Chimeric antigen receptor and cell modified by same

Abstract

The invention provides a chimeric antigen receptor (CAR), the CAR comprises a single-chain variable fragment (scFv) of a targeting antigen, a CD8 hinge region and a CD8 transmembrane region which are connected in sequence, and a heavy chain variable region (VH) of the single-chain variable fragment is connected with the CD8 hinge region. The CAR is suitable for mononuclear cells and macrophages, the prepared modified cells can specifically recognize specific antigens, and the phagocytic ability and the tumor killing ability are enhanced.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a chimeric antigen receptor suitable for monocytes or macrophages, and modified cells capable of expressing the chimeric antigen receptor. Background Art

[0002] In recent years, chimeric antigen receptor (CAR) T cells have achieved significant therapeutic effects in the treatment of hematological malignancies, but solid tumors are still difficult to conquer, for reasons including but not limited to the difficulty of solid tumors to infiltrate and their tumor microenvironment. CAR-T cells have difficulty infiltrating solid tumors, and a small number of CAR-T cells that enter the tumor will also be inhibited by the tumor microenvironment, resulting in impaired function and cell failure, leading to poor treatment efficacy.

[0003] In order to overcome the current difficulties faced by CAR-T cells in the treatment of solid tumors, macrophages have become a potential choice due to their natural characteristics. Through genetic engineering, they are endowed with specific antigen recognition capabilities. The main functions of macrophages derived from monocytes in tumors include infiltrating into tumor tissues, engulfing and degrading cancer cells, secreting cytokines and chemokines to recruit other immune cells, and presenting antigens to T cells to initiate adaptive immune responses.

[0004] CAR is an artificially designed transmembrane receptor consisting of three main parts: the extracellular domain, the transmembrane domain, and the intracellular domain. The CAR structure has undergone a series of evolutionary processes in T cell immunotherapy, and the second-generation CAR structure is currently the most commonly used. However, since macrophages have only recently entered the field of cell therapy, there is no clear and optimized CAR structure composition. Therefore, it is urgent to develop CAR suitable for macrophages. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention designs a CAR structure suitable for monocytes and macrophages. The modified cells prepared therefrom can specifically recognize specific antigens, thereby enhancing the in vitro phagocytic ability and the ability to kill tumors.

[0006] According to a first aspect of the present disclosure, a chimeric antigen receptor (CAR) is provided, wherein the CAR comprises a single-chain variable fragment (scFv) targeting an antigen, a CD8 hinge region and a CD8 transmembrane region connected in sequence, and the heavy chain variable region (VH) of the single-chain variable fragment is connected to the CD8 hinge region.

[0007] In some embodiments, the single chain variable fragment comprises a single chain variable fragment (scFv) targeting prostate specific membrane antigen (PSMA).

[0008] In some embodiments, the single-chain variable fragment includes a single-chain variable fragment (scFv) targeting human epidermal growth factor receptor-2 (HER2). The fusion protein corresponding to the CAR structure involved in the present disclosure can specifically bind to the HER2 protein and transmit signals to the intracellular domain to activate effector cells to exert phagocytic killing functions.

[0009] In some embodiments, the heavy chain variable region of the single-chain variable fragment has an amino acid sequence as shown in SEQ ID NO: 2 or 4, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 2 or 4, 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: 2 or 4 with one or more amino acids.

[0010] In some embodiments, the light chain variable region of the single-chain variable fragment has an amino acid sequence as shown in SEQ ID NO: 2 or 4, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with SEQ ID NO: 3 or 5, 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: 3 or 5 with one or more amino acids.

[0011] In some embodiments, the heavy chain variable region and the light chain variable region are connected by a linker.

[0012] In some embodiments, the linker comprises (G4S)n or (G3S)n, where n is an integer between 1 and 10. In some specific embodiments, the linker is (G4S)4, i.e., GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 6).

