Monoclonal antibodies targeting fap and uses thereof

Abstract

The application discloses a FAP-targeted antibody or antigen-binding fragment thereof, wherein the complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region have the amino acid sequences shown in SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 3 respectively, and the complementarity determining regions CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region have the amino acid sequences shown in SEQ ID No: 4, SEQ ID No: 5 and SEQ ID No: 6 respectively. The FAP-specific antibody of the application can combine with a human FAP antigen protein with high affinity and high specificity, has good physical and chemical stability and in-vivo circulation stability, and the FAP antibody and a derivative CAR structure thereof can be used for preparing a drug for treating malignant tumors and fibrosis diseases.

Description

Technical Field

[0001] This application relates to the biomedical field, specifically to antibodies targeting FAP, chimeric antigen receptors (CARs) containing such antibodies, and their use in treating FAP-related diseases. Background Technology

[0002] Cancer and fibrotic diseases are two major categories of intractable diseases that seriously threaten human health, characterized by high incidence, poor prognosis, and limited existing treatment options. Solid tumors (such as breast cancer, lung cancer, pancreatic cancer, gastric cancer, and colorectal cancer) account for the vast majority of human malignant tumors. Their tumor microenvironment (TME) contains a large number of activated stromal fibroblasts, forming a dense connective tissue proliferative stroma that accounts for 20%–60% of tumor mass. This constitutes a physical and immunosuppressive barrier, severely limiting the efficacy of chemotherapy, radiotherapy, and immunotherapy. Fibrotic diseases (such as liver fibrosis, pulmonary fibrosis, osteoarthritis-related fibrosis, and myocardial fibrosis) are characterized by excessive activation of fibroblasts and abnormal deposition of extracellular matrix (ECM) after tissue damage. They can progress to organ failure, and currently, there are no radical drugs available clinically, representing a significant unmet medical need.

[0003] Fibroblast activating protein (FAP) is a type II transmembrane serine protease with dipeptidase and collagenase activities, expressed as a homodimer on the cell membrane surface. FAP is highly expressed in tumor-associated fibroblasts (CAFs) of over 90% of human epithelial solid tumors (including breast cancer, lung cancer, pancreatic cancer, colorectal cancer, and gastric cancer), but is almost entirely absent in most normal resting tissues, only appearing transiently during physiological remodeling processes such as embryonic development and wound healing, constituting an ideal therapeutic window. Simultaneously, activated myofibroblasts in fibrotic lesions also specifically and highly express FAP, with its expression level positively correlated with the severity of fibrosis. FAP directly participates in ECM degradation, pro-inflammatory cytokine release, and tissue remodeling, driving the continuous progression of fibrosis. Therefore, FAP has been recognized as a common key target for both the tumor microenvironment and fibrotic lesions.

[0004] Chimeric antigen receptor T-cell (CAR-T) therapy has achieved breakthrough success in hematological malignancies, but it still faces significant challenges in solid tumors and fibrotic diseases: traditional CAR-T therapy often targets tumor cell autoantigens, making relapse likely due to antigen loss; furthermore, the tumor matrix barrier hinders T-cell infiltration, and the immunosuppressive microenvironment induces T-cell exhaustion. CAR-T targeting FAP can directly eliminate CAFs or activate myofibroblasts, disrupt the tumor matrix barrier, reshape the immune microenvironment, and block fibrosis progression. Preclinical models have shown significant therapeutic effects on breast cancer, pancreatic cancer, lung cancer, pulmonary fibrosis, and myocardial fibrosis. The recently developed in vivo CAR-T technology, through the delivery of CAR-encoding mRNA or viral vectors, generates transient CAR-T cells in situ within the patient, eliminating the need for in vitro cell separation and expansion. This reduces costs, shortens the preparation cycle, and decreases the risk of rejection, providing a novel strategy for FAP-targeted therapy.

[0005] Currently, targeted therapies for FAP still have significant shortcomings: small molecule inhibitors lack specificity and are prone to off-target toxicity; bispecific antibody molecules have limited stability and tissue penetration; existing FAP antibodies have inconsistent affinity, internalization efficiency, and development potential, making it difficult to meet the application requirements of in vivo CAR-T. In particular, there is a lack of a high-affinity, high-specificity, stable, and in vivo CAR-T targeted delivery anti-FAP antibody for the treatment of solid tumors and fibrotic diseases.

[0006] In summary, developing a highly active antibody targeting FAP for solid tumors and fibrotic diseases and applying it to in vivo CAR-T targeted therapy has significant clinical value and broad application prospects. However, there are still significant technological gaps in this field that urgently need to be addressed. Summary of the Invention

[0007] Given the limitations of existing anti-FAP antibodies, such as low affinity, poor specificity, and unsatisfactory physicochemical stability, and the difficulty of effectively applying existing CAR-T technology to solid tumors and fibrotic diseases, the present invention aims to provide a novel antibody that specifically targets human FAP, and the pharmaceutical use of this antibody in in vivo in situ CAR-T cell targeted therapy.

[0008] Specifically, this application relates to the following technical solutions: 1. An antibody targeting FAP or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region have amino acid sequences as shown in SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No: 3, respectively, and the complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region have amino acid sequences as shown in SEQ ID No: 4, SEQ ID No: 5, and SEQ ID No: 6, respectively.

[0009] 2. The antibody targeting FAP or its antigen-binding fragment according to claim 1, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 7 or has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequence shown in SEQ ID No: 7; and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 8 or has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequence shown in SEQ ID No: 8.

