A nanobody that binds to Trop2, a chimeric antigen receptor, and their applications

Abstract

The present invention relates to the technical field of antibodies, and particularly relates to a nanobody that binds to Trop2, a chimeric antigen receptor, and their applications. The nanobody that binds to Trop2 provided by the present invention can efficiently and specifically recognize and bind to the Trop2 protein, and can be used for diagnosing and treating diseases with Trop2 molecule as a biomarker. The present invention also provides a CAR and CAR-T cells that bind to the Trop2 protein. The CAR-T cells have a good immune clearance effect targeting Trop2, can be used to specifically recognize and kill cells expressing Trop2, and are used to improve and treat diseases with Trop2 molecule as a biomarker, and have good application prospects in the diagnosis and treatment of tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and particularly relates to a nanobody that binds to Trop2, a chimeric antigen receptor, and their applications. Background Art

[0002] Trop2 (Trophoblast cell surface antigen 2) is fully named trophoblast cell surface antigen, and is a single-pass transmembrane cell surface glycoprotein encoded and expressed by the TACSTD2 (Tumor associated calcium signal transducer 2) gene. Trop2 was first discovered as a surface marker of trophoblast cells more than 40 years ago, and was rediscovered as tumor associated calcium signal transducer 2 (TACSTD2), membrane component chromosome 1 surface marker 1 (M1S1), gastrointestinal tumor associated antigen (Gastrointestinal antigen 733-1, GA733-1), and epithelial glycoprotein-1 (EGP-1) in the following years. Trop2 belongs to the GA733 protein family and has a relatively high structural sequence similarity with epithelial cell adhesion molecule (EpCAM, also known as Trop1, TACSTD1), with a homology of 49%. Studies have found that Trop2 usually shows the lowest or relatively low levels of expression in normal epithelial tissues, while it is significantly highly expressed in a variety of epithelial malignancies, including pancreatic cancer, colorectal cancer, ovarian cancer, gastric cancer, breast cancer, prostate cancer, cervical cancer, head and neck cancer, etc. A large number of studies have clarified the key role of Trop2 in tumor proliferation, invasion, and metastatic spread, and its high expression is closely related to the shortening of the survival period and poor prognosis of tumor patients.

[0003] The high expression and mechanism characteristics of Trop2 in a variety of malignant tumors have attracted the attention of researchers in the field of tumor treatment. At present, the research and development of anti-tumor drugs targeting Trop2 mainly focuses on antibody-drug conjugates (ADCs). The clinical progress of Trop2 ADCs in the treatment of solid tumors highlights the potential of Trop2 as an immunotherapy tumor target. Compared with ADCs, the research on chimeric antigen receptor (CAR) modified immune cells targeting Trop2 is still in the early stage and lacks clinical progress, and there is still a need to develop effective chimeric antigen receptors targeting Trop2. Summary of the Invention

[0004] The present invention provides a nanobody that binds to Trop2, a chimeric antigen receptor, and their applications.

[0005] Specifically, the present invention provides the following technical solutions.

[0006] In a first aspect, the present invention provides a nanobody that binds to Trop2, wherein the nanobody comprises a heavy chain variable region, and the complementarity determining regions (CDRs) of the heavy chain variable region are any one of the following (1) to (6):

[0007] (1) When defined according to the IMGT numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs. 1, 2, and 3, respectively;

[0008] When defined according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs. 4, 5, and 6, respectively;

[0009] When defined according to the Chothia numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs. 7, 8, and 9, respectively;

[0010] When defined according to the Contact numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs. 10, 11, and 12, respectively;

[0011] (2) When defined according to the IMGT numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs. 13, 14, and 15, respectively;

[0012] When defined according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs. 16, 17, and 18, respectively;

[0013] When defined according to the Chothia numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs. 19, 20, and 21, respectively;

[0014] When defined according to the Contact numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs. 22, 23, and 24, respectively;

[0015] (3) When defined according to the IMGT numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs. 25, 26, and 27, respectively;

[0016] When defined according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 28, 29, and 30, respectively;

[0017] When defined according to the Chothia numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 31, 32, and 33, respectively;

[0018] When defined according to the Contact numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 34, 35, and 36, respectively;

[0019] (4) When defined according to the IMGT numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 37, 38, and 39, respectively;

[0020] When defined according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 40, 41, and 42, respectively;

[0021] When defined according to the Chothia numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 43, 44, and 42, respectively;

[0022] When defined according to the Contact numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 45, 46, and 47, respectively;

[0023] (5) When defined according to the IMGT numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 37, 38, and 48, respectively;

[0024] When defined according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 40, 41, and 49, respectively;

[0025] When defined according to the Chothia numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 43, 44, and 49, respectively;

[0026] When defined according to the Contact numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 45, 46, and 50, respectively;

[0027] (6) When defined according to the IMGT numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 37, 51, and 39 respectively;

[0028] When defined according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 40, 52, and 42 respectively;

[0029] When defined according to the Chothia numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 43, 53, and 42 respectively;

[0030] When defined according to the Contact numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 45, 54, and 47 respectively.

[0031] The heavy chain variable region of the nanobody described above further includes a framework region FR, and the framework region FR is any one of the following (1) to (11):

[0032] (1) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO. 55, 56, 57, and 58 respectively;

[0033] When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO. 59, 60, 61, and 58 respectively;

[0034] When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO. 55, 62, 63, and 58 respectively;

[0035] When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO. 64, 65, 66, and 67 respectively;

[0036] (2) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO. 55, 68, 69, and 58 respectively;

[0037] When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO. 70, 71, 72, and 58 respectively;

[0038] When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO. 55, 73, 74, 58 respectively;

[0039] When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO. 75, 76, 77, 67 respectively;

[0040] (3) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO.78, 79, 80, 81 respectively;

[0041] When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO. 82, 83, 84, 81 respectively;

[0042] When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO. 78, 85, 86, 81 respectively;

[0043] When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO. 87, 88, 89, 90 respectively;

[0044] (4) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO.91, 92, 93, 81 respectively;

[0045] When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO. 94, 95, 96, 81 respectively;

[0046] When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO. 91, 97, 98, 81 respectively;

[0047] When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO. 99, 100, 101, 90 respectively;

