Methods and applications for preparing nanobodies and conjugates that target tissue factors.

Abstract

This paper discloses novel tissue factor (TF)-targeting nanobodies (Nb) and nanobody-drug conjugates (NDCs), methods for their preparation, and applications. Monoclonal nanobodies and corresponding NDCs can efficiently and highly specifically bind to purified TF proteins and TFs on the surface of tumor cells that abnormally express multiple types of TF. They possess very high affinity and very low immunogenicity, and exhibit remarkable antitumor effects both in vivo and in vitro.

Description

[Technical Field]

[0001] This invention relates to medical components, and more particularly to nanobodies and nanobody-drug conjugates targeting tissue factor, methods for preparing them, and their uses. [Background technology]

[0002] Tissue factor (TF) is a transmembrane glycoprotein with a molecular weight of 47 kDa. Under physiological conditions, TF activates the exogenous blood coagulation reaction after vascular injury. However, TF is abnormally activated and expressed in many tumor tissues, and in breast cancer, pancreatic cancer, lung cancer, and esophageal cancer, the abnormal expression rate is very high. For example, the abnormal expression rate of TF in breast cancer is 85.8%, in pancreatic cancer it is 88.5%, in lung cancer it is 83.6%, and in esophageal cancer it is 91.3% (Blood, 2012, 119:924-932). The TF / FVIIa complex can also directly bind to and induce activation of the transmembrane G protein coupling receptor Protease-activated receptor 2 (PAR2). TF can influence a range of intracellular tumor function signals via PAR2. For example, TF-PAR2 induces gene expression of key growth factors, immunomodulators, and chemokines through MAPK / ERK phosphorylation, promoting angiogenesis and providing sufficient nutrients, energy, and a suitable microenvironment for tumor growth. Furthermore, TF can enhance tumor cell mobility and adhesion through interaction with Rac1, β1 family-related integrins, thereby increasing the overall hematogenous metastatic capacity of tumor cells (Journal of Thrombosis Research, 2012, 130:S84-S87; Journal of Thrombosis and Haemostasis, 2013, 11:285-293; International Journal of Cancer, 2014, doi:10.1002 / ijc.28959; Blood, 2012, 119:924-932). Therefore, given that international research is currently underway on the effects and functions of TF-related diseases, the development of specific therapeutic antibodies targeting TF would be extremely beneficial for the diagnosis, treatment, and prevention of pathological features caused by TF, such as angiogenesis and abnormal blood coagulation, in various diseases including cancer, thrombosis, and inflammation.Tivdak (TF-ADC Tisotumab Vedotin), the world's first TF-targeting antitumor drug, received marketing approval in 2021, and several TF-ADCs developed by multiple pharmaceutical companies are currently in clinical trials.

[0003] Monoclonal antibody drugs have brought about a groundbreaking advance in cancer treatment, leading to improvements in clinical therapy across multiple cancer areas. However, conventional monoclonal antibodies have a molecular weight of 150 kd, which limits their penetration into solid tumors and drug distribution, thus requiring further improvement in therapeutic efficacy. Nanobody (Nb) is an antibody consisting solely of heavy chains naturally produced in alpacas, where the target recognition module is composed of a single heavy chain variable region (VHH) with a molecular weight of only 12-14 kd, potentially overcoming the drawback of conventional monoclonal antibodies' poor penetration into solid tumors. Nanobody can be conjugated to functional domains such as Fc, other nanobody, polypeptide tags, or toxins. Due to their small volume and characteristics such as higher diffusion rate, vascular permeability, penetration of internal barriers such as the blood-brain barrier, and strong tumor penetration, they exhibit more uniform tissue distribution than conventional monoclonal antibodies. These characteristics make them particularly suitable for specific and effective tumor-targeted therapy in vivo. Nanobodies offer further advantages such as low immunogenicity, high stability, high resistance to degradation, low manufacturing costs due to prokaryotic expression, and ease of product characterization. However, despite many potential advantages, the development of nanobody-based oncology therapies remains in the early stages of exploratory research.

[0004] In recent years, antibody-drug conjugates (ADCs) have become a new trend in tumor treatment. ADCs utilize the property of monoclonal antibodies to specifically recognize certain antigens on the surface of tumor cells, thereby precisely delivering and releasing antitumor drugs (e.g., small molecule chemotherapeutic agents) to tumor target cells, achieving the goal of precisely killing tumors. However, due to limitations such as the large molecular weight of ADCs and their lower permeability into tumors, the absorption and distribution effect of drugs within tumors is limited and varies greatly, making their application to solid tumors less than ideal. Furthermore, because ADCs have a long terminal half-life in the body's circulatory system, the toxins they carry may exert undesirable toxicity on normal tissues or cells. Nanobody-drug conjugates (NDCs) prepared using nanobodies offer the advantages of conventional ADCs, plus high vascular permeability, excellent barrier penetration (such as the blood-brain barrier), potent tumor penetration, and rapid target cell delivery. This allows for improved drug accumulation in tumors while simultaneously enabling appropriate control of drug plasma exposure and half-life. This is expected to contribute to further improvements in therapeutic efficacy and the overall treatment window for solid tumors, making them the most promising new antitumor agents.

[0005] In summary, based on the crucial role of TF in the mechanisms of tumor progression, recurrence, and drug resistance, the development of drugs targeting TF-Nb and TF-NDC, either alone or in combination, may offer novel therapeutic strategies for the clinical treatment of tumor patients exhibiting abnormal TF expression. [Overview of the project] [Problems that the invention aims to solve]

[0006] The object of the present invention is to provide nanobodies and nanobody-drug conjugates that target TF, methods for preparing them, and applications. [Means for solving the problem]

[0007] The first aspect of the present invention provides a nanobody targeting TF, and the complementarity determining regions CDRs of the VHH chain of the nanobody are (1) CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, and CDR3 shown in SEQ ID NO.3, or (2) CDR1 shown in SEQ ID NO.5, CDR2 shown in SEQ ID NO.6, and CDR3 shown in SEQ ID NO.7, or (3) CDR1 shown in SEQ ID NO.9, CDR2 shown in SEQ ID NO.10, and CDR3 shown in SEQ ID NO.11, or (4) CDR1 shown in SEQ ID NO.13, CDR2 shown in SEQ ID NO.14, and CDR3 shown in SEQ ID NO.15, or (5) CDR1 shown in SEQ ID NO.17, CDR2 shown in SEQ ID NO.18, and CDR3 shown in SEQ ID NO.19, or (6) CDR1 shown in SEQ ID NO.21, CDR2 shown in SEQ ID NO.22, and CDR3 shown in SEQ ID NO.23, or (7) CDR1 shown in SEQ ID NO.25, CDR2 shown in SEQ ID NO.26, and CDR3 shown in SEQ ID NO.27, and is one or more selected from the group consisting of or In another preferred example, any one of the amino acid sequences may optionally further include a derivative sequence in which at least one amino acid is added, deleted, modified and / or substituted and can maintain the TF binding affinity.

[0008] In another preferred example, the CDR region of the nanobody VHH chain includes an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, still more preferably at least 99% sequence similarity to the CDR region described in any of the above. In another preferred example, the amino acid sequence of the CDR region of the nanobody VHH chain includes one or more amino acid substitutions, preferably conservative amino acid substitutions, as compared to any of the CDR regions described in any of the above.

[0009] In another preferred example, the VHH chain (1) Complementary determining regions CDR1, CDR2, CDR3 shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 (corresponding to the CDRs of nanobody 4-A02), or (2) Complementary determining regions CDR1, CDR2, CDR3 shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 (corresponding to the CDRs of nanobody F1-A01), or (3) Complementary determining regions CDR1, CDR2, CDR3 shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 (corresponding to the CDRs of nanobody 4-C08), or (4) Complementary determining regions CDR1, CDR2, CDR3 shown in SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15 (corresponding to the CDRs of nanobody 4-E04), or (5) Complementary determining regions CDR1, CDR2, CDR3 shown in SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19 (corresponding to the CDRs of nanobody 4-C05), or (6) Complementarity determination regions CDR1, CDR2, CDR3 (corresponding to the CDR of nanobody 5-C12) shown in SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, or (7) Includes CDR1, CDR2, and CDR3 selected from the group consisting of complementarity determination regions CDR1, CDR2, and CDR3 (corresponding to the CDR of nanobody 5-H3) shown in SEQ ID NO. 25, SEQ ID NO. 26, and SEQ ID NO. 27.

[0010] In another preferred example, CDR1, CDR2, and CDR3 are isolated by framework regions FR1, FR2, FR3, and FR4 of the VHH chain.

[0011] In another preferred example, the nanobody includes a human antibody, a camel-derived antibody, and a chimeric antibody.

[0012] In another preferred example, the VHH chain of the nanobody further comprises a framework region (FR).

[0013] In another preferred example, the framework region FR is derived from a human, mouse, rabbit, or camel.

[0014] In another preferred example, the framework region FR includes a human-derived FR region, a mouse-derived or camel-derived FR region.

[0015] In another preferred example, the VHH chain of the nanobody targeting the TF has an amino acid sequence selected from the group consisting of SEQ ID NO.4, SEQ ID NO.8, SEQ ID NO.12, SEQ ID NO.16, SEQ ID NO.20, SEQ ID NO.24, and SEQ ID NO.28.

[0016] In another preferred example, the VHH chain of the nanobody targeting the TF has an amino acid sequence such as that shown at positions 1-114 of SEQ ID NO.33, positions 1-114 of SEQ ID NO.34, or positions 1-114 of SEQ ID NO.35.

[0017] In another preferred example, the nanobody sequence includes amino acid sequences having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence similarity with SEQ ID NO.4, SEQ ID NO.8, SEQ ID NO.12, SEQ ID NO.16, SEQ ID NO.20, SEQ ID NO.24, and SEQ ID NO.28.

[0018] A second aspect of the present invention provides an antibody that targets TF, wherein the antibody comprises one or more VHH chains of the TF-targeting nanobody described in the first aspect of the present invention.

[0019] In another preferred example, the VHH chain of the nanobody targeting the TF has an amino acid sequence selected from the group consisting of SEQ ID NO.4, SEQ ID NO.8, SEQ ID NO.12, SEQ ID NO.16, SEQ ID NO.20, SEQ ID NO.24, and SEQ ID NO.28.

[0020] In another preferred example, the antibody is a monomer, a bivalent antibody, and / or a polyvalent antibody.

[0021] In another preferred example, the antibody is an animal-derived antibody, a human antibody, a chimeric antibody, or a chimeric antigen receptor antibody (CAR).

[0022] In another preferred example, the CDR region of the human antibody comprises one, two, or three amino acid changes.

[0023] In another preferred example, the animal is a non-human mammal, preferably a mouse, sheep, rabbit, or camel.

[0024] In another preferred example, the antibody is a double-chain antibody or a single-chain antibody.

[0025] In another preferred example, the antibody is a monoclonal antibody.

[0026] In another preferred example, the antibody is a partially or completely humanized monoclonal antibody.

[0027] In another preferred example, the antibody is a human antibody, and the VHH chain of the nanobody targeting the TF has the amino acid sequence shown at positions 1-114 of SEQ ID NO.33, positions 1-114 of SEQ ID NO.34, or positions 1-114 of SEQ ID NO.35.

[0028] In another preferred example, the number of added, deleted, modified and / or substituted amino acids does not exceed 40%, preferably not exceeding 20%, and more preferably not exceeding 10%, of the total number of amino acids in the initial amino acid sequence.

[0029] In another preferred example, the number of added, deleted, modified and / or substituted amino acids is 1 to 7, preferably 1 to 3, more preferably 1.

[0030] In another preferred example, the added, deleted, modified and / or substituted at least one amino acid sequence is an amino acid sequence having at least 80% homology.

[0031] In another preferred example, the derivative sequence in which at least one amino acid is added, deleted, modified, and / or substituted has a biological function that inhibits cell surface TF or recombinant TF protein.

[0032] In another preferred example, the antibody is in the form of a drug conjugate.

[0033] In another preferred example, the KD values ​​of the binding constants of the nanobody to the extracellular domain (TF-ECD) of the human TF protein, as measured by ForteBio surface plasmon resonance (SPR), are 0.202 to 7.04 nM.

[0034] In another preferred example, the antibody is (a) Specifically binds to tumor cells and / or immune cells / stromal cells in the tumor microenvironment, (b) Specifically inhibit the tumor TF-associated tumor cell signaling pathway, (c)Improve cytokine secretion disorders caused by TF overactivation, (d) Inhibit the migration or metastasis of tumor cells, (e) Inhibit tumor growth, thereby improving the efficacy of the combination therapy antitumor, (f) Promote the proliferation, survival and function of immune cells, thereby improving the effect of tumor immunity, (g) After binding to tumor cells, it is taken up by intracellular lysosomes, (h) It has the characteristic of being able to pass through the blood-brain barrier and be efficiently distributed within the brain, (i) It has excellent therapeutic effects against brain tumors, (j) Having one or more characteristics selected from the group including having excellent therapeutic effects against multiple types of solid tumors.

[0035] A third aspect of the present invention provides a multispecific antibody comprising a nanobody targeting a TF as described in the first aspect of the present invention or an antibody targeting a TF as described in the second aspect of the present invention.

[0036] In another preferred example, the multispecific antibody further comprises a second antigen-binding region that targets a target point selected from the group consisting of EGFR, TGFβ, BCMA, B7H6, GUCY2C, DLL3, CD38, CD123, CD19, CD20, CD22, B7-H3, GPC3, HER2, PMSA, CD28, 4-1BB, OX40, CD40, CD27, CD3, CTLA4, PD1, PDL1, CD73, BCMA, GLP-1, Trop2, TIGIT, LAG-3, FGL1, TLR7, or a combination thereof.

[0037] In another preferred example, the second antigen-binding region is a nanobody.

[0038] In another preferred example, the multispecific antibody comprises one or more secondary antigen-binding regions.

[0039] In another preferred example, the multispecific antibody further comprises an Fc segment of the antibody.

[0040] In another preferred example, the antigen-binding region is an antibody or antibody fragment, the antibody fragment comprising (i) a Fab fragment, (ii) an F(ab')2 fragment, (iii) an Fd fragment, (iv) an Fv fragment, (v) a single-stranded Fv(scFv) molecule, and (vi) an dAb fragment.

[0041] A fourth aspect of the present invention provides a recombinant protein, the recombinant protein is (i) Nanobodies that target TF, such as the nanobody that targets TF as described in the first aspect of the present invention, the anti-TF antibody as described in the second aspect of the present invention, or the multispecific antibody as described in the third aspect of the present invention, and (ii) Any polypeptide molecule or fragment having therapeutic function, and / or (iii) Having any functional domain that improves the physicochemical properties or drug potential of the protein. In another preferred example, improving the physicochemical properties or drug potential of the protein includes extending the half-life of nanobodies targeting the TF.

[0042] In another preferred example, the recombinant protein further comprises (iv) an optional tag sequence to aid in expression and / or purification.

[0043] In another preferred example, the tag sequence is selected from the group consisting of 6His tags, GGGS sequences, and FLAG tags.

[0044] In another preferred example, the recombinant protein is a monomer, a dimer, or a polymer.

[0045] In another preferred example, the polypeptide molecule or fragment having therapeutic function includes, but is not limited to, polypeptide molecules or fragments targeting EGFR, TGFβ, BCMA, B7H6, GUCY2C, DLL3, CD38, CD123, CD19, CD20, CD22, B7-H3, GPC3, HER2, PMSA, CD28, 4-1BB, OX40, CD40, CD27, CD3, CTLA4, PD1, PDL1, CD73, BCMA, GLP-1, Trop2, TIGIT, LAG-3, FGL1, and TLR7.

