WO2023160651A1 - 一种抗体及其药物偶联物和用途 - Google Patents

一种抗体及其药物偶联物和用途 Download PDF

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WO2023160651A1
WO2023160651A1 PCT/CN2023/078127 CN2023078127W WO2023160651A1 WO 2023160651 A1 WO2023160651 A1 WO 2023160651A1 CN 2023078127 W CN2023078127 W CN 2023078127W WO 2023160651 A1 WO2023160651 A1 WO 2023160651A1
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antibody
seq
amino acid
acid sequence
variable region
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PCT/CN2023/078127
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English (en)
French (fr)
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胡永韩
徐林峰
吴振伟
阮卡
王文贵
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苏州信诺维医药科技股份有限公司
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Publication of WO2023160651A1 publication Critical patent/WO2023160651A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors

Definitions

  • the present invention relates to antibodies and antibody-drug conjugates, more specifically to antibodies targeting tissue factor (Tissue Factor, TF) and antibody-drug conjugates (ADC) and compositions containing the antibodies or ADC and their Therapeutic applications.
  • tissue Factor Tissue Factor, TF
  • ADC antibody-drug conjugates
  • Tissue Factor is also called thromboplastin, CD142, or coagulation factor 3.
  • TF consists of an extracellular domain, a transmembrane region and an intracellular domain.
  • TF is an essential molecule for the initiation of exogenous coagulation events and is expressed on the cell surface in a functional form.
  • FVII coagulation factor VII
  • TF As a high-affinity receptor for coagulation factor VII (FVII, a serine protease) in plasma, TF, after forming a complex with the activated form of VIIa, triggers a catalytic event that activates factor IX or X through specific limited protein cleavage, Initiates the coagulation protease cascade.
  • FVII coagulation factor VII
  • TF is located on the adventitial cells of the blood vessel wall, fibroblasts surrounding blood vessels, etc., but it is very rare in the media or intima of the blood vessels.
  • TF is exposed to circulating blood only when the integrity of the vessel wall is compromised, exerting its hemostatic effect by activating the coagulation cascade. In this process, TF relies on its tight combination with the cell membrane to play an "anchor" role, so that physiological blood coagulation is limited to the injury site, and does not spread far from the initial site of blood coagulation.
  • TF is overexpressed on a variety of malignant tumors, including cervical cancer, pancreatic cancer, lung cancer, prostate cancer, bladder cancer, ovarian cancer, breast cancer, colorectal cancer and so on. Therefore, TF can be used as a target for the development of antibody drugs and ADC (antibody-drug conjugate) drugs.
  • ADC antibody-drug conjugate
  • Tivdak Teisotumab Vedotin
  • SGEN Seagen Inc.
  • GMAB Genemab A/S
  • the application in the indication is also in the clinical phase II.
  • the TF antibodies should have the ability to specifically target TF on the surface of tumor cells, but at the same time inhibit TF-mediated TF in normal tissues. Coagulation reactions have minimal impact in order to provide cancer patients with wider and better drug options.
  • the present inventors provided a new TF antibody and an antibody-drug conjugate (ADC) composed of it through intensive research.
  • the anti-TF antibody of the present invention not only exhibits high binding affinity and high specificity to TF-positive tumor cells, can be quickly and effectively endocytosed by tumor cells, and has little effect on TF-mediated coagulation.
  • the ADC composed of the TF antibody of the present invention not only has good physical properties and no obvious aggregation phenomenon, but also exhibits significant anti-tumor growth activity and good drug tolerance in animals in animal models .
  • the present invention provides an antibody-drug conjugate (ADC) having the following formula (I) or a pharmaceutically acceptable salt or solvate thereof: Ab-[LD] q (I)
  • D represents a cytotoxic or cytostatic drug, for example, a topoisomerase I inhibitor
  • the Ab comprises:
  • the CDR is defined according to Chothia, AbM, Kabat, IMGT or any combination thereof,
  • the Ab 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.
  • the invention provides a composition comprising an ADC of the invention, or a pharmaceutically acceptable salt or solvate thereof.
  • the present invention provides the use of the ADC of the present invention or its pharmaceutically acceptable salt or solvate and its composition in the treatment or prevention of TF-positive tumors, as well as the use in the preparation of drugs for the treatment or prevention use.
  • the present invention provides anti-TF antibodies, pharmaceutical compositions and uses thereof.
  • Figures 1A-1D show the detection of binding affinities of exemplary antibodies of the invention to human TF or monkey TF by antigenic protein-based ELSIA assays.
  • BM is the reference antibody.
  • Figures 2A-2D show the cell binding affinities of exemplary antibodies of the present invention to different TF-positive tumor cells (NCI-H358 and KYSE520) detected by flow cytometry.
  • BM is the reference antibody.
  • 3A-3C show that the blocking activity of the humanized antibody of the present invention on the downstream signaling pathway mediated by the TF/FVIIa complex was detected by the IL-8 release inhibition experiment.
  • Figures 4A-4B show the effect of the humanized antibody of the invention on coagulation compared to the reference antibody BM.
  • Figure 4A 2mM Ca 2+ , 50 ⁇ g/mL antibody concentration
  • Figure 4B 5 mM Ca 2+ , 20 ⁇ g/mL antibody concentration.
  • Figures 5A-5B show the test results of the serum stability of the humanized antibody of the present invention after incubation in human serum at 37°C for a period of time.
  • Figures 6A-6B show the changes in antibody affinity determined by ELISA before and after conjugation of the anti-TF humanized antibody to the toxin-Linker (B81, ie, DL-01 molecule).
  • Figures 7A-7B show the in vitro killing activity detection results of the ADC of the present invention.
  • Fig. 8 shows the anti-tumor effect of the ADCs of the present invention, 22F11H5-hz1 and 30G11B7 in mice after being coupled with a drug-linker (B81).
  • Figure 9 shows the effect of ADC conjugates comprising the antibodies 22F11H5-hz1 and 30G11B7 of the present invention on the body weight of mice after administration.
  • Fig. 10 shows the anti-tumor effect of the ADCs of the present invention, 2B12B10-hz1 and 27H8H3-hz1, after coupling with B81 in mice.
  • Figure 11 shows the effect of ADC conjugates comprising the antibodies 2B12B10-hz1 and 27H8H3-hz1 of the present invention on the body weight of mice after administration.
  • the trade name includes the product formulation, the generic drug and the active pharmaceutical ingredient for the product of the trade name.
  • the term “comprising” or “comprising” means including stated elements, integers or steps, but not excluding any other elements, integers or steps.
  • the term “comprising” or “comprises” is used, unless otherwise specified, it also covers the situation consisting of the mentioned elements, integers or steps.
  • an antibody variable region that "comprises” a particular sequence it is also intended to encompass an antibody variable region that consists of that particular sequence.
  • tissue factor or "TF” are used interchangeably and, unless otherwise stated, include any variant of human tissue factor, including sequence variants, especially naturally occurring variants, allelic variants, as well as post-translationally modified variants and conformational variants, and covers their species homologues. Furthermore, it is to be understood that the term encompasses not only TF natively or recombinantly expressed by or on a cell, but also recombinantly expressed fusion proteins comprising the ectodomain of TF.
  • tissue factor is the human TF protein comprising the amino acid sequence under UniProtKB-P13726, or a recombinant protein comprising the extracellular domain of said protein.
  • tissue factor is the monkey TF protein comprising the amino acid sequence under UniProtKB-A0A2K5VXA0, or a recombinant protein comprising the extracellular domain of said protein.
  • tissue factor or "TF” refers to human-derived tissue factor unless otherwise specified.
  • 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 expression level of TF on the cell surface can be determined by any conventional method known in the art for determining the expression level of a cell surface antigen, for example, FACS detection method or immunofluorescence staining method.
  • TF has significantly higher expression levels on various tumor cells than on normal tissues/cells, for example, MDA-MB-231 (breast cancer; >approximately 350,000 TF molecules/cell), BxPC-3 (pancreatic cancer; >approx. 350,000 TF molecules/cell).
  • the TF-positive cells are TF-positive tumor cells.
  • antibody refers to a polypeptide comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds an antigen.
  • the term encompasses various antibody structures including, but not limited to, monoclonal, polyclonal, single- or multi-chain antibodies, monospecific or multispecific (e.g., bispecific), chimeric, or human Antibodies, full-length antibodies, and antibody fragments are available as long as they exhibit the desired antigen-binding activity.