[0048] (5) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO.78, 92, 93, 81 respectively;

[0049] When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown as SEQ ID NO. 102, 95, 96, and 81, respectively;

[0050] When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown as SEQ ID NO. 78, 97, 98, and 81, respectively;

[0051] When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown as SEQ ID NO. 103, 100, 101, and 90, respectively;

[0052] (6) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown as SEQ ID NO.78, 92, 104, and 81, respectively;

[0053] When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown as SEQ ID NO. 102, 95, 105, and 81, respectively;

[0054] When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown as SEQ ID NO. 78, 97, 104, and 81, respectively;

[0055] When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown as SEQ ID NO. 103, 100, 106, and 90, respectively;

[0056] (7) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown as SEQ ID NO.78, 92, 107, and 81, respectively;

[0057] When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown as SEQ ID NO. 102, 95, 108, and 81, respectively;

[0058] When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown as SEQ ID NO. 78, 97, 109, and 81, respectively;

[0059] When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown as SEQ ID NO. 103, 100, 110, 90 respectively;

[0060] (8)When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown as SEQ ID NO.78, 92, 111, 81 respectively;

[0061] When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown as SEQ ID NO. 102, 95, 112, 81 respectively;

[0062] When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown as SEQ ID NO. 78, 97, 113, 81 respectively;

[0063] When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown as SEQ ID NO. 103, 114, 115, 90 respectively;

[0064] (9)When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown as SEQ ID NO.78, 92, 116, 81 respectively;

[0065] When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown as SEQ ID NO. 102, 95, 117, 81 respectively;

[0066] When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown as SEQ ID NO. 78, 97, 118, 81 respectively;

[0067] When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown as SEQ ID NO. 103, 114, 119, 90 respectively;

[0068] (10)When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown as SEQ ID NO.78, 92, 120, 81 respectively;

[0069] When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3, and FR4 are shown as SEQ ID NO. 102, 95, 121, 81 respectively;

[0070] When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO. 78, 97, 122, 81 respectively;

[0071] When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO. 103, 114, 123, 90 respectively;

[0072] (11) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO.78, 124, 125, 81 respectively;

[0073] When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO. 102, 126, 127, 81 respectively;

[0074] When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO. 78, 128, 129, 81 respectively;

[0075] When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO. 103, 76, 130, 90 respectively.

[0076] Preferably, the amino acid sequence of the heavy chain variable region of the nanobody is shown in SEQ ID NO.131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 or 142, or has at least 80% similarity with the sequence shown in SEQ ID NO.131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 or 142.

[0077] The above-mentioned sequence similarity is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 98.5%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, more preferably at least 99.9%.

[0078] In a second aspect, the present invention provides an antibody that binds to Trop2, and the antibody is a monovalent antibody, bispecific antibody, or multispecific antibody comprising one or more of the above-described nanobodies that bind to Trop2.

[0079] When the antibody that binds to Trop2 comprises two or more of the above-described nanobodies that bind to Trop2, the nanobodies can be linked by a linker peptide. The linker peptide is preferably a flexible peptide segment rich in glycine and serine.

[0080] In a third aspect, the present invention provides a fusion protein, which is obtained by fusing the above-described nanobody that binds to Trop2 with Fc.

[0081] Wherein, the Fc fragment includes the Fc fragment of human IgG and can be selected from the Fc fragments of IgG1, IgG2, IgG3, and IgG4.

[0082] In a fourth aspect, the present invention provides a chimeric antigen receptor (CAR) targeting TROP2, and the chimeric antigen receptor comprises the above-described nanobody that binds to Trop2 or the above-described antibody that binds to Trop2.

[0083] In the present invention, the chimeric antigen receptor can adopt the structure of the currently known chimeric antigen receptor.

[0084] Preferably, the chimeric antigen receptor comprises a signal peptide, the nanobody that binds to Trop2 or the antibody that binds to Trop2, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and an intracellular signal transduction domain.

[0085] Preferably, from the N-terminus to the C-terminus, the chimeric antigen receptor sequentially comprises a signal peptide, the nanobody that binds to Trop2 or the antibody that binds to Trop2, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and an intracellular signal transduction domain.

[0086] In the present invention, the signal peptide, hinge region, transmembrane region, intracellular co-stimulatory domain, and intracellular signal transduction domain can all be selected from the structural sequences of known CARs.

[0087] In some embodiments of the present invention, the CAR sequentially comprises a CD8α leader membrane receptor signal peptide, a (G4S)3 linker peptide, the nanobody that binds to Trop2 or the antibody that binds to Trop2, a CD8α Hinge hinge region, a CD8α TM transmembrane region, a CD28 co-stimulatory domain, and a CD3ζ intracellular signal transduction domain.

[0088] In a fifth aspect, the present invention provides a nucleic acid molecule encoding the Trop2-binding nanobody or the Trop2-binding antibody or the fusion protein or the chimeric antigen receptor.

[0089] Based on the amino acid sequences of the nanobody, antibody, fusion protein, and chimeric antigen receptor provided by the present invention, those skilled in the art can obtain the nucleotide sequences of the nucleic acid molecules encoding the above-mentioned nanobody, antibody, fusion protein, and chimeric antigen receptor. Due to the degeneracy of codons, the nucleotide sequences of nucleic acid molecules encoding an amino acid sequence are not unique, and all nucleic acid molecules capable of encoding and producing the above-mentioned nanobody, antibody, fusion protein, and chimeric antigen receptor are within the protection scope of the present invention.

[0090] In some specific embodiments of the present invention, the sequences of the nucleic acid molecules encoding the heavy chain variable regions of the nanobody are as shown in SEQ ID NO.143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, or 154.

[0091] In a sixth aspect, the present invention provides a biomaterial comprising the nucleic acid molecule; the biomaterial is an expression cassette, a vector, or a host cell.

[0092] The above-mentioned expression cassette can be obtained by connecting transcriptional or translational regulatory elements such as a promoter upstream of the nucleic acid molecule and / or connecting transcriptional or translational regulatory elements such as a terminator downstream thereof.

[0093] The above-mentioned vectors include but are not limited to plasmid vectors, lipid nanoparticle vectors, viral vectors, transposons, etc.

[0094] Among them, the viral vectors include lentiviral vectors, adenoviral vectors, AAV viral vectors, retroviral vectors, etc.

