Tissue factor targeting nano antibody and preparation method and application of conjugate

Abstract

The invention discloses a novel nano antibody (Nb) of a targeted tissue factor (TF), a nano antibody-drug conjugate (NDC), a preparation method of the nano antibody, and an application of the nano antibody, the nano antibody-drug conjugate, the preparation method of the nano antibody, the preparation method of the nano antibody, and the application of the nano antibody, the nano antibody-drug conjugate, the preparation method of the nano antibody, the preparation method of the nano antibody, the preparation method of the nano antibody, the preparation method of the nano antibody, the preparation method of the nano antibody and the application of the nano antibody. The monoclonal nano antibody and the corresponding NDC can be efficiently and highly specifically combined with purified TF protein and TF on the surfaces of various tumor cells with TF abnormally expressed, have very high affinity and very low immunogenicity, and have a remarkable in-vivo and in-vitro anti-tumor effect.

Description

Preparation method and use of nanoantibodies and conjugates targeting tissue factor Technical Field

[0001] The present invention relates to the field of medicine, and in particular to nano antibodies and nano antibody-drug conjugates targeting tissue factor, as well as preparation methods and uses thereof. Background Art

[0002] Tissue factor (TF) is a transmembrane glycoprotein with a molecular weight of 47 kd. Under physiological conditions, TF activates extrinsic coagulation after vascular trauma. However, TF is abnormally activated in many tumor tissues, with high rates of abnormal expression in breast, pancreatic, lung, and esophageal cancers. For example, the abnormal expression rate of TF in breast cancer is 85.8%, in pancreatic cancer is 88.5%, in lung cancer is 83.6%, and in esophageal cancer 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-coupled receptor Protease-activated receptor 2 (PAR2). TF, through PAR2, can influence a range of intracellular tumor signaling functions. For example, TF-PAR2 phosphorylation, through MAPK / ERK, induces the expression of key growth factors, immunomodulatory factors, and chemokines, promoting angiogenesis and providing sufficient nutrients, energy, and a suitable microenvironment for tumor growth. Furthermore, TF can enhance tumor cell migration and adhesion through interactions with Rac1 and β1 family integrins, thereby enhancing 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 the current international research on the role and function of TF in diseases related to TF, the development of specific therapeutic antibodies targeting TF is extremely beneficial for the diagnosis, treatment, and prevention of pathological features such as angiogenesis and abnormal coagulation caused by TF in various diseases such as cancer, thrombosis, and inflammation. Tivdak (tisotumab vedotin), the world's first TF-targeted anti-cancer drug, was approved for marketing in 2021, and several TF-ADCs developed by different pharmaceutical companies are in clinical trials.

[0003] Monoclonal antibody drugs have brought breakthroughs in cancer treatment and improved clinical treatment across multiple cancer areas. However, the molecular weight of traditional monoclonal antibodies is 150 kDa, which limits their penetration and drug distribution in solid tumors, and therapeutic efficacy needs to be further improved. Nanobodies (Nb) are naturally produced antibodies composed solely of heavy chains in alpacas. The target recognition module consists of a single heavy chain variable region (VHH) with a molecular weight of only 12-14 kDa, which is expected to overcome the low solid tumor penetration of traditional monoclonal antibodies. Nanobodies can be linked to functional domains such as Fc, other nanobodies, or peptide tags or toxins. Due to their small size, they have higher diffusion rates, better vascular permeability, better penetration of body barriers such as the blood-brain barrier, and stronger tumor penetration, resulting in more uniform tissue distribution than traditional monoclonal antibodies. These properties make them particularly suitable for specific and effective tumor-targeted therapy in vivo. Nanobodies also have advantages such as low immunogenicity, high stability, high resistance to degradation, low production costs through prokaryotic expression, and easy product characterization. However, despite the numerous potential advantages, the development of nanoantibody-based tumor therapeutics is still in the early exploratory research stage.

[0004] In recent years, antibody-drug conjugates (ADCs) have become a new trend in tumor treatment. ADCs utilize the ability of monoclonal antibodies to specifically recognize specific antigens on the surface of tumor cells, thereby accurately delivering and releasing anti-tumor drugs (such as small molecule chemotherapy drugs) to tumor target cells, achieving the purpose of precisely killing tumors. However, due to limitations such as the large molecular weight and weak intratumor penetration of ADCs, the absorption and distribution of drugs in tumors are limited and highly variable, making their application in solid tumors less than ideal. Furthermore, ADCs have a long terminal half-life in the body's circulatory system, which can cause the loaded toxins to produce unnecessary toxicity to normal tissues or cells. Nanobody-drug conjugates (NDCs) prepared using nanoantibodies not only retain the advantages of traditional ADCs, but also have high vascular permeability, good penetration of blood-brain barriers, strong tumor penetration, and rapid arrival at target cells. They can increase drug accumulation in tumors while also moderately controlling the plasma exposure and half-life of the drug, helping to further improve the therapeutic effect and overall therapeutic window for solid tumors, and are expected to become the most promising new class of anti-tumor drugs.

[0005] In summary, based on the important role of TF in tumor development, recurrence and drug resistance, the development of drugs targeting TF-Nb and TF-NDC can provide new treatment strategies for the clinical treatment of tumor patients with abnormal TF expression, either alone or in combination.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide a nanobody targeting TF and a nanobody-drug conjugate, as well as a preparation method and use thereof.

[0008] In a first aspect of the present invention, a Nanobody targeting TF is provided, wherein the complementarity determining region (CDR) of the VHH chain of the Nanobody is one or more selected from the following groups:

[0009] (1) CDR1 shown in SEQ ID NO. 1,

[0010] CDR2 shown in SEQ ID NO. 2, and

[0011] CDR3 shown in SEQ ID NO. 3;

[0012] or,

[0013] (2) CDR1 shown in SEQ ID NO. 5,

[0014] CDR2 shown in SEQ ID NO. 6, and

[0015] CDR3 shown in SEQ ID NO. 7;

[0016] or,

[0017] (3) CDR1 shown in SEQ ID NO. 9,

[0018] CDR2 shown in SEQ ID NO. 10, and

[0019] CDR3 shown in SEQ ID NO.11,

[0020] or,

[0021] (4) CDR1 shown in SEQ ID NO. 13,

[0022] CDR2 shown in SEQ ID NO. 14, and

[0023] CDR3 shown in SEQ ID NO.15,

[0024] or,

[0025] (5) CDR1 shown in SEQ ID NO. 17,

[0026] CDR2 shown in SEQ ID NO. 18, and

[0027] CDR3 shown in SEQ ID NO. 19,

[0028] or,

[0029] (6) CDR1 shown in SEQ ID NO. 21,

[0030] CDR2 shown in SEQ ID NO. 22, and

[0031] CDR3 shown in SEQ ID NO.23,

[0032] or,

[0033] (7) CDR1 shown in SEQ ID NO. 25,

[0034] CDR2 shown in SEQ ID NO. 26, and

[0035] CDR3 shown in SEQ ID NO. 27,

[0036] or,

[0037] In another preferred embodiment, any one of the above amino acid sequences further includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain TF binding affinity.

[0038] In another preferred example, the CDR region of the Nanobody VHH chain comprises an amino acid sequence that has at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence similarity to any of the CDR regions described above.

[0039] In another preferred example, the amino acid sequence of the CDR region of the Nanobody VHH chain comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to any of the CDR regions described above.

[0040] In another preferred embodiment, the VHH chain comprises CDR1, CDR2 and CDR3 selected from the group consisting of:

[0041] (1) the complementary determining regions CDR1, CDR2, and CDR3 shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 (corresponding to the CDRs of Nanobody 4-A02); or

[0042] (2) the complementary determining regions CDR1, CDR2, and CDR3 shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7 (corresponding to the CDRs of Nanobody F1-A01); or

[0043] (3) the complementary determining regions CDR1, CDR2, and CDR3 shown in SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11 (corresponding to the CDRs of Nanobody 4-C08); or

[0044] (4) the complementary determining regions CDR1, CDR2, and CDR3 shown in SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 15 (corresponding to the CDRs of Nanobody 4-E04); or

[0045] (5) the complementary determining regions CDR1, CDR2, and CDR3 shown in SEQ ID NO. 17, SEQ ID NO. 18, and SEQ ID NO. 19 (corresponding to the CDRs of Nanobody 4-C05); or

[0046] (6) the complementary determining regions CDR1, CDR2, and CDR3 shown in SEQ ID NO.21, SEQ ID NO.22, and SEQ ID NO.23 (corresponding to the CDRs of Nanobody 5-C12); or

[0047] (7) Complementarity determining regions CDR1, CDR2, and CDR3 shown in SEQ ID NO. 25, SEQ ID NO. 26, and SEQ ID NO. 27 (corresponding to the CDRs of Nanobody 5-H3)

[0048] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0049] In another preferred embodiment, the nanobodies include humanized antibodies, camel-derived antibodies, and chimeric antibodies.

[0050] In another preferred embodiment, the VHH chain of the Nanobody further comprises a framework region (FR).

[0051] In another preferred embodiment, the framework region FR is of human, mouse, rabbit or camel origin.

[0052] In another preferred embodiment, the framework region FR includes a human FR region, a mouse FR region or a camel FR region.

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

[0054] In another preferred example, the VHH chain of the Nanobody targeting TF has an amino acid sequence as shown in 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.

[0055] In another preferred example, the Nanobody sequence comprises an amino acid sequence that has at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence similarity to 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.

[0056] In the second aspect of the present invention, an antibody targeting TF is provided, wherein the antibody comprises one or more VHH chains of the Nanobody targeting TF as described in the first aspect of the present invention.

[0057] In another preferred example, the VHH chain of the TF-targeting Nanobody has an amino acid sequence selected from the following group: SEQ ID NO.4, SEQ ID NO.8, SEQ ID NO.12, SEQ ID NO.16, SEQ ID NO.20, SEQ ID NO.24, SEQ ID NO.28.

[0058] In another preferred embodiment, the antibody is a monomer, a bivalent antibody, and / or a multivalent antibody.

[0059] In another preferred embodiment, the antibody is an animal-derived antibody, a humanized antibody, a chimeric antibody or a chimeric antigen receptor antibody (CAR).

[0060] In another preferred embodiment, the CDR region of the humanized antibody comprises 1, 2, or 3 amino acid changes.

[0061] In another preferred embodiment, the animal is a non-human mammal, preferably a mouse, sheep, rabbit, or camel.

[0062] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0063] In another preferred embodiment, the antibody is a monoclonal antibody.

[0064] In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

[0065] In another preferred example, the antibody is a humanized antibody, and the VHH chain of the nanobody targeting TF has the amino acid sequence shown at positions 1-114 of SEQ ID NO.33, at positions 1-114 of SEQ ID NO.34, or at positions 1-114 of the amino acid sequence shown in SEQ ID NO.35.

[0066] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids does not exceed 40% of the total number of amino acids in the initial amino acid sequence, preferably 20%, and more preferably 10%.

[0067] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-7, preferably 1-3, and more preferably 1.

[0068] In another preferred embodiment, the at least one amino acid sequence that has been added, deleted, modified and / or substituted has an amino acid sequence with a homology of at least 80%.

[0069] In another preferred embodiment, the derivative sequence after addition, deletion, modification and / or substitution of at least one amino acid has the biological function of inhibiting cell surface TF or recombinant TF protein.

[0070] In another preferred embodiment, the antibody is in the form of a drug conjugate.

[0071] In another preferred example, the binding constant KD value of the nanobody to the extracellular region of human TF protein (TF-ECD) measured by ForteBio surface plasmon resonance (SPR) is 0.202~7.04nM.

[0072] In another preferred embodiment, the antibody has one or more characteristics selected from the following group:

[0073] (a) TF that specifically binds to tumor cells and / or immune / stromal cells in the tumor microenvironment;

[0074] (b) specifically inhibit tumor TF-related tumor cell signaling pathways;

[0075] (c) Improve the disorder of cytokine secretion caused by TF overactivation;

[0076] (d) inhibiting tumor cell migration or metastasis;

[0077] (e) inhibiting tumor growth and improving the anti-tumor efficacy of combined drug therapy;

[0078] (f) Promote the proliferation, survival and function of immune cells, thereby improving the effect of tumor immunity;

[0079] (g) After binding to tumor cells, it is internalized into intracellular lysosomes;

[0080] (h) It has the characteristics of penetrating the blood-brain barrier and being efficiently distributed in the brain;

[0081] (i) It has a good therapeutic effect on brain tumors;

[0082] (j) It has good therapeutic effects on various solid tumors.