[0046] In another preferred example, the polypeptide molecule or fragment having therapeutic function includes, but is not limited to, insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analogues, albumin, antibody fragments, and cytokines.

[0047] In another preferred example, the recombinant protein (or polypeptide) includes a fusion protein.

[0048] In another preferred example, the fusion protein includes a multispecific antibody and a chimeric antibody.

[0049] In another preferred example, the functional domain that enhances the physicochemical properties or drug potential of the protein includes an Fc segment, an anti-albumin nanobody (HLE), and an albumin-binding domain (ABD).

[0050] In another preferred example, the recombinant protein has the following structure from the NC terminus or CN terminus: (AB)m, Here, the A element is a nanobody that targets TF, The B element is an Fc segment, a human albumin-binding domain (ABD), or an anti-albumin nanobody (HLE). "-" represents a peptide bond or linker. Here, m is a positive integer.

[0051] In another preferred example, the linker is a rigid linker or a flexible linker.

[0052] In another preferred example, the B element further comprises any functional domain that enhances the physicochemical properties or drug potential of the protein, or any tag sequence that assists in expression and / or purification. In another preferred example, m is 1, 2, 3, or 4. In another preferred example, the nanobody targeting the TF is the nanobody targeting the TF described in the first aspect of the present invention, the anti-TF antibody described in the second aspect of the present invention, or the multispecific antibody described in the third aspect of the present invention.

[0053] In another preferred example, the VHH chain of the nanobody targeting the TF has an amino acid sequence selected from the group consisting of SEQ ID NO.4, SEQ ID NO.8, SEQ ID NO.12, SEQ ID NO.16, SEQ ID NO.20, SEQ ID NO.24, and SEQ ID NO.28.

[0054] In another preferred example, the Fc segment is a human IgG Fc segment, preferably derived from human IgG1 or human IgG2.

[0055] In another preferred example, the Fc segment is selected from FC1, FCWT, or FCLALAPG, where, The sequence of FC1 is as shown at positions 115-346 of SEQ ID NO. 29. The sequence of the aforementioned FCWT is as shown at positions 115-346 of SEQ ID NO.30. The sequence of FCLALAPG is as shown at positions 115-346 of SEQ ID NO. 31.

[0056] In another preferred example, the amino acid sequence of ABD is as shown at positions 130-198 of SEQ ID NO. 32.

[0057] In another preferred example, the recombinant protein has an amino acid sequence selected from the group consisting of SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, and SEQ ID NO. 32.

[0058] In another preferred example, the recombinant protein comprises a human antibody and preferably has an amino acid sequence selected from the group consisting of positions 1-114 of SEQ ID NO. 33, positions 1-114 of SEQ ID NO. 34, or positions 1-114 of SEQ ID NO. 35.

[0059] A fifth aspect of the present invention provides a CAR construct in which the antigen-binding region of the CAR construct is a VHH chain of the nanobody described in the first aspect of the present invention.

[0060] A sixth aspect of the present invention provides recombinant immune cells that express the exogenous CAR construct described in the fifth aspect of the present invention.

[0061] In another preferred example, the immune cells are selected from a group consisting of NK cells and T cells.

[0062] In another preferred example, the immune cells are derived from humans or non-human mammals (e.g., mice).

[0063] A seventh aspect of the present invention provides an immune complex, the immune complex is (a) an antibody portion which is a nanobody that targets TF as described in the first aspect of the present invention or an antibody that targets TF as described in the second aspect of the present invention, and (b) comprising a coupling portion coupled to the nanobody portion selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, an enzyme, a proteolytic agent, an immunomodulator (immunoagonist and inhibitor), an oligonucleotide, or a combination thereof.

[0064] In another preferred example, the immune complex is a nanobody drug conjugate (NDC).

[0065] In another preferred example, the nanobody portion and the coupling portion are coupled via a chemical bond or a linker.

[0066] In another preferred example, the coupling portion is a chemical label and a biomarker.

[0067] In another preferred example, the chemical label is an isotope, an immunotoxin, and / or a chemical drug.

[0068] In another preferred example, the biomarker is biotin, avidin, or enzyme-labeled.

[0069] In another preferred example, the coupling portion is a drug or a toxin.

[0070] In another preferred example, the drug is a cytotoxic drug.

[0071] In another preferred example, the cytotoxic drug is selected from the group consisting of antitubulin agents, DNA supraclavicular junction reagents, DNA replication inhibitors, alkylating reagents, antibiotics, folic acid antagonists, antimetabolites, chemotherapeutic sensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof. Particularly useful examples of cytotoxic drug systems include, for example, DNA supraclution binding reagents, DNA alkylation reagents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

[0072] In another preferred example, the toxin is Auristatin derivatives (e.g., auristatin E, auristatin F, MMAE, and MMAF), chlortetracycline, maytansinoids, lysine, lysine A-chain, combretastatin, duocalmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine The following are selected from the group consisting of dihydroxyanthracine dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modexin A chain, α-sarcin, geronin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria officinalis inhibitors, glucocorticoids, or combinations thereof.

[0073] In another preferred example, the coupling portion is a detectable marker.

[0074] In another preferred example, the detectable marker includes a radionuclide, and the radionuclide is (i) Diagnostic isotopes selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof, and / or (ii) A therapeutic isotope selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or a combination thereof.

[0075] In another preferred example, the proteolytic agent is a tumor-associated protein degrader, and the tumor-associated protein is selected from the group consisting of EGFR, NF-κB, RIPK2, BCR-ABL, HER2, c-Met, TBK1, CDK, ALK, Akt, CK2, ERK1 / 2, FLT3, PI3K, BTK, TRK, Fak, BRD, AR, ER, MetAp-2, BCL-XL, Sirt2, HDAC6, Pirin, SMAD3, ARNT, PCAF / GCN5, Tau, EZH2, IRAK4, STAT3FRS2, and RAS (e.g., KRAS, HRAS, and NRAS).

[0076] In another preferred example, the KRAS includes KRAS-G12C, KRAS-G12D, KRAS-G12V, etc.

[0077] In another preferred example, the proteolytic agent comprises a PROTAC (protein degradation target chimeric body).

[0078] In another preferred example, the PROTAC targets EGFR, KRAS (including KRAS-G12C, KRAS-G12D, KRAS-G12V, etc.).

[0079] In another preferred example, the oligonucleotide includes an antisense oligonucleotide (ASO), a small interfering RNA (siRNA), a microRNA (miRNA), a small activating RNA (saRNA), a messenger RNA (mRNA), or an RNA aptamer.

[0080] In another preferred example, the conjugate is selected from fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, enzymes that produce detectable products, radionuclides, biotoxins, cytokines (e.g., IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0081] In another preferred example, the immune complex comprises a polyvalent (e.g., bivalent) nanobody targeting the TF as described in the first aspect of the present invention or an antibody targeting the TF as described in the second aspect of the present invention.

[0082] In another preferred example, the polyvalent nature means that the amino acid sequence of the immune complex contains a plurality of repeating TF-targeting nanobodies according to the first aspect of the present invention or antibodies targeting TF according to the second aspect of the present invention.

[0083] In another preferred example, the detection is in vivo detection or in vitro detection.

[0084] In another preferred example, the immune complex is used to diagnose and / or treat tumors expressing the TF protein.

[0085] In another preferred example, the antibody-drug conjugate (ADC) is as shown in the following molecular formula: [ka] Here, nAb is a nanobody that targets TF or a recombinant protein according to the fourth aspect of the present invention. LU stands for Linker 2 (also known as Connexon), D is a drug, Additionally, the subscript 'p' is a value selected from 1 to 8.

[0086] In another preferred example, the nAb is a recombinant protein according to the fourth aspect of the present invention.

[0087] In another preferred example, the antibody-drug conjugate (ADC) is a monomer, dimer, or polymer.

[0088] In another preferred example, the recombinant protein has the following structure from the NC or CN terminus: (A-L1-B)m Here, the A element is a nanobody that targets TF, The B element is an Fc segment, a human albumin-binding domain (ABD), or an anti-albumin nanobody (HLE). "L1" represents none or linker 1. Here, m is a positive integer.

[0089] In another preferred example, m is 1, 2, 3, or 4.

[0090] In another preferred example, the drug is site-specifically bound to a terminal amino group, side-chain amino group, or thiol group of a nanobody targeting the TF.

[0091] In another preferred example, the drug binds site-specifically and / or randomly to the nanobodies targeting the TF.

[0092] In another preferred example, the drug binds site-specifically to the A element, the B element, or L1.

[0093] In another preferred example, the drug binds site-specifically to the B element and / or L1.

[0094] In another preferred example, the drug is site-specifically bound to the terminal amino group or thiol group of the B element and / or to the side chain amino group or thiol group of L1.

[0095] In another preferred example, the B element is an Fc segment, and the drug is site-specifically bound to the hinge region and / or the C-terminus of the Fc segment.

[0096] In another preferred example, the B element is a human albumin-binding domain (ABD), and the drug binds site-specifically to the C-terminus of the ABD.

[0097] In another preferred example, the B element is an HLE, and the drug is site-specifically bound to the C-terminus of the HLE.

[0098] In another preferred example, the antibody-drug conjugate (ADC) has a structure selected from those shown in Figure 24.

[0099] In another preferred example, LU is selected from maleimidocaproyl (MC), maleimide (MAL), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (SMCC) linker linked to the antibody moiety, and comprises one or more connexons of valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), alanine-alanine-alanine (AAA), p-aminobenzyloxycarbonyl (PAB), and polyethylene glycol (PEG).

[0100] In another preferred example, the antibody is covalently bonded to the linker by reacting with a moiety selected from the group consisting of maleimidocaproyl (MC), maleimide (MAL), and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (SMCC).

[0101] In another preferred example, D is (i) Tubulin inhibitors such as meitansine derivatives (DM1, DM4), monomethyl auristatin E (MMAE), and monomethyl auristatin F (MMAF), (ii) DNA-acting toxins such as duocarmycin and pyrrolobenzodiazepine (PBD), (iii) A compound having antitumor activity is selected from topoisomerase inhibitors such as camptothecin, SN38, extecan, and Dxd.

[0102] In another preferred example, the LU-D compound is selected from the group consisting of: [ka]

[0103] An eighth aspect of the present invention provides a pharmaceutical composition, the pharmaceutical composition is (i) a nanobody targeting TF as described in the first aspect of the present invention, an antibody targeting TF as described in the second aspect of the present invention, a multispecific antibody as described in the third aspect of the present invention, a recombinant protein as described in the fourth aspect of the present invention, a recombinant immune cell as described in the sixth aspect of the present invention, or an immune complex as described in the seventh aspect of the present invention, (ii) A pharmaceutically acceptable vector.

[0104] In another preferred example, the pharmaceutical composition includes a single-component drug, a combination drug, or a synergistic drug.

[0105] In another preferred example, the pharmaceutical composition further comprises other biologically active substances such as tumor treatment agents.

[0106] In another preferred example, the method of administering the pharmaceutical composition is selected from the group consisting of subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral / nasal spray and aerosol inhalation.

[0107] In another preferred example, the dosage form of the pharmaceutical composition is selected from the group consisting of liquids, solids, or gels.

[0108] In another preferred example, the pharmaceutical composition is a liquid formulation.

[0109] In another preferred example, the pharmaceutical composition is an injectable preparation.

[0110] A ninth aspect of the present invention provides an application for an active ingredient, the active ingredient being selected from the group consisting of a TF-targeting nanobody as described in the first aspect of the present invention, a TF-targeting antibody as described in the second aspect of the present invention, a multispecific antibody as described in the third aspect of the present invention, a recombinant protein as described in the fourth aspect of the present invention, a recombinant immune cell as described in the sixth aspect of the present invention, an immune complex as described in the seventh aspect of the present invention, or a combination thereof, the active ingredient being used for (a) the preparation of detection reagents, detection panels or kits, and / or (b) the preparation of drugs for the prevention and / or treatment of TF-related diseases.

[0111] In another preferred example, the detection reagent, detection panel, or kit is: (1) Detection of TF protein in the sample, and / or (2) Detection of endogenous TF protein in tumor cells, and / or (3) Used to detect tumor cells that express TF protein.

[0112] In another preferred example, the detection type includes, but is not limited to, flow cytometry, cellular immunofluorescence detection, enzyme-linked immunoadsorption detection, immunoblotting detection, and the like.

[0113] In another preferred example, the detection reagent, detection panel, or kit is used for the diagnosis of TF-related disease.

[0114] In another preferred example, the drug is used to treat or prevent TF-high-expressing tumors, tumor metastases, or tumor drug resistance.

[0115] In another preferred example, the tumor drug resistance includes drug resistance to tumor immunotherapy, drug resistance to tumor-targeted therapy, drug resistance to conventional tumor chemotherapy, and radiotherapy refractory.

[0116] In another preferred example, the drug is used for an application selected from the group consisting of: (a) Specifically binds to tumor cells and / or immune cells / stromal cells in the tumor microenvironment, (b) Specifically inhibit the tumor TF-associated tumor cell signaling pathway, (c)Improve cytokine secretion disorders caused by TF overactivation, (d) Inhibit the migration or metastasis of tumor cells, (e) Inhibit tumor growth, thereby improving the efficacy of the combination therapy antitumor, (f) Promote the proliferation, survival and function of immune cells, thereby improving the effect of tumor immunity, (g) After binding to tumor cells, it is taken up by intracellular lysosomes, (h) It has the characteristic of being able to pass through the blood-brain barrier and be efficiently distributed within the brain, (i) It has excellent therapeutic effects against brain tumors, (j) It has excellent therapeutic effects against multiple types of solid tumors.

[0117] In another preferred example, the TF-related disease is selected from the group consisting of cancer, thrombosis, autoimmune disease, metabolic disease, infection, or a combination thereof.

[0118] In another preferred example, the TF-related disease includes the development, growth, and / or metastasis of a tumor.

[0119] In another preferred example, the cancer includes solid tumors and hematological malignancies.

[0120] In another preferred example, the cancer is a TF-high-expression tumor.

[0121] In another preferred example, the TF-highly expressing tumor is selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, ovarian cancer, cervical cancer, rectal cancer, glioma, melanoma, leukemia, lymphoma, or a combination thereof.

[0122] In another preferred example, the cancer is a drug-resistant tumor.

[0123] In another preferred example, a TF-high-expressing tumor is defined as a tumor where the ratio of the level L1 of TF transcripts and / or proteins in tumor tissue to the level L0 of transcripts and / or proteins in normal tissue is L1 / L0 ≥ 2, preferably ≥ 3.

[0124] A tenth aspect of the present invention provides a polynucleotide, the polynucleotide being (1) A nanobody targeting TF as described in the first aspect of the present invention, an antibody targeting TF as described in the second aspect of the present invention, or a multispecific antibody as described in the third aspect of the present invention, (2) Recombinant protein according to the fourth aspect of the present invention, or (3) Encodes a polypeptide selected from the group consisting of CAR constructs described in the fifth aspect of the present invention.

[0125] In another preferred example, the polynucleotide includes RNA, DNA, or cDNA.

[0126] An eleventh aspect of the present invention provides a vector comprising a polynucleotide as described in the tenth aspect of the present invention.

[0127] In another preferred example, the vector may be a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as adenoviruses and retroviruses, or another vector.

[0128] A twelfth aspect of the present invention provides a genetically modified host cell, the host cell containing the vector described in the eleventh aspect of the present invention, or the genome incorporating the polynucleotide described in the tenth aspect of the present invention.

[0129] A thirteenth aspect of the present invention provides a method for detecting TF in a sample in vitro (including diagnostic and non-diagnostic methods), the method being (1) In vitro, the step of contacting the sample with a nanobody targeting TF as described in the first aspect of the present invention, an antibody targeting TF as described in the second aspect of the present invention, or an immunocomplex as described in the seventh aspect of the present invention, (2) The step includes detecting whether an antigen-antibody complex is formed, where the formation of the complex indicates the presence of TF in the sample.