[0095] The above-mentioned host cells include prokaryotic cells or eukaryotic cells. Among them, prokaryotic cells include but are not limited to Escherichia coli. Eukaryotic cells include but are not limited to yeast, insect cells, or other mammalian cells. Yeast includes but is not limited to Pichia pastoris, Saccharomyces cerevisiae, etc.; mammalian cells include but are not limited to immune cells (such as T cells, NK cells, etc.), stem cells or progenitor cells capable of differentiating into immune cells, Chinese hamster ovary cells (CHO), baby hamster kidney cells (BHK), mouse myeloma cells (SP0 / 2), African green monkey kidney cells (Vero), and human embryonic kidney 293 cells (HEK293), etc.

[0096] In a seventh aspect, the present invention provides a recombinant cell expressing the chimeric antigen receptor described above.

[0097] Preferably, the recombinant cell is an immune cell expressing the chimeric antigen receptor described above.

[0098] Preferably, the immune cell is a T cell or an NK cell.

[0099] In an eighth aspect, the present invention provides an antibody conjugate, which is obtained by conjugating the nanobody binding to Trop2, or the antibody binding to Trop2, or the fusion protein, or the chimeric antigen receptor with a detectable label, a drug, a toxin or a cytokine.

[0100] The above-mentioned labels are selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, colloidal gold labeling, and radioactive labeling.

[0101] The above-mentioned drug is a cytotoxic drug. The cytotoxic drugs include, but are not limited to, DNA replication inhibitors, topoisomerase inhibitors, alkylating agents, antibiotics, anti-tubulin drugs, folic acid antagonists, etc.

[0102] The above-mentioned toxins include, but are not limited to, ricin, paclitaxel, cisplatin, vincristine, vinblastine, colchicine, actinomycin, diphtheria toxin, abrin, etc.

[0103] In a ninth aspect, the present invention provides a method for producing the nanobody binding to Trop2, or the antibody binding to Trop2, or the fusion protein, or the chimeric antigen receptor, the method comprising: culturing a host cell containing the nucleic acid molecule, and collecting the nanobody binding to Trop2, or the antibody binding to Trop2, or the fusion protein, or the chimeric antigen receptor from the culture.

[0104] Among them, collecting from the culture includes steps such as separation and purification.

[0105] In a tenth aspect, the present invention provides any one of the following applications of the nanobody binding to Trop2, or the antibody binding to Trop2, or the fusion protein, or the chimeric antigen receptor, or the nucleic acid molecule, or the biological material, or the recombinant cell, or the antibody conjugate:

[0106] (1) Application in the preparation of a drug for preventing or treating a disease related to Trop2 expression;

[0107] (2) Application in the preparation of a CAR-T drug;

[0108] (3) Application in the preparation of a reagent for detecting the presence or level of Trop2 in a sample;

[0109] (4) Application in the preparation of a tumor detection reagent.

[0110] In the above (1), the disease related to Trop2 expression is preferably a disease with Trop2 molecule as a marker, preferably a tumor, more preferably a malignant tumor; preferably a tumor with high expression of TROP2, especially an epithelial malignant tumor with high expression of TROP2. The malignant tumors include at least one selected from breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, prostate cancer, cervical cancer, head and neck cancer, lung cancer, oral cancer, pharyngeal cancer, esophageal cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, glioma, glioblastoma, thyroid cancer, liver cancer, kidney cancer, urothelial cancer, skin cancer, melanoma, penile cancer, etc.

[0111] In the above (4), the tumor is a tumor expressing Trop2, preferably an epithelial malignant tumor with high expression of TROP2. The malignant tumors include at least one selected from breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, prostate cancer, cervical cancer, head and neck cancer, lung cancer, oral cancer, pharyngeal cancer, esophageal cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, glioma, glioblastoma, thyroid cancer, liver cancer, kidney cancer, urothelial cancer, skin cancer, melanoma, penile cancer, etc.

[0112] In the eleventh aspect, the present invention provides a pharmaceutical composition, which comprises the nanobody binding to Trop2 or the antibody binding to Trop2 or the fusion protein or the chimeric antigen receptor or the recombinant cell or the antibody conjugate.

[0113] Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0114] Preferably, the pharmaceutical composition is used for treating a disease related to Trop2 expression.

[0115] In the twelfth aspect, the present invention provides a detection reagent, which comprises the nanobody binding to Trop2 or the antibody binding to Trop2 or the fusion protein or the antibody conjugate.

[0116] The above-mentioned detection reagents include, but are not limited to, ELISA detection reagents or kits, chemiluminescence detection reagents or kits, radioimmunoassay detection reagents or kits, fluorescence immunoassay detection reagents or kits, etc.

[0117] In the thirteenth aspect, the present invention provides a method for treating a disease related to Trop2 expression, the method comprising: administering to a subject a therapeutically effective amount of the nanobody binding to Trop2 or the antibody binding to Trop2 or the fusion protein or the chimeric antigen receptor or the antibody conjugate.

[0118] In a fourteenth aspect, the present invention provides a method for detecting Trop2, the method comprising: contacting the nanobody that binds Trop2, or the antibody that binds Trop2, or the fusion protein, or the antibody conjugate with a sample to be tested, and determining the presence or level of Trop2 in the sample to be tested.

[0119] The beneficial effects of the present invention at least include: The antibody that binds Trop2 provided by the present invention is derived from alpaca and only contains one VHH, belonging to a nanobody, which is the antibody structure with the smallest protein molecular weight known at present; compared with traditional monoclonal antibodies and single-chain variable fragments (scFv) of antibodies, nanobodies have the advantages of small molecular weight, not easily aggregating and precipitating, and high stability, and while maintaining a high affinity for antigens, the long and flexible complementarity-determining region CDR3 of nanobodies allows binding to the cracks and cavities of target antigens, and can recognize some hidden epitopes. The anti-Trop2 nanobody provided by the present invention can efficiently and specifically recognize and bind to the Trop2 protein. Based on this nanobody, the present invention also provides drugs prepared therefrom, including but not limited to bispecific antibodies, antibody-drug conjugates, chimeric antigen receptors, oncolytic viruses, which can be used to specifically recognize or kill cells expressing Trop2, such as Trop2-positive cells in various malignant tumors such as breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, and ovarian cancer, for diagnosing and treating diseases with Trop2 molecules as markers.

