Targeted MSLN chimeric antigen receptor macrophage as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of cellular immunotherapy, and particularly relates to a targeted MSLN chimeric antigen receptor macrophage, a preparation method thereof and application of the targeted MSLN chimeric antigen receptor macrophage to treatment of MSLN positive tumors. According to the present invention, the chimeric antigen receptor modified engineering immune cell specifically targeting MSLN is prepared by using the chimeric antigen receptor modified macrophage technology, the preparation method has simple steps, and the obtained novel engineering immune cell can specifically recognize tumor cells, can more effectively target and attack the tumor cells, has a high tumor killing rate, and can be used for preparing the MSLN-targeted engineering immune cell. The compound can be used for preparing anti-tumor products, especially for preparing drugs for treating MSLN positive tumors, especially for preparing drugs for treating ovarian cancer malignant pleural mesothelioma, pancreatic cancer and part of lung cancer, and has good industrial application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of cellular immunotherapy, and particularly relates to a mesothelin (MSLN)-targeted chimeric antigen receptor macrophage, a preparation method thereof, and its use for treating MSLN-positive tumors, especially ovarian cancer and pancreatic cancer. Background Art

[0002] Ovarian cancer is a heterogeneous disease. Approximately 90% of ovarian cancers are classified as malignant epithelial ovarian cancers (EOCs), among which high-grade serous carcinoma (HGSC) accounts for 70% of the tumor types.

[0003] Mesothelin (MSLN) is a cell surface glycoprotein, and its normal expression is limited to mesothelial cells of the pleura, peritoneum, and pericardial lining, with a relatively low content. It is highly overexpressed in many cancers, including malignant mesothelioma, pancreatic cancer, ovarian cancer, lung adenocarcinoma, endometrial cancer, biliary tract cancer, pediatric acute myeloid leukemia, and gastric cancer. Since MSLN is only expressed in non-critical tissues, the risk of off-target toxicity is reduced. Mesothelin is expressed in most ovarian cancers but not in various normal adult tissues, indicating that mesothelin is an ideal marker for cancer diagnosis and targeted therapy.

[0004] With the rapid development of biotechnology, immunocyte therapy has become the fourth major therapy in the field of cancer treatment. Cancer immunotherapy mainly includes adoptive cell therapy, immunomodulators, tumor vaccines, and immune checkpoint blockade therapy, etc. Currently, the research and development targeting the MSLN target involve monoclonal antibodies, bispecific antibodies, and CAR-T therapy. Solid tumor indications have always been considered a difficult point for CAR-T therapy. However, the research and development of MSLN CAR-T cell therapy is currently in the ascendant. Many enterprises have laid out this target. On November 12, 2021, French company Cellectis announced the first preclinical data of the allogeneic CAR-T cell candidate product UCARTMESO targeting MSLN for the treatment of pancreatic cancer and mesothelioma at the annual meeting of the Society for Immunotherapy of Cancer (SITC). The study evaluated the in vivo activity of TGFβR2-knockout MESO CAR-T cells using an immunodeficient mesothelioma model mouse. The results showed that tumor regression and increased survival rate were observed in all mice receiving CAR-T treatment. In addition, a domestic self-developed clinical trial targeting the brand-new target MSLN for the treatment of recurrent and refractory epithelial ovarian cancer has officially started recruiting patients. Therefore, MSLN CAR-T is also a key direction in the field of solid tumor treatment research. Currently, there are 7 MSLN CAR-T clinical drug projects. 5 of them are about to enter the clinical stage, involving domestic pharmaceutical companies such as Shanghai Keji Biotechnology and Nanjing Legend Biotech Co., Ltd., as well as foreign pharmaceutical companies such as Novartis and Cellectis.

[0005] Chimeric antigen receptor (CAR) macrophages refer to macrophages that express a chimeric receptor on their surface that recognizes specific antigens and transmits signals intracellularly. The core is that the CAR molecule carries a single-chain antibody (scFv) that specifically recognizes cell surface antigens, thereby recognizing and killing cells that express specific antigens. Currently, CAR-M cell therapy is mainly applied to cancer treatment and has shown certain efficacy in some solid tumors. There are few reports on the application of CAR-M in ovarian cancer treatment. In summary, the development of engineered immune response cells modified with a specific chimeric antigen receptor that specifically targets MSLN has great application prospects. These engineered cells transmit activation signals and activate the immune system by highly specifically recognizing MSLN produced on the surface of tumor cells, exert a killing effect on tumor cells and have good safety, thereby achieving good clinical treatment effects. Summary of the Invention

[0006] To achieve the above object, the present invention adopts the following technical solutions.

[0007] In view of the existing problems and / or other problems in the related art, the object of the present invention is to overcome the problems of low specificity and low safety in the tendency of effector cells in the tumor microenvironment to bind and kill tumor cells in the existing tumor clinical technology, and to provide a chimeric antigen receptor that specifically targets MSLN, its gene and recombinant expression vector, engineered immune effector cells modified with a specifically targeted MSLN chimeric antigen receptor and their applications. The immune effector cells modified with the specifically targeted MSLN chimeric antigen receptor of the present invention highly specifically kill tumor cells and have good safety, thereby providing a new means for tumor treatment with application prospects.

