CAR-T cell and application thereof

Abstract

The invention discloses a CAR-T cell and an application thereof. According to the application, nano antibodies VHH1 and VHH2 capable of simultaneously recognizing human or mouse FAP are used as antigen binding regions to be constructed in a human or mouse CAR structure, and human or mouse FAP-CAR-T cells with a killing function are obtained. The mouse FAP-CAR-T cell or the human FAP-CAR-T cell provided by the invention has a good treatment effect on removal of FAP positive cells, and is a novel FAP-CAR-T cell which is expected to be applied to clinical solid tumor CAR-T therapy.

Description

[0001] This application is a divisional application with application number 2024116956771, invention name “Nanoantibodies, CAR-T cells and their applications targeting FAP”, and application date 2024.11.25. Technical Field

[0002] The present invention belongs to the field of biomedicine, and specifically relates to a CAR-T cell and its application. Background Art

[0003] Chimeric antigen receptor (CAR) T cell therapy is a promising new cancer therapy as an immunotherapy that involves genetically modifying the patient's T cells to recognize and attack cancer cells. The patient's own T cells are genetically engineered to express CARs that recognize and bind to specific antigens on the surface of cancer cells. In solid tumors, CAR-T therapy faces multiple challenges, including CAR-T cell targeting-off-tumor, limited CAR-T cell trafficking and penetration, immunosuppressive microenvironment, and inevitable related toxicity.

[0004] Fibroblast activation protein (FAP) is a membrane-bound serine protease that has been reported as one of the markers of cancer-associated fibroblasts (CAF). It is overexpressed in CAF cells of various tumor types, such as pancreatic cancer, breast cancer, colorectal cancer, skin cancer, and lung cancer, but rarely expressed in healthy human tissues. FAP is involved in the regulation of ECM, promoting angiogenesis and epithelial-mesenchymal transition and the secretion of immunosuppressive factors. It reshapes ECM through growth factors, chemokines, and cytokines. FAP can also inhibit IFN-γ and TNF-α, leading to immunosuppression and angiogenesis.

[0005] Nanobodies are the smallest antibody molecules currently available. Since their discovery, nanobodies have gradually attracted people's attention. Nanobodies have been used in autoimmune diseases, blood diseases, viral infection-related diseases and orthopedic diseases, and have also shown great advantages in anti-infection, anti-inflammatory diseases and anti-neurodegenerative diseases. The development of nanoantibody-based CAR-T cell therapy is crucial for the treatment of FAP-positive related diseases. Summary of the invention

[0006] To make up for the deficiencies of the prior art, the present invention provides a CAR-T cell and its application.

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

[0008] The first aspect of the present invention provides a chimeric antigen receptor, which comprises a nanobody targeting FAP, wherein the nanobody comprises VHH1 and VHH2, and the amino acid sequences of CDR1, CDR2, and CDR3 of VHH1 are respectively shown in SEQ ID NOs: 1-3, and the amino acid sequences of CDR1, CDR2, and CDR3 of VHH2 are respectively shown in SEQ ID NOs: 9-11.

[0009] Furthermore, the VHH1 also includes heavy chain variable region framework regions FR1, FR2, FR3, and FR4 having at least 80% sequence identity with the amino acid sequences shown in SEQ ID NOs: 4-7, and the VHH2 also includes heavy chain variable region framework regions FR1, FR2, FR3, and FR4 having at least 80% sequence identity with the amino acid sequences shown in SEQ ID NOs: 12-15.

[0010] Furthermore, the VHH1 has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:8, and the VHH2 has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:16.

[0011] Furthermore, the amino acid sequence of the nanobody VHH1 is shown in SEQ ID NO:8, and the amino acid sequence of the nanobody VHH2 is shown in SEQ ID NO:16.

[0012] Furthermore, the chimeric antigen receptor also includes a hinge region.

[0013] Furthermore, the chimeric antigen receptor also includes a transmembrane domain.

[0014] Furthermore, the chimeric antigen receptor also includes an intracellular domain.

[0015] Furthermore, the intracellular domain includes a co-stimulatory molecule domain and a signal transduction domain.

[0016] Furthermore, the chimeric antigen receptor also includes a tag protein.

[0017] Furthermore, the hinge region is selected from CD8 and CD28.

[0018] Furthermore, the transmembrane domain is selected from CD8 and CD28.

[0019] Furthermore, the co-stimulatory molecule domain is selected from 4-1BB.

[0020] Furthermore, the signal transduction domain is selected from CD3ζ.

[0021] Furthermore, the tag protein is selected from RQR8.

[0022] Furthermore, the chimeric antigen receptor also includes a self-cleaving peptide.

[0023] Furthermore, the self-cleaving peptide is selected from T2A.

[0024] Furthermore, the chimeric antigen receptor also includes a Linker.

[0025] Furthermore, the Linker is selected from (G4S)4.

[0026] Furthermore, the chimeric antigen receptor also includes a detection tag / auxiliary functional element.

[0027] Furthermore, the chimeric antigen receptor includes a human chimeric antigen receptor and a mouse chimeric antigen receptor.