[0010] 3. The antibody targeting FAP or its antigen-binding fragment according to claim 1 or 2, comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID No: 9; and / or the amino acid sequence of the light chain is as shown in SEQ ID No: 10.

[0011] 4. The antibody targeting FAP or its antigen-binding fragment according to claim 1 or 2, wherein the scFv fragment comprises a heavy chain variable region and a light chain variable region, the scFv fragment comprising a linker peptide connecting the heavy chain variable region and the light chain variable region, the amino acid sequence of the scFv fragment being shown in SEQ ID No: 11.

[0012] 5. The nucleic acid molecule encoding any one of items 1 to 4 that targets FAP or its antigen-binding fragment.

[0013] 6. A chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the antigen-binding domain comprises an antibody or antigen-binding fragment thereof targeting FAP as described in claim 4.

[0014] 7. The nucleic acid molecule encoding the chimeric antigen receptor (CAR) described in item 6.

[0015] 8. The nucleic acid molecule described in item 7 is an mRNA molecule.

[0016] 9. An immune cell comprising a chimeric antigen receptor (CAR) as described in item 6 or a nucleic acid molecule as described in item 7 or 8.

[0017] 10. The immune cells described in item 9 are T cells, NK cells, macrophages, antigen-presenting cells, or B cells.

[0018] 11. A nanoparticle, said nanoparticle encapsulating the nucleic acid molecule described in claim 7 or 8.

[0019] 12. The nanoparticles according to claim 11, wherein the nanoparticles are constructed from lipid aptamers, the lipid aptamers being composed of hydrophobic molecules and nucleic acid aptamers targeting cell surface molecules linked by chemical bonds.

[0020] 13. The nanoparticles according to claim 12, wherein the hydrophobic molecule in the lipid aptamer is selected from 1,2-dioleoyl-SN-glycerol-3-phosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), 1,2-distearyl-sn-propanetriyl-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine (sodium salt) (DOPS), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), hydrogenated lecithin (HSPC), 1,2-distearyl-sn-propanetriyl-3-phosphatidylethanolamine (DSPE), 1,2-distearyl-sn-glycerol-3-phosphatidylserine (DSPG), 1-palmitoyl-2-oleoyl-sn-glycerol- One or more of the following: 3-phosphatidylethanolamine (DPPE), 1,2-dilauroylphosphatidylethanolamine (DLPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylglycerol (DPPG), monophosphatidyllipid A (MPLA), 1,3-dilinolenin, polycaprolactone (PCL), SM-102, ALC-0315, DLin-MC3-DMA, DLin-KC2-DMA, cKK-E12-TREM2, arachidonic acid, linolenic acid, L319, cholesterol, DOTAP, DDAB, POPC, POPE, DPPE, DOPC, β-sitosterol, cholesterol, cholesterolone, palmitic acid, stearic acid, myristic acid, and arachidic acid.

[0021] 14. The nanoparticles according to item 12, wherein the nucleic acid aptamer is a nucleic acid aptamer targeting T cell surface molecules OSJ-T3, OX40, CD28Apt7, 4-1BB, CD5, CD62L or C2NP, or a nucleic acid aptamer targeting tumor-associated macrophage surface molecules CD32, MerTK, TLR4, LOX-1, MHC-II, CD68, A2, CD206, Dectin-1, CD163, TREM2 or WXY3, or a nucleic acid aptamer targeting NK cell surface molecules CD16a or CD56.

[0022] 15. The nanoparticles according to claim 12, wherein the hydrophobic molecule in the lipid aptamer is 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE), and the nucleic acid aptamer is a nucleic acid aptamer targeting the cell surface molecule CD62L.

[0023] 16. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof targeting FAP as described in any one of items 1-4, an immune cell as described in item 9 or 10 or a nanoparticle as described in any one of items 11-15, and a pharmaceutically acceptable carrier or excipient.

[0024] 17. Use of the nanoparticles described in any one of items 11 to 15 in the preparation of a medicament for generating chimeric antigen receptor immune cells in vivo.

[0025] 18. Use of the antibody or antigen-binding fragment thereof as described in any one of items 1 to 4, or the immune cell as described in item 9 or 10, or the nanoparticle as described in any one of items 11 to 15, in the preparation of a medicament for treating FAP-positive diseases.

[0026] 19. According to the use described in item 18, the FAP-positive disease is a malignant tumor or a fibrotic disease.

[0027] 20. According to the use described in item 19, the malignant tumor includes pancreatic cancer, gastric cancer, or colorectal cancer.

[0028] 21. According to the use described in item 19, the fibrotic disease includes liver fibrosis, pulmonary fibrosis, osteoarthritis fibrosis, myocardial fibrosis, or renal fibrosis.

[0029] The effects of the invention The fibroblast activation protein (FAP) specific antibody provided in this application can bind to human FAP antigen protein with high affinity and high specificity, and has good physicochemical stability and in vivo circulation stability. It can specifically recognize tumor-associated fibroblasts activated in the tumor microenvironment and abnormally activated fibroblasts in fibrotic lesions.

[0030] Using the FAP antibody molecule-encoded mRNA provided in this application, DOPE-LD201t1 nanoparticles loaded with FAP antibody molecule-encoded mRNA were successfully prepared, exhibiting uniform particle size and high mRNA encapsulation efficiency.

