  • the eighth aspect of the present application provides a method for treating a tumor, comprising administering any of the above immune cells, the product obtained by the method, or the pharmaceutical composition to an individual in need thereof.
  • the tumor is selected from a blood tumor or a solid tumor, and optionally, the blood tumor is selected from a B-cell lymphoma, a tumor expressing CD19, etc.; optionally, the solid tumor is selected from a non-small cell lung cancer, gastric cancer, colon adenocarcinoma, colon cancer, small cell lung cancer, a tumor expressing GCC or DLL3, etc.
  • the ninth aspect of the present application provides any of the above immune cells, the product obtained by the method, or the pharmaceutical composition, for use in preventing and/or treating tumors in an individual.
  • the tumor is selected from a blood tumor or a solid tumor, and optionally, the blood tumor is selected from a B-cell lymphoma, a tumor expressing CD19, etc.; optionally, the solid tumor is selected from non-small cell lung cancer, gastric cancer, colon adenocarcinoma, colon cancer, small cell lung cancer, a tumor expressing GCC or DLL3, etc.
  • the present application constructs mbIL15 armored T cells expressing GCC chimeric antigen receptors: CAR-1, CAR-2, CAR-3, CAR-4, CAR-5, CAR-6 and CAR-7.
  • the specific steps are as follows:
  • VHH antibody variant domain of heavy chain of heavy-chain antibody, VHH antibody
  • VHH antibody is an antibody targeting GCC
  • amino acid sequence is shown in SED ID NO: 2
  • CDR complementary region
  • CDR1 CDR2
  • CDR3 complementary region
  • the above antibody sequence numbering uses the IMGT numbering system.
  • the chimeric antigen receptor used is a second-generation chimeric antigen receptor, which has a CD28 hinge region (SEQ ID NO: 7), a CD28 transmembrane domain (SEQ ID NO: 8), a CD28 intracellular co-stimulatory domain (SEQ ID NO: 9), a CD3 ⁇ signaling domain (SEQ ID NO: 10, 11 or 12), a lytic peptide (SEQ ID NO: 13) and a membrane-bound IL15.
  • the amino acid sequence of IL15 is shown in SEQ ID NO: 1
  • the linker sequence between IL15 and the transmembrane domain is shown in SEQ ID NO: 14.
  • the transmembrane domain of membrane-bound IL15 is derived from NKG2D (SEQ ID NO: 15 or 16), OX40 (SEQ ID NO: 17), 2B4 (SEQ ID NO: 18) or EpCAM (SEQ ID NO: 19 or 33), among which mbIL15-IL15Ra (SEQ ID NO: 34) is derived from patent WO 2021/062281.
  • the constructed CAR structure and the amino acid sequence of each element are shown in Tables 1 and 2.
  • the plasmid was constructed by conventional molecular biological methods in the art. With the retroviral vector template, the retroviral plasmids P1, P2, P3, P4, P5, P6 and P7 expressing the second-generation chimeric antigen receptor were constructed with reference to the plasmid structure diagram shown in FIG1 . Each plasmid contains, from the 5' end to the 3' end, the following: CD8 ⁇ signal peptide, VHH, CD28 hinge region, CD28 transmembrane domain, CD28 intracellular co-stimulatory domain, CD3 ⁇ signal transduction domain and mbIL15.
  • the corresponding polynucleotide insertion sequences were synthesized respectively, and the retroviral vector was digested with restriction endonucleases EcoRI (Thermo, Cat#FD0274) and SalI (Thermo, Cat#FD0644), and the linearized vector was recovered and purified by agarose gel electrophoresis.
  • the polynucleotide sequences synthesized in the above steps were connected to the linearized vectors through the recombinase 5 ⁇ In-FusionHD enzyme (TaKaRa, Cat#ST0344).
  • the prepared reaction system was as follows: 2 ⁇ l synthetic polynucleotide fragments (50ng/ ⁇ l), 1 ⁇ l linearized plasmid (50ng/ ⁇ l), 2 ⁇ l 5 ⁇ HD InFusion enzyme, 5 ⁇ l ddH2O; pipette to mix, centrifuge briefly and place at 50°C for 15min. 10 ⁇ l of the recombinant reaction product was added to 100 ⁇ l of bacterial competent cells stbl3 (Frdbio, Cat#MCC0910), placed on ice for 5min, and the transformed bacterial solution was evenly spread on the LB plate containing 50 ⁇ g/ml kanamycin, and inverted in a constant temperature incubator for 12-16h. 3-5 clones were randomly selected from each plate for sequencing identification.
  • the correctly sequenced bacterial solution was transferred to 100 ml of LB liquid medium containing 50 ⁇ g/ml kanamycin, cultured overnight at 37°C, and the plasmid was extracted using the MN endotoxin-free plasmid extraction kit (MN, Cat#740420.50). After quantification, the plasmid was diluted to 1000 ng/ ⁇ l with endotoxin-free ultrapure water.
  • the amino acid sequence of the inserted CAR is shown in Table 3, and the amino acid sequence of membrane-bound IL-15 (mbIL15) is shown in Table 4.
  • 293T cells Cell Bank of Typical Culture Collection Committee of Chinese Academy of Sciences, Cat#GNHu17
  • DMEM medium Gibco, Cat#10566016
  • FBS Gibco, Cat#10099141
  • 293T cells cover about 70% of the surface of the culture dish, perform plasmid transfection: take the retroviral vector expressing CAR and the packaging plasmid, mix them and add them to 1.2ml Opti-MEM medium (Thermofisher Scientific, Cat#31985070), add 35 ⁇ l Fugene HD (Promega, Cat#04709691001) and mix them well.
  • This application constructs multiple strains of T cells expressing second-generation chimeric antigen receptors, which are named CAR-1, CAR-2, CAR-3, CAR-4, CAR-5, CAR-6 and CAR-7.
  • the specific steps are as follows:
  • T cell proliferation According to the instructions, the Stemcell Easy Sep Kit (Stemcell, Cat#19055) was used to isolate T cells from peripheral blood mononuclear cells (PBMC) of different healthy donors. The separated T cells were cultured in a culture dish pre-embedded with 1 ⁇ g/ml CD3/CD28 antibody (Thermo, Cat#11131D). The culture medium contained X-VIVO15 (Lonza, Cat#BEBP02-054Q), 5% human AB serum (Gemini, Cat#100-512), 100U/ml penicillin-streptomycin (Gibco, Cat#15140-122), and 200IU/ml human IL2 factor (Beijing Shuanglu, Cat#S19991007). Cell counts were performed twice a week, and the cells were subcultured and expanded when the cell density reached 2.5 ⁇ 10 6 /ml.
  • PBMC peripheral blood mononuclear cells
  • CAR-T cells Preparation of CAR-T cells: 24-well plates were coated with RetroNectin (Takara, Cat#T202) at a concentration of 7 ⁇ g/mL, 500 ⁇ l per well, and placed at 4°C overnight. On the second day, the corresponding retrovirus was added to the 24-well plate, followed by centrifugation at 2000g for 60 min at 4°C. After removing the supernatant, T cells were added to the 24-well plate at 3 ⁇ 10 5 cells/well and centrifuged at room temperature for 5 min at 400g. After centrifugation, the 24-well plate was placed in a constant temperature incubator for culture (37°C, 5% CO 2 ). On the third day, the cells were transferred to a 6-well plate for continued culture.
  • RetroNectin RetroNectin
  • Flow cytometry was used to detect the transfection efficiency of CAR: cells were washed twice with PBS buffer (Gibco, Cat#10010023), and then diluted to 2 ⁇ 10 6 /ml with PBS buffer after counting. 50 ⁇ l Fc receptor blocker (BioLegend, Cat#422302) was added and incubated at room temperature for 10 min, and then 100 ⁇ l was added to each well of the 96-well plate. After adding 100 ⁇ l of FITC-labeled human GUCY2C protein (Acro, Cat#GUC-HF255) at a concentration of 2 ⁇ g/ ⁇ l to each well, the 96-well plate was incubated on ice for 20 min.
  • FACS buffer Gibco, Cat#A1286301 was centrifuged and washed three times, and the cells were resuspended in 100 ⁇ l FACS buffer, and the results were detected and analyzed by FACS (BD, CANTOII).
  • CAR is continuously expressed on the surface of T cells.
  • IL15-armored CARs can be detected on the surface of T cells, while no CAR is detected on the surface of unactivated T cells (parental T cells).
  • the positive rates of CAR-2 and CAR-3 cells expressing only mbIL15 are similar to that of CAR-7, which is about 80%.
  • CAR-3 still maintains a positive rate similar to that of CAR-7, which remains at about 80%.
  • T cell differentiation was evaluated, including the changes in the proportions between CD8 + and CD4 + cell populations, the changes in the proportions between TCM, TSCM, TEM, and TE cell subsets, and the expression of inhibitory receptors LAG3 and TIM3.
  • the ratio between CD8 + cell population and CD4 + cell population on day 8 was between 2.45 and 2.61, and there was no significant difference among the CAR-T cells.
  • TSCM in CD8 + cell population The proportion of TCM was between 6.27% and 17.8%, of which the proportion of TCM of CAR-3 was 17.8% and that of CAR-7 was 6.27%; the sum of the proportions of TSCM and TCM was between 95.84% and 97.7%, and that of parental T cells was 95.84%;
  • the proportion of TCM in the CD4 + cell population was significantly higher, between 34.4% and 50.0%, of which the proportion of TCM of CAR-3 was 50.0% and that of CAR-7 was 34.4%, while that of TSCM was between 48.5% and 63.1%, and the sum of the proportions of TSCM and TCM was between 96.7% and 98.5%; as shown in Figure 3D,
  • TSCM was the majority in the CD8 + cell population, ranging from 68.3% to 82%, and the proportion of TCM was between 3.90% and 11.50%, of which the proportion of TCM of CAR-3 was 11.50% and that of CAR-7 was 3.90%, and the sum of the proportions of TSCM and TCM was between 79.2% and 89.29%;
  • the proportion of TCM in the CD4 + cell population increased significantly, ranging from 20.6% to 35.9%, of which the proportion of TCM of CAR-3 was 35.9% and that of CAR-7 was 20.6%, and the proportion of TSCM was between 41.9% and 60.3%, and the sum of the proportions of TSCM and TCM was between 77.7% and
  • LS174T-luc As target cells and mbIL15 CAR-T cells on days 8 and 11 as effector cells, the activity test was performed. LS174T-luc cells were washed with PBS buffer, digested with trypsin solution (Gibco, Cat#25200056), centrifuged at 1000 ⁇ rpm for 5min, the supernatant was removed and the cells were resuspended in fresh medium.
  • 20,000 cells/well were plated on a 96-well plate, and mbIL15 CAR-T cells or parental T cells were added for co-culture at a T cell: target cell ratio (E:T) of 1:1, 1:2, and 1:10, with two replicates for each group.
  • E:T target cell ratio
  • Samples were taken at 48h, and the firefly luciferase substrate D-Luciferin (Abcam, Cat#ab143655) was added.
  • the fluorescence value was read using a multifunctional microplate reader (Perkinelmer, Cat#EnSight), and the results are shown in Figure 4.
  • CAR-3 cells on days 8 and 11 showed higher killing activity than CAR-7 under different E:T ratio conditions, and more LS174T-luc cells were lysed.
  • Alexa 647 fluorescent Anti-CD3 antibody Abcam, Cat#ab253269
  • the ratio of T cells and target cells was detected by FACS and the cell lysis ratio was calculated.
  • resuspend the cells co-cultured for 72 hours take 1/2 volume of the cell suspension and co-culture with the same number of fresh target cells for the next round of killing experiment. Repeat the same method to complete 4 rounds of killing experiments.
  • mbIL15 armored T cells expressing DLL3 chimeric antigen receptors were constructed: CAR-8, CAR-9, CAR-10 and CAR-11.
  • the DLL3 single-chain antibody (scFv antibody) used is an antibody targeting DLL3, and the amino acid sequence is shown in SED ID NO: 35, with a heavy chain VH shown in SEQ ID NO: 36, a light chain VL shown in SEQ ID NO: 37, and a linker shown in SEQ ID NO: 38.
  • the heavy chain has a heavy chain determining cluster complementary region (Complementarity Determining Region of the Heavy Chain, referred to as HCDR) HCDR1, HCDR2, HCDR3 shown in SEQ ID NO: 39, 40, 41, and the light chain has a light chain determining cluster complementary region (Complementarity Determining Region of the Light Chain, referred to as LCDR) LCDR1, LCDR2, LCDR3 shown in SEQ ID NO: 42, 43, 44.