[0083] In the third aspect of the present invention, a multispecific antibody is provided, wherein the multispecific antibody comprises: the TF-targeting Nanobody according to the first aspect of the present invention or the TF-targeting antibody according to the second aspect of the present invention.

[0084] In another preferred embodiment, the multispecific antibody further comprises a second antigen binding region targeting a target 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.

[0085] In another preferred embodiment, the second antigen binding region is a nanobody.

[0086] In another preferred embodiment, the multispecific antibody comprises one or more second antigen binding regions.

[0087] In another preferred embodiment, the multispecific antibody further comprises an antibody Fc segment.

[0088] In another preferred embodiment, the antigen binding region is an antibody or antibody fragment, and the antibody fragment includes: (i) Fab fragment; (ii) F(ab')2 fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single-chain Fv (scFv) molecule; and (vi) dAb fragment.

[0089] A fourth aspect of the present invention provides a recombinant protein having:

[0090] (i) a Nanobody targeting TF, such as the Nanobody targeting TF as described in the first aspect of the invention, the anti-TF antibody as described in the second aspect of the invention, or the multispecific antibody as described in the third aspect of the invention; and

[0091] (ii) optionally a polypeptide molecule or fragment having therapeutic function; and / or

[0092] (iii) Optional functional domains that enhance the physicochemical properties or druggability of the protein.

[0093] In another preferred embodiment, the improving the physicochemical properties or drugability of the protein includes prolonging the half-life of the nanobody targeting TF.

[0094] In another preferred embodiment, the recombinant protein further comprises: (iv) an optional tag sequence for facilitating expression and / or purification.

[0095] In another preferred embodiment, the tag sequence is selected from the following group: 6His tag, GGGS sequence, FLAG tag.

[0096] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0097] In another preferred embodiment, the polypeptide molecules or fragments with therapeutic functions include but are 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.

[0098] In another preferred embodiment, the polypeptide molecules or fragments with therapeutic functions include but are not limited to: insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analogs, albumin, antibody fragments, and cytokines.

[0099] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0100] In another preferred embodiment, the fusion protein includes a multispecific antibody or a chimeric antibody.

[0101] In another preferred embodiment, the functional domains for improving the physicochemical properties or drugability of proteins include Fc segments, anti-albumin nanoantibodies (HLE), and albumin binding domains (ABD).

[0102] In another preferred embodiment, the recombinant protein has the following structure from the NC end or the CN end:

[0103] (AB)m;

[0104] Among them, the A element is a nanobody targeting TF;

[0105] The B element is an Fc segment, a human albumin binding domain (ABD), or an anti-albumin nanobody (HLE);

[0106] “-” represents a peptide bond or linker,

[0107] Wherein, m is a positive integer.

[0108] In another preferred embodiment, the joint is a rigid joint or a flexible joint.

[0109] In another preferred embodiment, the B element further includes an optional functional domain for improving the physicochemical properties or druggability of the protein, or an optional tag sequence for assisting expression and / or purification.

[0110] In another preferred embodiment, m is 1, 2, 3 or 4.

[0111] In another preferred example, the Nanobody targeting TF is the Nanobody targeting 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.

[0112] In another preferred embodiment, the VHH chain of the Nanobody targeting TF has an amino acid sequence selected from the following group:

[0113] SEQ ID NO.4, SEQ ID NO.8, SEQ ID NO.12, SEQ ID NO.16, SEQ ID NO.20, SEQ ID NO.24, SEQ ID NO.28.

[0114] In another preferred embodiment, the Fc segment is a human IgG Fc segment, preferably derived from human IgG1 or human IgG2.

[0115] In another preferred embodiment, the Fc segment is selected from FC1, FCWT or FCLALAPG; wherein,

[0116] The sequence of FC1 is shown in positions 115-346 of SEQ ID NO. 29.

[0117] The sequence of the FCWT is shown in positions 115-346 of SEQ ID NO.30.

[0118] The sequence of the FCLALAPG is shown in positions 115-346 of SEQ ID NO.31.

[0119] In another preferred embodiment, the amino acid sequence of the ABD is shown in positions 130-198 of SEQ ID NO.32.

[0120] In another preferred example, the recombinant protein has an amino acid sequence selected from the following group: SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.31, and SEQ ID NO.32.

[0121] In another preferred embodiment, the recombinant protein comprises a humanized antibody, preferably having an amino acid sequence selected from the following group: 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.

[0122] In a fifth aspect of the present invention, a CAR construct is provided, wherein the antigen binding region of the CAR construct is the VHH chain of the nanobody as described in the first aspect of the present invention.

[0123] The sixth aspect of the present invention provides a recombinant immune cell, which expresses an exogenous CAR construct as described in the fifth aspect of the present invention.

[0124] In another preferred embodiment, the immune cells are selected from the following group: NK cells and T cells.

[0125] In another preferred embodiment, the immune cells are from humans or non-human mammals (such as mice).

[0126] The seventh aspect of the present invention provides an immunoconjugate, wherein the immunoconjugate comprises:

[0127] (a) an antibody portion, which is the TF-targeting Nanobody described in the first aspect of the invention or the TF-targeting antibody described in the second aspect of the invention; and

[0128] (b) a coupling moiety coupled to said Nanobody portion, said coupling moiety being selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, an enzyme, a protein degrader, an immunomodulator (immune agonist and inhibitor), an oligonucleotide or a combination thereof.

[0129] In another preferred embodiment, the immunoconjugate is a nanobody drug conjugate (NDC).

[0130] In another preferred embodiment, the Nanobody portion and the coupling portion are coupled via a chemical bond or a linker.

[0131] In another preferred embodiment, the coupling moiety is a chemical label or a biological label.

[0132] In another preferred embodiment, the chemical label is an isotope, an immunotoxin and / or a chemical drug.

[0133] In another preferred embodiment, the biomarker is biotin, avidin or an enzyme label.

[0134] In another preferred embodiment, the coupling moiety is a drug or a toxin.

[0135] In another preferred embodiment, the drug is a cytotoxic drug.

[0136] In another preferred embodiment, the cytotoxic drug is selected from the following group: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or a combination thereof.

[0137] Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, 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.

[0138] In another preferred embodiment, the toxin is selected from the following group:

[0139] auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), chlortetracycline, maytansinoids, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, α-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis officinalis) inhibitors, glucocorticoids, or a combination thereof.

[0140] In another preferred embodiment, the coupling moiety is a detectable label.

[0141] In another preferred embodiment, the detectable marker comprises a radionuclide, and the radionuclide comprises:

[0142] (i) a diagnostic isotope 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 a combination thereof; and / or

[0143] (ii) therapeutic isotopes 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.

[0144] In another preferred embodiment, the protein degrader is a degrader of a tumor-associated protein, and the tumor-associated protein is selected from the following group: 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, RAS (such as KRAS, HRAS and NRAS).

[0145] In another preferred embodiment, the KRAS includes KRAS-G12C, KRAS-G12D, KRAS-G12V, etc.

[0146] In another preferred embodiment, the protein degrader includes PROTAC (protein degradation targeting chimera).

[0147] In another preferred embodiment, the PROTAC targets EGFR, KRAS (including KRAS-G12C, KRAS-G12D, KRAS-G12V, etc.).

[0148] In another preferred embodiment, 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.

[0149] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin) or any form of nanoparticles, etc.

[0150] In another preferred embodiment, the immunoconjugate contains: a multivalent (eg, bivalent) 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.

[0151] In another preferred embodiment, the multivalency means that the amino acid sequence of the immunoconjugate contains multiple repeats of the Nanobody targeting TF as described in the first aspect of the present invention or the Antibody targeting TF as described in the second aspect of the present invention.

[0152] In another preferred embodiment, the detection is in vivo detection or in vitro detection.

[0153] In another preferred embodiment, the immunoconjugate is used for diagnosing and / or treating tumors expressing TF protein.

[0154] In another preferred embodiment, the antibody-drug conjugate ADC has the following molecular formula:

[0155] in:

[0156] nAb is a nanobody targeting TF or a recombinant protein as described in the fourth aspect of the present invention,

[0157] LU is linker 2 (also called connector);

[0158] D is for medicine;

[0159] And the subscript p is a value selected from 1-8.

[0160] In another preferred embodiment, the nAb is the recombinant protein as described in the fourth aspect of the present invention.

[0161] In another preferred embodiment, the antibody-drug conjugate ADC is a monomer, dimer or multimer.

[0162] In another preferred embodiment, the recombinant protein has the following structure from the NC end or the CN end: (A-L1-B)m;

[0163] Among them, the A element is a nanobody targeting TF;

[0164] The B element is an Fc segment, a human albumin binding domain (ABD), or an anti-albumin nanobody (HLE);

[0165] "L1" stands for None or Connector 1,

[0166] Wherein, m is a positive integer.

[0167] In another preferred embodiment, m is 1, 2, 3 or 4.

[0168] In another preferred embodiment, the drug is site-specifically linked to the terminal amino group, side chain amino group or thiol group of the Nanobody targeting TF.

[0169] In another preferred embodiment, the drug is site-specifically and / or randomly linked to the TF-targeting Nanobody.

[0170] In another preferred embodiment, the drug is site-specifically linked to the A element, B element or L1.

[0171] In another preferred embodiment, the drug is site-specifically linked to the B element and / or L1.

[0172] In another preferred embodiment, the drug is site-specifically linked to the terminal amino group or thiol group of element B; and / or to the side chain amino group or thiol group of L1.

[0173] In another preferred embodiment, the B element is an Fc segment, and the drug is site-specifically linked to the hinge region of the Fc segment and / or the C-terminus of the Fc segment.

[0174] In another preferred embodiment, the B element is a human albumin binding domain (ABD), and the drug is site-specifically linked to the C-terminus of the ABD.

[0175] In another preferred embodiment, the B element is HLE, and the drug is site-specifically linked to the C-terminus of HLE.

[0176] In another preferred embodiment, the antibody-drug conjugate ADC has a structure selected from FIG. 24 .

[0177] In another preferred embodiment, LU is selected from maleimidocaproyl (MC), maleimide (MAL), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) (SMCC) linkers connected to the antibody portion, and comprises one or more linkers of valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), alanine-alanine-alanine (AAA), p-aminobenzyloxycarbonyl (PAB), and polyethylene glycol (PEG).

[0178] In another preferred embodiment, the antibody is covalently bound to the linker by reacting with a portion selected from the group consisting of maleimidocaproyl (MC), maleimide (MAL), and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (SMCC).

[0179] In another preferred embodiment, D is selected from the following group of compounds having anti-tumor activity:

[0180] (i) Tubulin inhibitors, such as maytansine derivatives (DM1, DM4), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF);

[0181] (ii) DNA-acting toxins, such as duocarmycin and pyrrolobenzodiazepine (PBD);

[0182] (iii) Topoisomerase inhibitors, camptothecin, SN38, exitecan, Dxd.

[0183] In another preferred embodiment, the LU-D compound is selected from the following group:

[0184] The eighth aspect of the present invention provides a pharmaceutical composition, comprising:

[0185] (i) the TF-targeting Nanobody according to the first aspect of the invention, the TF-targeting antibody according to the second aspect of the invention, the multispecific antibody according to the third aspect of the invention, the recombinant protein according to the fourth aspect of the invention, the recombinant immune cell according to the sixth aspect of the invention, or the immunoconjugate according to the seventh aspect of the invention;

[0186] (ii) a pharmaceutically acceptable carrier.

[0187] In another preferred embodiment, the pharmaceutical composition includes a single drug, a compound drug, or a synergistic drug.

[0188] In another preferred embodiment, the pharmaceutical composition further comprises other biologically active substances, such as drugs for treating tumors.

[0189] In another preferred embodiment, the administration method of the pharmaceutical composition is selected from the following group: subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral and nasal spraying and aerosol inhalation.

[0190] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: liquid, solid, or gel.

[0191] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0192] In another preferred embodiment, the pharmaceutical composition is an injection.

[0193] In the ninth aspect of the present invention, there is provided a use of an active ingredient, wherein the active ingredient is selected from the following group: the nanoantibody targeting TF as described in the first aspect of the present invention, the antibody targeting TF as described in the second aspect of the present invention, the multispecific antibody as described in the third aspect of the present invention, the recombinant protein as described in the fourth aspect of the present invention, the recombinant immune cell as described in the sixth aspect of the present invention, the immunoconjugate as described in the seventh aspect of the present invention, or a combination thereof, wherein the active ingredient is used for (a) preparing a detection reagent, a detection plate or a kit; and / or (b) preparing a drug for preventing and / or treating TF-related diseases.

