Targeted psma chimeric antigen receptor macrophages, methods of making and uses thereof

Abstract

The present application relates to the technical field of cellular immunotherapy, and particularly relates to a kind of targeting PSMA chimeric antigen receptor immune cells and preparation method and application thereof.A kind of targeting PSMA chimeric antigen receptor modified immune effector cell, the chimeric antigen receptor includes antigen binding domain, hinge region, transmembrane domain and signal transduction domain, and the antigen binding domain is anti-PSMA single-chain antibody.The chimeric antigen receptor modified immune effector cell provided in the present application can specifically recognize and combine the antigen binding domain of PSMA, and the prepared chimeric antigen receptor macrophage has specific killing effect on the target cells positive for PSMA protein, and can be used for preparing tumor drugs for treating or preventing PSMA positive.

Description

Technical Field

[0001] This invention relates to the field of cell immunotherapy technology, specifically to a PSMA-targeting chimeric antigen receptor immune cell, its preparation method, and its application. Background Technology

[0002] Prostate cancer is the most common malignant tumor of the male reproductive system. In the United States, it ranks first in incidence and second in mortality among all malignant tumors, after lung cancer. Although the incidence of prostate cancer in my country is much lower than in Western countries, it has shown a significant upward trend in recent years.

[0003] PSMA (Prostate-specific membrane antigen), officially named folic acid hydrolase 1 (FOLH1), is a biomarker located on the exterior of prostate cancer cells. It belongs to the M28 peptidase family and is a type II transmembrane glycoprotein containing 751 amino acids. It consists of an extracellular C-terminus, a helical transmembrane structure, and a cytoplasmic N-terminus. The first two amino acids at the N-terminus may be cleaved by proteolytic activity, and it lacks a signal peptide. PSMA is normally expressed in the renal tubules and duodenum, and specifically in prostate epithelial cells, playing a crucial role in the processing and uptake of dietary folic acid. In prostate cancer tissue, PSMA expression is significantly upregulated. PSMA is a non-secretory transmembrane glycoprotein located on the cell surface. It possesses carboxypeptidase and folic acid hydrolase activities and has a large extracellular domain, comprising an intracellular, transmembrane, and extracellular region. The extracellular region accounts for 95% of the PSMA protein and is the primary site of action for small molecules and antibodies. The extracellular region of PSMA can be recognized by antibodies, peptides, RNA aptamers, and small molecules, making it an ideal target for targeted therapy. Studies show that PSMA is overexpressed on the surface of over 90% of prostate cancer cells (100 to 1000 times higher than normal prostate cells), and its expression level is even higher in cancer cells from advanced and castration-resistant prostate cancer patients. According to current data analysis, PSMA-targeted therapy has become a hot research topic. Normal prostate cells have relatively little PSMA, but as cells become cancerous, grow faster, and begin to infiltrate surrounding tissues, more and more PSMA appears on the surface of tumor cells. Furthermore, research indicates that PSMA may become a new biomarker for early screening of prostate cancer and a new therapeutic target.

[0004] Currently, there are no highly effective treatments for malignant tumors like prostate cancer. Immunotherapy, which has developed in recent years, is the only method with the potential to completely eliminate cancer cells, overcoming the shortcomings of traditional therapies and considered one of the most promising treatment methods in the 21st-century comprehensive cancer treatment paradigm. Chimeric Antigen Receptor-Modified T Cells (CAR-T), as one of the latest immunotherapy technologies, has received widespread attention and research because it can activate the body's own immune system to continuously target and kill tumor cells, ultimately achieving complete elimination of malignant tumor cells. However, the current application of CAR-T technology is limited to hematologic malignancies; its application in various PSMA-positive solid tumors has not yet been studied. Summary of the Invention

[0005] Chimeric antigen receptor (CAR) macrophages are macrophages that express chimeric receptors on their surface that recognize specific antigens and transmit signals intracellularly. The core component is the CAR molecule carrying a single-chain antibody (scFv) that specifically recognizes cell surface antigens, thereby recognizing and killing cells expressing specific antigens. Currently, CAR-M cell therapy is mainly used in cancer treatment, showing some efficacy in certain solid tumors, but there are few reports of its application in treating prostate cancer. In summary, the development of engineered immune response cells modified with a specific chimeric antigen receptor that targets PSMA shows great promise. These engineered cells transmit activation signals and activate the immune system by highly specifically recognizing PSMA produced on the surface of tumor cells, exerting a killing effect on tumor cells with good safety, thus achieving good clinical therapeutic effects.

[0006] In view of the existing problems and / or other issues in related technologies, the purpose of this invention is to overcome the problems of low specificity and low safety of effector cells in the tumor microenvironment in binding to and killing tumor cells in existing clinical oncology techniques. This invention provides a chimeric antigen receptor specifically targeting PSMA, its gene and recombinant expression vector, engineered immune effector cells modified with the chimeric antigen receptor specifically targeting PSMA, and their applications. The immune effector cells modified with the chimeric antigen receptor specifically targeting PSMA of this invention kill tumor cells with high specificity and good safety, thus providing a promising new approach to tumor treatment.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] An immune effector cell targeting PSMA chimeric antigen receptor modification, wherein the chimeric antigen receptor includes an antigen-binding domain, a hinge region, a transmembrane domain, and a signal transduction domain, wherein the antigen-binding domain is an anti-PSMA single-chain antibody.

[0009] Furthermore, the hinge region includes CD8.