[0028] The second aspect of the present invention provides a nucleic acid molecule, which includes a nucleic acid molecule encoding the chimeric antigen receptor described in the first aspect of the present invention.

[0029] The third aspect of the present invention provides a vector, which comprises the nucleic acid molecule described in the second aspect of the present invention.

[0030] Furthermore, the vector also includes one or more regulatory sequences operably linked to the nucleic acid.

[0031] Furthermore, the regulatory sequence includes a promoter sequence, a transcription terminator sequence, and a leader sequence.

[0032] Furthermore, the vector includes a DNA vector, an RNA vector, a plasmid, and a vector of viral origin.

[0033] Furthermore, the virus-derived vectors include lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, poxvirus vectors, and herpesvirus vectors.

[0034] The fourth aspect of the present invention provides a host cell, wherein the host cell comprises the nucleic acid molecule described in the second aspect of the present invention or the vector described in the third aspect of the present invention.

[0035] Furthermore, the host cell is selected from eukaryotic cells.

[0036] Furthermore, the eukaryotic cells are selected from animal cells.

[0037] Furthermore, the animal cells are selected from mammalian cells.

[0038] Furthermore, the mammalian cells are selected from immune cells.

[0039] Furthermore, the immune cells are selected from T cells.

[0040] The fifth aspect of the present invention provides a pharmaceutical composition, which comprises the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the vector described in the third aspect of the present invention, or the host cell described in the fourth aspect of the present invention.

[0041] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0042] The sixth aspect of the present invention provides the use of the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the vector described in the third aspect of the present invention or the host cell described in the fourth aspect of the present invention in the preparation of a pharmaceutical composition for preventing / treating FAP-related diseases.

[0043] Furthermore, the FAP-related diseases include kidney disease, liver disease, lung disease, cardiovascular disease, immune-related diseases and tumors, as well as related conditions mediated by the above diseases.

[0044] Further, the tumor includes breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, cholangiocarcinoma, clear cell renal carcinoma, neuroendocrine tumor, carcinogenic osteomalacia, sarcoma, CUP, thymic carcinoma, glioma, glioma, astrocytoma, cervical cancer or prostate cancer.

[0045] Advantages and beneficial effects of the present invention:

[0046] This application constructs nanoantibodies VHH1 and VHH2 that can simultaneously recognize human or mouse FAP as antigen binding regions in human or mouse CAR structures, and obtains human or mouse FAP-CAR-T cells with killing function. The mouse FAP-CAR-T cells or human FAP-CAR-T cells provided by the present invention have a good therapeutic effect on clearing FAP-positive cells, and are new FAP-CAR-T cells that are expected to be applied to clinical solid tumor CAR-T therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 1A is a graph showing the results of nanobody screening and identification, wherein 1A is a graph showing the detection of FAP protein, 1B is a graph showing the detection of alpaca immune titer, 1C is a graph showing the construction of a yeast display library, 1D is a graph showing the detection of a yeast panning library FACS, 1E is a graph showing the detection of a yeast monoclonal FACS, 1F is a graph showing the FAP candidate antibody FACS binding detection, 1G is a graph showing the summary of the MFI values ​​of the FAP candidate antibody-FACS binding detection, and 1H is a graph showing the FAP antibody epitope competition detection;

[0048] Figure 22A is a schematic diagram of the CAR structure of FAP-CAR-T cells in mice, wherein 2A is a schematic diagram of the CAR structure of FAP-CAR-T 1 cells, and 2B is a schematic diagram of the CAR structure of FAP-CAR-T 2 cells;

[0049] Figure 3 3A is a flow cytometry result diagram of the CAR positivity rate of FAP-CAR-T cells in mice, wherein 3A is a flow cytometry result diagram of the CAR positivity rate of FAP-CAR-T 1 cells, and 3B is a flow cytometry result diagram of the CAR positivity rate of FAP-CAR-T 2 cells;

[0050] Figure 4 Figures verify the killing efficiency of mouse FAP-CAR-T cells, where 4A is a fluorescence microscopy image of FAP-CAR-T 1 cells, 4B is a Western blot image of FAP-CAR-T 1 cells, 4C is a flow cytometry image of FAP-CAR-T 1 cells, and 4D is a flow cytometry image of FAP-CAR-T 2 cells;

[0051] Figure 5 5A is a result diagram of the killing efficiency of FAP-CAR-T cells and NIH3T3-mFAP-GFP cells co-cultured with each other, and 5B is a result diagram of the killing efficiency of FAP-CAR-T 1 cells, and 5B is a result diagram of the killing efficiency of FAP-CAR-T 2 cells;

[0052] Figure 6 6A is a graph showing the killing efficiency of mouse FAP-CAR-T cells and NIH3T3-mFAP-GFP cells after co-culture for 24 hours, wherein 6B is a graph showing the killing efficiency of FAP-CAR-T 1 cells by flow cytometry, 6C is a graph showing the killing efficiency of FAP-CAR-T 2 cells by flow cytometry, and 6D is a graph showing the killing efficiency of FAP-CAR-T 2 cells by statistical results;

