PSMA-targeted chimeric antigen receptors and cells modified therefrom and their applications

Abstract

The present invention discloses a chimeric antigen receptor (CAR), which includes an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain includes a single-chain variable fragment (scFv) targeting prostate-specific membrane antigen (PSMA). By integrating the scFv targeting PSMA on the chimeric antigen receptor, the genetically modified macrophages containing the CAR have the effect of targeting PSMA and prostate cancer cells, thereby promoting the phagocytosis and killing effect of macrophages on prostate cancer cells.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a PSMA-targeted chimeric antigen receptor and cells modified therefrom, as well as applications thereof in treating prostate cancer. Background Art

[0002] Prostate cancer (PCa) ranks sixth in the global cancer incidence and is the second most common type of cancer in men, with high morbidity and mortality. Data show that almost all patients with advanced prostate cancer will progress to castration-resistant prostate cancer (CRPC) after endocrine therapy, with a median survival of approximately 14 months. Despite the increase in existing treatment options, the 5-year survival rate of patients with metastatic castration-resistant prostate cancer (mCRPC) remains low, and new treatments are urgently needed.

[0003] The breakthrough of adoptive immune cell therapy has changed the pattern of tumor drug development. So far, 11 CAR-T (Chimeric Antigen Receptor T cell) products have been approved by FDA / NMPA for the treatment of hematological malignancies and lymphomas, becoming an important milestone in the history of clinical tumor medicine. However, although CAR-T has shown good efficacy in hematological tumors, its efficacy in the treatment of solid tumors is still unsatisfactory. Considering the particularity of the microenvironment of solid tumors, macrophages have unique advantages as carriers of adoptive cell therapy.

[0004] Due to the high infiltration rate (~50%) of macrophages in the tumor microenvironment, using them as adoptive cell therapy carriers has its unique advantages: strong ability to penetrate solid tumors, strong phagocytic ability to quickly kill tumor cells and prevent immune escape, as antigen presenting cells can activate T cells, etc., thus having significant effects on reshaping the immune cell interaction network and reversing the immunosuppressive microenvironment. The construction of chimeric antigen receptor macrophages for prostate cancer targets is expected to meet the clinical needs of solid tumor treatment, mainly malignant prostate tumors. Summary of the invention

[0005] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a chimeric antigen receptor targeting prostate-specific membrane antigen (PSMA), and to provide a method for constructing macrophages targeting PSMA, so as to solve the problem that the treatment effects of prostate cancer in the prior art are not ideal.

[0006] According to one aspect of the present disclosure, a chimeric antigen receptor (CAR) is provided, which includes an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain includes a single-chain variable fragment (PSMA-scFv) targeting prostate-specific membrane antigen (PSMA).

[0007] In some embodiments, the extracellular domain includes a CD8α signal peptide and PSMA-scFv, the transmembrane domain includes a CD8 hinge region and a CD8 transmembrane region, and the intracellular domain includes an FcεR1γ intracellular region.

[0008] In some embodiments, the extracellular domain has an amino acid sequence as shown in SEQID NO: 1 or 3, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQID NO: 1 or 3, or an amino acid sequence with equivalent function formed by adding, deleting, replacing or modifying the amino acid sequence as shown in SEQ ID NO: 1 or 3 with one or more amino acids.

[0009] In some embodiments, the transmembrane domain has the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 5, or an amino acid sequence with equivalent function formed by adding, deleting, replacing or modifying the amino acid sequence shown in SEQ ID NO: 5 with one or more amino acids.

[0010] In some embodiments, the intracellular domain has the amino acid sequence shown in SEQID NO: 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQID NO: 7, or an amino acid sequence with equivalent function formed by adding, deleting, replacing or modifying the amino acid sequence shown in SEQ ID NO: 7 with one or more amino acids.

[0011] In some embodiments, the chimeric antigen receptor comprises a CD8α signal peptide, a PSMA-scFv, a CD8 hinge region, a CD8 transmembrane region, and an FcεR1γ intracellular region that are operably linked in sequence.

[0012] According to another aspect of the present disclosure, a nucleic acid molecule is provided, which encodes the chimeric antigen receptor described in the present disclosure.

[0013] In some embodiments, the nucleic acid molecule has the nucleotide sequence shown in SEQID NO: 9, or a nucleotide sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQID NO: 9.

