Nanometer antibody combined with Trop2, chimeric antigen receptor and application of nanometer antibody and chimeric antigen receptor

Abstract

The invention relates to the technical field of antibodies, in particular to a Trop2 combined nano antibody, a chimeric antigen receptor and application of the Trop2 combined nano antibody and the chimeric antigen receptor. The Trop2-binding nano antibody provided by the invention can efficiently and specifically recognize and bind Trop2 protein, and can be used for diagnosing and treating diseases taking Trop2 molecules as markers. The invention further provides CAR and CAR-T cells which are combined with the Trop2 protein, and the CAR-T cells have a good Trop2-targeting immune clearance effect, can be used for specifically recognizing and killing cells expressing Trop2, can be used for improving and treating diseases with Trop2 molecules as markers and have a good application prospect in tumor diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular to a Trop2-binding nano antibody, a chimeric antigen receptor and applications thereof. Background Art

[0002] Trop2 (Trophoblast cell surface antigen 2) is the full name of trophoblast cell surface antigen. It 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. In the following years, it 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 (EpCAM, also known as Trop1, TACSTD1). Trop2 belongs to the GA733 protein family and has a 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 lower level of expression in normal epithelial tissues, but 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 elucidated the key role of Trop2 in tumor proliferation, invasion and metastasis, and its high expression is closely related to the shortened survival and poor prognosis of tumor patients.

[0003] Trop2 has attracted the attention of researchers in the field of tumor treatment due to its high expression and mechanism of action in a variety of malignant tumors. At present, the development of anti-tumor drugs targeting Trop2 is mainly antibody-drug conjugate (ADC). The clinical progress of Trop2 ADC in the treatment of solid tumors highlights the potential of Trop2 as a tumor target for immunotherapy. Compared with ADC, the research on chimeric antigen receptor (CAR)-modified immune cells targeting Trop2 is still in the early stages and lacks clinical progress. It is still necessary to develop effective chimeric antigen receptors targeting Trop2. Summary of the invention

[0004] The present invention provides a Trop2-binding nano antibody, a chimeric antigen receptor and applications thereof.

[0005] Specifically, the present invention provides the technical solution described below.

[0006] In a first aspect, the present invention provides a nanobody that binds to Trop2, wherein the nanobody comprises a heavy chain variable region, wherein the complementarity determining region (CDR) of the heavy chain variable region is any one of the following (1) to (6): (1) When defined according to the IMGT numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NOs. 1, 2 and 3, respectively; 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; When defined according to the Chothia numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NOs. 7, 8 and 9, respectively; When defined according to the Contact numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NOs. 10, 11 and 12, respectively; (2) When defined according to the IMGT numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NOs. 13, 14 and 15, respectively; 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; When defined according to the Chothia numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NOs. 19, 20 and 21, respectively; When defined according to the Contact numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NOs. 22, 23 and 24, respectively; (3) When defined according to the IMGT numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NOs. 25, 26 and 27, respectively; When defined according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 28, 29 and 30, respectively; When defined according to the Chothia numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown as SEQ ID NOs. 31, 32, and 33, respectively; When defined according to the Contact numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NOs. 34, 35 and 36, respectively; (4) When defined according to the IMGT numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NOs. 37, 38 and 39, respectively; When defined according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NOs. 40, 41 and 42, respectively; When defined according to the Chothia numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NOs. 43, 44 and 42, respectively; When defined according to the Contact numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NOs. 45, 46 and 47, respectively; (5) When defined according to the IMGT numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NOs. 37, 38 and 48, respectively; When defined according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NOs. 40, 41 and 49, respectively; When defined according to the Chothia numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NOs. 43, 44 and 49, respectively; When defined according to the Contact numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NOs. 45, 46 and 50, respectively; (6) When defined according to the IMGT numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NOs. 37, 51 and 39, respectively; When defined according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown as SEQ ID NOs. 40, 52, and 42, respectively; When defined according to the Chothia numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown as SEQ ID NOs. 43, 53, and 42, respectively; When defined according to the Contact numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NOs. 45, 54 and 47, respectively.

[0007] The heavy chain variable region of the Nanobody described above further comprises a framework region FR, wherein the framework region FR is any one of the following (1) to (11): (1) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 55, 56, 57 and 58, respectively; When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 59, 60, 61, and 58, respectively; When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 55, 62, 63, and 58, respectively; When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 64, 65, 66 and 67, respectively; (2) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 55, 68, 69 and 58, respectively; When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 70, 71, 72, 58, respectively; When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 55, 73, 74, 58, respectively; When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 75, 76, 77, and 67, respectively; (3) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 78, 79, 80 and 81, respectively; When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 82, 83, 84, and 81, respectively; When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 78, 85, 86, and 81, respectively; When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 87, 88, 89, and 90, respectively; (4) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 91, 92, 93 and 81, respectively; When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 94, 95, 96, and 81, respectively; When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 91, 97, 98 and 81, respectively; When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 99, 100, 101, and 90, respectively; (5) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 78, 92, 93 and 81, respectively; When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 102, 95, 96, and 81, respectively; When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 78, 97, 98 and 81, respectively; When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 103, 100, 101, 90, respectively; (6) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 78, 92, 104 and 81, respectively; When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 102, 95, 105, 81, respectively; When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 78, 97, 104, and 81, respectively; When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 103, 100, 106, 90, respectively; (7) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 78, 92, 107 and 81, respectively; When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 102, 95, 108, 81, respectively; When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 78, 97, 109 and 81, respectively; When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 103, 100, 110, 90, respectively; (8) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 78, 92, 111 and 81, respectively; When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 102, 95, 112, and 81, respectively; When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 78, 97, 113 and 81, respectively; When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 103, 114, 115, and 90, respectively; (9) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 78, 92, 116 and 81, respectively; When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 102, 95, 117, 81, respectively; When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 78, 97, 118 and 81, respectively; When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 103, 114, 119, 90, respectively; (10) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 78, 92, 120 and 81, respectively; When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 102, 95, 121, 81, respectively; When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 78, 97, 122, and 81, respectively; When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 103, 114, 123, and 90, respectively; (11) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 78, 124, 125 and 81, respectively; When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 102, 126, 127, and 81, respectively; When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 78, 128, 129 and 81, respectively; When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 103, 76, 130 and 90, respectively.