[0053] Figure 7 7A is a schematic diagram of the structure of human FAP-CAR-T cell CAR, wherein 7A is a schematic diagram of the structure of human FAP-CAR-T 1 cell CAR, and 7B is a schematic diagram of the structure of human FAP-CAR-T 2 cell CAR;

[0054] Figure 8 8A is a flow cytometry result diagram of the CAR positivity rate of human FAP-CAR-T cells, wherein 8B is a flow cytometry result diagram of the CAR positivity rate of human FAP-CAR-T 2 cells;

[0055] Fig. 99A is a graph verifying the killing efficiency of human FAP-CAR-T cells, wherein 9A is a fluorescence microscopy graph of human FAP-CAR-T 1 cells, 9B is a Western blot graph of human FAP-CAR-T 1 cells, 9C is a flow cytometry graph of human FAP-CAR-T 1 cells, and 9D is a flow cytometry graph of human FAP-CAR-T2 cells;

[0056] Fig.10 10A is a result diagram of the killing efficiency of human FAP-CAR-T cells and SKOV3-hFAP-GF cells co-cultured, and 10B is a result diagram of the killing efficiency of human FAP-CAR-T 1 cells, and 10B is a result diagram of the killing efficiency of human FAP-CAR-T 2 cells;

[0057] Fig.11 11A is a graph showing the killing efficiency of human FAP-CAR-T cells and SKOV3-hFAP-GFP cells after co-culture for 24 hours, wherein 11A is a graph showing the killing efficiency of human FAP-CAR-T 1 cells detected by flow cytometry, 11B is a graph showing the statistical results of the killing efficiency of human FAP-CAR-T 1 cells, 11C is a graph showing the killing efficiency of human FAP-CAR-T 2 cells detected by flow cytometry, and 11D is a graph showing the statistical results of the killing efficiency of human FAP-CAR-T 2 cells;

[0058] Fig.12 This is the result of co-culture of Mock-t cells, FAP-CAR-T cells and mother NIH3T3 cells that do not express mouse FAP, and detecting the killing specificity of FAP-CAR-T cells by RTCA, among which 12A is FAP-CAR-T 1 cell and 12B is FAP-CAR-T 2 cell. DETAILED DESCRIPTION

[0059] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0060] The present invention provides a nanobody targeting FAP, wherein the nanobody comprises VHH1 and VHH2.

[0061] In some embodiments, the nanobody also includes a functional variant of nanobody VHH1 or VHH2, specifically, the functional variant includes but is not limited to: derivatives that are substantially similar in primary structural sequence but include chemical and / or biochemical modifications not present in the parent nanobody VHH1 or VHH2 of the invention, such as in vitro or in vivo. These modifications include, for example, acetylation, acylation, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, cross-linking, formation of disulfide bonds, glycosylation, hydroxylation, methylation, oxidative PEGylation, proteolytic treatment, phosphorylation, and such functional variants are also included in the protection scope of the present invention.

[0062] The functional variant may be a nanobody as follows: compared with the amino acid sequence of the parent nanobody VHH1 or VHH2, it includes an amino acid sequence containing one or more amino acid substitutions, insertions, deletions or a combination thereof. Further, the functional variant may include a truncation of the amino acid sequence at one or both ends of the amino or carboxyl terminus. Compared with the parent nanobody VHH1 or VHH2, the functional variant of the present application may have the same or different, higher or lower binding affinity, but can still specifically bind to FAP. For example, compared with the parent nanobody VHH1 or VHH2, the functional variant of the present application may have an increased or decreased binding affinity for FAP.

[0063] In some embodiments, identity refers to the amino acid residues between the sequences being identical at any particular position of the compared sequences. For example, leucine can be replaced with isoleucine or valine. Other amino acids that are usually replaced with each other include, but are not limited to, phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains), lysine, arginine and histidine (amino acids with basic side chains), aspartic acid and glutamic acid (amino acids with acidic side chains), asparagine and glutamine (amino acids with amide side chains), and cysteine ​​and methionine (amino acids with sulfur-containing side chains). Generally, the modification of one or more amino acids in a protein will not affect the function of the protein. Those skilled in the art will recognize that changing a single amino acid or a small percentage of amino acids or individual additions, deletions, insertions, and substitutions to an amino acid sequence are conservative modifications, wherein changes in proteins produce proteins with similar functions, and providing conservative substitution tables of amino acids with similar functions is well known in the art.

[0064] The present invention provides a chimeric antigen receptor, which comprises any one or two of the above-mentioned nanoantibodies or any other nanoantibodies targeting FAP.

[0065] In some embodiments, a chimeric antigen receptor (CAR) generally refers to a fusion protein comprising an extracellular domain capable of binding an antigen and at least one intracellular domain. CAR is a core component of a chimeric antigen receptor T cell (CAR-T), which may include an antigen (e.g., a tumor-specific antigen and / or a tumor-associated antigen) binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. CAR is an engineered receptor that can implant any specific receptor into immune effector cells, especially T cells. In CAR, a VHH fragment or a scFv fragment of a monoclonal antibody that specifically recognizes a tumor antigen can be implanted into a T cell or NK cell. For example, a retroviral vector can be used to introduce a nucleic acid encoding CAR into a T cell, a NK cell, or a NK T cell. In this way, a large number of cancer-specific T cells, NK cells, or NK T cells can be generated for adoptive cell transfer. In the present application, the CAR can be combined with a T cell receptor activation intracellular domain based on the antigen (e.g., FAP) specificity of the antibody. T cells genetically modified to express CAR can specifically recognize and eliminate malignant cells expressing the target antigen.