[0013] In some embodiments, the hinge region is preferably a CD8 hinge region. In some embodiments, the CD8 hinge region has an amino acid sequence as shown in SEQ ID 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 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity with SEQ ID NO: 7, or an amino acid sequence having equivalent function formed by adding, deleting, replacing or modifying the amino acid sequence as shown in SEQ ID NO: 7 with one or more amino acids.

[0014] In some embodiments, the CD8 transmembrane region has the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with SEQ ID NO:9, or an amino acid sequence with equivalent function formed by adding, deleting, replacing or modifying the amino acid sequence shown in SEQ ID NO:9 with one or more amino acids.

[0015] In some embodiments, the chimeric antigen receptor further comprises an intracellular region, and the intracellular region can be selected from any intracellular region commonly used in the art that can achieve intracellular signal transduction function, including but not limited to the FcεR1γ intracellular region.

[0016] In some embodiments, the FcεR1γ intracellular region has the amino acid sequence shown in SEQ ID NO:10, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO:10, or an amino acid sequence with equivalent function formed by adding, deleting, replacing or modifying the amino acid sequence shown in SEQ ID NO:10 with one or more amino acids.

[0017] In some embodiments, the single-chain variable fragment, hinge region, CD8 transmembrane region and / or intracellular signaling domain are directly connected or connected through a linker.

[0018] In some embodiments, the linker comprises (G4S)n or (G3S)n, where n is an integer between 1 and 10.

[0019] In some embodiments, the chimeric antigen receptor further comprises a signal peptide, and the signal peptide can be selected from any signal peptide commonly used in the art that can realize the function of a signal peptide, including but not limited to, a CD8α leader peptide.

[0020] In some embodiments, the CD8α leader peptide has the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with SEQ ID NO:1, or an amino acid sequence with equivalent function formed by adding, deleting, replacing or modifying the amino acid sequence shown in SEQ ID NO:1 with one or more amino acids.

[0021] In some embodiments, the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO: 12 or 14, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with SEQ ID NO: 12 or 14, or an amino acid sequence with equivalent function formed by adding, deleting, replacing or modifying the amino acid sequence shown in SEQ ID NO: 12 or 14 with one or more amino acids.

[0022] In some embodiments, the chimeric antigen receptor is expressed by a modified immune cell.

[0023] In some embodiments, the chimeric antigen receptor is expressed by a modified monocyte or a modified macrophage.

[0024] 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.

[0025] In some embodiments, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:16 or 18, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with SEQ ID NO:16 or 18.

[0026] According to another aspect of the present disclosure, a recombinant vector is provided, which contains the nucleic acid molecule described in the present disclosure.

[0027] In some embodiments, the recombinant vector is a viral vector, such as a retroviral vector, an adenoviral vector, a lentiviral vector or an adeno-associated viral vector. In some specific embodiments, the recombinant vector is preferably an adenoviral vector.

[0028] According to another aspect of the present disclosure, a modified cell is provided, which contains the nucleic acid molecule described in the present disclosure or the recombinant vector described in the present disclosure.

[0029] In some embodiments, the cells include monocytes and / or macrophages.

[0030] In some embodiments, the modified cells have at least one upregulated M1 marker.

[0031] In some embodiments, the modified cells have elevated expression of CD80.

[0032] According to another aspect of the present disclosure, a pharmaceutical composition is provided, which comprises the chimeric antigen receptor or the modified cell described in the present disclosure, 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 or the pharmaceutical composition described in the present disclosure in the preparation of a drug for preventing, treating or alleviating inflammatory diseases and / or tumors.

[0034] In some embodiments, the drug is used to inhibit the growth of tumor cells and / or induce apoptosis of tumor cells.