  • a “whole antibody” refers to an antibody comprising at least two heavy (H) chains and two light chains (L) Immunoglobulin molecule.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • Each light chain is composed 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 regions are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions.
  • the light chains of antibodies can be assigned to two types (termed kappa ( ⁇ ) and lambda ( ⁇ )) based on the amino acid sequence of their constant regions.
  • the heavy chains of antibodies can be divided into five main different classes based on the amino acid sequences of their constant regions: IgA, IgD, IgE, IgG, and IgM, and several of these classes It can be further divided into subclasses, eg, IgGl, IgG2, IgG3 and IgG4, IgAl and IgA2.
  • antibody fragment and “antigen-binding fragment” of an antibody are used interchangeably to refer to a molecule other than an intact antibody that comprises the portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • Antibody fragments typically comprise amino acid residues from "complementarity determining regions” or “CDRs” for antigen binding purposes, as understood by those of skill in the art.
  • Antibody fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.
  • 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 antibody, dia Chain antibody (diabody), triabody (triabody), tetrabody (tetrabody), minibody (minibody).
  • an antibody fragment comprises a portion of cysteine residues for formation of an interchain disulfide bond between a light chain and a heavy chain or between a heavy chain and a heavy chain, e.g., an IgG1 antibody Fab region and/or the cysteine residues in the hinge region to provide amino acid residue sites available for sulfhydryl coupling chemistry.
  • antibody fragments comprise cysteine residues introduced into the Fc region to provide amino acid residue sites available for sulfhydryl coupling chemistry.
  • immunoglobulin refers to a protein having the structure of a naturally occurring antibody.
  • IgG class immunoglobulins are heterotetrameric proteins of approximately 150,000 Daltons consisting of two light chains and two heavy chains that are disulfide bonded.
  • VH heavy chain variable region
  • CH1 heavy chain constant domain
  • CL light chain constant domain
  • an antibody is an IgG antibody means that the antibody is a heterotetrameric protein having an IgG-like immunoglobulin structure.
  • IgG antibodies usually the VH-CH1 of the heavy chain is paired with the VL-CL of the light chain to form a Fab fragment that specifically binds the antigen.
  • an IgG antibody essentially consists of two Fab molecules and two dimerized Fc regions linked by an immunoglobulin hinge region.
  • IgG immunoglobulins can be divided into subclasses based on the sequence of the heavy chain constant region, eg, gamma 1 (IgGl), gamma 2 (IgG2), gamma 3 (IgG3), and gamma 4 (IgG4).
  • the light chains of IgG immunoglobulins can also be divided into two types, called kappa and lambda, based on the amino acid sequence of their constant domains.
  • the antibody according to the invention is an IgG antibody, such as an IgGl, IgG2, IgG3 or IgG4 antibody.
  • antibodies according to the invention are IgGK or IgG ⁇ antibodies, eg, IgG1K or IgG1 ⁇ antibodies.
  • CDR region As used herein, the terms “complementarity determining region” or “CDR region” or “CDR” or “hypervariable region” are used interchangeably to refer to antibody variable domains that are highly variable in sequence and form structurally defined regions. (“hypervariable loops") and/or regions containing antigen contact residues ("antigen contact points").
  • the CDRs are primarily responsible for binding to antigenic epitopes.
  • the CDRs of antibody heavy and light chains are numbered sequentially starting from the N-terminus, and are generally referred to as CDR1, CDR2 and CDR3.
  • the CDRs located within the variable domain of an antibody heavy chain are also referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of an antibody light chain are referred to as LCDR1, LCDR2, and LCDR3.
  • various schemes known in the art can be used to determine its CDR sequence.
  • annotations of CDRs in a given light chain variable region or heavy chain variable region including those based on Kabat, AbM, Chothia, Contact, IMGT definitions, can be obtained at http://www.abysis.org/abysis/ CDR sequences.
  • CDRs can also be determined based on having the same Kabat numbering position as the reference CDR sequence.
  • residue positions in antibody variable regions including heavy chain variable region residues and light chain variable region residues
  • Kabat numbering system Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • a “variable region” or “variable domain” is a domain of an antibody's heavy or light chain that participates in the binding of the antibody to its antigen.
  • the heavy chain variable region (VH) and the light chain variable region (VL) can be further subdivided into hypervariable regions (HVR, also known as complementarity determining regions (CDR)), interspersed with more conserved regions (i.e., framework Region (FR)).
  • HVR hypervariable regions
  • FR framework Region
  • 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.
  • one or more residues in one or both of the two variable domains (i.e., VH and/or VL) of an antibody may be modified, for example, to one or more CDR regions and/or to One or more framework regions undergo residue modifications, especially conservative residue substitutions, to obtain antibody variants that still substantially retain at least one biological property (eg, antigen-binding ability) of the parent antibody prior to the alteration.
  • antibody variable regions can be modified by CDR grafting. Since the CDR sequences are responsible for most 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.
  • mutated and/or modified antibodies or ADC conjugates comprising the same, such as target antigen binding properties or other desired functional properties such as endocytic activity, coagulation effects, pharmacokinetics, can be assessed in in vitro or in vivo assays studies, and in vivo tumor-killing activity.
  • chimeric antibody refers to an antibody whose variable region sequences are derived from one species and constant region sequences are derived from another species, for example, An antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.
  • humanized antibody refers to an antibody in which CDR sequences derived from the germline of a non-human mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences, and/or additional amino acid modifications may be made in the CDR sequences, eg, for affinity maturation of antibodies.
  • the humanized antibodies of the invention have framework region sequences that are "derived from” specific human germline sequences.
  • “derived from” means that the amino acid sequence of the framework region of the antibody is at least 90%, more preferably at least 95%, and even more preferably At least 96%, 97%, 98% or 99% identity and the antibody retains antigen binding activity.
  • the term "isolated" antibody is one that has been separated from components of its natural environment.
  • antibodies are 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).
  • electrophoresis e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography e.g., ion exchange or reverse phase HPLC.
  • epitope refers to the region of an antigen to which an antibody binds. Epitopes can be formed from contiguous amino acids or from non-contiguous amino acids juxtaposed by the tertiary folding of the protein.
  • the antibody according to the present invention binds to the natural epitope of human TF, more preferably, binds to the natural epitope of the extracellular domain of human TF expressed on the cell surface.
  • affinity refers to the intrinsic 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 the antigenic protein or cell-based ELISA assay described in the examples herein, and another specific method is the flow cytometry method described in the examples herein. Dynamic affinity assessment of antibodies can also be performed using biolayer-based optical interferometry (BLI) techniques.
  • BBI biolayer-based optical interferometry
  • the term "binds" or “specifically binds”, in the context referring to the binding of an antibody to a cognate antigen (herein TF antigen), is used to mean binding with an affinity of about 10 ⁇ 6 M or less,
  • the KD value is about 10 -7 M or less, or about 10 -8 M or less.
  • the binding KD value of an antibody for its cognate antigen is preferably at least 100-fold or eg at least 1000-fold lower than the binding KD value for a non-specific antigen (eg, an unrelated antigen such as BSA).
  • the measurement of KD values is known in the art, for example based on the biolayer optical interferometry (BLI) technique. Available at e.g. ForteBio In the instrument, the assay is performed using an antibody as a ligand and an antigen (eg, a fusion protein comprising the ectodomain of TF, such as TF-His) as an analyte.
  • an antigen eg
  • KD (M) refers herein to the dissociation equilibrium constant for a particular antibody-antigen interaction. Affinity (affinity) is inversely correlated with the K D value, that is, the higher the affinity, the smaller the K D value; conversely, the lower the affinity, the higher the K D value. In general, the KD value depends on the dissociation rate constant (Kd or Kdis, sec ⁇ 1 ) and the association rate constant (Ka, M ⁇ 1 x sec ⁇ 1 ) between the interacting antibody-antigen pair.
  • antibodies that specifically bind human TF may be cross-reactive with TF proteins from other species.
  • cross-reactive refers to the ability of an antibody to bind TF from a different species.
  • antibodies according to the invention specific for human TF may also bind TF from other species (eg, cynomolgus TF).