[0130] In another preferred example, the detection may be for diagnostic or non-diagnostic purposes.

[0131] A fourteenth aspect of the present invention provides a detection panel comprising a substrate (support plate) and a test strip, wherein the test strip comprises a nanobody targeting TF as described in the first aspect of the present invention, or an antibody targeting TF as described in the second aspect of the present invention, or an immunocomplex as described in the seventh aspect of the present invention.

[0132] A fifteenth aspect of the present invention provides a kit, the kit is, (1) A first container comprising an antibody portion which is a nanobody targeting the TF as described in the first aspect of the present invention or an antibody targeting the TF as described in the second aspect of the present invention, and / or (2) A second container comprising a nanobody targeting the TF described in the first aspect of the present invention or a secondary antibody of an antibody targeting the TF described in the second aspect of the present invention, Alternatively, the kit includes the detection panel described in the 14th aspect of the present invention.

[0133] A sixteenth aspect of the present invention provides a method for preparing recombinant polypeptides, the method being: (a) A step of culturing the host cells according to the 12th aspect of the present invention under appropriate expression conditions, (b) a step of isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is a TF-targeting nanobody according to the first aspect of the present invention, a TF-targeting antibody according to the second aspect of the present invention, a multispecific antibody according to the third aspect of the present invention, or a recombinant protein according to the fourth aspect of the present invention.

[0134] A 17th aspect of the present invention provides a method for treating TF-related diseases, the method comprising the steps of providing, to a target of interest, a TF-targeting nanobody as described in the 1st aspect of the present invention, a TF-targeting antibody as described in the 2nd aspect of the present invention, a multispecific antibody as described in the 3rd aspect of the present invention, a recombinant protein as described in the 4th aspect of the present invention, a recombinant immune cell as described in the 6th aspect of the present invention, an immune complex as described in the 7th aspect of the present invention, a pharmaceutical composition as described in the 8th aspect of the present invention, or a combination thereof.

[0135] In another preferred example, the method includes the step of administering another drug to the target subject in question, or administering it in combination with another drug by means of a therapeutic method.

[0136] In another preferred example, the other drugs or treatment methods include antitumor immunotherapeutic agents, tumor targeting agents, tumor chemotherapy agents, and tumor radiotherapy.

[0137] In another preferred example, the antitumor immunotherapy comprises a PD-1, PD-L1 monoclonal antibody.

[0138] An eighteenth aspect of the present invention provides a method for preparing a chimeric antibody, The present invention includes the step of cloning the nucleotide sequence of the alpaca-derived VHH sequence of the TF-targeting nanobody described in the first aspect of the present invention into an expression vector of a nucleotide sequence containing a human antibody constant region, and then transfecting animal cells to express a human-alpaca chimeric antibody.

[0139] A 19th aspect of the present invention provides a method for preparing human antibodies, The present invention includes the step of transferring the nucleotide sequence of the CDR region of the VHH chain of a TF-targeting nanobody described in the first aspect of the present invention onto a nucleotide sequence template containing the FR region of a human antibody, cloning this into an expression vector containing the constant region of a human antibody, and then transfecting animal cells to express a human antibody.

[0140] A 20th aspect of the present invention provides a method for inhibiting the growth and migration of tumor cells, comprising the step of administering to a target subject of interest a TF-targeting nanobody described in the first aspect of the present invention, a TF-targeting antibody described in the second aspect of the present invention, or a nanobody-drug conjugate described in the present invention, or CAR-T cells of the nanobody, or a combination thereof.

[0141] A 21st aspect of the present invention provides a method for inhibiting tumor growth in a model animal, comprising the step of administering to a target subject of interest a TF-targeting nanobody described in the first aspect of the present invention, a TF-targeting antibody described in the second aspect of the present invention, a nanobody-drug conjugate described in the present invention, or CAR-T cells of the nanobody.

[0142] In another preferred example, the drug may be administered alone or in combination with tumor immunotherapy, tumor-targeting drugs, cytotoxic drugs, radiotherapy, etc.

[0143] A 22nd aspect of the present invention provides a method for preparing a nanobody-drug complex as described in the 7th aspect of the present invention. (1) The step of reacting the antibody with the reducing reagent in a buffer to obtain the reduced antibody, (2) The process includes the step of crosslinking (coupling) the DL connexon-drug conjugate and the reduced antibody obtained in step (1) in a mixture of buffer and organic solvent to obtain antibody-drug conjugates 1a, 1b, 1c, or 1d having different DAR values.

[0144] In another preferred example, the crosslinking reaction of the preparation method is as shown in Figure 23.

[0145] In another preferred example, the antibody in step (1) is reduced with a reducing reagent, thereby reducing the disulfide bonds between antibody chains to generate thiol groups.

[0146] In another preferred example, the reducing agent in step (1) is tris(2-carboxyethyl)phosphine hydrochloride (TCEP), beta-mercaptoethanol, beta-mercaptoethylamine hydrochloride, or dithiothreitol (DTT).

[0147] In another preferred example, the buffer solution is selected from the group consisting of potassium dihydrogen phosphate-sodium hydroxide (KH2PO4-NaOH) / sodium chloride (NaCl) / diethyltriaminepentaacetic acid (DTPA) buffer, disodium hydrogen phosphate-citric acid / sodium chloride (NaCl) / diethyltriaminepentaacetic acid (DTPA), boric acid-borax / sodium chloride (NaCl) / diethyltriaminepentaacetic acid (DTPA), histidine-sodium hydroxide / sodium chloride (NaCl) / diethyltriaminepentaacetic acid (DTPA), and PBS / diethyltriaminepentaacetic acid (DTPA).

[0148] In another preferred example, in step (2), the volume percentage of the organic solvent in the reaction solution does not exceed 15%.

[0149] In another preferred example, the organic solvent in step (2) is selected from the group consisting of acetonitrile (ACN), dimethylformamide (DMF), dimethylacetamide (DMA), and dimethyl sulfoxide (DMSO).

[0150] Furthermore, the Connexon drug (pre-dissolved at 10 mg / ml in acetonitrile (ACN), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), or diethylacetamide (DMA)) is added, and the volume percentage of organic solvent in the reaction mixture does not exceed 15%. The coupling reaction is carried out by stirring at 0-37°C for 2-4 hours. When using TCEP reduction, the substituted maleimide compound can be added directly for coupling without the need to remove the remaining TCEP.

[0151] Using a desalting column, the coupling reaction mixture is filtered and purified with sodium succinate / NaCl buffer or histidine-acetic acid / sucrose gel, and peak samples are collected based on UV280 ultraviolet absorption values. Alternatively, multiple ultrafiltrations are performed. The mixture is then filtered and sterilized, and the resulting product is stored at a low temperature.

[0152] The drug-antibody coupling ratio (DAR) of the obtained antibody-drug conjugates is relatively uniform. For NDCs with a certain difference in DAR, if a more uniform sample is required, further separation and purification can be performed using methods such as hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC), or ion exchange chromatography (IEC), but these methods are not limited to these. [Effects of the Invention]

[0153] It should be understood that, within the scope of the present invention, new or preferred technical solutions can be constructed by combining the above-described technical features of the present invention with the technical features specifically described below (e.g., in the examples). Due to space limitations, this will not be repeated here. [Brief explanation of the drawing]

[0154] [Figure 1] These are SDS-PAGE images of proteins expressed and purified using human FC1 chimeric nanobodies, showing fusion proteins of the heavy chain variable region (VHH) of 4-A02, F1-A01, 4-C08, 4-E04, 4-C05, 5-C12, and 5-H3 with FC1 fragments, respectively. [Figure 2] These are the binding affinity constants (KD) between the chimeric nanobodies 4-A02, F1-A01, 4-E04, 4-C05, 5-C12, and 5-H3 and human TF-ECD, as measured by surface plasmon resonance (SPR) analysis. [Figure 3] This shows the detection results of binding affinity (EC50) to TF on the cell surface of 4-A02, F1-A01, 4-C08, 4-E04, 4-C05, 5-C12, 5-H3, and the TF-high-expressing cell line MDA-MB-231. [Figure 4] This is hydrophobic interaction chromatography (HIC) of a VC-MMAE conjugate NDC coupled to 4-A02-FC1. [Figure 5]This is hydrophobic interaction chromatography (HIC) of a VC-MMAE conjugate NDC coupled to F1-A01-FC1. [Figure 6] This is hydrophobic interaction chromatography (HIC) of a VC-MMAE conjugate NDC coupled to 4-C08-FC1. [Figure 7] This is hydrophobic interaction chromatography (HIC) of a VC-MMAE conjugate NDC coupled to 4-C05-FC1. [Figure 8] This is hydrophobic interaction chromatography (HIC) of a VC-MMAE conjugate NDC coupled to 5-C12-FC1. [Figure 9] This is hydrophobic interaction chromatography (HIC) of a VC-MMAE conjugate NDC coupled to 4-E04-FC1. [Figure 10] This is hydrophobic interaction chromatography (HIC) of a VC-MMAE conjugate NDC coupled to 4-A02-ABD. [Figure 11] This is hydrophobic interaction chromatography (HIC) of VC-MMAE and GGFG-Dxd conjugate NDC coupled to 4-A02-FC1. [Figure 12] This report describes the detection of the growth inhibitory activity of TF-NDC 4-A02-MMAE, F1-A01-MMAE, 4-E04-MMAE, 4-C05-MMAE, 5-C12-MMAE, 5-H3-MMAE, and the control HuSC1-MMAE against TF-high-expressing triple-negative breast cancer (TNBC) HCC1806 cells. [Figure 13] This report describes the results of detecting the growth inhibitory activity of TF-NDC 4-A02-MMAE, F1-A01-MMAE, 4-E04-MMAE, 4-C05-MMAE, 5-C12-MMAE, 5-H3-MMAE, and the control HuSC1-MMAE against TF-high-expressing pancreatic ductal adenocarcinoma (PDAC) BxPC3 cells. [Figure 14]This report describes the detection of the growth inhibitory activity of TF-NDC 4-A02-MMAE, F1-A01-MMAE, 4-E04-MMAE, 4-C05-MMAE, 5-C12-MMAE, 5-H3-MMAE, and the control HuSC1-MMAE against TF-high-expressing pancreatic ductal adenocarcinoma (PDAC) HPAF-II cells. [Figure 15] This is the result of detecting the growth inhibitory activity of TF-NDC 4A02-FC1-MMAE, 4A02-FC1-DXd, and 4A02-ABD-MMAE against TF-high-expressing pancreatic ductal adenocarcinoma (PDAC) BxPC3 cells. [Figure 16] This is the result of detecting the growth inhibitory activity of TF-NDC 4A02-FC1-MMAE, 4A02-FC1-DXd, and 4A02-ABD-MMAE against TF-high-expressing pancreatic ductal adenocarcinoma (PDAC) HPAF-II cells. [Figure 17] This report describes the results of detecting the growth inhibitory activity of TF-NDC 4A02-FC1-MMAE, 4A02-FC1-DXd, and 4A02-ABD-MMAE against TF-abnormally expressing KRASG12C-mutated lung cancer (NSCLC) HCC44 cells. [Figure 18] This report describes the results of detecting the growth inhibitory activity of TF-NDC 4A02-FC1-MMAE, 4A02-FC1-DXd, and 4A02-ABD-MMAE against TF-abnormally expressing EGFRT790M mutant lung cancer NCI-H975 cells. [Figure 19] This report describes the detection of the growth inhibitory activity of TF-NDC 4A02-FC1-MMAE, 4A02-FCWT-MMAE, 4A02-FC1-DXd, and 4A02-ABD-MMAE against TF-abnormally expressing EGFR-mutated lung cancer PC9 cells. [Figure 20] This is an SDS-PAGE of proteins expressed and purified using mass-produced 4-A02-FC1, 4-A02-FCWT, 4-A02-FCLALAPG, and 4-A02-ABD, corresponding to SEQ NO. 29, SEQ NO. 30, SEQ NO. 31, and SEQ NO. 32. [Figure 21]This study investigated the therapeutic effect of TF-NDC in a nude mouse breast pad transplant tumor growth model of triple-negative breast cancer HCC1806. Tumor-bearing mice were divided into groups (n=8) on day 9, after which hIgG-MMAE, 4-A02-FC1-MMAE, F1-A01-FC1-MMAE, 4-C08-FC1-MMAE, 4-C05-FC1-MMAE, 5-C12-FC1-MMAE, and 4-E04-FC1-MMAE were administered via tail vein injection. The dose was 3 mg / kg, administered a total of one time during the study period. [Figure 22] This study investigated the therapeutic effect of TF-NDC in a nude mouse orthotopic lung transplant tumor growth model of KRASG12C lung cancer HCC44. Tumor-bearing mice were divided into groups (n=8) on day 13, after which hIgG-MMAE and 4-A02-FC1-MMAE were administered via tail vein injection. The dose was 3 mg / kg, administered a total of one time during the study period. [Figure 23] The crosslinking reaction equation for the nanobody-drug conjugate preparation method of the present invention is shown, where antibody-drug conjugates having different DAR values ​​are as shown in 1a, 1b, 1c, or 1d, respectively. [Figure 24] This is the structural formula of the fusion protein of the present invention. Here, (A) shows the structural formula of the VHH-FC segment fusion protein (approximately 40 kDa), (B) shows the structural formula of the VHH-HLE fusion protein (approximately 30 kDa), and (C) shows the structural formula of the VHH-ABD fusion protein (approximately 20 kDa). [Figure 25] This shows the results of detecting the growth inhibitory activity of 4A02-FC1-MMAE, 4A02-FCWT-MMAE, 4A02-FC1-DXd, and 4A02-ABD-MMAE against the TNBC cell line MDA-MB-231. [Figure 26] This shows the results of detecting the growth inhibitory activity of 4A02-FC1-MMAE, 4A02-FCWT-MMAE, 4A02-FC1-DXd, and 4A02-ABD-MMAE against the TNBC cell line HCC1806. [Figure 27]This shows the results of detecting the growth inhibitory activity of 4A02-FC1-MMAE, 4A02-FCWT-MMAE, 4A02-FC1-DXd, and 4A02-ABD-MMAE against the TNBC cell line HCC1954. [Figure 28] This shows the results of detecting the growth inhibitory activity of 4A02-FC1-MMAE, 4A02-FCWT-MMAE, 4A02-FC1-DXd, and 4A02-ABD-MMAE against the TNBC cell line HCC1937. [Figure 29] This is the result of detecting the growth inhibitory activity of 4A02-FC1-MMAE, 4A02-FCWT-MMAE, 4A02-FC1-DXd, and 4A02-ABD-MMAE against the TNBC cell line MDA-MB-468. [Figure 30] This describes the therapeutic effect of intravenous injection of 3 mg / kg of 4A02-FCWT-MMAE (once a week, for a total of two times) in an in vivo large tumor regression experiment involving HCC1806. [Figure 31] This study describes the therapeutic effects of intravenous injection of 3 mg / kg of 4A02-FC1-MMAE, 4A02-FCWT-MMAE, 4A02-ABD-MMAE, and 10 mg / kg of 4A02-FC1-DXd (once a week, for a total of two times) in an HCC1954 in vivo model. [Figure 32A] This shows the therapeutic effect of TF-NDC on KRASG12D pancreatic cancer HPAF-II in a nude mouse transplant tumor growth model. Figure 32A shows the therapeutic effect after a single intravenous injection of 3 mg / kg of 4A02-FC1-MMAE, 4A02-FCWT-MMAE, and 4A02-ABD-MMAE. [Figure 32B] This shows the therapeutic effect of TF-NDC on KRASG12D pancreatic cancer HPAF-II in a nude mouse transplant tumor growth model. Figure 32B shows the therapeutic effect after a single intravenous injection of 1 mg / kg of 4A02-FC1-MMAE and 4A02-FCWT-MMAE. [Figure 33] These are the binding affinity constants (KD) between humanized nanobodies 4A02-HM7, 4A02-HM8, 4A02-HM9, and the original antibody 4A02 and human TF-ECD, as measured by surface plasmon resonance (SPR) analysis. [Figure 34A] The measured binding affinities of the humanized nanobodies 4A02-HM7, 4A02-HM8, 4A02-HM9, and the original antibody 4A02 to the cell surface TF. Figure 34A shows the binding affinities of MDA-MB-231 cells detected by FACS. [Figure 34B] The measured binding affinities of the humanized nanobodies 4A02-HM7, 4A02-HM8, 4A02-HM9, and the original antibody 4A02 to the cell surface TF. Figure 34B shows the binding affinities of HPAF-II cells detected by FACS. [Figure 35A] This shows the binding affinity of TF-ECD to the humanized nanobodies 4A02-HM7, 4A02-HM8, 4A02-HM9, and the original antibody 4A02, as detected by ELISA. Figure 35A shows the ELISA results for human TF-ECD. [Figure 35B] Figure 35B shows the binding affinity of TF-ECD to the humanized nanobodies 4A02-HM7, 4A02-HM8, 4A02-HM9, and the original antibody 4A02, as detected by ELISA. [Modes for carrying out the invention]