[0010] Furthermore, the transmembrane structural domain includes the CD8 transmembrane region.

[0011] Furthermore, the signal conduction structure domain includes CD3ζ.

[0012] Furthermore, the signal transduction domain also includes any one or a combination of at least two of 4-1BB, CD28 intracellular region, DAP10, or OX40.

[0013] Preferably, the chimeric antigen receptor includes an anti-PSMA single-chain antibody, a CD8 hinge region, a CD8 transmembrane region, and CD3ζ.

[0014] Preferably, the antigen-binding domain of the chimeric antigen receptor includes the amino acid sequence shown in SEQ ID NO:2.

[0015] Preferably, the hinge region comprises the amino acid sequence shown in SEQ ID NO:1.

[0016] Preferably, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:3.

[0017] Preferably, the signal transduction domain includes the amino acid sequence shown in SEQ ID NO:4.

[0018] Furthermore, the immune effector cells are macrophages and peripheral blood mononuclear cells.

[0019] A nucleic acid molecule comprising the encoding gene of the chimeric antigen receptor.

[0020] An expression vector comprising the aforementioned nucleic acid molecule.

[0021] Furthermore, the expression vector is a lentiviral vector or adenovirus vector containing the aforementioned nucleic acid molecules.

[0022] A recombinant lentivirus or adenovirus, said recombinant lentivirus or adenovirus being prepared from mammalian cells transfected with the expression vector and helper plasmid.

[0023] The chimeric antigen receptor-modified immune effector cells have the aforementioned nucleic acid molecules integrated into their genome.

[0024] The chimeric antigen receptor-modified immune effector cells include the expression vector and / or the recombinant lentivirus and / or adenovirus.

[0025] The method for preparing chimeric antigen receptor-modified immune effector cells includes the step of introducing the expression vector and / or the recombinant lentivirus and / or adenovirus into immune effector cells.

[0026] Furthermore, the method for preparing chimeric antigen receptor-modified immune effector cells specifically includes the following steps.

[0027] (1) Synthesize the nucleic acid sequences of each domain of transmembrane expressed protein or polypeptide macromolecule.

[0028] (2) The target fragment is ligated with a lentivirus or adenovirus 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 comprising the chimeric antigen receptor-modified immune effector cells.

[0033] Furthermore, the pharmaceutical composition further includes any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient, or diluent.

[0034] The use of the chimeric antigen receptor-modified immune effector cells, the nucleic acid molecules, the expression vector, the recombinant lentivirus and / or adenovirus, or the pharmaceutical composition in the preparation of a drug for treating PSMA-positive tumors.

[0035] Furthermore, the PSMA-positive tumor is prostate cancer.

[0036] Through creative work, the inventors continuously designed amino acid sequences and performed sequence arrangements and screenings. They conducted random screening experiments and targeted function verification on more than 100 CAR molecule sequences (such as constructing viral vectors, further infecting macrophages to obtain modified macrophage cells, and testing the in vitro killing activity of the modified macrophages). After comparing the results of multiple random combinations, they adjusted the sequences and finally screened out one sequence with better performance, thus obtaining the high-potency PSMA-targeting scFv amino acid sequence and its functional variant of the present invention.

[0037] Genetic modification of immune effector cells (e.g., macrophages, monocytes, T cells, CTL cells, NK cells) can be achieved by transducing homologous cellular compositions using recombinant DNA or RNA constructs. In one embodiment, the vector is a viral vector (adenovirus, retrovirus, or lentivirus) that can introduce the DNA or RNA construct into the host cell genome. For example, polynucleotides that specifically target chimeric antigen receptors for PSMA can be cloned into retroviral vectors and can be driven for expression from their endogenous promoters, retroviral long terminal repeat sequences, or alternative internal promoters.

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

[0039] The vector is selected from retroviral vectors, lentiviral vectors, and adenovirus vectors.

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

[0041] In some embodiments, the immune effector cells are cytotoxic T lymphocytes, NK cells, NKT cells, 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, or retrovirus.

[0044] Preferably, the virus is an adenovirus.

[0045] An isolated modified immune response cell containing the chimeric antigen receptor, obtained by transformation of the recombinant vector or expression plasmid.

[0046] For the initial genetic modification of cells to provide chimeric antigen receptor modifications specifically targeting PSMA, transduction is typically performed using adenovirus or lentiviral vectors; however, any other suitable viral vector or non-viral delivery system may be used. For subsequent genetic modification of cells to provide cells containing an antigen-presenting complex containing at least one co-stimulatory ligand, retroviral gene transfer (transduction) has also proven effective. The combination of a retroviral vector and a suitable assembly line is also appropriate, wherein 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-culturing of cells with production cells. Transduction viral vectors can be used to express co-stimulatory ligands (e.g., 4-1BBL and IL-12) in immune-responding cells. Preferably, the selected vector exhibits high infection efficiency and stable integration and expression.

[0049] In some embodiments, preferably, the at least one costimulatory ligand is selected from 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14 and combinations thereof, or more preferably, the costimulatory 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 patients can be transduced using vectors for CAR expression.

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

[0053] Genetically modified chimeric antigen receptors can be prepared using the aforementioned specific targeting method and then stored under cold storage.

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

[0055] Step 1: The nucleic acid molecule is ligated into an expression vector by molecular cloning to obtain the expression vector of the chimeric antigen receptor that specifically targets PSMA.