[0008] To achieve the above object, the present invention adopts the following technical solutions.

[0009] An immune effector cell modified with a chimeric antigen receptor targeting MSLN, wherein the chimeric antigen receptor comprises an antigen-binding domain, a hinge region, a transmembrane domain and a signal transduction domain, and the antigen-binding domain is a single-chain antibody against MSLN.

[0010] Further, the hinge region comprises CD8.

[0011] Further, the transmembrane domain comprises a CD8 transmembrane region.

[0012] Further, the signal transduction domain comprises CD3ζ.

[0013] Further, the signal transduction domain further comprises any one or a combination of at least two of 4-1BB, the intracellular region of CD28, DAP10 or OX40.

[0014] Preferably, the chimeric antigen receptor comprises an anti-MSLN single-chain antibody, a CD8 hinge region, a CD8 transmembrane region, and CD3ζ.

[0015] Preferably, the antigen-binding domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:2.

[0016] Preferably, the hinge region comprises the amino acid sequence shown in SEQ ID NO:1.

[0017] Preferably, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:3.

[0018] Preferably, the signal transduction domain comprises the amino acid sequence shown in SEQ ID NO:4.

[0019] Furthermore, the immune effector cells are macrophages and peripheral blood mononuclear cells.

[0020] A nucleic acid molecule, which comprises the coding gene of the chimeric antigen receptor.

[0021] An expression vector, which comprises the nucleic acid molecule.

[0022] Furthermore, the expression vector is a lentiviral vector or an adenoviral vector containing the nucleic acid molecule.

[0023] A recombinant lentivirus or adenovirus, which is prepared from mammalian cells transfected with the expression vector and an auxiliary plasmid.

[0024] The immune effector cells modified with the chimeric antigen receptor have the nucleic acid molecule integrated into their genomes.

[0025] The immune effector cells modified with the chimeric antigen receptor comprise the expression vector and / or the recombinant lentivirus and / or adenovirus.

[0026] A method for preparing the immune effector cells modified with the chimeric antigen receptor, which comprises the step of introducing the expression vector and / or the recombinant lentivirus and / or adenovirus into immune effector cells.

[0027] Furthermore, the method for preparing the immune effector cells modified with the chimeric antigen receptor specifically comprises the following steps: (1) Synthesize the nucleic acid sequences of the respective domains of the transmembrane expression protein or polypeptide macromolecule.

[0028] (2) Connect the target fragment with a lentiviral or adenoviral overexpression vector to obtain a recombinant plasmid vector.

[0029] (3) Extraction and preparation of recombinant plasmid vectors and auxiliary plasmid vectors.

[0030] (4) Virus packaging.

[0031] (5) Construction of functional cell lines.

[0032] A pharmaceutical composition, which comprises the chimeric antigen receptor-modified immune effector cells as described above.

[0033] Furthermore, the pharmaceutical composition further comprises any one or a combination of at least two of pharmaceutically acceptable carriers, excipients or diluents.

[0034] Use of the chimeric antigen receptor-modified immune effector cells, the nucleic acid molecules, the expression vectors, the recombinant lentivirus and / or adenovirus or the pharmaceutical composition in the preparation of a medicament for treating MSLN-positive tumors.

[0035] Furthermore, the MSLN-positive tumors are mesothelioma, pancreatic cancer or ovarian cancer.

[0036] Through creative efforts, the inventors continuously carried out amino acid sequence design, sequence permutation and combination, and screening. Random screening tests and targeted function verification were performed on the sequences of more than a hundred CAR molecules (such as constructing viral vectors, and further infecting macrophages to obtain modified macrophages, and detecting the in vitro killing activity of the obtained modified macrophages, etc.). After comparing the results of multiple random combinations, sequence adjustment was carried out, and finally 1 sequence with better effects was screened out, and 1 scFv amino acid sequence with high titer targeting MSLN and its functional variants of the present invention were obtained.

[0037] Gene modification of immune effector cells (such as macrophages, monocytes, T cells, CTL cells, NK cells) can be achieved by transducing homologous cell compositions with recombinant DNA or RNA constructs. In one embodiment, the vector is a retroviral vector (such as γ-retrovirus or lentivirus), which can introduce the DNA or RNA construct into the host cell genome. For example, the polynucleotide of a chimeric antigen receptor specifically targeting MSLN can be cloned into a retroviral vector, and expression can be driven from its endogenous promoter, the retroviral long terminal repeat sequence or from an alternative internal promoter.

[0038] Non-viral vectors or RNAs can also be used. Random chromosomal integration or targeted integration (such as using nucleases, transcription activator-like effector nucleases TALEN, zinc finger nucleases ZFN and / or clustered regularly interspaced short palindromic repeats CRISPR) or transgenic expression (such as using natural or chemically modified RNAs) can be used.