[0035] The present disclosure provides a CAR structural sequence having a "VL-VH-CD8" structure, which is transfected into monocytes or macrophages to prepare CAR-monocytes / macrophages, thereby enhancing the phagocytic ability and tumor-killing ability of CAR-monocytes / macrophages, and providing support for the application of monocytes and macrophages in cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The schematic diagram of the structure of the CAR molecule is shown, which includes the antigen-specific scFv, CD28 hinge region / CD8 hinge region, CD8 transmembrane region and FcεRI-γ intracellular region.

[0037] Figure 2 A schematic diagram of the AD5 / F35 adenoviral plasmid structure is shown.

[0038] Figure 3 A schematic diagram of the plasmid structure of Pvax1 is shown.

[0039] Figure 4The figure shows the expression percentage of anti-HER2 CAR molecules on macrophages using adenovirus as a vector under different MOI conditions, where the UTD group is macrophages not transfected with adenovirus.

[0040] Figure 5 The expression percentage of anti-PSMA CAR molecules on macrophages using IVT RNA as a vector is shown.

[0041] Figure 6 The figure shows the expression percentage of anti-HER2 adenovirus CAR molecules on monocytes using adenovirus as a vector at MOI=500, where the UTD group is monocytes not transfected with adenovirus.

[0042] Figure 7 The experimental results of in vitro killing of OE19-Luci target cells by anti-HER2 CAR-macrophages at various effector-target ratios are shown, where the UTD group is macrophages that are not transfected with adenovirus.

[0043] Figure 8 The results of the in vitro killing of LnCap-Luci target cells by anti-PSMA CAR-macrophages at various effector-target ratios are shown, where the UTD group is macrophages that are not transfected with adenovirus.

[0044] Fig. 9 The results of the in vitro killing of OE19-Luci target cells by anti-HER2 CAR-monocytes at various effector-target ratios are shown, where the UTD group is macrophages that are not transfected with adenovirus.

[0045] Fig.10 The results of phagocytosis of OE19 tumor cells by anti-HER2 CAR-macrophages are shown, where the UTD group is macrophages that are not transfected with adenovirus.

[0046] Fig.11 The results of phagocytosis of OE19 tumor cells by anti-HER2 CAR-monocytes are shown, where the UTD group is monocytes not transfected with adenovirus.

[0047] Fig.12 The expression rate of CD80, an M1 marker of anti-HER2 CAR-macrophages at each effector-target ratio is shown. DETAILED DESCRIPTION

[0048] 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.

[0049] 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.

[0050] 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.

[0051] As used herein, the term "about" 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.

[0052] As used herein, the term "chimeric antigen receptor" (CAR) refers to a fusion protein including 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 in cells. "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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] As used herein, the term "linker" refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) or a non-polypeptide, such as an alkyl chain. In some embodiments, two or more linkers can be connected in series. When multiple linkers are present, each linker can be the same or different.

[0058] In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker may comprise at least about two amino acids. In some embodiments, the linker comprises one or more amino acids. In some embodiments, the linker comprises a Gly-Ser (GS) linker. In some embodiments, the GS linker comprises (G4S)n, where n is an integer between 1 and 10. In some embodiments, the GS linker comprises (G3S)n, where n is an integer between 1 and 10. In some embodiments, the peptide linker is synthetic, i.e., non-naturally occurring. In one aspect, the peptide linker comprises a peptide comprising an amino acid sequence that connects or genetically fuses a first linear amino acid sequence to a second linear amino acid sequence, wherein the first linear amino acid sequence is not naturally connected or genetically fused to the second linear amino acid sequence in nature.

[0059] 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.

[0060] 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, a gap present in the target sequence is not counted. Similarly, since the target sequence nucleotides or amino acids are counted, but the nucleotides or amino acids from the reference sequence are not counted, the gaps present in the reference sequence are not counted.

[0061] 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 determination of the sequence identity percentage between the comparison of the sequence and the two sequences can be completed using software that is easy to use and download online. Suitable software programs can be obtained from various sources for the comparison of protein and nucleotide sequences. A suitable program for determining the sequence identity percentage is bl2seq, which is a part of the BLAST program suite that can be obtained 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 for comparing nucleic acid sequences, and BLASTP is used for comparing 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.