[0155] Through extensive and thorough research, the inventors immunized alpaca, constructed libraries, and extensively screened yeast display libraries and phage display libraries to obtain multiple nanobodies such as 4-A02, F1-A01, 4-C08, 4-E04, 4-C05, 5-C12, and 5-H3. These antibodies can bind to human TF proteins with high specificity, and their binding constant KD values, measured by ForteBio surface plasmon resonance (SPR), are 0.2 nM to 7 nM. These antibodies exhibit excellent binding affinity to TF-highly expressing tumor cells, and EC 50 The values ​​range from 0.05 g / mL to 0.75 g / mL, and furthermore, nanobody-drug conjugates (TF-NDCs) designed based on TF nanobodies can specifically kill tumor cells highly expressed in TF, and IC50 The values ​​ranged from 0.0001 g / mL to 0.03 g / mL, and a single intravenous injection of 3 mg / kg of TF-NDC showed potent antitumor activity in vivo, suggesting the potential to improve the immune microenvironment. Based on this, the present invention was completed.

[0156] term As used herein, “the nanobody of the present invention,” “the anti-TF nanobody of the present invention,” and “the TF nanobody of the present invention” refer to nanobodies that are interchangeable and specifically identify and bind to TF (including human TF). Particularly preferred are nanobodies with VHH chain amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 8, SEQ ID NO. 12, SEQ ID NO. 16, SEQ ID NO. 20, SEQ ID NO. 24, and SEQ ID NO. 28.

[0157] As used herein, the terms “antibody” or “immunoglobulin” refer to a heterotetrameric glycoprotein of approximately 150,000 daltons having the same structural characteristics, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is attached to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes differs. Each heavy and light chain also has intrachain disulfide bonds arranged at regular intervals. Each heavy chain has a variable region (VH) at one end, followed by several constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end, with the constant region of the light chain facing the first constant region of the heavy chain, and the variable region of the light chain facing the variable region of the heavy chain. Certain amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0158] As used herein, the terms “single-domain antibody (VHH)” and “nanobody” have the same meaning and refer to the cloning of the variable region of a monoclonal antibody heavy chain to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with full function. Typically, after obtaining an antibody that naturally lacks the constant region 1 (CH1) of both the light and heavy chains, the variable region of the antibody's heavy chain is then cloned to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region.

[0159] As used herein, the term “variable” refers to a difference in the arrangement of specific portions of the variable region in an antibody, which forms the binding and specificity of different particular antibodies to a particular antigen. However, variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments called complementarity-determining regions (CDRs) or hypervariable regions of the light and heavy chain variable regions. The more conserved portion of the variable region is called the framework region (FR). The natural heavy and light chain variable regions each contain four FR regions, which are mostly in a β-folding configuration, connected by three CDRs that form a connecting ring, and can sometimes form a partial β-folding structure. The CDRs of each chain are closely adjacent by FR regions and, together with the CDRs of another chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). While the constant region is directly involved in antibody binding to antigens, it also exhibits various effector functions, such as involvement in antibody-dependent cytotoxicity.

[0160] As is known to those skilled in the art, immune complexes and fusion expression products include complexes formed by binding a drug, toxin, cytokine, radionuclide, enzyme, and other diagnostic or therapeutic molecule to the antibody or fragment thereof of the present invention. The present invention further includes a cell surface marker or antigen that binds to the anti-TF protein antibody or fragment thereof.

[0161] As used herein, “heavy chain variable region” and “V H " is used interchangeably.

[0162] As used herein, the “variable region” and the “complementarity determining region (CDR)” are interchangeable.

[0163] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity-determining regions CDR1, CDR2, and CDR3. In a preferred embodiment of the present invention, the heavy chain of the antibody includes the heavy chain variable region and the heavy chain constant region.

[0164] In the present invention, the terms "antibody of the present invention," "protein of the present invention," or "polypeptide of the present invention" are used interchangeably to refer to polypeptides that specifically bind to TF proteins, such as proteins or polypeptides having a heavy chain variable region. They may or may not contain initiation methionine.

[0165] The present invention further provides other proteins or fusion expression products having the antibody of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) of a heavy chain containing a variable region, provided that the variable region is identical to, or at least 90% identical to, the variable region of the heavy chain of the antibody of the present invention, or at least 95% identical.

[0166] Generally, the antigen-binding properties of an antibody can be explained by three specific regions located within the variable region of the heavy chain, called the variable region (CDR). This section is divided into four framework regions (FRs), and the amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a cyclic structure, and the β-folds formed by the FRs between them are spatially close. The CDRs of the heavy chain and their corresponding CDRs of the light chain constitute the antigen-binding site of the antibody. By comparing the amino acid sequences of similar antibodies, the amino acids that make up the FR or CDR region can be determined.

[0167] The heavy chain variable region of the antibody of the present invention is of particular interest because at least a portion of it is involved in binding to the antigen. Accordingly, the present invention includes molecules having an antibody heavy chain variable region having a CDR, insofar as its CDR has homology of 90% or more (preferably 95% or more, most preferably 98% or more) with the CDRs identified herein.

[0168] Anti-TF nanobody The present invention provides a variety of highly specific and highly affinity nanobodies that target TF, wherein the nanobodies consist solely of a heavy chain, and the heavy chain comprises the amino acid sequence of the heavy chain variable region (VH) (as shown in Tables 1 and 2).

[0169] Preferably, the amino acid sequence of the heavy chain variable region (VH) has the following polypeptide sequences CDR1, CDR2, and CDR3: a1) CDR1 is, SEQ ID NO.1: ETISSTYI, SEQ ID NO.5: GRAFSAYA, SEQ ID NO.9: GFSLSSYD, SEQ ID NO.13:EMISSTYI, SEQ ID NO.17:GFTLDTYA, SEQ ID NO.21: GRTFSTDA, SEQ ID NO.25: Selected from GFTLANYA, a2) CDR2 is, SEQ ID NO.2: ISGDGVTH, SEQ ID NO.6: ISWSGGST, SEQ ID NO.10: IHSSGGYP, SEQ ID NO.14: ISGDGVTH, SEQ ID NO.18:ISSTDGST, SEQ ID NO.22:INWSGGST, SEQ ID NO.26: Selected from ISRSDGDT, a3) CDR3 is, SEQ ID NO.3:YAAGRWNH, SEQ ID NO.7:NADSLLSLLDGSRGGPGTDSGS, SEQ ID NO.11:NLPPSRRWYKDY, SEQ ID NO.15:NAAGRRNH, SEQ ID NO.19:AAGPGTDCPLRFDY, SEQ ID NO.23:VADSLLALLDGRGGPGTDSDS, SEQ ID NO.27: Selected from RATEWCGVQDPHGY, a4) Any one of the above amino acid sequences, in which at least one amino acid is added, deleted, modified and / or substituted, has a TF-binding affinity sequence.

[0170] In another preferred example, the sequence formed by the addition, deletion, modification and / or substitution of the at least one amino acid sequence preferably has at least 80%, preferably at least 85%, more preferably at least 90%, and preferably at least 95% amino acid sequence homology.

[0171] Preferably, the antibody inhibits or inhibits the binding of the antibody to the cell surface and recombinant TF protein, and the antibody is rapidly endocytized and transported to the lysosome.

[0172] The antibody of the present invention may be a double-chain antibody or a single-chain antibody, and can be selected from animal-derived antibodies, chimeric antibodies, or human-animal chimeric antibodies, preferably a human antibody, and more preferably a fully human antibody. The antibody derivatives described in the present invention may be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2, or other antibody derivatives known in the art, as well as one or more of IgA, IgD, IgE, IgG, and IgM antibodies or other subtypes of antibodies.

[0173] Here, the animal is preferably a mammal such as a mouse.

[0174] The antibodies of the present invention may be chimeric antibodies targeting human TF, human antibodies, CDR-transplanted antibodies, and / or modified antibodies.

[0175] In preferred embodiments of the present invention, one or more oil sequences from among SEQ ID NO.1-3, SEQ ID NO.5-7, SEQ ID NO.9-11, NO.13-15, NO.17-19, NO.21-23, and NO.25-27, or sequences including the addition, deletion, modification and / or substitution of at least one amino acid, have a TF-binding affinity sequence and are located in the CDR region of the variable region (VH).

[0176] In the present invention described above, the number of added, deleted, modified and / or substituted amino acids is preferably not more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably not more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%.

[0177] In the present invention, more preferably, the number of added, deleted, modified and / or substituted amino acids may be 1 to 7, more preferably 1 to 5, more preferably 1 to 3, and more preferably 1 to 2.

[0178] In another preferred example, the original antibodies are human-alpaca chimeric antibodies 4-A02, F1-A01, 4-C08, 4-E04, 4-C05, 5-C12, and 5-H3.

[0179] In another preferred example, the amino acid sequence numbers of the heavy chain variable region (VH) of the chimeric antibody are shown in Table 1.

[0180] In another preferred example, the amino acid sequence numbers of the complementarity-determining region (CDR) of the antibody are shown in Table 2.

[0181] The antibodies of the present invention can be used in combination and are used in applications such as the construction of CAR constructs, recombinant immune cells containing CAR constructs, and antibody-drug conjugates, and are used for (a) the preparation of detection reagents, detection panels or kits, and / or (b) the preparation of drugs for the prevention and / or treatment of TF-related diseases.

[0182] [Table 1] TIFF2026509247000004.tif178169

[0183] [Table 2]

[0184] Recombinant protein (or fusion protein) The present invention further comprises the TF nanobody-containing recombinant protein (or fusion protein) of the present invention. A preferred fusion protein is a multispecific antibody, which further comprises a second antigen-binding region that targets a target point selected from the group consisting of EGFR, TGFβ, BCMA, B7H6, GUCY2C, DLL3, CD38, CD123, CD19, CD20, CD22, B7-H3, GPC3, HER2, PMSA, CD28, 4-1BB, OX40, CD40, CD27, CD3, CTLA4, PD1, PDL1, CD73, BCMA, GLP-1, Trop2, TIGIT, LAG-3, FGL1, TLR7, or a combination thereof.

[0185] Preferably, the multispecific antibody-inclusive second antigen-binding region or third antigen-binding region is further included.

[0186] Furthermore, the recombinant protein (or fusion protein) of the present invention further comprises, in addition to the CD73 nanobody of the present invention, any tag sequence that aids in expression and / or purification (e.g., 6His tag, GGGS sequence, FLAG tag), or a polypeptide molecule or fragment with therapeutic function, or a physicochemical or pharmacological protein function domain (e.g., a molecule that extends the in vivo half-life of nanobodies such as HLE and ABD).

[0187] The present invention includes not only complete antibodies, but also fragments of antibodies having immunoactivity or fusion proteins formed from antibodies with other sequences. Accordingly, the present invention further includes such antibody fragments, derivatives, and analogs.

[0188] As used herein, the terms “fragment,” “derivative,” and “analog” refer to polypeptides that possess essentially the same biological function or activity as the antibodies of the present invention. Polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted (the substituted amino acid residues may or may not be encoded by the genetic code), or (ii) polypeptides having substituents on one or more amino acid residues, or (iii) polypeptides formed by the fusion of a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by the fusion of an additional amino acid sequence to a polypeptide sequence (e.g., a leader sequence or secretion sequence or a sequence or protein sequence for purifying the polypeptide or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0189] The antibody of the present invention refers to a polypeptide having TF protein binding activity and containing the above-mentioned CDR region. The term further includes variant forms of the polypeptide containing the above-mentioned CDR region that have the same function as the antibody of the present invention. These variant forms include (but are not limited to) the deletion, insertion, and / or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and the addition of one or more (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids having similar or approximate performance usually does not alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and / or N-terminus also usually does not alter the function of the protein. The term further includes active fragments and active derivatives of the antibody of the present invention.

[0190] The variant forms of the polypeptide include homologous sequences, conserved variants, allelic variants, native mutants, induced mutants, proteins encoded by DNA that can hybridize to the DNA encoding the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0191] The present invention further provides other polypeptides, such as fusion proteins, that include nanobodies or fragments thereof. In addition to most full-length polypeptides, the present invention further includes fragments of the nanobodies of the present invention. Typically, such fragments have at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0192] In the present invention, "conservative variants of the antibody of the present invention" refers to polypeptides formed by the substitution of up to 10 amino acids, preferably up to 8, more preferably up to 5, and most preferably up to 3, amino acids with similar or equivalent properties compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are best produced by performing amino acid substitutions according to Table A. [Table A]

[0193] The present invention further provides polynucleotide molecules encoding the antibody or its fragment or its fusion protein. The polynucleotides of the present invention may be in DNA form or RNA form. The DNA form includes cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0194] The polynucleotides encoding mature polypeptides of the present invention include coding sequences that encode only mature polypeptides, coding sequences for mature polypeptides and various additional coding sequences, coding sequences for mature polypeptides (and any additional coding sequences) and non-coding sequences.

[0195] The term “polynucleotide encoding polypeptide” includes a polynucleotide that encodes this polypeptide, or further includes a polynucleotide that includes additional coding and / or non-coding sequences.

[0196] The present invention further relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the aforementioned polynucleotides under stringent conditions. In the present invention, “stringent conditions” means (1) hybridization and elution at lower ionic strength and higher temperature, for example, 0.2 × SSC, 0.1% SDS, 60°C, or (2) addition of a denaturing agent during hybridization, for example, 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, or (3) hybridization that occurs only when the identity between the two sequences is at least 90%, more preferably 95%. Furthermore, polypeptides encoded by hybridizable polynucleotides have the same biological functions and activities as mature polypeptides.

[0197] The full-length nucleotide sequence or fragments of the antibody of the present invention can typically be obtained by PCR amplification, recombination, or artificial synthesis. Especially when the fragment length is short, a viable method is to synthesize the relevant sequence using artificial synthesis. Generally, several small fragments are first synthesized and then joined together to obtain a very long sequence fragment. Furthermore, the heavy chain coding sequence and expression tag (e.g., 6His) can be fused to form a fusion protein.

[0198] Antibody preparation The DNA molecule sequences of the antibodies or fragments thereof of the present invention can be obtained using conventional techniques such as PCR amplification or genome library screening. Furthermore, single-chain antibodies can be formed by fusing the coding sequences of the light and heavy chains.