[0031] The FAP antibody and its derived CAR structure of this application can be used to prepare drugs for the treatment of malignant tumors and fibrotic diseases, including but not limited to pancreatic cancer, gastric cancer, colorectal cancer, liver fibrosis, pulmonary fibrosis, and osteoarthritis fibrosis. In a mouse model of pancreatic cancer, the in vivo CAR-T nanomedicine prepared using the FAP CAR mRNA of this application can disrupt the tumor matrix barrier and improve the tumor microenvironment as its core functions, with a tumor inhibition rate of approximately 20% to 40% (average 30.0 ± 6.1%), and can delay tumor progression. Attached Figure Description

[0032] Figure 1 Electron micrograph of DOPE-LD201t1 nanoparticles loaded with FAP CAR mRNA.

[0033] Figure 2 The in vitro killing effect of T cells reprogrammed with FAP CAR mRNA.

[0034] Figure 3 This is a schematic diagram of tumor inhibition rate in a mouse model of pancreatic cancer.

[0035] Figure 4 This is a schematic diagram illustrating the survival time of a mouse model of pancreatic cancer. Detailed Implementation

[0036] The present application will now be described in further detail with reference to specific embodiments. The embodiments given are intended to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0037] It should be noted that the terms "comprising" or "including" used throughout the specification and appendices are open-ended and should be interpreted as "comprising but not limited to". The subsequent descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appendices.

[0038] The term "antibody" as used in this application refers to an immunoglobulin molecule produced by B cells that specifically recognizes and binds to antigens. Natural antibodies are typically Y-shaped tetramers formed by two identical heavy chains (H chains) and two identical light chains (L chains) linked by disulfide bonds. Each heavy and light chain contains an N-terminal variable region (V region) and a C-terminal constant region (C region). The variable region is a key structural domain for antibody recognition and antigen binding. The variable region contains a conserved framework region (FR) and a highly variable complementarity determining region (CDR). Each heavy chain variable region contains three CDRs. In this application, from N-terminus to C-terminus, they are CDR-H1, CDR-H2, and CDR-H3; each light chain variable region contains three CDRs, in this application, CDR-L1, CDR-L2, and CDR-L3. These six CDRs together constitute the antigen-binding site of the antibody. Based on the different constant regions of the heavy chain, antibodies can be classified into types such as IgA, IgD, IgE, IgG, and IgM.

[0039] The “antibody targeting FAP” or “anti-FAP antibody” mentioned in this application refers to an antibody that can specifically bind to fibroblast activation protein (FAP).

[0040] The term "monoclonal antibody" as used in this application refers to a highly homogeneous antibody molecule produced from a single B cell clone that targets only a single antigenic epitope. Compared to polyclonal antibodies, it exhibits high specificity and homogeneity. The antibody targeting FAP in this application is preferably a monoclonal antibody. Monoclonal antibodies can be obtained using methods known in the art, such as hybridoma technology, phage display technology, or single-B cell sequencing technology.

[0041] The "chimeric antigen receptor" or "CAR" described in this application refers to a fusion protein constructed using genetic engineering techniques, typically comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain. CARs can endow immune cells (such as T cells) with antigen recognition capabilities independent of the major histocompatibility complex (MHC), enabling immune cells to directly recognize and kill target cells expressing target antigens. The CAR of this invention comprises an antigen-binding domain (preferably scFv) targeting FAP, a hinge region, a transmembrane domain, a co-stimulatory signaling domain, and a CD3ζ activation domain.

[0042] The "antigen-binding domain" described in this application refers to the extracellular functional region of a chimeric antigen receptor (CAR) responsible for specifically recognizing and binding to target antigens. Antigen-binding domains are typically derived from antibodies or their antigen-binding fragments, capable of directly recognizing native conformational antigens on the surface of target cells in a manner independent of the major histocompatibility complex (MHC). In this invention, the antigen-binding domain comprises an antibody or its antigen-binding fragment targeting fibroblast activation protein (FAP). The forms of antigen-binding domains applicable to this application include, but are not limited to, single-chain variable region fragments (scFv), single-domain antibodies (sdAb / VHH), and Fab fragments.

[0043] The "hinge region" mentioned in this application refers to a peptide segment that connects the antigen-binding domain and the transmembrane domain, providing a certain flexible space for antigen binding. Hinge regions that can be used for CAR construction include, but are not limited to: CD8α hinge region, CD28 hinge region, IgG1 hinge region, FcγRIII hinge region, etc.

[0044] The "transmembrane domain" described in this application refers to a peptide segment that spans the lipid bilayer of the cell membrane and is responsible for anchoring the CAR to the immune cell membrane. Transmembrane domains that can be used for CAR construction include, but are not limited to, the CD8α transmembrane domain, the CD28 transmembrane domain, the CD3ζ transmembrane domain, and the FcεRIγ transmembrane domain.

[0045] The "co-stimulatory signaling domain" mentioned in this application refers to an intracellular domain that provides the second signal required for the full activation of T cells. The co-stimulatory signaling domain in CARs typically originates from the intracellular segment of co-stimulatory molecules, including but not limited to: 4-1BB (CD137), CD28, OX40 (CD134), ICOS, CD27, GITR, etc.

[0046] The term "immune cells" as used in this application refers to cells involved in the immune response, including but not limited to T cells, B cells, natural killer cells (NK cells), natural killer T cells (NKT cells), invariant natural killer T cells (iNKT cells), γδT cells, monocytes, macrophages, and dendritic cells. T cells, macrophages, NK cells, or NKT cells are preferred in this application.