[0008] Preferably, the amino acid sequence of the heavy chain variable region of the Nanobody is as shown in SEQ ID NO.131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 or 142, or, has at least 80% similarity to the sequence shown in SEQ ID NO.131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 or 142.

[0009] The above 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%.

[0010] In a second aspect, the present invention provides an antibody that binds to Trop2, which is a monovalent antibody, a bispecific antibody or a multispecific antibody comprising one or more of the above-described nanobodies that bind to Trop2.

[0011] When the Trop2-binding antibody comprises two or more Trop2-binding Nanobodies, each Nanobodies may be connected by a connecting peptide, which is preferably a flexible peptide segment rich in glycine and serine.

[0012] In a third aspect, the present invention provides a fusion protein, wherein the fusion protein is obtained by fusing the above-mentioned Trop2-binding nanobody with Fc.

[0013] Wherein, the Fc fragment includes the Fc fragment of human IgG, which can be selected from: Fc fragments of IgG1, IgG2, IgG3, and IgG4.

[0014] In a fourth aspect, the present invention provides a chimeric antigen receptor (CAR) targeting TROP2, wherein the chimeric antigen receptor comprises the above-mentioned nanobody binding to Trop2 or the above-mentioned antibody binding to Trop2.

[0015] In the present invention, the chimeric antigen receptor may adopt the structure of a currently known chimeric antigen receptor.

[0016] Preferably, the chimeric antigen receptor comprises a signal peptide, the Trop2-binding Nanobody or the Trop2-binding antibody, a hinge region, a transmembrane region, an intracellular co-stimulatory domain and an intracellular signal transduction domain.

[0017] Preferably, from N-terminus to C-terminus, the chimeric antigen receptor comprises, in sequence, a signal peptide, the Trop2-binding nanobody or the Trop2-binding antibody, a hinge region, a transmembrane region, an intracellular co-stimulatory domain and an intracellular signal transduction domain.

[0018] 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 sequence of known CAR.

[0019] In some embodiments of the present invention, the CAR comprises, in sequence, a CD8α leader membrane receptor signal peptide, a (G4S)3 connecting peptide, the Trop2-binding nanobody or the Trop2-binding antibody, a CD8α Hinge hinge region, a CD8α TM transmembrane region, a CD28 co-stimulatory domain, and a CD3ζ intracellular signal transduction domain.

[0020] 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.

[0021] According to the amino acid sequences of nanobodies, antibodies, fusion proteins and chimeric antigen receptors provided by the present invention, those skilled in the art can obtain the nucleotide sequences of nucleic acid molecules encoding the above-mentioned nanobodies, antibodies, fusion proteins and chimeric antigen receptors. Due to the degeneracy of codons, the nucleotide sequence of a nucleic acid molecule encoding an amino acid sequence is not unique, and all nucleic acid molecules capable of encoding the above-mentioned nanobodies, antibodies, fusion proteins and chimeric antigen receptors are within the scope of protection of the present invention.

[0022] In some specific embodiments of the present invention, the sequence of the nucleic acid molecule encoding the heavy chain variable region of the Nanobody is as shown in SEQ ID NO.143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153 or 154.

[0023] In a sixth aspect, the present invention provides a biological material, wherein the biological material comprises the nucleic acid molecule; the biological material is an expression cassette, a vector or a host cell.

[0024] The above-mentioned expression cassette can be obtained by connecting a transcription or translation regulatory element such as a promoter upstream of the nucleic acid molecule and / or connecting a transcription or translation regulatory element such as a terminator downstream thereof.

[0025] The above-mentioned vectors include but are not limited to plasmid vectors, lipid nanoparticle vectors, viral vectors, transposons, etc.

[0026] Among them, the viral vectors include lentiviral vectors, adenoviral vectors, AAV viral vectors, retroviral vectors, etc.

[0027] The 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 that can differentiate into immune cells, Chinese hamster ovary cells (CHO), baby hamster embryonic kidney cells (BHK), mouse myeloma cells (SP0 / 2), African green monkey kidney cells (Vero) and human embryonic kidney 293 cells (HEK293), etc.

[0028] In a seventh aspect, the present invention provides a recombinant cell, wherein the cell expresses the chimeric antigen receptor described above.

[0029] Preferably, the recombinant cell is an immune cell expressing the chimeric antigen receptor described above.

[0030] Preferably, the immune cells are T cells or NK cells.

[0031] In an eighth aspect, the present invention provides an antibody conjugate, wherein the antibody conjugate is obtained by coupling the Trop2-binding nanobody or the Trop2-binding antibody or the fusion protein or the chimeric antigen receptor with a detectable marker, drug, toxin or cytokine.

[0032] The markers mentioned above are selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, colloidal gold labeling, and radioactive labeling.

[0033] The drugs mentioned above are cytotoxic drugs, including but not limited to DNA replication inhibitors, topoisomerase inhibitors, alkylating agents, antibiotics, anti-tubulin drugs, folic acid antagonists, and the like.

[0034] The toxins mentioned above include, but are not limited to, ricin, paclitaxel, cisplatin, vincristine, vinblastine, colchicine, actinomycin, diphtheria toxin, abrin, and the like.

