Nanobodies targeting fap, car-t cells and uses thereof

Abstract

The application discloses a nano antibody targeting FAP, a CAR-T cell and application thereof. The application provides a nano antibody VHH1 and VHH2 capable of simultaneously recognizing human or mouse FAP, and the nano antibody VHH1 and VHH2 are used as an antigen binding region to construct a human or mouse CAR structure to prepare human or mouse FAP-CAR-T cells with a killing function. The mouse FAP-CAR-T cell or the human FAP-CAR-T cell provided by the application has a good therapeutic effect on clearing FAP positive cells, and is a new type of FAP-CAR-T cell which is expected to be applied to clinical solid tumor CAR-T therapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to nanoantibodies, CAR-T cells targeting FAP and their applications. Background Art

[0002] Chimeric antigen receptor (CAR) T cell therapy, as a form of immunotherapy, is a promising new cancer treatment. It involves genetically modifying a 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 on-target de-tumoring, limited CAR-T cell trafficking and penetration, an immunosuppressive microenvironment, and inevitable associated toxicities.

[0003] Fibroblast activation protein (FAP), a membrane-bound serine protease, has been reported as a marker for cancer-associated fibroblasts (CAFs). It is overexpressed in CAFs in various tumor types, including pancreatic, breast, colorectal, skin, and lung cancers, while its expression in healthy human tissues is minimal. FAP participates in ECM regulation, promoting angiogenesis, epithelial-mesenchymal transition, and the secretion of immunosuppressive factors. It remodels the ECM through growth factors, chemokines, and cytokines. FAP can also inhibit IFN-γ and TNF-α, leading to immunosuppression and angiogenesis.

[0004] Nanobodies, currently the smallest antibody molecules, have garnered increasing attention since their discovery. They have been used in autoimmune diseases, blood disorders, viral infections, and orthopedic conditions, and have demonstrated significant advantages in combating infections, inflammatory diseases, and neurodegenerative diseases. The development of nanobody-based CAR-T cell therapy is crucial for treating FAP-related diseases. Summary of the Invention

[0005] To overcome the deficiencies of the prior art, the present invention provides nanobodies targeting FAP, CAR-T cells, and their applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions.

[0007] The first aspect of the present invention provides a Nanobody targeting FAP, which comprises VHH1 and VHH2, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of VHH1 are shown in SEQ ID NOs: 1-3, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of VHH2 are shown in SEQ ID NOs: 9-11, respectively.

[0008] Furthermore, the VHH1 further comprises 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 further comprises 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.

[0009] 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.

[0010] 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.

[0011] The second aspect of the present invention provides a chimeric antigen receptor, which comprises any one or two or any other nanoantibody targeting FAP described in the first aspect of the present invention.

[0012] Furthermore, the chimeric antigen receptor comprises the two nanobodies described in the first aspect of the present invention.

[0013] Furthermore, the chimeric antigen receptor further includes a hinge region.

[0014] Furthermore, the chimeric antigen receptor also includes a transmembrane domain.

[0015] Furthermore, the chimeric antigen receptor also includes an intracellular domain.

[0016] Furthermore, the intracellular domain includes a costimulatory molecule domain and a signal transduction domain.

[0017] Furthermore, the chimeric antigen receptor also includes a tag protein.

[0018] Furthermore, the hinge region is selected from CD8 and CD28.

[0019] Furthermore, the transmembrane domain is selected from CD8 and CD28.

[0020] Furthermore, the co-stimulatory molecule domain is selected from 4-1BB.

[0021] Furthermore, the signal transduction domain is selected from CD3ζ.

[0022] Furthermore, the tag protein is selected from RQR8.

[0023] Furthermore, the chimeric antigen receptor also includes a self-cleaving peptide.

[0024] Furthermore, the self-cleaving peptide is selected from T2A.

[0025] Furthermore, the chimeric antigen receptor also includes a linker.

[0026] Furthermore, the linker is selected from (G4S)4.

[0027] Furthermore, the chimeric antigen receptor also includes a detection tag / auxiliary functional element.