[0047] The "CAR-T cells" mentioned in this application refer to T cells that have been genetically engineered to express chimeric antigen receptors (CARs). CAR-T cells are able to specifically recognize and kill target cells expressing target antigens in an MHC-independent manner.

[0048] The "CAR-macrophage" mentioned in this application refers to macrophages that have been genetically engineered to express chimeric antigen receptors (CARs). CAR-macrophages can specifically recognize and engulf target cells expressing target antigens, or recruit other immune cells to participate in anti-tumor responses by secreting cytokines.

[0049] The "FAP" mentioned in this application refers to Fibroblast Activation Protein (FAP), a type II transmembrane serine protease. It is expressed at extremely low levels in normal adult tissues, but is highly expressed in cancer-associated fibroblasts (CAFs) in the tumor microenvironment and activated fibroblasts in fibrotic diseases. FAP is an important biomarker of the tumor and fibrotic microenvironments, and is also the antigen targeted by the antibodies and CARs described in this application.

[0050] The term "FAP-positive disease" as used in this application refers to a disease in which the number or proportion of cells expressing FAP (such as CAF or activated fibroblasts) in diseased tissues (such as tumor tissue or fibrotic tissue) is significantly higher than that in normal controls. FAP-positive diseases in this application include, but are not limited to, solid tumors (such as pancreatic cancer, liver cancer, colorectal cancer, gastric cancer, lung cancer, sarcoma, etc.) and fibrotic diseases (such as liver fibrosis, pulmonary fibrosis, myocardial fibrosis, renal fibrosis, etc.).

[0051] One aspect of this application provides an antibody or antigen-binding fragment thereof that targets FAP, and a nucleic acid molecule encoding said antibody or antigen-binding fragment thereof.

[0052] In one specific embodiment, the complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region of the antibody have amino acid sequences as shown in SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No: 3, respectively.

[0053] In one specific embodiment, the complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3 of the antibody's light chain variable region have amino acid sequences as shown in SEQ ID No: 4, SEQ ID No: 5, and SEQ ID No: 6, respectively.

[0054] In some specific embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID No: 7.

[0055] In some specific embodiments, the amino acid sequence of the heavy chain variable region of the antibody has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID No: 7.

[0056] In some specific embodiments, the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID No: 8.

[0057] In some specific embodiments, the amino acid sequence of the variable region of the light chain of the antibody has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID No: 8.

[0058] In some specific embodiments, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID No: 9.

[0059] In some specific embodiments, the amino acid sequence of the light chain of the antibody is shown in SEQ ID No: 10.

[0060] In one specific embodiment, the antibody is a monoclonal antibody that includes a linker peptide connecting the heavy chain variable region and the light chain variable region.

[0061] In one specific embodiment, the antibody or its antigen-binding fragment is an scFv fragment containing a heavy chain variable region and a light chain variable region.

[0062] In a specific implementation, the scFv fragment includes a linker peptide connecting the heavy chain variable region and the light chain variable region.

[0063] In one specific embodiment, the amino acid sequence of the scFv fragment is shown in SEQ ID No: 11.

[0064] Another aspect of this application provides the use of the antibody targeting FAP as a target molecule, prepared into in-vivoCAR-T, in the preparation of drugs for the treatment of cancer and fibrotic diseases.

[0065] In a specific embodiment, this application provides a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the antigen-binding domain contains an antibody or antigen-binding fragment of the FAP-targeting antibody described in this application.

[0066] In one specific embodiment, the antigen-binding domain comprises the scFv fragment, the sequence of which is shown in SEQ ID No: 11.

[0067] In one specific embodiment provided in this application, the amino acid sequence of the chimeric antigen receptor (CAR) is shown in SEQ ID No: 12.

[0068] This application further provides a nucleic acid molecule encoding the chimeric antigen receptor (CAR).

[0069] In a specific implementation, the nucleic acid molecule is mRNA.

[0070] In one specific embodiment provided in this application, the nucleotide sequence encoding the chimeric antigen receptor (CAR) is shown in SEQ ID No: 13.

[0071] The amino acid and nucleotide sequences involved in this application are shown below: SEQ ID No: 1(CDR-H1):GVSFNNYVIS SEQ ID No: 2 (CDR-H2):GILPAVGTTF SEQ ID No: 3 (CDR-H3): DAPGTNFNLPLHF SEQ ID No: 4 (CDR-L1):RASQGISSWLA SEQ ID No: 5 (CDR-L2): AASSLQS SEQ ID No: 6 (CDR-L3): QQANSFPPT SEQ ID No: 7 (heavy chain variable region): QVQLVQSGAEVKKPGSSVKVSCKSSGVSFNNYVISWVRQAPGQGLEWMGGILPAVGTTFYTPRFRGRVTFTADQSTTTAYMELGGLRSDDTAVYFCARDAPGTNFNLPLHFWGQGTPVTVSS SEQ ID No: 8 (light chain variable region): NIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTRLEIK SEQ ID NO: 9 (heavy chain: QVQLVQSGAEVKKPGSSVKVSCKSSGVSFNNYVISWVRQAPGQGLEWMGGILPAVGTTFYTPRFRGRVTFTADQSTTTAYMELGGLRSDDTAVYFCARDAPGTNFNLPLHFWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 10 (light chain): NIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 11 (scFv fragment): NIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTRLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKSSGVSFNNYVISWVRQAPGQGLEWMGGILPAVGTTFYTPRFRGRVTFTADQSTTTAYMELGGLRSDDTAVYFCARDAPGTNFNLPLHFWGQGTPVTVSS SEQ ID NO: 12 (FAP CAR sequence): MALPVTALLLPLALLLHAARPNIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTRLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKSSGVSFNNYVISWVRQAPGQGLEWMGGILPAVGTTFYTPRFRGRVTFTADQSTTTAYMELGGLRSDDTAVYFCARDAPGTNFNLPLHFWGQGTPVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR In a specific embodiment, this application provides nanoparticles constructed from lipid aptamers, wherein nucleic acid molecules encoding the chimeric antigen receptor (CAR) are encapsulated.