  • Methods of determining cross-reactivity include those described in the Examples as well as standard assays known in the art, for example by using flow cytometry or cellular ELISA techniques.
  • Species cross-reactivity of antibodies is advantageous in some circumstances. For example, when the target antibody has species cross-reactivity to preclinical experimental animals, such as primates, it will facilitate the preclinical safety and efficacy evaluation of the target antibody before human therapeutic or diagnostic application.
  • the term "isotype" refers to the class of an antibody determined by the constant region of the antibody's heavy chain.
  • 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 constants of said immunoglobulin type. district.
  • the antibody of the present invention may also be an IgG1 antibody having a human IgG1 constant region.
  • the present invention contemplates not only antibodies employing native sequence constant regions, but also antibodies comprising variant sequence constant regions.
  • the term "native sequence Fc region” encompasses naturally occurring Fc region sequences of various immunoglobulins, such as the Fc region sequences of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi: 10.3389/fimmu.2014.00520.).
  • the human IgG heavy chain Fc region has an amino acid sequence extending from Cys226 or from Pro230 to the carboxy-terminus of the heavy chain.
  • the C-terminal terminal lysine (Lys447) of the Fc region may or may not be present.
  • the human IgG heavy chain Fc region bears at the N-terminus a hinge sequence or a partial hinge sequence of a native immunoglobulin, for example a sequence from E216 to T225 or a sequence from D221 to T225 according to EU numbering.
  • variant sequence Fc region refers to an Fc region polypeptide comprising a modification relative to a native sequence Fc region polypeptide.
  • the modification may be addition, deletion or substitution of amino acid residues. Substitutions can include naturally occurring amino acids and non-naturally occurring amino acids.
  • the purpose of the modification may be to alter the binding of the Fc region to its receptor and the effector functions elicited thereby.
  • effector functions refers to those biological activities attributable to the Fc-region of an antibody, which vary with the antibody class. It is already known that the IgG Fc region can mediate several important effector functions, such as cytokine induction, ADCC, phagocytosis, complement-dependent cytotoxicity (CDC), and the half-life/clearance rate of antibodies and antigen-antibody complexes. In some cases, depending on the therapeutic purpose, these effector functions are desirable for a therapeutic antibody, but may be unnecessary in other cases.
  • the present invention provides antibodies having an Fc region that elicits effector functions such as ADCC or CDC, thereby inducing tumor cell apoptosis, cell lysis, and/or inhibiting antigen-bearing tumor cells in tumor cells bearing TF antigens. Proliferation, dissemination and/or metastasis of tumor cells of TF.
  • the invention provides antibodies having Fc regions with altered effector functions. Effector function can be altered by making sequence changes to the Fc region of an antibody.
  • antibodies can be prepared that have an altered type of glycosylation in the Fc region, such as a low or afucosylated antibody with a reduced number of fucosyl residues or an antibody with increased bisected GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies.
  • Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also contemplated, such antibody variants may have improved CDC function. Alteration of the glycosylation pattern of the Fc region is conveniently achieved by altering the amino acid sequence of the Fc region to create or remove one or more glycosylation sites.
  • receptor-mediated endocytosis refers to the internalization and delivery of ligand/receptor complexes into the cytosol or The process of translocation to the appropriate intracellular compartment.
  • antibodies of the invention trigger TF receptor-mediated endocytosis upon binding to TF expressed on the cell surface.
  • the endocytosis rate can be measured by, for example, the method described in the Examples to characterize the receptor-mediated endocytosis activity of the antibody.
  • antibodies of the invention having receptor-mediated endocytosis activity can be used as tools in ADCs of the invention to deliver anti-tumor drugs into cancer cells.
  • sequence identity refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino-acid-by-amino acid basis over a comparison window.
  • Percent sequence identity can be calculated by comparing two optimally aligned sequences over a comparison window and determining the presence of identical nucleic acid bases (e.g., A, T, C, G, I) in the two sequences ) or the same amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) Number of positions To obtain the number of matching positions, the number of matching positions was divided by the total number of positions in the comparison window (ie, window size) and the result multiplied by 100 to yield the percent sequence identity.
  • Optimal alignment for purposes of determining percent sequence identity can be achieved in various 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 over the full length of the sequences being compared or over a region of sequence of interest.
  • the percent amino acid sequence identity is determined by optimally aligning a candidate antibody sequence with a given antibody sequence, in a preferred embodiment according to the Kabat numbering rule .
  • the comparison window ie the target antibody region to be compared
  • it will apply to align over the full length of a given antibody sequence.
  • the reference antibody refers to the heavy chain and light chain variable region amino acid sequences (SEQ ID NOs: 107 and 108) from the Tisotumab antibody part published in the patent CN 103119065 B ) anti-TF antibody constructed.
  • the reference antibody will have the same antibody structure as that portion of the antibody to be compared outside the variable regions, for example where both have heavy and light chain constant region structures, have identical heavy and light chain constant region sequences.
  • halogen generally refers to fluorine, chlorine, bromine, iodine, for example, it may be fluorine, chlorine.
  • alkyl refers to a linear or branched saturated hydrocarbon group composed of carbon atoms and hydrogen atoms. Specifically, the alkyl group has 1-10 carbon atoms, such as 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 carbon atoms.
  • C 1 -C 6 alkyl refers to a straight or branched saturated hydrocarbon group having 1 to 6 carbon atoms, examples of which are methyl, ethyl, propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, sec-butyl or tert-butyl), pentyl (including n-pentyl, isopentyl, 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, 2-ethylbutyl), etc.
  • alkenyl refers to a linear or branched unsaturated hydrocarbon group consisting of carbon atoms and hydrogen atoms and containing at least one double bond.
  • alkenyl groups have 2-8, eg 2 to 6, 2 to 5, 2 to 4 or 2 to 3 carbon atoms.
  • C 2 -C 6 alkenyl refers to a straight or branched alkenyl group having 2 to 6 carbon atoms, such as ethenyl, propenyl, allyl, 1-but Alkenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentenyl Dienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl and the like.
  • alkynyl refers to a straight or branched unsaturated hydrocarbon group consisting of carbon atoms and hydrogen atoms and containing at least one triple bond.
  • alkynyl groups have 2-8, eg 2 to 6, 2 to 5, 2 to 4 or 2 to 3 carbon atoms.
  • C 2 -C 6 alkynyl refers to a straight or branched alkynyl group having 2 to 6 carbon atoms, such as ethynyl, propynyl, propargyl, 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.
  • alkylene refers to a divalent group obtained by removing two hydrogen atoms from the same or two different carbon atoms of a linear or branched saturated alkane. Specifically, the alkylene group has 1-10 carbon atoms, such as 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 carbon atoms.
  • C 1 -C 6 alkylene refers to a straight or branched chain alkylene group having 1 to 6 carbon atoms, including, but not limited to, methylene, ethylene group, propylene group, butylene group, etc.
  • alkenylene refers to a divalent group obtained by removing two hydrogen atoms from the same or two different carbon atoms of a linear or branched unsaturated olefin containing at least one double bond.
  • alkenylene has 2-8, eg 2 to 6, 2 to 5, 2 to 4 or 2 to 3 carbon atoms.
  • C 2 -C 6 alkenylene refers to a straight or branched chain alkenylene having 2 to 6 carbon atoms, such as ethenylene, propenylene, allene group, butenylene, pentenylene, and hexenylene.
  • alkynylene refers to a divalent group obtained by removing two hydrogen atoms from the same or two different carbon atoms of a linear or branched unsaturated alkyne containing at least one triple bond .
  • an alkynylene group has 2-8, such as 2 to 6, 2 to 5, 2 to 4 or 2 to 3 carbon atoms.
  • C 2 -C 6 alkynylene refers to a straight or branched chain alkynylene having 2 to 6 carbon atoms, such as ethynylene, propynylene, alkynylene Propyl, butynylene, pentynylene and hexynylene.
  • cycloalkyl refers to a monocyclic, fused polycyclic, bridged polycyclic or spiro non-aromatic monovalent hydrocarbon ring structure having the specified number of ring atoms, which may be saturated or unsaturated, for example comprising 1 or more double bonds.
  • a cycloalkyl group may contain 3 or more, such as 3-18, 3-10, or 3-8 carbon atoms in the ring, such as C 3-10 cycloalkyl, C 3-8 cycloalkyl, C 3-6 cycloalkyl, C 5-6 cycloalkyl.
  • Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl.
  • heterocycle refers to a 5-20 membered (eg 5-14 membered, 5- 8-membered, 5-6-membered) aromatic or non-aromatic monocyclic, bicyclic, or polycyclic ring systems.
  • One or more N, C or S atoms in the heterocycle may be oxidized.
  • the heterocycle is a 5-10 membered ring system, 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. It should be understood that this term includes heteroaryl as defined herein.
  • aryl refers to a monocyclic or polycyclic aromatic hydrocarbon group having 6-20, eg 6-12 carbon atoms in the ring portion.
  • aryl is (C 6 -C 10 )aryl.
  • Non-limiting examples include phenyl, biphenyl, naphthyl or tetrahydronaphthyl, each of which may be optionally substituted with 1-4 substituents such as alkyl, trifluoromethyl, cycloalkyl , halogen, hydroxyl, alkoxy, acyl, alkyl-C(O)-O-, aryl-O-, heteroaryl-O-, amino, mercapto, alkyl-S-, aryl-S- , nitro, cyano, carboxyl, alkyl-OC(O)-, carbamoyl, alkyl-S(O)-, sulfonyl, sulfonylamino, heterocyclyl, etc
  • heteroaryl refers to a 5-20 membered (eg 5-14, 5-8, 5-6) aromatic monocyclic ring containing 1-4 heteroatoms selected from N, O or S or polycyclic ring systems, which may be substituted or unsubstituted.
  • heteroaryl is a 5-10 membered ring system, monocyclic or fused bicyclic.
  • 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-iso Oxazolyl, 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, 2-, 4- or 5-pyrimidinyl.
  • heteroalkyl means a fully saturated or containing 1 to 3 degrees of unsaturation consisting of the indicated number of carbon atoms and one to ten, preferably one to three, heteroatoms selected from O, N, Si and S Stable linear or branched chain hydrocarbons in which the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatoms are optionally quaternized.
  • the heteroatoms O, N, Si and S can be located at any internal position of the heteroalkyl group or at the position where the heteroalkyl group is attached to the rest of the molecule.
  • Up to two heteroatoms may be consecutive, such as for example -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.
  • a C1 to C4 heteroalkyl or heteroalkylene has 1 to 4 carbon atoms and 1 or 2 heteroatoms
  • a C1 to C3 heteroalkyl or heteroalkylene has 1 to 3 carbon atoms and 1 or 2 a heteroatom.
  • heteroalkyl and heteroalkylene are saturated.
  • substituted used herein to define each group means that the corresponding group may be substituted by groups such as but not limited to: alkyl, alkenyl, alkynyl, cycloalkyl , aryl, heteroaryl, heterocyclyl, halogen, cyano, nitro, azido, carboxyl, hydroxyl, mercapto, amino, mono or dialkylamino, mono or dicycloalkylamino, mono or di Arylamino, mono- or diheterocyclylamino, mono- or diheteroarylamino, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl-oxy, alkyl- or cyclo Alkyl- or heterocyclyl- or heteroaryl- or aryl-thio, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl-acyl, alkyl-
  • substituents include, but are not limited to, one or more groups independently selected from the group consisting of halogen, OH, SH, CN, NH 2 , NHCH 3 , N(CH 3 ) 2 , NO 2 , N 3 , C( O)CH 3 , COOH, C(O)-amino, OCOCH 3 , methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, Methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methylsulfonylamino, SO, SO 2 , phenyl, piperidinyl, piperazinyl and pyrimidinyl.
  • substituted or “substituted” means that one or more (eg, 1, 2, 3, or 4) hydrogens on the designated atom are replaced by the designated group, provided that the designated group does not exceed the Atoms are bonded at their normal valences at the present situation and form stable compounds, and combinations of substituents and variables are permissible only if such combinations form stable compounds.
  • linker refers to a bifunctional moiety that links a drug to an antibody in a drug-antibody conjugate.
  • Linkers of the invention may have multiple components (eg, in some embodiments, a linker responsible for conjugation to the antibody; a degradable peptide unit; and optionally a spacer).
  • PEG unit refers to an organic moiety comprising repeating ethyleneoxy subunits (PEG or PEG subunits), which may be polydisperse, monodisperse, or discrete (i.e., having a discrete number of ethylene-oxyl subunit).
  • Polydisperse PEGs are a heterogeneous mixture of sizes and molecular weights, while monodisperse PEGs are usually purified from heterogeneous mixtures and thus have a single chain length and molecular weight.
  • Preferred PEG units comprise discrete PEGs, which are compounds synthesized in a stepwise fashion rather than via a polymerization process. Discrete PEGs provide a single molecule with a defined and specified chain length.
  • pharmaceutically acceptable salt means a salt that can maintain the biological effects and properties of the ADC conjugate of the present invention, and the salt is not biologically or otherwise undesirable.
  • the ADC conjugates of the present invention may exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts.
  • a pharmaceutically acceptable non-toxic acid addition salt refers to a salt formed between the ADC conjugate in the present invention and an organic or inorganic acid
  • the organic or inorganic acid includes but is not limited to hydrochloric acid, sulfuric acid, hydrobromic acid , hydriodic 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.
  • the pharmaceutically acceptable non-toxic base addition salt refers to the salt formed by the ADC conjugate in the present 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; salts of organic bases, such as ammonium salts formed with organic bases containing N groups.
  • alkali metal salts such as lithium, sodium or potassium salts
  • alkaline earth metal salts such as calcium or magnesium salts
  • salts of organic bases such as ammonium salts formed with organic bases containing N groups.
  • solvate means an association formed by one or more solvent molecules and the ADC conjugate of the present invention.
  • Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethylsulfoxide, and the like.
  • DAR drug:antibody ratio
  • DAR refers to the ratio of drug moiety (D) to Ab moiety conjugated to the Ab moiety described herein in an ADC conjugate.
  • DAR can be determined by q in formula I, for example, DAR can be 1 to 20, such as 2-18, 4-16, 5-12, 6-10, 2-8, 3- 8, 2-6, 4-6, 6-10, eg 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
  • DAR can also be calculated as the average DAR of the population of molecules in the product, i.e., the Ab moieties conjugated to the Ab moieties described herein, measured by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA assays, electrophoresis and/or HPLC).
  • detection methods e.g., by conventional methods such as mass spectrometry, ELISA assays, electrophoresis and/or HPLC.
  • the overall ratio of the small molecule drug fraction (D) to the Ab fraction, this DAR is referred to as the average DAR in the text.
  • the conjugates of the invention have an average DAR value of 1 to 20, such as 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4 -6, 6-10, such as 1.0-8.0, 2.0-6.0, such as 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.
  • drug 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 immunomodulators (such as immunosuppressants).
  • cytotoxic agent is used in the present invention to refer to a substance that inhibits or prevents cell function and/or causes cell death or destruction.
  • examples of cytotoxic agents include, but are not limited to, camptothecins, auristatin, chlortetracycline, maytansinoids, ricin, ricin A chain, combrestatin, duocarcinogen Doxorubicin, doxorubicin, daunomycin, taxazole, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine , vinblastine, colchicine, dihydroxyanthracendione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, lotus root toxin A Chain, ⁇ -sarcin, gelonin, mitogellin, retstrictocin
  • small molecule drug refers to low molecular weight organic compounds capable of modulating biological processes.
  • Small molecules are defined as molecules having 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, peptide mimetics, and antibody mimetics. As therapeutic agents, small molecules can be more cell permeable, less susceptible to degradation, and less prone to eliciting an immune response than larger molecules.
  • composition refers to a composition that allows the active ingredients contained therein to be present in a biologically effective form and does not contain additional substances that are unacceptably toxic to the subject to which the composition is administered. Element.
  • pharmaceutical excipient refers to a diluent, adjuvant (such as Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, etc., which are administered together with the active substance.
  • adjuvant such as Freund's adjuvant (complete and incomplete)
  • excipient carrier or stabilizer, etc.
  • non-fixed combination means that two or more active ingredients (e.g., (i) an ADC molecule of the invention or an antibody of the invention, and (ii) another therapeutic agent) are combined as separate entities simultaneously or sequentially. Preferably, it is administered to the patient without a specific time limit or at the same or different time intervals, wherein such administration provides prophylactically or therapeutically effective levels of the two or more active ingredients in the patient.