[0028] Furthermore, the chimeric antigen receptor includes a human chimeric antigen receptor and a mouse chimeric antigen receptor.

[0029] The third aspect of the present invention provides a nucleic acid molecule comprising a nucleic acid encoding the Nanobody of the first aspect of the present invention or the Chimeric Antigen Receptor of the second aspect of the present invention.

[0030] The fourth aspect of the present invention provides a vector comprising the nucleic acid molecule described in the third aspect of the present invention.

[0031] Furthermore, the vector also includes one or more regulatory sequences operably linked to the nucleic acid.

[0032] Furthermore, the regulatory sequence includes a promoter sequence, a transcription terminator sequence, and a leader sequence.

[0033] Furthermore, the vector includes a DNA vector, an RNA vector, a plasmid, and a vector of viral origin.

[0034] Furthermore, the virus-derived vectors include lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, poxvirus vectors, and herpesvirus vectors.

[0035] The fifth aspect of the present invention provides a host cell, which comprises the nucleic acid molecule described in the third aspect of the present invention or the vector described in the fourth aspect of the present invention.

[0036] Furthermore, the host cell is selected from eukaryotic cells.

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

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

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

[0040] Furthermore, the immune cells are selected from T cells.

[0041] The sixth aspect of the present invention provides an immunoconjugate, which comprises the Nanobody described in the first aspect of the present invention and a conjugate conjugated thereto.

[0042] Furthermore, the conjugate comprises 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.

[0043] The seventh aspect of the present invention provides a pharmaceutical composition, which comprises the Nanobody described in the first aspect of the invention, the Chimeric Antigen Receptor described in the second aspect of the invention, the nucleic acid molecule described in the third aspect of the invention, the vector described in the fourth aspect of the invention, the host cell described in the fifth aspect of the invention, or the immunoconjugate described in the sixth aspect of the invention.

[0044] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0045] The eighth aspect of the present invention provides a product for detecting FAP, which comprises the Nanobody described in the first aspect of the present invention or the immunoconjugate described in the sixth aspect of the present invention.

[0046] Furthermore, the product includes a test kit and a test paper.

[0047] Furthermore, the kit further comprises a solid support.

[0048] Furthermore, the kit further comprises a buffer, a blocking reagent, a washing reagent, and a detection substrate.

[0049] A ninth aspect of the present invention provides any of the following methods:

[0050] (1) A method for preparing the Nanobody of the first aspect of the present invention, comprising culturing the host cell of the fifth aspect of the present invention and recovering the Nanobody;

[0051] (2) A method for detecting FAP for non-diagnostic purposes, the method comprising contacting the Nanobody described in the first aspect of the present invention or the immunoconjugate described in the sixth aspect of the present invention with a sample to be detected, and detecting the level of FAP in the sample to be detected.

[0052] Furthermore, (1) also includes purifying the nanobody.

[0053] The tenth aspect of the present invention provides any one of the following applications:

[0054] (1) Use of the Nanobody described in the first aspect of the invention or the immunoconjugate described in the sixth aspect of the invention in detecting FAP protein for non-diagnostic purposes;

[0055] (2) Use of the Nanobody described in the first aspect of the invention or the immunoconjugate described in the sixth aspect of the invention in the preparation of a product for detecting FAP protein;

[0056] (3) Use of the nanobody described in the first aspect of the invention, the chimeric antigen receptor described in the second aspect of the invention, the nucleic acid molecule described in the third aspect of the invention, the vector described in the fourth aspect of the invention, the host cell described in the fifth aspect of the invention, or the immunoconjugate described in the sixth aspect of the invention in the preparation of a pharmaceutical composition for preventing / treating FAP-related diseases.

[0057] 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.

[0058] Furthermore, 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, bile duct cell carcinoma, clear cell renal carcinoma, neuroendocrine tumor, carcinogenic osteomalacia, sarcoma, CUP (cancer of unknown primary), thymic cancer, glioma, glioma, astrocytoma, cervical cancer or prostate cancer.