[0072] In some specific embodiments, the lipid aptamer is composed of a hydrophobic molecule and a nucleic acid aptamer that targets cell surface molecules linked by chemical bonds.

[0073] In some specific embodiments, the nucleic acid aptamer in the lipid aptamer is a nucleic acid aptamer that targets molecules on the surface of immune cells.

[0074] In some specific embodiments, the hydrophobic molecule in the lipid aptamer is selected from 1,2-dioleoyl-SN-glycerol-3-phosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), 1,2-distearyl-sn-propanetriyl-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phospho-L-serine (sodium salt) (DOPS), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), hydrogenated lecithin (HSPC), 1,2-distearyl-sn-propanetriyl-3-phosphatidylethanolamine (DSPE), 1,2-distearyl-sn-glycerol-3-phosphatidylserine (DSPG), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidyl ... - One or more of the following: phosphatidylethanolamine (DPPE), 1,2-dilauroylphosphatidylethanolamine (DLPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylglycerol (DPPG), monophosphatidyllipid A (MPLA), 1,3-dilinolenin, polycaprolactone (PCL), SM-102, ALC-0315, DLin-MC3-DMA, DLin-KC2-DMA, cKK-E12-TREM2, arachidonic acid, linolenic acid, L319, cholesterol, DOTAP, DDAB, POPC, POPE, DPPE, DOPC, β-sitosterol, cholesterol, cholesterolone, palmitic acid, stearic acid, myristic acid, and arachidic acid.

[0075] In some specific embodiments, the nucleic acid aptamer in the lipid aptamer is a nucleic acid aptamer that targets T cell surface molecules OSJ-T3, OX40, CD28Apt7, 4-1BB, CD5, CD62L or C2NP, or targets tumor-associated macrophage surface molecules CD32, MerTK, TLR4, LOX-1, MHC-II, CD68, A2, CD206, Dectin-1, CD163, TREM2 or WXY3, or targets NK cell surface molecules CD16a or CD56.

[0076] In one specific embodiment, the lipid aptamer includes the hydrophobic molecule 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE) and the nucleic acid aptamer LD201t1 that targets the cell surface molecule CD62L.

[0077] In one specific embodiment, the sequence of the nucleic acid aptamer is shown in SEQ ID No: 14 (TAGCCAAGGTAACCAGTACAAGGTGCTAAACGTAATGGCTTCGGCTTAC).

[0078] In another aspect of this application, an immune cell is provided that comprises the chimeric antigen receptor (CAR) or a nucleic acid molecule encoding the chimeric antigen receptor (CAR).

[0079] In some specific embodiments, the immune cells are selected from T cells, NK cells, macrophages, or B cells.

[0080] Another aspect of this application provides a pharmaceutical composition comprising the above-described antibody targeting FAP or its antigen-binding fragment, the immune cells or the nanoparticles, and a pharmaceutically acceptable carrier or excipient.

[0081] This application also provides the use of the nanoparticles in the preparation of a medicament for generating chimeric antigen receptor immune cells in vivo.

[0082] This application also provides the use of the antibody or its antigen-binding fragment, or the immune cell, or the nanoparticle in the preparation of a medicament for treating FAP-positive diseases.

[0083] In a specific implementation, the FAP-positive disease is a malignant tumor or a fibrotic disease.

[0084] In some specific embodiments, the malignant tumor includes pancreatic cancer, gastric cancer, or colorectal cancer.

[0085] In some specific embodiments, the fibrotic disease includes liver fibrosis, pulmonary fibrosis, osteoarthritis fibrosis, myocardial fibrosis, or renal fibrosis.

[0086] Example 1: Preparation of a monoclonal antibody targeting FAP (1) Plasmid construction After gene synthesis, homologous recombination, transformation, identification, sequencing, alignment, extraction and sequence alignment, the sequence (SEQ ID No: 11) was constructed into the pcDNA3.4 vector to obtain the protein expression plasmid.

[0087] (2) Expression and purification The constructed antibody plasmid was transfected into Expi CHO cells, and the antibody expression time was 7 days with an expression volume of 10 mL to 20 mL. After expression, the protein was purified and aliquoted to serve as samples for subsequent experiments (A162).

[0088] Example 2: Physicochemical properties and activity assay of FAP-targeting monoclonal antibodies 2.1 SDS-PAGE identification (1) Preparation of purified sample solution: According to the A280 determination, 1 μg of non-reduced sample was added to 4×LDS loading buffer and iodoacetamide (final concentration 40 mM), and heated in a dry bath at 75℃ for 10 min. 1 μg of reduced sample was added to 4×LDS loading buffer and DTT (final concentration 5 mM), and heated in a dry bath at 100℃ for 10 min.