  • HCDR heavy chain determining cluster complementary region
  • the chimeric antigen receptor used is a second-generation chimeric antigen receptor, having a CD8 ⁇ hinge region (SEQ ID NO: 14), a CD8 ⁇ transmembrane domain (SEQ ID NO: 45), a 4-1BB intracellular co-stimulatory domain (SEQ ID NO: 46), a CD3 ⁇ signal transduction domain (SEQ ID NO: 10, 11 or 12), a lytic peptide (SEQ ID NO: 13) and membrane-bound IL15.
  • a retroviral vector template with reference to the plasmid structure diagram shown in FIG6 , retroviral plasmids P8, P9, P10 and P11 expressing the second-generation chimeric antigen receptor were constructed.
  • Each plasmid contains from the 5' end to the 3' end: CD8 ⁇ signal peptide, scFv, CD8 ⁇ hinge region, CD8 ⁇ transmembrane domain, 4-1BB intracellular co-stimulatory domain, CD3 ⁇ signal transduction domain or membrane-bound IL15.
  • the amino acid sequence of each element is shown in Table 5, the connection order between each element is shown in Table 6, and the specific amino acid sequence of the inserted CAR is shown in Table 7, where matIL15-CD8 ⁇ -CD8 ⁇ is from patent WO2020/056045.
  • mbIL15 armored DLL3 CAR-T cells were prepared, and each group of cells on the 10th day of activation and each group of cells on the 11th day of activation were harvested and frozen.
  • CS10 Stemcell, Cat#07930
  • the cells were transferred to -80°C and slowly cooled at 1°C/min. After 4 hours, they were transferred and stored in liquid nitrogen.
  • the amino acid sequence of membrane-bound IL-15 (mbIL15) is shown in SEQ ID NO:48.
  • Flow cytometry was used to detect the transfection efficiency of CAR: the cells were washed twice with PBS buffer, and after counting the cells, the cells were diluted to 2 ⁇ 10 6 cells/ml with PBS buffer, 50 ⁇ l Fc blocking reagent was added, and the cells were incubated at room temperature for 10 min, and then 100 ⁇ l was added to each well of the 96-well FACS reaction plate, and 100 ⁇ l His-tagged DLL3 antigen protein (3 ⁇ g/ml, purchased from Acro, Cat#DL3-H52H4) was added, and the cells were incubated at 4°C for 30 min.
  • FACS Flow cytometry
  • the cells were washed twice by centrifugation with FACS buffer, and 100 ⁇ l THE TM His Tag Antibody (GenScript, Cat#A01802) and IL-15 Monoclonal Antibody (34559), PE (Invitrogen, Cat#MA5-23561) were added to each well, and the cells were incubated at 4°C for 20 min.
  • the cells were washed three times by centrifugation with FACS buffer, and the cells were suspended with 200 ⁇ l FACS buffer.
  • the results were detected and analyzed by FACS, and the results are shown in Figure 7.
  • FIG. 7A on days 8 and 10
  • the expression rate of CAR-T cells in each group was between 60% and 80%, indicating good viral transfection efficiency
  • FIG. 7B on days 8 and 10
  • the expression rate of IL-15 was between 50% and 80%, indicating good expression rate.
  • DLL3-targeted mbIL15 CAR-T cells To test the in vitro persistence of DLL3-targeted mbIL15 CAR-T cells, multiple rounds of killing experiments were performed using tumor cells SHP77-luc and NCI-H2171-luc expressing human DLL3.
  • SHP77 cells with high expression of DLL3 and NCI-H2171 cells with low expression of DLL3 were purchased from ATCC and transformed with bacterial luciferase to construct stable transfected cells SHP77-luc and NCI-H2171-luc.
  • SHP77-luc and NCI-H2171-luc were used as target cells, and fresh mbIL15 CAR-T cells on the 11th day after activation were used as effector cells for activity testing.
  • SHP77-luc and NCI-H2171-luc cells were washed with PBS buffer, centrifuged at 1000 ⁇ rpm for 5 minutes, the supernatant was removed and fresh culture medium was added to resuspend the cells.
  • 20,000 cells/well were plated on a 96-well plate, and the above-mentioned mbIL15 CAR-T cells or parental T cells were added for co-culture at a T cell: target cell ratio (E:T) of 1:2, with two replicates for each group.
  • E:T target cell ratio
  • SHP77-luc and NCI-H2171-luc were used as target cells, and mbIL15CAR-T cells frozen and revived on the 10th day after activation were used as effector cells for activity testing.
  • the cell cryopreservation tube was taken out from the liquid nitrogen tank and immediately placed in a 37°C water bath (Thermo Scientific, Cat#TSGP02) for rapid thawing; after the cell mixture in the cryopreservation tube was completely dissolved, the cell mixture was slowly added to a test tube containing 9mL of culture medium, mixed evenly, centrifuged at 200 ⁇ g for 5min, the supernatant was discarded, and the culture medium was used to resuspend and culture in a cell culture bottle. Multiple rounds of killing tests were performed according to the method described in Example 6.1, and the results are shown in Figure 8.
  • each group of cells that were frozen and revived showed excellent killing activity, and the killing activity from strong to weak was: CAR-10, CAR-9, CAR-11 and CAR-8, and mbIL15 CAR-T showed stronger killing activity than CAR-8;
  • the killing activity of frozen and revived mbIL15 CAR-T was similar, and it could still almost completely kill NCI-H2171-luc cells in the sixth round, while the killing activity of CAR-8 decreased significantly in the second round, with only 7.2% of cells lysed.
  • NPG mice combined immunodeficient mice, purchased from Beijing Weitongda Biotechnology Co., Ltd.
  • SHP77 cells were subcutaneously inoculated with 3 ⁇ 10 6 SHP77 cells in the logarithmic growth phase and in good growth condition.
  • mice with a tumor volume of about 30 mm 3 were randomly grouped according to the random number principle.
  • the CAR-T cells frozen and revived on the 10th day after activation were injected into the tail vein according to the method described in Examples 5 and 6.
  • the number of CAR-T cells was counted as CAR positive, and 2 ⁇ 10 6 CAR positive cells (ie, CAR+ cells) were injected per mouse.
  • the day of CAR-T cell injection was day 0 (D0).
  • the specific grouping and administration are shown in Table 8. Tumor volume, body weight changes and survival rate were continuously observed and measured, and the measurements were recorded twice a week. The test results are shown in Figure 9.
  • mbIL15 CAR-T cells targeting human CD19 were prepared and tested for killing activity against Nalm6-luc cells that highly expressed CD19.
  • Nalm6-luc cells were purchased from ATCC and bacterial luciferase was transferred to construct stably transfected cells Nalm6-luc.
  • mbIL15 armored T cells expressing CD19 chimeric antigen receptors were constructed: CAR-12, CAR-13 and CAR-14.
  • the CD19 single-chain antibody (scFv antibody) used is an antibody targeting CD19, and the amino acid sequence is shown in SED ID NO: 54, with a heavy chain VH shown in SEQ ID NO: 55, a light chain VL shown in SEQ ID NO: 56, and a linker shown in SEQ ID NO: 57.
  • the heavy chain has the heavy chain determinant cluster complementary regions HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 58, 59, and 60, and the light chain has the light chain determinant cluster complementary regions LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 61, 62, and 63.
  • the above antibody sequences are numbered using the Kabat numbering system.
  • the chimeric antigen receptor used is a second-generation chimeric antigen receptor, which has a CD28 hinge region (SEQ ID NO: 7), a CD28 transmembrane domain (SEQ ID NO: 8), a CD28 intracellular co-stimulatory domain (SEQ ID NO: 9), a CD3 ⁇ signal transduction domain (SEQ ID NO: 10, 11 or 12), a lytic peptide (SEQ ID NO: 13) and a membrane-bound IL15.
  • the constructed CAR structure and the amino acid sequence of each element are shown in Tables 9 and 10.
  • retroviral plasmids P12, P13 and P14 expressing second-generation chimeric antigen receptors were constructed.
  • Each plasmid contains, from the 5' end to the 3' end, the following: GM-CSF signal peptide, scFv, CD28 hinge region, CD28 transmembrane domain, CD28 intracellular co-stimulatory domain, CD3 ⁇ signal transduction structure and membrane-bound IL15.
  • the connection order is shown in Table 10, and the specific sequence is shown in Table 11.
  • the matIL15-CD8 ⁇ -CD8 ⁇ used in P14 is from patent WO2020/056045.
  • Example 1 mbIL15 armored CD19 CAR-T cells were prepared, and each group of cells was harvested on the 10th day after activation. And referring to the method of Example 1.4, the CAR transfection efficiency of each group of cells was detected, showing that the transfection of each group of cells was good.
  • Nalm6-luc was used as the target cell, and mbIL15 CAR-T cells on the 10th day after activation were used as effector cells for activity testing.
  • Nalm6-luc cells were washed with PBS buffer, centrifuged at 1000 ⁇ rpm for 5min, the supernatant was removed and the cells were resuspended in fresh medium.
  • 20,000 cells/well were plated on a 96-well plate, and mbIL15 CAR-T cells or parental T cells were added for co-culture at a T cell: target cell ratio (E:T) of 1:2, with two replicates for each group.