[0066] The chimeric antigen receptor also includes a hinge region.

[0067] In some embodiments, the hinge region includes but is not limited to CD8, CD28, CD34, OX40, CD3ε, IgG1, IgG4, PD-1, IL-2 receptor, IL-7 receptor, IL-11 receptor.

[0068] In a specific embodiment, the hinge region is selected from CD8, CD28.

[0069] The chimeric antigen receptor also includes a transmembrane domain.

[0070] In some embodiments, the transmembrane domain includes but is not limited to CD3ζ, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154 or GITR.

[0071] In a specific embodiment, the transmembrane domain is selected from CD8, CD28.

[0072] The chimeric antigen receptor also includes an intracellular domain, which includes a co-stimulatory molecule domain and a signal transduction domain.

[0073] In some embodiments, the co-stimulatory molecule domain includes but is not limited to 4-1BB, CD27, CD19, CD4, CD28, ICOS (CD278), CD8α, CD8β, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, and B7-H3.

[0074] In a specific embodiment, the co-stimulatory molecule domain is selected from 4-1BB.

[0075] In some embodiments, the signal transduction domain includes but is not limited to CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, and CD66d.

[0076] In a specific embodiment, the signal transduction domain is selected from CD3ζ.

[0077] The chimeric antigen receptor also includes a tag protein.

[0078] In some embodiments, the tag protein includes but is not limited to RQR8, EGFRt, CD20, c-MYC, 6×His, GST, Fc, Flag, and HA.

[0079] In a specific embodiment, the tag protein is selected from RQR8.

[0080] The chimeric antigen receptor also includes a self-cleaving peptide.

[0081] In some embodiments, the self-cleaving peptides include but are not limited to P2A, T2A, E2A, and F2A.

[0082] In a specific embodiment, the self-cleaving peptide is selected from T2A.

[0083] The chimeric antigen receptor also includes a Linker.

[0084] In some embodiments, the linker is a linker conventionally used in the art, and the linker can be (G4S) n 、(GGGS) n 、(SSSSG) n 、(GSGSA) n 、(GGSGG) n Or any one of the other linkers, wherein n can be any integer between 1-10.

[0085] In a specific embodiment, the linker is selected from (G4S)4.

[0086] The chimeric antigen receptor also includes auxiliary functional elements.

[0087] In some embodiments, the auxiliary functional elements include but are not limited to tEGFR, tCD34, tCD19, tCD20, tCD22, immune checkpoint inhibitors (CTLA-4, PD-1 / PD-L1, LAG-3, TIM-3, TIGIT, CD226, CD155, CD47, B7-H3, B7-H4), nanoantibodies, cytokines and their receptors (IL2, IL2 receptor, IL7, IL7 receptor, IL15, IL15 receptor).

[0088] The present invention provides a vector, which comprises the above nucleic acid molecule.

[0089] In some embodiments, the present application has no particular restrictions on the vector, and its selection depends on the desired function. Non-limiting examples of vectors include DNA vectors, RNA vectors, plasmid vectors, vectors of viral origin, phage vectors and other vectors conventionally used for, for example, genetic engineering. Various plasmids and vectors can be constructed based on methods well known to those skilled in the art. The vector according to the present application can guide the replication and expression of the nucleic acid molecules of the present application in the host, and thus ensure the expression of the nanobodies or chimeric antigen receptors targeting FAP described in the present application encoded thereby in the selected host. The expression vector may be, for example, a cloning vector, a binary vector or an integrative vector. Expression includes transcription of nucleic acid molecules, such as transcription into translatable mRNA.

[0090] The vector also includes one or more regulatory sequences operably linked to the nucleic acid.

[0091] The regulatory sequence includes a promoter sequence, a transcription terminator sequence, and a leader sequence.

[0092] In some embodiments, the promoter includes CMV promoter, EF-1α promoter, SV40 early promoter, MMTV promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, actin promoter, myosin promoter, heme promoter, creatine kinase promoter, metallothionein promoter, glucocorticoid promoter, progesterone promoter, tetracycline promoter.

[0093] In some embodiments, the transcription terminator includes CYC1 transcription terminator, T7 transcription terminator, rrnBT1 transcription terminator, rrnBT2 transcription terminator, ADH1 transcription terminator, TIF51A transcription terminator, ALG6 transcription terminator, AOD transcription terminator, AOX1 transcription terminator, ARG4 transcription terminator, PMA1 transcription terminator, TEF1 transcription terminator, TT1 transcription terminator, and TT2 transcription terminator.

[0094] The present invention provides a host cell, which comprises the above nucleic acid molecule or the above vector.

[0095] In some embodiments, the cell comprises a prokaryotic cell or a eukaryotic cell.

[0096] In some embodiments, the eukaryotic cells include, but are not limited to, protist cells, animal cells, or fungal cells, and the animal cells include mammalian cells, avian cells, and insect cells.

