Antibodies and drug conjugates thereof and uses thereof

Abstract

The present invention relates to antibodies and antibody-drug conjugates, more particularly to antibodies and antibody-drug conjugates (ADCs) that target tissue factor, as well as compositions comprising said antibodies or ADCs and therapeutic applications thereof.

Description

[Technical field]

[0001] The present invention relates to antibodies and antibody-drug conjugates, and more specifically to antibodies and antibody-drug conjugates (ADCs) that target tissue factor (TF), as well as compositions comprising said antibodies or ADCs and therapeutic applications thereof. [Background technology]

[0002] Tissue Factor (TF) is also called thromboplastin, CD142, or clotting factor 3. As a transmembrane glycoprotein, TF consists of an extracellular domain, a transmembrane region, and an intracellular domain.

[0003] TF is a molecule required for the initiation of extrinsic blood coagulation events expressed on the cell surface in a functional form. As a high-affinity receptor for blood coagulation factor VII (FVII, a serine protease) in plasma, TF forms a complex with the activated form of VII, VIIa, and then triggers a catalytic event to initiate the thrombin cascade through the cleavage of specific limited proteins, activating factor IX or X. Under normal physiological conditions, TF is located in adventitial cells of the vascular wall, fibroblasts surrounding blood vessels, etc., but rarely in the media or intima of blood vessels. Only when the integrity of the vascular wall is destroyed does TF become exposed to circulating blood and exert its hemostatic effect by activating the coagulation cascade reaction. In the process, TF exerts its "anchor" action by relying on its tight binding to the cell membrane, restricting physiological blood clots to the site of injury without diffusing distally from the initiation site of blood coagulation.

[0004] In contrast to limited expression in normal tissues and cells, TF has been demonstrated to be overexpressed in various malignant tumors, including cervical cancer, pancreatic cancer, lung cancer, prostate cancer, bladder cancer, ovarian cancer, breast cancer, colorectal cancer, etc. Therefore, TF can serve as a target for antibody drug and ADC (antibody-drug conjugate) drug development. Tivdak (Tisotumab Vedotin), an ADC drug targeting TF developed by Seagen Inc. (SGEN) / Genmab A / S (GMAB), has already been approved for the indication of cervical cancer in September 2021, and its application for other indications such as ovarian cancer has already entered clinical phase II.

[0005] However, although many anti-TF antibodies or antibody class drugs currently under development have shown a certain efficacy in cancer treatment, interference or inhibition of blood coagulation and associated side effects caused thereby have also been detected at the same time (Chenard-Poirier M, Hong DS, Coleman R, de Bono J, Mau-Sorensen M, Collins D, et al. A phase I / II safety study of tisotumab vedotin(HuMax-TFADC)in patients with solid tumors. Ann Oncol 2017, 28:v403-v27; Zhang X, Li Q, Zhao H, Ma L, Meng T, Qian J, et al. Pathological expression of tissue factor confers promising antitumor response to a novel therapeutic antibody SC1 in triple negative breast cancer and pancreatic adenocarcinoma. Oncotarget 2017, 8:59086-102.).

[0006] Therefore, there remains a need in the art to develop new TF antibodies and new ADC drugs containing same, which should have the ability to specifically target tumor cell surface TF, but at the same time have minimal effect on TF-mediated coagulation responses in normal tissues, so as to provide cancer patients with wider and better drug options. Summary of the Invention

[0007] In order to meet the above-mentioned needs in the field, the present inventors have conducted extensive research and provided a new TF antibody and an antibody-drug conjugate (ADC) made thereof. As shown in the Examples, the anti-TF antibody of the present invention not only exhibits high binding affinity and high specificity to TF-positive tumor cells, and can be rapidly and efficiently endocytosed by tumor cells, but also has little effect on TF-mediated blood coagulation. Furthermore, as shown in the Examples, the ADC made of the TF antibody of the present invention not only has good physical properties, no obvious aggregation phenomenon, but also shows significant tumor growth inhibition activity and good animal drug resistance in animal models.

[0008] Accordingly, in a first aspect, the present invention provides an antibody-drug conjugate (ADC) having formula (I) or a pharma- ceutically acceptable salt or solvate thereof: Ab-[LD] q (I) Among them, Ab stands for anti-TF antibody; L represents a linker, D represents a cytotoxic or cell inhibiting drug, such as a topoisomerase I inhibitor, and q=1 to 20, for example, q=1 to 10, 1 to 8, 3 to 8, 4 to 8, or 6 to 8; Among them, the above Ab is: - the three CDRs of the heavy chain variable region (VH) sequence of SEQ ID NO: 1 and the three CDRs of the light chain variable region (VL) sequence of SEQ ID NO: 2, - the three CDRs of the heavy chain variable region (VH) sequence of SEQ ID NO: 3 and the three CDRs of the light chain variable region (VL) sequence of SEQ ID NO: 4, - the three CDRs of the heavy chain variable region (VH) sequence of SEQ ID NO: 5 and the three CDRs of the light chain variable region (VL) sequence of SEQ ID NO: 6, or - comprising the three CDRs of the heavy chain variable region (VH) sequence of SEQ ID NO: 7 and the three CDRs of the light chain variable region (VL) sequence of SEQ ID NO: 8, Among them, preferably, the CDRs are defined according to Chothia, AbM, Kabat, IMGT, or any combination thereof, More preferably, said Ab comprises the three CDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:7 and the three CDRs of the light chain variable region (VL) sequence of SEQ ID NO:8.

[0009] In a second aspect, the invention provides a composition comprising an ADC of the invention, or a pharma- ceutically acceptable salt or solvate thereof.

[0010] In a third aspect, the present invention provides the use of an ADC of the invention or a pharma- ceutically acceptable salt or solvate thereof, and a composition thereof in the treatment or prevention of a TF-positive tumour, and in the preparation of a medicament for use in said treatment or prevention.

[0011] In a fourth aspect, the present invention provides anti-TF antibodies, and pharmaceutical compositions and uses thereof.

[0012] The present invention will be further described in the following figures and specific embodiments. However, these figures and specific embodiments should not be regarded as limiting the scope of the present invention, and any modifications that can be easily thought of by those skilled in the art will fall within the spirit of the present invention and the scope of protection of the appended claims. [Brief description of the drawings]

[0013] [Figure 1A] 1 shows the antigen protein-based ELSIA assay detecting the binding affinity of exemplary antibodies of the invention to human or monkey TF, BM is the reference antibody. [Figure 1B] This is a continuation of Figure 1A. [Figure 1C]This is a continuation of Figure 1B. [Figure 1D] This is a continuation of Figure 1C. [Figure 2A] Figure 1 shows the detection of cell binding affinity of exemplary antibodies of the invention to different TF positive tumor cells (NCI-H358 and KYSE520) by flow cytometry. BM is the reference antibody. [Figure 2B] This is a continuation of Figure 2A. [Figure 2C] This is a continuation of Figure 2B. [Figure 2D] This is a continuation of Figure 2C. [Figure 3A] The IL-8 release inhibition experiment demonstrates that the blocking activity of the humanized antibody of the present invention against the TF / FVIIa complex-mediated downstream signal pathway is detected. [Figure 3B] This is a continuation of Figure 3A. [Figure 3C] This is a continuation of Figure 3B. [Figure 4A] The effect of the humanized antibodies of the present invention on blood clotting compared to the reference antibody BM is shown in Fig. 4A, 2 mM Ca2+, 50 μg / mL antibody concentration, and Fig. 4B, 5 mM Ca2+, 20 μg / mL antibody concentration. [Figure 4B] This is a continuation of Figure 4A. [Figure 5A] 1 shows the results of detecting the serum stability of the humanized antibody of the present invention after incubation in human serum at 37° C. for a certain period of time. [Figure 5B] This is a continuation of Figure 5A. [Figure 6A] 1 shows the change in antibody affinity measured by ELISA before and after coupling of anti-TF humanized antibody to a toxin-Linker (B81, ie, DL-01 molecule). [Figure 6B] This is a continuation of Figure 6A. [Figure 7A] 1 shows the results of detecting the in vitro killing activity of the ADC of the present invention. [Figure 7B] This is a continuation of Figure 7A. [Figure 8]The ADCs of the present invention, 22F11H5-hz1 and 30G11B7, show tumor suppression effects in mice in vivo after coupling with a drug-linker (B81). [Figure 9] 1 shows the effect on mouse body weight following administration of an ADC conjugate comprising antibodies of the invention 22F11H5-hz1 and 30G11B7. [Figure 10] The ADCs of the present invention, 2B12B10-hz1 and 27H8H3-hz1, show tumor suppression effects in mice in vivo after coupling with B81. [Figure 11] 1 shows the effect on mouse body weight following administration of ADC conjugates comprising antibodies of the invention 2B12B10-hz1 and 27H8H3-hz1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For purposes of the present invention, the following terms are defined below.

[0015] When trade names are used herein, unless the context dictates otherwise, the trade names include trade names, product formulations of products, generic drugs, and active drug ingredients.

[0016] The term "about," when used in conjunction with numerical values, is meant to encompass numerical values ​​within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value.

[0017] The term "and / or" used in connection with two or more options is understood to mean any one of the options or a combination of any two or more of the options.

[0018] The term "comprising" or "including" as used herein means including the recited elements, integers, or steps, but not excluding any other elements, integers, or steps. When the term "comprising" or "including" is used herein, unless otherwise indicated, it also encompasses situations consisting of the recited elements, integers, or steps. For example, when referring to an antibody variable region that "comprises" a particular specific sequence, it is also intended to encompass an antibody variable region consisting of that particular specific sequence.

[0019] As used herein, the terms "tissue factor" or "TF" are used interchangeably and, unless otherwise indicated, include any variant of human tissue factor, including sequence variants, particularly naturally occurring variants, allelic variants, and post-translationally modified variants and conformational variants, and also include species homologues thereof. It should also be understood that the terms not only include TF naturally or recombinantly expressed by cells or TF expressed on naturally or recombinant cells, but also recombinantly expressed fusion proteins that include the TF extracellular domain. An example of tissue factor is a human TF protein comprising the amino acid sequence under UniProtKB-P13726, or a recombinant protein comprising the extracellular domain of said protein. Another example of tissue factor is a monkey TF protein comprising the amino acid sequence under UniProtKB-A0A2K5VXA0, or a recombinant protein comprising the extracellular domain of said protein. As used herein, unless otherwise indicated, the terms "tissue factor" or "TF" refer to tissue factor of human origin.

[0020] As used herein, the term "TF positive" cells refers to cells that are positive for TF cell surface expression, such as cancer cells, modified cancer cells, or modified non-tumor cells. The cell surface TF expression level can be determined by any conventional method known in the art for determining the expression level of cell surface antigens, such as FACS detection or immunofluorescence staining. TF has a significantly higher expression level in many types of tumor cells than in normal tissues / cells, such as MDA-MB-231 (breast cancer, >about 350,000 TF molecules / cell), BxPC-3 (pancreatic cancer, >about 350,000 TF molecules / cell). Preferably, as used herein, TF positive cells are TF positive tumor cells.

[0021] As used herein, the term "antibody" refers to a polypeptide comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigen. The term encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, single-chain or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), chimeric or humanized antibodies, full-length antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0022] As used herein, a "whole antibody" (which may be used interchangeably herein with "full-length antibody", "complete antibody", and "intact antibody") refers to an immunoglobulin molecule that comprises at least two heavy chains (H) and two light chains (L). Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The variable region is the domain of an antibody's heavy or light chain that is involved in binding the antibody to its antigen. The constant region is not directly involved in binding the antibody to the antigen, but exhibits several effector functions. The light chain of an antibody can be classified into two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant region. The heavy chains of antibodies can be classified into five main different types, namely IgA, IgD, IgE, IgG and IgM, based on the amino acid sequence of their constant region, and some of these types can be further classified into subclasses, e.g. IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2.

[0023] As used herein, the terms "antibody fragment" and "antigen-binding fragment" of an antibody may be used interchangeably and refer to a molecule that is not an intact antibody, but contains a portion of an intact antibody for binding to an antigen to which the intact antibody binds. As will be understood by those skilled in the art, to achieve the purpose of antigen binding, an antibody fragment generally contains amino acid residues from the "complementarity determining regions" or "CDRs". Antibody fragments can be prepared by recombinant DNA techniques or by enzymatic or chemical cleavage of whole antibodies. Examples of antibody fragments include, but are not limited to, Fab, scFab, disulfide-linked scFab, Fab', F(ab')2, Fab'-SH, Fv, scFv, disulfide-linked scFv, linear antibodies, diabodies, triabodies, tetrabodies, and minibodies. In some embodiments according to the invention, the antibody fragment comprises a cysteine ​​residue moiety for forming an interchain disulfide bond between the light and heavy chains or between the heavy and heavy chains, e.g., cysteine ​​residues in the Fab and / or hinge regions of an IgG1 antibody, thereby providing an amino acid residue site amenable for sulfhydryl coupling chemistry. In further embodiments according to the invention, the antibody fragment comprises a cysteine ​​residue introduced into the Fc region to provide an amino acid residue site amenable for sulfhydryl coupling chemistry.

[0024] As used herein, the term "immunoglobulin" refers to a protein having the structure of a naturally occurring antibody. For example, an IgG class immunoglobulin is a heterotetrameric protein of about 150,000 daltons consisting of two light chains and two heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has one heavy chain variable region (VH), also called the heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3). From the N-terminus to the C-terminus, each immunoglobulin light chain has one light chain variable region (VL), also called the light chain variable domain, followed by one light chain constant domain (CL). Thus, as used herein, a reference to an antibody being an IgG antibody means that the antibody is a heterotetrameric protein having the immunoglobulin structure of the IgG class. In an IgG antibody, the VH-CH1 of the heavy chain is usually paired with the VL-CL of the light chain to form a Fab fragment that specifically binds to an antigen. Thus, an IgG antibody is essentially composed of two Fab molecules linked with the aid of an immunoglobulin hinge region and two dimerized Fc regions. IgG immunoglobulins can be classified into subclasses, such as gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), and gamma 4 (IgG4), based on the sequence of the heavy chain constant region. The light chain of an IgG immunoglobulin can also be classified into two types, called kappa and lambda, based on the amino acid sequence of its constant domain. In some embodiments, the antibody according to the invention is an IgG antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody. In some other embodiments, the antibody according to the invention is an IgGκ or IgGλ antibody, such as an IgG1κ or IgG1λ antibody.

[0025] As used herein, the terms "complementarity determining region" or "CDR region" or "CDR" or "hypervariable region" may be used interchangeably and refer to the regions of an antibody variable domain that are highly variable in sequence and form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to an antigen epitope. As used herein, the CDRs of the heavy and light chains of an antibody are numbered sequentially from the N-terminus and are generally referred to as CDR1, CDR2 and CDR3. The CDRs located in the antibody heavy chain variable domain are also referred to as HCDR1, HCDR2 and HCDR3, and the CDRs located in the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR3. For a given amino acid sequence of a light chain variable region or a heavy chain variable region, its CDR sequences can be determined using a variety of methods known in the art. For example, annotation of CDRs in a given light or heavy chain variable region, including CDR sequences based on Kabat, AbM, Chothia, Contact, IMGT definitions, can be found at http: / / www.abysis.org / abysis / . CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence. Unless otherwise specified, in the present invention, when referring to a residue position in an antibody variable region (including heavy and light chain variable region residues), it refers to the numbering position according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0026] As used herein, a "variable region" or "variable domain" is a domain of an antibody's heavy or light chain that is involved in binding the antibody to an antigen. The heavy chain variable region (VH) and the light chain variable region (VL) can be further divided into hypervariable regions (HVRs, also called complementarity determining regions (CDRs)) between which relatively conserved regions (i.e., framework regions (FRs)) are inserted. Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, one or more residues in one or both of the two variable regions of an antibody (i.e., VH and / or VL) may be modified, for example, by modifying one or more CDR regions and / or one or more framework regions, particularly by conservative residue substitution, to obtain an antibody variant that still essentially maintains at least one biological property (e.g., antigen-binding ability) of the parent antibody before modification. In some further embodiments, antibody variable regions can be modified by CDR grafting. Since CDR sequences are responsible for the majority of antibody-antigen interactions, recombinant antibody variants can be constructed that mimic the properties of known antibodies. In such antibody variants, CDR sequences from a known antibody are grafted onto the framework regions of a different antibody with different properties. The properties of the mutated and / or modified antibody or ADC conjugates containing it, such as target antigen binding properties or other desirable functional properties, such as endocytosis activity, coagulation effects, pharmacokinetics, and in vivo tumor killing activity, can be evaluated in in vitro or in vivo measurement tests.

[0027] As used herein, the term "chimeric antibody" refers to an antibody whose variable region sequences are derived from one species and whose constant region sequences are derived from another species, e.g., an antibody whose variable region sequences are derived from a murine antibody and whose constant region sequences are derived from a human antibody.

[0028] The term "humanized antibody" refers to an antibody in which CDR sequences derived from a non-human mammalian species, such as a mouse species, are grafted onto human framework sequences. Additional framework region modifications can be made in the human framework sequences and / or additional amino acid modifications can be made in the CDR sequences, for example, for affinity maturation of the antibody. As used herein, in some embodiments, the humanized antibody of the invention has a framework region sequence "derived" from a particular human germline sequence. Here, "derived" means that the amino acid sequence of the antibody framework region has at least 90%, more preferably at least 95%, even more preferably at least 96%, 97%, 98% or 99% identity with the corresponding framework region amino acid sequence encoded by the human germline immunoglobulin gene, and the antibody retains antigen-binding activity.

[0029] As used herein, the term "isolated" antibody is an antibody that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 90%, 95% or 99% purity, as can be determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).

[0030] As used herein, the term "epitope" refers to an antigenic region to which an antibody binds. An epitope may be formed from contiguous amino acids or from non-contiguous amino acids juxtaposed by tertiary folding of a protein. In the present invention, preferably, the antibody according to the present invention binds to a native epitope of human TF, more preferably to a native epitope of the extracellular domain of human TF expressed on the cell surface.

[0031] As used herein, the term "affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. Affinity can be measured by common methods known in the art. One specific method for measuring affinity is an antigen protein or cell-based ELISA assay described in the Examples herein, and another specific method is a flow cytometry assay described in the Examples herein. Biolayer Interferometry (BLI)-based technology can also be used to assess the dynamic affinity of antibodies.

[0032] The terms "binding" or "specific binding" in the context of binding of an antibody to a relevant antigen (herein, the TF antigen) are intended to mean a binding site for the K D Value is about 10 -6 M or less, e.g. K D Value is about 10 -7 M or less, or about 10 -8 It is used to refer to binding with an affinity of M or less. Binding to a nonspecific antigen (e.g., an unrelated antigen such as BSA), K D The binding K of an antibody to its associated antigen compared to the D The value is preferably at least 100-fold, or for example at least 1000-fold lower. D Measurement of values ​​is known in the art, such as techniques based on biolayer interference (BLI). For example, such measurements can be performed in a ForteBio Octet® instrument using an antibody as ligand and an antigen (e.g., a fusion protein containing the TF extracellular domain, such as TF-His) as analyte.

[0033] "K D The term "(M)" as used herein refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Affinity is K D The affinity is inversely correlated with the K value, i.e., the higher the affinity, the higher the K D The lower the affinity, the smaller the K D Generally, K DThe value is the dissociation rate constant (Kd or Kdis, sec -1 ) and binding rate constants (Ka, M -1 ×sec -1 ) depending on

[0034] As will be appreciated by those skilled in the art, an antibody that specifically binds to human TF can have cross-reactivity with TF proteins from other species. As used herein, the term "cross-reactivity" refers to the ability of an antibody to bind to TF from different species. For example, in some embodiments, an antibody according to the invention that is specific for human TF can also bind to TF from other species (e.g., cynomolgus monkey TF). Methods for measuring cross-reactivity include those described in the Examples and standard measurements known in the art, for example, by using flow cytometry or cell ELISA techniques. Inter-species cross-reactivity of an antibody is advantageous in some cases. For example, if a target antibody has inter-species cross-reactivity to preclinical experimental animals, such as primate animals, it facilitates the preclinical evaluation of the safety and efficacy of the target antibody before human therapeutic or diagnostic application.

[0035] As used herein, the term "isotype" refers to the type of antibody determined by the heavy chain constant region of the antibody. For example, the antibodies according to the invention may be IgA (e.g., IgA1 or IgA2), IgG1, IgG2 (e.g., IgG2a or IgG2b), IgG3, IgG4, IgE, IgM and IgD antibodies and have heavy chain constant regions of the above immunoglobulin types. The antibodies of the invention may be IgG1 antibodies having a human IgG1 constant region. The invention also contemplates the use of antibodies with native sequence constant regions as well as antibodies with variant sequence constant regions.

[0036] As used herein, the term "native sequence Fc region" encompasses various naturally occurring immunoglobulin Fc region sequences, such as the Fc region sequences of various Ig subtypes and their isoforms (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520.). In some embodiments, a human IgG heavy chain Fc region has an amino acid sequence extending from Cys226 or Pro230 to the carboxy terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. In further embodiments, a human IgG heavy chain Fc region has a native immunoglobulin hinge sequence or partial hinge sequence at the N-terminus, such as the sequence from E216 to T225 or the sequence from D221 to T225 according to EU numbering.

[0037] As used herein, the term "variant sequence Fc region" is intended to encompass Fc region polypeptides that have been modified relative to a native sequence Fc region polypeptide. The modifications may be additions, deletions, or substitutions of amino acid residues. Substitutions may include naturally occurring and non-naturally occurring amino acids. The purpose of the modifications is intended to alter the binding of the Fc region to its receptor and the effector functions elicited thereby.

[0038] The term "effector function" refers to the biological activity attributable to the Fc region of an antibody, which varies depending on the class of the antibody. It is known that the IgG Fc region can mediate several important effector functions, such as cytokine induction, ADCC, phagocytosis, complement-dependent cytotoxicity (CDC), and half-life / clearance rate of antibody and antigen-antibody complexes. In some cases, depending on the therapeutic purpose, these effector functions are ideal for a therapeutic antibody, but in other cases may not be necessary. Thus, in one embodiment, the invention provides antibodies with Fc regions that trigger effector functions, such as ADCC or CDC, in tumor cells bearing the TF antigen, inducing tumor cell apoptosis, cell lysis, and / or inhibiting the proliferation, spread and / or metastasis of tumor cells bearing the antigen TF. In a further embodiment, the invention provides antibodies with Fc regions with altered effector functions. Effector functions can be altered by sequence alterations in the Fc region of the antibody. Alternatively, antibodies can be prepared in which the Fc region has an altered type of glycosylation, for example, hypo- or non-fucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased aliquot GlcNac structures. Such altered glycosylation modes have already been shown to be capable of increasing the ADCC ability of antibodies. Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also contemplated, and such antibody variants can have enhanced CDC function. Altering the Fc region glycosylation mode can be conveniently achieved by altering the amino acid sequence of the Fc region to generate or remove one or more glycosylation sites.

[0039] As used herein, the term "receptor-mediated endocytosis" refers to a process triggered by the binding of a ligand to a corresponding receptor on the cell surface, in which the ligand / receptor complex is internalized and delivered to the cytosol or transferred to an appropriate intracellular compartment. In some embodiments, the antibody of the present invention triggers TF receptor-mediated endocytosis after binding to TF expressed on the cell surface. Herein, the endocytosis rate can be measured, for example, by the method described in the Examples, to characterize the receptor-mediated endocytosis activity of the antibody. In some embodiments, the antibody of the present invention having receptor-mediated endocytosis activity can be used in the ADC of the present invention as a means of transporting antitumor drugs into cancer cells.

[0040] As used herein, "sequence identity" refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis in a comparison window. "Percent sequence identity" can be calculated by the following method: comparing two optimally aligned sequences in a comparison window, determining the number of positions in the two sequences where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occurs to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the size of the window), and multiplying the result by 100 to generate the percent sequence identity. Optimal alignment for determining percent sequence identity can be achieved in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment within the full length sequences or within a region of a target sequence being compared.

[0041] In the present invention, with respect to antibody sequences, percent amino acid sequence identity is determined after optimal alignment of a candidate antibody sequence with a given antibody sequence, in a preferred embodiment, after optimal alignment according to the Kabat numbering convention. In the present specification, if no comparison window (i.e., the target antibody region to be compared) is specified, it is applied to align over the entire length of the given antibody sequence.

[0042] In this specification, unless otherwise specified, the reference antibody (BM) refers to an anti-TF antibody constructed using the amino acid sequences of the heavy and light chain variable regions from the Tisotumab antibody portion disclosed in patent CN103119065B (SEQ ID NOs: 107 and 108). In the context of comparison with a reference antibody, the reference antibody has the same antibody structure as the portion of the antibody to be compared outside the variable region, e.g., has the same heavy and light chain constant region sequences when both have the same heavy and light chain constant region structure.

[0043] In this specification, the term "halogen" generally refers to fluorine, chlorine, bromine, and iodine, and may be, for example, fluorine or chlorine.

[0044] The term "alkyl group" as used herein refers to a linear or branched saturated hydrocarbon group consisting of carbon atoms and hydrogen atoms. Specifically, an alkyl group has 1 to 10 carbon atoms, for example, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. For example, as used herein, the term "C1-C6 alkyl group" refers to a straight or branched chain saturated hydrocarbon group having 1 to 6 carbon atoms, examples of which include, for example, methyl, ethyl, propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, sec-butyl, or t-butyl), pentyl (including n-pentyl, isopentyl, and neopentyl), hexyl (including n-hexyl, 2-methylpentyl, 3-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, and 2-ethylbutyl), and the like.