[0035] In the ninth aspect, the present invention provides a method for producing the nanobody that binds to Trop2, the antibody that binds to Trop2, the fusion protein, or the chimeric antigen receptor, the method comprising: culturing a host cell containing the nucleic acid molecule, and collecting the nanobody that binds to Trop2, the antibody that binds to Trop2, the fusion protein, or the chimeric antigen receptor from the culture.

[0036] Among them, collecting from culture includes steps such as separation and purification.

[0037] In a tenth aspect, the present invention provides any of the following uses of the Trop2-binding nanobody, the Trop2-binding antibody, the fusion protein, the chimeric antigen receptor, the nucleic acid molecule, the biomaterial, the recombinant cell, or the antibody conjugate: (1) Use in the preparation of drugs for preventing or treating diseases related to Trop2 expression; (2) Application in the preparation of CAR-T drugs; (3) Use in the preparation of a reagent for detecting the presence or level of Trop2 in a sample; (4) Application in the preparation of tumor detection reagents.

[0038] In the above (1), the disease associated with 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 tumor includes at least one selected from breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, bile duct cancer, prostate cancer, cervical cancer, head and neck cancer, lung cancer, oral cancer, pharyngeal cancer, esophageal cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, brain glioma, glioblastoma, thyroid cancer, liver cancer, kidney cancer, urothelial cancer, skin cancer, melanoma, penile cancer, etc.

[0039] In the above (4), the tumor is a tumor expressing Trop2, preferably an epithelial malignant tumor that highly expresses TROP2. The malignant tumor includes at least one selected from breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, bile duct cancer, prostate cancer, cervical cancer, head and neck cancer, lung cancer, oral cancer, pharyngeal cancer, esophageal cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, brain glioma, glioblastoma, thyroid cancer, liver cancer, kidney cancer, urothelial cancer, skin cancer, melanoma, penile cancer, etc.

[0040] In the eleventh aspect, the present invention provides a pharmaceutical composition, comprising the Trop2-binding nanobody or the Trop2-binding antibody or the fusion protein or the chimeric antigen receptor or the recombinant cell or the antibody conjugate.

[0041] Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0042] Preferably, the pharmaceutical composition is used to treat diseases associated with Trop2 expression.

[0043] In a twelfth aspect, the present invention provides a detection reagent, which comprises the Trop2-binding nanobody or the Trop2-binding antibody or the fusion protein or the antibody conjugate.

[0044] The detection reagents mentioned above include, but are not limited to, ELISA detection reagents or kits, chemiluminescence detection reagents or kits, radioimmunoassay reagents or kits, fluorescent immunoassay reagents or kits, etc.

[0045] In a thirteenth aspect, the present invention provides a method for treating a disease associated with Trop2 expression, the method comprising: administering to a subject a therapeutically effective amount of the Trop2-binding nanobody or the Trop2-binding antibody or the fusion protein or the chimeric antigen receptor or the antibody conjugate.

[0046] In a fourteenth aspect, the present invention provides a method for detecting Trop2, comprising: contacting the Trop2-binding nanobody or the Trop2-binding antibody 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.

[0047] The beneficial effects of the present invention include at least: the Trop2-binding antibody provided by the present invention is derived from alpaca, contains only one VHH, belongs to a nanobody, and is the antibody structure with the smallest protein molecular weight known so far; compared with traditional monoclonal antibodies and single-chain antibody variable regions (single chain variable fragment, scFv), nanobodies have the advantages of small molecular weight, low aggregation and precipitation, and high stability, and while maintaining a high affinity for the antigen, the long and flexible complementary determining region CDR3 of the nanobody allows binding to the cracks and cavities of the target antigen, and can recognize some hidden epitopes. The anti-Trop2 nanoantibody provided by the present invention can efficiently and specifically recognize and bind to the Trop2 protein. Based on the nanoantibody, the present invention also provides drugs prepared therefrom, including but not limited to bispecific antibodies, antibody-drug conjugates, chimeric antigen receptors, and 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, and to diagnose and treat diseases with Trop2 molecules as markers.

[0048] The present invention also provides CAR and CAR-T cells that bind to Trop2 protein. It has been verified by cell-level experiments that the CAR-T cells have a good immune clearance effect targeting Trop2, and can be used to specifically identify 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, and to improve and treat diseases with Trop2 molecules as markers.

[0049] The anti-Trop2 CAR of the present invention adopts a nanobody that binds to the Trop2 protein as the specific recognition structural component of the antigen. The nanobody has the advantage of a small molecular weight, which is conducive to the recombinant design to produce multi-specific nanobodies and multi-specific CARs, thereby improving the recognition specificity of 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

[0050] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0051] Figure 1 The OD values ​​of 12 anti-human Trop2 VHHs in the alpaca immune antibody library screened by phage ELISA in Example 1 of the present invention are as follows: 450 Reading results.

[0052] Figure 2 This is the flow cytometry identification of the expression of the target antigen Trop2 in the human pancreatic-related cell line in Example 2 of the present invention.

[0053] Figure 3 Schematic diagram of the molecular structure of the anti-Trop2 CAR in Example 3 of the present invention.

[0054] Figure 4 This is the positive rate of the anti-Trop2 CAR (T2-F09) transduced dual reporter cell line J-NN detected by flow cytometry in Example 4 of the present invention, wherein NC represents the negative control.

[0055] Figure 5 It is the detection of downstream signal activation level of anti-Trop2 CAR J-NN under different target cell stimulation and the peak comparison of GFP and mCherry signal activation in Example 4 of the present invention. Wherein, a and b are the changes in green fluorescence and red fluorescence signal intensity of anti-Trop2 CAR J-NN when co-cultured with Trop2-positive target cells CFPAC-1 and Trop2-negative target cells AsPC-1. c and d are the green fluorescence signal intensity and red fluorescence signal intensity of J-NN without CAR transduction and anti-Trop2 CAR J-NN after co-culture with four target cells for 12 hours. au represents arbitrary unit.