[0059] Advantages and beneficial effects of the present invention:

[0060] The present invention provides nanoantibodies VHH1 and VHH2 that can simultaneously recognize human or mouse FAP. These nanoantibodies, VHH1 and VHH2, can be incorporated into human or mouse CAR structures as antigen-binding regions to generate human or mouse FAP-CAR-T cells with anti-FAP activity. The mouse FAP-CAR-T cells or human FAP-CAR-T cells provided by the present invention have a promising therapeutic effect in clearing FAP-positive cells and represent a novel FAP-CAR-T cell candidate for clinical CAR-T therapy of solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1Figures 1A and 1B are graphs showing the results of nanobody screening and identification, wherein 1A is a graph showing FAP protein detection, 1B is a graph showing alpaca immune titer detection, 1C is a graph showing the construction of a yeast display library, 1D is a graph showing the FACS detection of a yeast panning library, 1E is a graph showing the FACS detection of yeast monoclonal antibodies, 1F is a graph showing the FAP candidate antibody FACS binding detection, 1G is a graph showing the MFI values ​​of the FAP candidate antibody-FACS binding detection, and 1H is a graph showing the FAP antibody epitope competition detection;

[0062] Figure 2 2A is a schematic diagram of the CAR structure of FAP-CAR-T cells in mice, wherein 2B is a schematic diagram of the CAR structure of FAP-CAR-T 1 cells, and 2A is a schematic diagram of the CAR structure of FAP-CAR-T 2 cells;

[0063] Figure 3 Figure 3A is a graph showing the CAR positivity rate of FAP-CAR-T cells in mice, wherein Figure 3A is a graph showing the CAR positivity rate of FAP-CAR-T 1 cells, and Figure 3B is a graph showing the CAR positivity rate of FAP-CAR-T 2 cells;

[0064] Figure 4 Figures verifying 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;

[0065] Figure 5 Figure 5 is the result of RTCA detection of killing efficiency of mouse FAP-CAR-T cells co-cultured with NIH3T3-mFAP-GFP cells, wherein 5A is the result of FAP-CAR-T 1 cell killing efficiency, and 5B is the result of FAP-CAR-T 2 cell killing efficiency;

[0066] Figure 6 Figure 6 is a graph showing the killing efficiency of mouse FAP-CAR-T cells after co-culture with NIH3T3-mFAP-GFP cells for 24 hours, wherein 6A is a graph showing the killing efficiency of FAP-CAR-T 1 cells detected by flow cytometry, 6B is a graph showing the statistical results of the killing efficiency of FAP-CAR-T 1 cells, 6C is a graph showing the killing efficiency of FAP-CAR-T 2 cells detected by flow cytometry, and 6D is a graph showing the statistical results of the killing efficiency of FAP-CAR-T 2 cells;

[0067] Figure 77A is a schematic diagram of the CAR structure of human FAP-CAR-T cells, wherein 7A is a schematic diagram of the CAR structure of human FAP-CAR-T 1 cells, and 7B is a schematic diagram of the CAR structure of human FAP-CAR-T 2 cells;

[0068] Figure 8 Figure 8 is a graph showing the results of flow cytometry detection of the CAR positivity rate of human FAP-CAR-T cells, wherein Figure 8A is a graph showing the results of flow cytometry detection of the CAR positivity rate of human FAP-CAR-T 1 cells, and Figure 8B is a graph showing the results of flow cytometry detection of the CAR positivity rate of human FAP-CAR-T 2 cells;

[0069] Figure 9 Figures 9A and 9B show the fluorescence microscopy of human FAP-CAR-T1 cells, 9B shows the Western blot of human FAP-CAR-T1 cells, 9C shows the flow cytometry of human FAP-CAR-T1 cells, and 9D shows the flow cytometry of human FAP-CAR-T2 cells.

[0070] Figure 10 Figure 10A shows the killing efficiency of human FAP-CAR-T cells and SKOV3-hFAP-GF cells co-cultured, and 10B shows the killing efficiency of human FAP-CAR-T 2 cells.

[0071] Figure 11 Figure 11 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;

[0072] Figure 12 The figure shows the results 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 them, 12A is FAP-CAR-T 1 cell and 12B is FAP-CAR-T 2 cell. DETAILED DESCRIPTION

[0073] The following provides definitions of some terms used in this specification. Unless defined otherwise, 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.