[0097] In a preferred embodiment, the cell is selected from eukaryotic cells.

[0098] In a more preferred embodiment, the eukaryotic cell is selected from animal cells.

[0099] In a more preferred embodiment, the animal cell is selected from mammalian cells.

[0100] In a more preferred embodiment, the mammalian cell is selected from immune cells.

[0101] In some embodiments, the immune cells include T cells, B cells, NK cells, iNKT cells, γδT cells, NK92 cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages, neutrophils, or any combination thereof.

[0102] In a specific embodiment, the immune cell is selected from T cells.

[0103] The present invention provides an immunoconjugate, which comprises the above-mentioned nano antibody and a conjugate conjugated thereto.

[0104] The conjugate includes a detectable marker, a cytokine, a therapeutic agent, a cytotoxin, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or VLP, or a combination thereof.

[0105] In some embodiments, the detectable marker comprises a fluorescent or luminescent marker, a radioactive marker, an MRI (magnetic resonance imaging) or CT (computer tomography) contrast agent. The radionuclide comprises 131 I.32 P. 89 Sr. 90 Y. 223 Ra, 125 I. 103 Pd. The cytokines include IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-14, IFN-γ, TNF-β, TNF-α, G-CSF, M-CSF. The therapeutic agents include alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, chromatin function inhibitors, anti-angiogenic agents, antiestrogens, antiandrogens, and immunomodulators.

[0106] The present invention provides a pharmaceutical composition, which comprises the above-mentioned nanoantibody, the above-mentioned chimeric antigen receptor, the above-mentioned nucleic acid molecule, the above-mentioned vector, the above-mentioned host cell or the above-mentioned immunoconjugate.

[0107] The pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0108] In some embodiments, a pharmaceutically acceptable excipient is generally any type of non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation aid.

[0109] In some embodiments, suitable pharmaceutical compositions are administered in forms suitable for parenteral administration, such as by injection or infusion, such as by rapid injection or continuous infusion, intravenous, inhalable or subcutaneous forms. Where the product is intended for injection or infusion, it may be in the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulation agents such as suspending agents, preservatives, stabilizers and / or dispersants.

[0110] In some embodiments, the pharmaceutical composition can be prepared into various dosage forms as needed, and the physician can determine the dosage that is beneficial to the patient based on factors such as the patient's type, age, weight, general disease condition, and administration method for administration. A skilled physician can usually easily determine the dosage and administration method that are effective for the desired treatment and / or prevention.

[0111] The present invention provides a product for detecting FAP, wherein the product comprises the above-mentioned nanoantibody or the above-mentioned immunoconjugate.

[0112] The products include test kits and test strips.

[0113] In some embodiments, the kit further comprises a solid support, a container, instructions for use, a buffer, etc. In other embodiments, the kit further comprises a lysis medium for dissolving the sample to be tested, universal reagents and buffers required for detection, such as various buffers, detection labels, blocking reagents, washing reagents, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0114] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0115] Example

[0116] Experimental methods

[0117] 1. Screening and identification of nanoantibodies

[0118] 1) Preparation and QC of FAP recombinant protein

[0119] FAP 26-760 Reduced SDS-PAGE detection of AA-His recombinant protein.

[0120] 2) Detection of alpaca immune titer: ELISA

[0121] Coated with FAP-His protein (1μg / mL); primary antibody: 100μL of serially diluted serum; secondary antibody: Goat anti-LlamaIgG ​​(H+L) Secondary Antibody [HRP] (1:50000 dilution).

[0122] 3) Construction of yeast display library: 103# VHH fragment cloning

[0123] Peripheral blood was collected, PBMC cells were isolated, RNA was extracted, and reverse transcribed into cDNA. The VHH sequence was amplified from the cDNA sample using the single-domain antibody cloning primer combination and subcloned into the yeast display vector pYDisplay, and electroporated into EBY100 competent cells to construct a single-domain antibody yeast display library.

[0124] 4) Yeast panning library FACS detection: 103#

[0125] The antigens were modified with Biotin, enriched by magnetic beads, and then detected by FACS.

[0126] Panning scheme: FAP antigen screening.

[0127] 5) Yeast monoclonal FACS detection: 103#

[0128] Use Biotin-FAP-His antigen detection to select positive clones for testing.

[0129] 6) FAP candidate antibody FACS binding assay

[0130] Cell amount: 3*10^5 / well; primary antibody: FAP target candidate antibody (10μg / ml), 100μl per well; secondary antibody: FC-PE (1:500 dilution).

[0131] 7) FAP candidate antibody-FACS binding assay (MFI value summary)

[0132] Cell amount: 3*10^5 / well; primary antibody: FAP target candidate antibody (10μg / ml), 100μl per well; secondary antibody: FC-PE (1:500 dilution).

[0133] 8) FAP antibody epitope competition assay

[0134] Human-FAP-His was immobilized using the HIS1K sensor at a concentration of 5 μg / ml and a curing time of 150 s.