[0039] The vector is selected from γ-retroviral vectors, lentiviral vectors, adenoviral vectors, and adenoviral vectors.

[0040] In one exemplary embodiment, the vector is an adenovirus.

[0041] In some embodiments, the immune effector cells are cytotoxic T lymphocytes, NK cells, NKT cells, or helper T cells, macrophages, or peripheral blood mononuclear cells.

[0042] Preferably, the immune response cells are macrophages.

[0043] In some embodiments, the virus is a lentivirus, adenovirus, adeno-associated virus, or retrovirus.

[0044] Preferably, the virus is an adenovirus.

[0045] An isolated modified immune response cell comprising the chimeric antigen receptor, which is obtained by transformation with the recombinant vector or expression plasmid.

[0046] For the initial genetic modification of cells to provide the chimeric antigen receptor-modified immune response cells specifically targeting MSLN, transduction is typically performed using adenovirus or lentiviral vectors, although any other suitable viral vector or non-viral delivery system can be used. For subsequent genetic modification of cells to provide cells comprising an antigen-presenting complex containing at least two co-stimulatory ligands, retroviral gene transfer (transduction) has also proven effective. The combination of retroviral vectors and a suitable packaging line is also suitable, where the capsid protein is functional for infecting human cells.

[0047] In some embodiments, the immune response cells further comprise at least one exogenous co-stimulatory ligand.

[0048] Possible transduction methods also include direct co-culture of the cells with producer cells. Transducing viral vectors can be used to express co-stimulatory ligands (such as 4-1BBL and IL-12) in immune response cells. Preferably, the selected vector exhibits high infection efficiency and stable integration and expression.

[0049] In some embodiments, preferably, the at least one co-stimulatory ligand is selected from 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, and combinations thereof, or more preferably, the co-stimulatory ligand is 4-1BBL.

[0050] In some embodiments, the immune effector cells are selected from macrophages, peripheral blood mononuclear cells, T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, human embryonic stem cells, and pluripotent stem cells that can differentiate into lymphoid cells, preferably peripheral blood mononuclear cells or macrophages.

[0051] Peripheral blood mononuclear cells or macrophages isolated from a patient can be transduced with a vector for CAR expression.

[0052] In an exemplary embodiment, the modified immune response cells are CAR-M cells.

[0053] The genetically modified specific-targeted chimeric antigen receptor can be prepared and then stored frozen.

[0054] The method for preparing the isolated chimeric antigen receptor-modified immune response cells includes the following steps.

[0055] Step 1: Connect the nucleic acid molecule to an expression vector by molecular cloning to obtain an expression vector for the chimeric antigen receptor specifically targeting MSLN.

[0056] Step 2: Transfect the obtained expression vector for the chimeric antigen receptor specifically targeting MSLN into 293T cells to obtain a viral solution.

[0057] Step 3: Infect immune response cells with the viral solution, and obtain chimeric antigen receptor-modified immune response cells expressing the chimeric antigen receptor specifically targeting MSLN from the infected cells.

[0058] In some non-limiting embodiments, the modified immune response cells of the present invention can be macrophages, peripheral blood mononuclear cells, or cells of the lymphoid lineage. The cells of the lymphoid lineage are selected from B, T, and natural killer (NK) cells, providing functions such as antibody production, regulation of the cellular immune system, detection of foreign substances in the blood, and detection of host foreign cells. Non-limiting examples of cells of the lymphoid lineage include T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, embryonic stem cells, and pluripotent stem cells (e.g., pluripotent stem cells that can differentiate into lymphoid cells).

[0059] In a preferred embodiment, the chimeric antigen receptor-modified immune effector cells are macrophages.

[0060] In some non-limiting embodiments, the chimeric antigen receptor-modified immune effector cells (e.g., macrophages or peripheral blood mononuclear cells or) can be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor cells or stem cells.

[0061] The pharmaceutical composition disclosed in the present application comprises isolated modified immune response cells expressing the chimeric antigen receptor specifically targeting MSLN and a pharmaceutically acceptable carrier.

[0062] The administration of the pharmaceutical composition can be autologous or allogeneic. For example, immune response cells expressing the chimeric antigen receptor specifically targeting MSLN and compositions containing the same can be obtained from one subject and administered to the same subject or a different compatible subject. The peripheral blood-derived cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) of the disclosed subject matter of the present invention can be administered by including catheter administration, intravenous injection or parenteral administration. When administering the pharmaceutical composition of the disclosed subject matter of the present invention (e.g., a pharmaceutical composition containing immune response cells expressing the chimeric antigen receptor specifically targeting MSLN), it is generally formulated into a unit dose injectable form (solution, suspension, emulsion).

[0063] The composition of the present application can be a formulation. The immune response cells expressing the chimeric antigen receptor (CAR) specifically targeting MSLN disclosed in the present application and compositions containing the same can be conveniently provided as a sterile liquid formulation, such as an isotonic aqueous solution, suspension, emulsion, dispersion or viscous composition, which can be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions and solid compositions. In addition, liquid compositions are more convenient to administer, especially by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide a longer contact time with a specific tissue. The liquid or viscous composition can contain a carrier, which can be a solvent or dispersion medium containing, for example, water, physiological saline, phosphate buffered saline, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof.