[0045] The term "alkenyl group" as used herein refers to a straight or branched chain unsaturated hydrocarbon group consisting of carbon atoms and hydrogen atoms, containing at least one double bond. Specifically, the alkenyl group has 2 to 8, for example, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. For example, as used herein, the term "C2-C6 alkenyl group" refers to a straight or branched chain alkenyl group having 2 to 6 carbon atoms, such as a vinyl group, a propenyl group, an allyl group, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadiene, 1,4-pentadiene, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, etc.

[0046] The term "alkynyl group" as used herein refers to a straight or branched chain unsaturated hydrocarbon group consisting of carbon atoms and hydrogen atoms, containing at least one triple bond. Specifically, the alkynyl group has 2 to 8, for example, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. For example, the term "C2-C6 alkynyl group" as used herein refers to a straight or branched chain alkynyl group having 2 to 6 carbon atoms, such as an ethynyl group, a propynyl group, a propargyl group, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-methyl-1-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 5-methyl-2-hexynyl, etc.

[0047] The term "alkylidene group" as used herein refers to a divalent group obtained by removing two hydrogen atoms from two identical or different carbon atoms of a linear or branched saturated alkane. Specifically, the alkylidene group has 1 to 10 carbon atoms, for example, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. For example, the term "C1-C6 alkylidene group" as used herein refers to a linear or branched alkylidene group having 1 to 6 carbon atoms, including, but not limited to, a methylene group, an ethylene group, a propylene group, a butylene group, and the like.

[0048] The term "alkenylene group" as used herein refers to a divalent group obtained by removing two hydrogen atoms from two identical or different carbon atoms of a straight-chain or branched-chain unsaturated olefin group containing at least one double bond. Specifically, the alkenylene group has 2 to 8, for example, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. For example, the term "C2-C6 alkenylene group" as used herein refers to a straight-chain or branched-chain alkenylene group having 2 to 6 carbon atoms, such as a vinylidene group, a propenylene group, an allylene group, a butenylene group, a pentenyl group, and a hexenylene group.

[0049] The term "alkynylene group" as used herein refers to a divalent group obtained by removing two hydrogen atoms from the same or two different carbon atoms of a straight or branched unsaturated alkyne containing at least one triple bond. Specifically, the alkynylene group has 2 to 8, for example, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. For example, the term "C2-C6 alkynylene group" as used herein refers to a straight or branched alkynylene group having 2 to 6 carbon atoms, such as an ethynylene group, a propynylene group, a propargylene group, a butynylene group, a pentynylene group, and a hexynylene group.

[0050] The term "cycloalkyl group" as used herein refers to a monocyclic, fused polycyclic, bridged polycyclic, or spirocyclic non-aromatic monovalent hydrocarbon ring structure having a specified number of ring atoms that may be saturated or unsaturated and contains, for example, one or more double bonds. A cycloalkyl group may contain three or more, for example, 3 to 18, 3 to 10, or 3 to 8 carbon atoms in the ring, such as, for example, C 3-10 Cycloalkyl groups, C 3-8 Cycloalkyl groups, C 3-6 Cycloalkyl groups, C 5-6 Cycloalkyl groups. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl groups.

[0051] The term "heterocycle" or "heterocyclyl group" as used herein refers to a 5- to 20-membered (e.g., 5- to 14-membered, 5- to 8-membered, 5- to 6-membered) aromatic or non-aromatic monocyclic, bicyclic, or polycyclic ring system having 1 to 4 heteroatom ring members independently selected from N, O, or S. One or more N, C, or S atoms in the heterocycle may be oxidized. Preferably, the heterocycle is a 5- to 10-membered ring system that is monocyclic or fused bicyclic. Representative examples include, but are not limited to, pyrrolidine, azetidine, piperidine, morpholine, tetrahydrofuran, tetrahydropyran, benzofuran, benzothiophene, indole, benzopyrazole, pyrrole, thiophene (thiophene), furan, thiazole, imidazole, pyrazole, pyrimidine, pyridine, pyrazine, pyridazine, isothiazole, and isoxazole. The term should be understood to include heteroaryl groups as defined herein.

[0052] The term "aryl group" refers to a monocyclic or polycyclic aromatic hydrocarbon group having 6 to 20, for example 6 to 12, carbon atoms in the ring portion. Preferably, an aryl group is (C6-C 10 ) aryl groups. Non-limiting examples include phenyl, biphenyl, naphthyl, or tetrahydronaphthyl groups, each of which may be optionally substituted with 1 to 4 substituents, such as alkyl, trifluoromethyl, cycloalkyl, halogen, hydroxyl, alkoxy, acyl, alkyl-C(O)-O-, aryl-O-, heteroaryl-O-, amino, thiol, alkyl-S-, aryl-S-, nitro, cyano, carboxyl, alkyl-OC(O)-, carbamoyl, alkyl-S(O)-, sulfonyl, sulfonylsemi, heterocyclyl, and the like.

[0053] The term "heteroaryl group" refers to a 5- to 20-membered (e.g., 5- to 14-membered, 5- to 8-membered, 5- to 6-membered) aromatic monocyclic or polycyclic ring system, which may be substituted or unsubstituted, containing 1 to 4 heteroatoms selected from N, O or S. Preferably, the heteroaryl group is a 5- to 10-membered ring system that is monocyclic or fused bicyclic. Representative heteroaryl groups include 2- or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl, 2-, 4- or 5-imidazolyl, 3-, 4- or 5-pyrazolyl, 2-, 4- or 5-thiazolyl, 3-, 4- or 5-isothiazolyl, 2-, 4- or 5-oxazolyl, 3-, 4- or 5-isoxazolyl, 3- or 5-1,2,4-triazolyl, 4- or 5-1,2,3-triazolyl, tetrazolyl, 2-, 3- or 4-pyridyl, 3- or 4-pyridazinyl, 3-, 4- or 5-pyrazinyl, 2-pyrazinyl, and 2-, 4- or 5-pyrimidinyl.

[0054] The term "heteroalkyl group" refers to a stable straight or branched chain hydrocarbon group that is fully saturated or has one to three degrees of unsaturation and consists of the indicated number of carbon atoms and one to ten, preferably one to three, heteroatoms selected from O, N, Si and S, of which the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatoms O, N, Si and S may be located at any interior position of the heteroalkyl group or at the position at which the heteroalkyl group is attached to the remainder of the molecule. Representative examples of heteroalkyl groups include -CH-CH-O-CH, -CH-CH-NH-CH, -CH-CH-N(CH)-CH, -CH-S-CH-CH, -CH-CH-S(O)-CH, -NH-CH-CH-NH-C(O)-CH-CH, -CH-CH-S(O)-CH, -CH=CH-O-CH, -Si(CH), -CH-CH=NO-CH and -CH=CH-N(CH). Up to two heteroatoms may be consecutive, such as, for example, -CH-NH-OCH and -CH-O-Si(CH). Typically, a C1-C4 heteroalkyl or heteroalkylene group has 1 to 4 carbon atoms and 1 or 2 heteroatoms, and a C1-C3 heteroalkyl or heteroalkylene group has 1 to 3 carbon atoms and 1 or 2 heteroatoms. In some embodiments, the heteroalkyl and heteroalkylene groups are saturated.

[0055] The term "substituted" as used in the definition of each group herein means, unless otherwise specified, that the corresponding group is, for example, but not limited to, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, a heteroaryl group, a heterocyclyl group, a halogen atom, a cyano group, a nitro group, an azido group, a carboxy group, a hydroxy group, a mercapto group, an amino group, a mono- or dialkylamino group, a mono- or dicycloalkylamino group, a mono- or diarylamino group, a mono- or diheterocyclylamino group, a mono- or diheteroarylamino group, an alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl-oxy group, an alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl-sulfenyl group, an alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- alkyl- or aryl-acyl, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl-acylamino, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl-acyloxy, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl-sulfonyl, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl-sulfonyloxy, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl-sulfonylsemi, or the above optionally substituted carbamoyl groups, each of which may be further substituted by any remaining substituents, wherein each group is as defined herein.Examples of substituents include, but are not limited to, one or more groups independently selected from halogen, OH, SH, CN, NH, NHCH, N(CH), NO, N, C(O)CH, COOH, C(O)-amino, OCOCH, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, cyclopropyl, methoxy, ethoxy, pentoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methylsulfonylamino, SO, SO, phenyl, piperidinyl, piperazinyl, and pyrimidinyl.

[0056] As used herein, the term "substituted" or "substituted" refers to the replacement of one or more (e.g., 1, 2, 3, or 4) hydrogens on the specified atom with the specified group, without exceeding the normal valence bonds of the specified atom and resulting in a stable compound, provided that combinations of substituents and variables are permissible only if such combinations result in stable compounds.

[0057] As used herein, the term "linker" refers to a bifunctional moiety that connects a drug and an antibody in a drug-antibody conjugate. The linkers of the present invention may have multiple compositions (e.g., in some embodiments, a linker group for coupling with an antibody, a degradable peptide unit, and an optional spacer group).

[0058] As used herein, the term "PEG unit" refers to an organic moiety that includes overlapping vinyloxy subunits (PEG or PEG subunits), which may be polydisperse, monodisperse, or discrete (i.e., having a discrete number of vinyloxy subunits). Polydisperse PEGs are a heterogeneous mixture of sizes and molecular weights, while monodisperse PEGs are usually purified from a heterogeneous mixture and therefore have a single chain length and molecular weight. Preferred PEG units include discrete PEGs, which are compounds that are synthesized by a stepwise method rather than a polymerization process. Discrete PEGs provide a single molecule with a defined and specified chain length.

[0059] The term "pharmaceutically acceptable salt" refers to a salt that can maintain the biological effectiveness and performance of the ADC conjugate of the invention, and that is not biologically or otherwise undesirable. The ADC conjugates of the invention may exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable non-toxic acid addition salt refers to a salt formed by the ADC conjugate of the invention with an organic or inorganic acid, including, but not limited to, hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, etc. A pharma- ceutically acceptable, non-toxic base addition salt refers to a salt formed with an ADC conjugate of the invention and an organic or inorganic base, including, but not limited to, alkali metal salts such as lithium, sodium or potassium salts, alkaline earth metal salts such as calcium or magnesium salts, and organic base salts such as ammonium salts formed with organic bases containing an N group. The term "solvate" refers to an association formed by one or more solvent molecules with an ADC conjugate of the invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethylsulfoxide, etc.

[0060] Unless contradictory by context, "pharmaceutically acceptable" and "medicinal" may be used interchangeably herein.

[0061] The term "drug:antibody ratio" or "DAR" refers to the ratio of drug moieties (D) coupled to Ab moieties described herein to Ab moieties in an ADC conjugate. In some embodiments described herein, the DAR can be determined by q in formula I, e.g., the DAR can be 1-20, e.g., 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The DAR can also be calculated as the average DAR of the molecular population in the product, i.e., the overall ratio of small molecule drug moieties (D) coupled to Ab moieties described herein to Ab moieties in the product as measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA measurement, electrophoresis, and / or HPLC), and this DAR is referred to herein as the average DAR. In some embodiments, the average DAR value of the conjugates of the invention is between 1 and 20, e.g., between 2 and 18, between 4 and 16, between 5 and 12, between 6 and 10, between 2 and 8, between 3 and 8, between 2 and 6, between 4 and 6, between 6 and 10, e.g., between 1.0 and 8.0, between 2.0 and 6.0, e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4 .6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0, with any two of these numbers being the endpoints of the range.

[0062] The term "drug" as used herein encompasses any substance effective in the prevention or treatment of tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents (e.g., immunosuppressants).

[0063] The term "cytotoxic agent" is used herein to refer to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. Illustrative cytotoxic agents are camptothecin class drugs, auristatins, aureomycins, maytansinoid alkaloids, ricin, ricin A chain, combrestatins, duocarmycins, aplysiatoxin, doxorubicin, daunomycin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracenedione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin. These include, but are not limited to, A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, restrictocin, phenomycin, enomycin, curicin, clotin, calicheamicin, Sapaonaria officinalis inhibitor, and glucocorticoids, as well as other chemotherapeutic agents, and radioisotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32, and radioisotopes of Lu, including Lu177.

[0064] The term "small molecule drug" refers to a low molecular weight organic compound that can regulate biological processes. A "small molecule" is defined as a molecule with a molecular weight of less than 10 kD, usually less than 2 kD, and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptidomimetics, and antibody mimetics. As therapeutic agents, small molecules are more cell permeable, less susceptible to degradation, and less likely to cause an immune response than large molecules.

[0065] The term "pharmaceutical composition" refers to a composition that is present in a form that permits the biological activity of the active ingredients contained therein to be effective, and that does not contain additional ingredients that have unacceptable toxicity to the subject to which the composition is administered.

[0066] The term "auxiliary material" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, vector, stabilizer, or the like, administered with an active substance.

[0067] The term "pharmaceutical combination" refers to a non-fixed combination product or a fixed combination product, including, but not limited to, a kit, a pharmaceutical composition. The term "non-fixed combination" means that two or more active ingredients (e.g., (i) the ADC molecule of the present invention or the antibody of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously in separate entities, without specific time restrictions, or sequentially at the same or different time intervals, whereby such administration provides a prophylactically or therapeutically effective level of said two or more active ingredients in the patient's body. The term "fixed combination" means that two or more active ingredients are administered to a patient simultaneously in the form of a single entity. In some cases regarding pharmaceutical combinations, preferably, the doses and / or time intervals of two or more active ingredients are selected so that the combined use of each ingredient produces a greater effect in treating a disease or condition than the use of any of the ingredients alone. Each ingredient included in the pharmaceutical combination may be in a single formulation, and the formulation may be the same or different. In some embodiments, the ADC molecules of the invention or antibodies of the invention and other therapeutic agents used in a pharmaceutical combination are administered at levels that do not exceed the levels at which they are used alone.

[0068] The term "combination therapy" or "therapeutic combination" refers to the administration of two or more therapeutic agents or therapeutic methods (e.g., radiation therapy or surgery) to treat a disease described herein. Such administration includes co-administration of these therapeutic agents in an essentially simultaneous manner, for example in a single capsule with a fixed ratio of the two or more active ingredients. Alternatively, such administration includes co-administration of two or more active ingredients in multiple formulations (e.g., tablets, capsules, powders and liquids) or in separate containers. The powders and / or liquids can be reconstituted or diluted to the desired dose before administration. Such administration also includes sequential use of each type of therapeutic agent at about the same time or at different times. In either case, the treatment regimen provides the beneficial effect of the therapeutic combination in treating a disease or condition described herein.

[0069] As used herein, the terms "individual," "subject," or "patient" may be used interchangeably and refer to a mammal. Mammals include, but are not limited to, livestock animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the subject is a human.

[0070] The terms "tumor" and "cancer" are used interchangeably herein to refer to a physiological disorder in mammals that is typically characterized by unregulated cell proliferation. The term encompasses primary and metastatic forms of tumors. The term also encompasses all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0071] The term "anti-tumor effect" refers to an anti-tumor biological effect that can be demonstrated by a variety of means, including, but not limited to, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell viability.

[0072] As used herein, "treatment" refers to slowing, interrupting, blocking, alleviating, halting, reducing, or reversing the progression or severity of an existing symptom, condition, pathology, or disease.

[0073] As used herein, "prevention" includes inhibition of the onset or progression of a disease or condition, or a symptom of a particular disease or condition. Typically, in the context of cancer, the term "prevention" refers to administration of a drug prior to the onset of a cancer condition or symptom, particularly in subjects at risk of cancer. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen.

[0074] As used herein, the term "effective amount" refers to an amount of drug (e.g., an antibody-drug conjugate of the invention or a pharma- ceutically acceptable salt or solvate thereof, or an antibody or antigen-binding fragment thereof of the invention, or a composition or pharmaceutical combination thereof) that produces a desired effect in a patient in need of treatment or prophylaxis after administration to the patient in one or more doses.

[0075] As used herein, the term "therapeutically effective amount" refers to an amount at a desired dosage and for a desired duration to effectively achieve a desired therapeutic result. A therapeutically effective amount is also an amount that, when administered to a subject, does not cause any toxic or adverse effects caused by said administration without causing any therapeutically beneficial effects. A "therapeutically effective amount" preferably achieves at least about 30%, more preferably at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100% improvement in a measurable parameter (e.g., tumor volume) relative to an untreated individual.

[0076] As used herein, the term "prophylactically effective amount" refers to an amount, at a desired dosage and for a desired period of time, to effectively achieve a desired prophylactic result. Typically, a prophylactic dose is used in subjects prior to or at a relatively early stage of disease, so that the prophylactically effective amount is less than the therapeutically effective amount.

[0077] Each aspect of the invention is described in further detail in the following sections.

[0078] Antibody-drug conjugates of the present invention I. Antibody-Drug Conjugates In one aspect, the present invention provides an antibody-drug conjugate (ADC) having formula (I) or a pharma- ceutically acceptable salt or solvate thereof: Ab-[LD] q (I) Among them, Ab stands for anti-TF antibody; L represents a linker, D represents a cytotoxic or cell inhibiting drug, such as a topoisomerase I inhibitor, and q represents an integer or a decimal number of 1 to 20, for example, q=1 to 10, 1 to 8, 3 to 8, 4 to 8, or 6 to 8.

[0079] The compositions of the ADC conjugates of the present invention and the ADC conjugates of the present invention comprising the same are described in detail below. Those skilled in the art can understand that any combination of any technical features of these compositions is within the contemplation of the present invention, unless the context clearly indicates otherwise. Furthermore, those skilled in the art can understand that the ADC conjugates of the present invention can include any such combination of features, unless the context clearly indicates otherwise.

[0080] Antibody Ab Unit In some aspects, the present invention provides an antibody-drug conjugate (ADC) comprising an antibody of the present invention that specifically binds to TF as an Ab unit of the conjugate of formula I of the present invention. The anti-TF antibody of the present invention used as an Ab unit may be a full-length antibody or an antibody fragment. In some embodiments, the Ab in formula (I) of the present invention is a full-length antibody comprising a heavy chain constant region and a light chain constant region. In some embodiments, the Ab in formula (I) of the present invention has a tetrameric structure formed from two light chains and two heavy chains. In some further embodiments, the Ab in formula (I) of the present invention is an IgG antibody, in particular an IgG1 antibody. In further embodiments, the Ab in formula (I) of the present invention is a humanized antibody.

[0081] Antibody properties The anti-TF antibodies of the invention used in the ADCs of the invention have one or more of the following properties: (i) binds to the human TF extracellular domain with high affinity; (ii) binds to TF-positive tumor cells with high affinity; (iii) cross-reactivity with cynomolgus monkey TF; (iv) having TF receptor-mediated endocytosis activity; (v) blocking TF / FVIIa complex-mediated downstream signaling pathways in TF-positive cells; and (vi) Coagulation initiated by cell surface TF has essentially no or reduced coagulation effect.

[0082] Preferably, said anti-TF antibodies and antibody-drug conjugates are capable of binding to TF-positive tumor cells with high affinity and have essentially no or reduced clotting effect on coagulation initiated by cell surface TF.

[0083] With regard to the properties of the anti-TF antibodies of the invention used in the ADCs of the invention, please also refer to the detailed explanation provided below in the section "Anti-TF antibodies of the invention".

[0084] Antibody CDR region CDRs are the regions of an antibody primarily responsible for binding to an antigen epitope. In some embodiments, the anti-TF antibodies used in the ADCs of the invention (i) the HCDR1, 2 and 3 sequences of the heavy chain variable region shown in SEQ ID NO: 1, and the LCDR1, 2 and 3 sequences of the light chain variable region shown in SEQ ID NO: 2; or (ii) the HCDR1, 2 and 3 sequences of the heavy chain variable region as set forth in SEQ ID NO: 3, and the LCDR1, 2 and 3 sequences of the light chain variable region as set forth in SEQ ID NO: 4; or (iii) the HCDR1, 2 and 3 sequences of the heavy chain variable region as set forth in SEQ ID NO:5, and the LCDR1, 2 and 3 sequences of the light chain variable region as set forth in SEQ ID NO:6; or (iv) HCDR1, 2, and 3 sequences of the heavy chain variable region shown in SEQ ID NO: 7, and LCDR1, 2, and 3 sequences of the light chain variable region shown in SEQ ID NO: 8 The CDR sequence includes:

[0085] Preferably, the CDRs are defined according to Chothia, AbM, Kabat, IMGT, or any combination thereof. More preferably, the CDRs are defined according to Kabat or IMGT, or any combination thereof, more preferably, the CDRs are defined according to IMGT. However, it should be understood that the CDRs may be defined in any other manner known in the art.

[0086] It is also known in the art that, although CDRs differ between antibodies, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. At least two of the Kabat, Chothia, AbM, and Contact methods can be used to determine the minimal overlap region, thereby providing a "minimal binding unit" for antigen binding. Such a minimal binding unit may be a sub-portion of the CDR. Meanwhile, the remaining residues of the CDR sequence can be determined by the structure and protein folding of the antibody, as will be apparent to those skilled in the art. Thus, the present invention also contemplates any variant of the CDRs provided herein. For example, in one variant of the CDR, the amino acid residues of the minimal binding unit may remain unchanged, and the remaining CDR residues may be substituted.

[0087] In some embodiments, the anti-TF antibodies used in the ADCs of the invention preferably comprise three complementarity determining regions HCDRs of a heavy chain variable region and three complementarity determining regions LCDRs of a light chain variable region, among which: (i) as defined according to Chothia, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:9, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:10, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:11, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:21, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:22, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:23; or (ii) as defined according to AbM, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 12, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 13, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 14, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 24, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 25, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 26, or (iii) as defined according to Kabat, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 15, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 16, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 17, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 27, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 28, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 29, or (iv) As defined according to IMGT, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 18, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 19, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 20, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 30, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 31, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 32.

[0088] In some embodiments, the anti-TF antibodies used in the ADCs of the invention preferably comprise three complementarity determining regions HCDRs of a heavy chain variable region and three complementarity determining regions LCDRs of a light chain variable region, among which: (i) as defined according to Chothia, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 33, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 34, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 35, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 45, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 46, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 47; or (ii) as defined according to AbM, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 36, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 37, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 38, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 48, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 49, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 50; or (iii) as defined according to Kabat, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 39, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 40, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 41, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 51, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 52, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 53; or (iv) As defined according to IMGT, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 42, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 43, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 44, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 54, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 55, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 56.

[0089] In some embodiments, the anti-TF antibodies used in the ADCs of the invention preferably comprise three complementarity determining regions HCDRs of a heavy chain variable region and three complementarity determining regions LCDRs of a light chain variable region, among which: (i) as defined according to Chothia, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 57, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 58, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 59, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 69, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 70, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 71; or (ii) as defined according to AbM, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 60, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 61, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 62, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 72, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 73, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 74; or (iii) as defined according to Kabat, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 63, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 64, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 65, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 75, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 76, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 77; or (iv) As defined according to IMGT, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 66, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 67, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 68, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 78, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 79, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 80.

[0090] In some embodiments, the anti-TF antibodies used in the ADCs of the invention preferably comprise three complementarity determining regions HCDRs of a heavy chain variable region and three complementarity determining regions LCDRs of a light chain variable region, among which: (i) as defined according to Chothia, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 81, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 82, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 83, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 93, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 94, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 95; or (ii) as defined according to AbM, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 84, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 85, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 86, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 96, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 97, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 98; or (iii) as defined according to Kabat, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 87, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 88, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 89, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 99, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 100, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 101; or (iv) As defined according to IMGT, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 90, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 91, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 92, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 102, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 103, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 104.

[0091] Antibody variable region A "variable region" or "variable domain" is a domain of an antibody's heavy or light chain that is involved in binding the antibody to an antigen. In some embodiments, the anti-TF antibody of the invention used in the ADC of the invention comprises any one of the heavy and light chain variable region sequences of the exemplary antibodies 2B12B10-hz1, 30G11B7-hz1, 22F11H5-hz1 and 27H8H3-hz1 of the invention or variants thereof, such as an antibody or fragment thereof, e.g., a humanized antibody, having the same CDR sequence as one of the above exemplary antibodies and having a homologous or different framework region sequence. For replacement of the framework region, a sequence similarity search tool such as Gapped BLAST is used to compare the parent antibody protein sequence with the protein sequences in the database and select a database framework sequence that has a high degree of structural similarity with the framework sequence of the parent antibody to be changed, e.g., has at least 80%, 85%, 90%, or 95%, 96%, 97%, 98%, 99% or more sequence identity for replacement of the framework region. In some cases, after substitution of the framework region, one or more residue mutations, such as back mutations, can be performed on the substituted framework sequence as necessary, and in vitro or in vivo measurement tests can be used to evaluate whether the antigen-binding properties or other functional properties of the antibody after the mutation are retained or improved.

[0092] In one embodiment, the anti-TF antibody of the invention used in the ADC of the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is (i) the amino acid sequence set forth in SEQ ID NO:1, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (ii) an amino acid sequence set forth in SEQ ID NO:3, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (iii) an amino acid sequence as set forth in SEQ ID NO:5, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; (iv) comprises an amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0093] In another embodiment, an anti-TF antibody for use in an ADC of the invention comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region is (i) the amino acid sequence set forth in SEQ ID NO:2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (ii) an amino acid sequence set forth in SEQ ID NO:4, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (iii) an amino acid sequence as set forth in SEQ ID NO:6, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; (iv) comprises an amino acid sequence set forth in SEQ ID NO:8, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0094] In some preferred embodiments, the anti-TF antibody comprises a heavy chain variable region and a light chain variable region, of which the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:1, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, and of which the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:2, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto. Preferably, the antibody or antigen-binding fragment comprises or consists of the heavy chain variable region of SEQ ID NO:1 and the light chain variable region of SEQ ID NO:2.

[0095] In some preferred embodiments, the anti-TF antibody comprises a heavy chain variable region and a light chain variable region, of which the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:3, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, and of which the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto. Preferably, the antibody or antigen-binding fragment comprises or consists of the heavy chain variable region of SEQ ID NO:3 and the light chain variable region of SEQ ID NO:4.