  • fixed combination means that two or more active ingredients are administered to a patient simultaneously as a single entity.
  • the doses and/or intervals of administration of two or more active ingredients are preferably selected such that the combined use of the ingredients produces a greater effect in the treatment of a disease or condition than either alone. effects of the ingredients.
  • the components contained in the pharmaceutical combination may each be in the form of separate preparations, and the preparation forms thereof may be the same or different.
  • ADC molecules of the invention or antibodies of the invention and other therapeutic agents used in a pharmaceutical combination are administered at levels no greater than when they are used alone.
  • combination therapy refers to the administration of two or more therapeutic agents or treatment modalities (eg, radiation therapy or surgery) to treat a disease described herein.
  • administration includes co-administration of the therapeutic agents in a substantially simultaneous manner, eg, administration of a single capsule having fixed ratios of two or more active ingredients.
  • administration includes for the Or co-administration of two or more active ingredients in separate containers.
  • Powders and/or liquids can be reconstituted or diluted to the desired dosage before administration.
  • administration also includes using each type of therapeutic agent in a sequential manner at about the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the therapeutic combination in treating the disorders or conditions described herein.
  • mammals include, but are not limited to, domesticated 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 rodents). mouse).
  • domesticated animals e.g., cows, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rodents.
  • rodents e.g., mice and rodents.
  • tumor and cancer are used interchangeably herein to refer to a physiological disorder in mammals that is typically characterized by unregulated cell growth.
  • the term encompasses both primary and metastatic forms of the tumor.
  • the term also covers all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • anti-tumor effect refers to anti-tumor biological effects that can be exhibited by various means, including but not limited to, for example, reduction in tumor volume, reduction in tumor cell number, reduction in tumor cell proliferation, or reduction in tumor cell survival.
  • treating means slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • prevention includes the inhibition of the occurrence or development of a disease or disorder, or a symptom of a particular disease or disorder.
  • prevention refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.
  • subjects with a family history of cancer are candidates for prophylactic regimens.
  • the term "effective amount” refers to a drug (e.g., an antibody drug conjugate or a pharmaceutically acceptable salt or solvate of the present invention, or an antibody or antigen-binding fragment thereof of the present invention, or a composition or pharmaceutical combination thereof) ) which, when administered to a patient in single or multiple doses, produces the desired effect in a patient in need of treatment or prophylaxis.
  • a drug e.g., an antibody drug conjugate or a pharmaceutically acceptable salt or solvate of the present invention, or an antibody or antigen-binding fragment thereof of the present invention, or a composition or pharmaceutical combination thereof
  • the term "therapeutically effective amount” refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired therapeutic result.
  • a therapeutically effective amount is also one in which, when administered to a subject, any toxic or detrimental effects resulting from such administration will outweigh the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably achieves at least about 30%, even more preferably at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or even 100% improvement.
  • prophylactically effective amount refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired prophylactic result. Typically, the prophylactically effective amount will be less than the therapeutically effective amount because the prophylactic dose is administered in the subject before or at an earlier stage of the disease.
  • the present invention provides an antibody-drug conjugate (ADC) having the following formula (I) or a pharmaceutically acceptable salt or solvate thereof: Ab-[LD] q (I)
  • D represents a cytotoxic or cytostatic drug, for example, a topoisomerase I inhibitor
  • the invention provides antibody-drug conjugates (ADCs) comprising an antibody of the invention that specifically binds TF as the Ab unit of a conjugate of Formula I of the invention.
  • the anti-TF antibody of the present invention used as the Ab unit may be a full-length antibody or an antibody fragment.
  • the Ab of formula (I) of the invention is a full-length antibody comprising a heavy chain constant region and a light chain constant region.
  • the Ab of formula (I) of the invention has a tetrameric structure formed by two light chains and two heavy chains.
  • the Ab in formula (I) of the present invention is an IgG antibody, especially an IgG1 antibody.
  • the Ab in formula (I) of the invention is a humanized antibody.
  • the anti-TF antibody of the present invention used in the ADC of the present invention has one or more of the following properties:
  • the anti-TF antibody and antibody-drug conjugate can bind to TF-positive tumor cells with high affinity, and have substantially no or reduced effect on coagulation initiated by cell surface TF.
  • anti-TF antibodies of the invention for use in the ADCs of the invention, see also the detailed description provided below in the section "Anti-TF antibodies of the invention”.
  • anti-TF antibodies for use in the ADCs of the invention comprise the following CDR sequences:
  • the CDRs are defined according to Chothia, AbM, Kabat, IMGT or any combination thereof. Still more preferably, the CDRs are defined according to Kabat or IMGT or a combination thereof, more preferably, the CDRs are defined according to IMGT. However, it should be understood that the CDRs may also be defined in any other manner known in the art.
  • CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding.
  • the region of minimal overlap can be determined, thereby providing a "minimum binding unit" for antigen binding.
  • Such a minimal binding unit may be a subsection of a CDR.
  • the remainder of the CDR sequence residues as will be apparent to those skilled in the art, can be determined from the structure and protein folding of the antibody. Accordingly, the invention also contemplates variations of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit can remain unchanged, while the remaining CDR residues can be substituted.
  • the anti-TF antibody used in the ADC of the present invention comprises 3 complementarity determining regions HCDR of the heavy chain variable region, and 3 complementarity determining regions LCDR of the light chain variable region, wherein:
  • 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 composition
  • LCDR1 contains SEQ the amino acid sequence of ID NO:21 or consists thereof
  • LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:22
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:23;
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:26; or
  • HCDR1 comprises the amino acid sequence of SEQ ID NO:15 or consists of it
  • HCDR2 comprises the amino acid sequence of SEQ ID NO:16 or consists of it
  • HCDR3 comprises the amino acid sequence of SEQ ID NO:17 or consists of it
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:29; or
  • 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 the amino acid sequence of SEQ ID NO:31 or consists of it
  • LCDR3 comprises the amino acid sequence of SEQ ID NO:32 or consists of it.
  • the anti-TF antibody used in the ADC of the present invention comprises 3 complementarity determining regions HCDR of the heavy chain variable region, and 3 complementarity determining regions LCDR of the light chain variable region, wherein:
  • HCDR1 comprises the amino acid sequence of SEQ ID NO:33 or consists of it
  • HCDR2 comprises the amino acid sequence of SEQ ID NO:34 or consists of it
  • HCDR3 comprises the amino acid sequence of SEQ ID NO:35 or consists of it
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:47; or
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:50; or
  • HCDR1 comprises the amino acid sequence of SEQ ID NO:39 or consists of it
  • HCDR2 comprises the amino acid sequence of SEQ ID NO:40 or consists of it
  • HCDR3 comprises the amino acid sequence of SEQ ID NO:41 or consists of it
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:53; or
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • the anti-TF antibody used in the ADC of the present invention comprises 3 complementarity determining regions HCDR of the heavy chain variable region, and 3 complementarity determining regions LCDR of the light chain variable region, wherein:
  • HCDR1 comprises the amino acid sequence of SEQ ID NO:57 or consists of it
  • HCDR2 comprises the amino acid sequence of SEQ ID NO:58 or consists of it
  • HCDR3 comprises the amino acid sequence of SEQ ID NO:59 or consists of it
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:71; or
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:74; or
  • HCDR1 comprises the amino acid sequence of SEQ ID NO:63 or consists of it
  • HCDR2 comprises the amino acid sequence of SEQ ID NO:64 or consists of it
  • HCDR3 comprises the amino acid sequence of SEQ ID NO:65 or consists of it
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:77; or
  • 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 the amino acid sequence of SEQ ID NO:79 or consists of it
  • LCDR3 comprises the amino acid sequence of SEQ ID NO:80 or consists of it.
  • the anti-TF antibody used in the ADC of the present invention comprises 3 complementarity determining regions HCDR of the heavy chain variable region, and 3 complementarity determining regions LCDR of the light chain variable region, wherein:
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:95; or
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:98; or
  • HCDR1 comprises the amino acid sequence of SEQ ID NO:87 or consists of it
  • HCDR2 comprises the amino acid sequence of SEQ ID NO:88 or consists of it
  • HCDR3 comprises the amino acid sequence of SEQ ID NO:89 or consists of it
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:101; or
  • 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 the amino acid sequence of SEQ ID NO:103 or consists of it
  • LCDR3 comprises the amino acid sequence of SEQ ID NO:104 or consists of it.
