Chimeric antigen receptors and modified cells thereof

Abstract

The present disclosure provides 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, a heavy chain variable region (VH) of the scFv being connected to the CD8 hinge region. The CAR is suitable for monocytes and macrophages, and the modified cells prepared therefrom can specifically recognize a specific antigen, and enhance phagocytic ability and tumor killing ability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a chimeric antigen receptor suitable for monocytes or macrophages, and a modified cell capable of expressing the chimeric antigen receptor. BACKGROUND

[0002] In recent years, chimeric antigen receptor (CAR) T cells have achieved remarkable efficacy in the treatment of hematological malignancies, but solid tumors are still difficult to conquer, and the reasons include but are not limited to the difficulty of infiltrating solid tumors and the tumor microenvironment. CAR-T cells are difficult to infiltrate solid tumors, and a small part of CAR-T cells that enter the tumor will be inhibited by the tumor microenvironment and functionally impaired and cell exhausted, thereby resulting in poor treatment efficacy.

[0003] To overcome the current problems of CAR-T cells in the treatment of solid tumors, macrophages have become a potential choice due to their natural characteristics. By genetically engineering them to have specific antigen recognition ability, macrophages derived from monocytes have the main functions in tumors, including penetrating into tumor tissues, phagocytosing 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, which includes three main parts: extracellular domain, transmembrane domain and intracellular domain. CAR structure has undergone a series of evolution processes in T cell immunotherapy, and the second generation CAR structure is currently used more. Macrophages have been in the field of cell therapy for a short time, and there is no clear optimized CAR structure composition. Therefore, it is urgent to develop a CAR suitable for macrophages. SUMMARY

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

[0006] According to a first aspect of the present disclosure, there is provided a chimeric antigen receptor (CAR) comprising, in sequence, a single chain variable fragment (scFv) targeting an antigen, a CD8 hinge region, and a CD8 transmembrane region, the heavy chain variable region (VH) of the single chain variable fragment being 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 comprises a single chain variable fragment (scFv) targeting human epidermal growth factor receptor-2 (HER2). The CAR structure corresponding to the fusion protein involved in the present disclosure can specifically bind to HER2 protein and transmit signals to the intracellular domain to activate effector cells to perform phagocytic killing function.

[0009] In some embodiments, the heavy chain variable region of the single chain variable fragment has the amino acid sequence set forth 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%, 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 one or more amino acids of the amino acid sequence set forth in SEQ ID NO: 2 or 4.

[0010] In some embodiments, the light chain variable region of the single chain variable fragment has the amino acid sequence set forth 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%, at least 99.5% sequence identity to SEQ ID NO: 3 or 5, or an amino acid sequence with equivalent function formed by adding, deleting, replacing or modifying one or more amino acids of the amino acid sequence set forth in SEQ ID NO: 3 or 5.

[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, 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 the amino acid sequence set forth 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 to SEQ ID NO: 7, or an amino acid sequence with equivalent functionality to the amino acid sequence set forth in SEQ ID NO: 7 with one or more amino acid additions, deletions, substitutions, or modifications.

[0014] In some embodiments, the CD8 transmembrane region has the amino acid sequence set forth 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%, at least 99.5% sequence identity to SEQ ID NO: 9, or an amino acid sequence with equivalent functionality to the amino acid sequence set forth in SEQ ID NO: 9 with one or more amino acid additions, deletions, substitutions, or modifications.

[0015] In some embodiments, the chimeric antigen receptor further comprises an intracellular region, which can be selected from any intracellular region commonly used in the art that is capable of achieving an intracellular signaling function, including but not limited to, an FcεR1 γ intracellular region.

[0016] In some embodiments, the FcεR1 γ intracellular region has the amino acid sequence set forth 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%, at least 99.5% sequence identity to SEQ ID NO: 10, or an amino acid sequence with equivalent functionality to the amino acid sequence set forth in SEQ ID NO: 10 with one or more amino acid additions, deletions, substitutions, or modifications.

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

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

[0019] In some embodiments, the chimeric antigen receptor further comprises a signal peptide, which can be selected from any signal peptide capable of achieving the function of a signal peptide commonly used in the art, including but not limited to, CD8a leader peptide.

[0020] In some embodiments, the CD8a leader peptide has the amino acid sequence set forth 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%, at least 99.5% sequence identity to SEQ ID NO: 1, or an amino acid sequence having equivalent function as the amino acid sequence set forth in SEQ ID NO: 1 with one or more amino acid additions, deletions, substitutions or modifications.

