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] This application is a divisional application of a patent application filed on January 10, 2025, with application number 2025100389431, entitled "A Nanobody Binding to Trop2, a Chimeric Antigen Receptor, and Applications Thereof." The present invention relates to the field of antibody technology, and more particularly to a Nanobody Binding to Trop2, a Chimeric Antigen Receptor, and applications thereof. Background Art

[0002] Trop2 (Trophoblast cell surface antigen 2), short for trophoblast surface antigen, is a single-pass transmembrane cell surface glycoprotein encoded by the TACSTD2 (Tumor associated calcium signal transducer 2) gene. Trop2 was first discovered as a surface marker of trophoblast cells over 40 years ago and subsequently rediscovered as tumor-associated calcium signal transducer 2 (TACSTD2), membrane component chromosome 1 surface marker 1 (M1S1), gastrointestinal tumor-associated antigen 733-1 (GA733-1), and epithelial glycoprotein-1 (EGP-1). Trop2 belongs to the GA733 protein family and shares a high degree of structural sequence similarity with epithelial cell adhesion molecule (EpCAM, also known as Trop1 or TACSTD1), with a homology of 49%. Studies have found that Trop2 typically exhibits minimal or low expression in normal epithelial tissues, but is significantly overexpressed in a variety of epithelial malignancies, including pancreatic cancer, colorectal cancer, ovarian cancer, gastric cancer, breast cancer, prostate cancer, cervical cancer, and head and neck cancer. Numerous studies have elucidated the key role of Trop2 in tumor proliferation, invasion, and metastasis, with its high expression being closely associated with shortened survival and poor prognosis in cancer patients.

[0003] Trop2's high expression in various malignancies and its unique mechanism of action have attracted the attention of researchers in the field of cancer treatment. Currently, the development of anti-tumor drugs targeting Trop2 focuses primarily on antibody-drug conjugates (ADCs). The clinical progress of Trop2 ADCs in the treatment of solid tumors highlights the potential of Trop2 as a tumor immunotherapy target. Compared to ADCs, research on immune cell modification with chimeric antigen receptors (CARs) targeting Trop2 is still in its early stages and lacks clinical progress. The development of effective CARs targeting Trop2 is still needed. Summary of the Invention

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

[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, 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 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 as 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 above-mentioned Nanobody 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 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 in 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 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 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 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 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 in 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 in 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 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 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 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 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 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 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 in 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 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 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 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, it 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, wherein the antibody 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] The Fc fragment includes the Fc fragment of human IgG, which can be selected from the 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 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 in sequence.

[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 costimulatory 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] Based on the amino acid sequences of the 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. 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 mentioned above include prokaryotic cells or eukaryotic cells. 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 and Saccharomyces cerevisiae. Mammalian cells include, but are not limited to, immune cells (e.g., T cells, NK cells, etc.), stem cells or progenitor cells capable of differentiating 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).

[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 labeling agent mentioned above is 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 a ninth aspect, the present invention provides a method for producing the Trop2-binding nanobody or the Trop2-binding antibody or the fusion protein or the chimeric antigen receptor, the method comprising: culturing a host cell containing the nucleic acid molecule, and collecting the Trop2-binding nanobody or the Trop2-binding antibody or the fusion protein or the chimeric antigen receptor from the culture.

[0036] The collection from the 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 reagents 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 using Trop2 as a marker, preferably a tumor, more preferably a malignant tumor; preferably a tumor that highly expresses TROP2, especially 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.

[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, is a nanobody, and is the antibody structure with the smallest protein molecular weight known to date; compared with traditional monoclonal antibodies and single-chain variable fragments (scFv), nanobodies have the advantages of small molecular weight, low aggregation and precipitation, and higher stability, and while maintaining high affinity for antigens, the long and flexible complementary determining region CDR3 of nanobodies allows binding to the cracks and cavities of the target antigen, and can recognize some hidden epitopes. The anti-Trop2 nanoantibodies provided by the present invention can efficiently and specifically recognize and bind to the Trop2 protein. Based on the nanoantibodies, 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 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 are used for the diagnosis and treatment of diseases with Trop2 molecules as markers.

[0048] The present invention also provides CAR and CAR-T cells that bind to the Trop2 protein. Cell-level experiments have verified that the CAR-T cells have a good immune clearance effect targeting Trop2 and can be used to specifically identify and kill Trop2-expressing cells, 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 are used to improve and treat diseases that use Trop2 molecules as markers.