  • an anti-TF antibody of the invention for use in an ADC of the invention comprises: the heavy and light chains of any of the exemplary antibodies 2B12B10-hz1, 30G11B7-hz1, 22F11H5-hz1, and 27H8H3-hz1 of the invention may be Variable region sequences or variants thereof, eg, antibodies or fragments thereof, eg, humanized antibodies, having the same CDR sequences as one of the exemplary antibodies, and having the same or different framework region sequences.
  • sequence similarity search tools such as Gapped BLAST, can be used to compare the protein sequence of the parental antibody with protein sequences in a database; select a framework sequence with a high degree of structural similarity to the parental antibody to be altered, e.g., with Database framework sequences with at least 80%, 85%, 90%, or 95%, 96%, 97%, 98%, 99% or more sequence identity are used for framework region substitution.
  • one to several residue mutations such as back mutations, can be performed on the replaced framework sequence based on the need, and assayed in vitro or in vivo In assessing the maintenance or improvement of the antigen-binding properties or other functional properties of the mutated antibody.
  • the anti-TF antibody of the present invention used in the ADC of the present invention comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises:
  • 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
  • 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
  • the anti-TF antibody used in the ADC of the present invention comprises a heavy chain variable region and a light chain variable region, wherein: said light chain variable region comprises:
  • 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
  • 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
  • amino acid sequence shown 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.
  • the anti-TF antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence described in SEQ ID NO: 1 or at least 95% thereof %, 96%, 97%, 98% or 99% sequence identity amino acid sequence, and wherein said light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2, or has at least 95%, 96% thereof , 97%, 98% or 99% sequence identity of amino acid sequences.
  • 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.
  • the anti-TF antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence described in SEQ ID NO: 3 or has at least 95 %, 96%, 97%, 98% or 99% sequence identity amino acid sequence, and wherein said light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 4, or has at least 95%, 96% thereof , 97%, 98% or 99% sequence identity of amino acid sequences.
  • 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.
  • the anti-TF antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence described in SEQ ID NO: 5 or has at least 95 %, 96%, 97%, 98% or 99% sequence identity amino acid sequence, and wherein said light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 6, or has at least 95%, 96% thereof , 97%, 98% or 99% sequence identity of amino acid sequences.
  • 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.
  • the anti-TF antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence described in SEQ ID NO: 7 or at least 95% thereof %, 96%, 97%, 98% or 99% sequence identity amino acid sequence, and wherein said light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, or has at least 95%, 96% thereof , 97%, 98% or 99% sequence identity of amino acid sequences.
  • the antibody or antigen-binding fragment comprises the heavy chain variable region of SEQ ID No: 7 and SEQ ID No: 7 The light chain variable region of ID No: 8, or consists of it.
  • the anti-TF antibody of the invention used as the Ab unit in the ADC of the invention may, in some cases, comprise a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region is a heavy chain constant region derived from human immunoglobulin.
  • the light chain constant region is a light chain constant region derived from human immunoglobulin.
  • the heavy chain constant region comprised in the anti-TF antibody may be of any isotype or subtype, such as an IgG1, IgG2, IgG3 or IgG4 isotype heavy chain constant region.
  • the anti-TF antibody preferably comprises an IgG1 heavy chain constant region, especially a human IgG1 heavy chain constant region, such as the amino acid sequence of SEQ ID NO: 106 or at least 90%, 95%, 96%, Amino acid sequences with 97%, 98% or 99% sequence identity.
  • the light chain constant region contained in the anti-TF antibody may be a kappa light chain constant region or a lambda light chain constant region.
  • the anti-TF antibody comprises a human-derived ⁇ light chain constant region or a ⁇ light chain constant region, preferably a human ⁇ light chain constant region, for example, the amino acid sequence of SEQ ID NO: 105 or a combination thereof Amino acid sequences having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • Antibody variants useful as Ab units according to the invention preferably retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding ability) of the parent antibody prior to the introduction of the alteration. More preferably, the alterations do not result in the antibody variant losing binding to the antigen, but optionally confer properties such as increased affinity for the antigen.
  • the heavy or light chain variable regions of an antibody, or each of the CDR or framework regions may be altered individually or in combination.
  • changes can also be made to the Fc region of the antibody. Alterations in the Fc region can be made alone, or in combination with changes in the framework and/or CDR regions.
  • the Fc region can be altered, eg, to alter one or more effector functions of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
  • antibodies of the invention may be chemically modified (eg, attached to PEG) or their glycosylation pattern altered.
  • antibodies according to the invention can be conjugated to toxin-linkers using some of the natural attachment sites thereon.
  • natural attachment sites are the sulfhydryl group of cysteine and the amino group of lysine.
  • DARs drug-to-antibody ratios
  • toxins are coupled to antibodies of the invention by sulfhydryl chemistry following reduction of the interchain disulfide bonds of the antibody to form ADC conjugates of formula (I).
  • the introduction of artificial linking sites in the antibody can also be considered to achieve more site-specific conjugation.
  • 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 can be any drug or its prodrug that is toxic or inhibitory to cells. Those skilled in the art can select a suitable drug molecule as the payload of the ADC according to the required ADC drug onset mechanism and cell killing effect.
  • Drug D is a cytotoxic agent.
  • cytotoxic agents suitable for use as payloads have been reported, including, but not limited to,
  • Microtubule inhibitory/destroying agents such as, but not limited to, auristatins (eg, MMAE or MMAF), maytansine derivatives (eg, DM2, DM4), tubulysins (Tubulysins), cryptocolistin (Cryptomycins), anti-mitotic EG5 inhibitors (eg, spindle kinesin KSP inhibitors);
  • DNA damaging agents such as, but not limited to, pyrrolobenzodiazepines (Pyrrolobenzodiazepines) (for example, pyrrolo[2,1-c][1,4]benzodiazepines (PBD )), Duocarmycin, Indolinobenzodiazepine; Duocarmycins; Calicheamicins;
  • Topoisomerase inhibitors for example, but not limited to, camptothecins (eg, exitecan and its derivative Dxd);
  • the drug D in the formula (I) of the present invention may be any compound selected from the above.
  • drug D is an anti-tumor growth inhibitor selected from maytansine derivatives, calicheamicin derivatives, and auristatin derivatives.
  • Drug D is a tubulin inhibitor/stability destabilizer such as vinca alkaloids, vincristine, paclitaxel and docetaxel.
  • drug D is a DNA synthesis inhibitor, eg, methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine.
  • Drug D is a DNA topoisomerase inhibitor, e.g., a topoisomerase I inhibitor (e.g. eg, camptothecins) and topoisomerase II inhibitors (eg, actinomycin D, doxorubicin, mitoxantrone).
  • a topoisomerase I inhibitor e.g. eg, camptothecins
  • topoisomerase II inhibitors eg, actinomycin D, doxorubicin, mitoxantrone
  • the drug D of the ADC according to the invention is a topoisomerase I inhibitor.
  • a typical example of a topoisomerase I inhibitor is a camptothecin class of drugs such as, but not limited to, camptothecin (CPT), hydroxycamptothecin, 9-aminocamptothecin, exatecan, topotecan, belotecan, irinotecan , SN-38, and FL118, and their derivatives.