[0056] Figure 6Comparison of downstream signal activation levels of 12 anti-Trop2 CAR J-NN under stimulation of positive target cells BxPC-3 in Example 4 of the present invention. Wherein, a and b are the green fluorescence signal intensity and red fluorescence signal intensity of J-NN without CAR transduction and 12 anti-Trop2 CAR J-NN after co-culture with BxPC-3 for 12 hours, respectively.

[0057] Figure 7 This is the positive rate of anti-Trop2 CAR (T2-F09) transduced human primary T cells detected by flow cytometry in Example 5 of the present invention, wherein NC represents the negative control.

[0058] Figure 8 The results of the in vitro killing kinetics of anti-Trop2 CAR-T (T2-F09) cells on different target cells in Example 6 of the present invention. Among them, a and c are the changes in the green fluorescence signal intensity of anti-Trop2 CAR-T when co-cultured with Trop2-positive target cells BxPC-3-GFP and Trop2-negative target cells hTERT-HPNE-GFP (normalized). b and d are the changes in the green fluorescence signal intensity of negative control T when co-cultured with Trop2-positive target cells BxPC-3-GFP and Trop2-negative target cells hTERT-HPNE-GFP (normalized).

[0059] Fig. 9 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. Wherein, a and b are the in vitro killing efficiency of anti-Trop2 CAR-T and control T cells after co-culture with Trop2-positive target cells BxPC-3-GFP (a) and CFPAC-1-GFP (b) for 72 hours; c and d are the in vitro killing efficiency of anti-Trop2 CAR-T and control T cells after co-culture with Trop2-negative target cells AsPC-1-GFP (c) and hTERT-HPNE-GFP (d) for 72 hours.

[0060] Fig.10 The figure compares the in vitro killing efficiency of 12 anti-Trop2 CAR-T cells against positive target cells BxPC-3-GFP under three effector-target ratios in Example 6 of the present invention.

[0061] Fig.11 The results of cytokine secretion of anti-Trop2 CAR-T (T2-F09) cells under stimulation of different target cells in Example 7 of the present invention are shown in Figure 7. Among them, a: IFNγ secretion level, b: TNFα secretion level, and c: IL-2 secretion level.

[0062] Fig.12The 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 result of the live cell real-time imager Incucyte S3, b: the in vitro killing kinetic results of anti-Trop2 CAR-T cells on tumor spheres. DETAILED DESCRIPTION

[0063] In a specific embodiment of the present invention, an antibody binding to Trop2 protein is provided, which 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, the alpaca is immunized with purified Trop2 protein. After successful immunization, the alpaca peripheral blood is collected to extract lymphocytes containing mature B cells, RNA is extracted and reversely transcribed into a cDNA library, and the heavy chain variable region of the alpaca antibody is amplified by primer PCR to obtain a specific gene fragment, which is then spliced ​​into a phage vector and transformed into Escherichia coli to obtain a nano antibody gene library, i.e., an immune library. Through phage display technology, after 4 rounds of panning, positive clones are identified by indirect ELISA, and the antibody sequence binding to the Trop2 antigen is confirmed by sequencing.

[0064] The Trop2 protein-binding antibodies obtained above comprise the variable domain of heavy chain of heavy-chain antibody (VHH), and the amino acid sequences of VHH of each antibody are shown in Table 1. The amino acid sequence of each antibody was numbered by Martin numbering scheme and defined by different CDR region definition schemes to obtain the CDR1, CDR2, CDR3 regions and framework regions FR1, FR2, FR3, FR4 of the antibody, wherein 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.

[0065] Table 1

[0066] Table 2

[0067] Table 3

[0068] The above-mentioned antibodies binding to Trop2 protein can be used to specifically identify 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 can be used to diagnose and treat diseases using Trop2 molecules as markers.

[0069] In a specific embodiment of the present invention, a chimeric antigen receptor (CAR) binding to Trop2 protein and an expression vector and a host cell comprising the chimeric antigen receptor are also provided. The CAR binding to Trop2 protein uses a nanobody binding to Trop2 protein as the specific recognition structural component of the antigen, that is, the antibody binding to Trop2 protein described above. The molecular structure of the anti-Trop2 CAR is SP-anti-Trop2 VHH-HINGE-TM-CD-SD, wherein 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 connecting peptide or peptide bond.

[0070] 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, the components of the CAR molecule are sequentially connected in series: CD8α leader membrane receptor signal peptide, (G4S) 3 connecting peptide, anti-Trop2 VHH, CD8α Hinge hinge region, CD8α TM transmembrane region, CD28 costimulatory domain, and CD3ζ intracellular signal transduction domain.

[0071] The chimeric antigen receptor binding to Trop2 protein described above uses a nano-antibody binding to Trop2 protein as an antigen-specific recognition structural component, and the chimeric antigen receptor is transduced into a host cell with an immune effector using a vector such as a lentivirus, which can target and recognize cells expressing Trop2, and can release various pro-apoptotic cytokines such as granzymes, perforins, and interferon-γ after antigen stimulation, causing target cell death and exerting an immune clearance function. Therefore, the chimeric antigen receptor binding to 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, and is used to improve and treat diseases with Trop2 molecules as markers.

[0072] In a specific embodiment of the present invention, an expression vector is also provided, which contains a nucleic acid molecule encoding the chimeric antigen receptor or antibody, and can be a plasmid vector, a lipid nanoparticle vector, or a viral vector, including but not limited to a lentiviral vector and a retroviral vector.

[0073] In a specific embodiment of the present invention, a host cell is also provided, and the host cell is an immune cell or a stem cell or progenitor cell that can differentiate into an immune cell. Immune cells include but are not limited to T cells, NK cells, NKT cells, macrophages, dendritic cells, and have killing or phagocytic functions, immunomodulatory functions, and cytokine release functions. 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 immune cells.