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

[0057] Step 3: Infect immune response cells with the viral fluid to obtain immune response cells expressing the chimeric antigen receptor modified to specifically target PSMA from the infected cells.

[0058] In some non-limiting embodiments, the immune response cells modified by the present invention may be macrophages, peripheral blood mononuclear cells, or cells of lymphoid lineages. The lymphoid lineage cells 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 foreign cells in the host. Non-limiting examples of lymphoid lineage cells 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 cell is a macrophage.

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

[0061] The pharmaceutical composition disclosed in this application comprises isolated modified immune response cells expressing the chimeric antigen receptor that specifically targets PSMA and a pharmaceutically acceptable carrier.

[0062] The administration of the pharmaceutical composition may be autologous or non-autologous. For example, immune response cells expressing the chimeric antigen receptor specifically targeting PSMA and the composition comprising them may be obtained from a subject and administered to the same subject or different compatible subjects. Peripheral blood-derived cells or their progeny (e.g., in vivo, in vitro, or in vitro derived) of the subject matter of this invention may be administered via catheter administration, intravenous injection, or parenteral administration. When administering a pharmaceutical composition of the subject matter of this invention (e.g., a pharmaceutical composition comprising the chimeric antigen receptor specifically targeting PSMA), it is typically formulated into a unit-dose injectable form (solution, suspension, emulsion).

[0063] The compositions of this application can be formulations. The immune response cells expressing the chimeric antigen receptor (CAR) specifically targeting PSMA disclosed in this application and the compositions comprising them can be readily provided as sterile liquid formulations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which can be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are more convenient to administer, particularly by injection. On the other hand, viscous compositions can be formulated within a suitable viscosity range to provide a longer contact time with a specific tissue. Liquid or viscous compositions may contain a carrier, which can be a solvent or dispersion medium comprising, for example, water, physiological saline, phosphate-buffered saline, polyols (e.g., 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 this application, any carrier, diluent, or additive used must be compatible with immune-responding cells expressing the specific PSMA-targeting chimeric antigen receptor (CAR) described in this invention.

[0066] If necessary, the viscosity of the composition can be maintained at a selected level using a pharmaceutically acceptable thickener. 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 or liquid-filled form).

[0067] The present invention relates to chimeric antigen receptor-modified immune response cells that specifically target PSMA. These engineered cells transmit activation signals and activate the immune system by recognizing PSMA 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] This invention utilizes chimeric antigen receptor-modified macrophage technology to prepare engineered immune cells that specifically target PSMA. The preparation method is simple, and the resulting novel engineered immune cells can specifically recognize tumor cells, more effectively target and attack tumor cells, exhibiting a high tumor-killing rate. Furthermore, these cells can be used to prepare anti-tumor products, particularly drugs for treating PSMA-positive tumors. This invention holds promise for the preparation of anti-tumor products, especially immunocellular therapy drugs for treating prostate cancer, and has excellent industrial application prospects. Attached Figure Description

[0070] Figure 1 This is a schematic diagram of the structure of the anti-PSMACAR carrier.

[0071] Figure 2 This is a map of the PSMACARM vector (pAd5F35-EF1a-CAR-PSMA-CD3ζ-PGK-mCherry).

[0072] Figure 3 This is a map of the PSMA overexpression vector (pCDH-CMV-MCS-PSMA-V5-puro).

[0073] Figure 4 The image shows the fluorescence detection results of the PSMA overexpression vector.

[0074] Figure 5 This is a WB detection result image of the PSMA overexpression vector.

[0075] Figure 6 This is a flow cytometry analysis of macrophage cell killing assays using chimeric antigen receptors that specifically target PSMA, as disclosed in this application.

[0076] Figure 7 BC represents the phenotypic results of BMDM infected with Ad5f35-PSMA-CAR, and A represents the killing effect of Ad5f35-PSMA-CAR on LNCaP in BMDM 6 hours after infection.

[0077] Figure 8 BC represents the phenotypic results of hMDM infected with Ad5f35-PSMA-CAR, and A represents the killing effect of hMDM infected with Ad5f35-PSMA-CAR on LNCaP 6h. Detailed Implementation

[0078] The present invention will be further illustrated by the following examples, but the present invention is not limited to these specific embodiments. Unless otherwise specified, the materials, reagents, etc. used in the following examples are commercially available.

[0079] One aspect of the present invention provides a chimeric antigen receptor (CAR) targeting PSMA. The chimeric antigen receptor generally comprises 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.

[0080] When expressed in cells, the chimeric antigen receptor polypeptide 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 directs the polypeptide to a desired site in the cell. In some embodiments, the signal peptide directs the polypeptide to a secretory pathway in the cell and will allow the polypeptide to integrate and anchor to the lipid bilayer.

[0081] The chimeric antigen receptor of the present invention comprises an antigen-binding region targeting PSMA. The antigen-binding region may be monovalent or multivalent (e.g., bivalent). The antigen-binding region may also be monospecific or multispecific (e.g., bispecific). Bispecificity may be against PSMA and another antigen, or it may be against two different epitopes of PSMA.

[0082] In some embodiments, the antigen-binding region used in this invention is the anti-PSMA antibody or antigen-binding fragment described above, particularly in the form of scFv.

[0083] 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 typically exists between two domains of a protein and allows for the flexibility of the protein and relative movement of the two domains relative to each other.

[0084] 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 IgG, IgA, IgM, IgE, or IgD antibodies) can also be used as the chimeric antigen receptor described herein. Non-naturally occurring peptides 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.