  • CPT camptothecin
  • CPT camptothecin
  • hydroxycamptothecin hydroxycamptothecin
  • 9-aminocamptothecin 9-aminocamptothecin
  • exatecan topotecan
  • belotecan belotecan
  • irinotecan irinotecan
  • SN-38 SN-38
  • FL118 FL118
  • the drug D of the ADC of the present invention is a camptothecin drug selected from the following:
  • RA is selected from, for example, hydrogen, optionally substituted alkyl, wherein substituents include but are not limited to hydroxy, amino, wherein the amino moiety or hydroxy moiety may be substituted or unsubstituted, e.g., by alkyl, alkylacyl , or alkylsulfonyl substitution;
  • substituents include but are not limited to hydroxy, amino, wherein the amino moiety or hydroxy moiety may be substituted or unsubstituted, e.g., by alkyl, alkylacyl , or alkylsulfonyl substitution;
  • the drug D is selected from, for example, hydrogen, optionally substituted alkyl, wherein substituents include but are not limited to hydroxy, amino, wherein the amino moiety or hydroxy moiety may be substituted or unsubstituted, e.g., by alkyl, alkylacyl , or alkylsulfonyl substitution;
  • the drug D is
  • R B is selected from, for example, hydrogen, cycloalkyl, phenyl, optionally substituted alkyl, wherein the substituents include, but are not limited to, halogen, hydroxy, optionally substituted alkoxy, cycloalkyl, heterocyclic Alkyl, phenyl, amino, wherein the amino moiety may be optionally substituted; in one embodiment, the drug D is
  • HCPT -10-hydroxy CPT
  • R C is selected from, for example, optionally substituted alkyl and cycloalkyl;
  • R' C is selected from, for example, H, alkanoyl or optionally substituted heterocycloalkyl acyl;
  • the drug D It is 7-ethyl-10-hydroxy CPT (SN-38) or its prodrug irinotecan (irinotecan, CPT-11), or the drug D is topotecan
  • the drug D is:
  • F118 ((20S)-10,11-methylenedioxycamptothecin) was obtained by high-throughput screening compound library. Compared with other camptothecin derivatives, FL118 has been demonstrated to have much higher in vivo and in vitro anticancer activity in many different cancer types. In addition to inhibiting topoisomerase I, FL118 can also selectively inhibit gene promoter activity and endogenous expression of anti-apoptotic proteins such as survivin, XIAP, cIAP2 and Mcl-1.
  • the conjugate formed by combining the compound with the antibody of the present invention and the linker not only effectively exerts this
  • the compound has a tumor-killing effect, and overcomes the defects of the compound, and the formed conjugate has a small degree of aggregation and has good animal tolerance.
  • the drug D of the ADC of the present invention is a FL118 derivative, especially a 7-substituted FL118.
  • the drug D in formula (I) of the present invention is a camptothecin drug comprising the following formula D structure:
  • R x , R y are each independently selected from H, halogen, -OH, C 1- C 6 alkyl, or R x and R y form together with the carbon atoms they are connected to have 1 or 2 selected from N, S and 5-6 membered heterocycle of O;
  • R a is selected from H, halogen, -OH, optionally substituted C 1 -C 8 alkyl or C 3 -C 8 alkynyl or C 3 -C 8 alkenyl, optionally substituted C 1 -C 8 alkoxy Base, optionally substituted C 3- C 8 cycloalkyl, optionally substituted phenyl, optionally substituted heterocycloalkyl, optionally substituted heteroaryl,
  • R a is selected from:
  • R 1 and R 2 are independently selected from
  • C 1 -C 8 alkyl optionally substituted with substituents selected from the group consisting of hydroxy, amino, amino substituted by one or two C 1 -C 4 alkyl, one or two C 1 -C 4 Amino groups substituted by hydroxyalkyl, amino groups substituted by (C 1 -C 4 hydroxyalkyl) and (C 1 -C 4 alkyl);
  • R and R combined with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered heterocyclic ring having 0 to 3 substituents selected from halogen, C 1 -C 4 alkyl , OH, C 1 -C 4 alkoxy, NH 2 , NH(C 1 -C 4 alkyl) and N(C 1 -C 4 alkyl) 2 ,
  • cycloalkyl, heterocycloalkyl, phenyl, and heteroaryl are each independently optionally substituted by 0-3 substituents selected from the group consisting of OH, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, NH 2 , NH(C 1 -C 4 alkyl) and N(C 1 -C 4 alkyl) 2 substitutions.
  • Rx and Ry are each independently H. In other preferred embodiments, Rx is F and Ry is methyl. In yet other preferred embodiments, R x and R y together with the carbon atoms to which they are attached form a 5 membered heterocyclic ring containing 2 O's.
  • the drug D in the formula (I) of the present invention is a camptothecin drug comprising the following formula D a or formula D b structure:
  • camptothecins of formula (D a ) are especially camptothecins of formula (D a ).
  • R a is selected from:
  • R 1 and R 2 are independently selected from
  • C 1 -C 8 alkyl optionally substituted with substituents selected from the group consisting of hydroxy, amino, amino substituted by one or two C 1 -C 4 alkyl, one or two C 1 -C 4 Amino groups substituted by hydroxyalkyl, amino groups substituted by (C 1 -C 4 hydroxyalkyl) and (C 1 -C 4 alkyl);
  • R and R combined with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered heterocyclic ring having 0 to 3 substituents selected from halogen, C 1 -C 4 alkyl , OH, C 1 -C 4 alkoxy, NH 2 , NH(C 1 -C 4 alkyl) and N(C 1 -C 4 alkyl) 2 ,
  • cycloalkyl, heterocycloalkyl, phenyl, and heteroaryl are each independently optionally substituted by 0-3 substituents selected from the group consisting of OH, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, NH 2 , NH(C 1 -C 4 alkyl) and N(C 1 -C 4 alkyl) 2 substitutions.
  • Ra is hydrogen
  • R a is C 3 -C 8 cycloalkyl (eg, cyclopropyl, cycloheptyl, cyclopentyl).
  • Ra is phenyl. In other embodiments, R a is phenyl substituted with NH 2 or NH(C 1 -C 4 alkyl).
  • R a is C 1 -C 6 alkyl, for example, methyl, ethyl, propyl, 2-methylpropyl, butyl, isobutyl, 2,2-dimethyl- Propyl, 2,2-Dimethyl-butyl, n-hexyl, n-pentyl, 3-ethyl-pentyl.
  • R a is C 1 -C 4 alkyl optionally substituted by hydroxy or C 1 -C 4 alkoxy, wherein alkoxy is optionally further replaced by -NH 2 , -NH( C 1 -C 4 alkyl) and -N(C 1 -C 4 alkyl) 2 substituted.
  • R a is C 2 -C 4 alkenyl or C 2 -C 4 alkynyl, optionally substituted with hydroxy or amino.
  • Ra is C 1 -C 4 alkyl substituted with 1 or 2 5-6 membered heterocycloalkyls selected from N, S, and O, wherein the heterocycloalkyl moiety is optionally Further substituted by C 1 -C 4 alkyl, preferably, the heterocycloalkyl is piperazinyl or morpholinyl.
  • R a is C 1 -C 4 alkyl substituted by -NR 1 R 2 , especially -methylene-NR 1 R 2 , wherein R 1 and R 2 are independently selected from each other:
  • Phenyl Halogenated phenyl
  • R a is C 1 -C 4 alkyl substituted by -NR 1 R 2 , especially -methylene-NR 1 R 2 , wherein R 1 and R 2 are combined with the nitrogen atom to which they are attached A 5-, 6- or 7-membered heterocycloalkyl ring is formed.
  • R 1 and R 2 combine with the nitrogen atom to which they are attached to form a 6-membered ring.
  • the 6-membered ring is morpholinyl or piperazinyl.
  • R a is -C 1 -C 4 alkylene-OH, -C 1 -C 4 alkylene-OC 1 -C 4 alkylene-NH 2 , or -C 1 -C 4 alkylene-NH 2 .
  • R a is -C 1 -C 4 alkylene-OH.
  • Ra is hydroxymethyl.
  • Ra is hydroxyethyl.
  • Ra is hydroxypropyl.
  • R a is -C 1 -C 4 alkylene-NH 2 .
  • Ra is aminomethyl.
  • Ra is aminoethyl.
  • Ra is aminopropyl.
  • the drug D is linked to the linker L through a free hydroxyl or amino group on it, so as to be coupled to the antibody. More preferably, the drug D of formula D or formula D a or D b is connected to the linker L through the hydroxyl or amino group on R a to be coupled to the antibody.
  • R a has a structure selected from the following after being connected with a linker:
  • R a after being connected with the linker has a structure selected from the following:
  • R a after linking with the linker has a structure selected from the following:
  • the wavy line on the left indicates the position connected with the linker L
  • the asterisk on the right indicates the position connected with the camptothecin mother nucleus.
  • the LD unit in formula (I) of the present invention comprises the following structures:
  • the LD unit in formula (I) comprises the following structure:
  • the linker L suitable for the present invention may be any linker capable of coupling the antibody of the present invention with a drug.