[0064] Various additives that enhance the stability and sterility of the composition can be added, including antimicrobial preservatives, antioxidants, chelating agents and buffers.

[0065] According to the present application, any carrier, diluent or additive used must be compatible with the immune response cells expressing the chimeric antigen receptor (CAR) specifically targeting MSLN of the disclosed subject matter of the present invention.

[0066] If necessary, the viscosity of the composition can be maintained at a selected level using a pharmaceutically acceptable thickening agent. The selection of a suitable carrier and other additives will depend on the exact route of administration and the nature of the specific dosage form, such as a liquid dosage form (e.g., whether the composition is formulated as a solution, suspension, gel or another liquid form, such as a time-release form or a liquid-filled form).

[0067] The chimeric antigen receptor-modified immune response cells specifically targeting MSLN of the present invention can transmit activation signals and activate the immune system by recognizing MSLN produced on the surface of tumor cells, thereby exerting a killing effect on tumor cells.

[0068] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0069] The present invention uses the technology of modifying macrophages with chimeric antigen receptors to prepare chimeric antigen receptor-modified engineered immune cells specifically targeting MSLN. The preparation method has simple steps. The obtained novel engineered immune cells can specifically recognize tumor cells, can more effectively target and attack tumor cells, have a high killing rate for tumors, and can be used to prepare anti-tumor products, especially drugs for treating MSLN-positive tumors. The present invention is expected to be used to prepare anti-tumor products, especially for preparing drugs for treating ovarian cancer, malignant pleural mesothelioma, pancreatic cancer, and some lung cancers, and has good industrial application prospects. Description of the Drawings

[0070] Figure 1 It is a schematic structural diagram of an anti-MSLN CAR vector.

[0071] Figure 2 It is a map of the CAR-MSLN adenovirus vector (pAd5F35-EF1a-CAR-MSLN-CD3ζ-PGK-mCherry).

[0072] Figure 3 It is a map of the MSLN overexpression vector (MSLN in pCDH-CMV-MCS-EF1-puro).

[0073] Figure 4 Fluorescence detection result diagram of the MSLN overexpression vector.

[0074] Figure 5 It is a WB detection result diagram of the MSLN overexpression vector.

[0075] Figures 6-7 It is a detection result diagram of the expression of the MSLN overexpression vector MARKER.

[0076] Figures 8-9 G4S detects the expression of CAR in hMDM-CAR-MSLN cells.

[0077] Figure 10 It is a 3-hour phagocytosis experiment of hMDM-CAR-MSLN targeting AsPC-1WT.

[0078] Figure 11 It is a 6-hour phagocytosis experiment of hMDM-CAR-MSLN targeting AsPC-1WT.

[0079] Figure 12 It is a 12-hour phagocytosis experiment of hMDM-CAR-MSLN targeting AsPC-1 cells Detailed implementation manners

[0080] The present invention will be further described below through examples, but the present invention is not limited to these specific implementation manners. Materials, reagents, etc. used in the following examples can be obtained from commercial channels without special instructions.

[0081] One aspect of the present invention provides a chimeric antigen receptor (CAR) targeting MSLN. The chimeric antigen receptor generally consists of an extracellular region, a transmembrane region, and an intracellular region. The extracellular region includes an antigen-binding region and, optionally, a hinge region. The intracellular region includes one or more signal transduction regions, including a co-stimulatory signal transduction region. When expressed in a cell, the polypeptide of the chimeric antigen receptor may also include a signal peptide, particularly a membrane-localization signal peptide.

[0082] When expressed in a cell, the polypeptide of the chimeric antigen receptor of the present invention may include a signal peptide (also referred to as a signal sequence) at the N-terminus of the polypeptide. Generally, a signal peptide is a peptide sequence that targets the polypeptide to a desired location in the cell. In some embodiments, the signal peptide targets the polypeptide to the secretory pathway of the cell and will allow the polypeptide to be integrated and anchored to the lipid bilayer.

[0083] The chimeric antigen receptor of the present invention includes an antigen-binding region targeting MSLN. The antigen-binding region can be monovalent or multivalent (e.g., bivalent). The antigen-binding region can also be monospecific or multispecific (e.g., bispecific). The bispecificity can be directed against MSLN and another antigen, or against two different epitopes of MSLN.

[0084] In some embodiments, the antigen-binding region used in the present invention is the anti-MSLN antibody or antigen-binding fragment described above, particularly in the form of a scFv.

[0085] Optionally, the chimeric antigen receptor of the present invention includes a hinge region located between the extracellular antigen-binding region and the transmembrane region. The hinge region is an amino acid segment that generally exists between two domains of a protein and can allow flexibility of the protein and relative movement of the two domains with respect to each other.