[0085] In some embodiments, the hinge region used in this invention is derived from CD8α. In some embodiments, the CD8α hinge region contains the amino acid sequence of SEQ ID NO: 1.

[0086] The chimeric antibody receptor of the present invention includes 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 translocating the cellular phospholipid bilayer. The transmembrane region can be of natural or synthetic origin. The transmembrane region can be derived from the α, β, or ζ chains of CD3ε, CD4, CD5, CD8α, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or the α, β, or ζ chains of the cellular receptor.

[0087] In some embodiments, the transmembrane region used in this invention is derived from CD8α. In some embodiments, the CD8α transmembrane region comprises the amino acid sequence of SEQ ID NO: 3.

[0088] The chimeric antigen receptor of the present invention comprises an intracellular region. The intracellular region includes one or more signal transduction regions, including a co-stimulatory signal transduction region.

[0089] Intracellular signaling regions are responsible for activating at least one normal effector function of immune effector cells expressing chimeric antigen receptors. For example, a cell's effector function could be cytolytic activity or cofactor activity, including cytokine secretion. While the entire intracellular signaling region can generally be used, in many cases, using the whole chain is unnecessary. Regarding the use of truncated portions of intracellular signaling regions, such truncated portions can be used in place of the whole chain as long as they transduce effector function signals. Therefore, intracellular signaling regions include any truncated form of an intracellular signaling region sufficient to transduce effector function signals. In some embodiments, the signaling region is derived from CD3ζ, FcRγ (FCER1G), FcRβ (FcεRib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0090] In some embodiments, the signal conduction region used in this invention is derived from CD3ζ. In some embodiments, The CD3ζ signal transduction region contains the amino acid sequence of SEQ ID NO: 4.

[0091] In some embodiments, the intracellular region of the chimeric antigen receptor of the present invention further includes one or more co-stimulatory signaling regions. In addition to stimulation by antigen-specific signals, many immune effector cells require co-stimulation to promote cell proliferation, differentiation, and survival, as well as to activate effector functions. 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 chaperone on an immune cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response mediated by the immune cell, such as, but not limited to, proliferation and survival. The co-stimulatory signal transduction region may originate 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.

[0092] In some embodiments, the co-stimulatory signaling regions used in this invention are derived from CD28 and / or 4-1BB. In some embodiments, the intracellular region of the chimeric antigen receptor of this invention comprises the aforementioned CD28 co-stimulatory signaling region and / or 4-1BB co-stimulatory signaling region and the aforementioned CD3ζ signaling region connected in an N-terminal to C-terminal direction.

[0093] One aspect of the invention provides at least one polynucleotide characterized in that it encodes a chimeric antigen receptor or polypeptide according to the invention. Another aspect of the invention provides a vector characterized in that it comprises the polynucleotide according to the invention. In one embodiment, the vector is a cloning vector or an expression vector. In one embodiment, the vector is a viral vector.

[0094] One aspect of the invention provides a vector for cloning and expressing the chimeric antigen receptor targeting PSMA of the invention. In some embodiments, the vector is adapted to replicate and integrate 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, adenovirus vectors, adeno-associated virus vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof.

[0095] Numerous virus-based systems have been developed for gene transfer into mammalian cells. In some embodiments, lentiviral vectors are used. In other embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying chimeric antigen receptor coding sequences can be packaged using methods known in the art. The resulting lentiviral vectors can be used to transduce mammalian cells using methods known in the art. Lentiviral vectors are suitable tools for achieving long-term gene transfer because they allow for the long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors also exhibit low immunogenicity and can transduce non-proliferating cells.

[0096] One aspect of the invention provides genetically modified cells, such as immune effector cells, that contain or express the chimeric antigen receptor targeting PSMA of the present invention. In some embodiments, the immune effector cells are cells cultured and differentiated from T cells, NK cells, peripheral blood mononuclear cells (PBMCs), macrophage hematopoietic stem cells, pluripotent stem cells, or 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.

[0097] "Immune effector cells" are immune cells capable of performing immune effector functions. In some implementations, 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.

[0098] Genetically modified immune effector cells are prepared by introducing chimeric antigen receptors into immune effector cells. In some embodiments, the chimeric antigen receptor is introduced into immune effector cells by transfecting a nucleic acid or vector containing a sequence encoding the chimeric antigen receptor. In some embodiments, the chimeric antigen receptor is introduced into immune effector cells by inserting a protein into the cell membrane while simultaneously allowing the cells to pass through a microfluidic system.

[0099] 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 methods 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 oil-in-water emulsions, micelles, mixed micelles, and liposomes). An exemplary colloidal system used as an in vitro delivery medium 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 cells, such as human cells.

[0100] In some embodiments, the transduced or transfected immune effector cells proliferate in vitro after the introduction of nucleic acids or vectors. In some embodiments, the transduced or transfected immune effector cells are further evaluated or screened to select modified immune effector cells.

[0101] One aspect of the invention provides a pharmaceutical composition comprising one or more antibodies or antigen-binding fragments of the invention, chimeric antigen receptors or modified immune effector cells, and one or more pharmaceutically acceptable carriers.

[0102] Pharmaceutical compositions can be prepared by mixing an active pharmaceutical agent of desired purity with an optional pharmaceutically acceptable carrier in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are non-toxic to the recipient at the dose and concentration used and include buffers, antioxidants, preservatives, isotonic agents, stabilizers, surfactants, etc.