[0049] The anti-Trop2 CAR of the present invention uses nanobodies that bind to the Trop2 protein as the specific recognition structural component of the antigen. Nanobodies have the advantage of 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, improving the safety of CAR in tumor immunotherapy, and obtaining 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, a brief introduction is given below to 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 any 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, where NC represents the negative control.

[0055] Figure 5The following are the detection of downstream signal activation levels of the anti-Trop2 CAR J-NN under stimulation of different target cells and the comparison of peak values ​​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 intensities of the anti-Trop2 CAR J-NN when co-cultured with Trop2-positive target cells CFPAC-1 and Trop2-negative target cells AsPC-1, respectively. c and d are the green fluorescence signal intensities and red fluorescence signal intensities of the non-CAR-transduced J-NN and the anti-Trop2 CAR J-NN after co-culture with the four target cells for 12 hours, respectively. au represents arbitrary unit.

[0056] Figure 6 Comparison of downstream signal activation levels of the 12 anti-Trop2 CAR J-NNs under stimulation of the positive target cells BxPC-3 in Example 4 of the present invention. References a and b represent the green fluorescence signal intensity and red fluorescence signal intensity of the untransduced CAR J-NN and the 12 anti-Trop2 CAR J-NNs, respectively, after co-culture with BxPC-3 for 12 hours.

[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, where NC represents the negative control.

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

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

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

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

[0062] Figure 12 The in vitro killing effect of anti-Trop2 CAR-T (T2-F09) cells on 3D tumor spheroids in Example 8 of the present invention is shown in Figure a, which is captured using the Incucyte S3 real-time imaging system; and Figure b, which is the in vitro killing kinetics of anti-Trop2 CAR-T cells on tumor spheroids. DETAILED DESCRIPTION

[0063] In a specific embodiment of the present invention, an antibody that binds to the Trop2 protein is provided. This antibody is a low-molecular-weight antibody derived from alpacas and is screened from an alpaca immune library using phage display technology. Alpacas are first immunized with purified Trop2 protein. After successful immunization, peripheral blood is collected to extract lymphocytes containing mature B cells. RNA is extracted and reverse-transcribed into a cDNA library. PCR primers are used to amplify the heavy chain variable regions of the alpaca antibodies to obtain specific gene fragments. These fragments are then spliced ​​into a phage vector and transformed into Escherichia coli to obtain a nanobody gene library, i.e., an immune library. After four rounds of panning using phage display technology, positive clones are identified using indirect ELISA, and the antibody sequences that bind to the Trop2 antigen are confirmed by sequencing.

[0064] The Trop2-binding antibodies obtained above comprise the heavy chain variable domain (VHH) of a heavy-chain antibody. The amino acid sequences of the VHHs of each antibody are shown in Table 1. The amino acid sequences of each antibody were numbered using the Martin numbering scheme and defined using different CDR region definition schemes to obtain the CDR1, CDR2, CDR3 regions and framework regions FR1, FR2, FR3, and FR4. The amino acid sequences of the CDR regions are shown in Table 2, and the amino acid sequences of FR1, FR2, FR3, and FR4 are shown in Table 3.

[0065] Table 1 Table 2 Table 3 The above-mentioned antibodies that bind 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 that use Trop2 molecules as markers.

[0066] 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 host cell comprising 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 the 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, wherein SP is a signal peptide, HINGE is a hinge region, TM is a transmembrane region, CD is a costimulatory domain, SD is a signal transduction domain, and "-" is a connecting peptide or peptide bond.

[0067] In the present invention, SP, HINGE, TM, CD, and SD can 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.

[0068] The chimeric antigen receptor binding to the Trop2 protein described above uses a nanobody that binds to the Trop2 protein as an antigen-specific recognition structural component. The chimeric antigen receptor is transduced into a host cell with an immune effector using a vector such as a lentivirus, and can target and recognize cells expressing Trop2. After being stimulated by an antigen, it can release various pro-apoptotic cytokines such as granzymes, perforins, and interferon-γ, causing target cell death and exerting an immune clearance function. Therefore, the chimeric antigen receptor binding 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, and is used to improve and treat diseases with Trop2 molecules as markers.

[0069] 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.