[0074] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0075] Example 1: Screening of Nanobodies Binding to Trop2 Protein Human TROP2 extracellular segment protein (Human TROP-2 / TACSTD2 Protein, His Tag, ACRO, Catalog No. TR2-H5223) was used as an antigen to immunize healthy adult alpacas. After four antigen immunizations, each with an interval of 20 days, the antibody titer of the alpaca peripheral blood serum was detected. The alpaca peripheral blood was collected and peripheral blood mononuclear cells (PBMC) were extracted, RNA was extracted, reverse transcribed to obtain cDNA, PCR amplified the heavy chain variable region to obtain the VHH gene fragment, and then spliced ​​into the phage vector and transformed into Escherichia coli to obtain the nanoantibody gene library.

[0076] Use phage display technology to bio-screen anti-Trop2 nanoantibodies. The specific steps are: 1) Antigen coating: coat the immunotube with TROP2 antigen (30µg / tube), rotate slowly at 4°C overnight, and coat 30µg 5% Non-fatmilk skim milk powder in parallel as a control; 2) Wash unbound antigen: discard the coating solution, add 2mL PBS buffer to wash the immunotube 3 times at room temperature, and rotate for 5 minutes each time; 3) Block: add 5mL blocking solution, rotate and block at room temperature for 2h; 4) Wash the blocking solution: discard the liquid in the blocked immunotube, and add 5mL PBS buffer to wash the immunotube 3 times at room temperature, and rotate for 5min each time; 5) Add phage display library for binding: discard the washing solution in the immunotube, add 2mL PBS buffer, add the prepared phage library as the first round of screening input phage library, and rotate and incubate at room temperature for 1h; 6) Wash unbound phage: discard the liquid in the immunotube, add 5mL PBST (1×PBS plus 0.1% 7) Elute the bound phages: add 1mL of pH 2.2 Glycine-HCl solution, rotate at room temperature for 30min, add 90μL of pH 8.8 Tris-HCl to terminate the elution, and transfer the solution in the immunotube to a new 1.5mL centrifuge tube, which is the first round of phage screening elution. 8) After the first round of phages are amplified, they are used for the subsequent second round of screening and detection. The above screening process is carried out for 3 rounds. In each round of screening, the phages are streaked to calculate the enrichment efficiency, and monoclones are selected for antigen-specific detection.

[0077] Phage ELISA was used to screen and identify positive monoclonal clones. The specific steps were as follows: after each round of phage eluate enrichment, the ELISA plate was coated with the TROP2 protein antigen, BSA was used as the control antigen, the antibody displayed by the selected cultured monoclonal phage was used as the antibody binding antigen to be tested, the unbound phage was eluted, the anti-phage M13 antibody labeled with HRP was used as the secondary antibody to bind the phage, the substrate was added for color development, and the microplate reader was used to read the data, and the OD values ​​corresponding to the TROP2 test group and the BSA control group were used. 450 The difference was used to preliminarily determine the positive clones.

[0078] 95 positive clones shown by phage ELISA results were selected and sent to Qingke Biotech Co., Ltd. for sequencing to identify the diversity of positive clone sequences. The sequenced sequences were converted into amino acid sequences, and the sequences were aligned and merged to obtain 12 positive antibody clones screened by Trop2 phage. The ELISA test results of the above 12 positive clones are as follows: Figure 1As shown. The coding nucleotide sequences corresponding to the amino acid sequences of the 12 positive antibody clones screened by Trop2 phage were synthesized respectively, and the coding nucleotide sequences were each provided with an amino acid tag / linker (GGGGS) × 3, which was convenient for subsequent experimental detection. The 12 Trop2 VHH genes with the above tags were synthesized respectively.

[0079] Example 2: Flow cytometry detection of Trop2-positive and -negative target cells The Trop2 protein expression on the surface of four human pancreatic cell lines was detected using Trop2 flow cytometry antibody (PE Mouse Anti-Human Trop-2, BD Biosciences, Cat. No. 564837). They are three pancreatic cancer cells AsPC-1, BxPC-3, CFPAC-1 and an immortalized human normal pancreatic ductal epithelial cell hTERT-HPNE. The detection steps are as follows: digest the cells to be tested with trypsin, resuspend the cells in culture medium after centrifugation and count them, and take 1×10 6 Cells were centrifuged and supernatant was removed. The cells were washed once with pre-prepared FACS buffer (1×PBS+2%FBS or 1×PBS+0.5%BSA). Trop2 flow cytometry antibody diluted in FACS buffer was added to the cells in the stained group and incubated at 4°C for 30 minutes. The supernatant was removed by centrifugation, and the cells were washed three times with FACS buffer and resuspended in 200μL for detection.

[0080] The flow cytometry results of four cell lines expressing Trop2 are shown in Figure 2. Figure 2 As shown, pancreatic cancer cells BxPC-3 and CFPAC-1 highly expressed Trop2 and were used as Trop2-positive target cells in subsequent experiments; pancreatic cancer cells AsPC-1 and pancreatic ductal epithelial cells hTERT-HPNE hardly expressed Trop2 and were used as Trop2-negative target cells in subsequent experiments.

[0081] Example 3: Plasmid construction of anti-Trop2 CAR and lentiviral packaging The anti-Trop2 VHH nanobody sequence obtained in Example 1 was amplified by PCR, and then spliced ​​with the gene-synthesized CAR skeleton by seamless cloning, and constructed into the PCDH lentiviral expression vector (the vector was modified, and the promoter was EF1α). The accuracy of the plasmid sequence was verified by sequencing, and the lentiviral master plasmid expressing the anti-Trop2 CAR was obtained. The molecular composition of the complete anti-Trop2 CAR is as follows Figure 3As shown, it includes a CD8α leader membrane receptor signal peptide, a (G4S)3 connecting 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 connected in series.