[0194] In another preferred embodiment, the detection reagent, detection plate or kit is used for:

[0195] (1) detecting TF protein in a sample; and / or

[0196] (2) detecting endogenous TF protein in tumor cells; and / or

[0197] (3) Detect tumor cells expressing TF protein.

[0198] In another preferred embodiment, the detection types include but are not limited to flow cytometry, cell immunofluorescence detection, enzyme-linked immunosorbent assay, immunoblotting detection, etc.

[0199] In another preferred embodiment, the detection reagent, detection plate or kit is used to diagnose TF-related diseases.

[0200] In another preferred embodiment, the drug is used to treat or prevent tumors with high TF expression, tumor migration, or tumor resistance.

[0201] In another preferred embodiment, the tumor drug resistance includes: resistance to tumor immunotherapy drugs, resistance to tumor targeted therapy drugs, resistance to conventional tumor chemotherapy, and insensitivity to radiotherapy.

[0202] In another preferred embodiment, the drug is used for a purpose selected from the group consisting of:

[0203] (a) TF that specifically binds to tumor cells and / or immune / stromal cells in the tumor microenvironment;

[0204] (b) specifically inhibit tumor TF-related tumor cell signaling pathways;

[0205] (c) Improve the disorder of cytokine secretion caused by TF overactivation;

[0206] (d) inhibiting tumor cell migration or metastasis;

[0207] (e) inhibiting tumor growth and improving the anti-tumor efficacy of combined drug therapy;

[0208] (f) Promote the proliferation, survival and function of immune cells, thereby improving the effect of tumor immunity;

[0209] (g) After binding to tumor cells, it is internalized into intracellular lysosomes;

[0210] (h) It has the characteristics of penetrating the blood-brain barrier and being efficiently distributed in the brain;

[0211] (i) It has a good therapeutic effect on brain tumors;

[0212] (j) It has good therapeutic effects on various solid tumors.

[0213] In another preferred embodiment, the TF-related disease is selected from the group consisting of cancer, thrombosis, autoimmune disease, metabolic-related disease, infectious disease, or a combination thereof.

[0214] In another preferred embodiment, the TF-related diseases include: tumor occurrence, growth and / or metastasis.

[0215] In another preferred embodiment, the cancer includes solid tumors and blood cancers.

[0216] In another preferred embodiment, the cancer is a tumor with high expression of TF.

[0217] In another preferred embodiment, the tumor with high TF expression 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.

[0218] In another preferred embodiment, the cancer is a drug-resistant tumor.

[0219] In another preferred embodiment, the tumor with high TF expression refers to the ratio of the level L1 of TF transcript and / or protein in tumor tissue to the level L0 of TF transcript and / or protein in normal tissue, L1 / L0 ≥ 2, preferably ≥ 3.

[0220] The tenth aspect of the present invention provides a polynucleotide, characterized in that the polynucleotide encodes a polypeptide selected from the following group:

[0221] (1) the TF-targeting Nanobody of the first aspect of the invention, the TF-targeting antibody of the second aspect of the invention, or the multispecific antibody of the third aspect of the invention;

[0222] (2) the recombinant protein according to the fourth aspect of the present invention; or

[0223] (3) The CAR construct as described in the fifth aspect of the present invention.

[0224] In another preferred embodiment, the polynucleotide includes RNA, DNA or cDNA.

[0225] The eleventh aspect of the present invention provides a vector, characterized in that the vector contains the polynucleotide as described in the tenth aspect of the present invention.

[0226] In another preferred embodiment, the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0227] The twelfth aspect of the present invention provides a genetically engineered host cell, characterized in that the host cell contains the vector described in the eleventh aspect of the present invention or the polynucleotide described in the tenth aspect of the present invention is integrated into its genome.

[0228] In a thirteenth aspect, the present invention provides a method for detecting TF in a sample (including diagnostic or non-diagnostic) in vitro, characterized in that the method comprises the steps of:

[0229] (1) in vitro, contacting the sample with the TF-targeting Nanobody of the first aspect of the invention, the TF-targeting antibody of the second aspect of the invention, or the immunoconjugate of the seventh aspect of the invention;

[0230] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of TF in the sample.

[0231] In another preferred embodiment, the detection includes diagnostic or non-diagnostic.

[0232] In the fourteenth aspect of the present invention, a detection plate is provided, which comprises: a substrate (support plate) and a test strip, wherein the test strip contains the nanoantibody targeting TF as described in the first aspect of the present invention or the antibody targeting TF as described in the second aspect of the present invention or the immunoconjugate as described in the seventh aspect of the present invention.

[0233] A fifteenth aspect of the present invention provides a kit, comprising:

[0234] (1) a first container, comprising the Nanobody targeting TF according to the first aspect of the invention or the Antibody targeting TF according to the second aspect of the invention; and / or

[0235] (2) a second container, wherein the second container contains a secondary antibody against the Nanobody targeting TF according to the first aspect of the present invention or the antibody targeting TF according to the second aspect of the present invention;

[0236] Alternatively, the kit contains the detection plate described in the fourteenth aspect of the present invention.

[0237] A sixteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, characterized in that the method comprises:

[0238] (a) culturing the host cell according to the twelfth aspect of the present invention under conditions suitable for expression;

[0239] (b) isolating the recombinant polypeptide from the culture, wherein the recombinant polypeptide is a Nanobody targeting TF as described in the first aspect of the invention, an antibody targeting TF as described in the second aspect of the invention, a multispecific antibody as described in the third aspect of the invention, or a recombinant protein as described in the fourth aspect of the invention.

[0240] In the seventeenth aspect of the present invention, a method for treating TF-related diseases is provided, characterized in that the method comprises: administering to a subject in need thereof the Nanobody targeting TF as described in the first aspect of the present invention, the antibody targeting TF as described in the second aspect of the present invention, the multispecific antibody as described in the third aspect of the present invention, the recombinant protein as described in the fourth aspect of the present invention, the recombinant immune cell as described in the sixth aspect of the present invention, the immunoconjugate as described in the seventh aspect of the present invention, the pharmaceutical composition as described in the eighth aspect of the present invention, or a combination thereof.

[0241] In another preferred embodiment, the method further comprises: administering other drugs or treatment methods to a subject in need for combined treatment.

[0242] In another preferred embodiment, the other drugs or treatment methods include: anti-tumor immunotherapy drugs, tumor-targeted drugs, tumor chemotherapy drugs, and tumor radiotherapy.

[0243] In another preferred embodiment, the anti-tumor immunotherapy drugs include PD-1 and PD-L1 monoclonal antibodies.

[0244] In an eighteenth aspect of the present invention, there is provided a method for preparing a chimeric antibody, comprising the steps of:

[0245] After the nucleotide sequence of the alpaca-derived VHH sequence of the TF-targeting nanobody described in the first aspect of the present invention is cloned into an expression vector containing the nucleotide sequence of the human antibody constant region, the human-alpaca chimeric antibody is expressed by transfecting animal cells.

[0246] In the nineteenth aspect of the present invention, there is provided a method for preparing a humanized antibody, comprising the steps of:

[0247] The nucleotide sequence of the CDR region of the VHH chain of the TF-targeting nanobody described in the first aspect of the present invention is implanted into a nucleotide sequence template containing the FR region of a human antibody, which is then cloned into an expression vector containing the constant region of a human antibody, and the humanized antibody is expressed by transfecting animal cells.

[0248] In the twentieth aspect of the present invention, a method for inhibiting tumor cell growth and migration is provided, comprising the steps of administering to a subject in need thereof the Nanobody targeting TF as described in the first aspect of the present invention, the antibody targeting TF as described in the second aspect of the present invention, or the Nanobody-drug conjugate of the present invention, or the CAR-T cell of the Nanobody, or a combination thereof.

[0249] In the twenty-first aspect of the present invention, a method for inhibiting tumor growth in a model animal is provided, comprising the steps of administering to a subject in need thereof the nanoantibody targeting TF as described in the first aspect of the present invention, the antibody targeting TF as described in the second aspect of the present invention, the nanoantibody-drug conjugate as described in the present invention, or the CAR-T cell of the nanoantibody.

[0250] In another preferred embodiment, the drug can be administered alone or in combination with tumor immunotherapy, tumor-targeted drugs, cytotoxic drugs, and radiotherapy.

[0251] In the twenty-second aspect of the present invention, there is provided a method for preparing the Nanobody-drug conjugate according to the seventh aspect of the present invention, comprising the steps of:

[0252] (1) reacting the antibody with a reducing agent in a buffer solution to obtain a reduced antibody;

[0253] (2) Cross-linking (coupling) the reduced antibody obtained in step (1) with the linker-drug conjugate of formula DL in a mixture of buffer and organic solvent to obtain antibody-drug conjugates 1a, 1b, 1c or 1d with different DAR values.

[0254] In another preferred embodiment, the cross-linking reaction of the preparation method is shown in FIG23 .

[0255] In another preferred embodiment, the antibody in step (1) is reduced with a reducing agent, thereby reducing the interchain disulfide bonds of the antibody to generate sulfhydryl groups.

[0256] In another preferred embodiment, the reducing agent in step (1) is tris(2-carboxyethyl)phosphine hydrochloride (TCEP), beta-mercaptoethanol, beta-mercaptoethylamine hydrochloride, or dithiothreitol (DTT).

[0257] In another preferred embodiment, the buffer is selected from the group consisting of potassium dihydrogen phosphate-sodium hydroxide (KH2PO4-NaOH) / sodium chloride (NaCl) / diethylenetriaminepentaacetic acid (DTPA) buffer, disodium hydrogen phosphate-citric acid / sodium chloride (NaCl) / diethylenetriaminepentaacetic acid (DTPA), boric acid-borax / sodium chloride (NaCl) / diethylenetriaminepentaacetic acid (DTPA), histidine-sodium hydroxide / sodium chloride (NaCl) / diethylenetriaminepentaacetic acid (DTPA), and PBS / diethylenetriaminepentaacetic acid (DTPA).

[0258] In another preferred embodiment, in step (2), the volume proportion of the organic solvent in the reaction solution does not exceed 15%.

[0259] In another preferred embodiment, the organic solvent in step (2) is selected from the group consisting of acetonitrile (ACN), dimethylformamide (DMF), dimethylacetamide (DMA), and dimethyl sulfoxide (DMSO).

[0260] Then, add the linker drug (previously dissolved in acetonitrile (ACN), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), or diethylacetamide (DMA) at 10 mg / ml), ensuring that the volume of organic solvent in the reaction solution does not exceed 15%. Stir the coupling reaction at 0-37°C for 2-4 hours. If TCEP reduction is used, the substituted maleimide compound can be added directly for coupling without removing the remaining TCEP.

[0261] The coupling reaction mixture is purified using a desalting column using sodium succinate / NaCl buffer or histidine-acetate / sucrose gel filtration. Peak samples are collected based on UV280 absorbance. Alternatively, ultrafiltration is performed several times. The product is then sterilized by filtration and stored at low temperatures.

[0262] The resulting antibody-drug conjugates have a relatively uniform drug-antibody ratio (DAR). For NDCs with varying DARs, further separation and purification using, but not limited to, the following methods can be used to obtain more uniform samples: hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC), and ion exchange chromatography (IEC).

[0263] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0264] Figure 1 is an SDS-PAGE of human FC1 chimeric nanobody after expression and purification, showing the fusion proteins of 4-A02, F1-A01, 4-C08, 4-E04, 4-C05, 5-C12, and 5-H3 heavy chain variable regions VHH with FC1 fragments, respectively.

[0265] Figure 2 shows the binding affinity constants (KD) of chimeric nanobodies 4-A02, F1-A01, 4-E04, 4-C05, 5-C12, and 5-H3 to human TF-ECD determined by surface plasmon resonance (SPR) analysis.

[0266] Figure 3 shows the binding affinity of 4-A02, F1-A01, 4-C08, 4-E04, 4-C05, 5-C12, and 5-H3 to TF on the surface of the TF-highly expressed cell line MDA-MB-231. 50 ) test results.

[0267] Figure 4 is the hydrophobic interaction chromatography (HIC) of 4-A02-FC1 coupled to VC-MMAE conjugate NDC.

[0268] Figure 5 is the hydrophobic interaction chromatography (HIC) of F1-A01-FC1 coupled VC-MMAE conjugate NDC.