[0103] For pharmaceutical compositions to be usable for in vivo administration, they must be sterile. Sterility of pharmaceutical compositions can be achieved through filtration using sterile filter membranes.

[0104] The pharmaceutical composition may contain more than one active pharmaceutical agent required for the specific indication to be treated, preferably those having complementary activities that do not adversely affect each other. Alternatively or additionally, the pharmaceutical composition may also contain cytotoxic agents, chemotherapeutic agents, cytokines, immunosuppressants, or growth inhibitors. Such molecules are appropriately combined in amounts effective for the intended purpose.

[0105] Example 1: Construction of PSMA overexpression vector.

[0106] The PSMA sequence was constructed in the synthetic overexpression vector pCDH-CMV-MCS-EF1-Puro, with a V5 tag added to the C-terminus. The resulting graph is shown below. Figure 3 As shown. Plasmid was extracted, with a mini-prep concentration of 284.276 ng / UI and OD... 260 / 280 =1.91.

[0107] The overexpression vector was transfected using 293T, and 2.1 μg of plasmid was transfected with calcium phosphate. Cells were harvested 48 h after transfection and Western blot was performed. The results are as follows: Figure 4 and 5 As shown, PMSA undergoes glycosylation, resulting in a protein size between 90-120 KD. PSMA overexpression vectors are effective. Calcium phosphate transfection method (using a 12-well plate as an example): 1) Pre-layer the 12-well plate (50% density); 2) Replace with 570 μl of serum-containing medium; 3) Place 36 μL of 1×HBS in a 1.5 ml EP tube, add 2.1 μg of plasmid, mix by pipetting, then add 2.1 μl of 2.5 M CaCl2, mix by pipetting, and incubate at room temperature for 15 min; 4) Pipette the mixture, holding the pipette close to the liquid surface, very slowly, and add it dropwise into the wells in a cloverleaf pattern, immediately gently shaking to mix, and incubate at 37°C; 5) Replace with 1 ml of fresh serum-containing medium after overnight incubation.

[0108] 293T lentivirus was packaged, and the virus was used to infect 293T cells. PSMA expression was detected by Western blotting.

[0109] The method for packaging lentiviruses with Lipo2000 is as follows.

[0110] Day 1 Transfection: (1) Before transfection, 293T cells were cultured in medium (DMEM, 10% FBS) to about 70%; (2) Change medium before transfection: Discard the old culture medium and add 1.5 mL of fresh serum-free DMEM medium. (3) Transfection (one 35 mm dish): ① Prepare a 1.5 mL EP tube, add 16 μL of Lipo 2000 and 250 μL of serum-free DMEM medium, mix well and label; prepare a 1.5 mL EP tube and add DNA mixes according to the following system: psPAX2 3 μg, pMD2.G 1 μg, target plasmid 4 μg, and 250 μL of serum-free DMEM medium. ② After adding DNA mixes, mix with pipette tip and let stand for 5 min; ③ Gently and slowly add DNA mixes to Lipo 2000 mixture, gently mix with pipette tip, and let stand at room temperature for 20 min; ④ Add Lipo-DNA mix dropwise to cell culture plate, gently mixing while adding; ⑤ Continue to keep cells at 37℃ for 5% Incubate in a CO2 incubator, and replace with serum-containing complete culture medium after 4-6 hours.

[0111] Day 2 Virus collection: 24h post-transfection (operated on ice): Collect culture medium into a 15mL centrifuge tube, add 2mL of culture medium to the original culture plate and continue culturing for 48h; centrifuge the collected culture medium at 4000g for 10min at 4℃ (to remove cell debris).

[0112] Day 3 Virus collection: 48 hours after transfection (operated on ice), collect the culture medium into a 15mL centrifuge tube, add 4mL of culture medium to the original culture plate and continue culturing for 72 hours; centrifuge the collected culture medium at 4000g for 10 minutes at 4℃ (to remove cell debris).

[0113] Day 4 Virus collection: 72 hours after transfection (operated on ice), collect the culture medium into a 15mL centrifuge tube, centrifuge at 4000g for 10min at 4℃.

[0114] After validation, LNCaP prostate cancer cells were infected with lentivirus. Five days later, puro screening was performed, and a monoclonal cell line overexpressing PSMA protein was constructed using the cell dilution method. Western blotting was used to detect PSMA protein expression in tumor cells.

[0115] Example 2: Construction of anti-PSMACAR viral vector.

[0116] This embodiment uses a PSMA single-chain antibody as the antigen-binding domain of the CAR molecule, which binds to the CD8α hinge region, CD8α transmembrane region, and CD3ζ to design an anti-PSMA antibody. The amino acid sequence is shown in SEQ ID NO:5, and the nucleic acid sequence is shown in SEQ ID NO:10. A schematic diagram is shown below. Figure 1 As shown.

[0117] The nucleic acid sequence of the CD8 hinge region is shown in SEQ ID NO:6.

[0118] The nucleic acid sequence of the anti-PSMA single-chain antibody is shown in SEQ ID NO:7.

[0119] The nucleic acid sequence of the CD8 transmembrane region is shown in SEQ ID NO:8.

[0120] The nucleic acid sequence of CD3ζ is shown in SEQ ID NO:9.