[0021] In some embodiments, the chimeric antigen receptor has the amino acid sequence set forth 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%, at least 99.5% sequence identity to SEQ ID NO: 12 or 14, or an amino acid sequence having equivalent function as the amino acid sequence set forth in SEQ ID NO: 12 or 14 with one or more amino acid additions, deletions, substitutions or modifications.

[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, there is provided a nucleic acid molecule encoding the chimeric antigen receptor described in the present disclosure.

[0025] In some embodiments, the nucleic acid molecule has the nucleotide sequence set forth 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%, at least 99.5% sequence identity to SEQ ID NO: 16 or 18.

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

[0027] In some embodiments, the recombinant vector is a viral vector, for example, 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 yet another aspect of the present disclosure, there is provided a modified cell containing the nucleic acid molecule of the present disclosure, or the recombinant vector of the present disclosure.

[0029] In some embodiments, the cell comprises monocytes and / or macrophages.

[0030] In some embodiments, the modified cell has at least one up-regulated M1 marker.

[0031] In some embodiments, the modified cell has elevated expression of CD80.

[0032] According to yet another aspect of the present disclosure, there is provided a pharmaceutical composition comprising the chimeric antigen receptor of the present disclosure or the modified cell of the present disclosure, and a pharmaceutically acceptable carrier.

[0033] According to yet another aspect of the present disclosure, there is provided use of the chimeric antigen receptor of the present disclosure, the modified cell of the present disclosure, or the pharmaceutical composition of the present disclosure, in the manufacture of a medicament for preventing, treating, or alleviating an inflammatory disease and / or a tumor.

[0034] In some embodiments, the medicament is for inhibiting the growth of tumor cells and / or inducing apoptosis of tumor cells.

[0035] The present disclosure provides a CAR structure sequence having a "VL-VH-CD8" structure, which, when transfected into monocytes or macrophages, produces CAR-monocyte / macrophage, enhances the phagocytic ability and killing ability of CAR-monocyte / macrophage against tumors, and provides assistance for the application of monocytes and macrophages in cell therapy. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 A schematic diagram showing the structure of a CAR molecule, which comprises an antigen-specific scFv, a CD28 hinge region / CD8 hinge region, a CD8 transmembrane region, and a FcεRI-γ intracellular region, respectively.

[0037] Figure 2 A schematic diagram showing the structure of an AD5 / F35 adenovirus plasmid.

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

[0039] Figure 4The percentage of expression of anti-HER2 CAR molecules on macrophages with adenovirus as a carrier under different MOI conditions is shown, wherein the UTD group is the macrophage not transfected with adenovirus.

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

[0041] Figure 6 The percentage of expression of anti-HER2 adenovirus CAR molecules on monocytes under the condition of MOI = 500 is shown, wherein the UTD group is the monocyte not transfected with adenovirus.

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

[0043] Figure 8 The experimental results of in vitro killing of LnCap-Luci target cells by anti-PSMA CAR-macrophages under different effector-to-target ratios are shown, wherein the UTD group is the macrophage not transfected with adenovirus.

[0044] Figure 9 The experimental results of in vitro killing of OE19-Luci target cells by anti-HER2 CAR-monocytes under different effector-to-target ratios are shown, wherein the UTD group is the macrophage not transfected with adenovirus.

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

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

[0047] Figure 12 The expression rate of M1 marker CD80 of anti-HER2 CAR-macrophages under different effector-to-target ratios is shown. DETAILED DESCRIPTION

[0048] For the purpose of clarity, technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the application pertains. Throughout the description and claims of this specification, the singular "a" and "an" can include plural references unless the context clearly dictates otherwise. As used herein, the term "another" is defined as at least a second or more.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following definitions are applied throughout this specification and claims, unless otherwise qualified. As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0050] The expressions "a" and "an" as used herein mean "one or more" unless otherwise defined. For example, reference to "a cell" includes a plurality of such cells, equivalents, and the like, as will be appreciated by those skilled in the art.

[0051] As used herein, the term "about" means a range of ±20% of the value that follows. In some embodiments, the term "about" means a range of ±10% of the value that follows. In some embodiments, the term "about" means a range of ±5% of the value that follows.