[0070] In a specific embodiment of the present invention, a host cell is also provided, wherein the host cell is an immune cell or a stem cell or progenitor cell capable of differentiating into an immune cell. Immune cells include, but are not limited to, T cells, NK cells, NKT cells, macrophages, and dendritic cells, and have killing or phagocytic functions, immunomodulatory functions, and cytokine release functions. The stem cell can be a hematopoietic stem cell or an induced pluripotent stem cell, and the stem cell can differentiate into one or more of the above-mentioned immune cells.

[0071] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0072] Example 1: Screening of Nanobodies Binding to Trop2 Protein Healthy adult alpacas were immunized with the human TROP-2 / TACSTD2 protein (His Tag, ACRO, Catalog No. TR2-H5223) as an antigen. After four immunizations, 20 days apart, the antibody titers in peripheral blood serum were measured. Peripheral blood was collected and peripheral blood mononuclear cells (PBMCs) were extracted. RNA was extracted and reverse transcribed to obtain cDNA. The heavy chain variable regions were amplified by PCR to obtain VHH gene fragments, which were then spliced ​​into a phage vector and transformed into Escherichia coli to generate a nanobody gene library.

[0073] Biopanning of anti-Trop2 nanobodies using phage display technology. The specific steps are as follows: 1) Antigen coating: Coat the immunotube with TROP2 antigen (30µg / tube), rotate slowly at 4°C overnight, and coat 30µg of 5% non-fat milk powder in parallel as a control; 2) Wash unbound antigen: Discard the coating solution, add 2 mL of PBS buffer and wash the immunotube 3 times at room temperature, rotating for 5 minutes each time; 3) Block: Add 5 mL of blocking solution and rotate at room temperature for 2 hours; 4) Wash the blocking solution: Discard the liquid in the blocked immunotube and add 5 mL of PBS buffer to wash the immunotube 3 times at room temperature, rotating for 5 minutes each time; 5) Add phage display library for binding: Discard the washing solution in the immunotube, add 2 mL of PBS buffer, add the prepared phage library as the first round of screening input phage library, and incubate at room temperature for 1 hour; 6) Wash unbound phage: Discard the liquid in the immunotube, add 5 mL of PBST (1×PBS plus 0.1% Tween20) buffer and wash the immunotube 20 times at room temperature, rotating for 5 minutes each time. 7) Elute bound phage: Add 1 mL of pH 2.2 Glycine-HCl solution and rotate at room temperature for 30 minutes. Terminate elution by adding 90 μL of pH 8.8 Tris-HCl. Transfer the solution from the immunotube to a new 1.5 mL centrifuge tube. This is the phage eluate from the first round of screening. 8) Amplify the phage from the first round and use them for the subsequent second round of screening and detection. Repeat this screening process for three rounds. After each round, streak the phages to calculate the enrichment efficiency and select single clones for antigen-specific detection.

[0074] Use phage ELISA to screen and identify positive monoclonal clones. The specific steps are as follows: after each round of phage eluate enrichment, the ELISA plate is coated with TROP2 protein antigen, BSA is used as the control antigen, the antibody displayed by the selected cultured monoclonal phage is used as the test antibody binding antigen, the unbound phage is eluted, and the anti-phage M13 antibody labeled with HRP is used as the secondary antibody to bind to the phage, the substrate is added for color development, and the microplate reader is used to read the OD value of the TROP2 test group and the BSA control group. 450 The difference was used to preliminarily determine the positive clones.

[0075] 95 positive clones from the phage ELISA results were selected and sent to Qingke Biotech Co., Ltd. for sequencing to identify the diversity of the positive clone sequences. The sequenced sequences were converted into amino acid sequences, and the same amino acid sequences were aligned and merged to obtain 12 positive antibody clones from Trop2 phage screening. The ELISA test results of the above 12 positive clones are as follows: Figure 1 As shown. For each of the 12 positive antibody clones screened with Trop2 phage, the coding nucleotide sequences corresponding to their amino acid sequences were synthesized, each with an amino acid tag / linker (GGGGS) × 3 coding nucleotide sequence to facilitate subsequent experimental detection. Each of the 12 Trop2 VHH genes with the aforementioned tags was synthesized.