[0135] The buffer was PBST (PBS + 0.02% tween 20), first bound to AB1 antibody (103-1-G1-FC), and then bound to AB2 antibody (103-1-D9-3-FC, 103-1-H7-FC and 103-1-G1-FC as controls for the same epitope) after equilibrium, with a concentration of 100 nM.

[0136] Affinity detection: equilibrium 60s, binding 180s, dissociation 180s, detection temperature 25℃.

[0137] 2. Construction of Lentiviral Vector

[0138] The CAR for expressing FAP was designed and constructed. The FAP-CAR structure consisted of an anti-FAP single-chain antibody (VHH1 and VHH2), a linker domain of (G4S)4 connecting VHH1 and VHH2, a hinge region and a transmembrane region of the CD8 molecule, an intracellular domain of 4-1BB, and a CD3ζ signaling domain, and was cloned into the lentiviral shuttle plasmid pHAGE. Figure 6 The schematic diagrams of the structures of the two CARs constructed in this example are provided.

[0139] 3. Construction of retroviral vector

[0140] The CAR for expressing FAP was designed and constructed. The FAP-CAR structure consisted of a linker domain containing an anti-FAP single-chain antibody (VHH1 and VHH2), a (G4S)4 linker domain connecting VHH1 and VHH2, a hinge region and a transmembrane domain and an intracellular domain of mouse CD28, and a mouse CD3ζ signal transduction domain, and was cloned into the retroviral shuttle plasmid pMIG. Figure 1 The schematic diagrams of the structures of the two CARs constructed in this example are provided.

[0141] 4. Preparation of human CAR-T cells

[0142] After obtaining informed consent, primary mononuclear cells (PBMCs) were isolated from peripheral blood of normal subjects or B-cell lymphoma patients using Ficoll density centrifugation. TM Human T Cell Isolation Kit was used to purify T lymphocytes and Human T-Expander CD3 / CD28 activates and expands T cells. The activated T cells are infected with lentivirus carrying CAR and then expanded in vitro for 10-12 days in the X-VIVO 15 culture system containing IL-2, IL-15 and IL-7 until the transduction efficiency reaches ≥40%, the cell viability reaches ≥70%, and there is no mycoplasma and bacterial contamination.

[0143] 5. Preparation of mouse CAR-T cells

[0144] Prepare mouse CAR-T cell culture medium containing RPMI1640, Hyclone, penicillin-streptomycin, HEPES solution, sodium pyruvate and β-hydroxyethanol. Day-1 Isolate the inguinal and mesenteric lymph nodes of mice, grind and wash them, and make single cell suspension. Anti-mouse CD3 (0.2 μg / mL) and anti-mouse CD28 (1 μg / mlL) antibodies are used to activate T cells, and 50 U / ml mouse cytokine IL-2 is added to adjust the T cell concentration to 2×10 6 / mlL. On Day 0, cells were infected with CAR retrovirus and centrifuged at 1500g for 2 hours. On Day 2, cells were infected with retrovirus for 48 hours and centrifuged at 400g for 5 minutes. 10ng / mL mouse cytokines IL-7 and IL-15 were added and some cells were aspirated to detect the CAR positive rate. Cells were replenished or subcultured according to the growth and number of cells to maintain the cell density.

[0145] 6. CAR positive rate detection

[0146] Take 1×10 6CAR-T cells were transferred to a 1.5 mL EP tube, and 1 mL of pre-cooled PBS solution containing 2% BSA was added to wash the cells. The cells were centrifuged at 4°C, 300 g for 5 minutes, and CAR was labeled with FITC-CD34 antibody after repeated washing twice. The cells were then washed with pre-cooled 2% BSA / PBS solution, and transferred to a flow tube and the CAR-T cell positivity was detected using a flow cytometer.

[0147] 7. Detection of mFAP or hFAP expression in target cells by flow cytometry and Western Blot

[0148] Take 1×10 6 Target cells (NIH3T3-mFAP, SKOV3-hFAP) were placed in a 1.5 mL EP tube, 1 mL of pre-cooled PBS solution containing 2% BSA was added to wash the cells, and the cells were centrifuged at 300 g for 5 minutes at 4°C. After the cells were fixed and permeabilized with fixative and permeabilization solution, anti-FAP primary antibody was added to label mFAP or hFAP, and the secondary antibody labeled with AF594 was incubated. The cells were washed with pre-cooled 2% BSA / PBS solution, transferred to a flow tube, and the expression of mFAP and hFAP in the target cells was detected by flow cytometry.

[0149] RIPA cell lysis buffer was used to extract target cell (NIH3T3-mFAP, SKOV3-hFAP) proteins, and these proteins were separated by SDS-PAGE electrophoresis. The proteins were then transferred to a PVDF membrane and incubated with anti-FAP antibody, followed by incubation with HRP-secondary antibody, and finally the presence and expression level of FAP protein were detected by chemiluminescence reaction.

[0150] 8. Cytotoxicity of tumor-specific CAR-T cells

[0151] Adherent cells (NIH3T3, NIH3T3-mFAP, SKOV3, SKOV3-hFAP) were cultured at 1×10 5 The cells were inoculated into a 48-well plate at a density of 1:1 / well. The next day, FAP-CAR-T or Mock-T cells were added to the corresponding wells at an effector-target ratio of 1:1, 1:3, or 1:5. After 24 hours, the cells were collected for flow cytometry detection of the killing ability of CAR-T cells, in which APC-anti-CD3 antibody labeled T cells and GFP-positive cells were labeled as residual tumor cells.