[0074] The present invention provides a nanobody targeting FAP, wherein the nanobody comprises VHH1 and VHH2.

[0075] In some embodiments, the Nanobodies further comprise functional variants of Nanobodies VHH1 or VHH2. Specifically, the functional variants include, but are not limited to, derivatives that are substantially similar in primary structural sequence but include modifications not present in the parent Nanobodies VHH1 or VHH2 of the invention, such as those that have been chemically and / or biochemically modified 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, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidative PEGylation, proteolytic treatment, and phosphorylation, and such functional variants are also encompassed within the scope of protection of the present invention.

[0076] Functional variants may be Nanobodies that comprise an amino acid sequence comprising one or more amino acid substitutions, insertions, deletions, or combinations thereof, compared to the amino acid sequence of the parent Nanobody VHH1 or VHH2. Furthermore, functional variants may comprise truncations of the amino acid sequence at one or both of the amino or carboxyl termini. Compared to the parent Nanobody VHH1 or VHH2, the functional variants of the present application may have the same or different, higher or lower binding affinity, but may still specifically bind to FAP. For example, compared to the parent Nanobody VHH1 or VHH2, the functional variants of the present application may have an increased or decreased binding affinity for FAP.

[0077] In some embodiments, identity refers to the fact that at any particular position in the compared sequences, the amino acid residues between the sequences are identical. For example, leucine can be replaced with isoleucine or valine. Other amino acids that are commonly substituted for one another 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). Typically, 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 altering a single amino acid or a small percentage of amino acids or individual additions, deletions, insertions, or substitutions to an amino acid sequence is a conservative modification, wherein alterations in proteins produce proteins with similar functions, and providing conservative substitution tables of amino acids with similar functions is well known in the art.

[0078] 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.

[0079] In some embodiments, a chimeric antigen receptor (CAR) generally refers to a fusion protein comprising an extracellular domain capable of binding to 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., tumor-specific antigen and / or 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. A nucleic acid encoding CAR can be introduced into a T cell, NK cell, or NK T cell using, for example, a retroviral vector. 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 the 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 target antigens.

[0080] The chimeric antigen receptor also includes a hinge region.

[0081] 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, and IL-11 receptor.

[0082] In a specific embodiment, the hinge region is selected from CD8, CD28.

[0083] The chimeric antigen receptor also includes a transmembrane domain.

[0084] 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.

[0085] In a specific embodiment, the transmembrane domain is selected from CD8, CD28.

[0086] The chimeric antigen receptor further includes an intracellular domain, which includes a costimulatory molecule domain and a signal transduction domain.

[0087] In some embodiments, the costimulatory 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.

[0088] In a specific embodiment, the costimulatory molecule domain is selected from 4-1BB.

[0089] 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.

[0090] In a specific embodiment, the signal transduction domain is selected from CD3ζ.

[0091] The chimeric antigen receptor also includes a tag protein.

[0092] In some embodiments, the tag protein includes but is not limited to RQR8, EGFRt, CD20, c-MYC, 6×His, GST, Fc, Flag, and HA.

[0093] In a specific embodiment, the tag protein is selected from RQR8.

[0094] The chimeric antigen receptor also includes a self-cleaving peptide.

[0095] In some embodiments, the self-cleaving peptide includes but is not limited to P2A, T2A, E2A, and F2A.

[0096] In a specific embodiment, the self-cleaving peptide is selected from T2A.

[0097] The chimeric antigen receptor also includes a linker.

[0098] In some embodiments, the linker is a linker commonly used in the art, and the linker can be (G4S) n 、(GGGS) n 、(SSSSG) n 、(GSGSA) n 、(GGSGG) n or any other linkers, wherein n can be any integer between 1-10.

[0099] In a specific embodiment, the linker is selected from (G4S)4.

[0100] The chimeric antigen receptor also includes auxiliary functional elements.

[0101] 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).

[0102] The present invention provides a vector comprising the above nucleic acid molecule.