[0269] Figure 6 is the hydrophobic interaction chromatography (HIC) of 4-C08-FC1 coupled to VC-MMAE conjugate NDC.

[0270] Figure 7 is the hydrophobic interaction chromatography (HIC) of 4-C05-FC1 coupled to VC-MMAE conjugate NDC.

[0271] Figure 8 is a hydrophobic interaction chromatography (HIC) spectrum of 5-C12-FC1 coupled VC-MMAE conjugate NDC.

[0272] Figure 9 is a hydrophobic interaction chromatography (HIC) of 4-E04-FC1 coupled to VC-MMAE conjugate NDC.

[0273] Figure 10 is a hydrophobic interaction chromatography (HIC) spectrum of 4-A02-ABD coupled to VC-MMAE conjugate NDC.

[0274] Figure 11 is the hydrophobic interaction chromatography (HIC) of 4-A02-FC1 coupled with VC-MMAE and GGFG-Dxd conjugate NDC.

[0275] Figure 12 shows the detection results of the proliferation 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 on the TF-highly expressed triple-negative breast cancer (TNBC) HCC1806 cells.

[0276] Figure 13 shows the detection results of the proliferation 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 on pancreatic adenocarcinoma (PDAC) BxPC3 cells with high TF expression.

[0277] Figure 14 shows the results of the detection of the proliferation 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 on pancreatic adenocarcinoma (PDAC) HPAF-II cells with high TF expression.

[0278] FIG15 shows the detection results of the proliferation inhibitory activity of TF-NDC 4A02-FC1-MMAE, 4A02-FC1-DXd, and 4A02-ABD-MMAE on pancreatic adenocarcinoma (PDAC) BxPC3 cells with high TF expression.

[0279] FIG16 shows the detection results of the proliferation inhibitory activity of TF-NDC 4A02-FC1-MMAE, 4A02-FC1-DXd, and 4A02-ABD-MMAE on pancreatic adenocarcinoma (PDAC) HPAF-II cells with high TF expression.

[0280] Figure 17 shows the effects of TF-NDC 4A02-FC1-MMAE, 4A02-FC1-DXd, and 4A02-ABD-MMAE on KRAS abnormally expressed by TF. G12C Results of the detection of proliferation inhibitory activity in mutant lung cancer (NSCLC) HCC44 cells.

[0281] Figure 18 shows the effects of TF-NDC 4A02-FC1-MMAE, 4A02-FC1-DXd, and 4A02-ABD-MMAE on abnormal expression of EGFR by TF T790M Results of the proliferation inhibitory activity test on mutant lung cancer NCI-H975 cells.

[0282] Figure 19 shows the detection results of the proliferation inhibitory activity of TF-NDC 4A02-FC1-MMAE, 4A02-FCWT-MMAE 4A02-FC1-DXd, and 4A02-ABD-MMAE on lung cancer PC9 cells with abnormal TF expression and EGFR mutation.

[0283] Figure 20 is the SDS-PAGE of the proteins after expression and purification of 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.

[0284] Figure 21 shows the therapeutic effect of TF-NDC in a nude mouse mammary pad xenograft model of triple-negative breast cancer HCC1806. Tumor-bearing mice were divided into groups (n=8) on day 9 and received tail vein injections of 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. The dose was 3 mg / kg, administered once throughout the study.

[0285] Figure 22 shows the expression of TF-NDC in KRAS G12C Therapeutic efficacy of HCC44 lung cancer in a nude mouse orthotopic lung xenograft model. Tumor-bearing mice were divided into groups (n=8) on day 13 and received a tail vein injection of hIgG-MMAE or 4-A02-FC1-MMAE at a dose of 3 mg / kg, administered once throughout the study.

[0286] Figure 23 is a cross-linking reaction formula for the preparation method of the Nanobody-drug conjugate of the present invention, wherein the antibody-drug conjugates with different DAR values ​​are shown as 1a, 1b, 1c or 1d respectively.

[0287] Figure 24 shows the structural formulas of the fusion proteins of the present invention. (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).

[0288] FIG25 shows the detection results of the proliferation inhibitory activity of 4A02-FC1-MMAE, 4A02-FCWT-MMAE, 4A02-FC1-DXd, and 4A02-ABD-MMAE on the TNBC cell line MDA-MB-231.

[0289] FIG26 shows the detection results of the proliferation inhibitory activity of 4A02-FC1-MMAE, 4A02-FCWT-MMAE, 4A02-FC1-DXd, and 4A02-ABD-MMAE on the TNBC cell line HCC1806.

[0290] FIG27 shows the detection results of the proliferation inhibitory activity of 4A02-FC1-MMAE, 4A02-FCWT-MMAE, 4A02-FC1-DXd, and 4A02-ABD-MMAE on the TNBC cell line HCC1954.

[0291] FIG28 shows the detection results of the proliferation inhibitory activity of 4A02-FC1-MMAE, 4A02-FCWT-MMAE, 4A02-FC1-DXd, and 4A02-ABD-MMAE on the TNBC cell line HCC1937.

[0292] FIG29 shows the detection results of the proliferation inhibitory activity of 4A02-FC1-MMAE, 4A02-FCWT-MMAE, 4A02-FC1-DXd, and 4A02-ABD-MMAE on the TNBC cell line MDA-MB-468.

[0293] Figure 30 shows the effect of intravenous injection of 3 mg / kg 4A02-FCWT-MMAE (once a week for a total of 2 times) on HCC1806

[0294] Effects of treatment in an in vivo large tumor regression assay.

[0295] Figure 31 shows the results of intravenous injection of 3 mg / kg 4A02-FC1-MMAE, 4A02-FCWT-MMAE, 4A02-ABD-MMAE, 10 mg / kg 4A02-FC1-DXd (once a week for a total of 2 times) in HCC1954

[0296] Therapeutic efficacy in in vivo models.

[0297] Figure 32 shows the effect of TF-NDC on KRAS G12D The therapeutic effect of HPAF-II in a nude mouse xenograft model of pancreatic cancer. Figure 32A shows the therapeutic effect after a single intravenous injection of 3 mg / kg 4A02-FC1-MMAE, 4A02-FCWT-MMAE, and 4A02-ABD-MMAE. Figure 32B shows the therapeutic effect after a single intravenous injection of 1 mg / kg 4A02-FC1-MMAE and 4A02-FCWT-MMAE.

[0298] FIG33 shows the binding affinity constants (KD) of humanized Nanobodies 4A02-HM7, 4A02-HM8, 4A02-HM9 and original antibody 4A02 to human TF-ECD determined by surface plasmon resonance (SPR) analysis.

[0299] [Corrected 08.05.2024 according to Rule 91] Figure 34 shows the binding affinity of humanized Nanobodies 4A02-HM7, 4A02-HM8, 4A02-HM9, and the original antibody 4A02 to cell surface TF. Figure 34A ... and 4A02-HM9 to cell surface TF. Figure 34B shows the binding affinity of humanized Nanobodies 4A02-HM7, 4A02-HM8, and 4A02-HM9 to cell surface TF. Figure 34A shows the binding affinity of humanized

[0300] Figure 35 shows the affinity of humanized Nanobodies 4A02-HM7, 4A02-HM8, 4A02-HM9 and the original antibody 4A02 for binding to TF-ECD as detected by ELISA. Figure 35A shows the ELISA results for human TF-ECD; Figure 35B shows the ELISA results for cynomolgus monkey TF-ECD. DETAILED DESCRIPTION

[0301] Through extensive and in-depth research, the inventors immunized and constructed libraries on alpaca, and screened yeast display libraries and phage display libraries, and obtained a number of nanobodies, including 4-A02, F1-A01, 4-C08, 4-E04, 4-C05, 5-C12, and 5-H3. These antibodies can bind to human TF protein with high specificity, and the binding constant KD value measured by ForteBio surface plasmon resonance (SPR) is 0.2nM to 7nM; these antibodies have excellent binding affinity for tumor cells with high TF expression, and EC 50 The value is 0.05μg / mL~0.75μg / mL; In addition, the nanobody-drug conjugate (TF-NDC) designed based on TF nanobody can specifically kill tumor cells with high expression of TF, IC 50 The value is 0.0001μg / mL to 0.03μg / mL; a single intravenous injection of 3mg / kg TF-NDC showed strong anti-tumor activity and the potential to improve the immune microenvironment in vivo. Based on this, the present invention was completed.

[0302] the term

[0303] As used herein, the terms "Nanobodies of the invention", "anti-TF Nanobodies of the invention", and "TF Nanobodies of the invention" are used interchangeably to refer to Nanobodies that specifically recognize and bind to TF (including human TF). Particularly preferred are Nanobodies whose VHH chain amino acid sequences are 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.

[0304] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric glycoproteins of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0305] As used herein, the terms "single-domain antibody (VHH)" and "nanobody" have the same meaning and refer to the variable region of the heavy chain of a monoclonal antibody. A single-domain antibody (VHH) consisting solely of a single heavy chain variable region is constructed, which is the smallest antigen-binding fragment with complete function. Typically, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting solely of a single heavy chain variable region.

[0306] As used herein, the term "variable" refers to certain parts of the variable region in an antibody that are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-pleated configuration and are connected by three CDRs that form a connecting loop, and in some cases can form a partial β-pleated structure. The CDRs in each chain are closely together through the FR region and form the antigen-binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume 1, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0307] As known to those skilled in the art, immunoconjugates and fusion products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens bound to the anti-TF protein antibodies or fragments thereof.

[0308] As used herein, the terms "heavy chain variable region" and "V H ” can be used interchangeably.

[0309] As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0310] 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.

[0311] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.

[0312] In the present invention, the terms "antibodies of the present invention," "proteins of the present invention," or "polypeptides of the present invention" are used interchangeably to refer to polypeptides that specifically bind to the TF protein, such as proteins or polypeptides having a heavy chain variable region. These may or may not contain an initial methionine.

[0313] The present invention also provides other proteins or fusion expression products comprising the antibodies 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) comprising a heavy chain containing a variable region, as long as the variable region is identical to or at least 90% homologous to the heavy chain variable region of the antibodies of the present invention, preferably at least 95% homologous.

[0314] Generally, an antibody's antigen-binding properties are described by three specific regions within the heavy chain variable region, known as the variable region (CDR). This region is divided into four framework regions (FR). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a loop structure, spatially close to each other through the β-sheet formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antibody's antigen-binding site. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0315] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because they are at least partially involved in antigen binding. Thus, the present invention includes molecules having antibody heavy chain variable regions with CDRs that are 90% or more (preferably 95% or more, and most preferably 98% or more) homologous to the CDRs identified herein.

[0316] Anti-TF nanobodies

[0317] The present invention provides a variety of high-specificity and high-affinity Nanobodies targeting TF, which only include heavy chains, and the heavy chains contain heavy chain variable region (VH) amino acid sequences (listed in Table-1 and Table-2).

[0318] Preferably, the heavy chain variable region (VH) amino acid sequence has CDR1, CDR2, and CDR3 of the following polypeptide sequence:

[0319] a1) CDR1 is selected from

[0320] a2) CDR2 is selected from

[0321] a3) CDR3 is selected from

[0322] a4) A sequence having TF binding affinity in any of the above amino acid sequences by addition, deletion, modification and / or substitution of at least one amino acid.

[0323] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or replacing at least one amino acid sequence is preferably an amino acid sequence with a homology of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

[0324] Preferably, the antibody has the function of inhibiting the binding or inhibition of cell surface and recombinant TF protein, and the antibody can be rapidly endocytosed into lysosomes.

[0325] The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, and human-animal chimeric antibodies, preferably humanized antibodies, and more preferably fully humanized antibodies.

[0326] The antibody derivatives of the present invention can 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 any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0327] The animal is preferably a mammal, such as a mouse.

[0328] The antibody of the present invention may be a chimeric antibody, a humanized antibody, a CDR-grafted and / or modified antibody targeting human TF.

[0329] In a preferred embodiment of the present invention, any one or more of the above-mentioned SEQ ID NOs. 1 to 3, SEQ ID NOs. 5 to 7, SEQ ID NOs. 9 to 11, NOs. 13 to 15, NOs. 17 to 19, NOs. 21 to 23, and NOs. 25 to 27, or sequences thereof having TF binding affinity after addition, deletion, modification, and / or substitution of at least one amino acid, are located in the CDR region of the variable region (VH).

[0330] In the above content of the present invention, 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%, more preferably 15-20%.

[0331] In the above content of the present invention, more preferably, the number of amino acids added, deleted, modified and / or substituted may be 1-7, more preferably 1-5, more preferably 1-3, more preferably 1-2.