[0152] 9. RTCA experiment

[0153] Adherent cells (NIH3T3, NIH3T3-mFAP, SKOV3, SKOV3-hFAP) were seeded into 96-well E-Plate (ACEA Biosciences, USA) at a density of 8000 / well and monitored overnight in the xCELLigence real-time cell analyzer (RTCA) system (ACEA Biosciences, USA) based on impedance. The next day, FAP-CAR-T or Mock-T cells were added to the corresponding wells at an effector-target ratio of 2:1, 1:1, 1:3 or 1:5. The RTCA system continuously monitored the cell impedance in the E-Plate for 96 hours and plotted the real-time impedance change curve.

[0154] 10. Statistical analysis

[0155] The results of flow cytometry experiments were analyzed using Flowjo V10 flow analysis software; the results of western blot were analyzed using ImageJ image analysis software. Statistical data were analyzed and processed using GraphPad Prism 7.0 statistical software, and statistical graphs were drawn. Quantitative data were expressed as mean ± standard deviation (mean ± SD). The t test was used for comparison between the two groups. One-way analysis of variance was used for comparison between multiple groups, and P < 0.05 indicated that the difference was statistically significant.

[0156] Experimental Results

[0157] The results are as follows Figure 1 As shown, FAP 26-760 AA-His purity>95% ( Figure 1 A), compared with negative serum, the OD value of FAP-His protein binding in alpaca hexa-rat serum was significantly higher ( Figure 1 B), and successfully cloned 103#VHH( Figure 1 C), after magnetic separation and flow separation using FAP-Biotin protein, V5 showed 44%, and the binding with FAP-Biotin accounted for 28.426% ( Figure 1 D), Biotin-FAP-His antigen detection positive clones such as Figure 1 As shown in E, both antibody 103-1-G1 (VHH1) and antibody 103-1-D9-3 (VHH2) can bind well to FAP antibody ( Figure 1 F), FACS combined with MFI value Figure 1 As shown in G, the results of the FAP antibody epitope competition assay showed that the two antibodies 103-1-D9-3 (VHH2) and 103-1-G1 (VHH1) have different binding epitopes ( Figure 1 H).

[0158] Based on the independent screening and verification of alpaca-derived nanoantibodies VHH1 and VHH2 that can bind to human or mouse FAP (the antibody sequences are shown in Table 1), VHH2 and VHH1 are connected through a linker, and connected to the CAR structure of the mouse CD28 hinge region, transmembrane region, intracellular co-stimulatory domain and CD3ζ intracellular signal transduction domain. The tag protein RQR8 is connected in series through the untranslated region T2A to detect CAR expression, and a shuttle plasmid (pMIG-FAP-CAR) of the retrovirus loaded with FAP-CAR is constructed, and the auxiliary plasmid pCL-Eco is used to package the retrovirus in 293T cells. Mouse Mock-T (non-transduced exogenous gene T cells) and mouse FAP-CAR-T cells ( Figure 2 ). In addition, the present invention simultaneously connects VHH2 and VHH1 through a linker, and connects to the hinge region and transmembrane region of human CD8, the co-stimulatory molecule 4-1BB and the intracellular signal transduction domain of CD3ζ on the basis of the CAR structure, and connects the tag protein RQR8 in series through the untranslated region T2A for detecting CAR expression, constructs a shuttle plasmid (pHAGE-FAP-CAR) of the lentivirus loaded with FAP-CAR, and uses the auxiliary plasmid pMD2.G and psPAX2 to package the lentivirus in 293T cells. By infecting human T cells, human Mock-T (non-transduced exogenous gene T cells) and human FAP-CAR-T cells ( Figure 7 ).

[0159] Table 1 Nanobody sequences

[0160]

[0161]

[0162] The present application provides a mouse FAP-CAR-T cell expressing FAP-CAR targeting mouse FAP-positive cells. In vitro experiments show that the CAR expression positive rate of FAP-CAR-T 1 cells is approximately 48.8% ( Figure 3 A), the CAR expression positive rate of FAP-CAR-T 2 cells is about 61.9% ( Figure 3B). The results of the co-culture experiment with NIH3T3-mFAP cells expressing mouse FAP showed that when the effector-target ratio (effector cells are FAP-CAR-T or Mock-T, target cells are NIH3T3-mFAP) was 1:1, 1:3 and 1:5, mouse FAP-CAR-T cells were able to significantly kill NIH3T3-mFAP cells compared with Mock-T cells, indicating that mouse FAP-CAR-T cells have the function of killing FAP-positive cells in vitro, and the killing efficiency is significantly higher than that of Mock-T cells. At the same time, the results of the co-culture experiment of mouse FAP-CAR-T cells or Mock-T cells with maternal NIH3T3 cells that do not express mouse FAP at an effector-target ratio of 2:1 and 1:1 showed that mouse FAP-CAR-T cells and Mock-T cells could not kill NIH3T3 cells, indicating that the killing effect of mouse FAP-CAR-T cells is target-dependent and is FAP-specific killing ( Figure 4-6 , Fig.12 ).