[0089] (2) Electrophoresis: 140 V, 75 min.

[0090] (3) Staining, destaining and scanning: Coomassie brilliant blue gel staining, destaining and scanning with EPSON V550 color scanner.

[0091] (4) Calculate purity: Use Image J to calculate the purity of the reduced band according to the peak area normalization method, or the purity of the reduced heavy chain plus the light chain.

[0092] The test results are shown in Table 1.

[0093] Table 1

[0094] 2.2 SEC Authentication (1) Preparation of mobile phase: Prepare 0.15 M PB + NaCl and adjust pH to 6.0.

[0095] (2) Sample preparation: Dilute the sample concentration to 0.5 mg / mL.

[0096] (3) Column conditions: XBridge BEH 200Å, SEC 3.5 µm, 7.8×300 mm, column temperature set at 20℃, and detection baseline was stable.

[0097] (4) Parameter settings: flow rate set to 0.8 mL / min; sample injection volume set to 20 μL; detection wavelength 280 nm, bandwidth 4 nm; reference wavelength 360 nm, bandwidth 100 nm; peak width (response time) > 0.1 min (2 s response time); slit 4 nm; negative absorbance baseline 100 mAU.

[0098] The test results are shown in Table 2.

[0099] Table 2

[0100] 2.3 ELISA Activity Assay (1) Plate preparation: Dilute the antigen sample with 1×PBS to a concentration of 2 μg / mL, add 30 µL / well to each well of a 96-well ELISA plate, and incubate overnight (more than 12 h) at 4°C.

[0101] (2) Blocking: Wash the plate 3 times with PBST, add blocking solution (5% PBSM) and block at room temperature for 2 h.

[0102] (3) Incubation: Wash the plate 3 times with PBST, add the primary antibody diluted with 1% PBSM at 30 µL / well, and incubate at room temperature for 60 min.

[0103] (4) Secondary antibody: Wash the plate 3 times with PBST, add the diluted secondary antibody, and incubate at room temperature for 60 min.

[0104] (5) Color development: Wash the plate 6 times with PBST and add 30 µL TMB to each well.

[0105] (6) Termination reading: Add 2 M stop solution to terminate the reaction and detect OD450, and perform data processing and analysis.

[0106] The experimental results are shown in Table 3-5.

[0107] Table 3 Results of antibody physicochemical property detection

[0108] Table 4. ELISA affinity assay results of antibody supernatant - EC50

[0109] Table 5. Antibody protein ELISA affinity assay results - EC50

[0110] Note: Y indicates that there is binding, but the EC50 value cannot be calculated. EC50 is measured in ug / mL. 2.4 FACS Binding Detection (1) Cell plating: Transfer cells from the culture flask to centrifuge tubes, centrifuge to remove the supernatant, resuspend in culture medium, count the cells, and adjust the cell density to 1×10⁶ cells / mL. Take a 96-well round-bottom plate and add 100 μL of cells to each well using a 100 μL pipette. Centrifuge at 500 g / min for 3 min. Discard the supernatant.

[0111] (2) Addition of antibody dilution buffer: Dilute the antibody to the appropriate concentration with FACS Buffer containing 2% FBS. Add 100 μL of antibody dilution buffer to each well of a 96-well cell plate using a 12-line pipette. Mix well and incubate at 4°C for 1 h.

[0112] (3) Addition of secondary antibody: The secondary antibody anti-Human IgG, Fcγ PE (Jackson) was diluted 1:200 with FACS Buffer containing 2% FBS. 100 μL of the secondary antibody dilution solution was added to the cell culture plate using a 100 μL 12-line pipette. The cell culture plate was placed in a refrigerator at 4℃ and incubated for 30 min. The supernatant was removed by centrifugation, and the plate was washed twice with FACS Buffer. The cells were resuspended in 120 μL of FACS Buffer in each well.

[0113] (4) Data acquisition: Turn on the flow cytometer and after the instrument is cleaned, perform FACS binding detection. After the detection is completed, collect the detection results as shown in Table 6 and Table 7.

[0114] Table 6 Results of FAC affinity assay in antibody supernatant

[0115] Table 7. Results of FACS affinity assay for antibody proteins (unit: nM)

[0116] Note: N represents no binding.

[0117] 2.5 Affinity kinetics detection (1) The SPR detection instrument was a Biacore T200 (Cytiva). The protein A chip was used to capture the stationary phase onto the chip, and the mobile phase was a gradient diluted analyte solution.

[0118] (2) The parameter settings are shown in Table 8.

[0119] Table 8 SPR Detection Parameter Settings

[0120] (3) Conduct the test and collect the test results, as shown in Table 9.

[0121] Table 9 Affinity kinetics test results

[0122] The FAP antibody provided in this application, after expression and purification, exhibits high protein purity, low aggregation rate, and stable biological activity, making it suitable for large-scale industrial production. Verification using various characterization methods, including SDS-PAGE, SEC-HPLC, and ELISA affinity assays, confirms that the antibody has uniform molecular weight, high monomer purity, low non-specific adsorption, and excellent antigen-binding activity.