[0086] The hinge region can be the hinge region of a naturally occurring protein or a portion thereof. The hinge region of an antibody (such as an IgG, IgA, IgM, IgE, or IgD antibody) can also be used for the chimeric antigen receptor described herein. A non-naturally occurring peptide can also be used as the hinge region of the chimeric antigen receptor described herein. In some embodiments, the hinge region is a peptide linker.

[0087] In some embodiments, the hinge region used in the present invention is derived from CD8α. In some embodiments, the CD8α hinge region comprises the amino acid sequence of SEQ ID NO: 1.

[0088] The chimeric antigen receptor of the present invention comprises a transmembrane region. The transmembrane region can form an α-helix, a complex of more than one α-helix, a β-barrel, or any other stable structure capable of spanning the cell phospholipid bilayer. The transmembrane region can be of natural or synthetic origin. The transmembrane region can be derived from CD3ε, CD4, CD5, CD8α, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, the α, β, or ζ chains of cell receptors.

[0089] In some embodiments, the transmembrane region used in the present invention is derived from CD8α. In some embodiments, the CD8α transmembrane region comprises the amino acid sequence of SEQ ID NO: 3.

[0090] The chimeric antigen receptor of the present invention comprises an intracellular region. The intracellular region comprises one or more signal transduction regions, including a co-stimulatory signal transduction region.

[0091] The intracellular signal transduction region is responsible for activating at least one normal effector function of the immune effector cell expressing the chimeric antigen receptor. For example, the effector function of the cell can be cell lysis activity or helper activity, including the secretion of cytokines. Although the entire intracellular signal transduction region can generally be utilized, in many cases, it is not necessary to use the entire chain. In terms of using a truncated portion of the intracellular signal transduction region, such a truncated portion can be used in place of the full chain as long as it transduces the effector function signal. Thus, the intracellular signal transduction region includes any truncated form of the intracellular signal transduction region that is sufficient to transduce the effector function signal. In some embodiments, the signal transduction region is derived from CD3ζ, FcRγ (FCER1G), FcRβ (Fcε Rib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0092] In some embodiments, the signal transduction region used in the present invention is derived from CD3ζ. In some embodiments, the CD3ζ signal transduction region comprises the amino acid sequence of SEQ ID NO: 4.

[0093] In some embodiments, the intracellular region of the chimeric antigen receptor of the present invention further comprises one or more co-stimulatory signaling regions. In addition to the stimulation by antigen-specific signals, many immune effector cells also require co-stimulation to promote cell proliferation, differentiation, and survival, as well as to activate the effector functions of the cells. The "co-stimulatory signaling region" can be the cytoplasmic portion of a co-stimulatory molecule. The term "co-stimulatory molecule" refers to an associated binding partner on an immune cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response, such as but not limited to proliferation and survival, by the immune cell. The co-stimulatory signaling region can be derived from the intracellular signaling regions of CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C, SLP76, TRIM, FcεRIγ, MyD88, and 41BBL; and / or.

[0094] In some embodiments, the co-stimulatory signaling region used in the present invention is derived from CD28 and / or 4-1BB. In some embodiments, the intracellular region of the chimeric antigen receptor of the present invention comprises the CD28 co-stimulatory signaling region and / or the 4-1BB co-stimulatory signaling region described above and the CD3ζ signaling region described above, connected in the N-terminal to C-terminal direction.

[0095] One aspect of the present invention provides at least one polynucleotide, characterized by encoding a chimeric antigen receptor or polypeptide according to the present invention. One aspect of the present invention provides a vector, characterized by comprising a polynucleotide according to the present invention. In one embodiment, the vector is a cloning vector or an expression vector. In one embodiment, the vector is a viral vector.

[0096] One aspect of the present invention provides a vector for cloning and expressing the MSLN-targeted chimeric antigen receptor of the present invention. In some embodiments, the vector is suitable for replication and integration in eukaryotic cells (such as mammalian cells). In some embodiments, the vector is a viral vector. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex virus vectors, and their derivatives.

[0097] Numerous virus-based systems have been developed for gene transfer into mammalian cells. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying a chimeric antigen receptor-encoding sequence can be packaged using protocols known in the art. The resulting lentiviral vectors can be used to transduce mammalian cells using methods known in the art. Vectors derived from lentiviruses are suitable tools for achieving long-term gene transfer because they allow for long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors also have low immunogenicity and can transduce non-proliferating cells.

[0098] One aspect of the invention provides cells that have been genetically modified to contain or express a chimeric antigen receptor of the invention that targets MSLN, such as immune effector cells. In some embodiments, the immune effector cells are T cells, NK cells, peripheral blood mononuclear cells (PBMCs), macrophages, hematopoietic stem cells, pluripotent stem cells, or cells differentiated from embryonic stem cells (e.g., immune cells). In some embodiments, the immune effector cells are autologous. In some embodiments, the immune effector cells are allogeneic.

[0099] An "immune effector cell" is an immune cell that can perform immune effector functions. In some embodiments, immune effector cells express at least FcγRIII and perform ADCC effector functions. Examples of immune effector cells that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, macrophages, cytotoxic T cells, neutrophils, and eosinophils.