  • E:T target cell ratio

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Abstract

提供了工程化的免疫细胞，具体而言，提供了经修饰以共表达嵌合抗原受体(CAR)和白介素-15(IL-15)的免疫细胞，及其药物组合物、使用方法和相关用途。所述工程化的免疫细胞具备优异的肿瘤细胞杀伤与抑制能力，可用于治疗癌症。

Description

工程化嵌合抗原受体免疫细胞及其应用
相关申请的引用
本申请要求2022年11月7日提交的中国专利申请号202211383548.X的优先权，通过援引加入的方式将该申请的内容全部并入本文，用于所有目的。
技术领域
本申请涉及免疫学领域，具体而言，涉及表达嵌合抗原受体和白细胞介素15(IL15)的免疫细胞。
背景技术
基于细胞的癌症免疫疗法已成为癌症治疗的革命性新支柱，例如使用表达嵌合抗原受体(chimeric antigen receptor，CAR)的工程化改造的T细胞(CAR-T细胞)和自然杀伤细胞(CAR-NK细胞)进行过继细胞疗法治疗。在体外激活自体或异体来源的免疫效应细胞并输注给患者，从而实现杀伤患者体内的肿瘤细胞的目的。然而在治疗某些恶性肿瘤时，CAR-T细胞和CAR-NK细胞会出现难以扩散和持久性较差的情况，特别是进行治疗实体瘤时。以CAR-T细胞为例，抑制性肿瘤微环境(tumor microenvironments，TME)可以抑制T细胞受体(T cell receptor,TCR)和共刺激信号传导，限制细胞运输，并使CAR-T细胞失活。目前，已有许多促进CAR-T细胞功能发挥的策略，例如利用刺激性细胞因子以增加免疫细胞的存活和扩增并逆转免疫抑制性肿瘤微环境。
白细胞介素15(Interleukin 15，IL15)是14～15kDa糖蛋白，氨基酸序列见SEQ ID NO：1(NP_000576.1，NCBI数据库)。IL15 mRNA存在于多种组织中，包括造血细胞(如单核细胞、巨噬细胞和树突细胞)和非造血细胞(如角质形成细胞、成纤维细胞、神经细胞、骨骼肌、和上皮细胞)，但IL15蛋白只集中于特定的细胞内表达。作为一种有效的免疫刺激细胞因子，IL15可增强T细胞和NK细胞免疫反应，进而提高细胞治疗的可靠性和疗效。IL15在结构上与白细胞介素2(Interleukin 2，IL2)相似，可以维持CD8+记忆T细胞(CD8+Memory T cell,CD8+TM)增殖，同时抑制IL-2诱导的T细胞死亡，从而更好地维持长期抗肿瘤免疫。共表达可溶性IL15(soluble IL15，sIL15)或膜结合IL15(membrane bound IL15，mbIL15)装甲的免疫细胞，已被证明具有增强的抗肿瘤作用和细胞持久性。
CAR-T细胞的治疗功效与T细胞亚群(CD4+或CD8+)的比例，分化(幼稚，记忆，效应子)，激活(激活标记的表达)和CAR-T细胞的功能状态有关。中央记忆型T细胞(Central Memory T cell，TCM,CD62L+CD45RO+)是初始T细胞(Naive T Cell)经过抗原激活后，产生的具有长期记忆性的，并能够归巢到淋巴结接受抗原再刺激的T细胞。在抗原的再次刺激之下可继续产生大量的携带同种抗原的克隆化的效应记忆型T细胞(Effective Memory T Cell，TEM,CD62L-CD45RO+)。干细胞样记忆型T细胞(stem cell-like memory T cell，TSCM,CD62L+CD45RO-)是记忆型T细胞的重要组成，具有干细胞样自我更新、多分化潜能和免疫重构特性。效应T细胞(Effector T cells，TE,CD62L-CD45RO-)是T细胞接受抗原刺激后， 经过增殖分化形成的细胞。T细胞的体外扩增是CAR-T细胞生产工艺的关键步骤。近期临床研究表明，功能记忆T细胞亚群，包括TSCM、TCM和其他分化程度较低的T细胞亚群是患者体内产生长期抗肿瘤反应的原因。因而体外T细胞扩增过程中，让T细胞产物中产生更高比例的TSCM和TCM对于细胞治疗的显著临床改善至关重要。
LAG3(Lymphocyte-activation gene 3，LAG3或CD223)是一种淋巴细胞活化基因，属于Ig家族，主要表达于活化的T细胞、NK细胞、B细胞和浆细胞样树突状细胞，是免疫负调控因子。TIM3(T cell immunoglobulin-3，TIM3或CD366)是一种活化诱导的抑制性分子，表达广泛，在CD4+Th1细胞、CD8+Tc1细胞、Th17细胞、调节性T细胞、树突状细胞、NK细胞和单核细胞中都有表达，参与慢性病毒感染和癌症的免疫耐受和T细胞耗竭。LAG3和TIM3可作为T细胞耗竭的标志物。
发明内容
本申请提供了只表达mbIL15的CAR-T细胞，以分子量较小的外源蛋白却依然实现了优异的增殖和杀伤活性。
本申请一方面提供了一种工程化的免疫细胞，其共表达嵌合抗原受体(CAR)和膜结合的IL15(mbIL15)的全部或其功能部分。
本申请的第二方面提供了一种融合蛋白，包含与跨膜蛋白的全部或一部分融合的IL15的全部或其功能部分，所述跨膜蛋白选自NKG2D、OX40、2B4或EpCAM。
本申请的第三方面提供了一种分离的核酸分子，其包含编码上述任一项所述的免疫细胞上共表达的CAR和mbIL15的核酸分子，或包含编码上述任一项所述的融合蛋白的核酸分子；优选地，所述核酸分子为DNA或RNA。
本申请的第四方面提供了一种载体(vector)，其包含上述核酸分子。
本申请的第五方面提供了一种制备上述任一项所述的免疫细胞的方法，其包括将所述核酸分子引入到所述免疫细胞中。
本申请的第六方面提供了一种药物组合物，其包含上述任一项所述的免疫细胞，上述任一项所述的融合蛋白，或上述核酸分子。
本申请的第七方面提供了上述任一项所述的免疫细胞，所述方法制备获得的产品，或所述药物组合物用于制备在个体中治疗肿瘤的药物的用途。
本申请的第八方面提供了一种在有需要的个体中治疗肿瘤的方法，包括向所述个体施用上述任一项的免疫细胞，所述方法制备获得的产品，或所述药物组合物。
本申请的第九方面提供了上述任一项所述的免疫细胞，所述方法制备获得的产品，或所述药物组合物，用于在个体中预防和/或治疗肿瘤的用途。
本申请公开的mbIL15装甲的CAR-T细胞与现有mbIL15-IL15Rα复合物装甲的CAR-T细胞相比，至少具有以下一种或多种优势：(1)IL15表达于细胞表面，并被限制在肿瘤微环境内；(2)具有较高的CAR表达阳性率或细胞增殖倍数，和表达稳定性；(3)扩增的T细胞中TCM比例更高；(4)针对特定的抗原表位，具有高度的特异性；(5)具有优异的 肿瘤细胞杀伤与抑制能力，包括但不限于：在长期杀伤试验或中低剂量时，显示更优异的肿瘤杀伤作用；(6)副作用较低，特异性强，更加安全。
附图说明
除非本申请另外定义，与本申请相关的科学和技术术语应具有本领域普通技术人员所理解的含义。
图1.mbIL15装甲的靶向人GCC的二代嵌合抗原受体逆转录病毒质粒简图。
图2.激活后第8天和第11天，mbIL15装甲的CAR在T细胞表面的表达情况。
图3.激活后第8天和第11天，mbIL15 CAR-T细胞中各亚群的比例。A.激活后第8天，CD8+细胞群和CD4+细胞群的检测结果；B.激活后第8天，CD8+细胞群中TSCM、TCM、TE和TEM亚群检测结果；C.激活后第8天，CD4+细胞群中TSCM、TCM、TE和TEM亚群检测结果；D.激活后第8天，CD8+细胞群中抑制性受体TIM3和LAG3的检测结果；E.激活后第8天，CD4+亚群中抑制性受体TIM3和LAG3的检测结果。F.激活后第11天，CD8+细胞群和CD4+细胞群的检测结果；G.激活后第11天，CD8+细胞群中TSCM、TCM、TE和TEM亚群检测结果；H.激活后第11天，CD4+细胞群中TSCM、TCM、TE和TEM亚群检测结果；I.激活后第11天，CD8+细胞群中抑制性受体TIM3和LAG3的检测结果。J.激活后第11天，CD4+细胞群中抑制性受体TIM3和LAG3的检测结果。
图4.mbIL15 CAR-T细胞对LS174T-luc细胞杀伤活性检测。A.激活后第8天，mbIL15 CAR-T细胞在不同的效靶比条件下对LS174T-luc细胞的杀伤活性结果；B.激活后第11天，mbIL15 CAR-T细胞对LS174T-luc细胞的杀伤活性结果。
图5.激活后第11天，mbIL15 CAR-T细胞的多轮杀伤活性检测。A.mbIL15 CAR-T细胞对HT55细胞杀伤活性的检测结果；B.mbIL15 CAR-T细胞对LS174T-luc细胞进行多轮杀伤活性的检测结果。
图6.mbIL15装甲的靶向人DLL3的二代嵌合抗原受体逆转录病毒质粒简图。
图7.激活后第8天和第10天，mbIL15装甲的CAR在T细胞表面的表达情况。A.靶向DLL3的CAR表达情况；B.IL15在细胞膜表面的表达情况。
图8.激活第11天mbIL15DLL3 CAR-T细胞和激活后第10天冻存复苏的mbIL15 DLL3CAR-T细胞的多轮杀伤活性检测。A和B分别为激活第11天mbIL15 DLL3 CAR-T细胞对SHP77-luc和NCI-H2171-luc细胞的多轮杀伤活性检测；C和D分别为激活第10天冻存复苏的mbIL15 DLL3 CAR-T细胞对SHP-77-luc和NCI-H2171-luc细胞的多轮杀伤活性检测。
图9.激活后第10天冻存复苏的mbIL15 DLL3 CAR-T细胞对SHP-77细胞小鼠皮下肿瘤模型抗肿瘤药效的检测。A.各治疗组小鼠体内肿瘤体积变化；B.各治疗组小鼠体重变化；C.各治疗组小鼠生存曲线。
图10.mbIL15装甲的靶向人CD19的二代嵌合抗原受体逆转录病毒质粒简图。
图11.激活后第10天，mbIL15 CD19 CAR-T细胞的多轮杀伤活性检测。
发明的详细描述
术语定义和说明
除非本申请另外定义，与本申请相关的科学和技术术语应具有本领域普通技术人员所理解的含义。
此外，除非本文另有说明，本文单数形式的术语应包括复数形式，复数形式的术语应包括单数形式。更具体地，如在本说明书和所附权利要求中所使用的，除非另外明确指出，否则单数形式“一种”和“这种”包括复数指示物。
本文中使用的“包括”、“包含”和“具有”之间可互换使用，旨在表示方案的包含性，意味着所述方案可存在除所列出的元素之外的其他元素。同时应当理解，在本文中使用“包括”、“包含”和“具有”描述，也提供“由……组成”方案。示例性地，“一种组合物，包括A和B”，应当理解为以下技术方案：由A和B组成的组合物，以及除A和B外，还含有其他组分的组合物，均落入前述“一种组合物”的范围内。
本文中使用的“和/或”在本文使用时，包括“和”、“或”和“由所属术语链接的要素的全部或任何其他组合”的含义。
本文中使用的“免疫细胞”是指与宿主免疫应答或炎症应答有关的任何细胞，包括但不限于T细胞、NK细胞、NKT细胞、经其他技术诱导生成的T细胞和NK细胞和其他免疫调节细胞。
本文中使用的“T细胞”表示T淋巴细胞，属于称为淋巴细胞的白细胞的一个组，并参与体液或细胞-介导的免疫。通过其细胞表面上特殊标记物比如T细胞受体(TCR)的存在，T细胞可与其他淋巴细胞类型比如B细胞和天然杀伤细胞(NK细胞)区分开。鉴定T细胞的其他标记物包括CD1a、CD3、CD4、CD8和被技术人员所知道的可能与T细胞状态和/或功能关联的其他标记物。
本文中使用的“自然杀伤细胞”(“NK细胞”)是指免疫系统的一种细胞毒性淋巴细胞。NK细胞对病毒感染的细胞提供迅速响应并且响应于转化细胞。通常来说，免疫细胞检测来自病原体的由被感染细胞表面上的主要组织相容性复合体(Major HistocompatibilityComplex，MHC)分子呈递的肽，引发细胞因子释放、引起溶胞(lysis)或凋亡。然而，NK细胞是独特的，因为其具有识别应激细胞的能力，而不管来自病原体的肽是否存在于MHC分子上。因为它们不需要预先活化以杀死目标的最初见解，故它们被命名为“自然杀伤细胞”。NK细胞是大颗粒淋巴细胞(large granular lymphocyte，LGL)，并且已知在骨髓(然后它们在此进入循环)中分化并成熟。
本文中使用的“NKT细胞”表示天然杀伤T细胞，指表达T细胞受体(TCR)的CD-ld限制性T细胞。不同于检测通过常规主要组织相容性复合物(MHC)分子呈递的肽抗原的常规T细胞，NKT细胞识别通过CD1d(非经典MHC分子)呈递的脂质抗原。目前已鉴定出两种NKT细胞。不变或I型NKT细胞表达非常有限的TCR库——典型的a链(人类中为Va24-Jal 8)，其与β链(人类中为ΛíβΙ1)有限的谱相关。第二NKT细胞群称为非经典型或非变异型II型NKT细胞，其展示出更多异质性TCRαβ使用。目前认为I型NKT细胞适合于免疫疗法。适应性或不变(I型)NKT细胞可通过下述至少一种或多种标志物的表达来鉴定：TCR Va24Jal 8、Vbl  l、CDl d、CD3、CD4、CD8、aGalCer、CD161和CD56。