[0199] Once the relevant sequence is obtained, it can be used to obtain large quantities of the relevant sequence using recombination. Typically, this is done by cloning it into a vector, then transforming it into cells, and then isolating the relevant sequence from host cells grown by conventional methods.

[0200] Furthermore, especially when the fragments are short, related sequences can be synthesized using artificial synthesis methods. Typically, very long fragments can be obtained by synthesizing multiple small fragments and then concatenating them.

[0201] Currently, DNA sequences encoding the antibody (or fragment thereof, or derivative thereof) of the present invention can be obtained entirely by chemical synthesis. These DNA sequences can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention by chemical synthesis.

[0202] The present invention further relates to vectors comprising the aforementioned appropriate DNA sequence and an appropriate promoter or regulatory sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.

[0203] The host cell may be a prokaryotic cell such as a bacterial cell, a lower eukaryotic cell such as a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Preferred animal cells include, but are not limited to, CHO-S and HEK-293 cells.

[0204] Under conditions suitable for the expression of the antibodies of the present invention, the obtained host cells can be cultured and transformed. Next, the antibodies of the present invention are produced and obtained using conventional separation and purification means well known to those skilled in the art, such as conventional immunoglobulin purification steps, for example, Protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography.

[0205] The obtained monoclonal antibodies can be identified by conventional means. For example, the binding specificity of monoclonal antibodies can be measured using immunoprecipitation or in vitro binding assays (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of monoclonal antibodies can be measured, for example, by Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0206] The antibodies of the present invention can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If necessary, various isolation methods can be used to isolate and purify the recombinant protein using physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional regeneration processes, treatment with protein precipitants (salting out), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

[0207] Nanobody-drug conjugate (NDC) The present invention further provides an immunocomplex (ADC) based on the antibody of the present invention, preferably a nanobody-drug conjugate (NDC).

[0208] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, and the antibody is coupled to the effector molecule, preferably by chemical coupling. Here, the effector molecule is preferably a drug having therapeutic activity. Further, the effector molecule can be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug or a radionuclide.

[0209] The antibody of the present invention can be coupled to the effector molecule via a coupling agent. Examples of the coupling agent can be any one or more of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond. The non-selective coupling agent refers to a compound that covalently binds an effector molecule and an antibody, such as glutaraldehyde. The coupling agent using a carboxyl group can be any one or more of a cis-aconitic anhydride coupling agent (e.g., cis-aconitic anhydride), an acylhydrazone coupling agent (the coupling site is an acylhydrazone).

[0210] Specific residues on the antibody (e.g., Cys or Lys, etc.) are used to bind to various functional groups, here including imaging reagents (e.g., chromophores and fluorophores), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., glycol polymers) as well as therapeutic agents. The antibody can be coupled to a functional agent to form an antibody-functional agent conjugate. The functional agent (e.g., a drug, a detection reagent, a stabilizer) is coupled (covalently) to the antibody. The functional agent can bind directly to the antibody or indirectly via a linker.

[0211] Nanobodies can form antibody-drug conjugates (NDCs) by coupling with drugs. Typically, these include a linker located between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers are usually susceptible to degradation in the intracellular environment, for example, at a site of interest that degrades the linker, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, such as peptidyl-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidases. Peptidyl linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, e.g., hydrazone linkers) and linkers that can be degraded under reducing conditions (e.g., disulfide linker). Non-degradable linkers typically release drugs under conditions where the antibody is hydrolyzed by a protease.

[0212] Before binding to the antibody, the linker has an active reactive group that can react with specific amino acid residues, and binding is achieved via this active reactive group. Sulfhydryl-specific active reactive groups are preferred and include maleimide compounds, halogenated amides (e.g., iodine, bromide, or chlorine), haloesters (e.g., iodine, bromide, or chlorine), halomethyl ketones (e.g., iodine, bromide, or chlorine), benzyl halides (e.g., iodine, bromide, or chlorine), vinyl sulfones, pyridyl disulfide, mercury derivatives such as 3,6-bis-(mercurymethyl)dioxane whose counterion is acetate, chloride ion, or nitrate, and polymethylenedimethyl sulfide thiosulfonates. The linker may include, for example, maleimide bound to the antibody via thiosuccinimide.

[0213] The drug may be any cytotoxic, cell growth inhibitory, or immunosuppressive drug. In embodiments, the linker binds to the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug may have an amino group, carboxyl group, sulfhydryl group, hydroxyl group, or keto group capable of forming a bond with the linker. If the drug is directly bound to the linker, the drug has reactive active groups before binding to the antibody.

[0214] Useful drug classes include, for example, antitubulins, DNA ligation reagents, DNA replication inhibitors, alkylating reagents, antibiotics, folic acid antagonists, antimetabolites, chemotherapeutic sensitizers, immunoagonists, topoisomerase inhibitors, and vinca alkaloids. Particularly useful examples of cytotoxic drug systems include, for example, DNA supraclution binding reagents, DNA alkylation reagents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines) and vinca alkaloids.

[0215] In the present invention, a drug-linker can form an NDC in one simple step. In other embodiments, a bifunctional linker compound can be used to form an NDC in two or multi-step steps. For example, a cysteine ​​residue is reacted with the reactive moiety of the linker in the first step, and then in a subsequent step, the functional group on the linker is reacted with the drug to form an NDC.

[0216] Typically, functional groups on the linker are selected to facilitate specific reactions with appropriate reactive groups on the drug moiety. A non-limiting example is the use of an azide-based moiety to specifically react with a reactive alkynyl group on the drug moiety. The drug is covalently bonded to the linker via a 1,3-dipolar cyclic addition between the azide and the alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reactions with hydrazides and alkoxyamines), phosphines (suitable for reactions with azides), isocyanates and isothiocyanates, and activated esters such as N-hydroxysuccinimidyl esters (suitable for reactions with amines and alcohols). These and other bonding strategies, as described in "Bioconjugation Techniques," second edition (Elsevier), are well known to those skilled in the art. Those skilled in the art should understand that, for selective reactions of the drug moiety and linker, if a complementary pair of reactive functional groups is selected, each member of that complementary pair can be used in both the linker and the drug.

[0217] Preferably, the coupling portion coupled to the nanobody portion in the nanobody drug complex NDC of the present invention contains a proteolytic agent or an oligonucleotide drug.

[0218] Preferably, the oligonucleotide drug is a nucleic acid drug that targets small molecules, including antisense nucleic acids (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), small activated RNAs (saRNAs), messenger RNAs (mRNAs), aptamers, nuclear enzymes (ribozymes), antibody-nucleotide coupling drugs (ARCs), etc.

[0219] As used herein, the proteolytic agent is a tumor-associated protein degrader, and the tumor-associated protein is (1) Kinase systems, such as RIPK2, BCR-ABL, EGFR, HER2, c-Met, TBK1, CDK2 / 4 / 6 / 9, ALK, Akt, CK2, ERK1 / 2, FLT3, PI3K, BTK, Fak, etc. (2) BET proteins, e.g., BRD2 / 4 / 6 / 9, (3) Nuclear receptors, such as AR, ER, etc. (4) Other proteins selected from the group consisting of MetAp-2, Bcl-xL, Sirt2, HDAC6, Pirin, SMAD3, ARNT, PCAF / GCN5, Tau, FRS2, RAS (KRAS, HRAS, and NRAS), etc.

[0220] Preferably, the proteolytic agent comprises a PROTAC (protein degradation target chimeric body), and more preferably, the PROTAC targets EGFR and KRAS (e.g., KRAS-G12C, KRAS-G12D, KRAS-G12V, etc.).

[0221] The present invention provides a method for preparing an antibody conjugate (NDC), which may further include the step of conjugating an antibody with a drug-linker compound under conditions sufficient to form an NDC.

[0222] In certain embodiments, the method of the present invention includes the step of binding an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the method of the present invention further includes the step of binding the antibody-linker conjugate to a drug moiety under conditions sufficient to bind the drug moiety to the antibody via the linker. In some embodiments, the nanobody-drug conjugate NDC is as shown in the following molecular formula, [Chemical Formula] where, nAb is a nanobody, LU is a linker / connexon, D is a drug, Also, the subscript p is a value selected from 1 to 8.

[0223] Drug As used herein, "drug" broadly refers to a compound having a desired biological activity and a reactive functional group useful for the preparation of the conjugates described in the present invention. The desired biological activity includes the diagnosis, cure, alleviation, treatment, and prevention of diseases in humans or other animals. Thus, as long as the necessary reactive functional groups are present, the compounds referred to by the term "drug" refer to the drugs described in the official Chinese Pharmacopoeia, the United States Homeopathic Pharmacopoeia, the official Chinese Formulary, or any of its supplementary materials, etc. Typical drugs are listed in the Physician's Desk Reference (PDR) and the Orange Book of the United States Food and Drug Administration (FDA). As new drugs continue to be discovered and developed, it should be understood that these drugs also include the "drugs" in the coupling drugs described in the present invention.

[0224] Drugs that can be used to construct the NDC of the present invention include, but are not limited to, cytotoxic drugs (e.g., cytotoxic small molecule drugs). The term "cytotoxic drug" refers to a substance that inhibits or blocks cell expression activity, cell function, and / or causes cell destruction. This term includes radioisotopes, chemotherapeutic agents, and toxins, such as low-molecular-weight toxins or enzymatically active toxins derived from bacteria, fungi, plants, or animals, and includes their fragments and / or variants. Examples of cytotoxic drugs include auristatin derivatives (e.g., auristatin E, auristatin F, MMAE, and MMAF), chlortetracycline, meitansinoids, lysine, lysine A-chain, combretastatin, duocalmycin, dorastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, and vinbrassic acid. This includes, but is not limited to, tin, colchicine, dihydroxyanthracine dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modexin A chain, α-sarcin, geronin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria officinalis inhibitors and glucocorticoids and other chemotherapeutic agents, and radioisotopes, such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32, and radioisotopes of Lu including Lu177. Antibodies can also couple prodrugs to their active form of anticancer prodrug activating enzymes.

[0225] The preferred drugs of the present invention are maytansine or maytansinoids. Maytansine compounds inhibit cell proliferation by inhibiting tubulin microtubule formation. Maytansinoids are derivatives of maytansine. Both maytansine and maytansinoids have efficient cytotoxicity, but their clinical application in cancer treatment has significant limitations, mainly due to the low selectivity of this type of molecule for tumors. However, due to such high cytotoxicity, they are preferred drug moieties in antibody-drug conjugates. The structure of desacetylmytansine is shown below. [ka]

[0226] Another preferred drug of the present invention is auristatin peptide drugs. Auristatin peptide drugs are analogues of dolastatin 10, the latter being a biologically active polypeptide isolated from the body of the marine mollusk sea hare. Dolastatin 10 binds to tubulin (a binding domain similar to vincristine) and inhibits tubulin polymerization. Dolastatin 10, auristatin peptide PE, and auristatin peptide E are all linear polypeptides containing four amino acids (where three amino acids are specific to dolastatin compounds) and a C-terminal amide group. Both monomethyl auristatin peptide E (MMAE) and monomethyl auristatin peptide F (MMAF), two representative auristatin peptide compounds, are preferred drugs for antibody-drug conjugates. [ka] Monomethyl Auristatin E (MMAE) [ka] Monomethyl Auristatin F (MMAF) [ka] Monomethyl Dolastatin 10(MMAD)

[0227] Another preferred drug of the present invention is tubulysin. Tubulysin, first isolated by the research team from myxobacteria cultures, is a highly effective cell growth inhibitor that induces cell apoptosis by inhibiting tubulin polymerization. Tubulysin D is the most synpotent of the tubulysin derivatives, exhibiting 10 to 100 times higher activity than most other tubulin regulators (including epotilon, vinblastine, and paclitaxel). Paclitaxel and vinblastine are currently used to treat several types of cancer, and epotilon derivatives are being evaluated for activity in clinical trials. Synthetic derivatives of tubulysin D provide the necessary information regarding inhibition and key binding interactions and can also possess excellent properties as anticancer agents, which can be used as isolated entities or as chemical warheads on target antibodies or ligands. Tubulicin D is a complex tetrapeptide that can be classified into four regions, including Mep (DN-methylpiperidine), Ile (isoleucine), Tuv (tubuvaline), and Tup (tubuphenylalanine), as shown in the following formula. [ka] Another preferred drug of the present invention is a cryptophycin derivative derived from microorganisms that inhibits microtubule polymerization. Cryptophycin is a novel antitumor active substance isolated from cyanobacteria cultures that can inhibit microtubule formation and is active against multiple types of tumors. Cryptophycin is a lipophilic compound containing two peptide bonds and two ester bonds, with five optically active centers and one epoxy group. Both the dipeptide bonds and diester bonds are in a macrocyclic structure. The structures of the Cryptophycin derivatives CP1 and CP2 are shown in the following formulas. [ka]

[0228] Another preferred drug of the present invention is the novel microtubule inhibitor Taltobulin (HTI-286, SPA-110). Taltobulin inhibits the polymerization of purified microtubules, interferes with the organization of intracellular microtubules, induces mitotic blockade, and induces cell apoptosis. Taltobulin is a potent inhibitor of cell proliferation and has an average IC against 18 human tumor cell lines. 50 Its concentration is 2.5 nM. Compared to currently applied microtubule inhibitors, Taltobulin is not a suitable substrate for p-glycoprotein, and the structure of Taltobulin is shown in the figure below. [ka]

[0229] In one embodiment, the drug is the camptothecin-based drug derivative SN-38. SN-38 is a biologically active metabolite of irinotecan hydrochloride (CPT-11) and is a topoisomerase inhibitor. SN-38 exerts the strongest inhibitory effect on DNA topoisomerase I, inhibiting DNA synthesis in a dose-dependent and time-dependent manner, and causing frequent DNA single-strand breaks. The structure of SN-38 is shown in the following figure. [ka]

[0230] In one embodiment, the drug is Exatecan, a camptothecin-based drug derivative. It is a synthetic analogue of the topoisomerase I inhibitor camptothecin, and its activity is higher than that of SN-38, causing the strongest inhibitory effect on DNA topoisomerase I, inhibiting DNA synthesis in a dose-dependent and time-dependent manner, and causing frequent DNA single-strand breaks. Here, the structure of Exatecan is shown in the following formula. [ka]

[0231] Another preferred drug of the present invention is an α-amanitin drug (α-amanitin), whose structure is shown in the following figure. α-amanitin is a mycotoxin derived from the poisonous mushroom Amanita phalloides, and its bicyclic octapeptide can inhibit the transcription of eukaryotic RNA polymerase II and RNA polymerase III. [ka]

[0232] Other preferred drugs of the present invention are benzodipyrrole antibiotics (duocarmycins, CC-1065, etc.) and other cyclopropapyrroloind-4-one (CPI) derivatives. Such compounds are effective DNA subgroove-linking alkylating reagents. The chemical structures of cyclopropabenzindol-4-one (CBI) analogues are more stable, have higher biological activity, and are more readily synthesized compared to the parent compound containing the natural CPI alkylating subunit. A typical CBI derivative is the phenolic hydroxyl-protected derivative CBI, which has reduced prodrug toxicity and enhanced water solubility (where the general formula of the CBI-seco system structure is shown in the figure below): [ka]

[0233] Another preferred drug of the present invention is pyrrolo[2,1-c][1,4]benzodi-azepines (PBDs) or PBD dimers. PBDs are natural products produced by bacteria of the genus Streptomyces, and their unique property is the formation of non-deformable covalent adducts in DNA subgrooves, particularly in purine-guanine-purine sequences. The application of PBDs as part of small molecule strategies targeting DNA sequences, and as novel anticancer and antibacterial agents, is attracting increasing attention. By linking the C8 / C8' hydroxyl groups of two PBD units with a flexible carbon chain, the resulting dimers have enhanced biological activity. PBD dimers are thought to exert their biological activity by generating sequence-selective DNA damage, such as inverted 5'-Pu-GATC-Py-3' interchain crosslinks. These compounds have proven to be very potent cytotoxic drugs and can be used as candidate drugs for antibody-drug conjugates. [ka]

[0234] PBD dimer Another preferred drug of the present invention is the PNU-159682 derivative, which is the major active metabolite of nemorubicin in human liver microsomes, exhibiting 3000-fold increased activity compared to MMDX and doxorubicin. [ka]

[0235] In another embodiment, the drugs are not limited to the classes described above and further include all drugs that can be used in antibody-drug conjugates. In particular, this includes drugs that can coordinate via an amide bond to a linker, such as cytotoxins having a basic amine group (primary or secondary amine), as exemplified by the structures of cytotoxins D1-D12 shown above.