[0120] The present invention also provides a CAR and CAR-T cells that bind to the Trop2 protein. Verified by cell-level experiments, the CAR-T cells have a good immune clearance effect targeting Trop2 and can be used to specifically recognize and kill cells expressing Trop2, such as Trop2-positive cells in various malignant tumors such as breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, and ovarian cancer, for improving and treating diseases with Trop2 molecules as markers.

[0121] The anti-Trop2 CAR of the present invention uses a nanobody that binds to the Trop2 protein as a specific recognition structural component of the antigen. The nanobody has the advantage of small molecular weight, which is conducive to recombinant design to produce multispecific nanobodies and multispecific CARs, improving the recognition specificity for the target antigen to avoid the on-target, off-tumor effect, and improving the safety of CAR in tumor immunotherapy to obtain a wider range of therapeutic application functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] To more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0123] Figure 1 OD reading results of screening 12 anti-human Trop2 VHHs in the alpaca immune antibody library by phage enzyme-linked immunosorbent assay (Phage ELISA) in Example 1 of the present invention 450 Reading results.

[0124] Figure 2 Flow cytometry identification of the expression of the target antigen Trop2 in human pancreatic-related cell lines in Example 2 of the present invention

[0125] Figure 3 Schematic diagram of the molecular structure of anti-Trop2 CAR in Example 3 of the present invention

[0126] Figure 4 Flow cytometry detection of the positive rate of anti-Trop2 CAR (T2-F09) transduced double reporter cell line J-NN in Example 4 of the present invention, where NC represents the negative control.

[0127] Figure 5 Detection of the downstream signal activation level of anti-Trop2 CAR J-NN under the stimulation of different target cells and comparison of the peak values of GFP and mCherry signal activation in Example 4 of the present invention. Among them, a and b are the changes in the green fluorescence and red fluorescence signal intensities of anti-Trop2 CAR J-NN when co-cultured with Trop2-positive target cell CFPAC-1 and Trop2-negative target cell AsPC-1, respectively. c and d are the green fluorescence signal intensities and red fluorescence signal intensities of J-NN without transduced CAR and anti-Trop2 CAR J-NN when co-cultured with four target cells for 12 hours. a.u. represents arbitrary unit, an arbitrary unit.

[0128] Figure 6 Comparison of the downstream signal activation levels of 12 anti-Trop2 CAR J-NNs under the stimulation of positive target cell BxPC-3 in Example 4 of the present invention. Among them, a and b are the green fluorescence signal intensities and red fluorescence signal intensities of J-NN without transduced CAR and 12 anti-Trop2 CAR J-NNs when co-cultured with BxPC-3 for 12 hours, respectively.

[0129] Figure 7To detect the positive rate of flow cytometry for anti-Trop2 CAR (T2-F09) transduced human primary T cells in Example 5 of the present invention, where NC represents the negative control.

[0130] Figure 8 It shows the in vitro killing kinetics results of anti-Trop2 CAR-T (T2-F09) cells against different target cells in Example 6 of the present invention. Among them, a and c are the changes in green fluorescence signal intensity (normalized) of anti-Trop2 CAR-T during co-culture with Trop2-positive target cells BxPC-3-GFP and Trop2-negative target cells hTERT-HPNE-GFP, respectively. b and d are the changes in green fluorescence signal intensity (normalized) of negative control T during co-culture with Trop2-positive target cells BxPC-3-GFP and Trop2-negative target cells hTERT-HPNE-GFP, respectively.

[0131] Figure 9 It shows the statistical results of the in vitro killing efficiency of anti-Trop2 CAR-T (T2-F09) cells against different target cells in Example 6 of the present invention. Among them, a and b are the in vitro killing efficiencies of anti-Trop2 CAR-T and control T after co-culturing with Trop2-positive target cells BxPC-3-GFP (a) and CFPAC-1-GFP (b) for 72 h, respectively; c and d are the in vitro killing efficiencies of anti-Trop2 CAR-T and control T after co-culturing with Trop2-negative target cells AsPC-1-GFP (c) and hTERT-HPNE-GFP (d) for 72 h, respectively.

[0132] Figure 10 It shows the comparison of the in vitro killing efficiencies of 12 anti-Trop2 CAR-Ts against the positive target cell BxPC-3-GFP under three effector-to-target ratios in Example 6 of the present invention.

[0133] Figure 11 It shows the cytokine secretion results of anti-Trop2 CAR-T (T2-F09) cells under stimulation by different target cells in Example 7 of the present invention. Among them, a: IFNγ secretion level, b: TNFα secretion level, c: IL-2 secretion level.

[0134] Figure 12 It shows the in vitro killing effect of anti-Trop2 CAR-T (T2-F09) cells on 3D tumor spheres in Example 8 of the present invention. Among them, a: the shooting results of the live cell imager Incucyte S3, b: the in vitro killing kinetics results of anti-Trop2 CAR-T cells on tumor spheres. Detailed implementation methods

[0135] In a specific embodiment of the present invention, an antibody that binds to Trop2 protein is provided. It is a small-molecular-weight antibody derived from alpaca and is obtained by screening from the immune library of alpaca through phage display technology. First, alpaca is immunized with purified Trop2 protein. After successful immunization, peripheral blood of alpaca is collected to extract lymphocytes containing mature B cells. RNA is extracted and reverse-transcribed into cDNA library. Specific gene fragments are obtained by PCR amplification of the variable region of the heavy chain of alpaca antibody using primers, and then spliced onto a phage vector and transformed into Escherichia coli to obtain a nanobody gene library, that is, an immune library. Through phage display technology, after 4 rounds of panning, positive clones are identified by indirect ELISA, and the antibody sequences that bind to Trop2 antigen are confirmed by sequencing.

[0136] The antibody that binds to Trop2 protein obtained above contains the variable domain of the heavy chain of the heavy-chain antibody (VHH). The amino acid sequences of the VHH of each antibody are shown in Table 1. The amino acid sequence of each antibody is numbered according to the Martin numbering scheme of the antibody numbering scheme and defined by different CDR region definition schemes to obtain the CDR1, CDR2, CDR3 regions and the framework regions FR1, FR2, FR3, FR4 of the antibody. Among them, the amino acid sequences of the CDR regions are shown in Table 2, and the amino acid sequences of FR1, FR2, FR3, FR4 are shown in Table 3.