[0014] According to another aspect of the present disclosure, a recombinant vector is provided, which contains the nucleic acid molecule described in the present disclosure.

[0015] According to another aspect of the present disclosure, a modified cell is provided, which contains the nucleic acid molecule or the recombinant vector described in the present disclosure.

[0016] In some embodiments, the cells include monocytes, macrophages, or dendritic cells, preferably macrophages.

[0017] In some embodiments, the modified cell is genetically modified to express the chimeric antigen receptor.

[0018] In some embodiments, the modified cells have at least one upregulated M1 marker and / or at least one downregulated M2 marker.

[0019] In some embodiments, the modified cells have elevated expression of CD80 and / or CD86.

[0020] In some embodiments, the modified cells have reduced expression of CD163.

[0021] According to another aspect of the present disclosure, a method for constructing a modified cell is provided, comprising introducing the chimeric antigen receptor, the nucleic acid molecule, or the recombinant vector described in the present disclosure into a cell.

[0022] In some embodiments, the method comprises transducing the cell with a viral vector comprising a nucleic acid sequence encoding the chimeric antigen receptor.

[0023] In some embodiments, the cells include monocytes, macrophages, or dendritic cells, preferably macrophages.

[0024] In some embodiments, the viral vector includes a retroviral vector, a lentiviral vector, an adenoviral vector and an adeno-associated viral vector, preferably an adenoviral vector.

[0025] In some embodiments, the multiplicity of infection (MOI) of the adenoviral vector is 500-2000, for example, 500, 1000, 1500, 2000 or any value therebetween.

[0026] In some embodiments, the method comprises using GM-CSF to induce the modified cells to assume an M1-like phenotype.

[0027] In some embodiments, the concentration of GM-CSF is 10-200 ng / mL, for example, 10 ng / mL, 20 ng / mL, 50 ng / mL, 80 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL or any value therebetween.

[0028] In some embodiments, the method comprises culturing the cells using X-VIVO10, X-VIVO15, or X-VIVO20 medium.

[0029] In some embodiments, the culture medium is supplemented with fetal bovine serum (FBS).

[0030] In some embodiments, the culture medium is supplemented with fetal bovine serum (FBS) and GM-CSF.

[0031] In some embodiments, the concentration of the fetal bovine serum is 8-15%, for example, 8%, 10%, 12%, 15% or any value therebetween.

[0032] According to another aspect of the present disclosure, a pharmaceutical composition is provided, which comprises the chimeric antigen receptor described in the present disclosure, the modified cell described or the modified cell obtained by the method described, and a pharmaceutically acceptable carrier.

[0033] According to another aspect of the present disclosure, provided is the use of the chimeric antigen receptor, the modified cell, the modified cell obtained by the method, or the pharmaceutical composition described in the present disclosure in the preparation of a drug for treating prostate cancer.

[0034] In some embodiments, the prostate cancer is metastatic castration-resistant prostate cancer.

[0035] According to another aspect of the present disclosure, a method for treating a disease or condition related to prostate cancer is provided, comprising administering to a subject in need thereof a therapeutically effective amount of the chimeric antigen receptor, the modified cell, the modified cell obtained by the method, or the pharmaceutical composition described in the present disclosure.

[0036] In some embodiments, the prostate cancer is metastatic castration-resistant prostate cancer.

[0037] The present disclosure provides a chimeric antigen receptor (CAR) targeting PSMA; by integrating scFv targeting PSMA on the chimeric antigen receptor, the CAR and genetically modified cells containing the CAR, especially macrophages, have the effect of targeting PSMA and prostate cancer cells, thereby promoting the phagocytosis and killing of prostate cancer cells by macrophages. The present disclosure further optimizes the CAR-M transfection and culture system to make CAR-M present an M1 phenotype, thereby enhancing the anti-tumor ability of CAR-M. The present disclosure proposes for the first time a CAR-M cell immunotherapy targeting PSMA, and experimentally verifies the effect of anti-PSMA CAR-M cell immunotherapy in alleviating the disease process and prolonging survival time, which is of great significance for the treatment of advanced prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The results of flow cytometry analysis of the expression of macrophage markers CD11b (A) and CD68 (B) in the prepared PBMac are shown.