[0236] Conexon Based on the mechanism of intracellular drug release, "connexons" or "connexons of antibody-drug conjugates" are classified into two categories: uncleaved connexons and cleaved connexons.

[0237] In the case of antibody-drug conjugates containing uncleaved connexons, the mechanism of drug release is as follows: After the conjugate binds to the antigen and is taken up into the cell, the antibody is enzymatically cleaved within the lysosome, releasing an active molecule consisting of a small-molecule drug, a connexon, and antibody amino acid residues. Although the resulting structural changes in the drug molecule do not reduce its cytotoxicity, the active molecule (amino acid residues) is electrically charged and therefore cannot penetrate adjacent cells. Consequently, such active drugs cannot kill adjacent tumor cells that do not express the target antigen (antigen-negative cells) (bystander effect).

[0238] As the name suggests, cleavage-type connexons are cleaved within target cells and release the active drug (the small molecule drug itself). Cleavage-type connexons are classified into two main categories: chemically unstable and enzymatically unstable. Chemically unstable connexons are selectively cleaved depending on differences in the properties of plasma and cytoplasm. These properties include pH value and glutathione concentration. pH-sensitive connexons are often called acid-cleavage-type connexons. Such connexons are relatively stable in the neutral environment of blood (pH 7.3-7.5), but are hydrolyzed in weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0). In first-generation antibody-drug conjugates, such connexons, mainly hydrazones, carbonates, acetals, and ketals, are used. Because acid-cleaved connexons have limited stability in plasma, antibody-drug conjugates based on such connexons typically have a short half-life (2-3 days). This short half-life limits, to some extent, the application of pH-sensitive connexons in next-generation antibody-drug conjugates.

[0239] Glutathione-sensitive connexons are also called disulfide-bonded connexons. Drug release is triggered by the difference between high intracellular glutathione concentrations (millimole range) and relatively low blood glutathione concentrations (micromolar range). In tumor cells in particular, their hypoxic content enhances reductase activity, which leads to higher glutathione concentrations. Disulfide bonds are thermodynamically stable and therefore have better stability in plasma.

[0240] Enzyme-unstable connexons, such as peptide connexons, allow for more precise control of drug release. Peptide connexons are efficiently cleaved by lysosomal proteases such as cathepsin B or plasmin (the content of such enzymes increases in some tumor tissues). Such peptide bonds are highly stable in plasma circulation because proteases are generally inactive extracellularly due to inappropriate pH values ​​and protease inhibitors in the serum. Due to their high plasma stability and excellent intracellular cleavage selectivity and efficacy, enzyme-unstable connexons are widely used as cleavage connexons in antibody-drug conjugates. Typical enzyme-unstable connexons include Val-Cit (VC), Val-Ala (VA), and Gly-Gly-Phe-Gly (GGFG).

[0241] Self-releasing connexons are generally inserted between a cleaved connexon and the active drug, or are themselves part of the cleaved connexon. The mechanism of action of self-releasing connexons is as follows: After a cleaved connexon is cleaved under appropriate conditions, the self-releasing connexon can spontaneously reconfigure its structure and thereby release the bound active drug. Common suicide connexons include p-aminobenzyl alcohol systems (PABs) and β-glucuronide conjugates.

[0242] application The present invention further provides uses for the antibodies of the present invention, such as in the preparation of diagnostic formulations or in the preparation of drugs for the prevention and / or treatment of TF-related diseases. The TF-related diseases include tumorigenesis, growth and / or metastasis, tumor drug resistance-related diseases, inflammation, metabolic diseases, and the like.

[0243] The uses of the antibodies, NDCs, or CAR-T cells of the present invention include (but are not limited to) the following:

[0244] (i) Diagnosis, prevention and / or treatment of tumor development, growth and / or metastasis, particularly of tumors with high TF expression. The tumors include, but are not limited to, breast cancer (e.g., triple-negative breast cancer), lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, malignant glioma, gastric cancer, liver cancer, esophageal cancer, kidney cancer, colorectal cancer, bladder cancer, prostate cancer, endometrial cancer, ovarian cancer, cervical cancer, leukemia, bone marrow cancer, angiosarcoma, etc., and are particularly limited to triple-negative breast cancer, non-small cell lung cancer, pancreatic cancer, and malignant glioma, and more preferably triple-negative breast cancer, non-small cell lung cancer, pancreatic cancer, etc.

[0245] (ii) Diagnosis, prevention and / or treatment of autoimmune diseases, including (but not limited to) systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, type 1 diabetes mellitus, psoriasis, and multiple sclerosis.

[0246] (iii) Diagnosis, prevention and / or treatment of inflammation. The inflammation includes, but is not limited to, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, gout, Reiter's syndrome, psoriatic arthritis, complete arthritis, tuberculous arthritis, viral arthritis, fungal arthritis, glomerulonephritis, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, acute lung injury, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis.

[0247] (iv) Diagnosis, prevention and / or treatment of metabolic disorders, including (but not limited to) diabetes mellitus, diet-induced obesity and adipose tissue inflammation.

[0248] Pharmaceutical composition The present invention further provides compositions. In a preferred example, the composition is a pharmaceutical composition comprising the antibody or its active fragment or its fusion protein or its NDC or corresponding CAR-T cell, and a pharmaceutically acceptable vector. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous vector medium, where the pH is typically about 5 to 8, preferably about 6 to 8, but the pH value can be varied depending on the properties of the substance being formulated and the disease being treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to) intratumoral, intraperitoneal, intravenous, or topical administration.

[0249] The antibodies described in the present invention can also be expressed in cells from their nucleotide sequences for use in cell therapy. For example, these antibodies can be used in chimeric antigen receptor T cell immunotherapy (CAR-T).

[0250] The pharmaceutical composition of the present invention can be used to directly bind to TF protein molecules and, therefore, can be used for the prevention and treatment of diseases such as tumors. Furthermore, other therapeutic agents can be used simultaneously.

[0251] The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001 to 99 wt%, preferably 0.01 to 90 wt%, more preferably 0.1 to 80 wt%) of the monoclonal antibody (or its conjugate) of the present invention and a pharmaceutically acceptable vector or excipient. Such vectors include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The drug formulation must be consistent with the method of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection by conventional methods, for example, using saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions must be prepared under sterile conditions. The dose of the active ingredient is a therapeutically effective dose, for example, about 1 μg / kg body weight to about 5 mg / kg body weight daily. Furthermore, the polypeptide of the present invention can also be used in combination with other therapeutic agents.

[0252] When using a pharmaceutical composition, a safe and effective amount of the immune complex is administered to the mammal, where this safe and effective amount is usually at least about 10 μg / kg body weight and in most cases does not exceed about 50 mg / kg body weight, preferably about 10 μg / kg body weight to about 20 mg / kg body weight. Of course, the specific dosage must take into account factors such as the route of administration and the patient's health condition, and these are all within the scope of the skills of a skilled physician.

[0253] In the case of NDCs, the nanobody-drug conjugates provided by the present invention can be used to treat targeted diseases by targeting specific cell populations, binding to specific proteins (antigens) on the cell surface, and thereby releasing the active drug into the cell by endocytosis or drug penetration. The antibody-drug conjugates can be administered to subjects (e.g., humans) in therapeutically effective doses via appropriate routes. Subjects requiring treatment may be patients at risk of or suspected of having a disease related to the activity or expression of a particular antigen. Such patients can be identified by conventional physical examinations.

[0254] When the nanobody-drug conjugate of the present invention is used for therapeutic purposes, it can be delivered by conventional methods in the art. For example, it can be introduced into cells using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the nucleic acid or vector can be delivered locally by direct injection or infusion pump.

[0255] The main advantages of this invention are as follows:

[0256] 1. The TF nanobodies of the present invention possess excellent biological activity and specificity, as well as high affinity. The range of the binding affinity constant KD values ​​between the chimeric nanobodies 4-A02, F1-A01, 4-E04, 4-C05, 5-C12, and 5-H3 and human TF-ECD, as measured by surface plasmon resonance (SPR) analysis, is 0.202 to 7.04 nM.

[0257] 2. It has excellent binding affinity to tumor cells' TF, and EC measured by FACS. 50 Since its concentration range is 0.05 μg / mL to 0.75 μg / mL and inhibits tumor cell TF-related function, it can be used as a targeted therapeutic antibody.

[0258] 3. Nanobody-drug conjugates TF-NDC (tubulin inhibitor, topoisomerase 1 inhibitor), designed based on TF nanobodies, can specifically kill high-expression tumor cells in TF and IC 50 The values ​​range from 0.0001 g / mL to 0.03 g / mL.

[0259] 4. The TF-NDC of the present invention possesses remarkable antitumor activity, and single doses of 3 mg / kg and 1 mg / kg demonstrate potent antitumor activity and the ability to modulate the immune microenvironment in vivo.

[0260] 5. According to the design of Figure 24 of the present invention, TF nanobody-drug complexes having a desired DAR value can be easily prepared, and their crosslinking reaction equations are as shown in Figure 23 1a, 1b, 1c, or 1d, respectively.

[0261] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are used solely for the purpose of illustrating the present invention and do not limit its scope. In the following examples, experimental methods that do not specify detailed conditions typically follow conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Experimental Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions suggested by the manufacturer. Unless otherwise specified, percentages and quantities are calculated by weight.

[0262] Example 1. Discovery and preparation of nanobodies targeting human tissue factor (TF). Human TF extracellular domain protein (TF-ECD, Met 1-Glu 251) is purchased from SinoBiological (catalog number: 13133-H08H). Alpaca are immunized using the TF extracellular domain protein prepared above, with an immunization interval of 21 days. Ten days after the final immunization, a portion of peripheral blood (PBMCs) is collected, the PBMCs are isolated, and VHH antibody fragments are cloned to construct yeast display libraries and phage display libraries. Through repeated panning, monoclonal ELISA, FACS screening, etc., seven highly active and sequence-independent nanobodies (VHH) 4-A02, F1-A01, 4-C08, 4-E04, 4-C05, 5-C12, and 5-H3 are obtained from the phage display library. Chimeric antibodies were prepared by fusing each of the seven VHH amino acid sequences with the human FC mutant fragment FC1. These were transiently transfected onto HEK293, and then suspended and cultured in serum-free medium for 6 days. The culture supernatant was collected, purified, and subjected to protein quantification and SDS-PAGE detection.

[0263] The results are shown in Figure 1, and the fusion proteins derived from 4-A02, F1-A01, 4-C08, 4-E04, 4-C05, 5-C12, and 5-H3 can all be efficiently expressed, produced, and purified.

[0264] Example 2. Measurement of SPR of affinity of TF nanobodies to human TF antigen. Antibody affinity was measured using the ForteBio OCTET R2 instrument. FDYKSD-01-His was immobilized on an NTA sensor (Sartorius, Cat#18-5101) for 200 seconds. The buffer was PBST (PBS + 0.02% tween20), and the nanobody samples were diluted to 25, 12.5, 6.25, 3.13, 1.56, and 0 nM. Affinity detection: equilibration 60 seconds, binding 180 seconds, dissociation 180 seconds, detection temperature 25°C. Kinetic characterization was performed using the ForteBio OCTET R2 system.

[0265] The detection results are shown in Figure 2. The 4-A02, F1-A01, 4-E04, 4-C05, 5-C12, and 5-H3 chimeric antibodies showed strong affinity for TF-ECD, with binding constants (KD) of 2.09 nM, 4.37 nM, 7.04 nM, 0.2 nM, 0.001 nM, and 0.45 nM, respectively.

[0266] Example 3. Measurement of the binding affinity of TF nanobodies to TF on the surface of tumor cells. Using TF-high-expressing triple-negative breast cancer cells MDA-MB-231, 100 μL of the test antibody was diluted from 10 μg / mL to 0.016 μg / mL using a 5-fold gradient. The test antibody was used as the primary antibody, and each was suspended in 100 μL of serum-free RPMI-1640 medium and measured 1 × 10⁶ 5The primary antibody was homogeneously mixed with individual tumor cells, diluted from 5 μg / mL to 0.008 μg / mL, incubated at 4°C for 1 hour, washed twice with PBS to remove unbound primary antibody, incubated the target cells with 200 μL of 2 μg / mL PE-labeled secondary antibody at 4°C for 30 minutes, washed twice with PBS to remove unbound secondary antibody, and finally resuspended the cells in 200 μL of PBS. The binding affinity of the test antibody to the corresponding cell surface TF was measured using flow cytometry.

[0267] The detection results are shown in Figure 3, and 4-A02, F1-A01, 4-C08, 4-E04, 4-C05, 5-C12, and 5-H3 have excellent binding affinity to MDA-MB-231, and EC 50 These are 0.08 μg / mL, 0.75 μg / mL, 0.05 μg / mL, 0.13 μg / mL, 0.7 μg / mL, 0.47 μg / mL, and 0.25 μg / mL, respectively.

[0268] Example 4. Preparation of TF nanobody fusion protein-vc-MMAE Protein stock solutions of FC1 chimeric nude antibodies (generally dimers) derived from 4-A02, F1-A01, 4-C08, 4-E04, 4-C05, 5-C12, and 5-H3, or 4-A02-ABD (monomer), are diluted to 5 mg / mL with 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH2PO4-NaOH) / 150 mM sodium chloride (NaCl) / 1 mM diethyltriaminepentaacetic acid (DTPA), pH 7.4 reaction buffer. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) is added in an excess molar ratio of 3-4 times, and the reaction mixture is stirred at 25°C for 2.5 hours.

[0269] The above reaction solution is cooled to 0-10°C, an appropriate amount of diethylacetamide (DMA) is added without purification, and mc-vc-MMAE (10 mg / ml dissolved in DMA beforehand) is added in an excess molar ratio of 6:1. The volume ratio of DMA in the reaction system is not more than 10%, and coupling is carried out with stirring at 10-25°C for 2 hours.

[0270] Using a desalting column, the coupling reaction mixture is filtered and purified from a pH 6.0 histidine-acetic acid / sucrose gel, and peak samples are collected based on UV280 ultraviolet absorption values. The samples are then sterilized using a 0.22 μm pore size filter and stored at -80°C.

[0271] For nude antibodies and MMAE conjugates, purity and coupling degree are detected using methods such as hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC). For specific conjugates of antibody II, the target drug loading (DAR) is 4, and for antibody-ABD monomer conjugates, the target drug loading (DAR) is 1.

[0272] The results are shown in Figure 4, indicating that the HIC detection analysis of the 4-A02-MMAE conjugate shows a DAR4 ratio of approximately 95%.

[0273] The results are shown in Figure 5, indicating that the HIC detection analysis of the F1-A01-MMAE conjugate shows a DAR4 ratio of approximately 91%.