[0332] In another preferred embodiment, the original antibodies are human-alpaca chimeric antibodies 4-A02, F1-A01, 4-C08, 4-E04, 4-C05, 5-C12, and 5-H3.

[0333] In another preferred embodiment, the amino acid sequence numbering of the heavy chain variable region (VH) of the chimeric antibody is listed in Table 1.

[0334] In another preferred embodiment, the amino acid sequence numbers of the complementarity determining regions (CDRs) of the antibody are listed in Table 2.

[0335] The antibodies of the present invention can be used in combination for the construction of CAR constructs, recombinant immune cells containing CAR constructs, antibody-drug conjugates, etc., and can also be used for (a) preparing detection reagents, detection plates or kits; and / or (b) preparing drugs for the prevention and / or treatment of TF-related diseases.

[0336] Table 1: Representative meanings of each sequence in the sequence list of the present invention

[0337] Table-2: CDR amino acid sequences of Nanobodies of the present invention

[0338] Recombinant protein (or fusion protein)

[0339] In the present invention, recombinant proteins (or fusion proteins) containing the TF nanobody of the present invention are also included. A preferred fusion protein is a multispecific antibody, which further includes a second antigen binding region targeting a target 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.

[0340] Preferably, the multispecific antibody comprises one or more second antigen-binding regions, or further comprises a third antigen-binding region.

[0341] In addition, the recombinant protein (or fusion protein) of the present invention, in addition to containing the CD73 nanobody of the present invention, also includes an optional tag sequence that assists in expression and / or purification (e.g., 6His tag, GGGS sequence, FLAG tag); or includes an optional polypeptide molecule or fragment with therapeutic function; or an optional protein functional domain that assists in physicochemical or pharmaceutical functions (e.g., molecules that can prolong the half-life of nanobodies in vivo, such as HLE, ABD).

[0342] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0343] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. A polypeptide fragment, derivative, or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by 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) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretory sequence, or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0344] The antibodies of the present invention refer to polypeptides that have TF protein binding activity and include the aforementioned CDR regions. The term also encompasses variants of polypeptides that include the aforementioned CDR regions and have the same function as the antibodies of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more (generally 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, as well as additions of one or more (generally within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. For example, substitutions with amino acids having similar or similar properties generally do not alter protein function. For another example, additions of one or more amino acids to the C-terminus and / or N-terminus generally do not alter protein function. The term also encompasses active fragments and active derivatives of the antibodies of the present invention.

[0345] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of 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.

[0346] The invention also provides other polypeptides, such as fusion proteins comprising Nanobodies or fragments thereof. In addition to nearly full-length polypeptides, the invention also encompasses fragments of the Nanobodies of the invention. Typically, the fragment will have at least about 50 consecutive amino acids of an antibody of the 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.

[0347] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides in which no more than 10, preferably no more than 8, more preferably no more than 5, and most preferably no more than 3 amino acids are replaced with amino acids having similar or similar properties, compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table A.

[0348] Table A

[0349] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies, fragments thereof, or fusion proteins thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0350] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0351] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0352] The present invention also relates to polynucleotides that hybridize to the above-mentioned 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 hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0353] The full-length nucleotide sequence of the antibody of the present invention or its fragments can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis methods. One feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then ligating them, very long fragments of sequence can be obtained. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0354] Antibody preparation

[0355] The sequence of the DNA molecule of the antibody of the present invention or its fragment can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and heavy chain can be fused together to form a single-chain antibody.

[0356] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0357] In addition, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0358] Currently, DNA sequences encoding the antibodies (or fragments thereof, or derivatives thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the present invention through chemical synthesis.

[0359] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0360] The host cell can 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.

[0361] Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention. The antibodies of the present invention are then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, among other conventional separation and purification methods well known to those skilled in the art.

[0362] The resulting monoclonal antibodies can be characterized using conventional methods. For example, the binding specificity of the monoclonal antibodies can be determined using immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, using the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0363] The antibodies of the present invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods utilizing its 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 renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock, ultrasonic treatment, 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.

[0364] Nanobody-drug conjugate (NDC)

[0365] The present invention also provides an immunoconjugate (ADC) based on the antibody of the present invention, preferably a nanobody-drug conjugate (NDC).

[0366] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0367] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent may include any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. The coupling agent utilizing a carboxyl group may include any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (where the coupling site is an acylhydrazone).

[0368] Certain residues on antibodies (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging agents (such as chromophores and fluorescent groups), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers) and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (such as drugs, detection reagents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

[0369] Nanobodies can be coupled to drugs to form antibody-drug conjugates (NDCs). Typically, NDCs contain a linker located between the drug and the antibody. The linker can be a degradable or non-degradable linker. Degradable linkers are typically easily degraded in the intracellular environment, for example, the linker degrades at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including linkers containing peptidyl groups that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, such as linkers containing glucuronides that can be degraded by glucuronidases. Peptide 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 (such as linkers that hydrolyze at a pH of less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (such as disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0370] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved via the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimides, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethylketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, where the counter ion is acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. Linkers may include, for example, maleimides attached to the antibody via thiosuccinimide.

[0371] The drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, a linker connects the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group capable of forming a bond with the linker. In cases where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0372] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, immunostimulants, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, 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.

[0373] In the present invention, drug-linkers can be used to form NDCs in a single step. In other embodiments, bifunctional linker compounds can be used to form NDCs in a two-step or multi-step process. For example, a cysteine ​​residue is reacted with a reactive moiety of a linker in a first step, and in a subsequent step, a functional group on the linker is reacted with a drug to form an NDC.

[0374] Typically, the functional group on the linker is selected to facilitate specific reaction with an appropriate reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Technology, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that, when a complementary pair of reactive functional groups is selected for selective reaction between the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0375] Preferably, the coupling portion coupled to the Nanobody portion in the Nanobody-drug conjugate NDC of the present invention comprises a protein degrader or an oligonucleotide drug.

[0376] Preferably, the oligonucleotide drug is a nucleic acid drug targeting small molecules, including antisense nucleic acid (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA), aptamer, ribozyme, antibody nucleic acid conjugate drug (ARC), etc.

[0377] As used herein, the protein degrader is a degrader of tumor-associated proteins, and the tumor-associated proteins are selected from the following group:

[0378] (1) Kinases, such as RIPK2, BCR-ABL, EGFR, HER2, c-Met, TBK1, CDK2 / 4 / 6 / 9, ALK, Akt, CK2, ERK1 / 2, FLT3, PI3K, BTK, Fak, etc.;

[0379] (2) BET proteins, such as BRD2 / 4 / 6 / 9;

[0380] (3) Nuclear receptors, such as AR and ER;

[0381] (4) Other proteins, such as MetAp-2, Bcl-xL, Sirt2, HDAC6, Pirin, SMAD3, ARNT, PCAF / GCN5, Tau, FRS2, RAS (KRAS, HRAS and NRAS), etc.

[0382] Preferably, the protein degrader includes PROTAC (protein degradation targeting chimera), and more preferably, the PROTAC targets EGFR, KRAS (e.g., KRAS-G12C, KRAS-G12D, KRAS-G12V, etc.).

[0383] The present invention also provides a method for preparing NDC, which may further comprise: combining an antibody and a drug-linker compound under conditions sufficient to form an antibody conjugate (NDC).

[0384] In certain embodiments, the methods of the present invention comprise conjugating an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the present invention further comprise conjugating the antibody-linker conjugate to a drug moiety under conditions sufficient to covalently attach the drug moiety to the antibody via the linker.

[0385] In some embodiments, the Nanobody Drug Conjugate NDC is represented by the following molecular formula:

[0386] in:

[0387] nAb is a nano antibody.

[0388] LU is the linker / connector;

[0389] D is for medicine;

[0390] And the subscript p is a value selected from 1 to 8.

[0391] Drug

[0392] As used herein, "drug" refers generally to any compound having a desired biological activity and a reactive functional group for preparing the conjugates of the present invention. The desired biological activity includes diagnosing, curing, alleviating, treating, or preventing a disease in humans or other animals. Therefore, as long as the necessary reactive functional groups are present, the term "drug" refers to compounds including those identified in official national pharmacopeias, as well as, for example, the official U.S. Homeopathic Pharmacopoeia, the official National Formulary, or any supplements thereof. Typical drugs are listed in the Physician's Desk Reference (PDR) and the Orange Book of the U.S. Food and Drug Administration (FDA). It should be understood that as new drugs are continuously discovered and developed, these drugs should also be included in the "drug" of the conjugates of the present invention.

[0393] Drugs that can be used to constitute the NDC of the present invention include, but are not limited to, cytotoxic agents (eg, cytotoxic small molecule drugs).

[0394] The term "cytotoxic agent" refers to a substance that inhibits or prevents cell expression activity, cell function and / or causes cell destruction. The term includes radioactive isotopes, chemotherapeutic agents, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof. Examples of cytotoxic agents include, but are not limited to, auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), chlortetracycline, maytansyl, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxycarbonyl esters. anthracnose dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, α-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria officinalis inhibitor, as well as glucocorticoids and other chemotherapeutic agents, and radioactive isotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32, and radioactive isotopes of Lu including Lu177. The antibody can also be conjugated to an anticancer prodrug activating enzyme capable of converting the prodrug into its active form.

[0395] A preferred drug of the present invention is maytansine or maytansine-like compounds. Maytansine compounds inhibit cell proliferation by inhibiting the formation of microtubules of tubulin. Maytansine-like compounds are derivatives of maytansine. Both maytansine and maytansine-like compounds have highly effective cytotoxicity, but they have significant limitations in their clinical application in cancer treatment, primarily due to the low selectivity of these molecules for tumors. However, this high cytotoxicity makes them the preferred drug component of antibody-drug conjugates. The structure of deacetyl maytansine is listed below.

[0396] Another preferred drug of the present invention is auriculariaxin peptide drug. Auriculariaxin peptide drugs are analogs of Dolastatin 10, which is a biologically active polypeptide isolated from the marine mollusk Aplysia. Dolastatin 10 inhibits tubulin polymerization by binding to tubulin (the same binding region as vincristine). Dolastatin 10, auriculariaxin peptide PE, and auriculariaxin peptide E are all linear polypeptides containing four amino acids (three of which are unique to Dolastatin compounds) and a C-terminal amide group. Two representative auriculariaxin peptide compounds, monomethyl auriculariaxin peptide E (MMAE) and monomethyl auriculariaxin peptide F (MMAF), are both first-choice drugs for antibody-drug conjugates.

[0397] Monomethyl Auristatin E(MMAE)

[0398] Monomethyl Auristatin F(MMAF)

[0399] Monomethyl Dolastatin 10(MMAD)

[0400] Another preferred medicine of the present invention is tubulysin. Tubulysin was first isolated from myxobacteria culture by a research team and is a very effective cell growth inhibitor that works by inhibiting tubulin polymerization and thereby inducing apoptosis. Tubulysin D among the tubulysins is the most effective, with an activity that exceeds 10 to 100 times that of most other tubulin regulators (including epothilone, vinblastine and paclitaxel). Paclitaxel and vinblastine are currently used for the treatment of various cancers, and epothilone derivatives are being evaluated for activity in clinical trials. Synthetic derivatives of tubulysin D will provide the necessary information relevant to inhibition and key binding interactions, and can have superior properties as anticancer agents, which are as isolated entities or as chemical warheads on targeting antibodies or ligands. Tubulysin D is a complex tetrapeptide that can be divided into four regions: Mep (DN-methylpiperidinic acid), Ile (isoleucine), Tuv (tubuvaline), and Tup (tubuphenylalanine), as shown in the following formula:

[0401] Another preferred drug of the present invention is a microbial-derived cryptophycin derivative that inhibits microtubule polymerization. Cryptophycin, isolated from a culture of cyanobacteria, is a novel anti-tumor substance that inhibits microtubule formation and is active against a variety of tumors. Cryptophycin is a fat-soluble compound containing two peptide bonds and two ester bonds, five optically active centers, and one epoxy group. The dipeptide and diester bonds are contained within a macrocyclic structure. The structures of the cryptophycin derivatives CP1 and CP2 are shown below:

[0402] Another preferred drug of the present invention is the novel antimicrotubule agent Taltobulin (HTI-286, SPA-110). Taltobulin inhibits the polymerization of purified microtubules, disrupts intracellular microtubule organization, induces mitotic arrest, and induces apoptosis. Taltobulin is a potent inhibitor of cell proliferation, with an average IC50 of 2.5 nM against 18 human tumor cell lines. Compared to currently used antimicrotubule agents, taltobulin is not a suitable substrate for P-glycoprotein. The structure of taltobulin is shown in the figure below.