[0100] Genetically modified immune effector cells are prepared by introducing a chimeric antigen receptor into the immune effector cells. In some embodiments, the chimeric antigen receptor is introduced into the immune effector cells by transfecting a nucleic acid or vector comprising a sequence encoding the chimeric antigen receptor. In some embodiments, the chimeric antigen receptor is introduced into the immune effector cells by inserting the protein into the cell membrane while passing the cells through a microfluidic system.

[0101] Methods for introducing nucleic acids or vectors into mammalian cells are known in the art. The vectors can be transferred into immune effector cells by physical, chemical or biological methods. Physical methods for introducing vectors into immune effector cells include calcium phosphate precipitation, liposome transfection, particle bombardment, microinjection, electroporation, and the like. Chemical means for introducing nucleic acids or vectors into immune effector cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (including water-in-oil emulsions, micelles, mixed micelles, and liposomes). An exemplary colloidal system used as an in vitro delivery vehicle is a liposome (e.g., an artificial membrane vesicle). Biological methods for introducing nucleic acids or vectors into immune effector cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.

[0102] In some embodiments, the transduced or transfected immune effector cells are propagated ex vivo after the introduction of the nucleic acid or vector. In some embodiments, the transduced or transfected immune effector cells are further evaluated or screened to select the engineered immune effector cells.

[0103] One aspect of the present invention provides a pharmaceutical composition comprising one or more antibodies or antigen-binding fragments, chimeric antigen receptors, or engineered immune effector cells of the present invention, and one or more pharmaceutically acceptable carriers.

[0104] The pharmaceutical composition can be prepared by mixing the active agent having the desired purity with an optional pharmaceutically acceptable carrier in the form of a lyophilized preparation or an aqueous solution. The pharmaceutically acceptable carrier is non-toxic to the recipient at the doses and concentrations used and includes buffers, antioxidants, preservatives, isotonic agents, stabilizers, surfactants, and the like.

[0105] In order for the pharmaceutical composition to be useful for in vivo administration, they must be sterile. The pharmaceutical composition can be made sterile by filtration through a sterile filtration membrane.

[0106] The pharmaceutical composition can contain more than one active agent required for the specific indication to be treated, preferably those having complementary activities that do not adversely affect each other. Alternatively or in addition, the pharmaceutical composition can further contain a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, or a growth inhibitor. Such molecules are present in appropriate combination in an amount effective for the intended purpose.

[0107] Example 1 Construction of anti-MSLN CAR viral vector.

[0108] In this example, the MSLN single-chain antibody was used as the antigen-binding domain of the CAR molecule, which was combined with the CD8α hinge region, the CD8α transmembrane region, and CD3ζ to design anti-MSLN CAR. The amino acid sequence is shown in SEQ ID NO:5, the nucleic acid sequence is shown in SEQ ID NO:10, and the schematic diagram is as Figure 1 shown.

[0109] The nucleic acid sequence of the CD8 hinge region is shown in SEQ ID NO:6.

[0110] The nucleic acid sequence of the anti-MSLN single-chain antibody is shown in SEQ ID NO:7.

[0111] The nucleic acid sequence of the CD8 transmembrane region is shown in SEQ ID NO:8.

[0112] The nucleic acid sequence of CD3ζ is shown in SEQ ID NO:9.

[0113] The anti-MSLN CAR coding gene was synthesized by total gene synthesis. The synthesized CAR molecule coding gene was cloned into the adenovirus vector through steps such as PCR, restriction enzyme digestion, and recombination, and then introduced into the host bacteria and stored in glycerol. Take 100 μL of glycerol bacteria and put them into a 50 mL centrifuge tube, add 25 mL of fresh LB medium, culture them in a shaker at 37 °C and 220 rpm for 16 hours, then extract the plasmid using a mid-scale plasmid miniprep kit, take a small amount of the plasmid for sequencing to check whether its sequence is consistent with the constructed target map as Figure 2 shown, and the remaining plasmids were stored at -20 °C.

[0114] Example 2 Construction of a cell line (SKOV3) overexpressing MSLN.

[0115] The synthesized overexpression vector pCDH-CMV-MSLN-V5-HA-EF1α-puro, the map is as Figure 3 shown. Transfect the overexpression vector into 293T cells, transfect 4 μg of plasmid with Lip2000, collect the cells 48 hours after transfection, and perform WB. The results are as Figure 4 and 5 shown, indicating that the MSLN overexpression vector is effective. Package the virus in 293T cells, collect the group virus to infect SKOV3 cells, and detect the expression of the target protein by WB. Infect SKOV3 cells with the verified lentivirus, add drugs 5 days later, and detect the target protein by WB.

[0116] Example 3 Packaging of anti-MSLN CAR virus and titer detection.

[0117] 1. Packaging of anti-MSLN CAR adenovirus and virus titer detection.

[0118] Example 4 Preparation of anti-MSLN CAR-M cells.

[0119] 1. Preparation of chimeric antigen receptor macrophages specifically targeting MSLN.