[0103] In some embodiments, the present application has no particular restrictions on vectors, and the choice thereof depends on the desired function. Non-limiting examples of vectors include DNA vectors, RNA vectors, plasmid vectors, viral-derived vectors, phage vectors, and other vectors conventionally used, for example, in genetic engineering. Various plasmids and vectors can be constructed based on methods well known to those skilled in the art. The vectors according to the present application are capable of directing the replication and expression of the nucleic acid molecules of the present application in a host, and thus ensuring the expression of the FAP-targeting Nanobodies or Chimeric Antigen Receptors described herein encoded thereby in a selected host. The expression vector may, for example, be a cloning vector, a binary vector, or an integrative vector. Expression includes transcription of the nucleic acid molecule, for example, into translatable mRNA.

[0104] The vector also includes one or more regulatory sequences operably linked to the nucleic acid.

[0105] The regulatory sequence includes a promoter sequence, a transcription terminator sequence, and a leader sequence.

[0106] In some embodiments, the promoter includes a CMV promoter, an EF-1α promoter, an SV40 early promoter, an MMTV promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an actin promoter, a myosin promoter, a heme promoter, a creatine kinase promoter, a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, or a tetracycline promoter.

[0107] In some embodiments, the transcription terminator includes a CYC1 transcription terminator, a T7 transcription terminator, a rrnBT1 transcription terminator, a rrnBT2 transcription terminator, an ADH1 transcription terminator, a TIF51A transcription terminator, an ALG6 transcription terminator, an AOD transcription terminator, an AOX1 transcription terminator, an ARG4 transcription terminator, a PMA1 transcription terminator, a TEF1 transcription terminator, a TT1 transcription terminator, or a TT2 transcription terminator.

[0108] The present invention provides a host cell, which comprises the above nucleic acid molecule or the above vector.

[0109] In some embodiments, the cells include prokaryotic cells and eukaryotic cells.

[0110] 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.

[0111] In a preferred embodiment, the cell is selected from eukaryotic cells.

[0112] In a more preferred embodiment, the eukaryotic cell is selected from animal cells.

[0113] In a more preferred embodiment, the animal cell is selected from mammalian cells.

[0114] In a more preferred embodiment, the mammalian cell is selected from immune cells.

[0115] 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.

[0116] In a specific embodiment, said immune cells are selected from T cells.

[0117] The present invention provides an immunoconjugate, which comprises the above-mentioned nanobody and a conjugate conjugated thereto.

[0118] The conjugate comprises a detectable label, 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.

[0119] In some embodiments, the detectable marker comprises a fluorescent or luminescent marker, a radioactive marker, an MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agent. The radionuclide comprises 131 I.32 P. 89 Sr. 90 Y. 223 Ra, 125 I. 103 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, and M-CSF. The therapeutic agents include alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, chromatin function inhibitors, anti-angiogenic agents, antiestrogens, antiandrogens, and immunomodulators.

[0120] 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.

[0121] The pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0122] In some embodiments, a pharmaceutically acceptable excipient is generally any type of non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary.

[0123] In some embodiments, suitable pharmaceutical compositions are administered in forms suitable for parenteral administration, such as by injection or infusion, for example, 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 may contain formulation agents such as suspending agents, preservatives, stabilizers, and / or dispersants.

[0124] 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. A skilled physician can usually easily determine the prescription and the dosage and administration method that are effective for the desired treatment and / or prevention.

[0125] The present invention provides a product for detecting FAP, which comprises the above-mentioned nanoantibody or the above-mentioned immunoconjugate.

[0126] The products include test kits and test strips.

[0127] 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, general 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.

[0128] The present invention will be further described below with reference to 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 present invention. The main features of the present invention may be applied to various embodiments without departing from the scope of the present invention.

[0129] Example

[0130] Experimental methods

[0131] 1. Screening and identification of nanoantibodies

[0132] 1) Preparation and QC of FAP recombinant protein

[0133] FAP 26-760 Reduced SDS-PAGE detection of AA-His recombinant protein.