[0403] On the one hand, the drug is SN-38, a camptothecin derivative. SN-38 is a bioactive metabolite of irinotecan hydrochloride (CPT-11) and a topoisomerase inhibitor. SN-38 has the strongest inhibitory effect on DNA topoisomerase I, inhibiting DNA synthesis in a dose- and time-dependent manner and causing frequent DNA single-strand breaks. The structure of SN-38 is shown in the figure below.

[0404] On the one hand, the drug is a camptothecin derivative, exatecan. It is a synthetic analog of the topoisomerase I inhibitor camptothecin and is more active than SN-38. It can cause the strongest inhibition of DNA topoisomerase I, inhibiting DNA synthesis in a dose- and time-dependent manner and causing frequent DNA single-strand breaks. The structure of exatecan is shown in the formula below.

[0405] Another preferred drug of the present invention is alpha-Amanitin, the structure of which is shown in the figure below. Alpha-Amanitin is a mycotoxin from the poisonous mushroom Amanita phalloides. It is a bicyclic octapeptide that inhibits transcription by eukaryotic RNA polymerase II and RNA polymerase III.

[0406] Another preferred drug of the present invention is benzodipyrrole antibiotics (duocarmycins, CC-1065, etc.) and other cyclopropapyrroloind-4-one (CPI) derivatives. These compounds are effective DNA minor groove binding-alkylating agents. Cyclopropabenzindol-4-one (CBI) analogs have more stable chemical structures, higher biological activity, and are easier to synthesize than their parent compounds containing the natural CPI alkylating subunit. A representative CBI derivative is the phenolic hydroxyl-protected derivative CBI, which has weakened prodrug toxicity and enhanced water solubility (the general structural formula of the CBI-seco class is shown below):

[0407] Another preferred drug of the present invention is pyrrolo[2,1-c][1,4]benzodi-azepines (PBDs) or PBD dimers. PBDs are a class of natural products produced by Streptomyces with the unique property of forming non-distorting covalent adducts in the minor groove of DNA, specifically at purine-guanine-purine sequences. The use of PBDs as part of small molecule strategies for sequence-targeted DNA and as novel anticancer and antibacterial agents has attracted increasing interest. A flexible carbon chain connects the C8 / C8' hydroxyl groups of two PBD units, resulting in dimers with enhanced biological activity. PBD dimers are believed to generate sequence-selective DNA lesions, such as the inverted 5'-Pu-GATC-Py-3' interstrand crosslink, leading to their biological activity. These compounds have been shown to be highly potent cytotoxic agents and are promising candidates for antibody-drug conjugates.

[0408] Another preferred drug of the present invention is a PNU-159682 derivative. PNU-159682 is the major active metabolite of Nemorubicin in human liver microsomes, and its activity is 3000 times higher than that of MMDX and doxorubicin.

[0409] On the other hand, drugs are not limited to the aforementioned categories, but include all drugs that can be used in antibody-drug conjugates, and in particular those that can be coordinated through an amide bond with the linker, such as cytotoxins that are coordinated through a basic amine group (primary or secondary amine), such as the structures of cytotoxins D1-D12 shown above.

[0410] Linker

[0411] Based on the mechanism of drug release within cells, “linkers” or “linkers of antibody-drug conjugates” can be divided into two categories: non-cleavable linkers and cleavable linkers.

[0412] For antibody-drug conjugates containing a non-cleavable linker, the drug release mechanism is as follows: After the conjugate binds to the antigen and is internalized by the cell, the antibody is enzymatically hydrolyzed in the lysosome, releasing the active molecule composed of the small molecule drug, the linker, and the antibody's amino acid residues. This resulting structural change in the drug molecule does not diminish its cytotoxicity, but because the active molecule is charged (amino acid residues), it is unable to penetrate neighboring cells. Consequently, such active drugs are unable to kill neighboring tumor cells that do not express the target antigen (antigen-negative cells) (a bystander effect).

[0413] Cleavable linkers, as the name suggests, can cleave within target cells to release the active drug (the small molecule drug itself). Cleavable linkers can be divided into two main categories: chemically labile linkers and enzymatically labile linkers. Chemically labile linkers can selectively cleave due to differences in plasma and cytoplasmic properties, including pH and glutathione concentration. pH-sensitive linkers are often referred to as acid-cleavable linkers. These linkers are relatively stable in the neutral environment of blood (pH 7.3-7.5), but are hydrolyzed in the weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0). First-generation antibody-drug conjugates (ADCs) primarily utilize this type of linker, such as hydrazones, carbonates, acetals, and ketals. Due to the limited plasma stability of acid-cleavable linkers, ADCs based on these linkers typically have a short half-life (2-3 days). This short half-life has, to some extent, limited the application of pH-sensitive linkers in newer ADCs.

[0414] Glutathione-sensitive linkers, also known as disulfide linkers, trigger drug release based on the difference between the high intracellular glutathione concentration (millimolar range) and the relatively low glutathione concentration (micromolar range) in blood. This is particularly true for tumor cells, where low oxygen levels lead to increased reductase activity, resulting in higher glutathione concentrations. Disulfide bonds are thermodynamically stable, resulting in good stability in plasma.

[0415] Enzyme-labile linkers, such as peptide linkers, enable better control of drug release. Peptide linkers can be effectively cleaved by lysosomal proteases, such as cathepsin B or plasmin (the levels of such enzymes are increased in some tumor tissues). This peptide linkage is considered very stable in the plasma circulation because the unfavorable extracellular pH and serum protease inhibitors usually render the protease inactive. Due to their high plasma stability and good intracellular cleavage selectivity and efficiency, enzyme-labile linkers are widely used as cleavable linkers in antibody-drug conjugates. Typical enzyme-labile linkers include Val-Cit (VC), Val-Ala (VA), Gly-Gly-Phe-Gly (GGFG), etc.

[0416] Self-releasing linkers are typically embedded between a cleavable linker and an active drug, or are themselves part of the cleavable linker. The mechanism of action of a self-releasing linker is that, upon cleavage of the cleavable linker under appropriate conditions, the self-releasing linker spontaneously rearranges its structure, releasing the attached active drug. Common suicide linkers include p-aminobenzyl alcohol (PAB) and β-glucuronide.

[0417] application

[0418] The present invention also provides uses of the antibodies of the present invention, such as for the preparation of diagnostic agents or medicaments for the prevention and / or treatment of TF-related diseases, including tumor development, growth, and / or metastasis, diseases associated with tumor drug resistance, inflammation, and metabolic diseases.

[0419] The uses of the antibodies, NDCs, or CAR-Ts of the present invention include (but are not limited to):

[0420] (i) Diagnosis, prevention, and / or treatment of tumor development, growth, and / or metastasis, particularly tumors with high TF expression. Such 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, and angiosarcoma; particularly 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, and pancreatic cancer.

[0421] (ii) Diagnosis, prevention and / or treatment of autoimmune diseases, including but not limited to systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, type I diabetes, psoriasis, and multiple sclerosis.

[0422] (iii) diagnosing, preventing and / or treating inflammation, including but not limited to rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, gout, Reiter's syndrome, psoriatic arthropathy, infectious 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.

[0423] (iv) diagnosing, preventing and / or treating metabolic diseases, including but not limited to diabetes, diet-induced obesity and adipose inflammation.

[0424] Pharmaceutical composition

[0425] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its NDC or corresponding CAR-T cell, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary with the properties of the formulated substance and the condition to be treated. The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0426] The antibody of the present invention can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used in chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

[0427] The pharmaceutical composition of the present invention can be used to directly bind to TF protein molecules, and thus can be used to prevent and treat diseases such as tumors. In addition, other therapeutic agents can also be used simultaneously.

[0428] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned monoclonal antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 μg / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents.

[0429] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 20 mg / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0430] For NDC, since the nano antibody-drug conjugates provided by the present invention can target specific cell populations and bind to specific cell surface proteins (antigens), the drugs are released into the cells in an active form through endocytosis of the conjugate or drug penetration. Therefore, the nano antibody-drug conjugates of the present invention can be used to treat target diseases. The above-mentioned antibody-drug conjugates can be administered to subjects (e.g., humans) in a therapeutically effective amount through a suitable route. Subjects in need of treatment can be patients who are at risk or suspected of having a disease related to the activity or expression of a specific antigen. Such patients can be identified by routine physical examinations.

[0431] When treating with the Nanobody-drug conjugates of the invention, delivery can be performed 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 by using an infusion pump.

[0432] The main advantages of the present invention include:

[0433] 1. The TF Nanobodies of the present invention possess excellent biological activity and specificity, and possess very high affinity. Surface plasmon resonance (SPR) analysis determined that the binding affinity constants (KD) of chimeric Nanobodies 4-A02, F1-A01, 4-E04, 4-C05, 5-C12, and 5-H3 to human TF-ECD ranged from 0.202 to 7.04 nM.

[0434] 2. It has good binding affinity to TF of tumor cells, and its EC is determined by FACS. 50 The concentration of the drug is 0.05μg / mL to 0.75μg / mL, and it inhibits TF-related functions of tumor cells and can be used as a targeted therapeutic antibody.

[0435] 3. The nanoantibody-drug conjugate TF-NDC (tubulin inhibitor, topoisomerase 1 inhibitor) designed based on TF nanoantibody can specifically kill tumor cells with high expression of TF. 50 The value is 0.0001μg / mL~0.03μg / mL.

[0436] 4. The TF-NDC of the present invention has significant anti-tumor activity. Single administration of 3 mg / kg and 1 mg / kg demonstrated extremely strong anti-tumor activity and the potential to regulate the immune microenvironment in vivo.

[0437] 5. According to the design of Figure 24 of the present invention, TF nanobody-drug conjugates with the required DAR value can be easily prepared, and their cross-linking reaction formulas are shown in Figure 23 1a, 1b, 1c or 1d respectively.

[0438] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which detailed conditions are not specified, were generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0439] Example 1 Discovery and preparation of nanobodies targeting human tissue factor (TF)

[0440] Human TF extracellular domain protein (TF-ECD, Met 1-Glu 251) was purchased from SinoBiological (Cat. No. 13133-H08H). Alpaca were immunized with this prepared TF extracellular domain protein, with a 21-day interval between immunizations. Peripheral blood microarray (PBMC) samples were collected 10 days after the final immunization. PBMCs were isolated and VHH antibody fragments were cloned to construct yeast and phage display libraries. Through repeated panning, monoclonal ELISA, and FACS screening, seven highly active, sequence-independent nanobodies (VHHs) were obtained from the phage display libraries: 4-A02, F1-A01, 4-C08, 4-E04, 4-C05, 5-C12, and 5-H3. Chimeric antibodies were prepared by fusion of each of the seven VHH amino acid sequences with the human Fc mutant fragment FC1. These antibodies were transiently transfected into HEK293 cells and then cultured in suspension in serum-free medium for 6 days. The culture supernatant was collected for purification, protein quantification and SDS-PAGE analysis.

[0441] 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 expressed, produced, and purified with high efficiency.

[0442] Example 2 SPR determination of affinity of TF nanobody to human TF antigen

[0443] Antibody affinity was determined using a ForteBio OCTET R2 instrument. FDYKSD-01-His was immobilized on an NTA sensor (Sartorius, Cat# 18-5101) for 200 seconds. Nanobody samples were diluted to 25, 12.5, 6.25, 3.13, 1.56, and 0 nM in PBST (PBS + 0.02% tween 20). Affinity assays were performed with a 60-second equilibrium, 180-second association, and 180-second dissociation cycle at 25°C. Kinetic characterization was performed using the ForteBio OCTET R2 system.

[0444] The test results are shown in Figure 2. The chimeric antibodies 4-A02, F1-A01, 4-E04, 4-C05, 5-C12, and 5-H3 have strong affinity for TF-ECD, and the binding constants (KD) are 2.09 nM, 4.37 nM, 7.04 nM, 0.2 nM, 0.001 nM, and 0.45 nM, respectively.