本文中使用的“白细胞介素15”(“IL15”)是指调节T和NK细胞活化和增殖的细胞因子。该细胞因子和白介素2共有许多生物活性。发现它们结合共同的受体亚基，并且可竞争相同的受体，并因此负调节彼此的活性。显示CD8+记忆细胞的数量由IL-15和IL-2之间的平衡控制。这种细胞因子诱导JAK激酶的活化以及转录活化剂STAT3、STAT5和STAT6的磷酸化和活化，并且可通过STAT6的转录活化活性来提高凋亡抑制剂BCL2L1/BCL-x(L)的表达，并因此防止凋亡。
IL-15的“功能部分”(“生物活性部分”)是指保持全长或成熟IL-15的一种或更多种功能的IL-15的一部分。这样的功能包括促进免疫细胞存活、调节NK细胞和T细胞活化和增殖以及维持来自造血干细胞的免疫细胞发育。
如本领域技术人员将理解的，多种IL-15分子的序列是本领域已知的。在一个方面，IL-15是野生型IL-15。在一些方面，IL-15是哺乳类IL-15(例如，人(Homo sapiens)白介素15(IL15)、转录物变体3、mRNA、NCBI参考序列：NP_000576.1；家犬(canis lupusfamiliaris)白介素15、mRNA、NCBI参考序列：NM_001197188.1；家猫(Felis catus)白介素15(IL15)、mRNA、NCBI参考序列：NM_001009207.1)。“哺乳类”或“哺乳动物”的实例包括灵长类(例如，人)、犬科动物、猫科动物、啮齿动物、猪、反刍动物等。具体实例包括人、狗、猫、马、牛、绵羊、山羊、兔、豚鼠、大鼠和小鼠。在一个具体方面，哺乳类IL-15是人IL-15。
本文中使用的“跨膜蛋白”或“膜蛋白”是位于膜(例如生物膜(例如，生物膜如细胞膜)的磷脂双层)处和/或膜内的蛋白质。膜蛋白使膜能够进行其独特的活动。附着到膜上的蛋白质的补足物(complement)根据细胞类型和亚细胞定位而不同。一些蛋白质仅结合到膜表面上，而另一些蛋白质具有一个或更多个埋入膜中的区域和/或膜一侧或两侧上的结构域。细胞外膜表面上的蛋白质结构域通常参与细胞-细胞信号传导或相互作用。位于膜的胞质表面的结构域具有广泛的功能，从锚定细胞骨架蛋白到膜至触发细胞内信号传导途径。本文中膜内的区域被称为“跨膜结构域”，特别是形成通道和孔使分子移动穿过膜的那些。“跨膜结构域”是三维蛋白质结构，其在膜(例如，囊泡(例如细胞)的膜)中是热力学稳定的。跨膜结构域的实例包括单个α螺旋、几个跨膜α螺旋的稳定复合物、跨膜β桶形结构(betabarrel)、短杆菌肽A的β螺旋或任何其他结构。跨膜螺旋通常为约20个氨基酸长。
通常来说，基于膜-蛋白质相互作用的性质，将膜蛋白分为两大类，整合的(内在的)和外周的(外在的)。大多数生物膜包含两种类型的膜蛋白。
整合膜蛋白(也称为内在蛋白)具有嵌入磷脂双层的一个或更多个片段。整合膜蛋白包括跨膜蛋白和脂质锚定蛋白。大多数整合蛋白包含具有疏水性侧链的残基，所述疏水性侧链与膜磷脂的脂肪酰基相互作用，从而将蛋白锚定到膜上。大多数整合蛋白跨越整个磷脂双层。这些跨膜蛋白包含一个或更多个跨膜结构域以及4至几百个残基长的延伸到双层每侧上的水性介质中的结构域。通常来说，跨膜结构域是一个或更多个(例如，约1、2、3、4、5、6、7、8、9、10或更多个)α螺旋和/或β链(βstrand)。跨膜α螺旋结构域通常通过与双层内部脂质的疏水相互作用以及还可能通过与磷脂(例如，血型糖蛋白)的极性头部基团的离子相互作用而嵌入到膜中。β链的结构通常是跨膜桶形结构(例如，孔蛋白)的形式。一些整合蛋白通过共价结合的脂肪酸锚定到膜小叶(leaflet)之一。在这些蛋白质中，结合的脂肪酸嵌入膜中，但多 肽链不进入磷脂双层。一些细胞表面蛋白通过连接到C末端的复合糖基化磷脂(例如，糖基磷脂酰肌醇、碱性磷酸酶)锚定到质膜的脂膜外面(exoplasmic face)。一些胞质蛋白通过共价连接到C末端附近的半胱氨酸的烃部分(例如，异戊二烯基、法尼基和牻牛儿基牻牛儿基基团)锚定到膜的胞质面。在另一组脂质锚定胞质蛋白中，脂肪酰基(例如，肉豆蔻酸酯或棕榈酸酯)通过酰胺键与N-末端甘氨酸残基连接。
外周膜蛋白或外源性蛋白不与磷脂双层的疏水核相互作用。相反，它们通常通过与整合膜蛋白的相互作用间接地或者通过与脂质极性头部基团的相互作用直接地结合到膜上。定位于质膜的胞质面的外周蛋白包括红细胞中的细胞骨架蛋白血影蛋白和肌动蛋白以及酶蛋白激酶C。这种酶在胞质溶胶和质膜的胞质面之间穿梭并在信号转导中发挥作用。其他外周蛋白(包括细胞外基质的某些蛋白质)定位于质膜的外(脂膜外)表面。
跨膜蛋白的实例包括受体、配体、免疫球蛋白、血型糖蛋白或其组合。跨膜蛋白的具体实例包括CD8α、CD4、CD3ε、CD3γ、CD3δ、CD3ζ、CD28、CD137、FcεRIγ、T细胞受体(TCR，例如TCRα和/或TCRβ)、烟碱型乙酰胆碱受体、GABA受体、NKG2D、OX40、2B4、EpCAM或其组合。免疫球蛋白的具体实例包括IgG、IgA、IgM、IgE、IgD或其组合。血型糖蛋白的具体实例包括血型糖蛋白A、血型糖蛋白D或其组合。
本文术语“嵌合抗原受体(CAR)”是指经改造以在免疫效应细胞上表达并且特异性结合抗原的人工细胞表面受体，其包含至少(1)细胞外抗原结合结构域，例如抗体的可变重链或轻链，(2)锚定CAR进入免疫效应细胞的跨膜结构域，和(3)胞内信号传导结构域。CAR能够利用细胞外抗原结合结构域以非MHC限制性的方式将T细胞和其它免疫效应细胞重定向至所选择的靶标，例如癌细胞。
本文术语“信号肽”是指蛋白或多肽中用于引导所述蛋白或多肽进入分泌路径，转移至细胞膜和/或细胞表面的片段。
如本文所用，嵌合抗原受体的“跨膜(TM)区”是指能够使嵌合抗原受体在免疫细胞(例如淋巴细胞、NK细胞或NKT细胞)表面上表达，并且引导免疫细胞针对靶细胞的细胞应答的多肽结构。跨膜结构域可以是天然或合成的，也可以源自任何膜结合蛋白或跨膜蛋白。当嵌合抗原受体与靶抗原结合时，跨膜结构域能够进行信号传导。
如本文所用，嵌合抗原受体的“铰链区”一般是指作用为连接跨膜区和抗原结合区的任何寡肽或多肽。具体地，铰链区用来为抗原结合区提供更大的灵活性和可及性。铰链区可以全部或部分源自天然分子，如全部或部分源自CD8、CD4或CD28的胞外区，或全部或部分源自抗体恒定区。或者，铰链区可以是对应于天然存在的铰链序列的合成序列，或可以是完全合成的铰链序列。
如本文所用，术语“胞内信号传导结构域”是指转导效应子功能信号并指导细胞进行指定功能的蛋白质部分。胞内信号传导结构域负责在抗原结合结构域结合抗原以后的细胞内初级信号传递，从而导致免疫细胞和免疫反应的活化。换言之，胞内信号传导结构域负责活化其中表达CAR的免疫细胞的正常的效应功能的至少一种。示例性的胞内信号传导结构域包括CD3ζ。
如本文所用，“共刺激信号结合域”包括在抗原呈递细胞(例如，aAPC，树突状细胞，B 细胞等)上的分子，其特异性结合T细胞上的同源共刺激分子，从而提供一种信号，该信号除了通过例如TCR/CD3复合物与载有肽的MHC分子结合提供的主要信号外，还介导T细胞反应，包括但不限于增殖，激活，分化等。共刺激信号结合域可包括但不限于CD7，B7-1(CD80)，B7-2(CD86)，PD-L1，PD-L2，4-1BBL，OX40L，诱导型共刺激配体(ICOS-L)，细胞间粘附分子(ICAM)，CD30L，CD40，CD70，CD83，HLA-G，MICA，MICB，HVEM，淋巴毒素β受体，3/TR6，ILT3，ILT4，结合Toll配体受体和特异性结合B7-H3的配体的激动剂或抗体。共刺激信号结合域还包括，除其他外，与T细胞上存在的共刺激分子特异性结合的抗体，例如但不限于，CD27，CD28，4-1BB，OX40，CD30，CD40，PD-1，ICOS，淋巴细胞功能相关抗原-1(LFA-1)，CD2，CD7，LIGHT，NKG2C，B7-H3，以及一种与CD83特异性结合的配体。
本文中使用的“GUCY2c”是指哺乳动物鸟苷酸环化酶C(GUCY2C)，优选人GUCY2C蛋白。术语“GUCY2C”可以与“STAR”、“GUC2C”、“GCC”或“ST受体”互换使用。人GUCY2C的核苷酸序列公开为GenBank登录号NM_004963，人GUCY2C的氨基酸序列公开为GenBank登录号NP_004954。通常，自然发生的等位基因变体具有与GenBank登录号NP.Sub.-004954中描述的蛋白质至少95％、97％或99％相同的氨基酸序列。GUCY2C蛋白是一种跨膜细胞表面受体蛋白，在维持肠液、电解质稳态和细胞增殖等方面起着重要作用。
本文中使用的“DLL3(δ样配体3，Delta-Like Ligand 3)”为一种附着在细胞表面的单次跨膜蛋白，属于Notch配体家族中的一员。人DLL3基因定位于染色体19q13，其开放阅读框长度约为1800bp。人DLL3蛋白由619个氨基酸组成，完整结构包含1个DSL结构域、1个胞内结构域和6个表皮生长因子样结构域。胞外结构域N端的DSL基因序列在配体家族中高度保守，是与Notch受体结合所必须的功能结构域。DLL3胞内结构域较短，其功能尚不清楚。研究发现DLL3在SCLC和其他神经内分泌肿瘤中高表达，而在正常组织中很少表达。DLL3的激活可以发挥促癌或抑癌作用。DLL3在人类癌症中广泛表达，包括小细胞肺癌、神经胶质瘤、胰腺癌、黑色素瘤、乳腺癌、垂体瘤、子宫内膜瘤、急性髓系白血病，肝癌、膀胱癌、结肠癌、前列腺癌、肾癌和食道癌等。
本文中使用的“CD19”指分化群19蛋白(the Cluster of Differentiation 19protein,CD19)，其是在白血病前体细胞上可检测到的抗原决定簇。CD19在大多数B谱系癌，例如急性淋巴母细胞白血病、慢性淋巴细胞白血病和非霍奇金淋巴瘤上表达。
本文中使用的“抗原结合片段”按最广义使用，是指特异性结合抗原的片段。示例性地，抗原结合分子包括但不限于抗体或抗体模拟物。“抗体模拟物”是指能够与抗原特异性结合，但与抗体结构无关的有机化合物或结合域，示例性地，抗体模拟物包括但不限于affibody、affitin、affilin、经设计的锚蛋白重复蛋白(DARPin)、核酸适体或Kunitz型结构域肽。
本文中使用的“抗体”按最广义使用，是指包含来自免疫球蛋白重链可变区的足够序列和/或来自免疫球蛋白轻链可变区的足够序列，从而能够特异性结合至抗原的多肽或多肽组合。本文“抗体”涵盖各种形式和各种结构，只要它们展现出期望的抗原结合活性。本文“抗体”包括具有移植的互补决定区(CDR)或CDR衍生物的替代蛋白质支架或人工支架。此类支架包括抗体衍生的支架(其包含引入以例如稳定化抗体三维结构的突变)以及包含例如生物相容性聚合物的全合成支架。参见，例如Korndorfer et al.,2003,Proteins:Structure,Function,and  Bioinformatics,53(1):121-129(2003)；Roque et al.,Biotechnol.Prog.20:639-654(2004)。此类支架还可以包括非抗体衍生的支架，例如本领域已知可用于移植CDR的支架蛋白，包括但不限于肌腱蛋白、纤连蛋白、肽适体等。
术语“抗体”包括完整抗体及其任何抗原结合片段(即“抗原结合部分”)或单链。“抗体”是指包含通过二硫键互相连接在一起的至少两条重(H)链和两条轻(L)链的糖蛋白，或其抗原结合部分。每条重链由重链可变区(在此缩写为VH)和重链恒定区组成。重链恒定区由三个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(在此缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可进一步再分为高变区，称为互补决定区(CDR)，CDR散布在被称为构架区(FR)的更加保守的区域中。每个VH和VL，均由三个CDR和四个FR组成，它们从氨基端向羧基端以如下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。重链和轻链的可变区含有可与抗原相互作用的结合结构域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合，该宿主组织或因子包括免疫系统的各种细胞(例如效应细胞)和经典补体系统的第一成分(C1q)。由于免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将本文“免疫球蛋白”分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4，IgA可分为IgA1和IgA2。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