[0082] Lentivirus was prepared by co-transfecting 293T with three plasmids. The transfection reagent was PEI MAX, i.e., linearized polyethyleneimine PEI 40000 (Polyethylenimine Max, MW40000, Polysciences, Cat. No. 24765-1). The three plasmids included the main lentiviral plasmid expressing anti-Trop2 CAR and two packaging plasmids psPAX2 and pMD2.G. 293T cells in good growth state were plated one day in advance, and the cell confluence was about 80% for transfection. The plasmids and transfection reagents were diluted with serum-free medium. The mass ratio of the three plasmids was lentiviral main plasmid: psPAX2: pMD2.G = 3:2:1, and the ratio of transfection reagent to plasmid was 2.5μL: 1μg. After mixing the diluted transfection reagent and plasmid, let it stand at room temperature for 20 minutes, and then add the transfection suspension to the 293T cells and gently shake to mix. After 8 hours of transfection, the medium was changed, and the supernatants of 48h and 72h culture were collected and centrifuged to remove cell debris. The supernatant was collected and added to the lentivirus concentrate (PEG8000 and NaCl), mixed and placed at 4°C overnight to concentrate the virus. Centrifuged at 4000g for 20min at 4°C, discarded the supernatant after centrifugation, and the virus precipitate was resuspended with an appropriate amount of PBS or culture medium to obtain a recombinant lentivirus vector containing anti-Trop2CAR. The lentivirus titer was verified to meet the requirements of subsequent experiments by the lentivirus titer rapid test card (Biodragon, catalog number BF06202-100).

[0083] Example 4: Expression of anti-Trop2 CAR transduced dual reporter cell line J-NN and downstream signal activation detection The dual reporter cell line J-NN (derived from Wei Ping's laboratory at the Chinese Academy of Sciences, see patent application CN115232216A) is a Jurkat dual reporter signal cell line (referred to as J-NN) with pNFAT-EGFP and pNFκB-mCherry. It can quickly and high-throughput complete the activation signal feature identification of the new CAR molecule design, and is an experimental means to quickly screen new CARs.

[0084] Anti-Trop2 CAR was transduced into J-NN cells using the lentiviral vector in Example 3 to construct anti-Trop2CAR J-NN cells. Cells were collected 48 hours after transduction, and the positive rate of anti-Trop2 CAR J-NN cells was detected using G4S antibody (Anti-(G4S)n-(B02H1)m-Ab (PE), Heyousheng Biotechnology, catalog number GS-ARPE100) with reference to the flow staining method in Example 2. Taking T2-F09 as an example, Figure 4 As shown, the positive expression rate of anti-Trop2 CAR was above 50%.

[0085] The above-mentioned anti-Trop2 CAR J-NN cells were co-cultured with Trop2-positive target cells BxPC-3, CFPAC-1 and Trop2-negative target cells AsPC-1, hTERT-HPNE, respectively. At the same time, an anti-Trop2 CAR J-NN cell group without target cells and a J-NN cell control group without CAR transduction were set up. One day in advance, the target cells were plated in a 96-well plate at a number of 5000-10000 cells per well, and three parallel wells were set up in each group. On the second day, anti-Trop2 CAR J-NN cells and J-NN cells were added at an effector-target ratio of 5:1. The changes in the green fluorescence and red fluorescence intensity of J-NN cells were dynamically monitored using the Incucyte S3 live cell analysis system (Sartorius). The shooting channel selected was BF+GFP+RFP, the shooting interval was 2h, and the shooting time was 3 days. The results are shown in Figure 5 As shown in the figure, the two Trop2-positive target cells BxPC-3 and CFPAC-1 can significantly activate the anti-Trop2 CAR J-NN cells to express green fluorescence and red fluorescence reporter genes. The Trop2-negative target cell group, the group without target cells and the J-NN cell control group did not produce green fluorescence and red fluorescence signals, indicating that the activation of the two downstream signals of the 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 the 12 anti-Trop2 CAR J-NN cells were co-cultured with BxPC-3. The results are shown in Figure 6 As shown, the anti-Trop2 CARs prepared from 12 cloned anti-Trop2 nanobodies were able to bind to the Trop2 antigen and activate J-NN.

[0086] Example 5: Preparation of anti-Trop2 CAR-T cells T cells were sorted from freshly isolated or resuscitated human peripheral blood mononuclear cells (PBMCs) using magnetic bead negative selection (Pan T Cell Isolation Kit, Miltenyi, Catalog No. 130-096-535). CD3 / CD28 magnetic beads (Dynabeads Human T-activator CD3 / CD28, Thermo Fisher Scientific, Catalog No. 11132D) were added at a ratio of Bead:cell = 1:1 to isolate the purified CD3 + T cells were activated and cultured in T cell serum-free medium (X-VIVO 15, Lonza, Catalog No. 04-418Q) with a final concentration of 200 IU / mL IL-2 (Sihuan Bio, Catalog No. S10970015). After 24 h of magnetic bead activation culture, the anti-Trop2 CAR lentivirus in Example 3 and polybrene (Merck, Catalog No. TR-1003-G) dyeing reagent with a final concentration of 6 μg / mL were added, and the medium was changed and cultured for 16 h after lentiviral infection.

[0087] After 4 days of activation culture, the magnetic beads were removed. After 72 hours of lentiviral infection, the positive rate of anti-Trop2 CAR-T cells was detected using the flow cytometry staining method in Example 2 using G4S antibody (Anti-(G4S)n-(B02H1)m-Ab (PE), Heyousheng Biotechnology, catalog number GS-ARPE100). Taking T2-F09 as an example, Figure 7 As shown in the figure, the positive expression rate of anti-Trop2 CAR-T was above 50%. T cells were expanded and cultured in serum-free medium containing 200 IU / mL IL-2 for subsequent CAR-T function testing.