[0062] 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.

[0063] 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.

[0064] 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, including 1-99.99% by weight or volume alone or in combination. 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.

[0065] The present invention designs different CAR structures, combines them with adenovirus vectors to transfect monocytes and macrophages, prepares chimeric antigen receptor-monocyte / macrophages expressing the target fusion protein, and evaluates their cellular function and anti-tumor activity.

[0066] The reagents and / or kits used in the following examples are all commercially available or can be synthesized by known methods.

[0067] Example 1 Construction of CAR molecules

[0068] like Figure 1As shown, an anti-HER2 CAR molecule and an anti-PSMA CAR molecule including a CD8α leader peptide, a HER2 antigen-specific scFv / PSMA antigen-specific scFv, a CD28 hinge region (CD28 hinge) / CD8 hinge region (CD8 hinge), a CD8 transmembrane region (CD8 TM), and a FcεRI universal γ subunit intracellular region (FcεRI-γ intracellular region) are constructed, and their structures are HER2-VH-VL-CD28, HER2-VL-VH-CD8, PSMA-VH-VL-CD8, and PSMA-VL-VH-CD8, respectively, and the sequences are shown in Table 1 below.

[0069] The CAR molecule gene fragments with sequences as shown in SEQ ID NO: 15-16 were cloned into adenovirus vectors. The adenovirus vector backbone was AD5 / F35 (purchased from Yunzhou Biotechnology, and the schematic diagram of the adenovirus vector structure is shown in Figure 2 As shown), a CAR molecule overexpression vector targeting HER2 is obtained, which can be used for the expression of CAR molecules. Virus liquid is obtained by linearization of adenovirus plasmid, packaging of adenovirus, concentration and purification of virus liquid.

[0070] The CAR molecule gene fragments with sequences as shown in SEQ ID NO: 17-18 were cloned into the IVT plasmid vector. The IVT vector plasmid was pVAX1 (purchased from Nanjing GenScript, the vector structure diagram is shown in Figure 3 As shown), an IVT vector expressing a CAR molecule targeting PSMA was obtained. The IVT plasmid was single-digested using BspQI restriction endonuclease (purchased from Takara), and the linearized DNA was purified and recovered using DNA magnetic beads (purchased from Shenji Biotechnology). The cap3011 RNA preparation kit (purchased from Shenji Biotechnology) was used to transcribe the linearized DNA for in vitro mRNA preparation, and the IVT RNA was purified and recovered using RNA magnetic beads (purchased from Shenji Biotechnology).

[0071] Table 1

[0072]

[0073]

[0074]

[0075]

[0076] Example 2. Isolation and sorting of peripheral blood mononuclear cells

[0077] The single blood sample obtained from the commercial company was transferred to a centrifuge tube and centrifuged at 700g for 10 minutes. After the centrifugation, the upper plasma was discarded and the blood cells were resuspended in sodium chloride solution to obtain a blood cell mixture. The obtained blood cell mixture was mixed with Ficoll-Paque TM PREMIUM sterile solution (Cytiva) was mixed and centrifuged at 400g for 30min. After centrifugation, the buffy coat layer was aspirated and sodium chloride solution was added. The cells were washed by centrifugation for 3 times at 500g, 400g, and 200g for 10min each. AutoMACS Running buffer (Miltenyi) was added to the blood cells to resuspend the cells.

[0078] CD14 magnetic beads (Miltenyi) were added to the cell resuspension, mixed and placed in a refrigerator at 2-8°C for 15 min, and CD14 was separated using LS columns and MidiMACS separation unit. + Cell separation and harvesting of CD14 + Monocytes.