[0039] Figure 2 Shown are the results of CD80 (A) and CD163 (B) expression of PBMac obtained with or without the addition of GM-CSF during PBMac induction.

[0040] Figure 3 The killing effect of PBMac on target cells 22Rv1 obtained by adding M-CSF (A) or GM-CSF (B) during PBMac induction is shown.

[0041] Figure 4 The results of cell viability (A) and cell phagocytic ability (B) of different culture systems of PSMA-targeting chimeric antigen receptor macrophages (PBMac) are shown.

[0042] Figure 5 The results of transfection efficiency of PSMA-targeted chimeric antigen receptor macrophages (PBMac) are shown (A) and CAR molecules are expressed on the cell membrane (B).

[0043] Figure 6 Tumor phagocytosis results (A) and in vitro killing results (B) of PSMA-targeting chimeric antigen receptor macrophages (PBMac) are shown.

[0044] Figure 7 The results of in vitro cytokine release of CAR-M targeting PSMA chimeric antigen receptor macrophages (PBMac) are shown.

[0045] Figure 8The results of the efficacy study of chimeric antigen receptor macrophages (PBMac) targeting PSMA in tumor-bearing immunodeficient mice are shown, where A is the image of the in vivo imaging of tumor-bearing immunodeficient mice during the treatment process, B is the statistical result of the residual tumor tissue in the tumor-bearing immunodeficient mice at the end point of the experiment, C is the quantitative statistical result of the in vivo imaging of tumor-bearing immunodeficient mice during the treatment process, and D is the statistical result of the survival time of tumor-bearing immunodeficient mice.

[0046] Fig. 9 The figure shows the safety evaluation results of chimeric antigen receptor macrophages (PBMac) targeting PSMA in tumor-bearing immunodeficient mice, where A is the pathological changes in the organs of tumor-bearing immunodeficient mice, and B is the blood biochemical test results of the serum of tumor-bearing immunodeficient mice. DETAILED DESCRIPTION

[0047] Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein expressed on the cell membrane. Compared with normal prostate tissue and other parts of the body, PSMA shows a specific high expression pattern in prostate cancer (PCa), and its expression level is highly correlated with the aggressiveness of PCa.

[0048] The present invention designs a chimeric antigen receptor nucleotide sequence targeting PSMA; uses adenovirus transfection to achieve efficient expression of CAR molecules in macrophages, adds GM-CSF during the CAR-M induction process, promotes CAR-M polarization to M1, and enhances CAR-M's killing and phagocytic function on tumor cells. In animal models, the efficacy of CAR-M was evaluated and it was found that CAR-M can significantly inhibit the proliferation of prostate cancer tumors, delay the progression of tumors, and increase the survival time of tumor-bearing mice.

[0049] The present disclosure provides a method for constructing a PSMA-targeted chimeric antigen receptor macrophage, which comprises the following steps performed in sequence:

[0050] Step 1: construct an adenovirus expression vector integrating a chimeric antigen receptor (CAR) targeting PSMA with a nucleotide sequence as shown in SEQ ID NO: 9, and obtain an adenovirus loaded with a CAR sequence targeting PSMA through packaging.

[0051] Step 2: Use adenovirus to infect macrophages, and obtain CAR-M cells targeting PSMA through screening.

[0052] Further, in step 1, the extracellular domain of the chimeric antigen receptor targeting PSMA includes a CD8α signal peptide with an amino acid sequence as shown in SEQ ID NO: 1 and a scFv targeting PSMA; the transmembrane domain of the chimeric antigen receptor targeting PSMA includes a hinge region and a transmembrane region of CD8 with an amino acid sequence as shown in SEQ ID NO: 5; and the intracellular domain of the chimeric antigen receptor targeting PSMA also includes an FcεR1γ intracellular region with an amino acid sequence as shown in SEQ ID NO: 7.

[0053] Furthermore, in step 2, granulocyte-macrophage colony-stimulating factor (GM-CSF) is used to induce CAR-M to present an M1-like phenotype with high expression of CD80 and CD86.

[0054] Further, in step 2, the working concentration of GM-CSF induction is 10-200 ng / mL.

[0055] The present disclosure also provides a use of a PSMA-targeted chimeric antigen receptor macrophage in the preparation of a drug for treating prostate cancer, which is mainly used to treat PSMA-positive prostate cancer, especially PSMA-positive advanced prostate cancer, and more preferably to treat advanced metastatic castration-resistant prostate cancer.