[0134] 2) Detection of alpaca immune titer: ELISA

[0135] Coated with FAP-His protein (1 μg / mL); primary antibody: 100 μL of serially diluted serum; secondary antibody: Goat anti-Llama IgG (H+L) Secondary Antibody [HRP] (1:50,000 dilution).

[0136] 3) Construction of yeast display library: 103# VHH fragment cloning

[0137] Peripheral blood was collected, PBMCs were isolated, RNA was extracted, and reverse transcribed into cDNA. VHH sequences were amplified from the cDNA samples using a single-domain antibody cloning primer set and subcloned into the yeast display vector pYDisplay. EBY100 competent cells were then electroporated to construct a single-domain antibody yeast display library.

[0138] 4) FACS detection of yeast panning library: 103#

[0139] The antigens were modified with Biotin, enriched by magnetic beads, and then detected by FACS.

[0140] Panning plan: FAP antigen screening.

[0141] 5) Yeast monoclonal FACS detection: 103#

[0142] Use Biotin-FAP-His antigen detection to select positive clones for testing.

[0143] 6) FACS binding assay of FAP candidate antibodies

[0144] Cell amount: 3*10^5 / well; primary antibody: FAP target candidate antibody (10μg / ml), 100μl per well; secondary antibody: FC-PE (1:500 dilution).

[0145] 7) FAP candidate antibody-FACS binding assay (MFI value summary)

[0146] Cell amount: 3*10^5 / well; primary antibody: FAP target candidate antibody (10μg / ml), 100μl per well; secondary antibody: FC-PE (1:500 dilution).

[0147] 8) FAP antibody epitope competition assay

[0148] Human-FAP-His was immobilized using the HIS1K sensor at a concentration of 5 μg / ml and a curing time of 150 s.

[0149] The buffer was PBST (PBS + 0.02% tween 20), and the AB1 antibody (103-1-G1-FC) was first bound, and after equilibrium, the AB2 antibody (103-1-D9-3-FC, 103-1-H7-FC, and 103-1-G1-FC as controls for the same epitope) was bound at a concentration of 100 nM.

[0150] Affinity detection: equilibrium 60 s, association 180 s, dissociation 180 s, detection temperature 25°C.

[0151] 2. Construction of Lentiviral Vector

[0152] A CAR for expressing FAP was designed and constructed. The FAP-CAR structure consists of an anti-FAP single-chain antibody (VHH1 and VHH2), a linker domain (G4S)4 connecting VHH1 and VHH2, a CD8 molecule hinge region and a transmembrane region, the intracellular domain of 4-1BB, and a CD3ζ signaling domain. The structure was then cloned into the lentiviral shuttle plasmid pHAGE. Figure 6 Schematic diagrams of the structures of the two CARs constructed in this example are provided.

[0153] 3. Construction of retroviral vector

[0154] A CAR for expressing FAP was designed and constructed. The FAP-CAR structure consists of an anti-FAP single-chain antibody (VHH1 and VHH2); a linker domain (G4S)4 connecting VHH1 and VHH2; the hinge region, transmembrane domain, and intracellular domain of mouse CD28; and a mouse CD3ζ signaling domain. The structure was then cloned into the retroviral shuttle plasmid pMIG. Figure 1 Schematic diagrams of the structures of the two CARs constructed in this example are provided.

[0155] 4. Preparation of human CAR-T cells

[0156] After obtaining informed consent, primary mononuclear cells (PBMCs) were isolated from peripheral blood of normal subjects or patients with B-cell lymphoma 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. Activated T cells are infected with a lentivirus carrying a CAR and then expanded in vitro for 10-12 days in an X-VIVO 15 culture system containing IL-2, IL-15, and IL-7, until transduction efficiency ≥40%, cell viability ≥70%, and freedom from mycoplasma and bacterial contamination are achieved.