[0052] As used herein, the term "chimeric antigen receptor" (CAR) refers to a fusion protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a different polypeptide than the extracellular domain is derived from, and at least one intracellular domain. A "chimeric antigen receptor" (CAR) is sometimes referred to as a "chimeric receptor," a "T-body," or a "chimeric immunoreceptor" (CIR). An "extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide that is capable of binding to some antigen. An "intracellular domain" refers to any oligopeptide or polypeptide known to function as a domain that sends a signal to cause activation or inhibition of a biological process within a cell. A "transmembrane domain" refers to any oligopeptide or polypeptide known to span the cell membrane and be capable of acting to link an extracellular domain and a signaling domain. A chimeric antigen receptor can optionally include a "hinge domain" that functions as a linker 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, this term includes, but is not limited to, single-, double- or multi- stranded DNA or RNA, DNA or RNA genomes, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0054] The term "encode", when applied to a nucleic acid sequence, refers to a polynucleotide recited to "encode" a polypeptide, which, 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 fragments thereof. 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 transfer between different hosts, including but not limited to plasmids, viruses, cosmids, phages, BACs, YACs, and the like. In some embodiments, plasmid vectors can be prepared from commercially available vectors. In other embodiments, viral vectors can be manufactured from baculovirus, retrovirus, adenovirus, AAV, and the like, 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 application. Molecules can be modified in a variety of ways, including chemically, structurally, and functionally. Cells can be modified by nucleic acid introduction.

[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 can 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., not naturally occurring. In one aspect, the peptide linker comprises a peptide comprising an amino acid sequence that links or genetically fuses a first linear amino acid sequence to a second linear amino acid sequence that are not naturally linked or genetically fused in nature.

[0059] As used herein, the term "transfection" or "transformation" or "transduction" refers to the process of transferring or introducing exogenous nucleic acids into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the original subject cell and its progeny.

[0060] A "percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences, for instance, refers to the number of identical matches between the sequences within a comparison window, taking into account any additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A match is any position wherein the same nucleotide or amino acid is present in both the target and reference sequences. Gaps are not counted as nucleotides or amino acids. Likewise, gaps present in the reference sequence are not counted, as are nucleotides or amino acids from the reference sequence, since the target sequence nucleotides or amino acids are counted.

[0061] The percentage of sequence identity can be determined by the following process: determining the number of positions at which the identical amino acid residue or nucleic acid base occurs in both sequences to be compared and dividing that number by the total number of positions in the comparison window. The percentage of sequence identity is calculated by multiplying the number of identical positions by 100 and dividing the product by the total number of positions in the comparison window. Comparison of sequences and determination of the percentage of sequence identity between two sequences can be accomplished using software programs commonly available online and for download. Suitable software programs are available from various sources for alignment of protein and nucleotide sequences. One suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST suite of programs available from the National Center for Biotechnology Information's BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparison between two sequences. BLASTN is used for comparing nucleic acid sequences, while BLASTP is used for comparing amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0062] Macrophages are a phenotypically heterogeneous population of immune cells that play important roles during both initiation and resolution of inflammation. Upon stimulation, macrophages can polarize into two phenotypes: (1) a classical activated (inflammatory) phenotype M1, which can be induced by lipopolysaccharide (LPS) or interferon gamma (IFN-γ), and produces proinflammatory cytokines such as TNFα, IL-1β, etc.; (2) an alternative activated (wound healing) phenotype M2, which can be induced by IL-4, IL-13, etc., and produces 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 exerts proinflammatory effects to counteract stimuli in the early phase of inflammation, but sustained M1 polarization causes tissue damage; M2 exerts anti-inflammatory effects to promote tissue repair and vascular regeneration.

[0063] The terms "subject," "host," "individual," and "patient" are used interchangeably herein to mean a human and veterinary subject, such as a human, an animal, a non-human primate, a dog, a cat, a sheep, a mouse, a horse, and a cow. 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 or non-naturally occurring carrier that is inert, e.g., a detectable agent or label, or active, e.g., an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant, and the like, and includes a pharmaceutically acceptable carrier. Carriers also include pharmaceutical excipients and additive proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derivatized sugars, e.g., sugar alcohols, aldonic acids, esterified sugars, and the like; and polysaccharides or sugar polymers), which can be present individually or in combination, comprising 1-99.99% by weight or volume, individually or in combination. Exemplary protein excipients include serum albumin (e.g., human serum albumin (HSA), recombinant human albumin (rHA)), gelatin, casein, and the like. 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, and the like. 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, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrine, dextran, starch, and the like; and sugar alcohols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), and inositol.