[0056] The present disclosure also provides a composition for treating metastatic castration-resistant prostate cancer. In some embodiments, the composition includes CAR-M cells targeting PSMA.

[0057] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0058] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0059] The term "about" as used herein refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0060] As used herein, the term "chimeric antigen receptor" (CAR) refers to a fusion protein that includes an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from the polypeptide from which the extracellular domain is derived, and at least one intracellular domain. "Chimeric antigen receptor (CAR)" is sometimes referred to as a "chimeric receptor", "T-body (Tbody)" or "chimeric immune receptor (CIR)". "Extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide capable of binding to an antigen. "Intracellular domain" refers to any oligopeptide or polypeptide known to be used as a domain for sending signals to cause activation or inhibition of biological processes within a cell. "Transmembrane domain" refers to any oligopeptide or polypeptide known to span the cell membrane and can act to connect the extracellular domain and the signaling domain. The chimeric antigen receptor may optionally include a "hinge domain" that serves as a connector between the extracellular domain and the transmembrane domain.

[0061] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, DNA genomes, cDNAs, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases.

[0062] The term "encoding" when applied to a nucleic acid sequence means that the polynucleotide stated to "encode" a polypeptide, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce mRNA for the polypeptide and / or its fragments. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be derived therefrom.

[0063] As used herein, the term "vector" refers to a nucleic acid construct designed for transmission between different hosts, including but not limited to plasmids, viruses, cosmids, phages, BACs, YACs, etc. In some embodiments, plasmid vectors can be prepared from commercially available vectors. In other embodiments, viral vectors can be made from baculovirus, retrovirus, adenovirus, AAV, etc. according to techniques known in the art. In one embodiment, the viral vector is an adenoviral vector.

[0064] As used herein, the term "modification" means a change in the state or structure of a molecule or cell of the invention. Molecules can be modified in a variety of ways, including chemical, structural and functional modifications. Cells can be modified by the introduction of nucleic acids.

[0065] The term "operably connected" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, when a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is operably connected to the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably connected to the coding sequence. In general, the DNA sequence dna that is operably connected is continuous and is in the same reading frame when connecting two protein coding regions.

[0066] As used herein, the term "transfection" or "transformation" or "transduction" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with an exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0067] Non-limiting exemplary polynucleotide sequences encoding components of each domain, for example: "Percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences within the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Since a gap is not a nucleotide or amino acid, gaps present in the target sequence are not counted. Similarly, since target sequence nucleotides or amino acids are counted, but nucleotides or amino acids from the reference sequence are not counted, gaps present in the reference sequence are not counted.

[0068] The sequence identity percentage can be calculated by the following process: determine the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the sequence identity percentage. The comparison of sequences and the determination of the sequence identity percentage between the two sequences can be completed using software that is easy to use and download online. Suitable software programs are available from various sources for the comparison of protein and nucleotide sequences. A suitable program for determining the sequence identity percentage is bl2seq, which is part of the BLAST program suite available from the BLAST website (blast.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information of the U.S. government. Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0069] Macrophages are phenotypically heterogeneous immune cells that play an important role during inflammation (both initiation and resolution). Macrophages can be polarized into two phenotypes upon stimulation: (1) one is the classical activation (inflammatory) phenotype M1, which can be induced by lipopolysaccharide (LPS) or interferon γ (IFN-γ) to produce proinflammatory cytokines such as TNFα and IL-1β; (2) the other is the alternative activation (wound healing) phenotype M2, which can be induced by IL-4, IL-13, etc. to produce anti-inflammatory cytokines such as IL-10, IL-13, Arg1, etc. The balance of M1 / M2 macrophage polarization determines the fate of an organ in inflammation or injury. M1 plays a proinflammatory role in counteracting stimuli in the early stages of inflammation, but continued inflammation will cause tissue damage; M2 plays an anti-inflammatory role, promoting tissue repair and angiogenesis.

[0070] The terms "subject," "host," "individual," and "patient" are used interchangeably herein to refer to human and veterinary subjects, such as humans, animals, non-human primates, dogs, cats, sheep, mice, horses, and cows. In some embodiments, the subject is a human.