[0120] Macrophages (RAW264.7 / miBMDM / hPBMC / THP-1) were infected with anti-MSLN CAR adenovirus to obtain the immune effector cells modified with the chimeric antigen receptor targeting MSLN disclosed in this application, namely anti-MSLN CAR-M.

[0121] 2. To verify that the anti-MSLN CAR-M disclosed in this application is of the M1 phenotype (i.e., the phenotype with phagocytic and killing effects on tumor cells), after adenovirus infection of human macrophages, gene expression was detected by RT-qPCR. Among them, compared with macrophages of the M0 phenotype, hIL-23a, hIL-27, hCXCL9, and hCD68 are up-regulated in macrophages of the M1 phenotype, and hIL-10, hTGF-β, hCCL22, and hCD206 are down-regulated in macrophages of the M2 phenotype. The experimental data are as Figures 6-7 shown.

[0122] As Figure 6 shown, the expression of four cytokines, hIL-23a, hIL-27, hCXCL9, and hCD68, was measured in wild-type, empty vector, and anti-MSLN CAR-M disclosed in this application. The four cytokine expression of the shaded part in No. 1 represents that of hMDM-WT, the four cytokine expression of the blank part in No. 2 represents that of hMDM-Ad5F35-mCheny, and the four cytokine expression of the blank part in No. 3 represents that of hMDM-Ad5F35-EF1a-CAR-MSLN-CD3C-PGK-mCherry. It can be seen that the four cytokine expression of hMDM-Ad5F35-EF1a-CAR-MSLN-CD3C-PGK-mCherry is significantly increased compared with hMDM-WT and hMDM-Ad5F35-mCheny.

[0123] As Figure 7As shown in the figure, the expression levels of four cytokines, namely hIL-10, hTGF-β, hCCL22, and hCD206, were measured in wild-type, empty vector, and anti-MSLN CAR-M disclosed in this application. The expression levels of the four cytokines in hMDM-WT are represented by the shaded part of serial number 1. The expression levels of the four cytokines in hMDM-Ad5F35-mCherry are represented by the blank part of serial number 2. The expression levels of the four cytokines in hMDM-Ad5F35-EF1a-CAR-MSLN-CD3C-PGK-mCherry are represented by the blank part of serial number 3. It can be seen that the expression levels of the four cytokines in hMDM-Ad5F35-EF1a-CAR-MSLN-CD3C-PGK-mCherry are decreased to varying degrees compared with hMDM-WT and hMDM-Ad5F35-mCherry.

[0124] Based on the above experimental data, it can be seen that anti-MSLN CAR-M disclosed in this application can be locked in the M1 state.

[0125] 3. Detect the expression of CAR in hMDM-CAR-MSLN cells using G4S, and the results are as Figures 8-9 shown.

[0126] Anti-MSLN CAR-M cell in vitro and in vivo anti-tumor activity evaluation experiment.

[0127] 1. In vitro killing evaluation.

[0128] (1) Based on the technical solution disclosed in this application, human macrophages (hMDM) were modified to obtain hMDM-CAR-MSLN cells disclosed in this application. A targeted phagocytosis experiment of the hMDM-CAR-MSLN cells on the human metastatic pancreatic cancer cell line (AsPC-1) was carried out, and at the same time, a control group was set up, that is, the phagocytosis experiments of wild-type human macrophages and empty vector human macrophages on the human metastatic pancreatic cancer cell line (AsPC-1). There were three groups in total, namely: hMDM vs AsPC-1 group, hMDM-CAR-MSLN vs ASPC-1 group, and hMDM-Ad-mCherry vs AsPC-1 group. The phagocytosis results were detected at 3h, 6h, and 12h respectively.

[0129] Immunofluorescence detection.

[0130] The flow cytometry detection results at 3h are as Figure 10As shown in the figure, the killing efficiency of the hMDM vs AsPC-1 group was 6.3%, the killing efficiency of the hMDM-CAR-MSLN vs ASPC-1 group was 48.2%, and the killing efficiency of the hMDM-Ad-mCherry vs AsPC-1 group was 12.9%;

[0131] The flow cytometry detection results at 6 h were as Figure 11 shown. The killing efficiency of the hMDM vs AsPC-1 group was 12.1%, the killing efficiency of the hMDM-CAR-MSLN vs ASPC-1 group was 42.8%, and the killing efficiency of the hMDM-Ad-mCherry vs AsPC-1 group was 17.1%;

[0132] The flow cytometry detection results at 12 h were as Figure 12 shown. The killing efficiency of the hMDM vs AsPC-1 group was 19.4%, the killing efficiency of the hMDM-CAR-MSLN vs ASPC-1 group was 37.5%, and the killing efficiency of the hMDM-Ad-mCherry vs AsPC-1 group was 26.3%. From the above experimental results, it can be seen that the hMDM-CAR-MSLN cells prepared based on the disclosed technical solution of the present application have a better killing effect on the human metastatic pancreatic cancer cell line (AsPC-1), achieving better killing of pancreatic cancer cells and providing an effective method for the treatment of pancreatic cancer.

[0133] Detect cytokine secretion.