[0079] Example 3. Preparation of CAR-macrophages and detection of CAR molecule expression intensity

[0080] The CD14 isolated in Example 2 + Monocytes were induced to differentiate into macrophages using granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0081] After the monocytes were induced to differentiate into macrophages, adenovirus vectors or IVT RNA of different CAR molecules constructed in Example 1 were added for transfection, and CAR-macrophages were harvested 48 hours after transfection. Among them, for the anti-HER2 CAR molecules, the adenovirus vector virus solution obtained in Example 1 was added to the macrophages at an MOI of 250, 500 and 750, respectively, to obtain anti-HER2 CAR-macrophages. For the anti-PSMA CAR molecules, IVT RNA and Lipofectamine TM MessengerMAX TM The transfection reagent (purchased from Thermo) was mixed at a plasmid ratio of 2:1, and after incubation at room temperature for 10 min, the mixture was added to the cell culture supernatant for transfection.

[0082] The harvested CAR-macrophages were mixed with PBS and centrifuged at 400g for 5 minutes. The cell pellet was mixed with PBS and HER2 protein (purchased from Acro) or PSMA protein (purchased from Acro) (4μl / test) was added and incubated at room temperature for 20 minutes. Unbound proteins were removed by centrifugation at 400g for 5 minutes. After washing, the cells were subjected to flow cytometry detection of CAR molecules targeting HER2 or PSMA, and the percentage of CAR molecules expressed on the macrophage membrane surface was analyzed.

[0083] Figure 4 It was shown that under a wide range of MOI conditions, adenoviral vectors were able to effectively deliver anti-HER2 CAR molecules into macrophages, and the effect of macrophage expression of CAR molecules by HER2-VL-VH-CD8 construct was significantly better than that by HER2-VH-VL-CD28 construct.

[0084] Figure 5 It showed that IVT RNA could effectively deliver anti-PSMA CAR molecules into macrophages, and the effect of macrophage expression of CAR molecules of PSMA-VL-VH-CD8 construct was significantly better than that of PSMA-VH-VL-CD8 construct.

[0085] Figure 4 and Figure 5 The results showed that the VL-VH tandem connection in the single-chain variable fragment (scFv) targeting HER2 or PSMA can significantly improve the effect of macrophage expression of CAR molecules. This result suggests that the VL-VH tandem CAR structure stability is better based on macrophages.

[0086] Example 4. Preparation of CAR-monocytes and detection of CAR molecule expression intensity

[0087] The adenovirus vector virus solution of the anti-HER2 CAR molecule constructed in Example 1 was added to the CD14+ monocytes sorted in Example 2 at an MOI of 500 for transfection, and the CAR-monocytes were harvested 48 hours after transfection.

[0088] The harvested CAR-monocytes were mixed with PBS and centrifuged at 400 g for 5 minutes. The cell pellet was mixed with PBS and HER2 protein (purchased from Acro) was added and incubated at room temperature for 20 minutes. Unbound proteins were removed by centrifugation at 400 g for 5 minutes. After washing, the cells were subjected to flow cytometry detection of CAR molecules targeting HER2, and the percentage of CAR molecules expressed on the monocyte membrane surface was analyzed.

[0089] Figure 6It was shown that under MOI = 500, adenoviral vectors effectively delivered anti-HER2 CAR molecules into monocytes, and the effect of monocyte expression of CAR molecules by HER2-VL-VH-CD8 construct was significantly better than that by HER2-VH-VL-CD28 construct.

[0090] Example 5. Tumor cell killing ability determination

[0091] Commercial luciferase lentivirus was purchased to transfect OE19 cells (human esophageal cancer cells, purchased from Kebai Bio) and LnCap cells (human prostate cancer cells, purchased from Kebai Bio), and OE19-luci cells and LnCap-luci cells that stably expressed luciferase were obtained after screening. OE19-luci cells expressed HER2 protein, and LnCap-luci cells expressed PSMA protein. OE19-luci cells and LnCap-luci cells were used as target cells for CAR-HER2 and CAR-PSMA killing experiments, respectively.