[0071] The term "pharmaceutical composition" or "composition" generally refers to a combination of an active agent (e.g., a compound or composition) and a naturally occurring or non-naturally occurring carrier, which is inert, such as a detectable agent or label, or active, such as an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant, etc., and includes a pharmaceutically acceptable carrier. Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids and carbohydrates (e.g., sugars, including monosaccharides, di-oligosaccharides, tri-oligosaccharides, tetra-oligosaccharides and oligosaccharides; derived sugars, such as sugar alcohols, aldonic acids, esterified sugars, etc.; and polysaccharides or sugar polymers), which may be present alone or in combination, alone or in combination, including 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin (e.g., human serum albumin (HSA), recombinant human albumin (rHA)), gelatin, casein, etc. Representative amino acid / antibody components that also have buffering capacity include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, etc. Carbohydrate excipients are also intended to be within the scope of the present technology, examples of which include, but are not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbitol, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), and inositol.

[0072] An "effective amount" refers to that amount of an agent or a combined amount of two or more agents, when administered to treat a mammal or other individual, that is sufficient to affect such treatment of a disease. An "effective amount" will vary depending on the agent, the disease and its severity, and the age, weight, etc., of the subject to be treated.

[0073] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0074] The reagents and / or kits used in the following examples are all commercially available or can be synthesized by known methods. Example

[0075] Example 1. Construction of expression plasmid and packaging of adenovirus

[0076] In this example, a chimeric antigen receptor plasmid targeting PSMA was constructed and packaged into adenovirus.

[0077] The chimeric antigen receptor includes a CD8α signal peptide, a PSMA-targeted scFv, a CD8 hinge region and a transmembrane region, and an FcεR1γ cytoplasmic domain, wherein the sequence information is shown in Table 1 below.

[0078] Table 1

[0079]

[0080] The gene fragment with the sequence shown in SEQ ID NO: 9 was connected to the multiple cloning site of the universal adenovirus expression vector Ad5 / F35 (purchased from Yunzhou Bio) to obtain an overexpression vector of the CAR molecule targeting PSMA, which can be used for the expression of the CAR molecule; the CAR sequence was obtained by connecting the sequence fragments of CD8α, PSMA-scFv-1, CD8 hinge region, CD8 transmembrane region, and FcεR1γ intracellular region.

[0081] Linearization of adenovirus plasmid: Use restriction endonuclease Pac I to digest the adenovirus plasmid at 37℃, dephosphorylate the linearized adenovirus plasmid to prevent vector self-ligation, incubate at 37℃ for 30 min, and then place at 4℃.

[0082] Adenovirus packaging: 293A cells were cultured at a density of 3 × 10 5 / mL was inoculated in a 10cm cell culture dish and cultured overnight in a 37℃, 5% CO2 incubator. During transfection, the plasmid solution and the transfection reagent jetPRIME® Transfection Reagent (purchased from Polyplus) were mixed evenly, placed at room temperature for 10 minutes, and the mixture was slowly added to the culture medium of 293A cells, mixed evenly, and cultured in a 37℃, 5% CO2 incubator. After 16 hours, the original culture medium was discarded, and preheated 10% FBS DMEM culture medium (purchased from Gibco) was slowly added to the cell wall and placed in the incubator.

[0083] Concentration and purification of virus liquid: After 293A cells become diseased and the culture medium turns yellow, add 3% FBS DMEM culture medium to continue culturing. When adding culture medium, be careful to add it slowly and gently to prevent cells from falling off. After the cells are completely diseased and dead, collect the virus supernatant, freeze it in a -80℃ refrigerator, and freeze and thaw it three times. Centrifuge at 4℃, 10000g for 15min to remove cell debris; filter the supernatant with a 0.45μm filter into a 40mL ultracentrifuge tube; centrifuge at 4℃, 25000g for 2h. After the centrifugation, discard the supernatant, try to remove the residual liquid, add PBS, and gently and repeatedly blow to resuspend; after sufficient dissolution, obtain the virus concentrate and detect the virus titer.

[0084] Example 2. Construction of chimeric antigen receptor macrophages targeting PSMA

[0085] The PSMA-targeted adenovirus constructed by the method in Example 1 was used to prepare chimeric antigen receptor macrophages (CAR-M).