[0137] Table 1

[0138]

[0139] Table 2

[0140]

[0141] Table 3

[0142]

[0143] The antibody that binds to Trop2 protein described above can be used to specifically recognize or kill cells expressing Trop2, such as Trop2-positive cells in various malignant tumors such as breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, and ovarian cancer, and is used for diagnosing and treating diseases with Trop2 molecule as a biomarker.

[0144] In a specific embodiment of the present invention, a chimeric antigen receptor (CAR) that binds to the Trop2 protein, as well as an expression vector and a host cell containing the chimeric antigen receptor, are also provided. The CAR that binds to the Trop2 protein uses a nanobody that binds to the Trop2 protein as a specific recognition structural component of the antigen, that is, the antibody that binds to the Trop2 protein described above. The molecular structure of the anti-Trop2 CAR is SP-anti-Trop2 VHH-HINGE-TM-CD-SD, where SP is a signal peptide, HINGE is a hinge region, TM is a transmembrane region, CD is a co-stimulatory domain, SD is a signal transduction domain, and "-" is a linker peptide or peptide bond.

[0145] In the present invention, SP, HINGE, TM, CD, and SD can all be selected from known sequences in the field of CAR technology. In a specific embodiment, each part of the CAR molecular composition is a CD8α leader membrane receptor signal peptide, a (G4S)3 linker peptide, an anti-Trop2 VHH, a CD8α Hinge hinge region, a CD8α TM transmembrane region, a CD28 co-stimulatory domain, and a CD3ζ intracellular signal transduction domain in series.

[0146] The chimeric antigen receptor that binds to the Trop2 protein described above uses a nanobody that binds to the Trop2 protein as a specific recognition structural component of the antigen. The chimeric antigen receptor is transduced into an immune effector host cell using a vector such as a lentivirus, and can specifically recognize cells expressing Trop2. After being stimulated by the antigen, various pro-apoptotic cytokines such as granzyme, perforin, and interferon-γ can be released, causing the death of target cells and exerting an immune clearance function. Therefore, the chimeric antigen receptor that binds to the Trop2 protein of the present invention can be used to specifically recognize and kill cells expressing Trop2, such as Trop2-positive cells in various malignant tumors such as breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, and ovarian cancer, for improving and treating diseases with Trop2 molecules as markers.

[0147] In a specific embodiment of the present invention, an expression vector is also provided. The expression vector contains a nucleic acid molecule encoding the chimeric antigen receptor or antibody, and can be a plasmid vector, a lipid nanoparticle vector, a viral vector, including but not limited to a lentiviral vector and a retroviral vector.

[0148] In a specific embodiment of the present invention, host cells are also provided, and the host cells are immune cells or stem cells or progenitor cells that can differentiate into immune cells. Immune cells include, but are not limited to, T cells, NK cells, NKT cells, macrophages, dendritic cells, which have functions of killing or phagocytosis, immune regulation, and releasing cytokines. The stem cells can be hematopoietic stem cells or induced pluripotent stem cells, and the stem cells can differentiate into one or more of the above-mentioned immune cells.

[0149] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the present invention. Obviously, the described embodiments are some, rather than all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0150] Example 1: Screening of Nanobodies Binding to Trop2 Protein

[0151] The extracellular domain protein of human Trop2 (Human TROP-2 / TACSTD2 Protein, His Tag, ACRO, product number TR2-H5223) was used as an antigen to immunize healthy adult alpacas. After four antigen immunizations, with a 20-day interval between each immunization, the antibody titer of the alpaca peripheral blood serum was detected. Alpaca peripheral blood was collected and peripheral blood mononuclear cells (PBMCs) were extracted. RNA was extracted, reverse transcribed to obtain cDNA, and the heavy chain variable region was amplified by PCR to obtain the VHH gene fragment, which was then spliced onto a phage vector and transformed into Escherichia coli to obtain a nanobody gene library.

[0152] Biopanning of anti-Trop2 nanobodies using phage display technology. The specific steps are as follows: 1) Coating antigen: Coat the immunization tubes with TROP2 antigen (30 μg / tube), slowly rotate at 4°C overnight, and simultaneously coat 30 μg of 5% non-fat milk skimmed milk powder in parallel as a control; 2) Washing unbound antigen: Discard the coating solution, add 2 mL of PBS buffer, and wash the immunization tubes 3 times at room temperature, rotating for 5 minutes each time; 3) Blocking: Add 5 mL of blocking solution, rotate and block at room temperature for 2 hours; 4) Washing the blocking solution: Discard the liquid in the blocked immunization tubes, and add 5 mL of PBS buffer to wash the immunization tubes 3 times at room temperature, rotating for 5 minutes each time; 5) Adding phage display library for binding: Discard the washing solution in the immunization tubes, add 2 mL of PBS buffer, add the prepared phage library as the input phage library for the first round of screening, and incubate by rotating at room temperature for 1 hour; 6) Washing unbound phages: Discard the liquid in the immunization tubes, add 5 mL of PBST (1×PBS plus 0.1% Tween20) buffer, wash the immunization tubes 20 times at room temperature, rotating for 5 minutes each time, discard the liquid in the immunization tubes, and try to remove the residual liquid as much as possible; 7) Eluting bound phages: Add 1 mL of pH 2.2 Glycine-HCl solution, elute by rotating at room temperature for 30 minutes, add 90 μL of pH 8.8 Tris-HCl to terminate the elution, transfer the solution in the immunization tube to a new 1.5 mL centrifuge tube, which is the phage eluate for the first round of screening. 8) The phages in the first round are amplified and used for the subsequent second round of screening and detection. The above screening process is carried out for 3 rounds. The enrichment efficiency is calculated by plating phages in each round of screening, and monoclonal colonies are selected for antigen specificity detection.