本文“抗体”还包括不包含轻链的抗体，例如，由单峰驼(Camelus dromedarius)、双峰驼(Camelus bactrianus)、大羊驼(Lama glama)、原驼(Lama guanicoe)和羊驼(Vicugna pacos)等骆驼科动物产生的重链抗体(heavy-chain antibodies,HCAbs)以及在鲨等软骨鱼纲中发现的免疫球蛋白新抗原受体(Ig new antigen receptor,IgNAR)。
如本文所用，术语“重链抗体”是指缺乏常规抗体的轻链的抗体。该术语具体包括但不限于在不存在CH1结构域的情况下包含VH抗原结合结构域以及CH2和CH3恒定结构域的同型二聚体抗体。
如本文所用，术语“纳米抗体”是指骆驼体内存在天然的缺失轻链的重链抗体，克隆其可变区可以得到只有重链可变区组成的单域抗体，也称为VHH(Variable domain of heavy chain of heavy chain antibody)，它是最小的功能性抗原结合片段。
本文中使用的“纳米抗体(nanobody)”、“单域抗体”(single domain antibody,sdAb)具有相同的含义并可互换使用，是指克隆重链抗体的可变区，构建仅由一个重链可变区组成的单域抗体，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的重链抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体。
关于“重链抗体”和“纳米抗体”的进一步描述可参见：Hamers-Casterman等，Nature.1993；363；446-8；Muyldermans的综述文章(Reviews inMolecular Biotechnology 74：277-302，2001)；以及以下专利申请，其被作为一般背景技术提及：WO 94/04678，WO 95/04079和WO 96/34103；WO94/25591，WO 99/37681，WO 00/40968，WO 00/43507，WO 00/65057，WO 01/40310，WO 01/44301，EP 1134231和WO 02/48193；WO97/49805，WO 01/21817，WO 03/035694，WO 03/054016和WO 03/055527；WO 03/050531；WO 01/90190；WO03/025020； 以及WO 04/041867，WO 04/041862，WO 04/041865，WO 04/041863，WO 04/062551，WO 05/044858，WO 06/40153，WO 06/079372，WO 06/122786，WO 06/122787和WO 06/122825以及这些申请中提到的其他现有技术。
本文“抗体”可以来源于任何动物，包括但不限于人和非人动物，所述非人动物可选自灵长类动物、哺乳动物、啮齿动物和脊椎动物，例如骆驼科动物、大羊驼、原鸵、羊驼、羊、兔、小鼠、大鼠或软骨鱼纲(例如鲨)。
本文中使用的“抗原结合片段”和“抗体片段”在本文中可互换使用，其不具备完整抗体的全部结构，仅包含完整抗体的局部或局部的变体，所述局部或局部的变体具备结合抗原的能力。本文“抗原结合片段”或“抗体片段”包括但不限于Fab、Fab’、Fab’-SH、F(ab’)2、Fd、Fv、scFv、双抗体(diabody)和单域抗体。
本文术语“可变区”是指抗体重链或轻链中牵涉使抗体结合抗原的区域，“重链可变区”与“VH”、“HCVR”可互换使用，“轻链可变区”与“VL”、“LCVR”可互换使用。天然抗体的重链和轻链的可变域(分别是VH和VL)一般具有相似的结构,每个域包含四个保守的框架区(FR)和三个高变区(HVR)。参见例如Kindt et al.,Kuby Immunology,6th ed.,W.H.Freeman and Co.,p.91(2007)。单个VH或VL域可足以赋予抗原结合特异性。本文术语“互补决定区”与“CDR”可互换使用，通常指重链可变区(VH)或轻链可变区(VL)的高变区(HVR)，该部位因在空间结构上可与抗原表位形成精密的互补，故又称为互补决定区，其中，重链可变区CDR可缩写为HCDR，轻链可变区CDR可缩写为LCDR。本术语“构架区”或“FR区”可互换，是指抗体重链可变区或轻链可变区中除CDR以外的那些氨基酸残基。通常典型的抗体可变区由4个FR区和3个CDR区按以下顺序组成：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
对于CDR的进一步描述，参考Kabat等人,J.Biol.Chem.,252:6609-6616(1977)；Kabat等人,美国卫生与公共服务部，“Sequences of proteins of immunological interest”(1991)；Chothia等人,J.Mol.Biol.196:901-917(1987)；Al-Lazikani B.等人,J.Mol.Biol.,273:927-948(1997)；MacCallum等人,J.Mol.Biol.262:732-745(1996)；Abhinandan和Martin,Mol.Immunol.,45:3832-3839(2008)；Lefranc M.P.等人,Dev.Comp.Immunol.,27:55-77(2003)；以及Honegger和Plückthun,J.Mol.Biol.,309:657-670(2001)。本文“CDR”可由本领域公知的方式加以标注和定义，包括但不限于Kabat编号系统、Chothia编号系统或IMGT编号系统，使用的工具网站包括但不限于AbRSA网站(http://cao.labshare.cn/AbRSA/cdrs.php)、abYsis网站(www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)和IMGT网站(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results)。本文CDR包括不同定义方式的氨基酸残基的重叠(overlap)和子集。
本文术语“保守氨基酸”通常是指属于同一类或具有类似特征(例如电荷、侧链大小、疏水性、亲水性、主链构象和刚性)的氨基酸。示例性地，下述每组内的氨基酸属于彼此的保守氨基酸残基，组内氨基酸残基的替换属于保守氨基酸的替换：
示例性地，以下六组是被认为是互为保守性置换的氨基酸的实例：
1)丙氨酸(A)、丝氨酸(S)、苏氨酸(T)；
2)天冬氨酸(D)、谷氨酸(E)；
3)天冬酰胺(N)、谷氨酰胺(Q)；
4)精氨酸(R)、赖氨酸(K)、组氨酸(H)；
5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；和
6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。
本文术语“同一性”可通过以下方式计算获得：为确定两个氨基酸序列或两个核酸序列的“同一性”百分数，将所述序列出于最佳比较目的比对(例如，可以为最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
考虑到为最佳比对这两个序列而需要引入的空位的数目和每个空位的长度，两个序列之间的同一性百分数随所述序列共有的相同位置变化而变化。
本文术语“核酸”包括包含核苷酸的聚合物的任何化合物和/或物质。每个核苷酸由碱基，特别是嘌呤或嘧啶碱基(即胞嘧啶(C)、鸟嘌呤(G)、腺嘌呤(A)、胸腺嘧啶(T)或尿嘧啶(U))、糖(即脱氧核糖或核糖)和磷酸基团组成。通常，核酸分子由碱基的序列描述，由此所述碱基代表核酸分子的一级结构(线性结构)。碱基的序列通常表示为5′至3′。在本文中，术语核酸分子涵盖脱氧核糖核酸(DNA)，包括例如互补DNA(cDNA)和基因组DNA、核糖核酸(RNA)，特别是信使RNA(mRNA)、DNA或RNA的合成形式，以及包含两种或更多种这些分子的混合的聚合物。核酸分子可以是线性的或环状的。此外，术语核酸分子包括有义链和反义链二者，以及单链和双链形式。而且，本文所述的核酸分子可含有天然存在的或非天然存在的核苷酸。非天然存在的核苷酸的例子包括具有衍生的糖或磷酸骨架键合或化学修饰的残基的修饰的核苷酸碱基。核酸分子还涵盖DNA和RNA分子，其适合作为载体用于在体外和/或体内，例如在宿主或患者中，直接表达本申请的构建体。此类DNA(例如cDNA)或RNA(例如mRNA)载体可以是未修饰的或修饰的。例如，可以对mRNA进行化学修饰以增强RNA载体的稳定性和/或被编码分子的表达，从而可以将mRNA注入到受试者内以在体内产生抗体(参见例如Stadler等人，Nature Medicine 2017，published online 2017年6月12日，doi：10.1038/nm.4356或EP 2 101 823 B1)。本文所述DNA或RNA可用于将核酸引入细胞内，例如通过脂质体如脂质体聚合物(LPR)、脂质体复合物(LP)、纳米颗粒(NP)或脂质纳米颗粒(LNP)等材料包封核酸，以将所述DNA或RNA引入细胞中。所述RNA分子可以是线性的，也可以是环状的，例如使用LNP将所述线性或环状RNA分子递送到细胞或受试者内部，利用受试者的表达系统，表达相应的融合蛋白或CAR，以在体内生产表达CAR的免疫细胞。本文“分离的”核酸指已经与其天然环境的组分分开的核酸分子。分离的核酸包括在下述细胞中含有的核酸分子，所述细胞通常含有该核酸分子，但该核酸分子存在于染色体外或存在于不同于其天然染色体位置的染色体位置处。
本文术语“载体”是指能够扩增与其连接的另一个核酸的核酸分子。该术语包括作为自我复制型核酸结构的载体以及整合入已引入该载体的宿主细胞的基因组中的载体。某些载体能够指导与它们可操作连接的核酸的表达。这样的载体在本文中称为“表达载体”。
本文术语“药物组合物”是指这样的制剂，其以允许包含在其中的活性成分的生物学活性 有效的形式存在，并且不含有对施用所述药物组合物的受试者具有不可接受的毒性的另外的成分。
本文术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如癌症的进展。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
本文中使用的“个体”是指动物，并且在一个具体方面是指哺乳动物。哺乳动物的实例包括灵长类、犬科动物、猫科动物、啮齿动物等。具体实例包括人、狗、猫、马、牛、绵羊、山羊、兔、豚鼠、大鼠和小鼠。术语“有需要的个体”是指如由研究者、兽医、医师或其他临床医生确定的需要治疗或预防的个体。在一个实施方案中，有需要的个体是哺乳动物，例如人。
具体实施方式
本申请公开的mbIL15装甲的CAR-T细胞与现有mbIL15-IL15Rα复合物装甲的CAR-T细胞相比，至少具有以下一种或多种优势：(1)IL15表达于细胞表面，并被限制在肿瘤微环境内；(2)具有较高的CAR表达阳性率或细胞增殖倍数，和表达稳定性；(3)扩增的T细胞中TCM比例更高；(4)针对特定的抗原表位，具有高度的特异性；(5)具有优异的肿瘤细胞杀伤与抑制能力，包括但不限于：在长期杀伤试验或中低剂量时，显示更优异的肿瘤杀伤作用；(6)副作用较低，特异性强，更加安全。
本申请一方面提供了一种工程化的免疫细胞，其共表达嵌合抗原受体(CAR)和膜结合的IL15(mbIL15)的全部或其功能部分。
在一些实施方案中，所述IL15的全部或其功能部分与跨膜蛋白的全部或一部分融合，所述跨膜蛋白选自NKG2D、OX40、2B4或EpCAM。
优选地，所述NKG2D包含与SEQ ID NO：15或16具有至少90％同一性的氨基酸序列。
优选地，所述OX40包含与SEQ ID NO：17具有至少90％同一性的氨基酸序列。
优选地，所述2B4包含与SEQ ID NO：18具有至少90％同一性的氨基酸序列。
优选地，所述EpCAM包含与SEQ ID NO：19或20具有至少90％同一性的氨基酸序列。
在一些实施方案中，所述IL15的全部或其功能部分与跨膜蛋白的全部或一部分通过连接子融合
在一些实施方案中，所述连接子为CD8α，并且优选的连接子包含与SEQ ID NO：14具有至少90％同一性的氨基酸序列。
在一些实施方案中，所述IL15的全部或其功能部分包含与SEQ ID NO：1或SEQ ID NO：47具有至少90％同一性的氨基酸序列。
在一些实施方案中，所述mbIL15包含与SEQ ID NO：27-32和48中的任一项具有至少90％同一性的氨基酸序列。
在一些实施方案中，所述CAR至少包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域。
在一些具体的实施方案中，所述细胞外抗原结合结构域包含靶向GCC的纳米抗体(VHH)或其抗原结合片段，所述纳米抗体或其抗原结合片段包含CDR1、CDR2和CDR3。