[0086] Sorting of peripheral blood mononuclear cells: Peripheral blood mononuclear cells (PBMCs) were isolated from fresh peripheral blood (purchased from Miaoshun Biotech) using lymphocyte separation solution (purchased from Yisheng Biotech) by density gradient centrifugation. PBMCs were resuspended in a low-adhesion 50 mL centrifuge tube and monocytes were sorted using CD14 magnetic beads (purchased from Miltenyi Biotec). Centrifuge at 400 g for 10 min, discard the supernatant, add MACS buffer (purchased from Miltenyi), and precipitate at 1×10 7 Add 80 μL of MACS buffer to the cells and gently disperse them. 7 Add 20 μL of CD14 magnetic beads to each 1×10 cells, blow gently to mix well, and incubate at 4°C for 15 min. 7 Add 1-2 mL of MACS buffer to the amount of cells and centrifuge at 400 g for 10 min. Place the column on the magnetic field and add 3 mL of MACS buffer to rinse the column. 8 Resuspend the cells in 500 μL MACS buffer and add the cell suspension to the sorting column. After the cell suspension has completely passed through the sorting column, add 3 mL MACS buffer to wash the centrifuge tube and add it to the sorting column. After elution, add 3 mL MACS buffer directly to the sorting column and repeat once. Add 5 mL MACS buffer to push the cells adsorbed by the magnetic beads into the centrifuge tube, count them, and count them according to 5×10 6 The cells were plated in a 10 cm culture dish and cultured in a 37°C, 5% CO2 incubator.

[0087] Adenovirus infection of peripheral blood monocytes: 5 days after sorting, add virus concentrate to peripheral blood monocytes at an MOI of 1000, change the medium every 6 hours, and place in a 37°C, 5% CO2 incubator for 2 days to culture. Chimeric antigen receptor macrophages targeting PSMA (PBMac) were obtained.

[0088] Example 3. Purity detection of PSMA-targeted chimeric antigen receptor macrophages (PBMac)

[0089] The PBMac obtained in Example 2 was subjected to flow cytometry using anti-human CD68 (purchased from Biolegend) and anti-human CD11b (purchased from Biolegend) flow cytometry antibodies to detect the expression of macrophage markers CD11b and CD68.

[0090] Figure 1 The results of A and B in Figure 2 showed that the purity of macrophages reached over 80%.

[0091] Example 4. Detection of CAR-M phenotype of PSMA-targeted chimeric antigen receptor macrophages (PBMac)

[0092] The chimeric antigen receptor macrophages targeting PSMA were constructed in the same manner as in Example 2, except that when the adenovirus infected the peripheral blood-derived monocytes, the culture medium used was a culture medium without GM-CSF (10% FBS + XVIVO15), that is, GM-CSF was added or not during the PBMac induction process.

[0093] PBMac obtained by adding / not adding GM-CSF during PBMac induction was subjected to flow cytometry using anti-human CD80 (purchased from Biolegend) and anti-human CD163 (purchased from Biolegend) flow cytometry antibodies to detect CAR-M macrophage polarization markers CD80 and CD163.

[0094] Figure 2 The results in A and B show that the use of GM-CSF can increase the expression of CD80 while reducing the expression of CD163, indicating that GM-CSF can promote the M1 polarization of CAR-M.

[0095] Example 5. Optimization of the induction system of PSMA-targeted chimeric antigen receptor macrophages (PBMac)

[0096] The same method as in Example 2 was used to construct chimeric antigen receptor macrophages targeting PSMA, with the only difference being that when adenovirus infected peripheral blood-derived monocytes, macrophage colony stimulating factor (M-CSF, purchased from Nearshore Protein) was used in the culture medium instead of GM-CSF, i.e., macrophage colony stimulating factor (M-CSF) was added instead of GM-CSF during PBMac induction.

[0097] After PBMac obtained by adding M-CSF or GM-CSF during the PBMac induction process and luciferase-expressing target cells 22Rv1 (purchased from Kebai Bio) were co-cultured in a 96-well plate at different effector-target ratios (1:1, 1:2, 1:5, 1:10) for 48 hours, the following operations were performed according to the instructions of the Steady-Glo® Luciferase Assay Kit (purchased from Promega): 100mlSteady-Glo® Luciferase Assay Buffer was added to 1 bottle of Steady-Glo® Luciferase Assay Substrate (lyophilized) and mixed; 50μL of the prepared Steady-Glo® Luciferase Assay reagent was added to each well of the 96-well plate to be tested. After 5 minutes of reaction, the reading was read on the microplate reader. The smaller the value, the stronger the killing ability.