[0153] Using phage ELISA to screen and identify positive monoclonal colonies. The specific steps are as follows: After enrichment of the phage eluate in each round, coat the ELISA plate with TROP2 protein antigen, use BSA as a control antigen, use the antibody displayed by the monoclonal phage selected and cultured as the antibody to be tested to bind the antigen, wash the unbound phages, use the anti-phage M13 antibody labeled with HRP as the secondary antibody to bind the phages, add the substrate for color development, read with an enzyme-linked immunosorbent assay reader, and initially determine the positive clones based on the OD 450 difference between the TROP2 test group and the BSA control group.

[0154] Select 95 positive clones shown by the phage ELISA results, send them to Tsingke Biotechnology Co., Ltd. for sequencing, identify the diversity of the positive clone sequences, convert the successfully sequenced sequences into amino acid sequences, align the sequences and merge the same amino acid sequences to obtain 12 positive antibody clones screened by Trop2 phages. The ELISA test results of the above 12 positive clones are as Figure 1As shown in the figure. The coding nucleotide sequences corresponding to the amino acid sequences of the positive antibody clones screened against 12 Trop2 phages were respectively synthesized, and each carried the coding nucleotide sequence of the amino acid tag / linker (GGGGS)×3, facilitating the detection of subsequent experiments. The 12 Trop2 VHH genes with tags were respectively synthesized.

[0155] Example 2: Flow cytometry detection of target cells with positive and negative Trop2 expression

[0156] The surface expression levels of Trop2 protein on the surfaces of four human pancreas-related cell lines were detected by surface staining with the Trop2 flow antibody (PE Mouse Anti-Human Trop-2, BD Biosciences, catalog number 564837). The four cell lines were three pancreatic cancer cells AsPC-1, BxPC-3, CFPAC-1 and an immortalized human normal pancreatic duct epithelial cell hTERT-HPNE. The detection steps were as follows: The cells to be tested were digested with trypsin, centrifuged, resuspended with medium and counted. 1×10 6 cells were taken from both the staining group and the blank control group, centrifuged to remove the supernatant, and washed once with pre-prepared FACS buffer (1×PBS + 2% FBS or 1×PBS + 0.5% BSA). The cells in the staining group were added with the Trop2 flow antibody diluted with FACS buffer and incubated at 4°C for 30 minutes. After centrifuging to remove the supernatant, the cells were washed three times with FACS buffer and resuspended with 200 μL for detection by flow cytometry.

[0157] The flow cytometry analysis results of the four cells expressing Trop2 are as Figure 2 shown. The pancreatic cancer cells BxPC-3 and CFPAC-1 highly expressed Trop2 and were used as Trop2-positive target cells in subsequent experiments; the pancreatic cancer cells AsPC-1 and the pancreatic duct epithelial cells hTERT-HPNE hardly expressed Trop2 and were used as Trop2-negative target cells in subsequent experiments.

[0158] Example 3: Plasmid construction of anti-Trop2 CAR and packaging of lentivirus

[0159] After the anti-Trop2 VHH nanobody sequence obtained in Example 1 was amplified by PCR, it was spliced with the CAR backbone synthesized by gene synthesis by seamless cloning and constructed into the PCDH lentiviral expression vector (the vector was modified and the promoter was EF1α). After verifying the accuracy of the plasmid sequence by sequencing, the lentiviral master plasmid expressing anti-Trop2 CAR was obtained. The molecular composition of the complete anti-Trop2 CAR is as Figure 3As shown, it includes a CD8α leader membrane receptor signal peptide, a (G4S)3 linker peptide, an anti-Trop2 VHH, a CD8α Hinge hinge region, a CD8α TM transmembrane region, a CD28 co-stimulatory domain, and a CD3ζ intracellular signal transduction domain, which are connected in series in sequence.

[0160] Lentivirus was prepared by the method of triple plasmid co-transfection of 293T cells. The transfection reagent was PEI MAX, that is, linearized polyethyleneimine PEI 40000 (Polyethylenimine Max, MW40000, Polysciences, catalog number 24765-1). The three plasmids included the lentivirus main plasmid expressing anti-Trop2 CAR and two packaging plasmids, psPAX2 and pMD2.G. 293T cells with good growth status were plated one day in advance, and transfection was carried out when the cell confluence was about 80%. The plasmids and the transfection reagent were diluted with serum-free medium. The mass ratio of the three plasmids was lentivirus main plasmid:psPAX2:pMD2.G = 3:2:1, and the ratio of the transfection reagent to the plasmid was 2.5 μL:1 μg. After mixing the diluted transfection reagent and the plasmid, it was allowed to stand at room temperature for 20 min, and then the transfection suspension was added to the 293T cells and gently shaken to mix. The medium was changed 8 h after transfection, and the culture supernatants were collected at 48 h and 72 h respectively, and the cell debris was removed by centrifugation. The supernatant was collected and mixed with the lentivirus concentrate (PEG8000 and NaCl), and then placed at 4°C overnight to concentrate the virus. Centrifuge at 4°C at 4000 g for 20 min, discard the supernatant after centrifugation, and resuspend the virus precipitate with an appropriate amount of PBS or medium to obtain the recombinant lentivirus vector containing anti-Trop2 CAR. The lentivirus titer was verified by a lentivirus titer rapid detection card (Biodragon, catalog number BF06202-100) to meet the requirements of subsequent experiments.

[0161] Example 4: Detection of the expression of anti-Trop2 CAR transduced double reporter cell line J-NN and activation of downstream signals

[0162] The double reporter cell line J-NN (derived from the laboratory of Wei Ping of the Chinese Academy of Sciences, see patent application CN115232216A) is a Jurkat double reporter signal cell line (abbreviated as J-NN) carrying pNFAT-EGFP and pNFκB-mCherry, which can quickly and high-throughput complete the identification of the activation signal characteristics of novel CAR molecules and is an experimental method for quickly screening novel CARs.

[0163] The anti-Trop2 CAR was transduced into J-NN cells using the lentiviral vector in Example 3 to construct anti-Trop2 CAR J-NN cells. Cells were collected 48 h after transduction. Referring to the flow cytometry staining method in Example 2, the positive rate of anti-Trop2 CAR J-NN cells was detected using the G4S antibody (Anti-(G4S)n-(B02H1)m-Ab(PE), Heyousheng Bio, product number GS-ARPE100). Taking T2-F09 as an example, as Figure 4 shown, the positive rate of anti-Trop2 CAR expression was above 50%.