[0157] 5. Preparation of Mouse CAR-T Cells

[0158] Prepare a 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 the mice, grind them, wash them, and prepare a single-cell suspension. Activate T cells with anti-mouse CD3 (0.2 μg / mL) and anti-mouse CD28 (1 μg / mL) antibodies, and add 50 U / mL of mouse cytokine IL-2 to adjust the T cell concentration to 2 × 10 6 On Day 0, cells were infected with CAR retrovirus and centrifuged at 1500 g for 2 hours. On Day 2, 48 hours after retrovirus infection, the cells were centrifuged at 400 g for 5 minutes and supplemented with 10 ng / mL of mouse cytokines IL-7 and IL-15. A portion of the cells was aspirated to detect CAR positivity. Depending on cell growth and number, the cells were rehydrated or passaged to maintain cell density.

[0159] 6. CAR positive rate detection

[0160] Take 1×10 6Transfer 10 CAR-T cells to a 1.5 mL EP tube, add 1 mL of pre-cooled PBS solution containing 2% BSA to wash the cells, centrifuge at 300 g for 5 minutes at 4°C, repeat washing twice, label CAR with FITC-CD34 antibody, then wash the cells with pre-cooled 2% BSA / PBS solution, transfer to a flow tube, and use flow cytometry to detect the CAR-T cell positive rate.

[0161] 7. Detection of mFAP or hFAP expression in target cells by flow cytometry and Western Blot

[0162] Take 1×10 6 Target cells (NIH3T3-mFAP, SKOV3-hFAP) were transferred to a 1.5 mL EP tube, and 1 mL of pre-chilled 2% BSA-containing PBS solution was added to wash the cells. The cells were centrifuged at 300 g for 5 minutes at 4°C. After fixation and permeabilization, the cells were fixed and permeabilized with fixative and permeabilization solution. Anti-FAP primary antibodies were added to label mFAP or hFAP. After incubation with AF594-labeled secondary antibodies, the cells were washed with pre-chilled 2% BSA / PBS solution, transferred to flow tubes, and flow cytometry was used to detect the expression of mFAP and hFAP in the target cells.

[0163] RIPA cell lysis buffer was used to extract target cell (NIH3T3-mFAP, SKOV3-hFAP) proteins, which 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. Finally, chemiluminescence reaction was used to detect the presence and expression level of FAP protein.

[0164] 8. Cytotoxicity of Tumor-Specific CAR-T Cells

[0165] Adherent cells (NIH3T3, NIH3T3-mFAP, SKOV3, SKOV3-hFAP) were cultured at 1×10 5 The cells were seeded at a density of 1:1 / well in a 48-well plate. 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 analysis of the killing ability of CAR-T cells, where APC-anti-CD3 antibodies labeled T cells and GFP-positive cells were labeled as residual tumor cells.

[0166] 9. RTCA experiment

[0167] Adherent cells (NIH3T3, NIH3T3-mFAP, SKOV3, and SKOV3-hFAP) were seeded at a density of 8,000 cells / well into 96-well E-Plate (ACEA Biosciences, USA) and monitored overnight using the impedance-based xCELLigence real-time cell analyzer (RTCA) system (ACEA Biosciences, USA). 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 real-time impedance curves were plotted.

[0168] 10. Statistical analysis

[0169] Flow cytometry results were analyzed using Flowjo V10 flow cytometry analysis software, and Western blot results 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 are expressed as mean ± standard deviation (mean ± SD). Comparisons between two groups were performed using the t-test. Comparisons between multiple groups were performed using one-way analysis of variance. P < 0.05 indicated statistical significance.

[0170] Experimental results

[0171] 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 of 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 ratio with FAP-Biotin was 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 the detection of MFI values 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) bind to different epitopes ( Figure 1 H).

[0172] This application is 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. The retrovirus is packaged in 293T cells using the auxiliary plasmid pCL-Eco. By infecting mouse-derived T 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 costimulatory 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-derived T cells, human Mock-T (non-transduced exogenous gene T cells) and human FAP-CAR-T cells ( Figure 7 ).

[0173] Table 1 Nanobody sequences

[0174]

[0175]

[0176] 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 was approximately 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) is 1:1, 1:3 and 1:5, mouse FAP-CAR-T cells can 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 parental NIH3T3 cells that do not express mouse FAP at an effector-target ratio of 2:1 and 1:1 showed that neither mouse FAP-CAR-T cells nor Mock-T cells could 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 , Figure 12 ).