[0096] In some preferred embodiments, the anti-TF antibody comprises a heavy chain variable region and a light chain variable region, of which the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:5, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, and of which the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:6, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto. Preferably, the antibody or antigen-binding fragment comprises or consists of the heavy chain variable region of SEQ ID NO:5 and the light chain variable region of SEQ ID NO:6.

[0097] In some preferred embodiments, the anti-TF antibody comprises a heavy chain variable region and a light chain variable region, of which the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:7, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, and of which the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto. Preferably, the antibody or antigen-binding fragment comprises or consists of the heavy chain variable region of SEQ ID NO:7 and the light chain variable region of SEQ ID NO:8.

[0098] Antibody Heavy and Light Chains The anti-TF antibody of the invention used in the ADC of the invention as an Ab unit may in some cases comprise a heavy chain constant region and / or a light chain constant region. Preferably, the heavy chain constant region is a heavy chain constant region derived from a human immunoglobulin. Preferably, the light chain constant region is a light chain constant region derived from a human immunoglobulin. In some embodiments, the heavy chain constant region comprised in the anti-TF antibody may be a heavy chain constant region of any isotype or subtype, for example, an IgG1, IgG2, IgG3 or IgG4 isotype. In some embodiments, the anti-TF antibody preferably comprises an IgG1 heavy chain constant region, in particular a human IgG1 heavy chain constant region, for example an amino acid sequence of SEQ ID NO: 106 or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some further embodiments, the light chain constant region comprised in the anti-TF antibody may be a κ light chain constant region or a λ light chain constant region. In some embodiments, the anti-TF antibody comprises a κ or λ light chain constant region of human origin, preferably a human κ light chain constant region, such as the amino acid sequence of SEQ ID NO: 105, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0099] The present invention also contemplates variants of any of the above antibody embodiments. The antibody variants used as Ab units according to the present invention preferably retain at least 60%, 70%, 80%, 90% or 100% of the biological activity (e.g., antigen binding ability) of the parent antibody introduced prior to the modification. More preferably, the modification does not result in loss of binding of the antibody variant to antigen, but optionally confers properties such as improved antigen affinity. It will be understood that the heavy or light chain variable regions, or each CDR or framework region of the antibody, may be modified alone or in combination. The Fc region of the antibody may also be modified. Modifications of the Fc region may be made alone or in combination with modifications to the framework and / or CDR regions. For example, the Fc region may be modified to modify one or more effector functions of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. The antibodies of the present invention may also be chemically modified (e.g., conjugated to PEG) or their glycosylation pattern may be modified.

[0100] To form an ADC conjugate, the antibody according to the present invention may be coupled to a toxin-linker using a part of its natural binding site. Such natural binding site has a sulfhydryl group of cysteine ​​and an amino group of lysine. Usually, a relatively well-defined drug-antibody ratio (DAR) can be achieved by using the interchain disulfide bridge of the antibody. Therefore, in one embodiment, after reducing the interchain disulfide bond of the antibody, a toxin is coupled to the antibody of the present invention by sulfhydryl chemistry to form the ADC conjugate of formula (I). It is also possible to consider introducing an artificial binding site into the antibody to achieve a more targeted conjugate.

[0101] Drug D Unit The drug D unit of the antibody-drug conjugate is also referred to herein as the payload of the ADC drug. The drug D that can be used in the ADC of the present invention is not particularly limited and may be any drug or its prodrug that has toxicity or inhibitory properties to cells. Those skilled in the art can select an appropriate drug molecule as the payload of the ADC based on the desired drug mechanism of action and cell killing effect of the ADC.

[0102] In some embodiments, drug D is a cytotoxic drug. Several types of cytotoxic drugs with different mechanisms have been reported in the art that are suitable for use as payloads: (1) Microtubule inhibitors / disruptors: for example, but not limited to, auristatins (e.g., MMAE or MMAF), maytansine derivatives (e.g., DM2, DM4), tubulysins, cryptomycins, anti-mitotic EG5 inhibitors (e.g., spindle kinesin KSP inhibitors), (2) DNA damaging agents: for example, but not limited to, pyrrolobenzodiazepines (e.g., pyrrolo[2,1-c][1,4]benzodiazepine (PBD)), duocarmycins, indolinobenzodiazepine, duocarmycins, calicheamicins, (3) Topoisomerase inhibitors: for example, but not limited to, camptothecins (e.g., exatecan and its derivative Dxd); (4) Others: including, but not limited to, apoptosis inducers (Bcl-xL inhibitors), thailanstatin and its analogs, amatoxins, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, and kanamycin.

[0103] Drug D in formula (I) of the present invention may be any compound selected from the above. In some embodiments, drug D is an antitumor growth inhibitor selected from maytansine derivatives, calicheamicin derivatives, and auristatin derivatives. In one embodiment, drug D is a tubulin inhibitor / stability disrupting agent such as vinca alkaloids, vincristine, paclitaxel, and docetaxel. In one embodiment, drug D is an inhibitor of DNA synthesis such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, and cladribine. In one embodiment, drug D is a DNA topoisomerase inhibitor such as topoisomerase I inhibitors (e.g., camptothecins) and topoisomerase II inhibitors (e.g., actinomycin D, doxorubicin, and mitoxantrone).

[0104] In some preferred embodiments, drug D of the ADC according to the present invention is a topoisomerase I inhibitor. Typical examples of topoisomerase I inhibitors include, but are not limited to, camptothecin class drugs, such as camptothecin (CPT), hydroxycamptothecin, 9-aminocamptothecin, exatecan, topotecan, belotecan, irinotecan, SN-38, and FL118, and derivatives thereof. For example, Vesela Kostova et al., The Chemistry Behind ADCs, Pharmaceuticals 2021, 14, 442. https: / / doi.org / 10.3390 / ph14050442, and WO2019 / 195665, which are incorporated herein by reference.

[0105] In some embodiments of the invention, drug D of an ADC of the invention is a camptothecin class drug selected from: Camptothecin and its derivatives, for example [ka] Among them, R Ais selected from, for example, hydrogen, an optionally substituted alkyl group, the substituents of which include, but are not limited to, a hydroxyl group, an amino group, the amino group moiety or the hydroxyl group moiety may be substituted or unsubstituted, for example, substituted with an alkyl group, an alkyl acyl group, or an alkyl sulfonyl group; in one embodiment, the drug D is [ka] and -10,11-methylenedioxy CPT (also abbreviated as MDCPT or FL118) and its derivatives, for example: [ka] Of these, R B is selected from, for example, hydrogen, a cycloalkyl group, a phenyl group, and an optionally substituted alkyl group, the substituents of which include, but are not limited to, halogen, a hydroxy group, an optionally substituted alkoxy group, a cycloalkyl group, a heterocycloalkyl group, a phenyl group, and an amino group, the amino group portion of which may be optionally substituted; in one embodiment, the drug D is [ka] and -10-hydroxy CPT (also abbreviated as HCPT) and its derivatives, for example: [ka] and Among them, R C is, for example, selected from optionally substituted alkyl and cycloalkyl groups; R' C is selected from, for example, H, an alkanoyl group, or an optionally substituted heterocycloalkyl acyl group, and in one embodiment, the drug D is 7-ethyl-10-hydroxy CPT (SN-38) or its prodrug irinotecan (CPT-11), or the drug D is topotecan [ka] Exatecan and its derivatives, for example [ka] Of these, R D is, for example, selected from H, an optionally substituted alkyl group, an optionally substituted alkyl-C(=O)-, the substituent being, for example, -OH or a substituted or unsubstituted amino group, and in one embodiment, the drug D is [ka] In another embodiment, the drug D is [ka] It is.

[0106] F118 ((20S)-10,11-methylenedioxycamptothecin) was obtained through high-throughput screening of a compound library. FL118 has been demonstrated to have much higher in vivo and in vitro anticancer activity in many different cancer types compared to other camptothecin derivatives. In addition to inhibiting topoisomerase I, FL118 can selectively inhibit gene promoter activity and endogenous expression of anti-apoptotic proteins, such as survivin, XIAP, cIAP2 and Mcl-1. See Xiang Ling et al., A Novel Small Molecule FL118 That Selectively Inhibits Survivin, Mcl-1, XIAP and cIAP2 in a p53-Independent Manner, Shows Superior Antitumor Activity, PLoS ONE 7(9):e45571.doi:10.1371 / journal.pone.0045571. The use of FL118 alone is significantly hindered by its extremely poor water solubility and toxic side effects, but as shown in the Examples, the conjugate formed by combining this compound with the antibody and linker of the present invention not only effectively exerts the tumor killing effect of the compound, but also overcomes the shortcomings of the compound, and the formed conjugate has a low degree of aggregation and good animal tolerance.

[0107] Thus, in some preferred embodiments of the invention, drug D of the ADC of the invention is a FL118 derivative, particularly a FL118 substituted at the 7-position.

[0108] In some embodiments, the drug D in formula (I) of the present invention is a camptothecin class drug having the structure of formula D: [ka] Among them, R x , R y are each independently selected from H, halogen, —OH, a C1-C6 alkyl group, or Rx and R y form, in combination with the carbon atom to which each is bonded, one or two 5- or 6-membered heterocycles selected from N, S, and O; R a is selected from H, halogen, -OH, an optionally substituted C1-C8 alkyl group, a C3-C8 alkynyl group, or a C3-C8 alkenyl group, an optionally substituted C1-C8 alkoxy group, an optionally substituted C3-C8 cycloalkyl group, an optionally substituted phenyl group, an optionally substituted heterocycloalkyl group, and an optionally substituted heteroaryl group; Of these, R a teeth, hydrogen, C3-C8 cycloalkyl group, Phenyl group, halogens, hydroxy groups, C1-C4 alkoxy groups optionally substituted with NH2, NH(C1-C4 alkyl) and N(C1-C4 alkyl)2, C3-C8 cycloalkyl groups, heterocycloalkyl groups, phenyl groups, and NR 1 R 2 A C1-C8 alkyl group, a C3-C8 alkynyl group, or a C3-C8 alkenyl group, optionally substituted with a substituent selected from the group consisting of: Among them, R 1 and R 2 are, independently of each other, hydrogen, a C1-C8 alkyl group optionally substituted with a substituent selected from the group consisting of a hydroxy group, an amino group, an amino group substituted with one or two C1-C4 alkyl groups, an amino group substituted with one or two C1-C4 hydroxyalkyl groups, an amino group substituted with (C1-C4 hydroxyalkyl) and a (C1-C4 alkyl) group; 1 or 2 C3-C 10 Cycloalkyl groups, C3-C 10 a C1-C4 alkyl group substituted with a heterocycloalkyl group, a phenyl group or a heteroaryl group; C3-C 10 Cycloalkyl groups, C3-C10 heterocycloalkyl groups, C2-C6 heteroalkyl group, Heteroaryl groups, a phenyl group optionally substituted with halogen; C1-C8 alkyl-C(=O)- optionally substituted with a hydroxy or amino group; Selected from Or, R 1 and R 2 form a 5-, 6- or 7-membered heterocycle in combination with the nitrogen atom to which they are bonded having 0 to 3 substituents, the substituents being selected from halogen, C1-C4 alkyl group, OH, C1-C4 alkoxy group, NH2, NH(C1-C4 alkyl) and N(C1-C4 alkyl)2; Among them, each occurrence of a cycloalkyl group, a heterocycloalkyl group, a phenyl group, and a heteroaryl group is independently optionally substituted with 0 to 3 substituents selected from the group consisting of OH, a C1-C4 alkyl group, a C1-C4 alkoxy group, NH2, NH(C1-C4 alkyl), and N(C1-C4 alkyl)2.

[0109] In some preferred embodiments, R x and R y are each independently H. In some further preferred embodiments, R x is F and R y is a methyl group. In some further preferred embodiments, R x and R y together with the carbon atom to which they are attached form a 5-membered heterocycle containing 2 O.

[0110] In some embodiments, the drug D in the formula (I) of the present invention is represented by the following formula D a Or formula D b is a camptothecin class drug having the structure [ka] In particular, the formula (D a ) camptothecin class drug, Among them, R a teeth, hydrogen, C3-C8 cycloalkyl group, Phenyl group, halogens, hydroxy groups, C1-C4 alkoxy groups optionally substituted with NH2, NH(C1-C4 alkyl) and N(C1-C4 alkyl)2, C3-C8 cycloalkyl groups, heterocycloalkyl groups, phenyl groups, and NR 1 R 2 A C1-C8 alkyl group, a C3-C8 alkynyl group, or a C3-C8 alkenyl group optionally substituted with a substituent selected from the group consisting of: Among them, R 1 and R 2 are, independently of each other, hydrogen, a C1-C8 alkyl group optionally substituted with a substituent selected from the group consisting of a hydroxy group, an amino group, an amino group substituted with one or two C1-C4 alkyl groups, an amino group substituted with one or two C1-C4 hydroxyalkyl groups, an amino group substituted with (C1-C4 hydroxyalkyl) and a (C1-C4 alkyl) group; 1 or 2 C3-C 10 Cycloalkyl groups, C3-C 10 a C1-C4 alkyl group substituted with a heterocycloalkyl group, a phenyl group or a heteroaryl group; C3-C 10 Cycloalkyl groups, C3-C 10 heterocycloalkyl groups, C2-C6 heteroalkyl group, Heteroaryl groups, a phenyl group optionally substituted with halogen; C1-C8 alkyl-C(=O)- optionally substituted with a hydroxy or amino group; Selected from Or, R 1 and R 2form a 5-, 6- or 7-membered heterocycle in combination with the nitrogen atom to which they are bonded having 0 to 3 substituents, the substituents being selected from halogen, C1-C4 alkyl group, OH, C1-C4 alkoxy group, NH2, NH(C1-C4 alkyl) and N(C1-C4 alkyl)2; Among them, each occurrence of a cycloalkyl group, a heterocycloalkyl group, a phenyl group, and a heteroaryl group is independently optionally substituted with 0 to 3 substituents selected from the group consisting of OH, a C1-C4 alkyl group, a C1-C4 alkoxy group, NH2, NH(C1-C4 alkyl), and N(C1-C4 alkyl)2.

[0111] In some embodiments, R a is hydrogen.

[0112] In some embodiments, R a is a C3-C8 cycloalkyl group (e.g., cyclopropyl, cycloheptyl, cyclopentyl).

[0113] In some embodiments, R a is a phenyl group. In some further embodiments, R a is a phenyl group substituted with NH2 or NH(C1-C4 alkyl).

[0114] In some embodiments, R a is a C1-C6 alkyl group, such as methyl, ethyl, propyl, 2-methylpropyl, butyl, isobutyl, 2,2-dimethyl-propyl, 2,2-dimethyl-butyl, n-hexyl, n-pentyl, 3-ethyl-pentyl.

[0115] In some embodiments, R a is a C1-C4 alkyl group, which is optionally substituted with a hydroxy group or a C1-C4 alkoxy group, of which the alkoxy group is further optionally substituted with -NH2, -NH(C1-C4 alkyl) and -N(C1-C4 alkyl)2.

[0116] In some embodiments, R a is a C2-C4 alkenyl group or a C2-C4 alkynyl group, which is optionally substituted with a hydroxy or amino group.

[0117] In some embodiments, R a is a C1-C4 alkyl group substituted with one or two 5- to 6-membered heterocycloalkyl groups selected from N, S, and O, in which the heterocycloalkyl group moiety is further optionally substituted with a C1-C4 alkyl group, and preferably the heterocycloalkyl group is a piperazinyl group or a morpholinyl group.

[0118] In some embodiments, R a Ha-NR 1 R 2 C1-C4 alkyl groups substituted with -methylene-NR 1 R 2 Of these, R 1 and R 2 are, independently of each other, hydrogen, -C1-C4 alkyl group, -OH, -NH2, -NH(C1-C4 alkyl), -N(C1-C4 alkyl)2, -phenyl group, -phenylene-NH2, -phenylene-C1-C4 alkoxy group, -diphenyl group, -C3-C 10 Cycloalkyl groups, -C3-C 10 Cycloalkylene-C1-C4 alkyl group, -C3-C 10 a -C1-C4 alkyl group substituted by a substituent selected from the group consisting of heterocycloalkyl groups; wherein the heterocycloalkyl group has 1 or 2 heteroatoms selected from N, S and O, and preferably the heterocycloalkyl group is a piperidinyl group; Phenyl group, halogen-substituted phenyl group, C3-C 10 Cycloalkyl groups, It is selected from -C(=O)-C1-C4 alkyl-OH and -C(=O)-C1-C4 alkyl-NH2.

[0119] In some embodiments, R a Ha-NR 1 R 2 C1-C4 alkyl groups substituted with, in particular, -methylene-NR 1 R 2 Of these, R 1 and R 2 and each are combined with the nitrogen atom to which they are attached to form a 5-, 6-, or 7-membered heterocycloalkyl group ring. 1 and R 2 combine with the nitrogen atom to which they are attached to form a 6-membered ring. In some embodiments, the 6-membered ring is a morpholinyl group or a piperazinyl group.

[0120] In some preferred embodiments, R a are -C1-C4 alkylidene-OH, -C1-C4 alkylidene-O-C1-C4 alkylidene-NH2, -C1-C4 alkylidene-NH2, -C1-C4 alkylidene-NH(C1-C4 hydroxyalkyl), -C1-C4 alkylidene-N(C1-C4 hydroxyalkyl)2, -C1-C4 alkylidene-N(C1-C4 aminoalkyl)(C1-C4 hydroxyalkyl), -C1-C4 alkylidene-NH( and -C1-C4 alkylidene-NH-C(=O)-C1-C4 alkylidene-OH, -C1-C4 alkylidene-NH-C(=O)-C1-C4 alkylidene-NH2, -C1-C4 alkylidene-N(C1-C4 alkyl)(C(=O)-C1-C4 alkylidene-OH), -C1-C4 alkylidene-N(C1-C4 alkyl)(C(=O)-C1-C4 alkylidene-NH2).

[0121] In some further preferred embodiments, R ais -C1-C4 alkylidene-NH-C(=O)-C1-C4 alkylidene-OH, -C1-C4 alkylidene-NH-C(=O)-C1-C4 alkylidene-NH2.

[0122] In some further preferred embodiments, R a is -C2-C4 alkenylene-NH2 or -C2-C4 alkenylene-OH, for example, -CH=CH-CH2-NH2 or -CH=CH-CH2-OH.

[0123] In some further preferred embodiments, R a is -C1-C4 alkylidene-OH, -C1-C4 alkylidene-O-C1-C4 alkylidene-NH2, or -C1-C4 alkylidene-NH2.

[0124] In some more preferred embodiments, R a is -C1-C4 alkylidene-OH. In some embodiments, R a is a hydroxymethyl group. In some embodiments, R a is a hydroxyethyl group. In some embodiments, R a is a hydroxypropyl group.

[0125] In some further preferred embodiments, R a is -C1-C4 alkylidene-NH2. In some embodiments, R a is an aminomethyl group. In some embodiments, R a is an aminoethyl group. In some embodiments, R a is an aminopropyl group.

[0126] Preferably, the drug D is coupled to the antibody via its free hydroxy or amino group by binding to the linker L. More preferably, the drug D is represented by the formula D or the formula D a Or D b Drug D is R ais bonded to a linker L via a hydroxy group or an amino group of R a has a structure selected from: [ka] wherein n is an integer of 1 to 4, preferably n=2 or 3; wherein R' is H or a C1-C4 alkyl group, preferably H; wherein the wavy line on the left side represents the position of attachment to the linker L, and the asterisk on the right side represents the position of attachment to the camptothecin parent core; More preferably, R after attachment to the linker a has a structure selected from: [ka] Most preferably, R after attachment to the linker a has a structure selected from: [ka] Among them, the wavy line on the left side indicates the position of binding to the linker L, and the asterisk on the right side indicates the position of binding to the camptothecin parent core.

[0127] In some preferred embodiments, the LD unit in formula (I) of the present invention comprises the following structure: [ka] More preferably, the LD unit in formula (I) comprises the following structure: [ka]

[0128] Linker L Unit A linker L suitable for the present invention may be any linker capable of coupling an antibody of the present invention to a drug. Suitably, the addition of a linker can ensure sufficient stability of the ADC of the present invention in the circulatory system while at the same time providing a rapid and efficient release of the active form of the toxic drug at the target site (e.g., tumor cells or tumor environment).

[0129] In some embodiments, the linker of formula (I) of the present invention is a non-degradable linker. Examples of non-degradable linkers include, but are not limited to, N-succinimide-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Usually, ADCs that contain such linkers must be internalized in cells, and the antibody portion of the ADC is degraded by intracellular lysosomal proteases to release the drug active molecule.

[0130] In some further embodiments, the linker of formula (I) of the present invention is a degradable linker. Drug release of ADCs containing such linkers is triggered by the nature of the cleavage site in the linker. Thus, the cleavage site of such linkers may be designed according to the characteristics of the target therapeutic site (e.g., tumor cell lysosomes and / or tumor environment). In most cases, the degradable linker may be composed of a conjugate moiety, a degradable moiety, and an optional spacer group moiety. The conjugate moiety is responsible for binding the antibody to the linker-drug and may be selected according to the desired antibody coupling chemistry. The degradable moiety includes peptides or peptide analogs recognized by enzymes under enzyme-based release mechanisms, for example oligopeptides or oligopeptide analogs such as Val-Ala, Val-Cit, Phe-Lys, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Gly-Gly-Phe-Gly, cyclobutyl-Ala, cyclobutyl-Cit, etc. that can be degraded by proteolytic enzymes. In some cases, the properties of the ADC, such as the stability of the ADC in the blood circulation and / or the efficacy of the ADC at the target site, can be improved by introducing modifications at the position of the peptide residue adjacent to the enzyme cleavage site in the linker. In some cases, if necessary, the release (particularly traceless release) of the drug active molecule from the rest of the conjugate can be promoted by introducing a spacer group between the degradable part of the linker and the drug D, such as para-aminobenzoic acid (PABA) or aminomethyl (-NHCH2-) spacers that can be removed spontaneously in acidic medium. Also, if the drug is highly hydrophobic, in some cases the addition of PEG units may be considered (but is not required) to improve the properties of the ADC, for example to reduce precipitation and aggregation. Linkers suitable for the present invention include, but are not limited to, those disclosed in WO2022 / 170971, which is incorporated herein by reference.

[0131] In some embodiments, the linker L in formula (I) of the present invention has the structure of formula (II): -ZYM- (II), Among them, Z is a linker group that attaches to the Ab, Y is a peptide of 2 to 5 amino acids, preferably a dipeptide, tripeptide or tetrapeptide; M is absent or is a spacer group for attachment to the drug D.

[0132] Z linker group Usually, the thio group of antibody cysteine ​​exists in the form of disulfide bond. The disulfide bond of antibody can be opened to provide free sulfhydryl group as a coupling site. One method of coupling to the sulfhydryl group of antibody to form ADC is to react free sulfhydryl group on antibody with heterocyclic (e.g., maleimide) linker by Michael addition reaction. Another method is to react linker containing heteroaromatic ring substituted with leaving group with free sulfhydryl group in antibody molecule by nucleophilic displacement to obtain antibody-drug conjugate. Both of the two methods are suitable for ADC conjugate of the present invention. Thus, the linker according to the present invention may include Z linker of heterocyclic (e.g., maleimide) or heteroaryl ring (e.g., pyrimidinyl) in some embodiments, and in some cases, preferably includes heteroaromatic (e.g., pyrimidinyl) linker to provide conjugate with higher stability in blood circulation.

[0133] In some embodiments, Z in formula (II) has the structure: -Z1-Z2-Z3-Z4-, Z1 is a sulfur atom in Ab; Z2 is a 5- to 10-membered heterocyclyl group, preferably containing 1 or 2 heteroatoms selected from N, S and O; Z3 is a bond, -C(=O)-, -C1-C 10 Alkylidene-C(=O)-, -C3-C 10 Alkynylene-C(=O)-, -C3-C 10Alkenylene-C(=O)-, -C1-C 10 Heteroalkylene-C(=O)-, -C3-C8 cycloalkylene-C(=O)-, -O-C1-C8 alkylidene-C(=O)-, -arylene-C(=O)-, -C1-C 10 Alkylidene-arylene-C(=O)-, -arylene-C1-C 10 Alkylidene-C(=O)-, -C1-C 10 Alkylidene-C3-C8 cycloalkylene-C(=O)-, -C3-C8 cycloalkylene-C1-C 10 Alkylidene-C(=O)-, -C3-C8 Heterocyclylene-C(=O)-, -C1-C 10 Alkylidene-C3-C8 heterocyclylene-C(=O)-, -C3-C8 heterocyclylene-C1-C 10 Alkylidene-C(=O)-, -heteroarylene-C(=O)-, -C1-C 10 Alkylidene-heteroarylene-C(=O)-, -heteroarylene-C1-C 10 alkylidene-C(=O)-; Z4 is a bond or a PEG unit represented by the formula: [ka] Of these, R5 is C 1-4 Alkylidene group, -NH-, -NH-C 1-4 alkylidene-heteroaryl-, wherein the heteroaryl group is a 5- or 6-membered nitrogen-containing heteroaryl group, preferably a triazolyl group; R6 is -C(=O)-, -C 1-4 Alkylidene group, -C 1-4 Alkylidene-C(=O)-, -NH-C(=O)-(CH2OCH2)-C(=O)-, -C 1-4 alkylidene-NH-C(=O)-(CH2OCH2)-C(=O)-, in which m is an integer from 2 to 12, for example, m=2, 4, 6 or 8.