[0076] Example 2: Flow cytometry detection of Trop2-positive and -negative target cells Surface staining with a Trop2 flow cytometry antibody (PE Mouse Anti-Human Trop-2, BD Biosciences, Cat. No. 564837) was used to detect the expression of Trop2 protein on the surface of four human pancreatic cell lines: three pancreatic cancer cells, AsPC-1, BxPC-3, and CFPAC-1, and an immortalized human normal pancreatic ductal epithelial cell line, hTERT-HPNE. The assay steps were as follows: the cells to be tested were digested with trypsin, centrifuged, resuspended in culture medium, and counted. 1×10 cells were collected for each of the stained and blank control groups. 6 Cells were centrifuged and the supernatant removed. Cells were washed once with pre-prepared FACS buffer (1× PBS + 2% FBS or 1× PBS + 0.5% BSA). For the stained cells, a Trop2 antibody diluted in FACS buffer was added and incubated at 4°C for 30 minutes. The supernatant was removed by centrifugation, and the cells were washed three times with FACS buffer. The cells were then resuspended in 200 μL of FACS buffer and analyzed.

[0077] The flow cytometry analysis results of Trop2 expression in four cell lines are as follows Figure 2As 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.

[0078] 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 seamlessly cloned with the gene-synthesized CAR backbone to construct a PCDH lentiviral expression vector (the vector was modified and the promoter was EF1α). The accuracy of the plasmid sequence was verified by sequencing to obtain the lentiviral master plasmid expressing the anti-Trop2 CAR. The molecular composition of the complete anti-Trop2 CAR is as follows: Figure 3 As 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 costimulatory domain, and a CD3ζ intracellular signal transduction domain connected in series.

[0079] Lentivirus was prepared by co-transfecting 293T cells with three plasmids. The transfection reagent used was PEI MAX (linearized polyethyleneimine PEI 40000) (Polyethylenimine Max, MW40000, Polysciences, Cat. No. 24765-1). The three plasmids included a master lentiviral plasmid expressing the anti-Trop2 CAR and two packaging plasmids, psPAX2 and pMD2.G. Well-grown 293T cells were plated one day in advance and transfected at approximately 80% confluence. The plasmids and transfection reagent were diluted in serum-free medium to a mass ratio of 3:2:1 for the master lentiviral plasmid:psPAX2:pMD2.G. The ratio of transfection reagent to plasmid was 2.5 μL:1 μg. The diluted transfection reagent and plasmids were mixed thoroughly and allowed to stand at room temperature for 20 minutes. The transfection suspension was then added to the 293T cells and gently shaken to mix thoroughly. 8 hours after transfection, the medium was changed. Supernatants from 48 and 72 hours of culture were collected and centrifuged to remove cell debris. The supernatant was added to a lentiviral concentrate (PEG8000 and NaCl), mixed thoroughly, and incubated at 4°C overnight to concentrate the virus. The virus was then centrifuged at 4000g for 20 minutes at 4°C. After centrifugation, the supernatant was discarded and the viral pellet was resuspended in an appropriate amount of PBS or culture medium to obtain the recombinant lentiviral vector containing the anti-Trop2 CAR. The lentiviral titer was verified to meet the requirements of subsequent experiments using a rapid lentiviral titer test kit (Biodragon, Cat. No. BF06202-100).

[0080] Example 4: Expression and downstream signal activation detection of anti-Trop2 CAR transduced dual reporter cell line J-NN 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 (abbreviated as J-NN) carrying pNFAT-EGFP and pNFκB-mCherry. It can quickly and high-throughput complete the activation signal characteristics of novel CAR molecular designs and is an experimental method for rapid screening of novel CARs.

[0081] The anti-Trop2 CAR was transduced into J-NN cells using the lentiviral vector described 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 the flow cytometry staining method described in Example 2 using the G4S antibody (Anti-(G4S)n-(B02H1)m-Ab (PE), Heyousheng Biotechnology, catalog number GS-ARPE100). Taking T2-F09 as an example, Figure 4 As shown in the figure, the positive expression rate of anti-Trop2 CAR was above 50%.

[0082] 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 for each group. The next 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 2. 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, 12 types of anti-Trop2 CAR J-NN cells were co-cultured with BxPC-3 to detect the activation of downstream signals. The results are shown in the figure. 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.