[0098] Figure 3 The results of A and B in FIG. 5 show that the ability of CAR-M obtained by culturing with GM-CSF to kill 22Rv1 was significantly enhanced, and there was a clear dose-effect relationship.

[0099] Example 6. Optimization of the culture system of PSMA-targeted chimeric antigen receptor macrophages (PBMac)

[0100] The same method as in Example 2 was used to construct chimeric antigen receptor macrophages targeting PSMA, with the only difference being that when adenovirus infected peripheral blood-derived monocytes, DMEM medium was used in the culture medium instead of XVIVO15 medium, or 10% FBS was not added, that is, DMEM medium or XVIVO15 medium was used during PBMac induction with / without 10% FBS.

[0101] The cell viability of induced PBMac was detected by cell counter. Figure 4 The results in A showed that culturing PBMac with XVIVO15+10% FBS could significantly increase the cell viability of harvested PBMac.

[0102] The phagocytic ability of PBMac harvested under different culture conditions was tested. After PBMac was incubated with fluorescently labeled phagocytic microspheres (purchased from Sigma) for 3 hours, the fluorescence intensity in PBMac was detected by flow cytometry. Figure 4 The results in B show that PBMac cultured with XVIVO15+10% FBS has the strongest phagocytic ability.

[0103] Example 7. Detection of the Positive Rate of CAR-PBMac Targeting PSMA Chimeric Antigen Receptor Macrophages (PBMac)

[0104] Using the PBMac obtained in Example 2, flow cytometry was used to detect the expression of CAR molecules using human PSMA antigen (purchased from ACROBiosystems). Figure 5 The results in A showed that the transfection efficiency reached 86.12%.

[0105] DIR and DAPI fluorescent dyes were used to label cell membranes and nucleic acids, and human PSMA antigens were used to label CAR molecules. Confocal microscopy was used to observe the expression of CAR molecules on the cell membrane. Figure 5 As shown in B.

[0106] Example 8. In vitro killing evaluation of CAR-M targeting PSMA chimeric antigen receptor macrophages (PBMac)

[0107] After the PBMac (CAR-M) obtained in Example 2 was co-cultured with the fluorescently labeled target cells 22Rv1 at a ratio of 1:2 for 4 hours, the phagocytic ability of CAR-M was detected by flow cytometry. The stronger the fluorescence in the macrophages, the stronger the phagocytic ability of the macrophages. Figure 6 The results in Figure A showed that CAR-M (42%) exhibited stronger tumor phagocytosis ability compared with Mac without adenovirus transfection (UTD, 19%).

[0108] After PBMac was co-cultured with target cells 22Rv1 expressing luciferase at different effector-target ratios (3:1, 1:1, 1:3, 1:5, and 1:10) for 48 hours, luciferase substrate was added and the fluorescence value was detected by a microplate reader. The smaller the value, the stronger the killing ability. Figure 6 The results of B in Figure 2 show that the ability of CAR-M to kill 22Rv1 is significantly enhanced, and there is a clear dose-effect relationship.

[0109] Example 9. In vitro cytokine release of CAR-M targeting PSMA chimeric antigen receptor macrophages (PBMac)

[0110] After the PBMac (CAR-M) obtained in Example 2 was incubated with PSMA-positive tumor cells (purchased from Kebai Bio) (1:1), the cell culture supernatant was collected and the contents of various cytokines in the cell culture supernatant were detected by ELISA.

[0111] Figure 7 The results showed that compared with the control group, the secretion levels of M1 cytokines IFN-γ, IL-2, IL-6 and TNFα increased, and the secretion levels of M2 cytokines IL-10 and IL-4 decreased.