[0134] In one embodiment, Z2 is a 5- to 10-membered heteroaryl group. In one embodiment, Z2 is one or more of hydrogen, halogen, nitro group, C 1-6 Alkyl groups and haloC 1-6 In some embodiments, Z2 is a heteroaryl group selected from the group consisting of pyrimidine, thiazole, benzothiazole, oxazole, quinazoline, and pyrrolo[2,3d]pyrimidine substituted with an alkyl group. [ka] In one embodiment, Z2 is linked to Z1 by the carbon atom adjacent to the heteroatom. In a preferred embodiment, Z2 is a pyrimidinylene group, preferably [ka] and more preferably [ka] where the wavy line on the left represents the position of binding to Z1, and the wavy line on the right represents the position of binding to Z3.

[0135] In some embodiments, Z2 is a maleimide group. [ka] Among them, the wavy line on the left side represents the position of binding to Z1, and the wavy line on the right side represents the position of binding to Z3.

[0136] In some embodiments, Z3 is -C3-C8 heterocyclylene-C(=O)-, -C1-C 10 Alkylidene-C3-C8 heterocyclylene-C(=O)-, -C3-C8 heterocyclylene-C1-C 10In some embodiments, Z3 is -heteroarylene-C(=O)-, -C1-C2-C3-C4-C5-C6-C7-C8-C9-C10-C11-C12-C13-C14-C15-C16-C17-C18-C29-C28-C39-C29-C39-C40-C19-C28-C39-C29-C39-C40-C19-C29-C3 ... 10 Alkylidene-heteroarylene-C(=O)-, -heteroarylene-C1-C 10 It is alkylidene-C(=O)-.

[0137] In some embodiments, Z3 is -heteroarylene-C1-C 10 Alkylidene-C(=O)-, in particular [ka] In the above, n' is 1 to 6, and preferably, Z3 is [ka] More preferably [ka] and Among them, the wavy line on the left side represents the position of bonding to Z2, and the wavy line on the right side represents the position of bonding to Z4, and among them, Z2 is preferably a pyrimidinylene group.

[0138] In some embodiments, Z3 is arylene- or heteroarylenyl-C(=O)-, particularly [ka] Among them, Z3 is bonded to Z4 by -C-(=O)-, and among them, preferably, Z2 is a pyrimidinylene group.

[0139] In some other embodiments, Z3 is -(C≡C)-C 1-5 Alkylidene-C(=O)-, -(CH=CH)-C 1-5 Alkylidene-C(=O)-, -C 1-6Alkylidene-C(=O)- or -C 3-8 It is cycloalkylene-C(=O)-, in which Z3 is bonded to Z4 via -C-(=O)-.

[0140] In some preferred embodiments, Z2 is a pyrimidinylene group and Z3 is -C(=O)-.

[0141] In some preferred embodiments, Z2 is a pyrimidinylene group and Z3 is -(C≡C)-C 1-5 Alkylidene-C(=O)- or -(CH=CH)-C 1-5 Alkylidene-C(=O)-, especially -(C≡C)-C 1-5 It is alkylidene-C(=O)-.

[0142] In some preferred embodiments, Z2 is a maleimide group and Z3 is -C 1-6 Alkylidene-C(=O)- or -C 3-8 Cycloalkylene -C(=O)-.

[0143] In some embodiments, Z4 is a bond and Z3 is directly attached to Y in formula (II).

[0144] In some embodiments, Z4 is a unit containing 2 to 12 PEG. [ka] wherein m=1 to 8, for example, 2, 3, 4, 5, 6, 7, or 8.

[0145] In some embodiments, Z4 is [ka] wherein the wavy line on the left side represents the position of binding to Z3, and the wavy line on the right side represents the position of binding to Y in formula II.

[0146] In some embodiments, Z of the present invention in formula (I) has the following structure: [ka] where x1=1 to 8, for example, x1=3.

[0147] In some embodiments, Z of the present invention in formula (I) has the following structure: [ka] where x2=1 to 6.

[0148] In some preferred embodiments, Z in formula (I) of the present invention has the following structure: [ka] In which, S is a sulfur atom in Ab, and R b is unsubstituted or substituted -C 3-10 Alkynyl-C(=O)- or -C 3-10 Alkenyl-C(=O)- or -heteroarylene-C 10 Alkylidene-C(=O)-, Preferably, R b teeth, [ka] and In which the wavy line on the left represents the position where it bonds to the pyrimidinyl group, and the wavy line on the right represents the position where it bonds to Y, More preferably, Z has the structure: [ka]

[0149] In some embodiments, Z of the present invention in formula (I) has the following structure: [ka] Among them, R E is hydrogen, C 1-6 Alkyl group, C 1-6 Aminoalkyl groups, C 1-6 Haloalkyl group, C 1-6 hydroxyalkyl groups, where y=0-4, e.g., 0, 2, or 5, and where the alkyl, amino, and hydroxy moieties are optionally substituted; in some embodiments, R E is an optionally substituted aminoalkyl group, for example, -C 1-4 Alkylidene-NH2, -C 1-4 Alkylidene NHR F and -C 1-4 Alkylidene N(R F )2, of which each R F is C 1-6 Alkyl group and C 1-6 haloalkyl groups, or two R F The groups combine with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl, or piperidinyl group.

[0150] In some embodiments, Z of the present invention in formula (I) has the following structure: [ka] has.

[0151] Y peptide-containing unit For the purpose of limiting or minimizing the non-targeted toxicity of ADCs while ensuring the release of the toxin molecule at the target tumor site, in some embodiments, it is advantageous to design the antibody-drug conjugate so that it can be degraded by proteolytic enzymes in the tumor cells or environment (especially enzymes that have significantly higher activity in tumor cells and / or the tumor environment compared to blood) while targeting tumor cells with high selectivity. To this end, oligopeptides or oligopeptide analogs that can be recognized and cleaved by such proteolytic enzymes can be included in the linker of such ADC conjugates.

[0152] In one embodiment, the Y unit, which is thus part of the linker of the invention, is a peptide-containing degradable moiety, e.g., a degradable peptide linker, which contains two or more (e.g., 2 to 12, e.g., 2, 3, 4, 5, or 6) consecutive or non-consecutive amino acids. Each amino acid of the peptide-containing unit can be independently selected from the group consisting of natural or non-natural amino acids, e.g., alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, substituted lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, cysteine, methionine, selenocysteine, ornithine, β-alanine, citrulline, and derivatives thereof. In some embodiments, each amino acid is independently selected from alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, cysteine, methionine, and derivatives thereof. In some embodiments, each amino acid is independently selected from alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, and valine, N-methylglycine, β-alanine, and derivatives thereof. In some embodiments, after the ADC is internalized into the tumor cell, the amide bond in the Y unit is recognized and degraded by enzymes in the lysosomes of the tumor cell, releasing the drug moiety D. In another embodiment, the amide bond in the Y unit can be recognized and degraded by enzymes in the tumor environment, releasing the drug moiety D. By designing antibody-drug conjugates with biased tumor distribution, linkers with such Y-peptide-containing units facilitate the release of the toxin molecule in tumor cells and the environment.

[0153] In some embodiments, Y comprises a unit selected from the group consisting of Val-Cit, Phe-Lys, Val-Ala, Val-Lys-Gly, Ala-Ala-Ala, Val-Ala, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Gly-Gly-Phe-Gly, cyclobutyl-Ala, and cyclobutyl-Cit. In some embodiments, Y comprises a unit selected from the group consisting of Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Cit-Val, Val-Ala, Lys-Val, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg, and Ala-Ala-Asn.

[0154] In some preferred embodiments, Y is a dipeptide, tripeptide, tetrapeptide, or pentapeptide comprising a substituted lysine. In one embodiment, the substituted lysine is: [ka] wherein R3 and R4 are independently selected from H, C 1-6 Alkyl groups, -CO-NH2, -CONH(C 1-6 alkyl), and -CONH(C 1-6 alkyl)2, wherein the alkyl group is selected from halogen, C 1-6 Alkoxy group, C 1-6 Haloalkyl group, C 3-6 It is optionally substituted with a group selected from a cycloalkyl group, a 6- to 10-membered aryl group, and a 5- to 14-membered heteroaryl group.

[0155] In some preferred embodiments, Y is a peptide having, from N-terminus to C-terminus, the amino acid sequence of the following formula: Xaa1-Xaa2-Xaa3-Xaa4-Xaa5, wherein Xaa1 is absent or is an amino acid selected from valine, glycine, alanine, and glutamic acid; Xaa2 is an amino acid selected from phenylalanine, leucine, alanine and valine, preferably valine; Xaa3 is an unsubstituted or substituted lysine; Xaa4 is an amino acid selected from leucine, glycine and alanine; Xaa5 is absent or is an amino acid selected from glycine and alanine; wherein the N-terminus of the amino acid sequence is linked to the Z unit of the linker, and the C-terminus is linked to the M unit or directly to the drug D; Preferably, Xaa3 is a lysine mono- or di-substituted at the ε-amino group with a C1-C3 alkyl group; More preferably, Y is a peptide selected from the group consisting of Phe-Lys-Gly, Leu-lys-Gly, Gly-Val-Lys-Gly, Val-Lys-Gly-Gly, Val-Lys-Gly, Val-Lys-Ala, Val-Lys-Leu, in which the Lys residue is unsubstituted or is lysine mono- or di-substituted with a C1-C3 alkyl group.

[0156] M spacer group In some embodiments, the ADC conjugates of the invention have a spacer group (M) between the releasable peptide-containing unit (Y) and the drug (D). The spacer group may be a functional group that facilitates attachment of the peptide-containing unit (Y) to the drug D, or it may provide an additional structural moiety (e.g., a self-degrading group, such as a p-aminobenzaldehyde (PAB) moiety) that further facilitates release of the drug D from the remainder of the conjugate.

[0157] In some embodiments, the M spacer group linking the Y and drug D units is [ka] and preferably [ka] and The wavy line on the left represents a bond to the Y unit, and the wavy line on the right represents a bond to the drug D unit.

[0158] In some other embodiments, M is a covalent bond, and Y in formula I is directly bonded to drug D via an amide bond.

[0159] In a preferred embodiment, the YM unit in the linker L comprises the following structure: [ka] And preferably, the YM unit is the following Z unit: [ka] Bind to.

[0160] Exemplary linkers L In some preferred embodiments, L in formula (I) of the present invention is a linker comprising the following structure: [ka] wherein R3 and R4 are each independently selected from a methyl group, an ethyl group, and a propyl group.

[0161] In some more preferred embodiments, R3 and R4 are both methyl groups, or R3 and R4 are both ethyl groups, or R3 and R4 are both propyl groups.

[0162] In some embodiments, the LD unit of formula I of the invention is attached to the antibody by formation of a thioether bond with a sulfhydryl group of a free cysteine ​​of the light and / or heavy chain of the Ab.

[0163] Exemplary ADC Conjugates In some embodiments, the invention provides an ADC conjugate having the structure: [ka] or [ka] or [ka] wherein q represents an average DAR value of 1 to 20, for example, an average DAR value of about 2, 3, 4, 6, and 8.

[0164] In some embodiments, an ADC according to the invention has at least one or more of the following advantages: - Significantly promote the distribution and concentration of ADC in the tumor environment with respect to the distribution of ADC in the blood circulation; -Effectively limit the premature release of payload in plasma and have relatively high blood circulation stability; - capable of providing an effective release of the active form of a toxic drug in the tumor environment, -provides highly efficient in vivo tumor killing in animals; and -Good animal tolerance.

[0165] In some further embodiments, the ADCs according to the invention may further have at least one or more of the following advantages: - the addition of a toxin-linker does not induce aggregation and can achieve high drug loading, with a DAR of up to 8; and -Acceptable PK characteristics.

[0166] II. Preparation of Antibody-Drug Conjugates The formation of antibody-drug conjugates can be accomplished by any technique known to those skilled in the art. In some embodiments, the coupling of the drug-linker to the antibody is accomplished by reaction with an amino acid residue of the antibody. In some embodiments, a heteroaryl linker L with a leaving group is applied to couple the drug D to a cysteine ​​residue of the antibody to prepare the conjugate of formula I of the present invention. In some embodiments, by controlling the conditions of treating the antibody with a reducing agent, such as tris(2-hydroxyethyl)phosphine (TCEP), the interchain disulfide bonds of the antibody can be broken and free sulfhydryl groups can be exposed for coupling with the heteroaryl linker-drug. In an IgG1 type antibody, up to four connected disulfide bonds can be reduced, thereby generating up to eight reactive sulfhydryl groups for coupling. The conjugates prepared by this method can contain 0, 1, 2, 3, 4, 5, 6, 7 or 8 drugs per antibody molecule.

[0167] When the prepared conjugates are conjugate compositions with different drug conjugate sites and / or numbers, the drug loading of the conjugates is represented by the average DAR, which is the average number of drug molecules per antibody. The average number of drugs per antibody in the preparation of the produced antibody-drug conjugate compositions can be characterized by conventional means such as mass spectrometry, ELISA measurement, and HPLC. In some other embodiments, the quantitative distribution of the antibody-drug conjugates, represented by q, can also be determined. Separation, purification, and characterization of a homogeneous antibody-drug conjugate with a particular value of q from antibody-drug conjugates with other drug loadings can be achieved by means such as reversed-phase HPLC or electrophoresis.

[0168] Thus, in one aspect of the invention, q represents the number of drug-linker (LD) moieties coupled to a single antibody (Ab), and is preferably an integer of 1 to 16, 1 to 12, 1 to 10, or 1 to 8. In this case, the single ADC conjugate can also be referred to as an ADC compound. In any embodiment herein, an ADC compound according to the present invention can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 drug linker moieties coupled to a single antibody.

[0169] In another embodiment of the invention, q represents the average DAR of the prepared conjugate composition. In this case, q may be an integer or decimal within the range of 1 to about 16, 1 to about 12, 1 to about 10, or 1 to about 8, 2 to about 16, 2 to about 12, 2 to about 10, or 2 to about 8. In some embodiments, q represents an average DAR of about 3. In some embodiments, q represents an average DAR of about 6. In some embodiments, q represents an average DAR of about 8.

[0170] III. Pharmaceutical Compositions In some embodiments, the invention provides a composition comprising any of the ADCs described herein, or a pharma- ceutically acceptable salt or solvate thereof, preferably the composition is a pharmaceutical composition or pharmaceutical formulation. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, e.g., a pharmaceutical composition, comprises a combination of an ADC of the invention, or a pharma- ceutically acceptable salt or solvate thereof, and one or more other therapeutic agents.

[0171] The invention further includes combinations (including pharmaceutical compositions) of the ADCs of the invention or pharma- ceutically acceptable salts or solvates thereof. These compositions may further include suitable pharmaceutical auxiliary materials, such as pharmaceutical vectors, pharmaceutical excipients, including buffering agents, as known in the art.

[0172] As used herein, "medicinal vectors" include any and all solvents, dispersion media, isotonic agents, absorption delaying agents, and the like that are physiologically compatible.

[0173] For a discussion of the use of pharmaceutical adjuvants and their applications, see also "Handbook of Pharmaceutical Excipients", 8th Edition, RC Rowe, PJ Eskey and SCOwen, Pharmaceutical Press, London, Chicago.

[0174] The compositions of the present invention may be in a variety of forms, including, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), powders or suspensions, liposomes and suppositories. The preferred form will depend on the intended mode of administration and therapeutic application.

[0175] Drugs comprising the ADCs described herein can be prepared by mixing an ADC of the invention, or a pharma- ceutically acceptable salt or solvate thereof, having the desired purity, preferably in the form of a lyophilized formulation or aqueous solution, with one or more optional pharmaceutical auxiliary materials.

[0176] The pharmaceutical composition or formulation of the present invention may further comprise one or more active ingredients with complementary activities, preferably not adversely affecting each other, as required for the specific indication to be treated.Ideally, other therapeutic agents are also provided, including, for example, chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists).The active ingredients are present in an amount effective for the intended use and in suitable combination.

[0177] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, such as films, or microcapsules.

[0178] IV. Pharmaceutical Combinations and Kits In some embodiments, the present invention further provides a pharmaceutical combination or pharmaceutical combination product comprising a compound of the present invention, or a pharma- ceutically acceptable salt or solvate thereof, and one or more other therapeutic agents.

[0179] Another object of the present invention is to provide a kit of parts comprising the pharmaceutical combination of the present invention, preferably said kit in the form of a drug dosage unit, whereby dosage units can be provided according to a dosing regimen or drug administration interval.

[0180] In one embodiment, the kit of parts of the invention comprises, in the same package: - a first container containing a pharmaceutical composition comprising an ADC of the invention, or a pharma- ceutically acceptable salt or solvate thereof; a second container containing a pharmaceutical composition comprising another therapeutic agent.

[0181] The method and order of administration of each component of the combination product can be determined by those skilled in the art depending on factors such as the disease to be treated and the individual's condition. The combination product of the present invention can be used in the treatment method of the present invention. In some embodiments, the present invention provides a combination product, in which the other therapeutic agent is, for example, a therapeutic agent, such as an antibody, that effectively stimulates the immune response, thereby further enhancing, stimulating, or upregulating the immune response of the subject. In some embodiments, the combination product is used for the prevention or treatment of TF-positive tumors.

[0182] Anti-TF antibodies of the present invention By screening for antibody sequences with sequence uniqueness and high antigen-binding specificity, and further evaluating the properties of the obtained antibodies, including affinity, endocytosis activity, signal pathway blocking, coagulation effect, stability, pharmacokinetics, etc., the inventors identify the humanized anti-TF monoclonal antibody of the present invention with excellent properties. The antibody of the present invention not only exhibits high binding affinity and high specificity to TF-positive tumor cells, is rapidly and efficiently endocytosed by tumor cells, and has little effect on TF-mediated coagulation, so that it is not only suitable for cancer treatment alone or in combination with other anti-cancer drugs, but also suitable as a molecular component for forming a novel anti-cancer molecule targeting cancer tissues.

[0183] Thus, in some aspects, the present invention further provides humanized anti-TF monoclonal antibodies with multiple superior properties, nucleic acids encoding same, vectors and host cells comprising said nucleic acids, as well as immunoconjugates, multispecific antibodies, pharmaceutical compositions and uses comprising said antibodies.

[0184] I. Antibodies of the Invention and Their Properties The anti-TF antibodies of the invention and their properties are described in detail below. As will be appreciated by those skilled in the art, unless the context clearly dictates otherwise, the technical features (including structural and characteristic features) of any antibody described in this section and any combination thereof are equally applicable to the antibodies of the invention that are the Ab units of the ADC conjugates of the invention.

[0185] Anti-TF antibodies of the present invention The present invention provides antibodies or antigen-binding fragments thereof, particularly humanized antibodies or antigen-binding fragments thereof, that specifically bind to TF, preferably human TF protein.

[0186] In some embodiments, the anti-TF antibodies or antigen-binding fragments thereof provided by the invention are (i) the HCDR1, 2 and 3 sequences of the heavy chain variable region shown in SEQ ID NO: 1, and the LCDR1, 2 and 3 sequences of the light chain variable region shown in SEQ ID NO: 2; or (ii) the HCDR1, 2 and 3 sequences of the heavy chain variable region as set forth in SEQ ID NO: 3, and the LCDR1, 2 and 3 sequences of the light chain variable region as set forth in SEQ ID NO: 4; or (iii) the HCDR1, 2 and 3 sequences of the heavy chain variable region as set forth in SEQ ID NO:5, and the LCDR1, 2 and 3 sequences of the light chain variable region as set forth in SEQ ID NO:6; or (iv) the HCDR1, 2 and 3 sequences of the heavy chain variable region shown in SEQ ID NO: 7, and the LCDR1, 2 and 3 sequences of the light chain variable region shown in SEQ ID NO: 8; or (v) A variant of any one of the CDR combinations (i) to (iv), preferably comprising a total of 1 to 10 amino acid alterations (including but not limited to insertions, deletions and / or substitutions, preferably amino acid substitutions, preferably conservative substitutions) across the six CDR regions, and preferably wherein the heavy chain CDR3 remains unchanged.

[0187] Preferably, the CDRs are defined according to Kabat or IMGT or a combination thereof, more preferably according to IMGT. The CDRs may also be defined in any other manner known in the art.

[0188] In some embodiments, an anti-TF antibody or antigen-binding fragment thereof according to the invention comprises three complementarity determining regions HCDRs of a heavy chain variable region and three complementarity determining regions LCDRs of a light chain variable region, among which: (i) as defined according to Chothia, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:9, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:10, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:11, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:21, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:22, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:23; or (ii) as defined according to AbM, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 12, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 13, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 14, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 24, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 25, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 26, or (iii) as defined according to Kabat, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 15, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 16, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 17, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 27, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 28, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 29, or (iv) As defined according to IMGT, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 18, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 19, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 20, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 30, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 31, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 32.

[0189] In some embodiments, an anti-TF antibody or antigen-binding fragment thereof according to the invention comprises three complementarity determining regions HCDRs of a heavy chain variable region and three complementarity determining regions LCDRs of a light chain variable region, among which: (i) as defined according to Chothia, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 33, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 34, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 35, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 45, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 46, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 47; or (ii) as defined according to AbM, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 36, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 37, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 38, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 48, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 49, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 50; or (iii) as defined according to Kabat, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 39, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 40, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 41, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 51, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 52, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 53; or (iv) As defined according to IMGT, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 42, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 43, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 44, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 54, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 55, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 56.

[0190] In some embodiments, an anti-TF antibody or antigen-binding fragment thereof according to the invention comprises three complementarity determining regions HCDRs of a heavy chain variable region and three complementarity determining regions LCDRs of a light chain variable region, among which: (i) as defined according to Chothia, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 57, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 58, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 59, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 69, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 70, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 71; or (ii) as defined according to AbM, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 60, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 61, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 62, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 72, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 73, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 74; or (iii) as defined according to Kabat, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 63, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 64, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 65, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 75, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 76, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 77; or (iv) As defined according to IMGT, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 66, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 67, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 68, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 78, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 79, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 80.

[0191] In some embodiments, an anti-TF antibody or antigen-binding fragment thereof according to the invention comprises three complementarity determining regions HCDRs of a heavy chain variable region and three complementarity determining regions LCDRs of a light chain variable region, among which: (i) as defined according to Chothia, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 81, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 82, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 83, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 93, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 94, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 95; or (ii) as defined according to AbM, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 84, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 85, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 86, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 96, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 97, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 98; or (iii) as defined according to Kabat, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 87, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 88, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 89, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 99, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 100, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 101; or (iv) As defined according to IMGT, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 90, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 91, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 92, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 102, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 103, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 104.

[0192] In some embodiments, an anti-TF antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is (i) the amino acid sequence set forth in SEQ ID NO:1, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (ii) an amino acid sequence set forth in SEQ ID NO:3, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (iii) an amino acid sequence as set forth in SEQ ID NO:5, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; (iv) comprises an amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0193] In some embodiments, an anti-TF antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region is (i) the amino acid sequence set forth in SEQ ID NO:2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (ii) an amino acid sequence set forth in SEQ ID NO:4, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (iii) an amino acid sequence as set forth in SEQ ID NO:6, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; (iv) comprises an amino acid sequence set forth in SEQ ID NO:8, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0194] In some embodiments, an anti-TF antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is (i) the amino acid sequence set forth in SEQ ID NO:1, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (ii) comprising an amino acid sequence as set forth in SEQ ID NO:3, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and / or wherein the light chain variable region comprises: (i) the amino acid sequence set forth in SEQ ID NO:2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (ii) comprises an amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0195] In some embodiments, an anti-TF antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is (i) the amino acid sequence set forth in SEQ ID NO:5, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (ii) comprising an amino acid sequence as set forth in SEQ ID NO: 7, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and / or wherein the light chain variable region comprises: (i) the amino acid sequence set forth in SEQ ID NO:6, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (ii) comprises an amino acid sequence set forth in SEQ ID NO:8, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0196] In some preferred embodiments, an anti-TF antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain variable region and a light chain variable region, of which the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, and of which the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto. Preferably, the antibody or antigen-binding fragment comprises the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 2.

[0197] In some preferred embodiments, an anti-TF antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:3, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, and wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto. Preferably, the antibody or antigen-binding fragment comprises the heavy chain variable region of SEQ ID NO:3 and the light chain variable region of SEQ ID NO:4.

[0198] In some preferred embodiments, an anti-TF antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:5, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, and wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:6, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto. Preferably, the antibody or antigen-binding fragment comprises the heavy chain variable region of SEQ ID NO:5 and the light chain variable region of SEQ ID NO:6.

[0199] In some preferred embodiments, an anti-TF antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain variable region and a light chain variable region, of which the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:7, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, and of which the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto. Preferably, the antibody or antigen-binding fragment comprises the heavy chain variable region of SEQ ID NO:7 and the light chain variable region of SEQ ID NO:8.

[0200] In some embodiments, the anti-TF antibody or antigen-binding fragment thereof according to the invention comprises the heavy and light chain variable region sequences of any one of the exemplary antibodies 2B12B10-hz1, 30G11B7-hz1, 22F11H5-hz1, and 27H8H3-hz1 of the invention or variants thereof, such as an antibody having the same CDR sequences as one of the exemplary antibodies above, and having homologous or different framework region sequences. Preferably, the antibody is a humanized antibody. More preferably, the heavy chain variable region of the antibody has framework region sequences derived from human germline.

[0201] The antibody according to the invention may comprise a heavy chain constant region and / or a light chain constant region. Preferably, the heavy chain constant region is a heavy chain constant region derived from a human immunoglobulin. Preferably, the light chain constant region is a light chain constant region derived from a human immunoglobulin. The heavy chain constant region comprised in the antibody of the invention may be a heavy chain constant region of any isotype or subtype, for example an IgG1, IgG2, IgG3 or IgG4 isotype. In some embodiments, the invention thus provides an anti-TF antibody comprising an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, preferably an IgG1 heavy chain constant region, in particular a human IgG1 heavy chain constant region, for example an amino acid sequence of SEQ ID NO: 106 or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. The light chain constant region comprised in the antibody of the invention may be a κ light chain constant region or a λ light chain constant region. In some embodiments, the invention therefore provides an anti-TF antibody comprising a κ or λ light chain constant region, preferably the antibody of the invention comprises a human κ light chain constant region, e.g., the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the heavy and / or light chain of the anti-TF antibody of the invention or a fragment thereof further comprises a signal peptide sequence.