[0134] Prime CD8-T cell detection.

[0135] 2. In vivo killing evaluation, that is, the anti-tumor effect of hMDM-CAR-MSLN cells in an animal model in vivo.

[0136] Male Balb / c-nuc mice at 4-5 weeks of age were selected and injected with MSLN-positive tumor cells (human metastatic pancreatic cancer cells) to establish a tumor model. After tumor modeling, hMDM-CAR-MSLN cells and Matrigel were subcutaneously injected for treatment at a ratio of 1:1. The tumor growth was detected by a small animal in vivo imaging system.

Claims

1. A chimeric antigen receptor-modified immune effector cell targeting MSLN, characterized in that, The chimeric antigen receptor comprises an antigen-binding domain, a hinge region, a transmembrane domain, and a signal transduction domain, and the antigen-binding domain is a single-chain antibody against MSLN.

2. The chimeric antigen receptor-modified immune effector cells targeting MSLN according to claim 1, characterized in that, The hinge region comprises CD8.

3. The chimeric antigen receptor-modified immune effector cell targeting MSLN according to claim 1, wherein The transmembrane domain comprises the CD8 transmembrane region.

4. The targeted MSLN chimeric antigen receptor-modified immune effector cells according to claim 1, characterized in that, The signal transduction domain comprises CD3ζ.

5. The chimeric antigen receptor-modified immune effector cell targeting MSLN according to claim 1, wherein The signal transduction domain further comprises any one or a combination of at least two of 4-1BB, the intracellular region of CD28, DAP10, or OX40.

6. The chimeric antigen receptor-modified immune effector cells targeting MSLN according to claim 1, wherein The chimeric antigen receptor comprises a single-chain antibody against MSLN, a CD8 hinge region, a CD8 transmembrane region, and CD3ζ.

7. The chimeric antigen receptor-modified immune effector cells targeting MSLN according to claim 1, wherein The antigen-binding domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:

2.

8. The chimeric antigen receptor-modified immune effector cell targeting MSLN according to claim 1, wherein The hinge region comprises the amino acid sequence shown in SEQ ID NO:

1.

9. The targeted MSLN chimeric antigen receptor-modified immune effector cell according to claim 1, wherein The transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:

3.

10. The chimeric antigen receptor-modified immune effector cell targeting MSLN according to claim 1, wherein The signal transduction domain comprises the amino acid sequence shown in SEQ ID NO:

4.

11. The chimeric antigen receptor-modified immune effector cell targeting MSLN according to claim 1, wherein The immune effector cells are macrophages and peripheral blood mononuclear cells.

12. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises the coding gene of the chimeric antigen receptor according to any one of claims 1-11.

13. An expression vector, characterized in that, The expression vector comprises the nucleic acid molecule according to claim 12.

14. The expression vector according to claim 13, characterized in that, The expression vector is a lentiviral vector or an adenoviral vector containing the nucleic acid molecule according to claim 12.

15. A recombinant lentivirus or adenovirus, characterized in that, The recombinant lentivirus or adenovirus is prepared from mammalian cells transfected with the expression vector according to claim 13 or 14 and an auxiliary plasmid.

16. The chimeric antigen receptor-modified immune effector cell according to any one of claims 1-11, characterized in that, The nucleic acid molecule according to claim 12 is integrated into its genome.

17. The chimeric antigen receptor-modified immune effector cell according to any one of claims 1-11, characterized in that, The chimeric antigen receptor macrophages comprise the expression vector according to claim 13 or 14 and / or the recombinant lentivirus and / or adenovirus according to claim 15.

18. The method for preparing the chimeric antigen receptor-modified immune effector cells according to claim 17, wherein The preparation method comprises the step of introducing the expression vector according to claim 12 or 13 and / or the recombinant lentivirus and / or adenovirus according to claim 14 into immune effector cells.

19. The method for preparing the chimeric antigen receptor-modified immune effector cells according to claim 18, characterized in that, The preparation method specifically comprises the following steps: (1) Synthesize the nucleic acid sequences of the respective domains of the transmembrane expression protein or polypeptide macromolecule; (2) ligate the target fragment with a lentiviral or adenoviral overexpression vector to obtain a recombinant plasmid vector; (3) extraction and preparation of the recombinant plasmid vector and the auxiliary plasmid vector; (4) virus packaging; (5) construction of a functional cell line.

20. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the immune effector cells modified with the chimeric antigen receptor according to any one of claims 1-11, 16, and 17.

21. The pharmaceutical composition according to claim 20, wherein It further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient, or diluent.

22. Use of the immune effector cells modified with the chimeric antigen receptor according to any one of claims 1-11, 16, and 17, the nucleic acid molecule according to claim 11, the expression vector according to claim 13 or 14, the recombinant lentivirus and / or adenovirus according to claim 15, or the pharmaceutical composition according to claim 20 or 21 in the preparation of a drug for treating MSLN-positive tumors.

23. The application according to claim 22, wherein The MSLN-positive tumors are mesothelioma, pancreatic cancer, or ovarian cancer.

Images