在优选的实施方案中，所述CDR1、CDR2和CDR3分别选自SEQ ID NO：2所示的VHH的CDR1、CDR2和CDR3。
优选地，所述CDR1、CDR2和CDR3分别含有SEQ ID NO：3-5所示的氨基酸序列。优选地，所述纳米抗体或其抗原结合片段包含SEQ ID NO：2所示的氨基酸序列，或者包含与SEQ ID NO：2所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，或与SEQ ID NO：2所示的氨基酸序列相比具有至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的氨基酸序列。
在一些具体的实施方案中，所述细胞外抗原结合结构域包含靶向DLL3的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3。
优选地，所述HCDR1、HCDR2和HCDR3分别选自SEQ ID NO：36所示的VH中包含的HCDR1、HCDR2和HCDR3；并且所述LCDR1、LCDR2、LCDR3分别选自SEQ ID NO：37所示的VL中包含的LCDR1、LCDR2和LCDR3。
优选地，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：39-44所示的氨基酸序列。
优选地，所述抗体或其抗原结合片段包含SEQ ID NO：36和SEQ ID NO：37所示的氨基酸序列，或者包含与SEQ ID NO：36和37所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，或与SEQ ID NO：36和37所示的氨基酸序列相比具有至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的氨基酸序列。
在一些具体的实施方案中，所述细胞外抗原结合结构域包含靶向CD19的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3。
优选地，所述HCDR1、HCDR2和HCDR3分别选自SEQ ID NO：55所示的VH中包含的HCDR1、HCDR2和HCDR3；并且所述LCDR1、LCDR2和LCDR3分别选自SEQ ID NO：56所示的VL中包含的LCDR1、LCDR2和LCDR3。
优选地，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：58-63所示的氨基酸序列。
优选地，所述抗体或其抗原结合片段包含SEQ ID NO：55和SEQ ID NO：56所示的氨 基酸序列，或者包含与SEQ ID NO：55和56所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，或与SEQ ID NO：55和56所示的氨基酸序列相比具有至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的氨基酸序列。
在一些实施方案中，所述突变选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
在一些实施方案中，所述免疫细胞选自T细胞、NK细胞、NKT细胞或经其他技术诱导生成的T细胞和NK细胞。
本申请的第二方面提供了一种融合蛋白，包含与跨膜蛋白的全部或一部分融合的IL15的全部或功能部分，所述跨膜蛋白选自NKG2D、OX40、2B4或EpCAM。
在一些实施方案中，所述融合蛋白还包含CAR，优选上述CAR。
优选地，所述NKG2D包含与SEQ ID NO：15或16具有至少90％同一性的氨基酸序列。
优选地，所述OX40包含与SEQ ID NO：17具有至少90％同一性的氨基酸序列。
优选地，所述2B4包含与SEQ ID NO：18具有至少90％同一性的氨基酸序列。
优选地，所述EpCAM包含与SEQ ID NO：19或20具有至少90％同一性的氨基酸序列。
在一些实施方案中，所述IL15的全部或其功能部分与跨膜蛋白的全部或一部分通过连接子融合。
在一些实施方案中，所述连接子为CD8α，并且优选的连接子包含与SEQ ID NO：14具有至少90％同一性的氨基酸序列。
优选地，所述IL15的全部或其功能部分包含与SEQ ID NO：1或SEQ ID NO：47具有至少90％同一性的氨基酸序列。
优选地，所述融合蛋白包含与SEQ ID NO：27-32和48中的任一项具有至少90％同一性的氨基酸序列。
本申请的第三方面提供了一种分离的核酸分子，其包含编码上述任一项所述的免疫细胞上共表达的CAR和mbIL15的核酸分子，或包含编码上述任一项所述的融合蛋白的核酸分子；优选地，所述核酸分子为DNA或RNA。
本申请的第四方面提供了一种载体，其包含上述核酸分子。优选地，所述载体是病毒载体。
本申请的第五方面提供了一种制备上述任一项所述的免疫细胞的方法，其包括将所述核酸分子引入到所述免疫细胞中。
本申请的第六方面提供了一种药物组合物，其包含上述任一项所述的免疫细胞，上述任一项所述的融合蛋白，或上述核酸分子。
本申请的第七方面提供了上述任一项所述的免疫细胞，所述方法制备获得的产品，或所述药物组合物用于制备在个体中治疗肿瘤的药物的用途。任选地，所述肿瘤选自血液瘤或实体瘤，任选地，所述血液瘤选自B细胞淋巴瘤，表达CD19的肿瘤等；任选地，所述实体瘤 选自非小细胞肺癌，胃癌，结肠腺癌，结肠癌，小细胞肺癌，表达GCC或DLL3的肿瘤等。
本申请的第八方面提供了一种治疗肿瘤的方法，包括向有需要的个体施用上述任一项的免疫细胞，所述方法制备获得的产品，或所述药物组合物。任选地，所述肿瘤选自血液瘤或实体瘤，任选地，所述血液瘤选自B细胞淋巴瘤，表达CD19的肿瘤等；任选地，所述实体瘤选自非小细胞肺癌，胃癌，结肠腺癌，结肠癌，小细胞肺癌，表达GCC或DLL3的肿瘤等。
本申请的第九方面提供上述任一项的免疫细胞，所述方法制备获得的产品，或所述药物组合物，用于在个体中预防和/或治疗肿瘤的用途。任选地，所述肿瘤选自血液瘤或实体瘤，任选地，所述血液瘤选自B细胞淋巴瘤，表达CD19的肿瘤等；任选地，所述实体瘤选自非小细胞肺癌，胃癌，结肠腺癌，结肠癌，小细胞肺癌，表达GCC或DLL3的肿瘤等。
实施例
下面结合具体实施例来进一步描述本申请，本申请的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本申请实施例仅是范例性的，并不对本申请的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本申请的精神和范围下可以对本申请技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本申请的保护范围内。
实施例1 IL15装甲的表达二代嵌合抗原受体的T细胞(CAR-T细胞)的构建
本申请分别构建了mbIL15装甲的表达GCC嵌合抗原受体的T细胞：CAR-1、CAR-2、CAR-3、CAR-4、CAR-5、CAR-6和CAR-7。具体步骤如下：
1.1 质粒构建
本实施例采用的VHH抗体(variable domain of heavy chain of heavy-chain antibody，VHH antibody)为靶向GCC的抗体，氨基酸序列如SED ID NO：2所示，具有SEQ ID NO：3、4和5所示的决定簇互补区(Complementarity Determining Region，简称CDR)CDR1、CDR2和CDR3。上述抗体序列编号使用IMGT编号系统。所采用的嵌合抗原受体为二代的嵌合抗原受体，具有CD28铰链区(SEQ ID NO：7)、CD28跨膜结构域(SEQ ID NO：8)，CD28胞内共刺激域(SEQ ID NO：9)、CD3ζ信号转导结构域(SEQ ID NO：10、11或12)、裂解肽(SEQ ID NO：13)及膜结合型IL15。IL15氨基酸序列见SEQ ID NO：1，IL15与跨膜域间的连接子序列见SEQ ID NO：14。膜结合型IL15的跨膜域分别来自NKG2D(SEQ ID NO：15或16)、OX40(SEQ ID NO：17)、2B4(SEQ ID NO：18)或者EpCAM(SEQ ID NO：19或33)，其中mbIL15-IL15Ra(SEQ ID NO：34)来源于专利WO 2021/062281。构建的CAR结构和各元件的氨基酸序列见表1和表2。
表1.膜结合型IL-15装甲的二代嵌合抗原受体结构

表2.膜结合型IL-15装甲的二代嵌合抗原受体各元件序列表

采用本领域常规分子生物学方法，进行质粒构建。以逆转录病毒载体模板，参照图1所示的质粒结构简图，构建了表达二代嵌合抗原受体的逆转录病毒质粒P1、P2、P3、P4、P5、P6和P7。每个质粒从5`端到3`端依次包含：CD8α信号肽、VHH、CD28铰链区、CD28跨膜结构域，CD28胞内共刺激域、CD3ζ信号转导结构域及mbIL15。分别合成相应的多聚核苷酸插入序列，使用限制性内切酶EcoRI(Thermo，Cat#FD0274)和SalI(Thermo，Cat#FD0644)对逆转录病毒载体进行酶切，琼脂糖凝胶电泳回收纯化线性化载体。将以上步骤中合成的多聚核苷酸序列分别和线性化载体通过重组酶5×In-FusionHD酶(TaKaRa，Cat#ST0344)连接，配制的反应体系如下：2μl合成多聚核苷酸片段(50ng/μl)，1μl线性化质粒(50ng/μl)，2μl5×HD InFusion酶，5μl ddH2O；用移液器吹打混匀，短暂离心并置于50℃反应15min。将10μl重组反应产物加入到100μl细菌感受态细胞stbl3(Frdbio，Cat#MCC0910)中，在冰上放置5min，将转化菌液均匀涂布在含有50μg/ml卡那霉素的LB平板上，在恒温培养箱中倒置培养12～16h。随机从每个平板上挑取3～5个克隆进行测序鉴定。将测序正确的菌液转接于100ml含50μg/ml卡那霉素的LB液体培养基中，37℃培养过夜，用MN无内毒素质粒中抽试剂盒(MN，Cat#740420.50)抽提质粒，定量后用无内毒素超纯水稀释至1000ng/μl。插入的CAR氨基酸序列见表3，膜结合型IL-15(mbIL15)的氨基酸序列见表4。
表3.膜结合型IL-15装甲的GCC嵌合抗原受体氨基酸序列



表4膜结合型IL-15氨基酸序列
1.2逆转录病毒制备
在100mm培养皿内接种293T细胞(中国科学院典型培养物保藏委员会细胞库， Cat#GNHu17)，添加含10％FBS(Gibco，Cat#10099141)的DMEM培养基(Gibco，Cat#10566016)进行培养。待293T细胞覆盖培养皿表面约70％时进行质粒转染：分别取表达CAR的逆转录病毒载体与包装质粒混合加入到1.2ml Opti-MEM培养基(Thermofisher Scientific，Cat#31985070)中，加入35μl Fugene HD(Promega，Cat#04709691001)混匀，室温孵育15min，将混合物加入状态良好的293T细胞培养基内，移入恒温培养箱(37℃，5％CO2)内培养。48h后收集293T细胞上清，用0.45μm滤膜过滤，并浓缩备用。
1.3表达mbIL15装甲的嵌合抗原受体的T细胞(mbIL15 CAR-T)的制备
本申请构建了多株表达二代嵌合抗原受体的T细胞，分别命名为CAR-1、CAR-2、CAR-3、CAR-4、CAR-5、CAR-6和CAR-7。具体步骤如下：
T细胞增殖：按照说明书，使用Stemcell Easy Sep Kit试剂盒(Stemcell,Cat#19055)从不同健康捐献者的外周血单个核细胞(Peripheral blood mononuclear cell，简称PBMC)中分离T细胞。将分离后的T细胞置于1μg/ml CD3/CD28抗体(Thermo，Cat#11131D)预先包埋的培养皿中培养。培养基含有X-VIVO15(Lonza，Cat#BEBP02-054Q)，5％人AB血清(Gemini，Cat#100-512)，100U/ml青霉素-链霉素(Gibco，Cat#15140-122)，200IU/ml人IL2因子(北京双鹭，Cat#S19991007)。每周进行2次细胞计数，当细胞密度到2.5×106个/ml时传代扩培。
CAR-T细胞制备：用浓度为7μg/mL的RetroNectin(Takara，Cat#T202)包被24孔板，每孔500μl，并置于4℃过夜。第2天，向24孔板中添加相应逆转录病毒，随后在2000g条件下4℃离心60min。随后移除上清后，按3×105个/孔添加T细胞至24孔板，并在400g条件下室温离心5min。将离心后24孔板放入恒温培养箱中培养(37℃，5％CO2)。第3天，将细胞移入6孔板中继续培养。