[0088] Example 6: In vitro killing efficiency of anti-Trop2 CAR-T cells on different target cells 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 T cell control group without CAR transduction and a simple target cell group without effector cells were set up. One day in advance, the target cells were plated in a 96-well plate at a number of 2000-5000 cells per well, and three parallel wells were set up in each group. On the second day, anti-Trop2 CAR-T cells and T cells were added at effector-target ratios of 1:2, 1:1, 2:1, and 4:1, respectively. The changes in the green fluorescence intensity of the target cells were dynamically monitored using the Incucyte S3 live cell analysis system (Sartorius). The shooting channel selected was BF+GFP, the shooting interval was 4h, and the shooting time was 3 days. The results are as follows Figure 8 As shown in the figure, compared with the T cell control group, anti-Trop2 CAR-T cells have a higher specific killing effect on Trop2-positive target cells BxPC-3-GFP, and the in vitro killing efficiency increases with the increase of effector-target ratio and time, while there is no killing effect on Trop2-negative target cells hTERT-HPNE-GFP.

[0089] The in vitro killing efficiency at 72 h was calculated according to the following formula: In vitro killing efficiency (Cytotoxicity) = (normalized single target group fluorescence intensity - normalized experimental group fluorescence intensity) / normalized single target group fluorescence intensity × 100%. The results are shown in Fig. 9 As shown in the figure, compared with the T cell control group, anti-Trop2 CAR-T cells have a higher specificity to kill two Trop2 positive target cells BxPC-3-GFP and CFPAC-1-GFP, and show a significant dose effect. The in vitro killing efficiency increases with the increase of the effector-target ratio, while there is no killing effect on two 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 48h was statistically analyzed, and the results are shown in Fig.10 , which verified that anti-Trop2 CAR-T has a good specific killing effect on Trop2-positive target cells at the in vitro cell level.

[0090] Example 7: Cytokine release detection of anti-Trop2 CAR-T cells The plate was plated according to the in vitro killing experiment with a target to effect ratio of 4:1 in Example 6, and the culture medium for the effector cells was a serum-free T cell culture medium without IL-2. After the target cells and effector cells were co-cultured for 24 hours, the supernatant was collected and the secretion of three cytokines, IL-2 (Human IL-2 Precoated ELISA Kit, Dakoway, catalog number 1110202), IFNγ (HumanIFN-γ Precoated ELISA Kit, Dakoway, catalog number 1110002), and TNFα (Human TNF-α PrecoatedELISA Kit, Dakoway, catalog number 1117202), was detected by ELISA. The results are shown in Fig.11 As shown, compared with the T cell control group, anti-Trop2 (T2-F09) CAR-T cells released a large amount of IFNγ, TNFα and IL-2 after co-incubation with two Trop2-positive target cells BxPC-3-GFP and CFPAC-1-GFP for 24 hours. The cytokines of the two Trop2-negative target cells AsPC-1-GFP and hTERT-HPNE-GFP groups were at a lower level. It was verified that anti-Trop2 CAR-T can be stimulated and activated by Trop2-positive target cells to produce a series of cytokines related to immune activation.

[0091] Example 8: In vitro killing efficiency of anti-Trop2 CAR-T cells on tumor spheres Trop2-positive target cells BxPC-3 with GFP fluorescence were plated in a 96-well transparent round-bottom ultra-low adsorption microplate (Corning, Cat. No. 7007) at a number of 3,000 cells per well. Tumor spheres with a diameter of 200-500 μm were observed 1-3 days after inoculation. Anti-Trop2 (T2-F09) CAR-T cells and T cells were added at an effector-target ratio of 1:1, 2:1, and 4:1, respectively. The changes in the green fluorescence intensity of the tumor spheres were dynamically monitored using the Incucyte S3 live cell analysis system (Sartorius). The shooting mode was selected as Spheroid, the shooting channel was selected as BF+GFP, the shooting interval was 4h, and the shooting time was 3 days. The results are shown in Fig.12 As shown, compared with the T cell control group, anti-Trop2 CAR-T cells can effectively eliminate the GFP fluorescence of tumor spheres and achieve a three-dimensional in vitro killing effect on tumor cells.

[0092] 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions 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 region (CDR) of the heavy chain variable region is any one of the following (1) to (6): (1) When defined according to the IMGT numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NO. 1, 2 and 3, respectively; When defined according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown as SEQ ID NOs. 4, 5, and 6, respectively; When defined according to the Chothia numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown as SEQ ID NOs. 7, 8, and 9, respectively; When defined according to the Contact numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NOs. 10, 11 and 12, respectively; (2) When defined according to the IMGT numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NOs. 13, 14 and 15, respectively; 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; When defined according to the Chothia numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NOs. 19, 20 and 21, respectively; When defined according to the Contact numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NOs. 22, 23 and 24, respectively; (3) When defined according to the IMGT numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NOs. 25, 26 and 27, respectively; When defined according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs. 28, 29, and 30, respectively; When defined according to the Chothia numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown as SEQ ID NOs. 31, 32, and 33, respectively; When defined according to the Contact numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NOs. 34, 35 and 36, respectively; (4) When defined according to the IMGT numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NOs. 37, 38 and 39, respectively; When defined according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown as SEQ ID NOs. 40, 41, and 42, respectively; When defined according to the Chothia numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NOs. 43, 44 and 42, respectively; When defined according to the Contact numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NOs. 45, 46 and 47, respectively; (5) When defined according to the IMGT numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NOs. 37, 38 and 48, respectively; When defined according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown as SEQ ID NOs. 40, 41, and 49, respectively; When defined according to the Chothia numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NOs. 43, 44 and 49, respectively; When defined according to the Contact numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NOs. 45, 46 and 50, respectively; (6) When defined according to the IMGT numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NOs. 37, 51 and 39, respectively; When defined according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown as SEQ ID NOs. 40, 52, and 42, respectively; When defined according to the Chothia numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 are shown as SEQ ID NOs. 43, 53, and 42, respectively; When defined according to the Contact numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NOs. 45, 54 and 47, respectively.