[0274] The results are shown in Figure 6, and the HIC detection analysis of the 4-C08-MMAE conjugate shows that the DAR4 and DAR2 ratios are approximately 41.8% and 42.7%, respectively.

[0275] The results are shown in Figure 7, and HIC detection analysis of the 4-C05-MMAE conjugate indicates that the nude antibody is substantially converted to the conjugate.

[0276] The results are shown in Figure 8, and the HIC detection analysis of the 5-C12-MMAE conjugate shows that the DAR4 and DAR2 ratios are approximately 66.2% and 21.9%, respectively.

[0277] The results are shown in Figure 9, and HIC detection analysis of the 4-E04-MMAE conjugate indicates that the coupling reaction did not complete as expected, resulting in a very low proportion of coupling products.

[0278] The results are shown in Figure 10, and HIC detection analysis of the 4-A02-ABD-MMAE conjugate shows that the nude antibody is substantially converted to the conjugated DAR1.

[0279] Example 5. Preparation of TF nanobody-GGFG-DXd The 4-A02-FC1 nude antigen solution is replaced with 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate (NaH2PO4-Na2HPO4) / 150 mM sodium chloride (NaCl) / 2 mM ethylenediaminetetraacetic acid (EDTA) in a pH 7.0 reaction buffer. The concentration of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) is 10 mg / mL and 2.2 times the molar ratio. The reaction mixture is stirred at 25°C for 4-6 hours. The reaction mixture is cooled to room temperature, an appropriate amount of diethylacetamide (DMA) is added, and compound GGFG-DXd (purchased from Shanghai Haoyuan Chemexpress, 10 mg / mL pre-dissolved in DMA) is added at a 4 times molar ratio. The volume ratio of DMA in the reaction system is not more than 10%, and coupling is performed at 25°C for 1-2 hours with stirring. Excess low molecular weight is removed using a G25 desalting column, or the excess low molecular weight is ultrafiltered directly through a filter tube, replaced with PBS / 8% sucrose, pH 7.4 buffer, then sterilized using a 0.22 μm pore size filter, stored at -80°C, and the resulting antibody conjugate is named 4-A02-DXd. The results are as shown in Figure 11, and the HIC detection analysis of the 4-A02-DXd conjugate shows that the naked antibody is completely converted to conjugate DAR4.

[0280] Example 6. In vitro antitumor activity of TF-NDC against tumor cells with high and medium levels of TF expression The cell lines used in this example were purchased from the American Type Culture Collection (ATCC), the Cell Bank of the Chinese Academy of Sciences, and Nanjing COBIOER, and cultured according to the corresponding instructions. They included triple-negative breast cancer HCC1806, MDA-MB-231, HCC1954, HCC1937, MDA-MB-468, pancreatic cancer BxPC3, HPAF-II, non-small cell lung cancer HCC44, NCI-H1975, PC9, etc. The cells in the logarithmic growth phase were inoculated into a 96-well cell culture plate at a density of 200 - 2000 cells per well (determined by the growth rate of different cells) at 150 μL / well, cultured at 37°C and 5% CO2 for about 5 hours, and then different concentrations of TF-NDCs (15 μg / mL - 0.00019 μg / mL) were added. For each drug concentration, 2 - 4 duplicate wells, as well as corresponding solvent control and blank control wells, were set. After culturing for 5 - 9 days (to ensure sufficient cell division times according to the cell growth rate), the culture medium was removed, 100 μL / well of MTS reaction solution (purchased from Promega, cat#G3581) was added, and the reaction was carried out at 37°C until the desired color development intensity was reached. The cell viability (OD490nm) of each group was measured, and the cell viability was calculated according to the following formula. Viability = (OD administered - OD blank) / (OD control - OD blank) × 100%. The above data was analyzed using GraphPad Prism 8 software, and the IC 50 values of the above TF-NDC in different cell lines were calculated respectively.

[0281] The in vitro antitumor activity results for the preferred TF-NDC4-A02-MMAE, F1-A01-MMAE, 4-C08-MMAE, 4-C05-MMAE, 5-C12-MMAE, 4-E04-MMAE, and HuSC1-MMAE as a control are shown in Figures 12, 13, and 14, respectively.

[0282] As shown in Figure 12, TF-NDCs exhibit potent growth inhibitory activity against TF-high-expressing triple-negative breast cancer HCC1806 cells, and IC 50 The values ​​range from 0.0001 to 0.0326 g / mL.

[0283] As shown in Figure 13, TF-NDCs exhibit potent growth inhibitory activity against TF-high-expressing pancreatic cancer BxPC3 cells, and IC 50 The values ​​range from 0.0001 to 0.0288 g / mL.

[0284] As shown in Figure 14, TF-NDCs exhibit potent growth inhibitory activity against TF-high-expressing pancreatic cancer HPAF-II cells, and IC 50 The values ​​range from 0.0007 to 0.0243 g / mL.

[0285] Table 3 shows the IC scores for six types of TF-NDC cell proliferation inhibition tests. 50 Combine the values. [Table 3]

[0286] Based on the completion of the above antitumor activity detection, 4-A02 was selected for design and mass production, and the proteins 4-A02-FC1, 4-A02-FCWT, 4-A02-FCLALAPG, and 4-A02-ABD were purified. The results are shown in Figure 20. Here, the molecular weights of 4-A02-FC1, 4-A02-FCWT, and 4-A02-FCLALAPG are approximately 39 kDa, and the molecular weight of the 4-A02-ABD protein is approximately 20 kDa.

[0287] Using the batch-purified proteins described above, 4-A02-MMAE was repeatedly prepared as shown in Figures 10 and 11 to design and prepare 4-A02-ABD-MMAE and 4-A02-DXd.

[0288] The results are shown in Figures 15, 16, 17, 18, and 19. 4-A02-MMAE and 4-A02-ABD-MMAE exhibit potent growth inhibitory activity in TF-high or moderately expressing HPAF-II, BxPC3, HCC44, PC9, and H1975 cells. IC 50 The values ​​are 0.0002–0.022 g / mL and 0.0003–0.163 g / mL, respectively.

[0289] The results are shown in Figures 15, 16, 17, 18, and 19. 4-A02-DXd exhibited excellent growth inhibitory activity in HPAF-II, BxPC3, and HCC44 cells, and IC 50 The value is approximately 0.0024 to 0.019 g / mL. The inhibitory activity of 4-A02-DXd in lung cancer PC9 and H1975 cells is relatively weak, and IC 50 The values ​​are 5.63 g / mL and >15 g / mL, respectively.

[0290] Table 4 shows the IC scores for three types of TF-NDC cell proliferation inhibition tests. 50 Combine the values. [Table 4]

[0291] Further evaluation of the in vitro antitumor efficacy of 4-A02-FC1-Dxd, 4-A02-FC1-MMAE, 4-A02-FCWT-MMAE, and 4-A02-ABD-MMAE in multiple triple-negative breast cancer (TNBC) cell lines. As shown in Figures 25 (MDA-MB-231), 26 (HCC1806), 27 (HCC1954), 28 (HCC1937), and 29 (MDA-MB-468), 4-A02-Dxd inhibits TNBC cell proliferation (IC). 50The values ​​were 0.001 to 0.008 g / mL, and similarly, MMAE-coupling type 4-A02-NDC also showed a similarly potent antitumor effect, IC 50 The value is between 0.0003 and 0.004 g / mL.

[0292] Example 7. Batch expression, production, and purification of TF nanobodies and fusion proteins. Nanobody variable region sequences were cloned into FC1, FCWT, FCLLALAPG, and ABD expression vectors (4-A02-FC1, 4-A02-FCWT, 4-A02-FCLALAPG, 4-A02-ABD), transiently transfected with HEK293, and then suspended and cultured in serum-free medium for 6 days. The culture supernatant was collected, purified, and subjected to protein quantification and SDS-PAGE detection.

[0293] As shown in Figure 20, both chimeric nanobodies and recombinant fusion proteins derived from 4-A02 can be efficiently expressed, produced, and purified.

[0294] Example 8. In vivo antitumor effect of TF nanobodies (triple-negative breast cancer breast pad transplant tumor model) Triple-negative breast cancer (TNBC) HCC1806 cells in the logarithmic growth phase were divided into 3 × 10⁶ cells per 200 μL of serum-free medium. 6 At a density of [number], the breastfeeding pads of 6-week-old female Balb / c nude mice (Balb / c nude mice are purchased from Shanghai SIPPR-BK LAB ANIMAL Co., Ltd.) were inoculated, and the tumors grew to 100-200 mm. 3 After growing to maturity, the animals are randomly divided into groups, with eight tumors placed in each group. Six preferred TF-NDCs of the present invention are administered intravenously at a dose of 3 mg / kg on the day of group division. hIgG1-MMAE is used as a negative control drug and is administered a total of one time during the study period. Tumor volume and nude mouse weight are measured and recorded 2-3 times per week to create a tumor growth curve. The formula for calculating tumor volume (V) is V = 1 / 2 × a × b 2 Here, a and b represent the length and width of the tumor, respectively.

[0295] The results are shown in Figure 21. Compared to the hIgG1-MMAE control group, the 3 mg / kg doses of 4-A02-MMAE, F1-A01-MMAE, 4-C08-MMAE, 4-C05-MMAE, and 5-C12-MMAE all showed potent antitumor activity and led to tumor regression. Furthermore, the conjugate 4-E04-MMAE showed only partial tumor inhibition, which may be related to its low coupling efficiency (Figure 9).

[0296] Further evaluation of the in vivo antitumor efficacy of 4-A02-FC1-Dxd, 4-A02-FC1-MMAE, 4-A02-FCWT-MMAE, and 4-A02-ABD-MMAE in a triple-negative breast cancer model.

[0297] The results are shown in Figure 30, and in the HCC1806 tumor regression experiment, the 3 mg / kg dose of 4-A02-MMAE resulted in complete tumor regression within 21 days.

[0298] The results are shown in Figure 31. In the HCC1954 breast milk pad transplant tumor experiment, 10 mg / kg of 4-A02-FC1-Dxd, 3 mg / kg of 4-A02-MMAE, and 3 mg / kg of 4-A02-ABD-MMAE all significantly inhibited tumor growth and induced tumor regression.

[0299] Example 9. In vivo antitumor effect of TF nanobodies (lung cancer orthotopic transplant tumor model) Nude mice aged 4-5 weeks are purchased and acclimatized for one week in an SPF animal laboratory. HCC44-luc cells in the logarithmic growth phase are digested, centrifuged in a pre-cooled centrifuge at 4°C, and the cells are resuspended in PBS according to the cell number, with a cell density of 40 × 10⁶. 6Adjust the concentration to cells / mL and add an equal volume of high-concentration Matrigel, i.e., cell suspension:Matrigel = 1:1. After anesthetizing the mice with Aveltin (400 μL / 20 g), make a small incision in the right flank near the axilla (a total of 3 layers of skin and muscle) to expose the pink lung, taking care to avoid the Y-shaped blood vessel on the skin during the incision, and insert the needle at the midpoint of the Y-shape. Draw a line 3 mm from the tip of the insulin needle, i.e., the depth of needle insertion, and slowly insert it into the cells, inoculating 50 μL into each mouse, for a total of 1 × 10⁶ 6 After injection, the needle is held for 5-10 seconds, rotated, removed, and the wound sutured. On day 13, images are taken using a live imaging device, and the mice are grouped based on signal values. On day 14, 3 mg / kg of hIgG1-MMAE and 4-A02-MMAE (n=6-8) are administered intraveinally, for a total of one dose during the study period. On days 20, 27, and 34, images are taken using a live imaging device, and fluorescence values ​​are detected and recorded.

[0300] The results are shown in Figure 22. Compared to the hIgG1-MMAE control group, the 3 mg / kg dose of 4-A02-MMAE demonstrated superior antitumor activity, resulting in complete tumor regression in 5 out of 6 mice.

[0301] Example 10. In vivo antitumor effect of TF nanobodies (pancreatic cancer transplant tumor model) Pancreatic cancer (PDAC) HPAF-II cells in the logarithmic growth phase were divided into 2.5 × 10⁶ cells per 200 μL of serum-free medium. 6 Inoculate subcutaneously into the dorsal region of 5-week-old female Balb / c nude mice at the specified density. (Balb / c nude mice are purchased from Shanghai SIPPR-BK LAB ANIMAL Co., Ltd.) When the tumor is 100-200 mm 3After growth, the animals are randomly divided into groups, with eight tumors placed in each group. TF-NDCs such as the preferred 4-A02-MMAE and 4-A02-ABD-MMAE of the present invention are administered intraveinally at doses of 3 mg / kg and 1 mg / kg respectively on the day of group division. hIgG1-MMAE is used as a negative control drug and is administered a total of one time during the study period. Tumor volume and nude mouse weight are measured and recorded twice a week to create a tumor growth curve. The formula for calculating tumor volume (V) is V = 1 / 2 × a × b 2 Here, a and b represent the length and width of the tumor, respectively.

[0302] As shown in Figure 32A, compared to the hIgG1 group, the single-dose groups of 3 mg / kg 4-A02-FC1-MMAE, 4-A02-FCWT-MMAE, and 4-A02-ABD-MMAE all resulted in complete tumor regression. As shown in Figure 32B, compared to the hIgG1 group, the single-dose groups of 1 mg / kg 4-A02-FC1-MMAE and 4-A02-FCWT-MMAE also showed superior antitumor activity, resulting in complete inhibition of tumor growth and partial regression.

[0303] Example 11. Humanization modification and activity detection of TF nanobodies Based on the original antibody sequence information, an antibody homology model was obtained by modeling, and framework amino acids in the 5-angstrom range were analyzed from the CDRs. These amino acid sites typically affect the conformation or antigen-binding activity of the CDRs. Humanized germlines were obtained by IMGT analysis, and after joining the selected humanized germline framework with the antibody's CDRs, the designed human antibody and the framework region sequence of the original antibody were aligned. By analyzing the homology modeling results of the parent antibody, amino acids similar to those on the human antibody surface were selected and replaced while maintaining antibody activity and reducing heterogeneity. This resulted in the acquisition of human antibodies 4A02-HM7, 4A02-HM8, and 4A02-HM9, with corresponding sequences SEQ ID NO.33, SEQ ID NO.34, and SEQ ID NO.35.

[0304] The affinity of human antibodies to the antigen was detected using the surface plasmon resonance (SPR) measurement method of Example 2. The results are shown in Figure 33, and 4A02-HM7, 4A02-HM8, and 4A02-HM9 had antigen-binding affinities very similar to the original antibody 4A02, with binding constants of 0.62 nM, 0.3 nM, 1.64 nM, and 0.29 nM, respectively, in the same detection experiment.

[0305] The binding activity between human antibodies and cells was detected using the method for measuring TF binding affinity on the surface of tumor cells described in Example 3. The results are shown in Figure 34A, and the EC of the binding affinity between 4A02-HM7, 4A02-HM8, 4A02-HM9, and the original antibody 4A02 and triple-negative breast cancer MDA-MB-231 cells was measured. 50 The values ​​were 0.18-0.21 g / mL, and the results are shown in Figure 3B, showing the binding affinity of 4A02-HM7, 4A02-HM8, 4A02-HM9, and the original antibody 4A02 to pancreatic cancer HPAF-II cells. 50 The concentration is 0.24-0.32 g / mL.