[0123] Example 3: In vitro cell killing study of DOPE-LD201t1-encapsulated FAP CAR mRNA nanoparticles (1) Preparation and performance study of DOPE-LD201t1-encapsulated FAP CAR mRNA nanoparticles (DOR NPs) First, activated DOPE was prepared using the method described in Chinese patent CN120249289B. Thiol-modified nucleic acid aptamer LD201t1 (SPDP:LD201t1 molar ratio 1:1) was added to the activated DOPE, and the mixture was reacted with shaking at room temperature for 24 h. After dialysis purification, DOPE-LD201t1 lipid aptamer molecules were obtained. The DOPE-LD201T1 lipid aptamer molecules were then mixed with cationic lipid materials (such as DOTAP) at a mass ratio of 3:1, and an appropriate amount of PBS buffer was added. Blank nanoparticles were prepared using the liposome film dispersion method. Subsequently, FAP CAR mRNA solution and blank nanoparticles were mixed using the ethanol dilution method at an N / P ratio of 6:1 and incubated at room temperature for 30 min to complete mRNA encapsulation, obtaining uniformly sized DOPE-LD201T1-encapsulated FAP CAR mRNA DOR NPs.

[0124] The particle size was determined using a Malvern particle size analyzer (electron microscopy image shown). Figure 1 As shown in the figure, the average particle size of the nanoparticles is 142.3±3.15 nm, and the dispersibility is 0.11±0.07. Agarose gel electrophoresis results show that the mRNA encapsulation efficiency is 52.7%±2.4%, which meets the requirements for in vivo delivery.

[0125] (2) In vitro cell killing study of DOPE-LD201t1-encapsulated FAP CAR mRNA nanoparticles Following the above method, DOR NPs carrying mRNA were prepared using molecular self-assembly technology at a mass ratio (DOPE-LD201t1:FAP CAR mRNA = 2:1). These NPs were then transfected into T cells and combined with FAP CAR mRNA. + Fibroblasts were co-incubated. The in vitro efficacy of DOPE-LD201t1 nanoparticles loaded with FAP CAR mRNA against FAP was evaluated using an LDH cytotoxicity assay kit. + The killing effect on fibroblasts.

[0126] The experiment was conducted in four groups: a PBS control group (untreated T cells), a DO NPs group (nanoparticles without mRNA loading), a Naked mRNA group (free FAP CAR mRNA), and a DOR NPs group (nanoparticles loaded with FAP CAR mRNA prepared above). Tumor cells were cultured to the logarithmic growth phase and the density was adjusted to 6 × 10⁻⁶ cells / year. 4 After being seeded at / mL, cells were transferred to 96-well plates and co-cultured at effector-to-target ratios (E:T, CAR-T cells: fibroblasts) of 1:1, 1:2, 1:4, and 1:8. The killing efficiency was assessed by detecting LDH release after culture. Results are as follows: Figure 2 As shown.

[0127] Depend on Figure 2 It was found that, compared with the other three groups, the DOR NPs group significantly killed tumor cells. The DOR NPs group achieved the highest killing efficiency of 53.4% ​​± 2.8% at an effector-to-target ratio of 1:1, which was significantly higher than that of the free mRNA group (31.2% ± 1.6%) and the DOR NPs group (43.6% ± 3.1%), and also better than that of the PBS control group (33.5% ± 2.9%). As the effector-to-target ratio decreased from 1:1 to 1:8, the killing efficiency of the DOR NPs group showed a dose-dependent decrease, but it still maintained 30.8% ± 5.1% at an effector-to-target ratio of 1:8, which was 2.2 times that of the free mRNA group (13.9% ± 2.7%).

[0128] The above experimental results demonstrate that T cells reprogrammed with the FAP CAR mRNA of this invention have the ability to recognize and kill FAP+ cells in vitro at different effector-to-target ratios, with the best in vitro killing effect at an effector-to-target ratio of 1:1.

[0129] Example 4: Study on the therapeutic effect of DOPE-LD201T1 nanoparticles loaded with FAP CAR mRNA on pancreatic cancer mice and its impact on survival. 6-8 week old BALB / c nude mice were selected, and PANC-1 pancreatic cancer cells (5×10⁻⁶) were subcutaneously injected into the right side. 6 (each tumor cell / animal), until the tumor volume grows to approximately 100 mm.3 At that time, the tumors were randomly divided into 4 groups (n=6): PBS group (blank control group), Naked FAP CAR mRNA group (free FAP CAR mRNA), chemotherapy drug group (positive control group), and DOR NPs group (nanoparticles loaded with FAP CAR mRNA prepared in Example 3). The PBS group received the drug via tail vein at a dose of 10 mg / kg, once every 7 days for a total of 3 doses; the chemotherapy drug group received the drug via intraperitoneal injection at a clinically equivalent dose. The long diameter (L) and short diameter (W) of the tumor were measured every 2 days using calipers, and the tumor volume was calculated according to the formula. Tumor growth curves were plotted, and the results are shown in […]. Figure 3 The survival time of mice in each group was continuously recorded, survival curves were plotted, and the mean survival time was calculated. The results are shown in [Table missing]. Figure 4 .

[0130] According to the experimental results, compared with the PBS group, the Naked FAP CAR mRNA group, and the chemotherapy group, the tumor volume in the DOR NPs group showed a continuous decreasing trend, and tumor growth in some mice was completely inhibited. At the treatment endpoint, the average tumor inhibition rate in this group of mice reached 62.8% ± 3.7%, which was significantly better than other control groups (P < 0.01). The survival time of mice in the DOR NPs group was significantly prolonged, with a median survival time more than 45% longer than that in the PBS group, and the difference was statistically significant (P < 0.01).