[0445] Example 3 Determination of the binding affinity of TF nanobody to TF on the surface of tumor cells

[0446] The triple-negative breast cancer cells MDA-MB-231 with high TF expression were used. 100 μL of the test antibody diluted 5-fold from 10 μg / mL to 0.016 μg / mL was used as the primary antibody and then incubated with 1×10 5 Tumor cells were mixed to a final concentration of 5 μg / mL and diluted to 0.008 μg / mL. The cells were then incubated at 4°C for 1 hour. The cells were washed twice with PBS to remove unbound primary antibody. The target cells were then incubated with 200 μL of 2 μg / mL PE-labeled secondary antibody at 4°C for 30 minutes. The cells were washed twice with PBS to remove unbound secondary antibody. Finally, the cells were resuspended in 200 μL PBS. The binding affinity of the test antibody to the corresponding cell surface TF was measured by flow cytometry.

[0447] The test results are shown in Figure 3. 4-A02, F1-A01, 4-C08, 4-E04, 4-C05, 5-C12, and 5-H3 have excellent binding affinity to MDA-MB-231. 50 They are 0.08μg / mL, 0.75μg / mL, 0.05μg / mL, 0.13μg / mL, 0.7μg / mL, 0.47μg / mL and 0.25μg / mL respectively.

[0448] Example 4 Preparation of TF Nanobody Fusion Protein-vc-MMAE

[0449] Protein stock solutions of FC1 chimeric naked antibodies (generally dimers) derived from 4-A02, F1-A01, 4-C08, 4-E04, 4-C05, 5-C12, or 5-H3, or 4-A02-ABD (monomer), were diluted to 5 mg / mL with 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH2PO4-NaOH) / 150 mM sodium chloride (NaCl) / 1 mM diethylenetriaminepentaacetic acid (DTPA), pH 7.4 reaction buffer. A 3-4-fold molar excess of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added, and the reaction solution was stirred at 25°C for 2.5 hours.

[0450] The reaction solution was cooled to 0-10°C, and an appropriate amount of diethylacetamide (DMA) was added without purification. Then, a 6-fold molar excess of mc-vc-MMAE (10 mg / ml pre-dissolved in DMA) was added to ensure that the volume of DMA in the reaction system did not exceed 10%. The coupling was carried out by stirring at 10-25°C for 2 hours.

[0451] The coupling reaction mixture was purified by gel filtration using a desalting column with histidine-acetate / sucrose at pH 6.0, and the peak sample was collected based on UV280 absorbance. The sample was then sterilized through a 0.22 μm pore size filter and stored at -80°C.

[0452] For naked antibodies and MMAE conjugates, purity and degree of conjugation are tested using methods such as hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC). For specific antibody-ABD conjugates, the target drug loading rate (DAR) is 4; for antibody-ABD monomer conjugates, the target drug loading rate (DAR) is 1.

[0453] The results are shown in FIG4 . HIC analysis of the 4-A02-MMAE conjugate showed that the DAR4 ratio was approximately 95%.

[0454] The results are shown in FIG5 . HIC analysis of the F1-A01-MMAE conjugate showed that the DAR4 ratio was approximately 91%.

[0455] The results are shown in FIG6 . HIC analysis of the 4-C08-MMAE conjugate showed that the ratios of DAR4 and DAR2 were approximately 41.8% and 42.7%, respectively.

[0456] The results are shown in FIG7 . HIC analysis of the 4-C05-MMAE conjugate showed that the naked antibody had been substantially converted into the conjugate.

[0457] The results are shown in FIG8 . HIC analysis of the 5-C12-MMAE conjugate showed that the ratios of DAR4 and DAR2 were approximately 66.2% and 21.9%, respectively.

[0458] The results are shown in FIG9 . HIC analysis of the 4-E04-MMAE conjugate showed that the coupling reaction was not completed as expected, and the ratio of the coupling product was very low.

[0459] The results are shown in FIG10 . HIC analysis of the 4-A02-ABD-MMAE conjugate showed that the naked antibody had been substantially converted into the conjugate DAR1.

[0460] Example 5 Preparation of TF Nanobody-GGFG-DXd

[0461] The 4-A02-FC1 naked antigen solution was replaced with 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate (NaH2PO4-Na2HPO4) / 150mM sodium chloride (NaCl) / 2mM ethylenediaminetetraacetic acid (EDTA), pH 7.0 reaction buffer to a concentration of 10 mg / mL, and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added in a 2.2-fold excess molar ratio. The reaction solution was stirred at 25°C for 4-6 hours. The reaction solution was cooled to room temperature, an appropriate amount of diethylacetamide (DMA) was added, and then a 4-fold excess molar ratio of the compound GGFG-DXd (purchased from Shanghai Haoyuan Chemical, 10 mg / mL pre-dissolved in DMA) was added to ensure that the volume of DMA in the reaction system did not exceed 10%, and the coupling was carried out by stirring at 25°C for 1-2 hours. Excess small molecules were removed using a G25 desalting column or directly ultrafiltered using a filter tube, and the mixture was replaced with a PBS / 8% sucrose, pH 7.4 buffer solution. The mixture was then sterilized through a 0.22 μm pore size filter and stored at -80°C. The resulting antibody conjugate was named 4-A02-DXd.

[0462] The results are shown in FIG11 . HIC analysis of the 4-A02-DXd conjugate showed that the naked antibody had been completely converted into the conjugate DAR4.

[0463] Example 6 In vitro antitumor activity of TF-NDC against tumor cells with high and medium TF expression

[0464] The cell lines used in the examples were purchased from the American Type Culture Collection (ATCC), the Cell Bank of the Chinese Academy of Sciences, and Nanjing Kebai Biotechnology Co., Ltd., and cultured according to the corresponding instructions, including: 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 above-mentioned cells in the logarithmic growth phase were seeded into 96-well cell culture plates at a density of 200-2000 cells per well (depending on the growth rate of different cells). 150 μL / well was cultured at 37°C, 5% CO2 for approximately 5 hours. Then, different concentrations of TF-NDCs (15 μg / mL to 0.00019 μg / mL) were added. Two to four replicate wells were set up for each drug concentration, along with corresponding vehicle and blank control wells. After 5-9 days of culture (depending on the cell growth rate to ensure a sufficient number of cell divisions), the culture medium was decanted and MTS reaction solution (purchased from Promega, cat# G3581) was added at 100 μL / well. The reaction was incubated at 37°C until the desired color depth was reached. The cell viability (OD490nm) of each group was measured, and the cell survival rate was calculated according to the following formula: Survival rate = (ODdrug - ODblank) / (ODcontrol - ODblank) × 100%. The above data were analyzed by GraphPad Prism 8 software, and the IC of the above TF-NDC in different cell lines was calculated. 50 value.

[0465] The in vitro anti-tumor activity results of the preferred TF-NDC4-A02-MMAE, F1-A01-MMAE, 4-C08-MMAE, 4-C05-MMAE, 5-C12-MMAE, 4-E04-MMAE of the present invention and HuSC1-MMAE as a control are shown in Figures 12, 13 and 14, respectively.

[0466] As shown in Figure 12, TF-NDCs have a strong inhibitory activity on the proliferation of triple-negative breast cancer HCC1806 cells with high TF expression. 50 The value is 0.0001~0.0326μg / mL.

[0467] As shown in Figure 13, TF-NDCs have a strong anti-proliferation activity against pancreatic cancer BxPC3 cells with high TF content, IC 50 The value is 0.0001~0.0288μg / mL.

[0468] As shown in Figure 14, TF-NDCs have a strong inhibitory activity on the proliferation of pancreatic cancer HPAF-II cells with high TF expression. 50 The value is 0.0007~0.0243μg / mL.

[0469] Table 3 summarizes the IC values ​​of 6 TF-NDC cell proliferation inhibition tests 50 value.

[0470] Table-3: In vitro antitumor activity of TF nanobody-drug conjugates

[0471] Based on the above anti-tumor activity testing, 4-A02 was selected for design, mass production, and purification of the proteins 4-A02-FC1, 4-A02-FCWT, 4-A02-FCLALAPG, and 4-A02-ABD. The results are shown in Figure 20. The molecular weights of 4-A02-FC1, 4-A02-FCWT, and 4-A02-FCLALAPG are approximately 39 kDa, while the molecular weight of 4-A02-ABD is approximately 20 kDa.

[0472] Using the above-mentioned batch purified protein, 4-A02-MMAE was repeatedly prepared as shown in Figures 10 and 11, and 4-A02-ABD-MMAE and 4-A02-DXd were designed and prepared.

[0473] The results are shown in Figures 15, 16, 17, 18, and 19. 4-A02-MMAE and 4-A02-ABD-MMAE have strong proliferation inhibitory activity in HPAF-II, BxPC3, HCC44, PC9, and H1975 cells with high or medium TF expression. 50 The values ​​were 0.0002~0.022μg / mL and 0.0003~0.163μg / mL respectively.

[0474] The results are shown in Figures 15, 16, 17, 18, and 19. 4-A02-DXd has excellent proliferation inhibitory activity in HPAF-II, BxPC3, and HCC44 cells. 50 The inhibitory activity of 4-A02-DXd in lung cancer PC9 and H1975 cells was relatively weak, with an IC 50 The values ​​were 5.63μg / mL and >15μg / mL, respectively.

[0475] Table 4 summarizes the IC values ​​of three TF-NDC cell proliferation inhibition tests 50 value.

[0476] Table-4: In vitro antitumor activity of TF nanobody-drug conjugates

[0477] 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 was further evaluated. As shown in Figures 25 (MDA-MB-231), 26 (HCC1806), 27 (HCC1954), 28 (HCC1937), and 29 (MDA-MB-468), 4-A02-Dxd inhibited TNBC cell proliferation with an IC of 0. 50 The value was between 0.001 and 0.008 μg / mL. Similarly, MMAE-conjugated 4-A02-NDC also showed a strong anti-tumor effect, IC 50 The value is between 0.0003 and 0.004 μg / mL.

[0478] Example 7 Batch Expression, Production and Purification of TF Nanobodies and Fusion Proteins

[0479] The variable region sequences of the nanobodies were cloned into the FC1, FCWT, FCLALAPG, and ABD expression vectors (4-A02-FC1, 4-A02-FCWT, 4-A02-FCLALAPG, and 4-A02-ABD), transiently transfected into HEK293 cells, and then cultured in suspension in serum-free medium for 6 days. The culture supernatants were collected for purification, protein quantification, and SDS-PAGE analysis.

[0480] As shown in Figure 20, the chimeric Nanobody and recombinant fusion protein derived from 4-A02 can be expressed, produced and purified with high efficiency.

[0481] Example 8 In vivo anti-tumor effect of TF nanobody (triple-negative breast cancer breast pad transplant model)

[0482] Triple-negative breast cancer (TNBC) HCC1806 cells in the logarithmic growth phase were cultured in 200 μL serum-free medium containing 3 × 10 6 The cells were inoculated into the mammary pads of 6-week-old Balb / c female nude mice (Balb / c nude mice were purchased from Shanghai Xipu-Bikai Experimental Animal Co., Ltd.) at a density of 100-200 mm. 3 The animals were then randomly divided into groups, with 8 tumors in each group. The six preferred TF-NDCs of the present invention were administered via the tail vein at a dose of 3 mg / kg on the day of grouping. hIgG1-MMAE was used as a negative control drug, and the drug was administered once throughout the entire experiment. Tumor volume and nude mouse weight were measured 2-3 times a week and recorded to draw a tumor growth curve. The formula for calculating tumor volume (V) is: V = 1 / 2 × a × b 2 Where a and b represent the length and width of the tumor, respectively.

[0483] As shown in Figure 21, compared to the hIgG1-MMAE control group, the 3 mg / kg dose of 4-A02-MMAE, F1-A01-MMAE, 4-C08-MMAE, 4-C05-MMAE, and 5-C12-MMAE groups all demonstrated strong anti-tumor activity, leading to tumor regression. In addition, the conjugate 4-E04-MMAE only exhibited a partial tumor suppression effect, which may be related to the lower conjugation efficiency (Figure 9).

[0484] The in vivo antitumor efficacy of 4-A02-FC1-Dxd, 4-A02-FC1-MMAE, 4-A02-FCWT-MMAE, and 4-A02-ABD-MMAE in the triple-negative breast cancer model was further evaluated.

[0485] The results are shown in FIG30 . In the HCC1806 tumor regression experiment, the 3 mg / kg dose of 4-A02-MMAE resulted in complete regression of large tumors within 21 days.

[0486] The results are shown in FIG31 . In the HCC1954 breast pad transplant tumor experiment, 10 mg / kg 4-A02-FC1-Dxd, 3 mg / kg 4-A02-MMAE, and 3 mg / kg 4-A02-ABD-MMAE all significantly inhibited tumor growth and caused tumor regression.