[0202] In some embodiments, the present invention further provides a variant of any of the antibodies according to the invention described herein, in particular a variant of any one of the exemplary antibodies of the invention 2B12B10-hz1, 30G11B7-hz1, 22F11H5-hz1, and 27H8H3-hz1.

[0203] The term "variant" in reference to an antibody, as used herein, refers to an antibody comprising a region of interest having at least one, e.g., 1-30, or 1-20, or 1-10, e.g., 1 or 2 or 3 or 4 or 5, amino acid substitutions, deletions and / or insertions, as compared to a given parent antibody, where the variant essentially retains at least one biological property (e.g., antigen binding ability) of the parent antibody molecule. The antibody region of interest may be a full-length antibody, or a heavy chain variable region or a light chain variable region or a combination thereof, or a heavy chain CDR region(s) or a light chain CDR region(s) or a combination thereof. Such variants of specific exemplary antibodies of the invention are included in the contemplation of the present invention.

[0204] In some embodiments, the antibody variants of the invention preferably retain at least 60%, 70%, 80%, 90% or 100% of the biological activity of the parent antibody prior to the introduction of the alterations or modifications (e.g., essentially the same antigen binding ability and / or receptor endocytosis activity, or essentially the same effect on coagulation). It should be understood that the heavy or light chain variable regions or respective CDR or framework regions of the antibodies according to the invention can be altered, either alone or in combination, to generate the antibody variants of the invention. The Fc region of the antibodies according to the invention can also be altered. Alterations in the Fc region can be made alone or in combination with alterations in the framework regions and / or CDR regions and / or heavy and / or light chain variable regions. The Fc region can be selectively altered, for example, to confer one or more altered effector functions on the antibody, such as altered serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity, depending on the specific application of the antibody molecule. The antibodies of the invention may also be chemically modified (eg, conjugated to PEG) or their glycosylation patterns altered.

[0205] In some embodiments, the antibody variant according to the invention has amino acid changes in the heavy and / or light chain sequences compared to any one of the exemplary antibodies of the invention. Preferably, the variant comprises at least one, two or three, but not more than 20, 10 or 5 amino acid changes in the heavy or light chain sequences or both, compared to the corresponding antibody, or an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity. Preferably, the amino acid changes do not occur in the CDR regions, and more preferably, do not occur in the variable regions. In some embodiments, at least some or all of the amino acids contained in the antibody variant are changed to conservative amino acid substitutions. "Conservative substitution" refers to an amino acid change that results in the replacement of an amino acid with a chemically similar amino acid. To achieve conservative substitutions, amino acid modifications such as substitutions can be introduced into the antibodies of the invention by standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0206] In some further aspects, the present invention further provides anti-Tissue Factor (TF) antibodies or antigen-binding fragments thereof that have one or more properties, compared to any given antibody according to the present invention described above (particularly any one of the exemplary antibodies of the present invention, 2B12B10-hz1, 30G11B7-hz1, 22F11H5-hz1, and 27H8H3-hz1), such as (i) binding to the same or overlapping epitope as the given antibody according to the present invention; (ii) competitively binding to human tissue factor (TF) with the given antibody according to the present invention; (iii) inhibiting (e.g., competitively inhibiting) binding of the given antibody according to the present invention to cells expressing human tissue factor on the cell surface, and preferably exhibiting the same or similar binding affinity and / or specificity as the given antibody according to the present invention.

[0207] As used herein, an antibody that "competitively binds to a TF antigen" with a given antibody refers to an antibody that blocks 50% or more of the binding of the given antibody to the antigen TF in a competitive assay, and conversely, an antibody that blocks 50% or more of the binding of the given antibody to the antigen TF in a competitive assay. Exemplary competitive assays are described in "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Competitively binding antibodies can bind to the same epitopic region as the given antibody, e.g., the same epitope, adjacent epitopes, or overlapping epitopes.

[0208] As used herein, an antibody that inhibits (e.g., competitively inhibits) the binding of a given antibody to its antigen refers to an antibody that inhibits 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the given antibody to its antigen, and conversely, the given antibody also inhibits 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the antibody to its antigen. The binding of an antibody to its antigen can be determined using binding affinity. Methods for measuring affinity are known in the art.

[0209] As used herein, an antibody that exhibits the same or similar binding affinity and / or specificity as a given antibody refers to an antibody that can have at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding affinity and / or specificity of the given antibody, which can be measured by any method known in the art for measuring binding affinity and / or specificity.

[0210] In some embodiments, the antibody according to the invention is an IgG1 antibody, more particularly an IgG1κ or IgG1λ isotype.More preferably, the IgG1 antibody according to the invention has a human IgG1 constant region, more preferably a human IgG1κ isotype.

[0211] Properties of the anti-TF antibodies of the invention The anti-TF antibodies of the invention have one or more of the following properties: (i) binds to the human TF extracellular domain with high affinity; (ii) binds to TF-positive tumor cells with high affinity; (iii) cross-reactivity with cynomolgus monkey TF; (iv) having TF receptor-mediated endocytosis activity; (v) blocking TF / FVIIa complex-mediated downstream signaling pathways in TF-positive cells; and (vi) Coagulation initiated by cell surface TF has essentially no or reduced coagulation effect.

[0212] In some embodiments, the antibodies of the invention have high binding affinity and high binding specificity to TF. The binding EC50 value of the antibodies of the invention to the TF extracellular domain and / or TF-positive tumor cells can be determined by FACS or ELSA measurements (e.g., measurements described in the Examples). Alternatively, the binding kinetic parameters of the antibodies can be determined by biofilm layer interference techniques to characterize the binding affinity and specificity of the antibodies. In some embodiments, the anti-TF antibodies of the invention have high binding affinity and high binding specificity to TF at 10 -7 K is smaller than M D For example, about 10 -8 M or smaller K D value, preferably 5 x 10 -8 In another embodiment, the antibodies of the invention have a biofilm layer interference (BIL) assay of less than about 10 -1 ~10 -3 s -1 K d A value, for example about 10 -2 Or about 10 -3 s -1 K d Preferably, the K value is determined using the ForteBio measurement method described in the Examples. D value and K dA binding kinetic parameter of the antibody, such as a binding kinetic K value, is determined and, optionally, compared to a reference antibody in the same assay. In some embodiments, the antibody has the same or similar or lower binding kinetic K value compared to the reference antibody. D In some embodiments, the antibody has a relatively fast dissociation rate (K d Previous studies have shown that in some cases, antibodies with relatively fast dissociation rates can penetrate tumors more efficiently to promote distribution of antibodies in solid tumors. (See Influence of Affinity and Antigen Internalization on the Uptake and Penetration of Anti-HER2 Antibodies in Solid Tumors, DOI:10.1158 / 0008-5472.CAN-10-2277)

[0213] In some embodiments, the anti-TF antibodies of the present invention bind strongly to TF-positive tumor cells. The cell-binding EC50 value (i.e., half the maximum effective concentration for cell binding) and / or maximum binding amount of the antibody can be measured by cell ELISA or flow cytometry measurement and compared with a reference antibody, thereby reflecting the cell-binding affinity of the antibody. In some embodiments, the antibodies of the present invention have an EC50 value (i.e., half the maximum effective concentration for cell binding) and / or maximum binding amount of the antibody for at least one TF-positive tumor cell, such as NIC-H358 cells or KYSE50 cells. 50 Values ​​are the EC 50 The value is about 50% to about 160%, for example, about 80%, about 90%, about 100%, about 110%, about 120%, or about 130%.

[0214] In some embodiments, the anti-TF antibodies of the invention exhibit cross-reactivity to human and monkey TF. In some embodiments, the binding K of the antibodies of the invention to human TF D The values ​​are the binding K D For example, the K DThe ratio is between 1 and 10, for example between 1 and 5, more preferably between about 1 and 3. In a further embodiment, the binding K d The values ​​are the binding K d For example, the K d The ratio is between 1 and 10, for example between 1 and 5, more preferably between about 1 and 3.

[0215] In some embodiments, the antibody of the present invention has TF receptor-mediated endocytosis activity. In a cell-based assay, such as the assay described in the Examples, the endocytosis activity of the antibody can be evaluated. In some embodiments, in a TF-positive cell-based assay, to determine the endocytosis rate of the antibody, the antibody to be assayed is incubated with TF-positive cells (especially TF-positive tumor cells) for a certain time (e.g., 2 hours or 4 hours) at 37°C, and then the change in the amount of TF antibody bound to the cell surface is fluorescently detected by flow cytometry, compared to a negative control kept for the same time at 4°C. In some embodiments, the antibody of the present invention has an endocytosis rate that is essentially the same or better than that of the reference antibody. In another embodiment, under equivalent measurement conditions, the endocytosis rate of the antibody of the present invention is about 50% to 200% or more of the endocytosis rate of the reference antibody, for example, about 70% or more, for example, about 80% or more, preferably about 100% or more, or about 110% or more, more preferably about 120% or more, for example, about 130% or more, about 150% or more, or about 200% or more. In further embodiments, the antibody of the present invention exhibits faster endocytosis compared to the reference antibody, for example, reaching about 30%, 40% or 50% endocytosis in a shorter time. In further embodiments, in the measurement test for evaluating the above-mentioned endocytosis activity described in the Examples, the endocytosis rate of the antibody of the present invention is, for example, at least 10%, for example, 15% to 20%, preferably at least 25%, for example, 20% to 35%, more preferably 35% or more, for example, about 40% or 45% or more, after incubation at 37°C for 2 hours.

[0216] In some embodiments, the antibodies of the invention have essentially no effect on clotting or have a reduced effect on clotting. 2+The interference of the antibody with clotting initiated by TF-positive cells in the presence of ions and human plasma can be detected to determine the effect of the antibody on clotting. In some embodiments, the antibody of the invention has essentially no effect on clotting initiated by cell surface TF compared to a negative control without antibody or has a reduced clotting effect compared to a reference antibody. A change in clotting time caused by the antibody can be detected compared to a negative control without antibody and / or a reference antibody, thereby reflecting the effect of the antibody on clotting. Preferably, the antibody is detected in the presence of TF-positive cells and high or low concentrations of free Ca according to the clotting measurement test described in the examples. 2+ The effect of the antibody on the TF-mediated clotting process is characterized by measuring the time course of absorbance at 405 nm after addition of human plasma to a solution containing ions and the antibody to be measured, and estimating the clotting time required to reach a half-maximum absorbance value of OD405 (V50). 2+ The concentration of the ion may be 1 mM to 5 mM, for example, one or more concentrations selected from 2 mM and 5 mM. The concentration of the antibody used for detection may be 10 μg / mL to 50 μg / mL, for example, one or more concentrations selected from 20 μg / mL and 50 μg / mL. In some embodiments, the effect of the antibody on blood clotting is evaluated using 5 mM Ca 2+ and 20 μg / mL antibody. 2+ and 50 μg / mL antibody. In some embodiments, the effect of the antibody on clotting is measured under at least one measurement condition, e.g., in the presence of 5 mM Ca 2+ In some embodiments, an antibody is considered to have essentially no effect on clotting or a reduced effect on clotting if, in the presence of 20 μg / mL antibody, the delay in clotting time (V50) caused by the antibody is not more than 50 seconds, preferably not more than 40 seconds, not more than 30 seconds, not more than 20 seconds, not more than 10 seconds, not more than 5 seconds, in comparison to a negative control without antibody. 2+An antibody is considered to have essentially no effect on clotting or a reduced effect on clotting if, in the presence of 20 μg / mL antibody, the antibody does not cause a delay in clotting time (V50) or, preferably, the clotting time (V50) is reduced by about 10% or more, or more preferably about 20% or 30% or 40% or more, and even more preferably about 50% or 60% or more, compared to a reference antibody. In some further embodiments, the antibody is considered to have essentially no effect on clotting or a reduced effect on clotting if, in the presence of 20 μg / mL antibody, the antibody does not cause a delay in clotting time (V50) or, preferably, the clotting time (V50) is reduced by about 10% or more, or, more preferably, about 20% or 30% or 40% or more, and even more preferably, about 50% or 60% or more, compared to a reference antibody. 2+ An antibody is considered to have essentially no effect on clotting or a reduced effect on clotting if, in the presence of 50 μg / mL antibody, the antibody does not cause a delay in clotting time (V50), or preferably the clotting time (V50) is reduced by about 10%, 20%, or 30% or more, compared to a reference antibody.

[0217] In some further embodiments, the antibody of the present invention has the ability to block the TF / FVIIa complex-mediated downstream signal pathway in TF-positive tumor cells. The blocking activity of the anti-TF antibody against the TF downstream signal pathway can be determined by detecting the level of IL-8 release caused by activation of the pathway. In some embodiments, the antibody of the present invention exhibits an inhibitory effect on the TF signal pathway according to the evaluation test of signal pathway blocking activity described in the Examples. In some embodiments, the antibody of the present invention exhibits TF signal pathway blocking activity IC 50 In some embodiments, the IC value of the antibody of the present invention is less than 500 ng / mL or 150 ng / mL, and the maximum inhibition rate reaches 90% or more, preferably 95% or more. 50 Values ​​are comparable (±10%) to the reference antibody or less.

[0218] In some further embodiments, the anti-TF antibodies or antigen-binding fragments thereof of the invention further have one or more of the following properties: (vii) good productivity, (viii) good stability, and (ix) advantageous pharmacokinetic properties.

[0219] In some embodiments, the antibodies of the present invention purified by one-step purification from antibody products produced by recombinant mammalian cells using Protein A affinity chromatography, followed by SEC-HPLC, can reach a purity of 90% or more and show good hydrophilicity in HIC measurement.

[0220] In some further embodiments, the antibodies of the present invention have good stability, including thermal stability and repeated freeze-thaw stability. In some embodiments, after standing at 37°C for 3 to 14 days according to the accelerated stability measurement in the Examples, the SEC-HPLC purity of the antibodies of the present invention (i.e., antibody monomer percentage measured by SEC-HPLC) is maintained at 90% or more, or preferably 95% or more, or the decrease is not more than 5% or preferably not more than 3%. In some further embodiments, the CE-SDS non-reduced purity (i.e., main peak percentage) is maintained at 85% or more, or preferably 90% or more, and the CE-SDS reduced purity (i.e., LC+HC percentage) is 90% or preferably 95% or more, or the decrease in the non-reduced purity and reduced purity is not more than 5% or 4%. In some further embodiments, according to the repeated freeze-thaw measurements in the Examples, after 4 to 8 repeated freeze-thaw cycles, the SEC purity of the antibodies of the present invention is maintained at 95% or more, or the purity does not decrease by more than 5%, or preferably does not decrease by more than 3%, in any case.

[0221] In some further embodiments, the antibody of the present invention has serum stability. According to the serum stability measurement test in the examples, the change in affinity of the antibody to TF antigen after incubation in serum at 37°C for a certain period of time can be measured to determine the serum stability of the antibody. If the change in affinity is not significant, the antibody is considered to have serum stability. In some embodiments, the antibody of the present invention can be maintained in human serum for at least 14 days and maintains 90% or more of its antigen binding affinity.

[0222] II. Polynucleotides, Vectors and Hosts In a further aspect, the invention provides a nucleic acid encoding any of the above anti-TF antibodies or fragments thereof. Further provided are a vector comprising such a nucleic acid, and a host cell comprising such a nucleic acid or such a vector.

[0223] In some embodiments, the invention provides nucleic acid molecules comprising polynucleotides encoding at least one CDR region, and all three common CDR regions, from the heavy chain VH or light chain VL sequence of an antibody that binds to TF as described above. In some further embodiments, the invention provides nucleic acid molecules comprising polynucleotides encoding the complete or essentially complete variable region sequence of the heavy and / or light chain of an anti-TF antibody as described above. As will be apparent to one of skill in the art, due to codon degeneracy, the amino acid sequence of each antibody or polypeptide can be encoded by many different nucleic acid sequences.

[0224] In one embodiment, the present invention provides one or more vectors comprising the nucleic acid of the present invention, including cloning vectors and expression vectors. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors that can be used in the present invention include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs).

[0225] In one embodiment, the invention provides a host cell comprising the vector of the invention. Suitable host cells for vectors for cloning or expressing the encoded antibody include prokaryotic or eukaryotic cells. For example, the antibody may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. After expression in bacteria such as E. coli, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified. In a further embodiment, the host cell is a eukaryotic cell. In another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the preparation of antibodies or antigen-binding fragments thereof. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (293HEK or 293 cells), Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, and myeloma cell lines such as Y0, NS0, and Sp2 / 0.

[0226] III. Preparation of Antibodies In a further aspect, the invention provides a method for preparing an anti-TF antibody of the invention.

[0227] In one embodiment, the method of the invention comprises culturing a host cell comprising a nucleic acid encoding an antibody of the invention under conditions suitable for expression of the antibody, and optionally recovering said antibody from said host cell (or host cell medium). For recombinant production of an anti-TF antibody, an isolated, artificially synthesized or recombinantly synthesized nucleic acid encoding an antibody (e.g., an antibody as described above) can be inserted into one or more vectors for further cloning and / or expression in a host cell.

[0228] In further embodiments, the invention further provides anti-TF antibodies or antigen-binding fragments thereof, having one or more of the following characteristics, and methods for their preparation: (i) binds to the same or overlapping epitope as any one of the antibodies of the invention; (ii) competitively binding any one of the antibodies according to the present invention to human TF; (iii) inhibiting (e.g. competitively inhibiting) the binding of any one of the antibodies according to the invention to cells expressing human TF on their cell surface. Among them, said antibodies according to the invention are in particular the antibodies 2B12B10-hz1, 30G11B7-hz1, 22F11H5-hz1 or 27H8H3-hz1, Preferably, the preparation method includes screening an antibody mixture for an antibody having the above-mentioned properties in a binding affinity measurement test using the antibody according to the present invention as a reference, where usable antibody mixtures include, but are not limited to, antisera from TF-immunized animals, or antibody libraries for yeast or mammals.

[0229] IV.Measurement method The anti-TF antibodies provided herein can be identified, screened or otherwise characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art.

[0230] Suitable methods that can be used to determine the binding of an antibody to a TF antigen are known in the art. For example, the affinity of an antibody to a protein (e.g., a fusion protein) comprising a TF extracellular domain or to a TF-positive tumor cell can be easily determined using conventional techniques known to the skilled artisan. Such methods include, but are not limited to, ELISA-based measurement tests, BIAcore2000 or ForteBio Octet® instruments, or radioimmunoassay tests using radiolabeled target antigens, or cell ELISA or FACS techniques or biofilm layer interference techniques as described in the examples of the present application. Preferably, the measurement of affinity parameters herein is performed according to or essentially according to the methods described in the examples.

[0231] Suitable methods for measuring the endocytosis activity of antibody are known in the art.For example, in cell-based assays, particularly in TF-positive tumor cell-based assays, the endocytosis rate can be determined after antibody binding to the cell surface.Preferably, in this specification, the endocytosis activity of antibody is measured according to or essentially according to the method described in the examples.

[0232] Suitable methods for measuring the effect of an antibody on blood clotting are known in the art. For example, the change in blood clotting parameters, such as blood clotting time, in the presence of an antibody compared to a control (e.g., a negative control or a positive control) can be measured to reflect the effect of an antibody on blood clotting. In the present specification, the effect of an antibody on blood clotting is preferably performed according to or essentially according to the method described in the Examples.

[0233] V. Immunoconjugates and Immunofusions In a further aspect, the present invention provides immunofusions or immunoconjugates produced by fusing or conjugating an antibody of the invention to a heterologous molecule.

[0234] In one embodiment, in the immunofusion, the antibody (or antigen-binding fragment thereof) of the invention is linked directly or via an amino acid linker to a heterologous peptide or polypeptide molecule. Heterologous peptides or polypeptides that may be mentioned include, but are not limited to, proteins or polypeptides that confer another functional activity to the fusion, or tag peptides that facilitate purification or detection of the immunofusion.

[0235] In one embodiment, the antibody (or antigen-binding fragment thereof) of the present invention is conjugated to a therapeutic agent or a diagnostic or detectable agent in an immunoconjugate. In some embodiments, the antibody of the present invention can be conjugated to a heterologous molecule in the form of a full-length antibody or an antibody fragment. In the conjugate, the different entities of the conjugate can be covalently linked by a linker. Suitable linkers include chemical linkers or peptide linkers. Advantageously, the linker is a "cleavable linker" that facilitates the release of the polypeptide after delivery to the target site. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker or a disulfide-containing linker can be used.

[0236] In embodiments where a therapeutic agent is conjugated, suitable therapeutic agents for conjugation include, but are not limited to, a cytotoxin (eg, a cell growth inhibitor or cell killing agent), a drug, or a radioisotope.

[0237] In embodiments conjugated to a diagnostic or detectable agent, such conjugates may be used as part of a clinical testing methodology to monitor or predict the onset, formation, progression and / or severity of a disease or condition (e.g., to determine the efficacy of a particular therapy). Such diagnosis and detection can be accomplished by coupling the antibody to a detectable agent, including, but not limited to, several enzymes such as horseradish peroxidase, prosthetic groups such as streptavidin / biotin and avidin / biotin, fluorescent materials, luminescent materials, radioactive materials, and positron-emitting metals and non-radioactive paramagnetic metal ions for use in various positron emission imaging techniques.

[0238] VI. Multispecific antibodies In a further aspect, the invention provides a multispecific (including bispecific) antibody molecule that specifically binds to TF. In one embodiment, in a multispecific antibody, an antibody of the invention (or an antigen-binding fragment thereof) forms a first binding specificity for TF. In a further embodiment, the multispecific antibody further comprises a second binding specificity, or further comprises a second and a third binding specificity for two different molecules. The second and third binding specificities may, for example, be for another antigen expressed on the surface of a tumor cell, or for an antigen expressed on the surface of a T cell.

[0239] In such multispecific molecules, the binding specificity is preferably provided by an antibody "combining site" or "antigen-binding site" (the region of an antibody molecule that actually binds to the antigen). More preferably, the antigen-binding site consists of a VH / VL pair consisting of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0240] VII. Pharmaceutical Compositions and Formulations The present invention further provides compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising anti-TF antibodies or immunoconjugates / fusions or multispecific antibodies thereof, as well as compositions comprising polynucleotides encoding anti-TF antibodies or immunoconjugates / fusions or multispecific antibodies thereof. These compositions may optionally further comprise suitable pharmaceutical auxiliary materials, such as pharmaceutical vectors, pharmaceutical excipients, including buffers, known in the art. For the use of excipients and their applications, see "Handbook of Pharmaceutical Excipients", 5th Edition, RC Rowe, PJ Eskey and SCOwen, Pharmaceutical Press, London, Chicago. In some embodiments, pharmaceutical formulations comprising the present invention can be prepared by mixing the anti-TF antibodies, immunoconjugates or multispecific antibodies of the present invention having the desired purity with one or more optional pharmaceutical auxiliary materials (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980)).

[0241] In the pharmaceutical compositions and pharmaceutical formulations of the present invention, the antibody of the present invention may be the only active agent or may be combined with other therapeutic agents. The therapeutic agents that can be combined with the antibody of the present invention include, but are not limited to, therapeutic agents that have beneficial therapeutic effects on the disease and / or condition being treated. For example, the active ingredient may be one necessary for the particular indication being treated, and preferably, is an active ingredient that has complementary activities that do not adversely affect each other, such as other drug ingredients that can provide anti-cancer activity. The antibody of the present invention is present in the pharmaceutical compositions and pharmaceutical formulations in an amount effective for the intended use in suitable combination with the active ingredient.

[0242] VIII. Combination Products In a further aspect, the present invention further provides a combination product comprising the antibody or antigen-binding fragment thereof, multispecific antibody or immunoconjugate of the present invention, and one or more other therapeutic agents (e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, antitumor drugs, etc.). The components constituting the combination product, such as the antibody of the present invention and the other therapeutic agents, may be prepared in different formulations, and are preferably contained in different containers. The administration method and administration order of each component of the combination product can be determined by those skilled in the art depending on factors such as the disease to be treated and the individual's condition. The combination product of the present invention can be used in the treatment method of the present invention. In some embodiments, the present invention provides a combination product, in which the other therapeutic agent is, for example, a therapeutic agent, such as an antibody, that effectively stimulates the immune response, thereby further enhancing, stimulating, or upregulating the immune response of the subject. In some embodiments, the combination product is used for the prevention or treatment of TF-positive tumors.

[0243] IX. Methods and Uses In one aspect, the invention provides methods and uses of the TF antibodies or antigen-binding fragments thereof of the invention, e.g. in vivo and in vitro: (1) targeting TF-positive tumor cells; and / or (2) blocking TF / VIIa-mediated downstream signaling pathways in TF-positive tumor cells; and (3) Used to inhibit and / or kill TF-positive tumor cells.

[0244] In some embodiments, the methods and uses of the present invention relate to the treatment of a disease in a subject individual. In some further embodiments, the methods and uses of the present invention relate to detecting the presence of TF, for example, in a sample derived from a subject. In some further embodiments, the present invention further provides the use of an anti-TF antibody or an antigen-binding fragment thereof of the present invention in the preparation of a product for the above uses (for example a pharmaceutical composition, or a pharmaceutical product, or a combination product, or a detection product).