[0177] In vitro experiments on 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 was approximately 65.6% ( Figure 8 B). The results of co-culture experiments 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 were able to 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 ).

[0178] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. A nanobody targeting FAP, characterized in that The nanobody is VHH1, and the amino acid sequences of CDR1, CDR2, and CDR3 of VHH1 are shown in SEQ ID NOs: 1-3, respectively.

2. The Nanobody according to claim 1, characterized in that The VHH1 further comprises 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.

3. The Nanobody according to claim 1, characterized in that The VHH1 has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:

8.

4. The Nanobody according to claim 3, characterized in that The amino acid sequence of the nanobody VHH1 is shown in SEQ ID NO:

8.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a sequence encoding the Nanobody according to any one of claims 1 to 4.

6. A carrier, characterized in that The vector comprises the nucleic acid molecule of claim 5.

7. The carrier according to claim 6, characterized in that The vector also includes one or more regulatory sequences operably linked to the nucleic acid molecule.

8. The carrier according to claim 7, characterized in that The regulatory sequence includes a promoter sequence, a transcription terminator sequence, and a leader sequence.

9. The carrier according to claim 6, characterized in that The vectors include DNA vectors and RNA vectors.

10. A host cell, characterized in that The host cell comprises the nucleic acid molecule of claim 5 or the vector of any one of claims 6 to 9.

11. The host cell according to claim 10, characterized in that The host cell is selected from eukaryotic cells.

12. The host cell according to claim 11, characterized in that The eukaryotic cells are selected from animal cells.

13. The host cell according to claim 12, characterized in that The animal cells are selected from mammalian cells.

14. The host cell according to claim 13, characterized in that The mammalian cells are selected from immune cells.

15. The host cell according to claim 14, characterized in that The immune cells are selected from T cells.

16. An immunoconjugate, characterized in that The immunoconjugate comprises the Nanobody according to any one of claims 1 to 4 and a conjugate conjugated thereto.

17. The immunoconjugate according to claim 16, characterized in that The conjugate includes a detectable label, an enzyme, gold nanoparticles / nanorods, nanomagnetic particles, viral coat proteins or VLPs, or a combination thereof.

18. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the Nanobody according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the vector according to any one of claims 6 to 9, the host cell according to any one of claims 10 to 15, or the immunoconjugate according to any one of claims 16 to 17.

19. The pharmaceutical composition according to claim 18, characterized in that The pharmaceutical composition further includes pharmaceutically acceptable excipients.

20. A product for detecting FAP, characterized in that: The product comprises the nanobody according to any one of claims 1 to 4 or the immunoconjugate according to any one of claims 16 to 17.

21. The product according to claim 20, characterized in that The products include test kits and test strips.

22. The product according to claim 21, characterized in that The kit also includes a solid support.

23. The product according to claim 21, characterized in that The kit further comprises a buffer, a blocking reagent, a washing reagent, and a detection substrate.

24. Any of the following methods: (1) A method for preparing the nanobody according to any one of claims 1 to 4, characterized in that: The method comprises culturing the host cell according to any one of claims 10 to 15, and recovering the Nanobody; (2) A method for detecting FAP for non-diagnostic purposes, characterized in that the method comprises contacting the Nanobody according to any one of claims 1 to 4 or the Immunoconjugate according to any one of claims 16 to 17 with a sample to be detected, and detecting the level of FAP in the sample to be detected.

25. Any of the following applications: (1) Use of the Nanobody according to any one of claims 1 to 4 or the Immunoconjugate according to any one of claims 16 to 17 in detecting FAP protein for non-diagnostic purposes; (2) Use of the Nanobody according to any one of claims 1 to 4 or the Immunoconjugate according to any one of claims 16 to 17 in the preparation of a product for detecting FAP protein; (3) Use of the Nanobody according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the vector according to any one of claims 6 to 9, the host cell according to any one of claims 10 to 15, or the immunoconjugate according to any one of claims 16 to 17 in the preparation of a pharmaceutical composition for preventing / treating FAP-related diseases; The FAP-related disease is ovarian cancer.

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