[0306] Example 12. ELISA measurement of affinity of humanized TF nanobodies to human TF and cynomolgus monkey TF antigens. Using a coating solution, dilute the TF protein extracellular domain (human TF-ECD, cynomolgus monkey TF-ECD) to 1 μg / mL, coat an ELISA plate with 100 μL / well, and incubate overnight at 4°C. Wash off excess antigen, block with 1% BSA at room temperature for 2 hours, then add each nanobody diluted with a 5-fold gradient at 100 μL / well, incubate at room temperature for 1 hour, wash off unbound antibody, add 100 μL / well of appropriate concentration of HRP-labeled anti-Fc segment secondary antibody, incubate at room temperature, wash off unbound secondary antibody, add TMB chromogenic solution and react for approximately 15 minutes, add 50 μL / well of 1 M HCl to stop the color reaction, then measure absorbance at 450 nm and analyze the data.

[0307] According to the ELISA detection results, the binding affinity of 4A02-HM7, 4A02-HM8, 4A02-HM9, and the original antibody 4A02 to human TF-ECD was as follows: 50 The EC binding affinity was 5.91-6.89 ng / mL (Figure 35A), and the binding affinity of 4A02-HM7, 4A02-HM8, 4A02-HM9, and the original antibody 4A02 to cynomolgus monkey TF-ECD was EC 50 This indicates a concentration of 5.96–9.78 ng / mL (Figure 35B).

[0308] In summary, the efficacy studies of TF-NDC-related examples are clearly shown as follows:

[0309] 1. TF-NDC 4-A02-MMAE, F1-A01-MMAE, 4-C08-MMAE, 4-C05-MMAE, and 5-C12-MMAE all possess excellent TF-specific tumor cell killing activity, meaning they exert potent inhibitory effects on the proliferation of TF-moderately and highly expressed tumor cells, while showing no significant toxicity to the proliferation of TF-low expressing cells.

[0310] 4-A02-ABD-MMAE, prepared with 2.4-A02-ABD, also exhibits similarly excellent antitumor activity.

[0311] 3.4-A02-DXd exhibits very strong or excellent in vitro antitumor activity in preferred pancreatic cancer, multiple triple-negative breast cancer, and lung cancer cells.

[0312] 4. In in vivo drug efficacy models for triple-negative breast cancer, lung cancer, and pancreatic cancer, the preferred TF-NDC of the present invention exhibits excellent antitumor therapeutic effects after a single intravenous administration.

[0313] Nanobody array of the present invention SEQ ID NO.1:4-A02 CDR1 ETISSTYI SEQ ID NO.2:4-A02 CDR2 ISGDGVTH SEQ ID NO.3:4-A02 CDR3 YAAGRWNH SEQ ID NO.4:4-A02 VH QVQLVESGGGLVQPGGSLSLSCTAS ETISSTYI MGWHRRSPGKERELVAV ISGDGVTH YADFVKGRFVISRDNAKNAVYLKMNFLTPEDTAVYYC YAAGRWNH WGQGTQVTVSS SEQ ID NO.5:F1-A01 CDR1 GRAFSAYA SEQ ID NO.6:F1-A01 CDR2 ISWSGGST SEQ ID NO.7:F1-A01 CDR3 NADSLLSLLDGSRGGPGTDSGS SEQ ID NO.8:F1-A01 VH EVQLVESGGGLVQAGGSLRLSCAAS GRAFSAYA MGWFRQAPGKEREFVAA ISWSGGST YYADSVRGRFTISRDNTKNTVYLQMNSLKPEDTAVYYC NADSLLSLLDGSRGGPGTDSGS WGQGTQVTVSS SEQ ID NO.9:4-C08 CDR1 GFSLSSYD SEQ ID NO.10:4-C08 CDR2 IHSSGGYP SEQ ID NO.11:4-C08 CDR3 NLPPSRRWYKDY SEQ ID NO.12:4-C08 VH DVQLVESGGGLVQPGGSLRLSCAVS GFSLSSYD MNWYRQGPGKEREWVAN IHSSGGYP DYADAVKGRFTISRDNAKNMVYLQMNSLKPEDTAVYYC NLPPSRRWYKDY WGQGTQVTVSS SEQ ID NO.13:4-E04 CDR1 EMISSTYI SEQ ID NO.14:4-E04 CDR2 ISGDGVTH SEQ ID NO.15:4-E04 CDR3 NAAGRRNH SEQ ID NO.16:4-E04 VH AVQLVESGGGLVQPGGSLSLSCAAS EMISSTYI MGWYRRSPGKERELVAV ISGDGVTH YGDFVKGRFAISRDNVKNAVYLKMNFLTPEDTAVYYC NAAGRRNH WGQGTQVTVSS SEQ ID NO.17:4-C05 CDR1 GFTLDTYA SEQ ID NO.18:4-C05 CDR2 ISSTDGST SEQ ID NO.19:4-C05 CDR3 AAGPGTDCPLRFDY SEQ ID NO.20:4-C05 VH QVQLVESGGGLVQPGGSLRLSCAAS GFTLDTYA IGWIRQAPGKEREGVSC ISSTDGST YYAASVKGRFTINRDNAKNTVYLQMNSLKPEDTAVYYC AAGPGTDCPLRFDY WGQGIQVTVSS SEQ ID NO.21:5-C12 CDR1 GRTFSTDA SEQ ID NO.22:5-C12 CDR2 INWSGGST SEQ ID NO.23:5-C12 CDR3 VADSLLALLDGSRGGPGTDSDS SEQ ID NO.24:5-C12 VH QVQLVESGGGLAQAGGSLRLSCAGS GRTFSTDA MGWFRQAPGKEREFVAA INWSGGST YYADSVKGRFTISRDNAKNTVYLQMNSVKPEDTAVYYC VADSLLALLDGSRGGPGTDSDS WGQGTQVTVSS SEQ ID NO.25:5-H3 CDR1 GFTLANYA SEQ ID NO.26:5-H3 CDR2 ISRSDGDT SEQ ID NO.27:5-H3 CDR3 RATEWCGVQDPHGY SEQ ID NO.28:5-H3 VH QVQLVESGGGLVQPGGSLRLSCVAS GFTLANYA IGFYRRAPGKGREGVSC ISRSDGDT YHVDSVKGRFTVSRDNAKNTAYLQMNSLVPEDTGVYYC RATEWCGVQDPHGY WGQGTQVTVSS SEQ ID NO.29:4-A02-FC1 QVQLVESGGGLVQPGGSLSLSCTASETISSTYIMGWHRRSPGKERELVAVISGDGVTHYADFVKGRFVISRDNAKNAVYLKMNFLTPEDTAVYYCYAAGRWNHWGQGTQVTVSSEPKSSDKTHT C PP C PAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.30:4-A02-FCWT QVQLVESGGGLVQPGGSLSLSCTASETISSTYIMGWHRRSPGKERELVAVISGDGVTHYADFVKGRFVISRDNAKNAVYLKMNFLTPEDTAVYYCYAAGRWNHWGQGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.31:4-A02-FCLALAPG QVQLVESGGGLVQPGGSLSLSCTASETISSTYIMGWHRRSPGKERELVAVISGDGVTHYADFVKGRFVISRDNAKNAVYLKMNFLTPEDTAVYYCYAAGRWNHWGQGTQVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.32:4-A02-ABD QVQLVESGGGLVQPGGSLSLSCTASETISSTYIMGWHRRSPGKERELVAVISGDGVTHYADFVKGRFVISRDNAKNAVYLKMNFLTPEDTAVYYCYAAGRWNHWGQGTQVTVSSGGGGSGGGGSGGGGSQHDEAVDANSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPAAACAAAHHHHHH SEQ ID NO.33:4A02-HM7-FCWT QVQLVESGGGLVQPGGSLRLSCTASETISSTYIMGWHRQAPGKGRELVAVISGDGVTHYADFVKGRFTISRDNAKNTVYLQMNFLRAEDTAVYYCYAAGRWNHWGQGTMVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.34:4A02-HM8-FCWT QVQLVESGGGLVKPGGSLRLSCTASETISSTYIMGWHRQAPGKGRELVAVISGDGVTHYADFVKGRFTISRDNAKNTVYLQMNFLRAEDTAVYYCYAAGRWNHWGQGTMVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.35:4A02-HM9-FCWT QVQLVESGGGLVQPGGSLRLSCTASETISSTYIMGWHRQAPGKGLELVAVISGDGVTHYADFVKGRFTISRDNAKNAVYLQMNFLRAEDTAVYYCYAAGRWNHWGQGTMVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0314] All documents referenced in this invention are cited as references in this application, as if each document were cited individually. Furthermore, after reading the above teachings of this invention, persons skilled in the art can make various changes or modifications to the invention, and these equivalent forms are also included within the scope defined by the claims appended to this application.

Claims

1. A nanobody that targets tissue factor (TF), The complementarity determination region CDR of the VHH chain of the nanobody is (1) CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3, indicated by SEQ ID NO. 3, Or, (2) CDR1 shown in SEQ ID NO. 5, CDR2 shown in SEQ ID NO. 6, and CDR3, indicated in SEQ ID NO. 7 Or, (3) CDR1 shown in SEQ ID NO. 9, CDR2 shown in SEQ ID NO. 10, and CDR3, indicated in SEQ ID NO. 11 Or, (4) CDR1 shown in SEQ ID NO. 13, CDR2, shown in SEQ ID NO. 14, and CDR3, indicated in SEQ ID NO. 15, Or, (5) CDR1 shown in SEQ ID NO. 17, CDR2 shown in SEQ ID NO. 18, and CDR3, indicated in SEQ ID NO. 19 Or, (6) CDR1 shown in SEQ ID NO. 21, CDR2 shown in SEQ ID NO. 22, and CDR3 shown in SEQ ID NO. 23, Or, (7) CDR1 shown in SEQ ID NO. 25, CDR2, shown in SEQ ID NO. 26, and One or more types selected from the group consisting of CDR3 shown in SEQ ID NO. 27, The nanobody is characterized in that any one of the above amino acid sequences further comprises a derivative sequence in which at least one amino acid is optionally added, deleted, modified, and / or substituted, and which maintains maintenance TF binding affinity.

2. The VHH chain of the nanobody is characterized by further comprising a framework region (FR). The nanobody according to claim 1.

3. The VHH chain of the nanobody targeting the TF has an amino acid sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 8, SEQ ID NO. 12, SEQ ID NO. 16, SEQ ID NO. 20, SEQ ID NO. 24, and SEQ ID NO. 28, or the VHH chain of the nanobody targeting the TF has an amino acid sequence as shown at positions 1 to 114 of SEQ ID NO. 33, positions 1 to 114 of SEQ ID NO. 34, or positions 1 to 114 of SEQ ID NO.

35. The nanobody according to claim 1.

4. An antibody that targets TF, The antibody is characterized in that it comprises a VHH chain of a nanobody that targets tissue factor as described in one or more claims 1 to 3, and is an antibody that targets the TF.

5. It is a multispecific antibody, The multispecific antibody is characterized by comprising a nanobody that targets TF as described in any one of claims 1 to 3 or an antibody that targets TF as described in claim 4. Multispecific antibodies.

6. The multispecific antibody is characterized by further comprising a second antigen-binding region that targets a target point selected from the group consisting of EGFR, TGFβ, BCMA, B7H6, GUCY2C, DLL3, CD38, CD123, CD19, CD20, CD22, B7-H3, GPC3, HER2, PMSA, CD28, 4-1BB, OX40, CD40, CD27, CD3, CTLA4, PD1, PDL1, CD73, BCMA, GLP-1, Trop2, TIGIT, LAG-3, FGL1, TLR7, or a combination thereof. The multispecific antibody according to claim 5.

7. Recombinant protein, The recombinant protein is (i) Nanobody that targets TF as described in any one of claims 1 to 3, nanobody that targets TF as described in claim 4, or nanobody that targets TF as described in claim 5, and (ii) Any polypeptide molecule or fragment having therapeutic function, and / or (iii) The recombinant protein having an optional functional domain that improves the physicochemical properties or drug potential of the protein.

8. The recombinant protein has the following structure from the N-C terminus or C-N terminus: (A - B) m, Here, element A is a nanobody that targets TF, The B element is an Fc segment, a human albumin-binding domain (ABD), or an anti-albumin nanobody (HLE). The "-" symbol represents a peptide bond or linker. Here, m is a positive integer. The recombinant protein according to claim 7.

9. It is a CAR structure, The CAR construct is characterized in that the antigen-binding region of the CAR construct is a VHH chain of a nanobody according to any one of claims 1 to 3.

10. Recombinant immune cells, The immune cells are characterized by expressing the exogenous CAR construct described in claim 9. Recombinant immune cells.

11. It is an immune complex, The aforementioned immune complex (a) an antibody portion which is a nanobody that targets a TF as described in any of claims 1 to 3 or an antibody that targets a TF as described in claim 4, and (b) The immune complex comprising a coupling portion coupled to the nanobody portion selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, an enzyme, a proteolytic agent, an immunomodulator (immunoagonist and inhibitor), an oligonucleotide, or a combination thereof.

12. The aforementioned immune complex is an antibody-drug conjugate (ADC) represented by the following molecular formula: 【Chemistry 17】 Here, nAb is a nanobody that targets TF or the recombinant protein according to claim 5. LU stands for Linker 2 (also known as Connexon), D is a drug, Furthermore, the subscript 'p' is characterized by being a value selected from 1 to 8. The immune complex according to claim 11.

13. The LU is characterized by being selected from maleimidocaproyl (MC), maleimide (MAL), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (SMCC) linker linked to the antibody portion, and containing one or more connexons of valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), alanine-alanine-alanine (AAA), p-aminobenzyloxycarbonyl (PAB), and polyethylene glycol (PEG). The immune complex according to claim 12.

14. D is, (i) Tubulin inhibitors such as meitansine derivatives (DM1, DM4), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), (ii) DNA-acting toxins such as duocarmycin and pyrrolobenzodiazepine (PBD), (iii) Characterized by being selected from the following compounds that have antitumor activity with topoisomerase inhibitors such as camptothecin, SN38, extecan, and Dxd. The immune complex according to claim 12.

15. The aforementioned proteolytic agent is a tumor-related protein degrader, and the tumor-related proteins include EGFR, NF-κB, RIPK2, BCR-ABL, HER2, c-Met, TBK1, CDK, ALK, Akt, CK2, ERK1 / 2, FLT3, PI3K, BTK, TRK, Fak, BRD, AR, ER, MetAp-2, BCL-XL, Sirt2, HDAC6, Pirin, SMAD3, ARNT, PCAF / GCN5, Tau, and EZH. ２ The proteolytic agent is selected from the group consisting of IRAK4, STAT3FRS2, and RAS (e.g., KRAS, HRAS, and NRAS), and preferably comprises PROTAC (a proteolytic target chimeric body). The immune complex according to claim 11.

16. A pharmaceutical composition, The aforementioned pharmaceutical composition, (i) a nanobody that targets TF according to any one of claims 1 to 3, an antibody that targets TF according to claim 4, a multispecific antibody according to claim 5, a recombinant protein according to claim 7, a recombinant immune cell according to claim 10, or an immune complex according to claim 11, (ii) The pharmaceutical composition comprising a pharmaceutically acceptable vector.

17. Uses of the active ingredient, The active ingredient is selected from the group consisting of a TF-targeting nanobody according to any one of claims 1 to 3, a TF-targeting antibody according to claim 4, a multispecific antibody according to claim 5, a recombinant protein according to claim 7, a recombinant immune cell according to claim 10, or an immune complex according to claim 11, or a combination thereof, and the active ingredient is (a) Preparation of reagents, detection panels or kits for detecting TF-related diseases, and / or (b) The use characterized by being used in the preparation of drugs for the prevention and / or treatment of TF-related diseases.

18. The aforementioned TF-related disease is selected from the group consisting of cancer, thrombosis, autoimmune disease, metabolic disease, infectious disease, or a combination thereof, and is preferably selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, ovarian cancer, cervical cancer, rectal cancer, glioma, melanoma, leukemia, lymphoma, or a combination thereof. The use described in claim 17.

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