[0065] The present disclosure designs different CAR structures, which are combined with adenovirus vector to transfect monocytes and macrophages, to prepare chimeric antigen receptor-monocyte / macrophage expressing the fusion protein of interest, to evaluate its cell function and anti-tumor activity.

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

[0067] Example 1 Construction of CAR molecules

[0068] As Figure 1As shown, the CAR molecules against HER2 and against PSMA were constructed, including CD8 alpha leader peptide, HER2 antigen-specific scFv / PSMA antigen-specific scFv, CD28 hinge region (CD28 hinge) / CD8 hinge region (CD8 hinge), CD8 transmembrane region (CD8 TM), and Fc epsilon R1 universal gamma subunit intracellular region (Fc epsilon R1- gamma intracellular region), the structures of which are HER2-VH-VL-CD28, HER2-VL-VH-CD8, PSMA-VH-VL-CD8, and PSMA-VL-VH-CD8, respectively, as shown in Table 1 below.

[0069] The gene fragments of the CAR molecules with sequences as shown in SEQ ID NOs: 15-16 were cloned into an adenovirus vector. The adenovirus vector skeleton was AD5 / F35 (purchased from Yunzhou Biology, and the schematic diagram of the adenovirus vector structure is as shown in Figure 2 As shown), to obtain a CAR molecule overexpression vector expressing a HER2-targeting CAR molecule, which can be used for expression of the CAR molecule. After linearization of the adenovirus plasmid, adenovirus packaging, and virus liquid concentration and purification, the virus liquid was obtained.

[0070] The gene fragments of the CAR molecules with sequences as shown in SEQ ID NOs: 17-18 were cloned into an IVT plasmid vector. The IVT vector plasmid was pVAX1 (purchased from Nanjing Kingsrui, and the schematic diagram of the vector structure is as shown in Figure 3 As shown), to obtain a CAR molecule IVT vector expressing a PSMA-targeting CAR molecule. The IVT plasmid was single-cut using a BspQI restriction enzyme (purchased from Takara), and the linearized DNA was purified and recovered using a DNA magnetic bead (purchased from Shenji Biology) purification method. The linearized DNA was used to prepare in vitro mRNA using a cap3011 RNA preparation kit (purchased from Shenji Biology), and the IVT RNA was purified and recovered using an RNA magnetic bead (purchased from Shenji Biology) purification method.

[0071] Table 1

[0072]

[0073]

[0074]

[0075]

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

[0077] Apheresis blood obtained from commercial companies was transferred to centrifuge tubes and centrifuged at 700g for 10 minutes. After centrifugation, the supernatant 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 then mixed with Ficoll-Paque. TM Mix with PREMIUM sterile solution (Cytiva) and centrifuge at 400g for 30 minutes. After centrifugation, aspirate the white membrane layer and add sodium chloride solution. Wash three times by centrifugation at 500g, 400g, and 200g for 10 minutes each. Resuspend the cells in autoMACS running buffer (Miltenyi) in the blood cells.

[0078] CD14 magnetic beads (Miltenyi) were added to the cell resuspension, mixed well, and incubated at 2–8°C for 15 min. CD14 separation was then performed using an LS column and a MidiMACS separation unit. + Cell isolation and CD14 harvest + Mononuclear cells.

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

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

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

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

[0083] Figure 4 It was shown that under a wide range of MOI conditions, the adenovirus vector could effectively deliver the anti-HER2 CAR molecules into macrophages, and the macrophage expression of CAR molecules of the HER2-VL-VH-CD8 construct was significantly better than that of the HER2-VH-VL-CD28 construct.

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

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

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

[0087] The CD14+ monocytes sorted in Example 2 were transfected with the adenovirus vector virus liquid of the anti-HER2 CAR molecules constructed in Example 1 at an MOI of 500. The CAR-monocytes were harvested 48 hours after transfection.

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

[0089] Figure 6It is shown that the adenovirus vector delivers the anti-HER2 CAR molecule into monocytes effectively under the condition of MOI = 500, and the effect of monocyte expression of the CAR molecule of the HER2-VL-VH-CD8 construct is obviously better than that of the HER2-VH-VL-CD28 construct.

[0090] Example 5. Tumor cell killing ability assay

[0091] Commercial luciferase lentivirus was used to transfect OE19 cells (human esophageal cancer cells, purchased from Keygen Biotech) and LnCap cells (human prostate cancer cells, purchased from Keygen Biotech), and OE19-luci cells and LnCap-luci cells stably expressing luciferase were obtained after screening. OE19-luci cells express HER2 protein, and LnCap-luci cells express 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 with X-VIVO15 (Lonza) + 10% FBS (gibco) medium. 1E4 target cells were added to each well of the 96-well plate.