[0121] The anti-PSMACAR encoding gene (CAR-PSMA-CD3ζ-, specifically Xba I-SP(hCD8)-Myc-EcoR-anti-Hpsma-scFv-Sbfl-Hinge / TM(hCD8)-Xho I-CD3ζ-BamHI) was synthesized in its entirety. The vector was linearized by enzyme digestion and recovered via ethanol precipitation. The plasmid was then extracted using a plasmid mini-prep kit, and a small sample was sequenced to verify its sequence consistency with the constructed target map. Figure 2 Consistent with the above, the remaining plasmids were stored at -20℃. Extraction of cellular genomic DNA – Ethanol precipitation method: (Preparation: Thaw PK on ice beforehand (approximately 2 hours). Adjust the metal bath to 56℃ and observe whether the lysis buffer crystallizes; if crystallization occurs, thaw it beforehand. 75% ethanol should be prepared fresh for use.) 1) Collect cells into 1.5ml EP tubes and centrifuge at 500rcf for 5 minutes at room temperature. 2) Resuspend in 500μl PBS and centrifuge again at 500rcf for 5 minutes at room temperature (purpose: to wash away residual culture medium). 3) Resuspend in PBS and aliquot into new EP tubes according to cell quantity and requirements. 4) Add 400μl lysis buffer and 10μl PK to each tube, vortex for 30 seconds, and incubate in a 56℃ metal bath for 4 hours. 5) Remove and allow to stand; adjust the metal bath to 95℃. Once the temperature reaches 95℃, transfer the EP tubes to the metal bath and incubate again for 10 minutes. 6) Remove and allow to stand until returned to room temperature. 7) Add 200 μl NaCl (5M), vortex for 1 min, and incubate on ice for 10 min. 8) Centrifuge at 12000 rcf at room temperature for 10 min. 9) Carefully pour the supernatant into a new EP tube, centrifuge again at room temperature, 12000 rcf, for 5 min. 10) Take 500 μl of the supernatant and add 1 ml (twice the volume) of anhydrous ethanol. 11) Mix well, centrifuge at 12000 rcf at room temperature for 10 min. 12) Discard the supernatant, add 1 ml of 75% ethanol to wash, and centrifuge at 12000 rcf for 10 min. 13) Discard the supernatant and allow to air dry for 6 min. 14) Add 30 μl of sterile water and incubate for 30 min. 15) Measure the concentration: small-particle concentration 348.740 ng / UI, OD 260 / 280 =1.828.

[0122] Example 3: Packaging, purification, and titer detection of anti-PSMACAR virus.

[0123] The specific steps for adenovirus packaging and concentration are as follows.

[0124] Step 1: Prepare the linearized Ad5F35 plasmid and 293A cells in advance. 24 hours before transfection, seed 293A cells onto 35mm cell culture dishes, ensuring 80-90% confluence and good cell condition. Use the following transfection plasmid: 4μg of the empty Ad5F35 vector plasmid. Remove Lipo2000 from the 4℃ freezer and allow it to reach room temperature before use, shaking well before application. Replace the medium with 1mL of serum-free medium before transfection. Add 4μg of plasmid to 125μL of serum-free medium, mix well, and incubate at room temperature for 5 minutes. Add 10μL of Lipo2000 to 125μL of serum-free medium, mix well, and incubate at room temperature for 5 minutes. Add the plasmid solution to the Lipo2000 solution, mix thoroughly, and incubate at room temperature for 20 minutes. Then, add the above transfection reagent to the 293A cells. Replace with fresh complete medium 6 hours after transfection. Observe the cells. Change the medium every other day. When viral plaques appear (generally after about 10 days), collect the cells. Centrifuge at 1000 rpm for 5 min at room temperature, discard the supernatant, add 2 mL of fresh complete culture medium, and store at -80°C. Repeat the freeze-thaw cycle of the above cell suspension at -80°C / water three times, thoroughly Vortexing each time to obtain crude virus. Centrifuge at 7000 rpm for 5 min at 4°C, discard the supernatant, add 2 mL of fresh complete culture medium, and store at -80°C for later use.

[0125] Step 2: Collect adenovirus-infected cells (three 10cm dish volumes of culture medium per column): Transfer 10mL of culture supernatant to a new 15mL centrifugal tube, leaving 5mL of supernatant. Collect the cells from the culture dish with a scraper, and transfer the cells and supernatant to another new 15mL centrifugal tube. Freeze in a dry ice / ethanol mixture, then thaw at 37°C, repeating this process three times. Mix the lysis buffer with the 10mL supernatant. Centrifuge at 3,000rpm for 10min at 4°C, collect the supernatant, and filter through a 0.45μm filter. The filtered supernatant can be used for purification or stored at -80°C for later use.

[0126] Step 3: Equilibrate the column: Dilute 10×AVWash Buffer with ddH2O to make 1×AV Wash Buffer; dilute 2×AV Elution Buffer with ddH2O to make 1×AV Elution Buffer; place the AV Mini Column in a 15 mL Centrifugal Tube and centrifuge at 500×g for 2 min at 4°C. Secure the AV Mini Column with clamps or other supports, break off the bottom tip, loosen the cap, and allow the liquid to drain from the AV Mini Column by gravity. Once the liquid stops dripping, slowly add 2 mL of ddH2O. After the solution flows out of the AV Mini Column, add 5 mL of 1×AV Wash Buffer and continue to allow the solution to flow away.

[0127] Step 4, Column Loading: Transfer 5 mL of supernatant to the AV Mini Column and allow it to flow through under gravity. Continue adding supernatant to the AV Mini Column until all supernatant has passed through.