2. The Trop2-binding Nanobody according to claim 1, characterized in that The heavy chain variable region further comprises a framework region FR, and the framework region FR is any one of the following (1) to (11): (1) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 55, 56, 57 and 58, respectively; When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 59, 60, 61 and 58, respectively; When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown as SEQ ID NOs. 55, 62, 63 and 58, respectively; When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown as SEQ ID NOs. 64, 65, 66 and 67, respectively; (2) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 55, 68, 69 and 58, respectively; When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown as SEQ ID NOs. 70, 71, 72 and 58, respectively; When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 55, 73, 74 and 58, respectively; When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown as SEQ ID NOs. 75, 76, 77 and 67, respectively; (3) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 78, 79, 80 and 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 NOs. 82, 83, 84 and 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 NOs. 78, 85, 86 and 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 NOs. 87, 88, 89 and 90, respectively; (4) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 91, 92, 93 and 81, respectively; When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 94, 95, 96 and 81, respectively; When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 91, 97, 98 and 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 NOs. 99, 100, 101, and 90, respectively; (5) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 78, 92, 93 and 81, respectively; When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 102, 95, 96 and 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 NOs. 78, 97, 98 and 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 NOs. 103, 100, 101, and 90, respectively; (6) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 78, 92, 104 and 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 NOs. 102, 95, 105, and 81, respectively; When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 78, 97, 104 and 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 NOs. 103, 100, 106 and 90, respectively; (7) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 78, 92, 107 and 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 NOs. 102, 95, 108 and 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 NOs. 78, 97, 109 and 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 NOs. 103, 100, 110 and 90, respectively; (8) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 78, 92, 111 and 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 NOs. 102, 95, 112, and 81, respectively; When defined according to the Chothia numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 78, 97, 113 and 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 NOs. 103, 114, 115 and 90, respectively; (9) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 78, 92, 116 and 81, respectively; When defined according to the Kabat numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 102, 95, 117 and 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 NOs. 78, 97, 118 and 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 NOs. 103, 114, 119 and 90, respectively; (10) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 78, 92, 120 and 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 NOs. 102, 95, 121, and 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 NOs. 78, 97, 122 and 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 NOs. 103, 114, 123 and 90, respectively; (11) When defined according to the IMGT numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are as shown in SEQ ID NOs. 78, 124, 125 and 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 NOs. 102, 126, 127, and 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 NOs. 78, 128, 129 and 81, respectively; When defined according to the Contact numbering system, the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs. 103, 76, 130 and 90, respectively.

3. The Trop2-binding Nanobody according to claim 1 or 2, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 or 142.

4. An antibody that binds to Trop2, characterized in that The antibody is a monovalent antibody, a bispecific antibody or a multispecific antibody comprising one or more Trop2-binding Nanobodies according to any one of claims 1 to 3.

5. A fusion protein, characterized in that The fusion protein is obtained by fusing the Trop2-binding nanobody according to 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 Trop2-binding Nanobody according to any one of claims 1 to 3 or the Trop2-binding antibody according to claim 4.

7. The chimeric antigen receptor according to claim 6, characterized in that The chimeric antigen receptor comprises a signal peptide, a Trop2-binding nanobody according to any one of claims 1 to 3 or an antibody binding to Trop2 according to 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 Trop2-binding nanobody according to any one of claims 1 to 3, the Trop2-binding antibody according to claim 4, the fusion protein according to claim 5, or the chimeric antigen receptor according to claim 6 or 7.

9. Biomaterial, characterized in that The biological material comprises the nucleic acid molecule according to 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 according to claim 6 or 7.

11. An antibody conjugate, characterized in that: The antibody conjugate is obtained by coupling the Trop2-binding nanobody according to any one of claims 1 to 3, the Trop2-binding antibody according to claim 4, the fusion protein according to claim 5, or the chimeric antigen receptor according to claim 6 or 7 with a detectable marker, drug, toxin or cytokine.

12. A method for producing the Trop2-binding nanobody according to any one of claims 1 to 3, the Trop2-binding antibody according to claim 4, the fusion protein according to claim 5, or the chimeric antigen receptor according to claim 6 or 7, characterized in that: The method comprises: culturing a host cell comprising the nucleic acid molecule of 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.

13. Any of the following uses of the Trop2-binding Nanobody according to any one of claims 1 to 3, the Trop2-binding antibody according to claim 4, the fusion protein according to claim 5, the chimeric antigen receptor according to claim 6 or 7, the nucleic acid molecule according to claim 8, the biomaterial according to claim 9, the recombinant cell according to claim 10, or the antibody conjugate according to claim 11: (1) Use in the preparation of drugs for preventing or treating diseases related to Trop2 expression; (2) Application in the preparation of CAR-T drugs; (3) Use in the preparation of a reagent for detecting the presence or level of Trop2 in a sample; (4) Application in the preparation of tumor detection reagents.

14. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the Trop2-binding nanobody according to any one of claims 1 to 3, the Trop2-binding antibody according to claim 4, the fusion protein according to claim 5, the chimeric antigen receptor according to claim 6 or 7, the recombinant cell according to claim 10, or the antibody conjugate according to claim 11.

15. A detection reagent, characterized in that: The detection reagent comprises the Trop2-binding nanobody according to any one of claims 1 to 3, the Trop2-binding antibody according to claim 4, the fusion protein according to claim 5, or the antibody conjugate according to claim 11.

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