[0163] In vitro experiments of human FAP-CAR-T cells expressing FAP-CAR targeting human FAP-positive cells showed that the CAR expression positive rate of FAP-CAR-T 1 cells was approximately 57.9% ( Figure 8 A), the CAR expression positive rate of FAP-CAR-T 2 cells is about 65.6% ( Figure 8 B). The results of the co-culture experiment with SKOV3-hFAP cells expressing human FAP showed that when the effector-target ratio (effector cells are FAP-CAR-T or Mock-T, target cells are SKOV3-hFAP) was 1:1, 1:3 and 1:5, human FAP-CAR-T cells could significantly kill SKOV3-hFAP cells compared with Mock-T cells, indicating that human FAP-CAR-T cells have the function of killing FAP-positive cells ( Figure 9-10 ).

[0164] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises a nanobody targeting FAP, the nanobody comprises VHH1 and VHH2, the amino acid sequences of CDR1, CDR2 and CDR3 of VHH1 are respectively as shown in SEQ ID NOs: 1-3, and the amino acid sequences of CDR1, CDR2 and CDR3 of VHH2 are respectively as shown in SEQ ID NOs: 9-11; Preferably, the VHH1 further comprises heavy chain variable region framework regions FR1, FR2, FR3, FR4 having at least 80% sequence identity with the amino acid sequences shown in SEQ ID NOs: 4-7, and the VHH2 further comprises heavy chain variable region framework regions FR1, FR2, FR3, FR4 having at least 80% sequence identity with the amino acid sequences shown in SEQ ID NOs: 12-15; Preferably, the VHH1 has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 8, and the VHH2 has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 16; preferably, the amino acid sequence of the Nanobody VHH1 is as shown in SEQ ID NO: 8, and the amino acid sequence of the Nanobody VHH2 is as shown in SEQ ID NO:

16.

2. The chimeric antigen receptor according to claim 1, characterized in that The chimeric antigen receptor further comprises a hinge region; Preferably, the chimeric antigen receptor further comprises a transmembrane domain; Preferably, the chimeric antigen receptor further comprises an intracellular domain; Preferably, the intracellular domain includes a co-stimulatory molecule domain and a signal transduction domain; Preferably, the chimeric antigen receptor further comprises a tag protein; Preferably, the hinge region is selected from CD8, CD28; Preferably, the transmembrane domain is selected from CD8, CD28; Preferably, the co-stimulatory molecule domain is selected from 4-1BB; Preferably, the signal transduction domain is selected from CD3ζ; Preferably, the tag protein is selected from RQR8; Preferably, the chimeric antigen receptor further comprises a self-cleaving peptide; Preferably, the self-cleaving peptide is selected from T2A; Preferably, the chimeric antigen receptor further comprises a Linker; Preferably, the Linker is selected from (G4S)4; Preferably, the chimeric antigen receptor further comprises a detection tag / auxiliary functional element; Preferably, the chimeric antigen receptor comprises a human chimeric antigen receptor and a mouse chimeric antigen receptor.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleic acid encoding the chimeric antigen receptor of claim 1 or 2.

4. A carrier, characterized in that The vector comprises the nucleic acid molecule of claim 3; Preferably, the vector further comprises one or more regulatory sequences operably linked to the nucleic acid; preferably, the regulatory sequences comprise a promoter sequence, a transcription terminator sequence, and a leader sequence; Preferably, the vector includes a DNA vector, an RNA vector, a plasmid, or a vector of viral origin; Preferably, the virus-derived vector includes a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a poxvirus vector, and a herpesvirus vector.

5. A host cell, characterized in that The host cell comprises the nucleic acid molecule of claim 3 or the vector of claim 4.

6. The host cell according to claim 5, characterized in that The host cell is selected from eukaryotic cells; preferably, the eukaryotic cells are selected from animal cells; Preferably, the animal cell is selected from mammalian cells; Preferably, the mammalian cell is selected from immune cells; Preferably, the immune cells are selected from T cells.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the chimeric antigen receptor of claim 1 or 2, the nucleic acid molecule of claim 3, the vector of claim 4 or the host cell of any one of claims 5-6.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition also includes pharmaceutically acceptable excipients.

9. Use of the chimeric antigen receptor according to claim 1 or 2, the nucleic acid molecule according to claim 3, the vector according to claim 4, or the host cell according to any one of claims 5 to 6 in the preparation of a pharmaceutical composition for preventing / treating FAP-related diseases.

10. The use according to claim 9, characterized in that: The FAP-related diseases include kidney disease, liver disease, lung disease, cardiovascular disease, immune-related diseases and tumors, as well as related conditions mediated by the above diseases; Preferably, the tumor comprises breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, cholangiocarcinoma, clear cell renal carcinoma, neuroendocrine tumor, carcinogenic osteomalacia, sarcoma, CUP, thymic carcinoma, glioma, glioma, astrocytoma, cervical cancer or prostate cancer.

Images