[0164] The above anti-Trop2 CAR J-NN cells were co-cultured with the Trop2-positive target cells BxPC-3, CFPAC-1 and the Trop2-negative target cells AsPC-1, hTERT-HPNE, respectively. At the same time, a group of anti-Trop2 CAR J-NN cells without adding target cells and a control group of J-NN cells not transduced with CAR were set up. The target cells were plated in 96-well plates at a quantity of 5000 - 10000 cells per well one day in advance, and three parallel wells were set for each group. The next day, anti-Trop2 CAR J-NN cells and J-NN cells were added according to an effector-to-target ratio of 5:1. The Incucyte S3 live cell analysis system (Sartorius) was used to dynamically monitor the changes in the green fluorescence and red fluorescence intensities of J-NN cells. The imaging channels were selected as BF + GFP + RFP, the imaging interval was 2 h, and the imaging duration was 3 days. The results were as Figure 5 shown. Both of the two Trop2-positive target cells, BxPC-3 and CFPAC-1, could significantly activate the anti-Trop2 CAR J-NN cells to express the green fluorescence and red fluorescence reporter genes. No green fluorescence and red fluorescence signals were produced in the Trop2-negative target cell group, the group without adding target cells, and the J-NN cell control group, indicating that the activation of the two downstream signals of anti-Trop2 CAR J-NN cells depends on the specific binding of Trop2 antigen and anti-Trop2 CAR. Similarly, the downstream signal activation was detected after co-culturing 12 anti-Trop2 CAR J-NN cells with BxPC-3. The results were as Figure 6 shown. The anti-Trop2 CARs prepared from 12 clones of anti-Trop2 nanobodies were all able to bind to the Trop2 antigen and activate J-NN.

[0165] Example 5: Preparation of anti-Trop2 CAR-T cells

[0166] T cells were sorted from freshly isolated or revived human peripheral blood mononuclear cells (PBMCs) using the magnetic bead negative selection method (Pan T Cell Isolation Kit, Miltenyi, catalog number 130-096-535). CD3 / CD28 magnetic beads (Dynabeads Human T-activator CD3 / CD28, Thermo Fisher Scientific, catalog number 11132D) were added at a ratio of Bead:cell = 1:1 to activate the purified CD3 + T cells. The cells were cultured in serum-free T cell medium (X-VIVO 15, Lonza, catalog number 04-418Q) supplemented with IL-2 (Sihuan Bio, catalog number S10970015) at a final concentration of 200 IU / mL. After 24 h of magnetic bead activation culture, the anti-Trop2 CAR lentivirus from Example 3 and the transfection reagent polybrene (Merck, catalog number TR-1003-G) at a final concentration of 6 μg / mL were added. After 16 h of lentivirus infection, the medium was changed and the culture was continued.

[0167] After 4 days of activation culture, the magnetic beads were removed. After 72 h of lentivirus infection, the positive rate of anti-Trop2 CAR-T cells was detected using the G4S antibody (Anti-(G4S)n-(B02H1)m-Ab(PE), Heyousheng Bio, catalog number GS-ARPE100) according to the flow cytometry staining method in Example 2. Taking T2-F09 as an example, as Figure 7 shown, the positive expression rate of anti-Trop2 CAR-T was above 50%. The cells were continuously amplified in serum-free T cell medium containing 200 IU / mL IL-2 for subsequent CAR-T function detection.

[0168] Example 6: In vitro killing efficiency of anti-Trop2 CAR-T cells against different target cells

[0169] The above anti-Trop2 CAR-T cells were co-cultured with Trop2-positive target cells BxPC-3, CFPAC-1 and Trop2-negative target cells AsPC-1, hTERT-HPNE respectively. All four target cells carried GFP fluorescence. At the same time, a control group of T cells not transduced with CAR and a group of pure target cells without effector cells were set up. One day in advance, the target cells were plated in 96-well plates at a density of 2000 - 5000 cells per well, and three parallel wells were set for each group. The next day, anti-Trop2 CAR-T cells and T cells were added respectively according to the effector-to-target ratios of 1:2, 1:1, 2:1, and 4:1. The Incucyte S3 live cell analysis system (Sartorius) was used to dynamically monitor the change in the green fluorescence intensity of the target cells. The shooting channel was selected as BF+GFP, the shooting interval was 4 h, and the shooting duration was 3 days. The results are as Figure 8 shown. Compared with the T cell control group, the anti-Trop2 CAR-T cells had higher specific killing of the Trop2-positive target cell BxPC-3-GFP. The in vitro killing efficiency increased with the increase of the effector-to-target ratio and time, while there was no killing effect on the Trop2-negative target cell hTERT-HPNE-GFP.

[0170] The in vitro killing efficiency at 72 h was calculated according to the following formula: In vitro killing efficiency (Cytotoxicity) = (fluorescence intensity of the single-target group after normalization - fluorescence intensity of the experimental group after normalization) / fluorescence intensity of the single-target group after normalization × 100%. The results are as Figure 9 shown. Compared with the T cell control group, the anti-Trop2 CAR-T cells had higher specific killing of both Trop2-positive target cells BxPC-3-GFP and CFPAC-1-GFP, and showed an obvious dose effect. The in vitro killing efficiency increased with the increase of the effector-to-target ratio, while there was no killing effect on both Trop2-negative target cells AsPC-1-GFP and hTERT-HPNE-GFP. Similarly, the in vitro killing efficiency of 12 anti-Trop2 CAR-T cells against BxPC-3-GFP at 48 h was statistically analyzed, and the results are shown in Figure 10 , verifying that anti-Trop2 CAR-T had good specific killing effect on Trop2-positive target cells at the in vitro cell level.