[0083] Example 5: Preparation of anti-Trop2 CAR-T cells T cells were isolated from freshly isolated or revived human peripheral blood mononuclear cells (PBMCs) using magnetic bead negative selection (Pan T Cell Isolation Kit, Miltenyi, Cat. No. 130-096-535). CD3 / CD28 magnetic beads (Dynabeads Human T-activator CD3 / CD28, Thermo Fisher Scientific, Cat. No. 11132D) were added at a bead:cell ratio of 1:1 to isolate the purified CD3 + T cells were activated and cultured in serum-free T cell medium (X-VIVO 15, Lonza, Catalog No. 04-418Q) supplemented with IL-2 (Sihuan Biopharmaceuticals, Catalog No. S10970015) at a final concentration of 200 IU / mL. After 24 hours of magnetic bead activation and culture, the anti-Trop2 CAR lentivirus described in Example 3 and polybrene (Merck, Catalog No. TR-1003-G) staining reagent at a final concentration of 6 μg / mL were added. After 16 hours of lentiviral infection, the medium was changed and culture continued.

[0084] After 4 days of activation and 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 No. GS-ARPE100). Taking T2-F09 as an example, Figure 7 As shown, the positive expression rate of anti-Trop2 CAR-T cells was above 50%. T cells were then expanded and cultured in serum-free medium containing 200 IU / mL IL-2 for subsequent CAR-T function testing.

[0085] Example 6: In vitro killing efficiency of anti-Trop2 CAR-T cells against different target cells The above-mentioned 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 for each group. The next 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 shown in Figure 2. 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.

[0086] The in vitro cytotoxicity at 72 h was calculated according to the following formula: In vitro cytotoxicity = (normalized fluorescence intensity of the single target group - normalized fluorescence intensity of the experimental group) / normalized fluorescence intensity of the single target group × 100%. Figure 9 As shown in the figure, compared with the T cell control group, anti-Trop2 CAR-T cells have a high specificity for killing 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. The results are shown in Figure 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.

[0087] Example 7: Cytokine Release Detection of Anti-Trop2 CAR-T Cells The cells were plated with the reference to the in vitro killing experiment in Example 6 with a target to effect ratio of 4:1. The culture medium for the effector cells was T cell serum-free culture medium without IL-2. After co-culturing the target cells and effector cells for 24 hours, the supernatant was collected and the secretion of three cytokines, IL-2 (Human IL-2 Precoated ELISA Kit, Dakoway, Catalog No. 1110202), IFNγ (Human IFN-γ Precoated ELISA Kit, Dakoway, Catalog No. 1110002), and TNFα (Human TNF-α Precoated ELISA Kit, Dakoway, Catalog No. 1117202), was detected by ELISA. The results are shown in Figure 5. Figure 11 As shown, compared to the T cell control group, anti-Trop2 (T2-F09) CAR-T cells co-incubated with two Trop2-positive target cells, BxPC-3-GFP and CFPAC-1-GFP, released significant amounts of IFNγ, TNFα, and IL-2 after 24 hours of incubation. In contrast, cytokine levels were lower in the two Trop2-negative target cells, AsPC-1-GFP and hTERT-HPNE-GFP. This demonstrates that anti-Trop2 CAR-T cells can be activated by Trop2-positive target cells, producing a series of cytokines associated with immune activation.

[0088] Example 8: In vitro killing efficiency of anti-Trop2 CAR-T cells against tumor spheres Trop2-positive target cells BxPC-3 with GFP fluorescence were plated in a 96-well clear round-bottom ultra-low attachment microplate (Corning, Cat. No. 7007) at a density 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 effector-target ratios 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 Spheroid mode was selected, the BF+GFP channel was selected, the shooting interval was 4 hours, and the shooting duration was 3 days. The results are shown in Figure 2. Figure 12 As shown in the figure, 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.

[0089] 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 various embodiments of the present invention.

Claims

1. A nanobody that binds to Trop2, characterized in that 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 (2): (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. 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; (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. 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.

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 (7): (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. 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 as 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 as 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; (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. 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 as 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; (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, 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 as 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; (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. 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; (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, 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 as 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; (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, 116, 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, 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; (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, 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 in SEQ ID NOs. 103, 114, 123 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.134, 135, 136, 137, 138, 139, 140 or 141.

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, the Trop2-binding nanobody according to any one of claims 1 to 3 or the Trop2-binding antibody 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 reagents 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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