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

[0094] After 48 hours, the 96-well plate was removed and 50uL of prepared Steady-Glo Luciferase Assay reagent (Promega) was added to each well. After 5 minutes of reaction, the fluorescence value was read using a microplate reader.

[0095] Figures 7-9 It is shown that the anti-HER2 CAR-macrophages, anti-PSMA CAR-macrophages, and anti-HER2 CAR-monocytes can all specifically lyse the corresponding tumor cells at various effector-to-target ratios, while the untransfected UTD group only has low-level non-specific lysis of tumor cells at high doses. The HER2-VL-VH-CD8 construct has a higher tumor cell killing rate than the HER2-VH-VL-CD28 construct, and the PSMA-VL-VH-CD8 has a higher tumor cell killing rate than the PSMA-VH-VL-CD8.

[0096] Figures 7-9 The results show 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. Phagocytosis function assay

[0098] The OE19 cells were suspended in DPBS, and Dir fluorescent probe (yeasen) dye was added to the cell suspension. After mixing evenly, the mixture was incubated in a cell incubator for 30 min. The staining was terminated by adding preheated complete medium (X-VIVO 15 + 10% FBS) at 37°C, and the cells were resuspended and adjusted to a cell density of 7.5E5 / mL.

[0099] The harvested anti-HER2 CAR-macrophage cells prepared in Example 3 and the anti-HER2 CAR-monocyte cells prepared in Example 4 were suspended in complete medium, and the cell density was adjusted to 7.5E5 / mL.

[0100] The Dir-labeled OE19 and CAR-M cells / CAR-monocytes were mixed at a ratio of 1:1 and incubated in a cell incubator for 2 h.

[0101] 3 μL of FITC-CD11b was added for staining, and CD11b was analyzed by flow cytometry. + The cells labeled with DiR in the group were considered to be cells phagocytosing OE19.

[0102] The results show that the anti-HER2 CAR-macrophage cells ( Figure 10 ) and the anti-HER2 CAR-monocyte cells ( Figure 11 ) can phagocytose OE19 tumor cells expressing HER2, while the UTD group without transfection only shows a low level of non-specific phagocytosis, and the HER2-VL-VH-CD8 construct has a higher phagocytosis rate than the HER2-VH-VL-CD28 construct.

[0103] Figures 10-11 The results show 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.

[0104] Example 7. Macrophage M1 phenotype detection

[0105] The anti-HER2 CAR-macrophage cells prepared in Example 3 were mixed with PBS, and CD80 antibody (purchased from BD) was added. After incubation at room temperature for 30 min, the unbound antibody was removed by centrifugation at 400g for 5 min, and the cells were washed and subjected to flow cytometry detection.

[0106] The results are shown inFigure 12 It is shown that the M1 marker CD80 of macrophages is up-regulated in an adenovirus MOI dose-dependent manner. Relative to UTD, both HER2-VL-VH-CD8 and HER2-VH-VL-CD28 CAR-macrophages show good expression rates of M1 macrophage marker CD80, with good M1 maintenance, but the CD80 expression rate of HER2-VL-VH-CD8 is higher.

[0107] The technical solutions of the present application are not limited to the above specific embodiments, and any technical variations made according to the technical solutions of the present application fall within the scope of protection of the present application.

Claims

1. A chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus, a signal peptide, a single-chain variable fragment (scFv) targeting an antigen, a CD8 hinge region, a CD8 transmembrane region, and a FcsR1y intracellular region, in sequence, a heavy chain variable region (VH) of the single-chain variable fragment is connected to the CD8 hinge region; the single-chain variable fragment is a single-chain variable fragment (scFv) targeting human epidermal growth factor receptor-2 (HER2); the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO:

12.

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

3. A recombinant vector containing the nucleic acid molecule of claim 2.

4. A modified cell containing the nucleic acid molecule of claim 2, or the recombinant vector of claim 3.

5. The modified cell of claim 4, wherein, the cell comprises monocytes and / or macrophages.

6. A pharmaceutical composition comprising the chimeric antigen receptor of claim 1 or the modified cell of claim 4 or 5, and a pharmaceutically acceptable carrier.

7. Use of the chimeric antigen receptor of claim 1, the modified cell of claim 4 or 5, or the pharmaceutical composition of claim 6 in the preparation of a medicament for treating a tumor, the tumor being human esophageal cancer.

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