1.4流式细胞实验(FACS)检测CAR转染效率
流式细胞实验(FACS)检测CAR转染效率：用PBS缓冲液(Gibco，Cat#10010023)洗涤细胞2次，计数后用PBS缓冲液稀释细胞至2×106个/ml，加入50μl Fc受体阻断剂(BioLegend，Cat#422302)室温孵育10min，然后按每孔100μl加入96板内。向每孔加入100μl浓度为2μg/μl的FITC-标记的人GUCY2C蛋白(Acro，Cat#GUC-HF255)后，将96孔板置于冰上孵育20min。FACS缓冲液(Gibco，Cat#A1286301)离心洗涤3次，100μl FACS缓冲液重悬细胞，用FACS(BD，CANTOII)检测和分析结果。
如图2所示，CAR在T细胞表面持续表达。在激活后第8天，IL15装甲的CAR在T细胞表面均可被检测到，而未激活的T细胞(parental T细胞)表面无CAR被检测到。相较表达mbIL15-IL15Rα的CAR-7，仅表达mbIL15的CAR-2和CAR-3细胞阳性率与其相近，约为80％。培养到第11天，CAR-3依然保持与CAR-7相近的阳性率，保持在80％左右。
实施例2 CAR-T细胞亚群检测
在使用FACS检测CAR表达情况的同时确定T细胞亚群的比例，并评估了T细胞的分化。包括CD8+和CD4+细胞群间比例变化，TCM、TSCM、TEM和TE细胞亚群间比例变化，以及抑制性受体LAG3和TIM3的表达情况。
如图3中A所示，除parental T细胞(3.44)外，第8天CD8+细胞群和CD4+细胞群间的比值在2.45～2.61之间，各CAR-T细胞间无显著差异。如图3中B所示，CD8+细胞群中TSCM 居多，在79.5％～90.8％之间，TCM的比例在6.27％～17.8％之间，其中CAR-3的TCM比例为17.8％而CAR-7为6.27％；TSCM和TCM的比例之和在95.84％～97.7％之间，parental T细胞为95.84％；而如图3中C所示，CD4+细胞群中TCM比例显著更高，在34.4％～50.0％之间，其中CAR-3的TCM比例为50.0％而CAR-7为34.4％，TSCM则在48.5％～63.1％之间，TSCM和TCM的比例之和在96.7％～98.5％之间；如图3中D所示，CD8+细胞群中同时表达LAG3和TIM3的细胞比例在11.7％～18.8％之间，而如图3中E所示，CD4+细胞群中同时表达LAG3和TIM3的细胞比例在2.43％～5.85％之间。
如图3中F所示，除parental T细胞(6.58)外，第11天CD8+细胞群和CD4+细胞群间的比值在4.17～5.36之间，各CAR-T细胞间无显著差异。如图3中G所示，CD8+细胞群中TSCM居多，在68.3％～82％之间，TCM的比例在3.90％～11.50％之间，其中CAR-3的TCM比例为11.50％而CAR-7为3.90％，TSCM和TCM的比例之和在79.2％～89.29％之间；而如图3中H所示，CD4+细胞群中TCM比例显著提高，在20.6％～35.9％之间，其中CAR-3的TCM比例为35.9％而CAR-7为20.6％，TSCM则为41.9％～60.3％之间，TSCM和TCM的比例之和在77.7％～82.7％之间；如图3中I所示，CD8+细胞群中同时表达LAG3和TIM3的细胞，除parental(13.5％)外余下在3.51％～5.49％之间，而如图3中J所示，CD4+细胞群中同时表达LAG3和TIM3的细胞，除parental(2.08％)和CAR-7(1.14％)外，余下均低于1％。
实施例3 mbIL15 CAR-T细胞杀伤活性检测
为了测试mbIL15 CAR-T细胞的体外杀伤活性，向低表达GCC的LS174T细胞(ATCC，Cat#CL-188)转入细菌荧光素酶(Luciferase)，构建了稳转细胞LS174T-luc。使用LS174T-luc作为靶细胞，第8天和第11天的mbIL15 CAR-T细胞作为效应细胞，进行活性测试。用PBS缓冲液清洗LS174T-luc细胞，加入胰蛋白酶溶液(Gibco，Cat#25200056)进行消化，1000×rpm离心5min，移除上清并加入新鲜培养基重悬细胞。按20000个/孔铺在96孔板上，并按照T细胞：靶细胞比例(E：T)1：1、1：2和1：10加入mbIL15 CAR-T细胞或parental T细胞共培养，每组设置两个重复。48h取样，加入萤火虫荧光素酶底物D-Luciferin(Abcam，Cat#ab143655)，使用多功能酶标仪(Perkinelmer，Cat#EnSight)读取荧光值，结果如图4所示。相较parental T细胞，mbIL15 CAR-T细胞对LS174T-luc细胞均显示出较强的杀伤活性：第8天和第11天的CAR-3细胞在不同的E：T比条件下均表现出比CAR-7更高的杀伤活性，有更多的LS174T-luc细胞裂解。单因素方差分析(One-Way ANOVA)分析结果表明，在第11天CAR-3细胞杀伤活性显著高于CAR-7(调整的p值＝0.006，t＝0.05)。
实施例4 mbIL15 CAR-T细胞多轮杀伤活性检测
为了测试mbIL15 CAR-T细胞的体外杀伤持久性，使用高表达GCC的HT55细胞(南京科佰，Cat#CBP60012)和低表达GCC的LS174T-luc细胞进行多轮杀伤实验。分别使用LS174T-luc和HT55作为靶细胞，第11天的mbIL15 CAR-T细胞作为效应细胞，进行测试。按20000个细胞/孔分别将两种靶细胞铺于96孔板内，按照T细胞：靶细胞比例＝1：2与靶细胞共培养。72h时分别取样，使用Alexa647荧光Anti-CD3抗体(Abcam，Cat#ab253269)进行染色，洗涤后，用FACS检测T细胞以及靶细胞比例并计算细胞裂解比 例。同时，将共培育72h的细胞重悬，取1/2体积细胞悬浮液再与相同数量新鲜靶细胞共培育进行下一轮杀伤实验。重复使用相同的方法，共完成4轮杀伤实验。
结果如图5所示。相较parental T细胞，mbIL15 CAR-T细胞对LS174T-luc和HT55细胞均显示出较强的杀伤活性：多轮杀伤中，CAR-3细胞对两株细胞均表现出比CAR-7更高的杀伤活性，有更多的细胞裂解。
实施例5靶向DLL3的mbIL15 CAR-T细胞制备
5.1质粒构建和CAR-T细胞制备
参照实施例1的实验方法，构建了mbIL15装甲的表达DLL3嵌合抗原受体的T细胞：CAR-8、CAR-9、CAR-10和CAR-11。采用的DLL3单链抗体(single-chain variable fragment，scFv antibody)为靶向DLL3的抗体，氨基酸序列如SED ID NO：35所示，具有SEQ ID NO：36所示的重链VH，SEQ ID NO：37所示的轻链VL，以及SEQ ID NO：38所示的连接子。重链具有SEQ ID NO：39、40、41所示的重链决定簇互补区(Complementarity Determining Region of the Heavy Chain，简称HCDR)HCDR1、HCDR2、HCDR3，而轻链具有SEQ ID NO：42、43、44所示的轻链决定簇互补区(Complementarity Determining Region of the Light Chain，简称LCDR)LCDR1、LCDR2、LCDR3。上述抗体序列编号使用Kabat编号系统。所采用的嵌合抗原受体为二代的嵌合抗原受体，具有CD8α铰链区(SEQ ID NO：14)、CD8α跨膜结构域(SEQ ID NO：45)，4-1BB胞内共刺激域(SEQ ID NO：46)、CD3ζ信号转导结构域(SEQ ID NO：10、11或12)、裂解肽(SEQ ID NO：13)和膜结合型IL15。以逆转录病毒载体模板，参照图6所示的质粒结构简图，构建了表达二代嵌合抗原受体的逆转录病毒质粒P8、P9、P10和P11。每个质粒从5`端到3`端依次包含：CD8α信号肽、scFv、CD8α铰链区、CD8α跨膜结构域，4-1BB胞内共刺激域、CD3ζ信号转导结构域或膜结合型IL15，各元件氨基酸序列见表5，各元件间连接顺序见表6，插入的CAR的具体氨基酸序列见表7，其中matIL15-CD8α-CD8α来自专利WO2020/056045。参照实施例1的方法，制备mbIL15装甲的DLL3 CAR-T细胞，收获并冻存激活第10天的各组细胞和激活第11天的各组细胞。冻存时，离心并使用CS10(Stemcell，Cat#07930)重悬混匀待冻存细胞，移入-80℃以1℃/min缓慢降温，4小时后转移储存于液氮中。膜结合型IL-15(mbIL15)的氨基酸序列如SEQ ID NO：48所示。
表5.膜结合型IL-15装甲的二代嵌合抗原受体序列表

表6.膜结合型IL-15装甲的二代嵌合抗原受体结构
表7.膜结合型IL-15装甲的DLL3嵌合抗原受体氨基酸序列


5.2流式细胞实验(FACS)检测CAR转染效率
流式细胞实验(FACS)检测CAR转染效率：用PBS缓冲液洗涤细胞2次，进行细胞计数后将细胞用PBS缓冲液稀释至2×106细胞/ml，加入50μl Fc封闭试剂，室温孵育10min，然后按每孔100μl加入到96孔FACS反应板中，加入100μl His标签标记的DLL3抗原蛋白(3μg/ml，购自Acro，Cat#DL3-H52H4)，4℃孵育30min。用FACS缓冲液离心洗涤2次，加入每孔100μl THETMHis Tag Antibody(GenScript，Cat#A01802)和IL-15Monoclonal Antibody(34559)，PE(Invitrogen，Cat#MA5-23561)，4℃孵育20min。用FACS缓冲液离心洗涤3次，用200μl FACS缓冲液悬浮细胞，用FACS检测和分析结果，结果见图7。如图7中A所示，在第8和第10天，各组CAR-T细胞的表达率在60％～80％之间，显示良好的病毒转染效率；如图7中B所示，在第8和第10天，IL-15表达率在50％～80％之间，显示出良好的表达率。
实施例6靶向DLL3的mbIL15 CAR-T细胞多轮杀伤活性检测
为了测试靶向DLL3的mbIL15 CAR-T细胞的体外杀伤持久性，使用表达人DLL3的肿瘤细胞SHP77-luc和NCI-H2171-luc进行多轮杀伤实验。高表达DLL3的SHP77细胞和低表达DLL3的NCI-H2171细胞购自ATCC，转入细菌荧光素酶，构建了稳转细胞SHP77-luc和NCI-H2171-luc。
6.1新鲜mbIL15 CAR-T细胞的多轮杀伤活性检测
分别使用SHP77-luc和NCI-H2171-luc作为靶细胞，激活后第11天的新鲜mbIL15 CAR-T细胞分别作为效应细胞，进行活性测试。用PBS缓冲液清洗SHP77-luc和NCI-H2171-luc细胞，1000×rpm离心5min，移除上清并加入新鲜培养基以重悬细胞。按20000个/孔铺在96孔板上，并按照T细胞：靶细胞比例(E：T)1：2加入上述mbIL15 CAR-T细胞或parental T细胞共培养，每组设置两个重复。每隔72h取样，加入D-Luciferin后使用多功能酶标仪读取荧光值，换算以表征靶细胞裂解比例。同时，对每孔细胞的数量进行计数，按照1：2比例 与新鲜靶细胞共培育进行下一轮杀伤实验。重复使用相同的方法，共完成多轮杀伤实验，结果见图8。
如图8中A所示，在高表达人DLL3细胞系SHP-77上，激活后第11天的mbIL15装甲的CAR-9和CAR-10均显示出比CAR-11更强杀伤活性；如图8中B所示，在低表达人DLL3细胞系NCI-H2171-luc上，激活后第11天的mbIL15装甲的CAR-9和CAR-11杀伤活性相似，到第4轮时依然可以杀伤约90％的NCI-H2171-luc细胞，而CAR-8在第2轮时杀伤活性明显下降，到第3轮时便无杀伤活性。
6.2冻存复苏后mbIL15 CAR-T细胞的多轮杀伤活性检测
分别使用SHP77-luc和NCI-H2171-luc作为靶细胞，激活后第10天冻存复苏的mbIL15CAR-T细胞作为效应细胞，进行活性测试。复苏时，从液氮罐中取出细胞冷冻保存管，立即放入37℃水浴(Thermo Scientific，Cat#TSGP02)中快速解冻；待冻存管中细胞混合液完全溶化后，将细胞混合液缓慢加入含有9mL培养基的试管中，混合均匀后在200×g条件下，离心5min，弃掉上清后使用培养基重悬，移入细胞培养瓶内培养。参照实施例6.1所述方法进行多轮杀伤试验，结果见图8。
如图8中C所示，在SHP-77细胞上，冻存复苏的各组细胞均显示出优异的杀伤活性，杀伤活性由强到弱依次为：CAR-10、CAR-9、CAR-11和CAR-8，mbIL15 CAR-T均显示出比CAR-8更强杀伤活性；如图8中D所示，在NCI-H2171-luc细胞上，冻存复苏的mbIL15 CAR-T杀伤活性相似，到第6轮时依然可以近乎完全杀伤NCI-H2171-luc细胞，而CAR-8在第2轮时杀伤活性明显下降，仅有7.2％的细胞裂解。