[0092] OE19-luci cells or LnCap-luci cells were plated, and the target cell pellet was resuspended in X-VIVO15 (Lonza) + 10% FBS (gibco) culture medium. The cell suspension was added to each well of a 96-well plate until there were 1E4 target cells in each well.

[0093] The anti-HER2 / anti-PSMA CAR-macrophages prepared in Example 3 and the anti-HER2 CAR-monocytes (effector cells) prepared in Example 4 were gradiently diluted according to the effector-target ratio (E:T) requirements, and the effector cells of different cell densities were added to the 96-well plate inoculated with target cells at a ratio of 1:1 and cultured in a cell culture incubator.

[0094] After 48 h, the 96-well plate was removed and 50 uL of the prepared Steady-Glo Luciferase Assay reagent (Promega) was added to each well. After reacting for 5 min, the fluorescence value was read using an enzyme reader.

[0095] Figure 7-Figure 9 The results showed that anti-HER2 CAR-macrophages, anti-PSMA CAR-macrophages and anti-HER2 CAR-monocytes were able to specifically lyse the corresponding tumor cells at each effector-target ratio, while the untransfected UTD group only nonspecifically lysed tumor cells at a low level at high doses, and the HER2-VL-VH-CD8 construct had a higher tumor cell killing rate than the HER2-VH-VL-CD28 construct, and PSMA-VL-VH-CD8 had a higher tumor cell killing rate than PSMA-VH-VL-CD8.

[0096] Figure 7-Figure 9 The results showed that the VL-VH tandem connection in the single-chain variable fragment (scFv) targeting HER2 or PSMA can significantly improve the killing rate of tumor cells.

[0097] Example 6. Phagocytic function assay

[0098] Suspend OE19 cells in DPBS, add Dir fluorescent probe (yeasen) dye to the cell suspension, mix well and place in a cell culture incubator for 30 minutes. Add complete culture medium (X-VIVO15 + 10% FBS) preheated at 37°C to terminate the staining, resuspend the cells, and adjust the cell density to 7.5E5 / mL.

[0099] The harvested anti-HER2 CAR-macrophages prepared in Example 3 and the anti-HER2 CAR-monocytes prepared in Example 4 were added to complete culture medium to suspend the cells, and the cell density was adjusted to 7.5E5 / mL.

[0100] Dir-labeled OE19 and CAR-M cells / CAR-monocytes were mixed evenly in a 1:1 ratio and cultured in a cell culture incubator for 2 h.

[0101] Add 3 μL FITC-CD11b for staining and analyze CD11b by flow cytometry + The cells in the group labeled with DiR are considered to be cells that phagocytize OE19.

[0102] The results showed that anti-HER2 CAR-macrophages ( Fig.10 ) and anti-HER2 CAR-monocytes ( Fig.11 ) were able to phagocytose HER2-expressing OE19 tumor cells, while the untransfected UTD group showed only a low level of nonspecific phagocytosis, and the HER2-VL-VH-CD8 construct had a higher phagocytosis rate than the HER2-VH-VL-CD28 construct.

[0103] Figure 10-11 The results showed that the VL-VH tandem connection mode in the single-chain variable fragment (scFv) targeting HER2 can significantly improve the phagocytic level of tumor cells.

[0104] Example 7. Detection of Macrophage M1 Phenotype

[0105] The anti-HER2 CAR-macrophages prepared in Example 3 were mixed with PBS and then CD80 antibody (purchased from BD) was added, incubated at room temperature for 30 minutes, centrifuged at 400g for 5 minutes to remove unbound antibodies, and the cells were washed and subjected to flow cytometry.

[0106] The results are as follows Fig.12 The results showed that the M1 marker CD80 of macrophages was upregulated in adenovirus MOI dose-dependent manner. Compared with UTD, HER2-VL-VH-CD8 and HER2-VH-VL-CD28 CAR-macrophages both showed a good expression rate of M1 macrophage marker CD80, and had a good M1 type maintenance effect, but the CD80 expression rate of HER2-VL-VH-CD8 was higher.