[0171] Example 7: Detection of cytokine release of anti-Trop2 CAR-T cells

[0172] Plating was carried out according to the in vitro killing experiment with an effector-to-target ratio of 4:1 in Example 6. The culture medium for effector cells was T cell serum-free medium without IL-2. After co-culturing target cells and effector cells for 24 h, the supernatant was collected, and the secretion of three cytokines, IL-2 (Human IL-2 Precoated ELISA Kit, Dakewei, catalog number 1110202), IFNγ (Human IFN-γ Precoated ELISA Kit, Dakewei, catalog number 1110002), and TNFα (Human TNF-α Precoated ELISA Kit, Dakewei, catalog number 1117202), was detected by ELISA method. The results are as Figure 11 shown. Compared with the T cell control group, after co-incubating anti-Trop2 (T2-F09) CAR-T cells with two Trop2-positive target cells, BxPC-3-GFP and CFPAC-1-GFP, for 24 h, a large amount of IFNγ, TNFα, and IL-2 was released. While the cytokine levels in the two Trop2-negative target cell groups, AsPC-1-GFP and hTERT-HPNE-GFP, were at a relatively low level. It was verified that anti-Trop2 CAR-T could be stimulated and activated by Trop2-positive target cells, generating a series of cytokines related to immune activation.

[0173] Example 8: In vitro killing efficiency of anti-Trop2 CAR-T cells against tumor spheres

[0174] Trop2-positive target cells BxPC-3 with GFP fluorescence were plated in a 96-well clear round-bottom ultra-low attachment microplate (Corning, catalog number 7007) at a density of 3000 cells per well. Tumor spheres with a diameter of 200 - 500 μm could be observed 1 - 3 days after inoculation. Anti-Trop2 (T2-F09) CAR-T cells and T cells were added according to the effector-to-target ratios of 1:1, 2:1, and 4:1 respectively. The Incucyte S3 live cell analysis system (Sartorius) was used to dynamically monitor the change in the green fluorescence intensity of the tumor spheres. The shooting mode was selected as Spheroid, the shooting channel was selected as BF+GFP, the shooting interval was 4 h, and the shooting duration was 3 days. The results are as Figure 12 shown. Compared with the T cell control group, anti-Trop2 CAR-T cells could effectively eliminate the GFP fluorescence of the tumor spheres, achieving an in vitro killing effect on tumor cells at the three-dimensional level.

[0175] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nanobody that binds to Trop2, characterized in that, The nanobody comprises a heavy chain variable region, and the complementarity determining regions CDR of the heavy chain variable region are as follows: When defined according to the IMGT numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NO.37, 51, 39 respectively; When defined according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NO.40, 52, 42 respectively; When defined according to the Chothia numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NO.43, 53, 42 respectively; When defined according to the Contact numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NO.45, 54, 47 respectively.

2. The nanobody combined with Trop2 according to claim 1, characterized in that The heavy chain variable region further comprises a framework region FR, and the framework region FR is as follows: When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown as SEQ ID NO.78, 124, 125, 81 respectively; When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown as SEQ ID NO.102, 126, 127, 81 respectively; When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown as SEQ ID NO.78, 128, 129, 81 respectively; When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown as SEQ ID NO.103, 76, 130, 90 respectively.

3. The nanobody conjugated with Trop2 according to claim 1 or 2, characterized in that, The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.

142.

4. An antibody that binds to Trop2, characterized in that, The antibody is a monovalent antibody or a multispecific antibody comprising one or more nanobodies that bind to Trop2 as described in any one of claims 1 to 3.

5. A fusion protein, characterized in that, The fusion protein is obtained by fusing the nanobody that binds to Trop2 as described in any one of claims 1 to 3 with Fc.

6. A chimeric antigen receptor targeting Trop2, characterized in that, The chimeric antigen receptor comprises the nanobody that binds to Trop2 as described in any one of claims 1 to 3 or the antibody that binds to Trop2 as described in claim 4.

7. The chimeric antigen receptor according to claim 6, wherein The chimeric antigen receptor comprises a signal peptide, the nanobody that binds to Trop2 as described in any one of claims 1 to 3 or the antibody that binds to Trop2 as described in claim 4, a hinge region, a transmembrane region, an intracellular co-stimulatory domain and an intracellular signal transduction domain.

8. A nucleic acid molecule, characterized in that, It encodes the nanobody that binds to Trop2 as described in any one of claims 1 to 3, or the antibody that binds to Trop2 as described in claim 4, or the fusion protein as described in claim 5, or the chimeric antigen receptor as described in claim 6 or 7.

9. A biomaterial, characterized in that, The biological material comprises the nucleic acid molecule as described in claim 8; the biological material is an expression cassette, a vector or a host cell.

10. A recombinant cell, characterized in that, The recombinant cell expresses the chimeric antigen receptor as described in claim 6 or 7.

11. A method for generating a nanobody that binds to Trop2 according to any one of claims 1 to 3, or an antibody that binds to Trop2 according to claim 4, or a fusion protein according to claim 5, or a chimeric antigen receptor according to claim 6 or 7, characterized in that, The method includes: culturing a host cell containing the nucleic acid molecule described in claim 8, and collecting the Trop2-binding nanobody or the Trop2-binding antibody or the fusion protein or the chimeric antigen receptor from the culture.

12. The following applications of the Trop2-binding nanobody according to any one of claims 1 to 3, or the Trop2-binding antibody according to claim 4, or the fusion protein according to claim 5, or the chimeric antigen receptor according to claim 6 or 7, or the nucleic acid molecule according to claim 8, or the biological material according to claim 9, or the recombinant cell according to claim 10: (1) Application in the preparation of a drug for preventing or treating malignant tumors; (2) Application in the preparation of a reagent for detecting the presence or level of Trop2 in a sample; (3) Application in the preparation of a malignant tumor detection reagent; Among them, The malignant tumor is pancreatic cancer.

13. The application of the Trop2-binding nanobody according to any one of claims 1 to 3, or the Trop2-binding antibody according to claim 4, or the fusion protein according to claim 5, or the chimeric antigen receptor according to claim 6 or 7, or the nucleic acid molecule according to claim 8, or the biological material according to claim 9, or the recombinant cell according to claim 10 in the preparation of a CAR-T drug for treating malignant tumors; Among them, The malignant tumor is pancreatic cancer.

14. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the Trop2-binding nanobody according to any one of claims 1 to 3, or the Trop2-binding antibody according to claim 4, or the fusion protein according to claim 5, or the chimeric antigen receptor according to claim 6 or 7, or the recombinant cell according to claim 10.

15. A detection reagent, characterized in that, The detection reagent contains the Trop2-binding nanobody according to any one of claims 1 to 3, or the Trop2-binding antibody according to claim 4, or the fusion protein according to claim 5.

Images