[0112] Example 10: Application of PSMA-targeted chimeric antigen receptor macrophages (PBMac) in the treatment of prostate cancer

[0113] Resuspend human prostate cancer cells 22Rv1 (purchased from Kebai Bio) and adjust the cell density to 5×10 7 6-8 weeks old male immunodeficient NCG mice (purchased from Jicui Yaokang) were intraperitoneally injected with 200 μL of cell suspension (the total amount of cells injected was 1×10 7 6 hours after modeling, mice injected intraperitoneally with PBS buffer were designated as the PBS group, and 1×10 7 UTD cells or CAR-M cells (cell density was 5×10 7 The mice with 40 cells / mL of CAR-M were divided into UTD group and CAR-M group, with 5 mice in each group. The tumor burden was continuously observed and recorded during the treatment. Tumor tissues were collected for histological processing 25 days after treatment, and the survival curve was recorded.

[0114] (1) Efficacy study of CAR-M in tumor-bearing immunodeficient mice

[0115] During the treatment, live imaging technology was continuously used to analyze the tumor burden of the three groups of mice based on fluorescence intensity. Figure 8 As shown in A and C.

[0116] After some mice were killed at the end of the experiment, the remaining tumor tissues in the mice were collected and weighed to calculate the size of the tumor tissue blocks. Figure 8 As shown in B.

[0117] The other part of the mice continued to be raised until they died or reached the euthanasia standard, and the survival time of the mice was counted. Figure 8 As shown in D.

[0118] Figure 8 The results of AD showed that during the experimental observation period, compared with the PBS and UTD groups, CAR-M injection had a significant inhibitory effect on tumor growth and significantly prolonged the survival time of mice.

[0119] (2) Safety evaluation of CAR-M in tumor-bearing immunodeficient mice

[0120] At the end of the experiment, the main organs of the mice (heart, liver, spleen, lung, and kidney) were collected and fixed for HE staining, and histopathological examination was performed to observe the pathological changes of the organs. Fig. 9 As shown in A.

[0121] Serum samples of mice were collected for blood biochemical tests: alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), total bile acid (TBA), albumin (ALB), alkaline phosphatase (ALP), urea nitrogen (UREA), and creatinine (CREA). The results were as follows: Fig. 9 As shown in B.

[0122] Fig. 9 The results of AB in the experiment showed that there were no drug-related abnormalities in the experimental results of organ pathology and blood biochemistry tests of tumor-bearing immunodeficient mice.

[0123] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain and an intracellular domain, wherein: The extracellular domain includes a CD8α signal peptide and a single-chain variable fragment targeting prostate-specific membrane antigen, the transmembrane domain includes a CD8 hinge region and a CD8 transmembrane region, and the intracellular domain includes an FcεR1γ intracellular region. The amino acid sequence of the extracellular domain is shown in SEQ ID NO: 1 or 3, the amino acid sequence of the transmembrane domain is shown in SEQ ID NO: 5, and the amino acid sequence of the intracellular domain is shown in SEQ ID NO:

7.

2. A nucleic acid molecule encoding the chimeric antigen receptor according to claim 1.

3. A recombinant vector comprising the nucleic acid molecule according to claim 2.

4. A modified cell comprising the nucleic acid molecule of claim 2 or the recombinant vector of claim 3, wherein: The cells include macrophages.

5. The modified cell according to claim 4, characterized in that The modified cells have increased expression of CD80 and / or CD86; and / or the modified cells have decreased expression of CD163.

6. A method for constructing the modified cell according to claim 4 or 5, comprising introducing the chimeric antigen receptor according to claim 1, the nucleic acid molecule according to claim 2, or the recombinant vector according to claim 3 into the cell.

7. The method according to claim 6, characterized in that The method comprises using GM-CSF to induce the modified cells to exhibit an M1-like phenotype; and / or The method comprises culturing the cells using X-VIVO10, X-VIVO15 or X-VIVO20 culture medium; and adding fetal bovine serum to the culture medium.

8. The method according to claim 7, characterized in that The concentration of GM-CSF is 50-80 ng / mL; and / or The concentration of the fetal bovine serum is 8-15%.

9. A pharmaceutical composition comprising the modified cell according to claim 4 or 5 or the modified cell obtained by the method according to any one of claims 6 to 8, and a pharmaceutically acceptable carrier.

10. Use of the modified cell according to claim 4 or 5 in the preparation of a medicament for treating prostate cancer.

11. Use of the modified cells obtained by the method according to any one of claims 6 to 8 in the preparation of a drug for treating prostate cancer.

12. Use of the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating prostate cancer.

13. The use according to any one of claims 10 to 12, characterized in that: The prostate cancer is metastatic castration-resistant prostate cancer.

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