[0245] In one aspect, the present invention provides a method and use for preventing and / or treating TF positive tumors in a subject by application of the antibody or antigen-binding fragment of the present invention, comprising administering the antibody or antigen-binding fragment of the present invention in a prophylactically and / or therapeutically effective amount. In another aspect, the present invention further provides a method and use for treating or preventing other diseases associated with elevated expression of cell membrane TF in a subject by application of the antibody or antigen-binding fragment of the present invention, comprising administering to a subject in need thereof an anti-TF antibody or antigen-binding fragment thereof of the present invention.

[0246] In further aspects, the present invention provides a method and a reagent kit for detecting TF in a sample, the method comprising: (a) contacting the sample with an antibody or antigen-binding fragment thereof or an immune complex / fusion of the present invention; and (b) detecting the formation of a complex between the antibody or antigen-binding fragment thereof or immune complex and a TF protein. In some aspects, the sample is from a cancer patient. The detection may be in vitro or in vivo.

[0247] The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads conjugated with antibody molecules, ELISA assays, PCR-techniques (e.g., RT-PCR). In some embodiments, the biological sample is blood, serum or other liquid sample from a living organism. In some embodiments, the biological sample comprises cells or tissues. In some embodiments, the biological sample is from a hyperproliferative or cancerous lesion. In some embodiments, the TF to be measured is human TF.

[0248] In one embodiment, the anti-TF antibodies are used to select subjects suitable for treatment with anti-TF antibodies. In a further embodiment, the antibodies of the invention can be used to diagnose cancer or tumors, e.g., to assess (e.g., monitor) the treatment or progression, diagnosis and / or stage of a disease described herein (e.g., a hyperproliferative or cancerous disease) in a subject.

[0249] In some embodiments, a labeled anti-TF antibody is provided. Labels include, but are not limited to, directly detected labels or moieties (e.g., fluorescent labels, chromophore labels, electron dense labels, chemiluminescent labels and radioactive labels) and indirectly detected moieties, such as enzymes or ligands, such as by enzymatic reactions or molecular interactions. Exemplary labels include the radioisotopes 32P, 14C, 125I, 3H, and 131I, rare earth chelates or fluorophores, such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases, such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), fluorescein, 2,3-dihydrophthalazinedione, horseradish peroxidase (HR), alkaline phosphatase, and the like. Examples of suitable enzymes include, but are not limited to, sugar oxidases such as catalyse, β-galactosidase, glucoamylase, lytic enzymes, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, and enzymes that utilize hydrogen peroxide to oxidize dye precursors such as HR, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, phage labels, and stabilized radicals.

[0250] Therapeutic methods and uses of the present invention Based on the good targeting properties of the anti-TF antibody of the present invention or the ADC of the present invention against TF and the above-mentioned excellent properties, the present invention further provides applications and methods of the ADC of the present invention and the antibody of the present invention in the treatment and prevention of diseases associated with TF.

[0251] TF is overexpressed on the surface of cancer tissue cells of many origins, making it a suitable target for the development of cancer immunotherapy. In one aspect, the present invention therefore provides the application of the ADC of the present invention or a pharma- ceutically acceptable salt or solvate thereof, and the antibody or antigen-binding fragment thereof of the present invention, for preventing and / or treating TF-positive tumors in a subject. In the above applications, the antibody-drug conjugate of the present invention or a pharma- ceutically acceptable salt or solvate thereof, or the antibody or antigen-binding fragment thereof of the present invention is administered to a subject as the sole active agent or in combination with other therapies or therapeutic agents. The above other therapies and therapeutic agents include, for example, drugs that target antigens on the surface of tumor cells and eliminate tumors by binding to and / or blocking these molecules, and drugs that activate the subject's immune system and promote it to eliminate tumors spontaneously.

[0252] In a further aspect, the invention further provides a method of preventing or treating a TF positive tumor in a subject, the method comprising administering to a subject in need thereof an ADC of the invention, or a pharma- ceutically acceptable salt or solvate thereof, or administering to a subject in need thereof an anti-TF antibody of the invention, or an antigen-binding fragment thereof.

[0253] TF positive tumors suitable for the methods and applications of the present invention may be selected from myeloma (e.g. multiple myeloma), acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia (AML) and non-Hodgkin's lymphoma, central nervous system tumors, gliomas, brain cancer, head and neck cancer such as head and neck squamous cell carcinoma, gastrointestinal cancer, genitourinary cancer, lung cancer such as NSCLC, esophageal cancer, gastric cancer, hepatobiliary cancer, pancreatic cancer, colorectal cancer, rectal cancer, bladder cancer, renal cancer, breast cancer, especially triple negative breast cancer, prostate cancer, endometrial cancer, ovarian cancer, cervical cancer, melanoma, sarcoma, and skin cancer.

[0254] TF positive tumors suitable for the methods and applications of the present invention may be early, intermediate or late stage, or metastatic cancers, and may also be previously treated immune evasive tumors.

[0255] In some embodiments, the TF positive tumor treated by the methods of the invention may be selected from cervical cancer, pancreatic cancer, lung cancer, prostate cancer, bladder cancer, ovarian cancer, breast cancer, colorectal cancer, esophageal cancer, head and neck cancer, gastric cancer, including primary or advanced or metastatic cancer. In further embodiments, the TF positive tumor is cervical cancer. In another embodiment, the TF positive tumor is non-small cell lung cancer. In further embodiments, the TF positive tumor is esophageal squamous cell carcinoma. In further embodiments, the TF positive tumor is breast cancer.

[0256] In some embodiments, TF-positive tumors treated by the methods of the invention have at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100% TF-positive cells. Tissue factor expression levels in tumor biopsies can be assessed by immunohistochemistry. High percentages of TF-positive cells have already been detected in biopsies of many types of cancer, e.g., cervical cancer (100%), non-small cell lung cancer (34%-88%), endometrial cancer (14%-100%), prostate cancer (47%-75%), ovarian cancer (75%-100%), esophageal cancer (43%-91%), and bladder cancer (78%).

[0257] Preferably, in some embodiments, the method according to the invention is used to treat tumors with a high percentage of TF positive cells, e.g., tumors with at least 25%, 50%, 75% or 100% TF positive cells, e.g., tumors with a range of 25%-50% to 75%-100% TF positive cells, such as bladder cancer, lung cancer, pancreatic cancer, prostate cancer, ovarian cancer, and cervical cancer, etc. In some embodiments, application of the method according to the invention in the above cancers induces tumor regression.

[0258] In some further embodiments, the method according to the invention can be used to treat cancers in which the percentage of TF positive cells is lower than 25% or 20%. In some embodiments, application of the method of the invention in said cancers results in inhibition of tumor growth.

[0259] In a further aspect, the invention further provides a method for preventing or treating other diseases associated with elevated cell membrane TF expression, comprising administering to a subject in need thereof an anti-TF antibody or antigen-binding fragment thereof of the invention, or an ADC of the invention, or a pharma- ceutically acceptable salt or solvate thereof, including, but not limited to, benign tumors, vascular diseases such as neurofibromas, hemangiomas, atherosclerosis, retinopathy and macular degeneration, inflammatory diseases such as rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, and autoimmune inflammation such as multiple sclerosis.

[0260] In any one of the above embodiments of the method of the invention, the administration of the antibody or binding fragment thereof according to the invention and the ADC or pharma- ceutically acceptable salt or solvate thereof according to the invention can include 1) a therapeutic measure to cure, alleviate, relieve symptoms of a diagnosed pathological condition or disease and / or halt the progression of the diagnosed pathological condition or disease, or 2) a prophylactic or preventative measure to prevent and / or alleviate the progression of a pathological condition or disease. Thus, in the method of the invention, the subject may be an individual already suffering from a disease, an individual susceptible to a disease, or an individual for whom prevention of a disease is intended. The individual will benefit from the therapeutic or preventative measure and will show a reduction or improvement in the occurrence, recurrence, or progression of a disease, condition, pathology, and / or symptom compared to an individual not subjected to the treatment. In some embodiments, the invention relates to the treatment of a disease or condition, and in some other embodiments, the invention relates to the prevention of a disease or condition.

[0261] The antibodies or binding fragments thereof according to the invention and the ADCs or pharma- ceutically acceptable salts or solvates thereof according to the invention, and other therapeutic agents optionally used in combination therewith, can be administered by any suitable method, including parenteral administration, intratumoral administration, and intranasal administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. The present specification includes various administration schedules, including, but not limited to, a single dose or multiple doses at multiple time points, bolus administration, and pulse infusion.

[0262] Appropriate doses of the antibodies or binding fragments thereof and ADCs according to the invention, or pharma- ceutically acceptable salts or solvates thereof, according to the invention for preventing or treating a disease will depend (when used alone or in combination with one or more other therapeutic agents) on the type of disease being treated, the specific type of drug used, the severity and progression of the disease, whether the drug is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibodies or binding fragments thereof and ADCs according to the invention, or pharma- ceutically acceptable salts or solvates thereof, according to the invention can be administered to the patient in one treatment or over a series of treatments.

[0263] In some embodiments, prior to receiving treatment according to the methods of the invention, the subject is undergoing or has undergone other treatments, such as chemotherapy and / or radiation therapy, or other immunotherapy.

[0264] In the above-described embodiments of the methods and applications of the invention, a composition, multispecific antibody or immunoconjugate / fusion, or combination product of the invention can be administered instead of an antibody or antigen-binding portion thereof of the invention, or instead of an ADC of the invention or a pharma- ceutically acceptable salt or solvate thereof. Alternatively, in these methods, in addition to administration of an antibody or antigen-binding portion thereof, or an ADC of the invention or a pharma- ceutically acceptable salt or solvate thereof, a composition, multispecific antibody or immunoconjugate / fusion, or combination product of the invention can be further administered.

[0265] In some embodiments, the invention further provides the use of an ADC of the invention, or a pharma- ceutically acceptable salt or solvate thereof, in the preparation of a medicament for use in the above-mentioned treatment and prophylaxis methods. In some further embodiments, the invention further provides the use of an antibody or antigen-binding fragment, composition, immunoconjugate / fusion, multispecific antibody of the invention in the preparation of a medicament for use in the above-mentioned treatment and prophylaxis methods.

[0266] Any or all of the features described above and throughout this application may be combined in various embodiments of the invention. The following examples further illustrate the invention, but it should be understood that the examples are for illustrative purposes and should not be construed as limiting in any way.

[0267] Working Example Example I Preparation and characterization of anti-TF humanized antibodies Common methods Recombinant expression of cytokine antigens The amino acid sequence of human tissue factor was referenced to P13726 in the Uniprot protein database, and the extracellular segment was aa 33-aa 251. The amino acid sequence of cynomolgus tissue factor was referenced to A0A2K5VXA0 in the Uniprot protein database, and the extracellular segment was aa 33-aa 252.

[0268] The amino acid sequences of the extracellular segments were codon-optimized by General Biology (Anhui) Company and synthesized into the expression vector pTT5-His. HEK293-EBNA cells were transiently transfected with PEImax (Polysciences, 24765-1), and after 7 days of expression, human TF-his (h.TF-His) and cynomolgus TF-his (c.TF-His) antigens were obtained by purification through nickel column. The recombinantly expressed and purified TF antigens were used as immunogens or in ELISA assays in the subsequent examples.

[0269] Antigen protein-based ELISA TF antigen (h.TF-His or c.TF-His antigen) in buffer (CBS coating solution) was coated with 50 mM carbonate, 1 μg / mL coating concentration, 100 μL volume, 4 °C overnight on ELISA plates. The next day, the coating solution was removed, each well was washed once with 300 μL PBS, 100 μL of 2% BSA (Biotopped, A6020) in PBS was added to each well, and blocked at 37 °C for 2 h. The samples to be measured were gradient diluted with 2% BSA in PBS to establish 11 concentration points. 100 μL of the gradient diluted samples were added to the plate and incubated at 37 °C for 2 h, the supernatant was removed, each well was washed three times with 300 μL PBST, 100 μL of HRP-conjugated secondary antibody in 2% BSA was added to each well, and incubated at 37 °C for 1 h. For the hybridoma-derived mouse antibodies, HRP-labeled anti-mouse secondary antibodies (Jackson, 115-035-003) were used, and for the humanized antibodies, HRP-labeled anti-human secondary antibodies (Jackson, 109-035-088) were used. After the incubation, the secondary antibodies were removed and each well was washed five times with 300 μL of PBST, followed by adding 100 μL of TMB substrate (Huzhou InnoReagents, TMB-S-004) to each well and allowing the color to develop for 5 to 20 minutes. The reaction was stopped by adding 50 μL of 2N HCl to each well, and the readings at OD450 nM were read using a microplate reader (manufacturer: MD, model number: SpectraMax), which was then introduced into GraphPad software to perform fitting mapping and determine the binding of the sample to the TF antigen protein.

[0270] Cell-based ELISA On the day before the experiment, TF antigen-positive cells (MDA-MB-231 or NCI-H358 cells) were added at 5 × 10 4Cells / well were seeded in a 96-well plate. On the day of the experiment, the culture supernatant was removed, each well was washed once with 300 μL of PBS, and 100 μL of 4% paraformaldehyde was added to each well and fixed at room temperature for 20 minutes. Paraformaldehyde was removed, each well was washed twice with 300 μL of PBS, and 100 μL of 2% BSA in PBS was added to each well and blocked at 37°C for 2 hours. After blocking was completed, 30 μL of samples to be measured (hybridoma supernatant or purified antibody) was added to each well and incubated at 37°C for 2 hours. The liquid was removed, each well was washed three times with 300 μL of PBST, and 100 μL of HRP-conjugated secondary antibody in 2% BSA was added to each well and incubated at 37°C for 1 hour. If the sample to be measured was a hybridoma or mouse antibody, an HRP-labeled anti-mouse secondary antibody (Jackson, 115-035-003) was used, and if the sample to be measured was a humanized antibody, an HRP-labeled anti-human secondary antibody (Jackson, 109-035-088) was used. The secondary antibody was removed, and each well was washed five times with 300 μL of PBST, followed by adding 100 μL of TMB substrate (Huzhou InnoReagents, TMB-S-004) to each well and allowing the color to develop for 5 to 20 minutes. The reaction was stopped by adding 50 μL of 2N HCl to each well, and the values ​​at OD450 nm were read using a microplate reader (manufacturer: MD, model number: SpectraMax) to determine the binding of the sample to TF-positive cells.

[0271] Detection of antibody purity by SEC-HPLC For recombinantly expressed and purified antibodies, SEC-HPLC was used to detect antibody purity. The detection method was as follows. Instrument: Waters Alliance e2695 HPLC; Column: Thermo MabPac SEC-1, 5 μm, 7.8×300 mm, Mobile phase: 61 mmol / L Na2HPO4, 39 mmol / L NaH2PO4, 200 mmol / L NaCl, 5% IPA, Instrument parameters: sample chamber temperature: 8°C, column temperature: 30°C, flow rate: 0.5 mL / min, injection volume: 20 μg, detection wavelength: 280 nm, isocratic operation: 30 min.

[0272] Detection of antibody hydrophilicity and hydrophobicity by HIC The hydrophilicity or hydrophobicity of the recombinantly expressed and purified antibodies was detected by HIC as follows.

[0273] Hydrophilicity and hydrophobicity were detected on an Agilent HPLC system using a Tosoh hydrophobic column (TOSOH Tskgel Buty-NPR (2.5), 4.6 × 100). Mobile phase A was 1.5 M (NH4)2SO4, and mobile phase B was 25 mM Na2HPO4 (pH = 7.0) + 25% IPA. The instrument parameters were set as follows: sample chamber temperature 8 °C, column temperature 30 °C, flow rate 0.5 mL / min, and detection wavelength 280 nm. The sample to be measured was diluted with mobile phase A to a final concentration of 1 mg / mL, and 20 μL was injected to perform gradient elution. The elution gradient was as follows: [Table 1A]

[0274] Three female mice aged 5 to 6 weeks were selected from each of three breeds: Balb / C, Kunming (KM), and CD01. They were immunized with human TF-his protein and cynomolgus monkey TF-his protein. The immunization protocol is shown in Table 1. [Table 1B]

[0275] After two boosts, the serum titers of the immunized animals were detected by antigen protein-based ELISA measurement using ELISA plates coated with h.TF-His and c.TF-His antigens, respectively, as described in the general method. Based on the detection results, taking into consideration the affinity of human and monkey TF comprehensively, CD1-immunized mice and Balb / c-immunized mice were selected for hybridoma fusion, and the final boost was performed before hybridoma fusion.

[0276] Cell suspensions were prepared from mouse spleens and lymph nodes, mixed with SP2 / 0 mouse myeloma cells at a 1:1 ratio, resuspended in cell electrofusion buffer, and electrofusion reaction was performed using a BTX-ECM2001 cell electrofusion device. After electrofusion reaction, the cells were resuspended in complete fusion medium (RPMI160 + 15% FBS + 1xHAT), sorted into 96-well cell culture plates at 20,000 to 25,000 cells / well, and cultured at 37℃, 5% CO2.

[0277] After 7 days of culture, ELISA rescreening was performed sequentially using ELISA plates coated with human TF-his and cynomolgus TF-his, and the cells were ELISA rescreened using TF-positive tumor cells (MDA-MB-231 or NCI-H358).Positive parental clones were selected and used for subcloning.

[0278] For each parent clone, 75 cells were sorted into a 96-well plate and subclone cultured. Then, ELISA and cell ELISA screening were performed sequentially by taking the subclone supernatant and coating human TF-his, cynomolgus monkey TF-his, rat TF-his protein and NCI-H358 cells. Based on the screening results, a total of 119 positive clones were selected and expanded in serum-free medium. The monoclonal supernatants of the cultured hybridomas were collected and purified by antibody purification medium ProA (GE, Mabselect XL) packing to obtain mouse antibodies.

[0279] The purified mouse antibodies were successively evaluated by ELISA and cell ELISA by coating human TF-his, cynomolgus monkey TF-his protein and NCI-H358 cells. The experimental method was as described in the general method, in which the mouse antibodies were started at 10 μg / mL and diluted three-fold to establish 11 concentration points. Based on the measurement results, 13 hybridoma clones with high affinity were selected and used for antibody sequencing and humanization.

[0280] Example 2 Sequencing and humanization of anti-TF mouse monoclonal antibody Approximately 1×10 5 Candidate hybridoma cells were collected, and RNA was extracted by Trizol (Invitrogen, 15596026), and then reverse transcribed by PolyA using PrimeScript RT reagent kit (TAKARA, RR047A) to obtain cDNA. Upstream primers were designed for the heavy chain and light chain upstream, respectively, and downstream primers were designed for the heavy chain CH1 region and light chain CL region. After amplifying the product by PCR, the fragments were recovered using an agarose gel recovery reagent kit, and the samples were sent to Beijing Tsingke Biotech Co., Ltd. for sequencing to obtain the mouse antibody sequence.

[0281] The mouse antigen sequence was humanized using the CDR grafting method. First, the human germline sequence with the highest homology to the original mouse sequence was found by the conventional BLAST method and used as a template. The CDR of the mouse antibody was grafted onto the human template to construct a chimera. The FR amino acids that can maintain the original conformation in the mouse antibody were analyzed according to structure, and the corresponding amino acids in the chimera were mutated back to mouse amino acids to maintain the original affinity. The constructed humanized antibody was subjected to calculation and immunogenicity analysis, and highly immunogenic fragments were found and replaced with low immunogenic fragments. The sequences of 17 types of humanized antibodies were obtained.

[0282] Example 3 Expression and purification of humanized antibodies The amino acid sequences of the heavy and light variable regions of each of the above humanized antibodies and reference antibodies (Benchmark, abbreviated as BM, Tisotumab of Genmab Company, the sequences of which are derived from SEQ ID NO: 5 (VH) and SEQ ID NO: 45 (VL) of patent CN103119065B) were provided to a common organism for gene synthesis, codon optimization, and then constructed into a PTT5 vector. After plasmid synthesis, HEK293E cells were transfected with PEImax, expressed for about 7 days, and the supernatant was collected by centrifugation. The supernatant was purified with MabSelectSure ProActive affinity packing (GE, catalog number Parr17547401). The expressed reference antibody had the same human IgG1 heavy chain constant region (SEQ ID NO: 106) and human Kappa light chain constant region (SEQ ID NO: 105) as the humanized antibody. All purified antibodies were ultrafiltered into PBS buffer, the concentration was measured, and stored at -20°C.

[0283] Example 4 ELISA affinity evaluation of humanized antibodies Antigen protein-based ELISA detection The detection was carried out as described in the general method. Briefly, the ELISA plate coated with human TF-his protein or cynomolgus monkey TF-his protein, respectively, was blocked with 2% BSA at 37°C for 2 hours, and the gradient-diluted antibody (starting from 10 μg / mL, 3-fold gradient dilution, 11 concentration points) was added and incubated at 37°C for 2 hours. Then, anti-human secondary antibody (Peroxidase-AffiniPure Goat Anti-Human IgG(H+L), Jackson, 109-035-088) was added and incubated at 37°C for 1 hour, and TMB substrate (Huzhou InnoReagents, TMB-S-004) was added for color development. After the color development reaction was completed, the absorbance value at 450 nM was read on the instrument, and the curve was fitted using GraphPad software.

[0284] Cell-based ELISA detection: The detection was carried out as described in General Methods. Briefly, TF antigen-positive NCI-H358 cells were collected by pancreatin digestion, and 5 × 10 4 Cells / well were seeded in a 96-well plate overnight, and then the cells were fixed with 4% paraformaldehyde. After removing the paraformaldehyde and washing the cells with PBS, the plate was blocked with 2% BSA at 37°C for 2 hours. Gradient-diluted antibodies (starting from 10 μg / mL, 3-fold gradient dilution, 11 concentration points) were added and incubated at 37°C for 2 hours. Then, anti-human secondary antibody (Peroxidase-AffiniPure Goat Anti-Human IgG(H+L), Jackson, 109-035-088) was added and incubated at 37°C for 1 hour, and TMB substrate (Huzhou InnoReagents, TMB-S-004) was added to develop color. After the color reaction was completed, the absorbance value at 450 nM was read on the instrument, and the curve was fitted using GraphPad software.

[0285] The experimental results are shown in Table 2 and Figure 1. Humanized antibodies 2B12B10-hz1, 4F6C3-hz1, 30G11B7-hz1, 22F11H5-hz1, 27B9F2-hz1 and 27H8H3-hz1 that showed good binding activity to human / cynomolgus monkey TF-his and TF positive cells were selected for further evaluation. [Table 2]

[0286] The humanized antibodies recombinantly expressed and purified with Protein A affinity as described above were subjected to SEC-HPLC to identify the purity of the antibodies, and HIC to detect the hydrophilicity of the antibodies, as described in the general method. The SEC and HIC detection results are shown in Table 3. The SEC results showed that the purity of the selected six humanized antibodies was relatively high, all of which was >90%, and the HIC results showed that the selected six humanized antibodies had relatively weak binding to the hydrophobic chromatography column, relatively short retention time, and excellent hydrophilicity. [Table 3]

[0287] TF low-expressing cell line NCI-H358 (source: Nanjing Kebai, CBP60136) and TF high-expressing cell line KYSE520 cells (source: Nanjing Kebai, CBP60658) were collected by pancreatin digestion and centrifugation, washed three times with pre-chilled PBS, and resuspended in 1% BSA to obtain 3 × 10 5 Cells were seeded in a 96-well V-bottom plate at 100 cells / well. Gradient-diluted antibodies were added and incubated at 4°C for 1 h, and then washed three times with pre-chilled PBS. Anti-human fluorescent secondary antibodies (APC anti-human IgG Fc Antibody, Biolegend, 410712) were added at 1 μL / well and incubated at 4°C for 0.5 h. After washing three times with pre-chilled PBS, the cells were resuspended and detected by a flow cytometer (Sony, LE-SA3800GA), and the data was imported into GraphPad software for curve fitting.

[0288] The experimental results are shown in Table 4 and FIG. 2, and all six candidate humanized antibodies strongly bound to tumor cells. [Table 4]

[0289] The dynamic affinity of the humanized antibodies was measured by ForteBio. The experimental steps are as follows: Take out the ProA sensor (18-5010, Octet) and pre-wet the sensor with PBST diluent (PH7.4) for 10 minutes. The antibody that needs to be immobilized was diluted to 5 μg / mL, and the antigens h.TF-His and c.TF-His were diluted 2-fold with 4 concentration points starting from 75 nM and a zero concentration point was set. The program was set up, the sensor plate and the sample plate were inserted, the program was started, and the sensor was regenerated with 20 mM glycine solution (pH1.7). The data was analyzed using Octet analysis software, and a result diagram was derived. The experimental results are shown in Table 5, and all six candidate humanized antibodies had high affinity for human and monkey TF. [Table 5]

[0290] The endocytosis activity of the humanized monoclonal antibodies was detected by flow cytometry (FACS). The experimental steps were as follows: NCI-H358 cells were collected by pancreatin digestion and centrifugation, and incubated with the antibody to be detected at a concentration of 10 μg / mL at 4°C for 1 h. After washing three times with PBS, the cells were resuspended in RPMI1640+10% FBS, and incubated in three portions at 37°C for 0, 2, and 4 h, respectively. After washing three times with PBS, 1 μL of anti-human fluorescent secondary antibody (APC anti-human IgG Fc Antibody, Biolegend, 410712) was added and incubated at 4°C for 0.5 h, and the cells were resuspended after washing three times with PBS and detected by the instrument. Isotype control negative antibody HLE IgG1 was used as a negative control. The endocytosis rate was calculated according to the formula: Endocytosis rate (%) = {1 - (mean fluorescence value of the sample detected at that time point - mean fluorescence value of the negative control sample at that time point) / (mean fluorescence value of the sample detected at 0 hours - mean fluorescence value of the negative control sample at 0 hours)} x 100.