[0487] Example 9 In vivo anti-tumor effect of TF nanobody (lung cancer orthotopic transplant tumor model)

[0488] 4-5 week old nude mice were purchased and bred for one week in an SPF animal laboratory. HCC44-luc cells in the logarithmic growth phase were digested and centrifuged using a 4°C pre-cooled centrifuge. The cells were resuspended in PBS according to the cell number and the cell density was adjusted to 40×10 6 Cells / mL were added with an equal volume of high-concentration Matrigel, resulting in a cell suspension:Matrigel ratio of 1:1. After anesthetizing the mouse with Avertin (400 μL / 20 g), a small incision was made on the right side of the rib cage near the armpit (three layers of skin and muscle). The pink lungs were visible. Care was taken to avoid the Y-shaped blood vessels in the skin when making the incision. The needle was inserted in the middle of the Y-shaped pattern. A line was drawn 3 mm from the insulin needle tip, representing the insertion depth. The cells were then slowly inserted. Each mouse was inoculated with 50 μL, for a total of 1 × 10 6 After the injection, pause for 5-10 seconds, remove the needle, and suture the wound. On day 13, images were taken using a live imaging device and mice were divided into groups based on signal levels. On day 14, 3 mg / kg of hIgG1-MMAE or 4-A02-MMAE (n=6-8) were injected intravenously into the tail vein. A total of one dose was administered throughout the entire study. Fluorescence levels were measured and recorded using a live imaging device on days 20, 27, and 34.

[0489] As shown in FIG22 , the 3 mg / kg dose of 4-A02-MMAE group showed excellent anti-tumor activity compared with the hIgG1-MMAE control group, resulting in complete regression of tumors in 5 / 6 mice.

[0490] Example 10 In vivo anti-tumor effect of TF nanobody (pancreatic cancer transplant model)

[0491] Pancreatic cancer (PDAC) HPAF-II cells in the logarithmic growth phase were cultured in 200 μL serum-free medium containing 2.5×10 6 The cells were inoculated subcutaneously on the back of 5-week-old female Balb / c nude mice (Balb / c nude mice were purchased from Shanghai Xipu-Bikai Laboratory Animal Co., Ltd.). When the tumor grew to 100-200 mm, 3 The animals were then randomly divided into groups with 8 tumors in each group. The preferred TF-NDCs of the present invention, such as 4-A02-MMAE and 4-A02-ABD-MMAE, were administered via the tail vein at doses of 3 mg / kg and 1 mg / kg on the day of grouping. hIgG1-MMAE was used as a negative control drug, and the drug was administered once during the entire experiment. The tumor volume and nude mouse weight were measured twice a week and recorded to draw a tumor growth curve. The formula for calculating tumor volume (V) is: V = 1 / 2 × a × b 2 Where a and b represent the length and width of the tumor, respectively.

[0492] As shown in Figure 32A , compared with the hIgG1 group, the 3 mg / kg 4-A02-FC1-MMAE, 4-A02-FCWT-MMAE, and 4-A02-ABD-MMAE single-dose groups all resulted in complete tumor regression; as shown in Figure 32B , compared with the hIgG1 group, the 1 mg / kg 4-A02-FC1-MMAE and 4-A02-FCWT-MMAE single-dose groups also showed excellent anti-tumor activity, resulting in complete inhibition of tumor growth and partial regression.

[0493] Example 11 Humanization modification and activity detection of TF nanobody

[0494] Based on the original antibody sequence information, the homology model of the antibody is obtained by modeling and CDRs are analyzed. A range of framework amino acids is required, as these amino acid positions typically affect the conformation or antigen-binding activity of the CDRs. Humanized germlines were obtained through IMGT analysis. The selected humanized germline framework was spliced ​​with the antibody's CDRs. The framework sequences of the designed humanized antibodies were then compared with those of the original antibody. Homology modeling results from the parental antibody were analyzed, and amino acid substitutions similar to surface residues in human antibodies were selected while maintaining antibody activity and minimizing heterogeneity. This resulted in humanized antibodies 4A02-HM7, 4A02-HM8, and 4A02-HM9, with corresponding sequences as SEQ ID NO. 33, SEQ ID NO. 34, and SEQ ID NO. 35.

[0495] The affinity of the humanized antibodies for the antigen was determined using the surface plasmon resonance (SPR) assay method of Example 2. As shown in Figure 33 , 4A02-HM7, 4A02-HM8, and 4A02-HM9 exhibited very similar antigen-binding affinities 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 assay.

[0496] The binding activity of humanized antibodies to cells was detected according to the method for determining the binding affinity of TF on the surface of tumor cells in Example 3. The results are shown in Figure 34A. The binding affinity of 4A02-HM7, 4A02-HM8, 4A02-HM9 and the original antibody 4A02 to triple-negative breast cancer MDA-MB-231 cells was EC 50 The results are shown in Figure 3B. The binding affinity EC values ​​of 4A02-HM7, 4A02-HM8, 4A02-HM9, and the original antibody 4A02 to pancreatic cancer HPAF-II cells are 0.18-0.21 μg / mL. 50 It is 0.24~0.32μg / mL.

[0497] Example 12 ELISA determination of affinity of humanized TF nanobody to human TF and cynomolgus monkey TF antigens

[0498] The TF protein extracellular domain (human TF-ECD, cynomolgus monkey TF-ECD) was diluted to 1 μg / mL using coating buffer and coated onto ELISA plates at 100 μL / well at 4°C overnight. Excess antigen was washed away, and the plates were blocked with 1% BSA at room temperature for 2 hours. Five-fold serial dilutions of each nanobody were then added at 100 μL / well and incubated at room temperature for 1 hour. Unbound antibody was washed away, and an appropriate concentration of horseradish peroxidase-labeled anti-Fc secondary antibody was added at 100 μL / well. After incubation at room temperature, unbound secondary antibody was washed away, and TMB color development solution was added for approximately 15 minutes. The color reaction was terminated by adding 1 M HCl at 50 μL / well. The absorbance was then measured at 450 nm, and the data analyzed.

[0499] ELISA test results showed that the binding affinity of 4A02-HM7, 4A02-HM8, 4A02-HM9 and the original antibody 4A02 to human TF-ECD was 50 The binding affinity EC for cynomolgus monkey TF-ECD was 5.91-6.89 ng / mL ( FIG. 35A ). 50 The serum levels of 5.96 to 9.78 ng / mL were observed (Figure 35B).

[0500] In summary, the efficacy studies of TF-NDC-related examples clearly show that:

[0501] 1. TF-NDC 4-A02-MMAE, F1-A01-MMAE, 4-C08-MMAE, 4-C05-MMAE, and 5-C12-MMAE all exhibited excellent TF-specific tumor cell killing activity, demonstrating a strong inhibitory effect on the proliferation of tumor cells with moderate to high TF expression, while exhibiting no significant toxicity against the proliferation of cells with low TF expression.

[0502] 2. 4-A02-ABD-MMAE prepared from 4-A02-ABD also has excellent anti-tumor activity;

[0503] 3. 4-A02-DXd has strong or good in vitro anti-tumor activity in selected pancreatic cancer, multiple triple-negative breast cancer, and lung cancer cells;

[0504] 4. In the in vivo efficacy models of triple-negative breast cancer, lung cancer, and pancreatic cancer, the preferred TF-NDC of the present invention showed excellent anti-tumor therapeutic effects after a single intravenous administration.

[0505] Nanobody sequences of the present invention

[0506] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A nanobody targeting tissue factor (TF), characterized in that: The complementarity determining region CDR of the VHH chain of the Nanobody is one or more selected from the following groups: (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; Any of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and is able to retain TF binding affinity.

2. The Nanobody according to claim 1, characterized in that The VHH chain of the Nanobody also comprises a framework region (FR).

3. The Nanobody according to claim 1, characterized in that The VHH chain of the nanobody targeting TF has an amino acid sequence selected from the following group: SEQ ID NO.4, SEQ ID NO.8, SEQ ID NO.12, SEQ ID NO.16, SEQ ID NO.20, SEQ ID NO.24, SEQ ID NO.28; or the VHH chain of the nanobody targeting TF has an amino acid sequence as shown in positions 1-114 of SEQ ID NO.33, as shown in positions 1-114 of SEQ ID NO.34, or as shown in positions 1-114 of SEQ ID NO.

35.

4. An antibody targeting TF, characterized in that: The antibody comprises one or more VHH chains of the nanobody targeting tissue factor as described in any one of claims 1-3.

5. A multispecific antibody, characterized in that: The multispecific antibody comprises: the TF-targeting nanobody according to any one of claims 1 to 3 or the TF-targeting antibody according to claim 4.

6. The multispecific antibody according to claim 5, characterized in that The multispecific antibody also includes a second antigen binding region targeting a target 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.

7. A recombinant protein, characterized in that The recombinant protein has: (i) a Nanobody targeting TF, a Nanobody targeting TF as claimed in any one of claims 1 to 3, an antibody targeting TF as claimed in claim 4, or a multispecific antibody as claimed in claim 5; and (ii) optional polypeptide molecules or fragments having therapeutic functions; and / or (iii) Optional functional domains that enhance the physicochemical properties or druggability of the protein.

8. The recombinant protein according to claim 7, characterized in that The recombinant protein has the following structure from the NC end or the CN end: (AB)m; Among them, the A element is a nanobody targeting 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, Wherein, m is a positive integer.

9. A CAR construct, characterized in that: The antigen binding region of the CAR construct is the VHH chain of the Nanobody as described in any one of claims 1-3.

10. A recombinant immune cell, characterized in that: The immune cells express the exogenous CAR construct as described in claim 9.

11. An immunoconjugate, characterized in that: The immunoconjugate contains: (a) an antibody portion, wherein the antibody portion is a TF-targeting Nanobody according to any one of claims 1 to 3 or an antibody targeting TF according to claim 4; and (b) a coupling moiety coupled to the Nanobody portion, the coupling moiety being selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, an enzyme, a protein degrader, an immunomodulator (immune agonist and inhibitor), an oligonucleotide or a combination thereof.

12. The immunoconjugate according to claim 11, characterized in that The immunoconjugate is an antibody-drug conjugate ADC shown in the following molecular formula: in: nAb is a nanobody targeting TF or a recombinant protein as claimed in claim 5, LU is linker 2 (also called connector); D is for medicine; And the subscript p is a value selected from 1-8.

13. The immunoconjugate according to claim 12, characterized in that LU is selected from maleimidocaproyl (MC), maleimide (MAL), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) (SMCC) linkers connected to the antibody portion, and contains one or more linkers of valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), alanine-alanine-alanine (AAA), p-aminobenzyloxycarbonyl (PAB), and polyethylene glycol (PEG).

14. The immunoconjugate according to claim 12, characterized in that Wherein D is selected from the following group of compounds having anti-tumor activity: (i) Tubulin inhibitors, such as maytansine derivatives (DM1, DM4), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF); (ii) Acting on DNA toxins, such as duocarmycin and pyrrolobenzodiazepine (PBD); (iii) Topoisomerase inhibitors, camptothecin, SN38, exitecan, Dxd.

15. The immunoconjugate according to claim 11, characterized in that The protein degrader is a degrader of tumor-associated proteins, and the tumor-associated proteins are selected from the following group: 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, RAS (such as KRAS, HRAS and NRAS); preferably, the protein degrader includes PROTAC (protein degradation targeting chimera).

16. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (i) a Nanobody targeting TF as described in any one of claims 1 to 3, an antibody targeting TF as described in claim 4, a multispecific antibody as described in claim 5, a recombinant protein as described in claim 7, a recombinant immune cell as described in claim 10, or an immunoconjugate as described in claim 11; (ii) a pharmaceutically acceptable carrier.

17. A use of an active ingredient, characterized in that: The active ingredient is selected from the following group: a nanobody targeting TF as described in any one of claims 1 to 3, an antibody targeting TF as described in claim 4, a multispecific antibody as described in claim 5, a recombinant protein as described in claim 7, a recombinant immune cell as described in claim 10, or an immunoconjugate as described in claim 11, or a combination thereof, wherein the active ingredient is used (a) preparing reagents, detection panels or kits for detecting TF-related diseases; and / or (b) preparing drugs for preventing and / or treating TF-related diseases.

18. The use according to claim 17, characterized in that The TF-related disease is selected from the group consisting of cancer, thrombosis, autoimmune disease, metabolic-related disease, infectious disease, or a combination thereof; preferably, selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, ovarian cancer, cervical cancer, colorectal cancer, brain glioma, melanoma, leukemia, lymphoma, or a combination thereof.

Images