[0128] Step 5, Washing and Elution: Add 5 mL of 1×AV Wash Buffer to the AV Mini Column and repeat once. Centrifuge by gravity flow or at 4°C, 1,000×g for 5 min; add 4 mL of 1×AV Elution Buffer to elute the virus and collect 4 mL of eluent.

[0129] Step 6, Desalting and Buffer Exchange: Transfer the 4 mL sample collected in the previous step to a Centrifugal Filter. Centrifuge at 3000 rpm for 10-15 min at 4°C until approximately 500 μL of solution remains in the Centrifugal Filter. Discard the filtrate, add 3.5 mL of PBS to the Centrifugal Filter, and centrifuge at 3000 rpm for 10-15 min at 4°C until approximately 400-500 μL of solution remains in the Centrifugal Filter. Pipette the solution up and down several times in the Centrifugal Filter and transfer the virus-containing solution to a clean tube.

[0130] Step 7, Regeneration of the purification column: After purification, add 5 mL of Regeneration Buffer to the column, allowing the solution to flow through the column by gravity. Add 5 mL of 1×AV Wash Buffer, tighten the bottom cap, seal the column with sealing film, seal it in a bag, and store at 4°C for later use.

[0131] Step 8, Adenovirus Titer Determination: Dilute the virus 20-fold (5 μl of virus added to 95 μl of lysis buffer: 0.1% SDS, 10 mM Tris, pH 7.4, 1 mM EDTA), incubate at 55°C for 10 min, and measure OD260. Titer. =OD260*20*1.1x10 12 (vp / ml). Titer is 1.5 × 10⁻⁶. 11 .

[0132] Example 4: Preparation of anti-PSMACAR-M cells.

[0133] anti-PSMACAR adenovirus infection of macrophages (hPBMCs) and Western blot analysis of Myc tag expression in the CAR vector.

[0134] Peripheral blood mononuclear cells (PBMCs) were collected from healthy donors using a blood cell separator. CD14 microbeads were then sorted from the PBMCs to obtain CD14. + Mononuclear cells; efficiency approximately 15%. Specific steps are as follows: Step 1: Resuspend peripheral blood mononuclear cells (PBMCs) in 10 ml PBS, pass through a cell sieve to remove large cell clusters, centrifuge at 300g for 10 min, and discard the supernatant. Step 2: Resuspend the obtained cell pellet in buffer (buffer volume is 10 ml). 7 (80 μl / cell), then add MicroBeads (10 μl of MicroBeads). 7 (20 μl of cells / cell), mix well by pipetting, and incubate at 4°C in the dark for 15 min. Step 3: After incubation, add buffer (10 μL of buffer used) to the centrifuge tube. 7Step 4: Add 500 μl of buffer to resuspend the cells (1-2 ml of cells per 1 ml). Step 5: Sorting on an MS sorting column: Install the sorting column on the sorter. First, add 500 μl of buffer to rinse the sorting column, being careful not to create visible air bubbles to avoid clogging the column. After the buffer has flowed out, add the sample while it is suspended. At this point, the positive cells labeled with magnetic beads will remain on the column. After the buffer has flowed out, add 500 μl of buffer to wash the sorting column. Repeat this process 3 times, making sure the previous wash is almost empty before the next wash to avoid leaving any residue on the column. Elution of target cells: After the last wash buffer has flowed out, place the sorting column on a new positive collection tube (15 ml centrifuge tube), add 1 ml of buffer to the sorting column, and quickly push it down with the stopcock. The positive collection tube now contains the target cells. The sorted cells were counted using a hematology analyzer to calculate the sorting yield. The cells were then seeded into culture plates / dishes (RPMI-1640 + 10% FBS + 1% P / S + 100 ng / ml M-CSF). The CD14-positive rate of monocytes after magnetic bead sorting was 97.84%.

[0135] Mononuclear cells were sorted, resuspended in culture medium, and seeded into culture plates. Purified Ad5F35-hPGK-mCherry and EF1A-Her2-CAR-hPGK-mCherry adenoviruses were added directly, along with 10 ng / ml M-CSF. The plates were placed in an incubator, and timing was started 10 minutes after virus addition. The plates were then removed, the supernatant was aspirated, and the cells were centrifuged at 4000 rpm for 5 minutes, discarding the supernatant. The plates were washed once with PBS, and the PBS was transferred to the centrifuge tubes from the previous centrifuge. The cell pellet was resuspended, centrifuged again for 5 minutes, and the PBS was discarded. The cells were resuspended in 1640 mL of medium containing 10% FBS and 100 ng / ml M-CSF, reseeded into culture plates, and cultured in a CO2 incubator for 2 hours to obtain CAR-expressing mononuclear cells. CAR protein expression was analyzed by flow cytometry.

[0136] Example 5: Evaluation of the in vitro and in vivo antitumor effect of anti-PSMACAR-M cells.

[0137] 1. In vitro experimental evaluation - phagocytosis effect.

[0138] (1) Immunofluorescence detection.

[0139] (2) Flow cytometry detection.

[0140] like Figure 6As shown, the four experimental groups represent the loss analysis results for RAW+293ft co-culture for 0h, RAW+293ft co-culture for 6h, RAW-CAR+293ft co-culture for 6h, and RAW-Ad empty + 293ft co-culture for 6h. In all four experiments, the 293ft cells expressed PSMA. The phagocytic rate of RAW+293ft co-culture for 0h was 10.14%, the phagocytic rate of RAW+293ft co-culture for 6h was 26.53%, the phagocytic rate of RAW-CAR+293ft co-culture for 6h was 52.26%, and the phagocytic rate of RAW-Ad empty+293f co-culture for 6h was 22.22%.