[0291] The experimental results are shown in Table 6. The endocytosis rates of 2B12B10-hz1, 4F6C3-hz1, 30G11B7-hz1, 22F11H5-hz1, and 27B9F2-hz1 were basically equivalent to those of the reference antibody, and the endocytosis rate of 27H8H3-hz1 was superior to that of the reference antibody. [Table 6]

[0292] Binding of FVIIa to tissue factor on the surface of TF-positive tumor cells MDA-MB-231 activated the downstream signal pathway of TF, resulting in the release of IL-8. Therefore, detection of the release of IL-8 can reflect the blocking activity of anti-TF humanized antibodies against the TF signal pathway.

[0293] The experimental steps were as follows: FVIIa (40 nM, Haematologic Technologies Inc, HCVIIA-0031), gradient-diluted anti-TF humanized antibody (starting at 60 μg / mL, 3-fold gradient dilution, 11 concentration points) was incubated with MDA-MB-231 cells (5 × 10 4After co-incubation with 100 μg / well of 1000 μg / mL ... The IL-8 detection steps are as follows: the night before the experiment, coat 96-well plates with 200 ng / well of capture antibody MT8H6 in PBS, leave at 4°C overnight, remove coating solution, wash once with 300 μL PBST, add 100 μL of 2% BSA in PBS to each well of all 96-well plates, block at 37°C for 2 h, add 50 μL of cell culture supernatant directly, incubate at 37°C for 2 h, remove supernatant, wash each well three times with 300 μL PBST, add 100 μL (1 μg / mL) of MT8F19-biotin secondary antibody (in 2% BSA) to each well, incubate at 37°C for 1 h, remove supernatant, wash each well four times with 300 μL PBST, and add 100 μL of 1 μg / mL ... 1 μL of peroxidase-conjugated streptavidin (diluted 1:10000) was added and incubated at 37°C for 1 hour, the supernatant was removed, each well was washed 5 times with 300 μL of PBST, then 100 μL of TMB substrate was added to each well, color was developed for 5-20 minutes, 50 μL of 2 N HCl was added to each well to stop the reaction, and the OD450 mm was read on a microplate reader. The results were imported into GraphPad and plotted, and the inhibition rate and IC50 were calculated.

[0294] The experimental results are shown in Table 7 and Figure 3. In addition to 27B9F2-hz1, the remaining five humanized antibodies all had relatively high inhibitory effects on the TF signal pathway. [Table 7]

[0295] Anti-TF humanized antibody was diluted in HEPES solution containing CaCl2, then incubated with 50,000 TF positive MDA-MB-231 cells at 37°C for 30 min, then 50 μL human plasma was added, mixed quickly and homogenously, and the absorbance value at 405 nm was immediately and continuously read to calculate the anticoagulant effect of the blood clot initiated from cell surface TF. The results were imported into GraphPad and plotted. A total of two experiments were performed: the first experiment with a CaCl2 concentration of 2 mM and a final antibody concentration of 50 μg / mL, and the second experiment with a CaCl2 concentration of 5 mM and a final antibody concentration of 20 μg / mL. The experimental results are shown in Figure 4 (Figure 4A: 2 mM Ca 2+ , antibody concentration 50 μg / mL, Figure 4B: 5 mM Ca 2+ , antibody concentration 20 μg / mL) and are shown in Table 8 below. Taking the results of the two experiments together, among the antibodies tested, 27H8H3-hz1 and 2B12B10-hz1 had the least effect on human blood coagulation and were superior to the reference antibodies. [Table 8]

[0296] Example 11. Stability evaluation of humanized antibodies Accelerated stability assessment of humanized antibodies The anti-TF humanized antibody samples were ultrafiltered into PBS (pH=7.4) at a concentration of 5 mg / mL, filtered to sterilize, and then left at 37°C for 0, 3, 7, and 14 days, respectively, and subjected to four and eight freeze-thaw cycles.Then, the SEC-HPLC and CE-SDS purity of the samples were detected as described in General Methods.

[0297] The experimental results showed that the 22F11H5-hz1 and 27H8H3-hz1 humanized antibodies had good stability when detected in the case of accelerated stability and repeated freeze-thawing. After the accelerated stability and repeated freeze-thawing tests, the SEC purity of the two humanized antibodies was maintained above 95%, the CE-SDS non-reduced purity was maintained above 87%, and the CE-SDS reduced purity was above 95%.

[0298] Measurement of Tm value of humanized antibody The Tm value of the humanized antibody was measured using DSF to reflect the thermal stability of the antibody. The experimental steps are as follows: the antibody sample to be measured was diluted to 1 mg / mL with PBS, and the dye SYPRO Orange dye (Thermo#56651) was diluted 40 times with ddH2O. The reaction system was: 12.5 μL of sample + 2.5 μL of 40× dye + 5 μL of ddH2O, the membrane was sealed, centrifuged briefly, and detected by Q-PCR, and the Q-PCR parameters were set as follows: Target (ROX), program (25℃, 3 min, speed 1%, 95℃, 95℃, 2 min).

[0299] The results showed that the five candidate humanized antibodies 2B12B10-hz1, 4F6C3-hz1, 30G11B7-hz1, 22F11H5-hz1, and 27H8H3-hz1 all had relatively high Tm values ​​(all higher than 70°C), indicating that they had relatively good thermal stability.

[0300] Example 12 Serum stability evaluation of humanized antibodies The anti-TF humanized antibody was diluted to 20 μg / mL in human serum and left at 37°C for 14 days, and then the affinity change was detected by ELISA based on the antigen protein. The experimental steps of the ELISA method are described in General Methods. The experimental results are shown in Figures 5A and 5B, and the affinity of 22F11H5-hz1 and 27H8H3-hz1 did not obviously change after leaving them in human serum for 14 days.

[0301] Example 13 Detection of in vivo PK of humanized antibodies in rats The samples awaiting detection were administered to male rats (Chengdu Dossy) at a dose of 10 mg / kg, 3 rats per group, via tail vein injection. After administration, serum samples were collected at 30 min, 1 h, 2 h, 6 h, 24 h, 48 h, 96 h, 168 h, and 336 h. The corresponding antibodies were diluted with 2% BSA containing negative rat serum, starting from 1 μg / mL, and diluted 2-fold gradient, resulting in 11 concentration points, which were used as standard curve samples. Blood drug concentrations were detected by ELISA based on antigen protein, and the ELISA method was described in General Methods. The experimental results showed that the half-life of humanized antibodies 22F11H5-hz1 and 27H8H3-hz1 in rats was about 120 hours or more.

[0302] Based on the above antibody characterization, four candidate humanized antibodies, 2B12B10-hz1, 30G11B7-hz1, 22F11H5-hz1 and 27H8H3-hz1, were selected and used for subsequent ADC construction and performance characterization. The sequences of the four selected antibodies are shown in the sequence listing.

[0303] Example II Preparation and Characterization of Antibody Conjugated Drugs (ADCs) Example 1 Preparation of Antibody Conjugate Drugs (ADCs) Example 1.1: Synthesis of intermediates used in the synthesis of "Drug-Linker Compounds" Example 1.1.1: Synthesis of (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1) [ka]

[0304] Step 1: Synthesis of (S)-7-ethyl-7-hydroxy-14-(3-chloropropyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1B) [ka]

[0305] Under ice bath conditions, compound (S)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1A, Cas No.:151636-76-9, 500 mg) was added to a 75% sulfuric acid solution (5 mL) of iron(II) sulfate heptahydrate (570 mg of iron(II) sulfate heptahydrate was dissolved in 1 mL of water) and 4,4-dimethoxychlorobutane (3.89 g) and the reaction solution was stirred for 3 minutes, and then hydrogen peroxide solution (29%, 2.5 mL) was added dropwise. The reaction solution was stirred at 0°C for 5 minutes, then warmed to room temperature, and stirred for 3 hours to react. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (80 mL x 2). The organic phase was washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was further purified using a C18 column (acetonitrile / 0.05% formic acid aqueous solution: 5% to 60%) to obtain the target compound A1B (yellow solid, 400 mg, yield: 67%). LCMS (ESI) [M+H] + :468.9, 1 H NMR (400 MHz, DMSO-d6) δ 7.65 (s, 1H), 7.51 (s, 1H), 7.24 (s, 1H), 6.50 (s, 1H), 6.30 (s, 2H), 5.42 (s, 2H), 5.26 (s, 2H), 3.81 (d, J = 5.9 Hz, 2H), 3.22 (s, 2H), 1.98 (d, J = 6.7 Hz, 4H), 0.88 (t, J = 7.2 Hz, 3H).

[0306] Step 2: Synthesis of (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1) [ka]

[0307] The compound (S)-7-ethyl-7-hydroxy-14-(3-chloropropyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (100 mg, 0.213 mmol) was dissolved in 10% sulfuric acid (5 mL) solution, and the reaction was allowed to react for 48 hours at 110° C. Saturated sodium bicarbonate (30 mL) solution was added to the reaction solution, extracted with dichloromethane (10 mL×5), dried over anhydrous sodium sulfate, suction filtered, and concentrated under reduced pressure to obtain a crude product. Purification by preparative high-performance liquid chromatography (acetonitrile / water containing 0.05% formic acid) gave (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1, 1.78 mg). LCMS (ESI) [M+H] + :451.0, 1 H NMR (400 MHz, DMSO-d6) δ 7.63 (s, 1H), 7.50 (s, 1H), 7.24 (s, 1H), 6.48 (s, 1H), 6.28 (s, 2H), 5.47 - 5.37 (m, 2H), 5.32 - 5.19 (m, 2H), 3.51 - 3.46 (m, 2H), 3.17 - 3.13 (m, 2H), 1.92 - 1.76 (m, 4H), 0.90 - 0.84 (m, 3H).

[0308] Example 1.1.2: Synthesis of (S)-2-amino-N-((3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)propoxy)methyl)acetamide (B1) [ka]

[0309] Step 1: Compound (B1A) (Cas No.: 1599440-06-8, 368 mg), compound (A1) (440 mg) and pyridinium p-toluenesulfonate (PPTS) (25 mg) were refluxed in dichloromethane (20 mL) for 20 hours, then washed with aqueous sodium bicarbonate and aqueous hydrochloric acid, respectively, and the organic solvent was removed under reduced pressure to obtain a crude product. The crude product was isolated and purified by column chromatography (dichloromethane:methanol = 10 / 1) to obtain the target compound B1B (240 mg). LCMS (ESI) [M+H] + :759.5.

[0310] Step 2: B1B (240 mg) was dissolved in DMF (5 mL), piperidine (1 mL) was added, the compound was stirred for 20 min, the solution was decompressed to remove low boiling components, and the residue was directly used for the synthesis of the next step. After a small amount of the crude product was purified by reverse phase chromatography (acetonitrile / 0.05% FA in water: 5%-50%), the target compound B1 was obtained. ESI-MS (m / z): 537.4 [M+H] + , 1 H NMR (400 MHz, DMSO-d6) 。 δ 。9.13 (t, 1H), 8.04 (br, 2H), 7.58(s, 1H), 7.51(s, 1H), 7.25(s, 1H), 6.29(s, 2H), 5.43(S, 2H), 5.21(s, 2H), 4.65(d, 2H), 3.63(m, 2H), 3.53(m, 2H), 3.11(m, 2H), 1.87(m, 4H), 0.88(t, 3H).

[0311] Example 1.1.3: N 6 ,N 6 -Dimethyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valine)-L-lysine (C1) [ka]

[0312] 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (268 mg, Cas No. 2356229-58-6), compound C1A (328 mg), and triethylamine (322 mg) were dissolved in N,N-dimethylformamide (5 mL). Then, 1-hydroxybenzotriazole (HOBT, 162 mg) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 229 mg) were further added, and the reaction solution was stirred at room temperature for 16 hours. The reaction solution was directly purified using a C18 column reverse phase (acetonitrile and 0.05% formic acid aqueous solution system) to obtain the target compound N. 6 ,N 6 -Dimethyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valine)-L-lysine (C1, white solid, 327 mg) was obtained. LCMS (ESI) [M+H] + :524.4. 1H NMR (400 MHz, ) δ 9.13 (s, 2H), 7.95 (t, J = 8.8 Hz, 2H), 4.21 (dd, J = 8.8, 6.9 Hz, 1H), 4.08 - 4.03 (m, 1H), 3.41 (s, 3H), 2.55 (t, J = 7.0 Hz, 2H), 2.42 - 2.32 (m, 4H), 2.27 (s, 6H), 1.98 (dd, J = 13.6, 6.8 Hz, 1H), 1.86 - 1.77 (m, 2H), 1.74 - 1.55 (m, 2H), 1.47 - 1.37 (m, 2H), 1.31 - 1.23 (m, 2H), 0.85 (dd, J = 12.8, 6.8 Hz, 6H).

[0313] Example 1.1.4: N 6 ,N 6 -Diethyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valine)-L-lysine (C2) [ka]

[0314] Step 1: Compound C2A (5.0 g, 12.05 mmol, Cas: 1872-06-8) was dissolved in dichloromethane (100 mL), acetaldehyde (3.2 g, 72.3 mmol) was added to the reaction solution, and the reaction was stirred at room temperature for 10 minutes to react, sodium triacetoxyborohydride (12.8 g, 60.25 mmol) was further added to the reaction solution, and the reaction was stirred at room temperature for 1 hour to react. LCMS showed that the reaction was complete. Saturated aqueous ammonium chloride solution was added to the reaction solution and stirred for 1 hour, then rotary dried and filtered, and the filtrate was isolated and purified by C18 column reverse phase (acetonitrile vs. 0.05% formic acid aqueous solution: 5%-55%) to obtain the target compound C2B (4.57 g, 82.0% yield) as a white solid. LCMS (ESI) [M+H] + = 436.4, 1 H NMR (400 MHz, DMSO-d6) δ 7.70 (d, J = 7.0 Hz, 1H), 7.41 (d, J = 9.0 Hz, 1H), 7.38 - 7.26 (m, 5H), 5.08 - 4.99 (m, 2H), 4.00 (dd, J = 12.6, 6.5 Hz, 1H), 3.86 (dd, J = 8.6, 6.8 Hz, 1H), 2.74 (dd, J = 14.0, 6.9 Hz, 4H), 2.64 - 2.54 (m, 2H), 2.05 - 1.94 (m, 1H), 1.72 - 1.52 (m, 2H), 1.52 - 1.38 (m, 2H), 1.38 - 1.18 (m, 2H), 1.04 (t, J = 7.1 Hz, 6H), 0.87 - 0.81 (m, 6H).

[0315] Step 2: Compound C2B (1.6 g, 3.68 mmol) was dissolved in methanol (80 mL) at room temperature, then Pd / C (0.16 g) was added to the reaction solution and the reaction was stirred at room temperature for 12 hours under hydrogen gas. LCMS showed the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to give the desired compound C2C (900 mg, 82% yield) as an off-white solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.04 (br s, 1H), 4.02 - 3.99 (m, 1H), 3.10 (d, J = 4.5 Hz, 1H), 2.65 (q, J = 7.1 Hz, 4H), 2.55 - 2.51 (m, 2H), 2.06 - 1.93 (m, 1H), 1.73 - 1.54 (m, 2H), 1.47 - 1.38 (m, 2H), 1.30 - 1.21 (m, 2H), 1.01 (t, J = 7.1 Hz, 6H), 0.89 (d, J = 6.9 Hz, 3H), 0.79 (d, J = 6.8 Hz, 3H).

[0316] Step 3: Compound 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (268 mg, 1 mmol) was dissolved in DMF (8 mL), HATU (380 mg, 1 mmol) and triethylamine (322 mg, 2.5 mmol) were added in sequence, and the mixture was stirred at room temperature for 20 minutes. Compound C2C (301 mg, 1 mmol) was then added and the mixture was stirred at room temperature for 30 minutes. After the completion of the reaction was detected by LCMS, the reaction solution was directly purified by C18 column reverse phase (acetonitrile and 0.05% formic acid aqueous solution system) to obtain the target compound C2 (280 mg, 51% yield) as a white solid. LCMS (ESI) [M+H] + = 552.3, 1 H NMR (400 MHz, DMSO-d6) δ 9.13 (s, 2H), 7.95 (d, J = 8.9 Hz, 1H), 7.86 (d, J = 7.2 Hz, 1H), 4.18 (dd, J = 8.8, 6.8 Hz, 1H), 4.02 (dd, J = 12.8, 7.2 Hz, 1H), 3.41 (s, 3H), 2.74 - 2.69 (m, 4H), 2.62 - 2.52 (m, 4H), 2.44 - 2.29 (m, 2H), 2.04 - 1.94 (m, 1H), 1.86 - 1.77 (m, 2H), 1.72 - 1.54 (m, 2H), 1.51 - 1.39 (m, 2H), 1.33 - 1.23 (m, 2H), 1.02 (t, J = 7.2 Hz, 6H), 0.87 - 0.82 (m, 6H).

[0317] Example 1.1.5: N 6 ,N 6 -Di-n-propyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valine)-L-lysine (C3) [ka]

[0318] Step 1: Compound C2A (5.0 g, 12 mmol) was dissolved in dichloromethane (100 mL), n-propanal (4.2 g, 72.3 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 10 minutes to react. Sodium triacetoxyborohydride (12.8 g, 60.25 mmol) was further added to the reaction solution, and the mixture was stirred at room temperature for 1 hour to react. LCMS showed that the reaction was complete. Saturated aqueous ammonium chloride solution was added to the reaction solution, and the mixture was stirred for 1 hour, rotary dried, and filtered. The filtrate was isolated and purified by C18 reverse phase column (acetonitrile vs. 0.05% formic acid aqueous solution: 5%-55%) to obtain the target compound C3A (4.57 g, 82.0% yield) as a white solid. LCMS (ESI) [M+H] + = 464.0, 1 H NMR (400 MHz, DMSO-d6) δ 7.81 (d, J = 7.3 Hz, 1H), 7.38 - 7.28 (m, 5H), 5.04 (d, J = 1.7 Hz, 2H), 4.12 - 4.02 (m, 1H), 3.94 - 3.82 (m, 1H), 2.65 - 2.52 (m, 6H), 2.06 - 1.94 (m, 1H), 1.76 - 1.64 (m, 1H), 1.64 - 1.53 (m, 1H), 1.52 - 1.40 (m, 6H), 1.34 - 1.18 (m, 2H), 0.92 - 0.80 (m, 12H).

[0319] Step 2: At room temperature, compound C3A (2.0 g, 4.32 mmol) was dissolved in methanol (80 mL), and then Pd / C (0.16 g) was added to the reaction solution, and the reaction was stirred at room temperature under hydrogen gas for 12 hours. LCMS showed that the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound C3B (1.2 g, 85.5% yield) as a white solid.

[0320] Step 3: Compound 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (100 mg, 0.373 mmol) was dissolved in N,N-dimethylformamide (1 mL), then HATU (142 mg, 0.373 mmol) and N,N-diisopropylethylamine (120 mg, 0.93 mmol) were added, the system was stirred for 30 minutes, then compound C3B (122 mg, 0.371 mmol) was added, and the reaction solution was stirred at room temperature for 1 hour. After the completion of the reaction was detected by LCMS, the reaction solution was directly purified by C18 column reverse phase (acetonitrile and 0.05% formic acid aqueous system) to obtain the target compound N. 6 ,N 6 -Di-n-propyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valine)-L-lysine (C3, 50 mg, 28% yield) was obtained as a pale yellow solid. LCMS (ESI) [M+H] + = 580.0, 1 H NMR (400 MHz, DMSO-d6) δ 8.24 (s, 2H), 7.98 - 7.93 (m, 2H), 4.24 - 4.16 (m, 1H), 4.10 (d, J = 5.2 Hz, 1H), 3.41 (s, 3H), 2.79 - 2.64 (m, 6H), 2.55 (t, J = 7.1 Hz, 2H), 2.45 - 2.26 (m, 2H), 2.06 - 1.91 (m, 1H), 1.89 - 1.78 (m, 2H), 1.76 - 1.66 (m, 1H), 1.64 - 1.57 (m, 1H), 1.57 - 1.42 (m, 6H), 1.37 - 1.24 (m, 2H), 0.93 - 0.78 (m, 12H).

[0321] Example 1.2 Synthesis of Drug-Linker Compounds Example 1.2.1: N-((11S,14S)-11-(4-(di-n-propylamino)butyl)-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxeno[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadecan-14-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (DL-01) [ka]

[0322] Compound C3 (43 mg, 0.074 mmol) and compound B1 (40 mg, 0.075 mmol) were dissolved in N,N-dimethylformamide (1 mL), and then HBTU (28 mg, 0.075 mmol) and N,N-diisopropylethylamine (24 mg, 0.187 mmol) were added in that order, and the reaction solution was stirred at room temperature for 1 hour. After the completion of the reaction was detected by LCMS, the reaction solution was directly purified by preparative chromatography (0.01% aqueous trifluoroacetic acid solution, acetonitrile) to obtain the target compound (DL-01, 8.5 mg, 10% yield) as a yellow solid. LCMS (ESI) [M+H] + = 1098.6, 1H NMR (400 MHz, DMSO-d6) δ 9.10 (s, 2H), 9.03 (s, 1H), 8.64 (t, J = 6.4 Hz, 1H), 8.19 (t, J = 5.9 Hz, 1H), 8.08 (d, J = 7.4 Hz, 1H), 7.92 (d, J = 8.5 Hz, 1H), 7.59 (s, 1H), 7.51 (s, 1H), 7.24 (s, 1H), 6.49 (s, 1H), 6.29 (s, 2H), 5.42 (s, 2H), 5.24 (s, 2H), 4.66 - 4.52 (m, 2H), 4.31 - 4.21 (m, 1H), 4.19 - 4.10 (m, 1H), 3.74 (d, J = 5.5 Hz, 2H), 3.49 - 3.48 (m, 2H), 3.40 (s, 3H), 3.15 - 3.06 (m, 2H), 3.02 - 2.95 (m, 6H), 2.59 - 2.52 (m, 3H), 2.41 - 2.29 (m, 2H), 2.05 - 1.90 (m, 2H), 1.91 - 1.77 (m, 6H), 1.63 - 1.57 (m, 6H), 1.31 - 1.29 (m, 2H), 0.92 - 0.80 (m, 15H).

[0323] Example 1.2.2: N-((11S,14S)-11-(4-(diethylamino)butyl)-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadecan-14-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (DL-02) [ka]

[0324] Compound C2 (40 mg, 0.075 mmol) and compound B1 (41 mg, 0.075 mmol) were dissolved in DMF (1 mL), then HOBt (15.2 mg, 0.113 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (21.5 mg, 0.113 mmol) were added, then triethylamine (23 mg, 0.225 mmol) was added, and the reaction solution after the addition was stirred at room temperature for 16 hours. After the completion of the reaction was detected by LCMS, the reaction solution was concentrated to obtain a crude product. The crude product was purified by preparative chromatography (0.01% TFA aqueous solution, acetonitrile) to obtain the target compound (DL-02, 16 mg, 20% yield) as a yellow solid. LCMS (ESI) [M+H] + = 1070.6, 1 H NMR (400 MHz, DMSO-d6) δ 9.13 - 9.08 (m, 2H), 9.01 (s, 1H), 8.64 (t, J = 6.5 Hz, 1H), 8.20 (t, J = 5.7 Hz, 1H), 8.09 (d, J = 7.4 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.59 (s, 1H), 7.51 (s, 1H), 7.24 (s, 1H), 6.50 (s, 1H), 6.29 (s, 2H), 5.43 (s, 2H), 5.24 (s, 2H), 4.65 - 4.53 (m, 2H), 4.26 (d, J = 6.5 Hz, 1H), 4.20 - 4.10 (m, 1H), 3.74 (d, J = 5.5 Hz, 2H), 3.50 (t, J = 5.8 Hz, 2H), 3.41 (s, 3H), 3.14 - 3.07 (m, 6H), 2.98 (s, 2H), 2.55 (d, J = 7.3 Hz, 2H), 2.41 - 2.29 (m, 2H), 2.03 - 1.89 (m, 2H), 1.89 - 1.77 (m, 7H), 1.61 - 1.56 (m, 2H), 1.32 (s, 2H), 1.16 (t, J = 7.2 Hz, 6H), 0.91 - 0.78 (m, 9H).

[0325] Example 1.2.3: N-((11S,14S)-11-(4-(dimethylamino)butyl)-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadecan-14-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (DL-03) [ka]

[0326] Compound B1 (100 mg, 0.186 mmol) and compound N 6 ,N 6 -Dimethyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valine)-L-lysine (C1, 100 mg, 0.191 mmol) was dissolved in DMF (2 mL). Then, 1-hydroxybenzotriazole (38 mg, 0.280 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (53 mg, 0.280 mmol) were added, and triethylamine (56 mg, 0.559 mmol) was further added, and the reaction solution after addition was stirred at room temperature for 16 hours. After the completion of the reaction was detected by LCMS, the reaction solution was purified by preparative chromatography (0.01% TFA aqueous solution, acetonitrile) to obtain the target compound (DL-03, 20 mg, 10% yield) as a yellow solid. LCMS (ESI) [M+H] + = 1042.5, 11H NMR (400 MHz, DMSO-d6) δ 9.28 (s, 1H, TFA), 9.10 (s, 2H), 8.64 (br s, 1H), 8.19 (br s, 1H), 8.09 (d, J = 6.4 Hz, 1H), 7.92 (d, J = 7.8 Hz, 1H), 7.59 (s, 1H), 7.51 (s, 1H), 7.24 (s, 1H), 6.50 (s, 1H), 6.29 (s, 2H), 5.43 (s, 2H), 5.24 (s, 2H), 4.67 - 4.52 (m, 2H), 4.30 - 4.10 (m, 2H), 3.74 (br s, 2H), 3.50 (br s, 2H), 3.41 (s, 3H), 3.17 - 3.07 (m, 2H), 3.04 - 2.91 (m, 2H), 2.75 (s, 6H), 2.46 - 2.24 (m, 3H), 2.04 - 1.66 (m, 9H), 1.63 - 1.46 (m, 3H), 1.32 - 1.29 (m, 2H), 0.87 - 0.82 (m, 9H).