实施例7靶向DLL3的mbIL15 CAR-T细胞对SHP77小鼠皮下肿瘤模型抗肿瘤药效的检测
为了测试靶向DLL3的mbIL15 CAR-T细胞对体内肿瘤杀伤活性，使用小鼠皮下瘤CDX模型评估DLL3 CAR-T细胞的抗肿瘤效果。具体如下：
NPG小鼠(联合免疫缺陷小鼠，购自北京维通达生物技术有限公司)皮下接种3×106个处于对数生长期且生长状态良好的SHP77细胞。
接种后第7天测量小鼠肿瘤的长短径a和b，计算小鼠肿瘤体积V(mm3)＝1/2×(a×b2)，根据随机数原则选择肿瘤体积在30mm3左右的小鼠随机分组。接种肿瘤后第4天，尾静脉注射按照实施例5和6所述方法冻存复苏的激活后第10天冻存复苏的CAR-T细胞。CAR-T细胞数量以CAR阳性计数，每只注射2×106CAR阳性细胞(即CAR+细胞)，CAR-T细胞注射日为第0天(D0)。具体分组给药情况见表8。持续观察并测量瘤体积、体重变化和生存率，每周测量记录2次，检测结果如图9所示。
表8.SHP-77模型体内抗肿瘤实验分组情况
如图9中A所示，mbIL15装甲的CAR-10和CAR-11均显示出比未装甲的CAR-8更强肿瘤抑制活性，在第15天便抑制肿瘤的生长；CAR-10显示出比CAR-11更优的抑制活性，在第23天差异显著(P<0.05)。如图9中B所示，未装甲的CAR-8治疗组小鼠体重变化较为剧烈。如图9中C所示，在第23天，CAR-11治疗组有2只小鼠死亡。
实施例8靶向CD19的mbIL15 CAR-T细胞的制备和多轮杀伤活性检测
为了测试靶向其他免疫原的mbIL15 CAR-T细胞的体外杀伤活性，制备了靶向人CD19的mbIL15 CAR-T细胞，并检测了对高表达CD19的Nalm6-luc细胞的杀伤活性。Nalm6-luc细胞购自ATCC，转入细菌荧光素酶，构建了稳转细胞Nalm6-luc。
8.1质粒构建和CAR-T细胞制备
参照实施例1的实验方法，构建了mbIL15装甲的表达CD19嵌合抗原受体的T细胞：CAR-12、CAR-13和CAR-14。采用的CD19单链抗体(single-chain variable fragment，scFv antibody)为靶向CD19的抗体，氨基酸序列如SED ID NO：54所示，具有SEQ ID NO：55所示的重链VH，SEQ ID NO：56所示的轻链VL，以及SEQ ID NO：57所示的连接子。重链具有SEQ ID NO：58、59、60所示的重链决定簇互补区HCDR1、HCDR2、HCDR3，轻链具有SEQ ID NO：61、62、63所示的轻链决定簇互补区LCDR1、LCDR2、LCDR3。上述抗体序列编号使用Kabat编号系统。所采用的嵌合抗原受体为二代的嵌合抗原受体，具有CD28铰链区(SEQ ID NO：7)、CD28跨膜结构域(SEQ ID NO：8)，CD28胞内共刺激域(SEQ ID NO：9)、CD3ζ信号转导结构域(SEQ ID NO：10、11或12)、裂解肽(SEQ ID NO：13)和膜结合型IL15。构建的CAR结构和各元件氨基酸序列见表9和表10。以逆转录病毒载体模板，参照图10所示的质粒结构简图，构建了表达二代嵌合抗原受体的逆转录病毒质粒P12、P13和P14。每个质粒从5`端到3`端依次包含：GM-CSF信号肽、scFv、CD28铰链区、CD28跨膜结构域，CD28胞内共刺激域、CD3ζ信号转导结构和膜结合型IL15，连接顺序分别见表10，具体序列见表11，其中P14使用的matIL15-CD8α-CD8α来自专利WO2020/056045。参照实施例1的方法，制备mbIL15装甲的CD19 CAR-T细胞，收获激活后第10天的各组细胞。并参照实施例1.4的方法，检测各组细胞CAR转染效率，显示各组细胞转染情况良好。
表9.膜结合型IL-15装甲的二代嵌合抗原受体序列表

表10.膜结合型IL-15装甲的二代嵌合抗原受体结构
表11.膜结合型IL-15装甲的CD19嵌合抗原受体氨基酸序列

8.2多轮杀伤活性检测
使用Nalm6-luc作为靶细胞，激活后第10天的mbIL15 CAR-T细胞作为效应细胞，进行活性测试。用PBS缓冲液清洗Nalm6-luc细胞，1000×rpm离心5min，移除上清并加入新鲜培养基重悬细胞。按20000个/孔铺在96孔板上，并按照T细胞：靶细胞比例(E：T)1：2加入mbIL15 CAR-T细胞或parental T细胞共培养，每组设置两个重复。每隔72h取样，加入D-Luciferin后使用多功能酶标仪读取荧光值，换算以表征细胞裂解比例。同时，对每孔细胞的数量进行计数，与按照1：2比例与新鲜靶细胞共培育进行下一轮杀伤实验。重复使用相同的方法，共完成多轮杀伤实验，结果见图11。如图11所示，CAR-12和CAR-13杀伤活性优于CAR-14。到第4轮时，CAR-13显示出最强的杀伤活性。

Claims (22)

  1. 一种工程化的免疫细胞，其共表达嵌合抗原受体(CAR)和膜结合的IL15(mbIL15)的全部或其功能部分。
  2. 如权利要求1所述的免疫细胞，其中，所述IL15的全部或其功能部分与跨膜蛋白的全部或一部分融合，所述跨膜蛋白选自NKG2D、OX40、2B4或EpCAM。
  3. 如权利要求2所述的免疫细胞，其中，所述NKG2D包含与SEQ ID NO：15或16具有至少90％同一性的氨基酸序列，或者所述OX40包含与SEQ ID NO：17具有至少90％同一性的氨基酸序列，或者所述2B4包含与SEQ ID NO：18具有至少90％同一性的氨基酸序列，或者所述EpCAM包含与SEQ ID NO：19或20具有至少90％同一性的氨基酸序列。
  4. 如权利要求2-3任一项所述的免疫细胞，其中，所述IL15的全部或其功能部分与所述跨膜蛋白的全部或一部分通过连接子融合，优选地，所述连接子为CD8α，优选地，所述连接子包含与SEQ ID NO：14具有至少90％同一性的氨基酸序列。
  5. 如权利要求1-4任一项所述的免疫细胞，其中，所述IL15的全部或其功能部分包含与SEQ ID NO：1或SEQ ID NO：47具有至少90％同一性的氨基酸序列。
  6. 如权利要求1-5任一项所述的免疫细胞，其中，所述mbIL15包含与SEQ ID NO：27-32和48中的任一项具有至少90％同一性的氨基酸序列。
  7. 如权利要求1-6任一项所述的免疫细胞，其中，所述CAR至少包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，优选地，所述细胞外抗原结合结构域包含靶向GCC的纳米抗体(VHH)或其抗原结合片段，所述纳米抗体或其抗原结合片段包含CDR1、CDR2和CDR3，优选地，所述CDR1、CDR2和CDR3分别选自SEQ ID NO：2所示的VHH中包含的CDR1、CDR2和CDR3；
    优选地，所述细胞外抗原结合结构域包含靶向DLL3或CD19的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，优选地，所述HCDR1、HCDR2和HCDR3分别选自SEQ ID NO：36或55所示的VH中包含的HCDR1、HCDR2和HCDR3；所述LCDR1、LCDR2和LCDR3分别选自SEQ ID NO：37或56所示的VL中包含的LCDR1、LCDR2和LCDR3。
  8. 如权利要求7所述的免疫细胞，其中所述CDR1、CDR2和CDR3分别具有SEQ ID NO：3-5所示的氨基酸序列；所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：39-44或SEQ ID NO：58-63所示的氨基酸序列。
  9. 如权利要求7-8任一项所述的免疫细胞，其中，所述纳米抗体或其抗原结合片段包含SEQ ID NO：2所示的氨基酸序列，或者包含与SEQ ID NO：2所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列或与SEQ ID NO：2所示的氨基酸序列相比具有至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的氨基酸序列；；或者
    所述抗体或其抗原结合片段包含SEQ ID NO：36和SEQ ID NO：37所示的氨基酸序列，或包含SEQ ID NO：55和SEQ ID NO：56所示的氨基酸序列，或者包含与SEQ ID NO：36和 37，或SEQ ID NO：55和56所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，或与SEQ ID NO：36和37，或SEQ ID NO：55和56所示的氨基酸序列相比具有至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的氨基酸序列；
    优选地，所述突变选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
  10. 如权利要求1-9任一项所述的免疫细胞，其中，所述免疫细胞选自T细胞、NK细胞、NKT细胞或诱导生成的T细胞和NK细胞。
  11. 一种融合蛋白，包含与跨膜蛋白的全部或一部分融合的IL15的全部或其功能部分，所述跨膜蛋白选自NKG2D、OX40、2B4或EpCAM，优选地，所述融合蛋白还包含CAR，优选权利要求7-9中任一项中所记载的CAR。
  12. 如权利要求11所述的融合蛋白，其中，所述NKG2D包含与SEQ ID NO：15或16具有至少90％同一性的氨基酸序列，或者所述OX40包含与SEQ ID NO：17具有至少90％同一性的氨基酸序列，或者所述2B4包含与SEQ ID NO：18具有至少90％同一性的氨基酸序列，或者所述EpCAM包含与SEQ ID NO：19或20具有至少90％同一性的氨基酸序列。
  13. 如权利要求11或12所述的融合蛋白，其中，所述IL15的全部或其功能部分与所述跨膜蛋白的全部或一部分通过连接子融合，优选地，所述连接子为CD8α，优选地，所述连接子包含与SEQ ID NO：14具有至少90％同一性的氨基酸序列。
  14. 如权利要求11-13任一项所述的融合蛋白，其中，所述IL15的全部或其功能部分包含与SEQ ID NO：1或SEQ ID NO：47具有至少90％同一性的氨基酸序列。
  15. 如权利要求11-14任一项所述的融合蛋白，其中，所述融合蛋白包含与SEQ ID NO：27-32和48中的任一项具有至少90％同一性的氨基酸序列。
  16. 一种分离的核酸分子，其包含编码权利要求1-10任一项所述的免疫细胞上共表达的CAR和mbIL15的核酸分子，或包含编码权利要求11-15任一项所述的融合蛋白的核酸分子；优选地，所述核酸分子为DNA或RNA。
  17. 一种载体，其包含权利要求16所述的核酸分子，优选地，所述载体是病毒载体。
  18. 一种制备权利要求1-10任一项所述的免疫细胞的方法，其包括将权利要求16的核酸分子引入到所述免疫细胞中。
  19. 一种药物组合物，其包含权利要求1-10任一项所述的免疫细胞，权利要求11-15任一项所述的融合蛋白，权利要求16所述的核酸分子。
  20. 权利要求1-10任一项所述的免疫细胞，权利要求18所述方法制备获得的产品，或权利要求19所述的药物组合物用于制备在个体中治疗肿瘤的药物的用途；任选地，所述肿瘤选自血液瘤或实体瘤；任选地，所述血液瘤选自B细胞淋巴瘤，表达CD19的肿瘤等；任选地，所述实体瘤选自非小细胞肺癌，胃癌，结肠腺癌，结肠癌，小细胞肺癌，表达GCC或DLL3的肿瘤等。
  21. 一种在个体中治疗肿瘤的方法，包括向所述有需要的个体施用权利要求1-10任一项所述 的免疫细胞，权利要求18所述方法制备获得的产品，或权利要求19所述的药物组合物；任选地，所述肿瘤选自血液瘤或实体瘤；任选地，所述血液瘤选自B细胞淋巴瘤，表达CD19的肿瘤等；任选地，所述实体瘤选自非小细胞肺癌，胃癌，结肠腺癌，结肠癌，小细胞肺癌，表达GCC或DLL3的肿瘤等。
  22. 权利要求1-10任一项所述的免疫细胞，权利要求18所述方法制备获得的产品，或权利要求19所述的药物组合物，用于在个体中预防和/或治疗肿瘤的用途；任选地，所述肿瘤选自血液瘤或实体瘤；任选地，所述血液瘤选自B细胞淋巴瘤，表达CD19的肿瘤等；任选地，所述实体瘤选自非小细胞肺癌，胃癌，结肠腺癌，结肠癌，小细胞肺癌，表达GCC或DLL3的肿瘤等。
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