[0141] In summary, the phagocytosis rate of the RAW-CAR+293ft co-culture group for 6 hours was 52.26%, which was higher than the other three groups. This shows that the phagocytosis rate of the CAR-M disclosed in this application on PSMA-expressing cells is much higher than that of wild-type and empty vector, which is sufficient to prove the prospect of the CAR-M disclosed in this application in treating tumors with high PSMA expression, especially for the preparation of drugs that phagocytose and kill prostate cancer tumor cells.

[0142] like Figure 7 As shown, BMDM infected with Ad5f35-PSMA-CAR tends to be M1 type, and BMDM infected with Ad5f35-PSMA-CAR for 6 hours has a significant killing effect on LNCaP. Figure 8 As shown, hMDM infected with Ad5f35-PSMA-CAR tends to be M1 type, and hMDM infected with Ad5f35-PSMA-CAR for 6 hours has a significant killing effect on LNCaP, reaching 51.8%. Therefore, the macrophages designed with the CAR disclosed in this application have achieved a strong targeted killing ability.

Claims

1. An immune effector cell targeting PSMA chimeric antigen receptor modification, characterized in that, The chimeric antigen receptor includes an antigen-binding domain, a hinge region, a transmembrane domain, and a signal transduction domain, wherein the antigen-binding domain is an anti-PSMA single-chain antibody.

2. The immune effector cells targeting PSMA chimeric antigen receptor modification as described in claim 1, characterized in that, The hinge area includes CD8.

3. The immune effector cells targeting PSMA chimeric antigen receptor modification as described in claim 1, characterized in that, The transmembrane structural domain includes the CD8 transmembrane region.

4. The immune effector cells targeting PSMA chimeric antigen receptor modification as described in claim 1, characterized in that, The signal conduction structure domain includes CD3ζ.

5. The immune effector cells targeting PSMA chimeric antigen receptor modification as described in claim 1, characterized in that, The signal transduction domain further includes any one or a combination of at least two of 4-1BB, CD28 intracellular region, DAP10, or OX40.

6. The immune effector cells targeting PSMA chimeric antigen receptor modification as described in claim 1, characterized in that, The chimeric antigen receptor includes an anti-GPC3 single-chain antibody, a CD8 hinge region, a CD8 transmembrane region, and CD3ζ.

7. The immune effector cells targeting PSMA chimeric antigen receptor modification as described in claim 1, characterized in that, The antigen-binding domain of the chimeric antigen receptor includes the amino acid sequence shown in SEQ ID NO:

2.

8. The immune effector cells targeting PSMA chimeric antigen receptor modification as described in claim 1, characterized in that, The hinge region includes the amino acid sequence shown in SEQ ID NO:

1.

9. The immune effector cells targeting PSMA chimeric antigen receptor modification as described in claim 1, characterized in that, The transmembrane domain includes the amino acid sequence shown in SEQ ID NO:

3.

10. The immune effector cells targeting PSMA chimeric antigen receptor modification as described in claim 1, characterized in that, The signal transduction domain includes the amino acid sequence shown in SEQ ID NO:

4.

11. The immune effector cells targeting PSMA chimeric antigen receptor modification as described in claim 1, characterized in that, The immune effector cells mentioned are macrophages and peripheral blood mononuclear cells.

12. A nucleic acid molecule, characterized in that, The nucleic acid molecule includes the encoding gene of the chimeric antigen receptor as described in any one of claims 1-11.

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

14. The expression vector as described in claim 13, characterized in that, The expression vector is a lentiviral vector or adenoviral vector containing the nucleic acid molecule described in 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 and helper plasmid as described in claim 13 or 14.

16. The chimeric antigen receptor-modified immune effector cells according to any one of claims 1-11, characterized in that, Its genome integrates the nucleic acid molecule described in claim 12.

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

18. The method for preparing chimeric antigen receptor-modified immune effector cells as described in claim 17, characterized in that, The preparation method includes the step of introducing the expression vector of claim 12 or 13 and / or the recombinant lentivirus and / or adenovirus of claim 15 into immune effector cells.

19. The method for preparing chimeric antigen receptor-modified immune effector cells as described in claim 18, characterized in that, The preparation method specifically includes the following steps: (1) Synthesize the nucleic acid sequences of each domain of transmembrane expressed proteins or polypeptide macromolecules; (2) Ligate the target fragment with a lentivirus or adenovirus overexpression vector to obtain a recombinant plasmid vector; (3) Extraction and preparation of recombinant plasmid vectors and auxiliary plasmid vectors; (4) Virus packaging; (5) Construction of functional cell lines.

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

21. The pharmaceutical composition of claim 20, characterized in that, It also includes any one or a combination of at least two of pharmaceutically acceptable carriers, excipients or diluents.

22. Use of chimeric antigen receptor-modified immune effector cells targeting PSMA in the preparation of drugs for treating PSMA-positive tumors, wherein the chimeric antigen receptor-modified immune effector cells targeting PSMA include the nucleic acid molecule of claim 11, the expression vector of claim 13 or 14, the recombinant lentivirus and / or adenovirus of claim 15.

23. The application as described in claim 22, characterized in that, The PSMA-positive tumor is prostate cancer.

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