[0107] 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 (CAR), comprising a single-chain variable fragment (scFv) targeting an antigen, a CD8 hinge region and a CD8 transmembrane region connected in sequence, wherein the heavy chain variable region (VH) of the single-chain variable fragment is connected to the CD8 hinge region.

2. The chimeric antigen receptor according to claim 1, characterized in that The single-chain variable fragment includes a single-chain variable fragment targeting prostate-specific membrane antigen (PSMA) and / or human epidermal growth factor receptor-2 (HER2); Preferably, the heavy chain variable region of the single-chain variable fragment has an amino acid sequence as shown in SEQ ID NO: 2 or 4, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 2 or 4, or an amino acid sequence having equivalent function formed by adding, deleting, replacing or modifying the amino acid sequence as shown in SEQ ID NO: 2 or 4 with one or more amino acids; Preferably, the light chain variable region of the single-chain variable fragment has an amino acid sequence as shown in SEQ ID NO: 3 or 5, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 3 or 5, or an amino acid sequence having equivalent function formed by adding, deleting, replacing or modifying the amino acid sequence as shown in SEQ ID NO: 3 or 5 with one or more amino acids; Preferably, the heavy chain variable region and the light chain variable region are connected by a linker. More preferably, the linker comprises (G4S)n or (G3S)n, n being an integer between 1 and 10.

3. The chimeric antigen receptor according to claim 1 or 2, characterized in that The CD8 hinge region has the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 7, or an amino acid sequence having 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; and / or The CD8 transmembrane region has the amino acid sequence shown in SEQ ID NO:8, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:8, or an amino acid sequence with equivalent function formed by adding, deleting, replacing or modifying the amino acid sequence shown in SEQ ID NO:8 with one or more amino acids; and / or The chimeric antigen receptor further comprises an intracellular region, preferably, the intracellular region comprises an FcεR1γ intracellular region; more preferably, the FcεR1γ intracellular region has an amino acid sequence as shown in SEQ ID NO: 10, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 10, or an amino acid sequence having equivalent function formed by adding, deleting, replacing or modifying the amino acid sequence as shown in SEQ ID NO: 10 with one or more amino acids; and / or The single-chain variable fragment, hinge region, CD8 transmembrane region and / or intracellular region are directly connected or connected through a linker; Preferably, the linker comprises (G4S)n or (G3S)n, n is an integer between 1 and 10; Preferably, the chimeric antigen receptor further comprises a signal peptide; More preferably, the chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO: 12 or 14, or an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 12 or 14, 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: 12 or 14 with one or more amino acids.

4. The chimeric antigen receptor according to any one of claims 1 to 3, characterized in that The chimeric antigen receptor is expressed by a modified immune cell; Preferably, the chimeric antigen receptor is expressed by a modified monocyte or a modified macrophage.

5. A nucleic acid molecule encoding the chimeric antigen receptor according to any one of claims 1 to 4. A recombinant vector comprising the nucleic acid molecule according to claim 5.

7. A modified cell comprising the nucleic acid molecule of claim 5 or the recombinant vector of claim 6; Preferably, the cells include monocytes and / or macrophages; Preferably, the modified cells have at least one upregulated M1 marker; More preferably, the modified cells have elevated expression of CD80.

8. A pharmaceutical composition comprising the chimeric antigen receptor according to any one of claims 1 to 4 or the modified cell according to claim 7, and a pharmaceutically acceptable carrier.

9. Use of the chimeric antigen receptor according to any one of claims 1 to 4, the modified cell according to claim 7 or the pharmaceutical composition according to claim 8 in the preparation of a medicament for preventing, treating or alleviating inflammatory diseases and / or tumors.

10. The use according to claim 9, characterized in that: The drug is used for inhibiting the growth of tumor cells and / or inducing apoptosis of tumor cells.

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