[0327] Example 1.3 Preparation of Anti-TF Antibody-Drug Conjugate [Chemical Structure] Ab is an anti-TF antibody.

[0328] 1.3.1 Preparation of TF-ADC (DAR8) Sample 30 mg of anti-TF antibody prepared according to Example I was diluted with diluent (20 mM PB, pH 7.5), sodium edetate solution with a final concentration of 1 mM was added, mixed uniformly, TCEP solution with 6.0 times the equivalent of antibody was added, mixed uniformly, and left at room temperature for 60 minutes, DL-01 with 15 times the equivalent of antibody dissolved in dimethyl sulfoxide was added to the above solution system, mixed uniformly, and left at room temperature for 1 hour to obtain a sample after coupling, and after the reaction was completed, the sample was replaced with 20 mM histidine buffer with pH 6.0 using a 30 KDa ultrafiltration tube, and low molecular weight substances were removed, and finally the sample was concentrated to obtain a solution containing an ADC composition of anti-TF antibody, TF-ADC (DAR8). The DAR value was 8.0 as measured by mass spectrometry in Example 1.4.

[0329] 1.3.2 Preparation of TF-ADC(DAR3) samples 30 mg of the anti-TF antibody prepared in Example I was diluted with a diluent (20 mM PB, pH 7.5), and a sodium edetate solution with a final concentration of 1 mM was added, mixed uniformly, and a TCEP solution with an amount equivalent to 6.0 times the antibody was added, mixed uniformly, and left at room temperature for 60 minutes. DL-01 with an amount equivalent to 4.0 times the antibody dissolved in dimethyl sulfoxide was added to the above solution system, mixed uniformly, and left at room temperature for 1 hour to obtain a sample after coupling. After the reaction was completed, the sample was replaced with a 20 mM histidine buffer solution with a pH of 6.0 using a 30 KDa ultrafiltration tube, and low molecular weight substances were removed. Finally, the sample was concentrated to obtain a solution containing an anti-TF antibody-ADC composition, TF-ADC (DAR3). The DAR value was 3.0 when measured by mass spectrometry.

[0330] Example 1.4 Measurement of DAR value of sample after coupling by mass spectrometry 1.4.1 LC-MS molecular weight and DAR value analysis of TF-ADC (DAR3) was performed Chromatography conditions: Column: PLRP-S, 2.1×50 mm, 5 μm, Mobile phase A: 0.1% FA / H2O, Mobile phase B: 0.1% FA / ACN Column temperature: 30℃ Sample chamber temperature: 8℃ Flow rate: 0.6 mL / min Injection volume: 2 μL [Table 9A]

[0331] Sample treatment: 50 μg of each sample was taken, 2 μL of 1 M DTT was added, and ultrapure water was added up to 50 μL to dilute to a concentration of approximately 1.0 mg / mL, mixed uniformly, and reduced at room temperature for 30 min. LC / MS model: Agilent 1290-6545XT Q-TOF Mass spectrometry conditions: Gas temp: 320℃, Drying Gas: Nitrogen, Nebulizer: 35 psi, Sheath Gas Temp: 350℃, sheath Gas Flow: 11 L / min, m / z 500~3000 The results are shown in Table 9 below.

[0332] [Table 9B]

[0333] In Table 9, mAb represents an uncoupled monoclonal antibody, LC represents an antibody light chain, HC represents an antibody heavy chain, DAR1 represents a conjugate containing a light chain or a heavy chain and coupled with one toxin molecule, DAR2 represents a conjugate containing a light chain or a heavy chain and coupled with two toxin molecules, and DAR3 represents a conjugate containing a light chain or a heavy chain and coupled with three toxin molecules, among which the theoretical molecular weight of the monoclonal antibody is calculated based on the G0F glycotype. The following explanations of mAb, LC, HC, DAR1, DAR2, and DAR3 are as above.

[0334] The detection results showed that the antibody light chain in TF-ADC(DAR3) was coupled to 0 to 1 toxin molecule (LC, DAR1 ratios were 53.8% and 46.2%, respectively), and the heavy chain was coupled to 0 to 3 toxin molecules (mAb, DAR1, DAR2, DAR3 ratios were 30.6%, 42.4%, 20.2%, and 6.8%, respectively). Based on this, the coupling ratio (average DAR value) of the TF-ADC(DAR3) sample was calculated to be 3.0.

[0335] 1.4.2 Analysis of LC-MS molecular weight and DAR value for TF-ADC (DAR8) Chromatography conditions: Column: Xbridge Protein BEH SEC (2.5 μm, 4.6×150 mm) Mobile phase A: 0.1% FA / H2O, Mobile phase B: 0.1% FA / ACN Column temperature: 30℃ Sample chamber temperature: 8℃ Flow rate: 0.3 mL / min Injection volume: 1 μL [Table 10A]

[0336] Sample treatment: 50 μg of each sample was taken, 2 μL of 1 M DTT was added, and ultrapure water was added up to 50 μL to dilute to a concentration of approximately 1.0 mg / mL, mixed uniformly, and reduced at room temperature for 30 min. LC / MS model number: UPLC (AB SCIEX), high resolution mass spectrometer (AB SCIEX) Mass spectrometry conditions: Gas 1: 45, Gas 2: 45, CUR: 30, TEM: 450, ISVF: 5000, DP: 120, CE: 12, mass range: 600-4000

[0337] The results are shown in Table 10 below. [Table 10B]

[0338] The detection results showed that the antibody light chain in TF-ADC(DAR8) was coupled to 0 to 1 toxin molecule (LC and DAR1 percentages were 0% and 100.0%, respectively), and the heavy chain was coupled to 0 to 3 toxin molecules (mAb, DAR1, DAR2, and DAR3 percentages were 0%, 0%, 0%, and 100.0%, respectively). Therefore, the coupling ratio (average DAR value) of the TF-ADC(DAR8) sample was calculated to be 8.0.

[0339] Example 2: Evaluation of anti-TF antibody ADC Example 2.1 Detection of Anti-TF ADC Affinity According to the method in the above Example for preparing the ADC sample TF-ADC (DAR8), ADC conjugates of four candidate antibodies of the present invention (2B12B10-hz1, 30G11B7-hz1, 22F11H5-hz1, 27H8H3hz1) and a reference antibody (BM) against the toxin-Linker (B81, i.e., DL-01 molecule) were prepared.

[0340] Before and after coupling of the anti-TF humanized antibody with a toxin-Linker (B81, i.e., DL-01 molecule), the change in antibody affinity was measured by antigen protein-based ELISA according to a similar method described in Example I.

[0341] The experimental results are shown in Table 11 and Figures 6A and 6B, and show that the four humanized antibodies did not show a significant change in affinity after coupling with B81. [Table 11]

[0342] Example 2.2 In vitro killing activity detection of ADCs TF-positive tumor cells KYSE520 cells were collected by pancreatin digestion, resuspended in RPMI1640+2% FBS, and seeded at 2000 cells per well. The ADC to be measured was gradient-diluted in RPMI1640+2% FBS, starting from 50 μg / mL, and diluted 3-fold, resulting in 11 concentration points. The diluted ADC was added to the plate and incubated at 37°C for 120 hours. After incubation was completed, CCK8 reagent was added and reacted at 20 μL / well for 2 to 4 hours. The data was read on a microplate reader at 450 nm, and then introduced into Graphpad Prism for curve fitting.

[0343] The experimental results are shown in Figures 7A and 7B. 2B12B10-hz1, 30G11B7-hz1, 22F11H5-hz1, and 27H8H3-hz1 could all effectively kill KYSE520 cells after coupling with B81, and the killing IC50 values ​​were essentially equivalent to those of the ADCs after coupling with the reference antibody.

[0344] Example 2.3 In vivo efficacy of ADCs To verify the in vivo efficacy of anti-TF humanized ADC drugs and evaluate the antitumor activity of the test drugs in the KYSE520 subcutaneous xenograft female Balb / c nude mouse model, 5- to 6-week-old female Balb / c nude mice were purchased. KYSE520 cells grown to logarithmic growth phase were digested with pancreatin and resuspended in PBS, and 5 × 10 cells were injected into each mouse. 6 The cells were inoculated subcutaneously. 3 When the tumors grew to a size of 100 mm, the ADC to be measured was intravenously administered once a week at 1 mg / kg or 3 mg / kg each time, and the specific methods are shown in Tables 13 and 14 below. The main observation indicators of this experiment are 1) TGI (%), formula: TGI (%) = (1-T / C) x 100% (T and C are the relative tumor volumes of the treatment group and the control group at a certain time point, respectively); 2) the size, photo, and weight of the tumor volume at the end of the experiment; and 3) the effect of the ADC drug on the mouse body weight. The experiment was conducted twice in total.

[0345] The administration method and results of the first experiment are shown in Table 12 and Figure 8. After coupling with B81, 22F11H5-hz1 and 30G11B7 both had good tumor suppression effects in mice in vivo, and the TGI at a dose of 3 mg / kg was equivalent to that of BM-B81, and at a dose of 1 mg / kg, both were superior to BM-B81. The changes in mouse body weight are shown in Figure 9, which shows that the two ADCs did not affect the mouse body weight during the entire administration process. [Table 12]

[0346] The experimental design and results of the second experiment are shown in Table 13 and Figure 10. After coupling with B81, 2B12B10-hz1 and 27H8H3-hz1 both had good tumor suppression effects in mice in vivo, and both were superior to BM-B81 at a dose of 1 mg / kg. The changes in mouse body weight are shown in Figure 11, which shows that the two ADCs did not affect mouse body weight. [Table 13]

[0347] Sequence Listing: [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4] [Table 14-5] [Table 14-6] [Table 14-7]

Table 14-8

Claims

1. An antibody-drug conjugate (ADC) having the following formula (I), or a pharmaceutically acceptable salt or solvate thereof: Ab-[L-D] q (I) Among them, Ab represents anti-TF antibody; L represents a linker, D represents a cytotoxic or cell-inhibiting drug, such as a topoisomerase I inhibitor, and q=1 to 20, 1 to 10, 1 to 8, 3 to 8, 4 to 8, or 6 to 8; Among them, the Ab is - the three CDRs of the heavy chain variable region (VH) sequence of SEQ ID NO: 7 and the three CDRs of the light chain variable region (VL) sequence of SEQ ID NO: 8, - the three CDRs of the heavy chain variable region (VH) sequence of SEQ ID NO: 1 and the three CDRs of the light chain variable region (VL) sequence of SEQ ID NO: 2, the three CDRs of the heavy chain variable region (VH) sequence of SEQ ID NO: 3 and the three CDRs of the light chain variable region (VL) sequence of SEQ ID NO: 4, or - comprising the three CDRs of the heavy chain variable region (VH) sequence of SEQ ID NO: 5 and the three CDRs of the light chain variable region (VL) sequence of SEQ ID NO: 6, wherein the CDRs are defined according to Chothia, AbM, Kabat, IMGT, or any combination thereof; An antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt or solvate thereof.

2. The Ab contains three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs), among which: (i) as defined according to IMGT, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 90, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 91, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 92, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 102, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 103, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 104; or (ii) as defined according to IMGT, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 18, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 19, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 20, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 30, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 31, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 32; or (iii) as defined according to IMGT, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 42, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 43, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 44, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 54, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 55, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 56; or (iv) The antibody-drug conjugate of claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein, as defined according to IMGT, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 66, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 67, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 68, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 78, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 79, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:

80.

3. The Ab is a heavy chain variable region comprising SEQ ID NO: 7, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region comprising SEQ ID NO: 8, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; or a heavy chain variable region comprising SEQ ID NO: 1 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a light chain variable region comprising SEQ ID NO: 2 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or a heavy chain variable region comprising SEQ ID NO: 3 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a light chain variable region comprising SEQ ID NO: 4 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or - a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 5 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 6 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, wherein preferably said Ab is an IgG1 antibody.

2. The antibody-drug conjugate of claim 1, or a pharmaceutically acceptable salt or solvate thereof.

4. D represents a camptothecin-class drug, preferably FL118 or a derivative thereof, for example, a camptothecin-class drug having the following structure: 【Chemistry 1】 Among them, R a teeth, hydrogen, C 3 -C 8 cycloalkyl groups, phenyl group, Halogen, hydroxy group, optionally NH 2 , NH(C 1 -C 4 alkyl) and N(C 1 -C 4 alkyl) 2 C replaced with 1 -C 4 Alkoxy group, C 3 -C 8 Cycloalkyl groups, heterocycloalkyl groups, phenyl groups, NR 1 R 2 C optionally substituted with a substituent selected from the group consisting of 1 -C 8 alkyl groups, Selected from Among them, R 1 and R 2 are, independently of each other, hydrogen, Hydroxy group, amino group, 1 or 2 C 1 -C 4 Amino group substituted with alkyl group, one or two C 1 -C 4 Hydroxyalkyl-substituted amino groups (C 1 -C 4 hydroxyalkyl) and (C 1 -C 4 C optionally substituted with a substituent selected from the group consisting of amino groups substituted with alkyl 1 -C 8 alkyl groups, 1 or 2 C 3 -C 10 Cycloalkyl groups, C 3 -C 10 C substituted with heterocycloalkyl, phenyl or heteroaryl groups 1 -C 4 alkyl groups, C 3 -C 10 cycloalkyl groups, C 3 -C 10 heterocycloalkyl groups, C 2 -C 6 heteroalkyl groups, heteroaryl groups, a phenyl group optionally substituted with halogen; C optionally substituted with a hydroxy or amino group 1 -C 8 Alkyl-C(=O)-, Selected from Or, R 1 and R 2 are combined with the nitrogen atom to which they are attached to form a 5-, 6- or 7-membered heterocycle having 0 to 3 substituents, the substituents being halogen, C 1 -C 4 Alkyl groups, OH, C 1 -C 4 Alkoxy group, NH 2 , NH(C 1 -C 4 alkyl) and N(C 1 -C 4 alkyl) 2 Selected from Among these, each occurrence of a cycloalkyl group, a heterocycloalkyl group, a phenyl group, and a heteroaryl group independently represents OH, C 1 -C 4 Alkyl group, C 1 -C 4 Alkoxy group, NH 2 , NH(C 1 -C 4 alkyl) and N(C 1 -C 4 alkyl) 2 optionally substituted with 0 to 3 substituents selected from the group consisting of Preferably, R a -C 1 -C 4 Alkyl-OH, -C 1 -C 4 Alkyl-OC 1 -C 4 Alkyl-NH 2 , or -C 1 -C 4 Alkyl-NH 2 and Preferably, the drug D unit is bonded to the linker L unit via a hydroxy or amino group present therein.

2. The antibody-drug conjugate of claim 1, or a pharmaceutically acceptable salt or solvate thereof.

5. The LD unit of formula (I) has the following structure: 【Chemistry 2】 , or 【Transformation 3】 2. The antibody-drug conjugate of claim 1, or a pharmaceutically acceptable salt or solvate thereof, comprising:

6. L is a peptide-containing linker and comprises the structure of formula (II): -ZYM-, (II) Among them, Z is a linker group that attaches to the Ab, Y is a peptide of 2 to 5 amino acids, preferably a dipeptide, tripeptide or tetrapeptide, M is absent or is a spacer group for attachment to drug D; 2. The antibody-drug conjugate of claim 1, or a pharmaceutically acceptable salt or solvate thereof.

7. Y is a peptide having, from the N-terminus to the C-terminus, the amino acid sequence of the following formula: Yes 1 -Yes 2 -Yes 3 -Yes 4 -Yes 5 、 Among them, Xaa 1 is absent or is an amino acid selected from valine, glycine, alanine and glutamic acid, Xaa 2 is an amino acid selected from phenylalanine, leucine, and valine, preferably valine; Xaa 3 is unsubstituted or substituted lysine, Xaa 4 is an amino acid selected from leucine, glycine and alanine, Xaa 5 is absent or is an amino acid selected from glycine and alanine, wherein the N-terminus of the amino acid sequence is linked to Z, and the C-terminus is linked to M (if M is present) or directly to drug D; Preferably, Xaa 3 The ε-amino group is C 1 -C 3 lysine mono- or di-substituted with alkyl groups; More preferably, Y is a peptide selected from the group consisting of Phe-Lys-Gly, Leu-lys-Gly, Gly-Val-Lys-Gly, Val-Lys-Gly-Gly, Val-Lys-Gly, Val-Lys-Ala, Val-Lys-Leu, wherein the Lys residue is unsubstituted or C 1 -C 3 lysine mono- or di-substituted with alkyl groups; The antibody-drug conjugate of claim 6, or a pharmaceutically acceptable salt or solvate thereof.

8. Y is 【Chemistry 4】 and Among them, R 3 and R 4 are each independently selected from a methyl group, an ethyl group, and a propyl group; Preferably, R 3 and R 4 are the same, and more preferably, R 3 and R 4 are each independently a propyl group; The wavy line on the left represents the position where Z binds, and the wavy line on the right represents the position where M binds. The antibody-drug conjugate of claim 6, or a pharmaceutically acceptable salt or solvate thereof.

9. Z has the structure: -WITH 1 -WITH 2 -WITH 3 -WITH 4 -、 Among them, Z 1 is the sulfur atom in Ab, Z 2 is a 5- to 10-membered heterocyclyl group, preferably containing 1 or 2 heteroatoms selected from N, S and O, Z 3 is a bond, -C(=O)-, -C 1 -C 10 Alkylidene-C(=O)-, -C 3 -C 10 Alkynylene-C(=O)-, -C 3 -C 10 Alkenylene-C(=O)-, -C 1 -C 10 Heteroalkylene-C(=O)-, -C 3 -C 8 Cycloalkylene-C(=O)-, -OC 1 -C 8 Alkylidene-C(=O)-, -arylene-C(=O)-, -C 1 -C 10 Alkylidene-arylene-C(=O)-, -arylene-C 1 -C 10 Alkylidene-C(=O)-, -C 1 -C 10 Alkylidene-C 3 -C 8 Cycloalkylene-C(=O)-, -C 3 -C 8 Cycloalkylene-C 1 -C 10 Alkylidene-C(=O)-, -C 3 -C 8 Heterocyclylene-C(=O)-, -C 1 -C 10 Alkylidene-C 3 -C 8 Heterocyclylene-C(=O)-, -C 3 -C 8 Heterocyclylene-C 1 -C 10 alkylidene-C(=O)-; Z 4 is a bond or a PEG unit represented by the formula: 【Transformation 5】 Among them, R 5 is C 1-4 Alkylidene group, -NH-, -NH-C 1-4 alkylidene-heteroaryl-, wherein the heteroaryl group is a 5- or 6-membered nitrogen-containing heteroaryl group, preferably a triazolyl group; R 6 is -C(=O)-, C 1-4 Alkylidene group, C 1-4 Alkylidene-C(=O)-, -NH-C(=O)-(CH 2 OCH 2 )-C(=O)-, C 1-4 Alkylidene-NH-C(=O)-(CH 2 OCH 2 )-C(=O)-, wherein m is an integer from 2 to 12, for example, m=2, 4, 6, or 8; Preferably, Z has the structure: 【Transformation 6】 Among them, R b is alkynylene-C(═O)- or alkenylene-C-(═O)-, Preferably, R b teeth, 【Transformation 7】 and The wavy line on the left represents the site where antibody (Ab) binds, and the wavy line on the right represents the site where Y binds. More preferably, Z has the structure: 【Transformation 8】 The antibody-drug conjugate of claim 6, or a pharmaceutically acceptable salt or solvate thereof.

10. M is absent or amino-C 1 -C 3 Alkylidene groups, such as -NH-CH 2 - or amino-phenyl-C 1 -C 3 alkylidene-OC(=O)-, preferably M is 【Chemistry 9】 and The wavy line on the left represents a bond to Y, and the wavy line on the right represents a bond to the drug D unit. The antibody-drug conjugate of claim 6, or a pharmaceutically acceptable salt or solvate thereof.

11. L is a linker comprising the structure: 【Chemistry 10】 Among them, R 3 and R 4 are each independently selected from a methyl group, an ethyl group, and a propyl group; The antibody-drug conjugate of claim 6, or a pharmaceutically acceptable salt or solvate thereof.

12. R 3 and R 4 are both methyl groups, or R 3 and R 4 are both ethyl groups, or R 3 and R 4 are both propyl groups, 12. The antibody-drug conjugate of claim 11, or a pharmaceutically acceptable salt or solvate thereof.

13. The LD unit of formula I binds to the antibody by forming a thioether with a sulfhydryl group of a cysteine ​​in the light and / or heavy chain of the Ab.

2. The antibody-drug conjugate of claim 1, or a pharmaceutically acceptable salt or solvate thereof.

14. The ADC 【Chemistry 11】 or 【Chemistry 12】 or 【Chemistry 13】 Selected from wherein q is an average DAR value between 1 and 8, about 2, about 3, about 4, about 5, about 6, about 7, or about 8; Preferably, the Ab is a full-length IgG1 antibody and comprises three CDRs of the heavy chain variable region (VH) sequence of SEQ ID NO: 7 and three CDRs of the light chain variable region (VL) sequence of SEQ ID NO: 8, or more preferably comprises the heavy chain variable region of SEQ ID NO: 7 and the light chain variable region of SEQ ID NO:

8.

2. The antibody-drug conjugate of claim 1, or a pharmaceutically acceptable salt or solvate thereof.

15. 15. An antibody-drug conjugate according to any one of claims 1 to 14, or a pharmaceutically acceptable salt or solvate thereof, and optionally an auxiliary pharmaceutical material. Pharmaceutical compositions.

16. An antibody or antigen-binding fragment thereof that binds to tissue factor (TF), (i) the HCDR1, 2, and 3 sequences of the heavy chain variable region shown in SEQ ID NO: 7, and the LCDR1, 2, and 3 sequences of the light chain variable region shown in SEQ ID NO: 8; or (ii) the HCDR1, 2, and 3 sequences of the heavy chain variable region shown in SEQ ID NO: 1 and the LCDR1, 2, and 3 sequences of the light chain variable region shown in SEQ ID NO: 2; or (iii) the HCDR1, 2, and 3 sequences of the heavy chain variable region shown in SEQ ID NO: 3 and the LCDR1, 2, and 3 sequences of the light chain variable region shown in SEQ ID NO: 4; or (iv) the HCDR1, 2, and 3 sequences of the heavy chain variable region shown in SEQ ID NO: 5, and the LCDR1, 2, and 3 sequences of the light chain variable region shown in SEQ ID NO: 6; Including, Preferably, the CDRs are defined according to Chothia, Abm, Kabat or IMGT or a combination thereof. An antibody or antigen-binding fragment thereof.

17. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, in which (i) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; or (ii) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; or (iii) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; or (iv) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 5, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 6, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; or wherein the antibody or antigen-binding fragment thereof is (i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8; or (ii) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; or (iii) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 4; or (iv) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 5 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 6; a heavy chain variable region and a light chain variable region selected from The antibody or antigen-binding fragment thereof of claim 16.

18. the antibody comprises an Fc region, preferably a human Fc region; the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody, and preferably has a human IgG1 heavy chain constant region; the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single chain antibody, scFv, scFab, disulfide-linked scFv, disulfide-linked scFab, (Fab')2 fragment or linear antibody; and / or The antibody is a murine antibody, or a chimeric antibody, or a humanized antibody, preferably a humanized antibody; The antibody or antigen-binding fragment thereof of claim 16.

19. Encoding the anti-TF antibody or antigen-binding fragment thereof according to any one of claims 16 to 18. Isolated nucleic acid.

20. 20. The nucleic acid of claim 19, preferably an expression vector. vector.

21. 20. The method of claim 19, wherein the vector comprises the nucleic acid of claim 19 or the nucleic acid of claim 19, and the vector is a mammalian cell. host cell.

22. An immunoconjugate or immunofusion or multispecific antibody comprising the antibody or antigen-binding fragment thereof according to any one of claims 16 to 18.

23. 19. A method for the treatment of a cancer, comprising administering to a patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 16 to 18, or an immunoconjugate or immunofusion or multispecific antibody comprising said antibody or antigen-binding fragment thereof, and optionally a pharmaceutical auxiliary material. Pharmaceutical compositions.

24. Use of an antibody or antigen-binding fragment thereof according to any one of claims 16 to 18, or an immunoconjugate or immunofusion or multispecific antibody comprising said antibody or antigen-binding fragment thereof, In vivo or in vitro (1) Target binding to TF-positive tumor cells; (2) blocking TF / VIIa-mediated downstream signaling pathways in TF-positive tumor cells; or (3) used to inhibit and / or kill TF-positive tumor cells; Alternatively, it is used for preparing the drug used in any one of the above. use.

25. Use of the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 14 in the manufacture of a medicament for preventing or treating a TF-positive tumor, preferably wherein the tumor is cervical cancer, pancreatic cancer, lung cancer, prostate cancer, bladder cancer, ovarian cancer, breast cancer, colorectal cancer, esophageal cancer, head and neck cancer, or gastric cancer, including primary, advanced, or metastatic cancer.

26. A pharmaceutical composition for use in the prevention or treatment of TF-positive tumors, comprising the antibody-drug conjugate of any one of claims 1 to 14 or a pharmaceutically acceptable salt or solvate thereof, preferably wherein the tumor is cervical cancer, pancreatic cancer, lung cancer, prostate cancer, bladder cancer, ovarian cancer, breast cancer, colorectal cancer, esophageal cancer, head and neck cancer, or gastric cancer, including primary, advanced, or metastatic cancer.

27. 1. A method for detecting TF in a sample, comprising: (a) contacting a sample with the antibody or antigen-binding fragment thereof of any one of claims 16 to 18; (b) detecting the formation of a complex between the antibody or antigen-binding fragment thereof and TF, wherein optionally the antibody is detectably labeled; method.

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