[0131] This application provides an in vivo CAR-T application method for the aforementioned FAP antibody. A nucleic acid vector containing the antibody sequence is delivered via nanoparticles to express the CAR structure in situ within T cells, achieving immediate in vivo generation of functional CAR-T cells. This eliminates the need for in vitro cell sorting, expansion, and culture, simplifying the preparation process and reducing the risk of immune rejection and production costs. These CAR-T cells can precisely identify and eliminate tumor-associated fibroblasts, disrupt the dense stromal barrier of solid tumors, improve the tumor immune microenvironment, and simultaneously kill activated myofibroblasts within fibrotic lesions, inhibiting abnormal extracellular matrix deposition.

[0132] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. An antibody or antigen-binding fragment thereof targeting FAP, comprising a heavy chain variable region and a light chain variable region, characterized in that, The amino acid sequences of the complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region are shown in SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No: 3, respectively, and the amino acid sequences of the complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region are shown in SEQ ID No: 4, SEQ ID No: 5, and SEQ ID No: 6, respectively.

2. The FAP-targeting antibody or antigen-binding fragment thereof of claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 7; and / or the amino acid sequence of the light chain variable region is shown in SEQ ID No:

8.

3. The FAP-targeting antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising a heavy chain and a light chain, characterized in that, The amino acid sequence of the heavy chain is shown in SEQ ID No: 9; and / or the amino acid sequence of the light chain is shown in SEQ ID No:

10.

4. The FAP-targeting antibody or antigen-binding fragment thereof of claim 1 or 2, characterized in that, It is an scFv fragment containing a heavy chain variable region and a light chain variable region, the scFv fragment containing a linker peptide connecting the heavy chain variable region and the light chain variable region, and the amino acid sequence of the scFv fragment is shown in SEQ ID No:

11.

5. A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof targeting FAP as described in any one of claims 1 to 4.

6. A chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, characterized in that, The antigen-binding domain is the antibody targeting FAP or its antigen-binding fragment as described in claim 4.

7. A nucleic acid molecule encoding the chimeric antigen receptor (CAR) of claim 6.

8. The nucleic acid molecule according to claim 7, characterized in that, The nucleic acid molecule is an mRNA molecule.

9. An immune cell, characterized in that, The immune cells comprise the chimeric antigen receptor (CAR) as described in claim 6 or the nucleic acid molecule as described in claim 7 or 8, and the immune cells are T cells, NK cells or macrophages.

10. A nanoparticle, characterized in that, The nanoparticles are loaded with the nucleic acid molecules as described in claim 7 or 8.

11. The nanoparticles according to claim 10, characterized in that, The nanoparticles are constructed from lipid aptamers, which are composed of hydrophobic molecules and nucleic acid aptamers that target cell surface molecules linked by chemical bonds.

12. The nanoparticles according to claim 11, characterized in that, The hydrophobic molecules in the lipid aptamers are selected from 1,2-dioleoyl-SN-glycerol-3-phosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), 1,2-distearyl-sn-propyltriyl-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine (sodium salt) (DOPS), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), hydrogenated lecithin (HSPC), 1,2-distearyl-sn-propyltriyl-3-phosphatidylethanolamine (DSPE), 1,2-distearyl-sn-glycerol-3-phosphatidylserine (DSPG), and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylethanolamine. One or more of the following: amine (DPPE), 1,2-dilauroyl phosphatidylethanolamine (DLPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylglycerol (DPPG), monophosphatidyllipid A (MPLA), 1,3-dilinolenin, polycaprolactone (PCL), SM-102, ALC-0315, DLin-MC3-DMA, DLin-KC2-DMA, cKK-E12-TREM2, arachidonic acid, linolenic acid, L319, cholesterol, DOTAP, DDAB, POPC, POPE, DPPE, DOPC, β-sitosterol, cholesterol, cholesterolone, palmitic acid, stearic acid, myristic acid, and arachidic acid.

13. The nanoparticles according to claim 11, characterized in that, The nucleic acid aptamer is a nucleic acid aptamer that targets T cell surface molecules OX40, 4-1BB, CD5 or CD62L, or a nucleic acid aptamer that targets tumor-associated macrophage surface molecules CD32, MerTK, TLR4, LOX-1, MHC-II, CD68, A2, CD206, Dectin-1, CD163 or TREM2, or a nucleic acid aptamer that targets NK cell surface molecules CD16a or CD56.

14. The nanoparticles according to claim 11, characterized in that, The nucleic acid aptamer is OSJ-T3, CD28Apt7, C2NP, or WXY3.

15. The nanoparticles according to claim 11, characterized in that, The hydrophobic molecule in the lipid aptamer is 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE), and the nucleic acid aptamer is a nucleic acid aptamer that targets the cell surface molecule CD62L.

16. A pharmaceutical composition, characterized in that, It comprises an antibody or antigen-binding fragment thereof targeting FAP as described in any one of claims 1-4, an immune cell as described in claim 9, or nanoparticles as described in any one of claims 10-15, and a pharmaceutically acceptable carrier or excipient.

17. Use of the nanoparticles according to any one of claims 10 to 15 in the preparation of a medicament for generating chimeric antigen receptor immune cells in vivo.

18. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 4, or the immune cell of claim 9, or the nanoparticle of any one of claims 10 to 15 in the preparation of a medicament for treating pancreatic cancer.

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