  • the linker should be added to ensure sufficient stability of the ADC of the invention in the circulation while providing rapid and efficient release of the active form of the toxic drug at the target site (eg tumor cells or tumor environment).
  • the linker in formula (I) of the present invention is a non-degradable linker.
  • a non-degradable linker includes, but is not limited to, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).
  • SMCC N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • ADCs containing such linkers must be internalized by the cell, and the antibody portion of the ADC is degraded by intracellular lysosomal proteases, releasing the pharmaceutically active molecule.
  • the linker in formula (I) of the present invention is a degradable linker.
  • Drug release from ADCs comprising such linkers is triggered by the nature of the cleavage site in the linker.
  • the cleavage site of such linkers can be designed according to the characteristics of the target therapeutic site (eg, tumor cell lysosome and/or tumor environment).
  • a degradable linker can consist of a coupling moiety, a degradable moiety, and optionally a spacer moiety.
  • the conjugation moiety is responsible for the attachment of the antibody to the linker-drug and can be selected according to the desired antibody conjugation chemistry.
  • the degradable moieties will comprise peptides or peptide analogs that are recognized by enzymes under an enzyme-based release mechanism, for example, Val-Ala, Val-Cit, Phe-Lys, Gly-Phe-Leu- Gly, Ala-Leu-Ala-Leu, Gly-Gly-Phe-Gly, cyclobutyl-Ala, cyclobutyl-Cit and other oligopeptides or oligopeptide analogues.
  • ADC properties can be improved by introducing modifications in the linker at peptide residue positions adjacent to the enzymatic cleavage site, for example, the stability of the ADC in blood circulation and/or the drug resistance of the ADC at the target site.
  • a spacer can also be introduced between the degradable part of the linker and the drug D to facilitate the release of the drug active molecule from the rest of the conjugate (especially, traceless release), for example , a p-aminobenzyl carbamate (PABA) or aminomethyl (-NHCH 2 -) spacer that can be eliminated spontaneously in acidic medium.
  • PABA p-aminobenzyl carbamate
  • -NHCH 2 - aminomethyl
  • Linkers applicable to the present invention include, but are not limited to, those disclosed in WO2022/170971 (this document is hereby incorporated by reference).
  • the linker L in formula (I) of the present invention has the structure of formula (II): -ZYM- (II),
  • Z is the linker connected with Ab
  • Y is a peptide of 2-5 amino acids, preferably a dipeptide, tripeptide or tetrapeptide,
  • M is absent, or is a spacer for attachment of drug D.
  • the sulfur group of antibody cysteine exists in the form of a disulfide bond. Opening of antibody disulfide bonds provides free sulfhydryl groups as conjugation sites.
  • One way to form ADC by conjugating antibody sulfhydryl groups is to make Michael addition reaction between the free sulfhydryl groups on the antibody and heterocyclic (eg maleimide) linkers.
  • Another way is to make a nucleophilic substitution reaction between a linker comprising a heteroaryl ring substituted with a leaving group and a free sulfhydryl group in an antibody molecule to obtain an antibody-drug conjugate. Both approaches are applicable to the ADC conjugates of the present invention.
  • linkers according to the invention may in some aspects comprise a Z linker of the heterocyclic type (e.g. maleimide type) or heteroaromatic ring type (e.g. pyrimidine type); Also, in some cases it may be preferable to include a heteroaromatic (eg, pyrimidine) linker to provide a conjugate with greater stability in blood circulation.
  • heterocyclic type e.g. maleimide type
  • heteroaromatic ring type e.g. pyrimidine type
  • Z in formula (II) has the following structure: -Z 1 -Z 2 -Z 3 -Z 4 -,
  • Z is a 5-10 membered heterocyclic group, preferably containing 1 or 2 heteroatoms selected from N, S and O;
  • Z 4 is a bond or a PEG unit represented by the formula
  • R 5 is selected from C 1-4 alkylene, -NH-, -NH-C 1-4 alkylene-heteroaryl-, wherein heteroaryl is 5-membered or 6-membered nitrogen-containing heteroaryl , preferably triazolyl;
  • Z is 5-10 membered heteroaryl.
  • Z is selected from pyrimidine, thiazole, benzene optionally substituted with one or more independently selected from hydrogen, halogen, nitro, C1-6 alkyl and halogenated C1-6 alkyl Prothiazole, oxazole, quinazoline and pyrrolo[2,3d]pyrimidine.
  • Z is heteroaryl selected from the group consisting of,
  • Z2 is linked to Z1 through a carbon atom adjacent to the heteroatom.
  • Z is pyrimidinylidene, preferably more preferred Among them, the wavy line on the left indicates the position connected with Z 1 ; the wavy line on the right indicates the position connected with Z 3 .
  • Z is maleimide, Among them, the wavy line on the left indicates the position connected with Z 1 ; the wavy line on the right indicates the position connected with Z 3 .
  • n' is 1-6, preferably, Z 3 is more preferred
  • the wavy line on the left side represents the position connected with Z2
  • the wavy line on the right side represents the position connected with Z4
  • Z2 is a pyrimidinyl group
  • Z 2 is pyrimidinylene
  • Z 2 is maleimide
  • Z 4 is a bond and Z 3 is directly linked to Y in formula (II).
  • Z4 is a unit comprising 2-12 PEG. In some embodiments, Z is
  • n 1-8, eg, 2, 3, 4, 5, 6, 7 or 8.
  • Z is selected from: Wherein the wavy line on the left represents the position connected with Z3 ; the wavy line on the right represents the position connected with Y in formula II.
  • Z in formula (I) of the present invention has the following structure:
  • Z in formula (I) of the present invention has the following structure:
  • Z in formula (I) of the present invention has the following structure:
  • S is a sulfur atom in Ab
  • R b is
  • Z has the structure:
  • Z in formula (I) of the present invention has the following structure:
  • Z in formula (I) of the present invention has the following structure:
  • antibody-drug conjugates For the purpose of limiting or minimizing the off-target toxicity of ADCs while ensuring release of the toxin molecules at the target tumor site, in some aspects it is advantageous to design antibody-drug conjugates so that they are highly selective in targeting Simultaneously to tumor cells, it can be degraded by proteolytic enzymes in tumor cells or in the environment (in particular, enzymes with significantly higher activity in tumor cells and/or in the tumor environment relative to blood).
  • proteolytic enzymes in tumor cells or in the environment (in particular, enzymes with significantly higher activity in tumor cells and/or in the tumor environment relative to blood).
  • oligopeptides or oligopeptide analogs that can be recognized and cleaved by such proteolytic enzymes can be included in the linker of such ADC conjugates.
  • the Y unit that is part of the linker of the invention is a peptide-containing degradable moiety, e.g., containing two or more (e.g., 2-12, e.g. 2,3,4,5, or 6) degradable peptide linkers of continuous or discontinuous amino acids.
  • Each amino acid of the peptide-containing unit may independently of one another be selected from natural amino acids or unnatural amino acids, for example from: 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, beta-alanine, citrulline, and their derivatives.
  • natural amino acids or unnatural amino acids for example from: alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid , glutamine, phenylalanine, lysine, substituted lysine, leucine, serine, tyrosine, threonine,
  • each amino acid is independently selected from alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine amino acid, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, cysteine, methionine and their derivatives.
  • each amino acid is independently selected from alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine amino acid, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan and valine, N-methylglycine, ⁇ -alanine, and their derivatives .
  • the amide bond in the Y unit will be recognized and degraded by enzymes in tumor cell lysosomes, releasing the drug moiety D.
  • the amide bond in the Y unit can be recognized and degraded by enzymes in the tumor environment, releasing the drug moiety D.
  • Linkers with such Y-containing peptide units will facilitate the release of toxin molecules in tumor cells and the environment by designing antibody-drug conjugates that favor tumor distribution.
  • Y is a unit comprising 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, Cyclobutyl-Cit.
  • Y is a unit comprising 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, Ala-Ala-Asn.
  • Y is a dipeptide, tripeptide, tetrapeptide or pentapeptide comprising substituted lysines.
  • the substituted lysine is:
  • R 3 and R 4 are independently selected from: H, C 1-6 alkyl, -CO-NH 2 , -CONH (C 1-6 alkyl), and -CONH (C 1-6 alkyl) 2 , wherein the alkyl group is optionally substituted by a group selected from the group consisting of halogen, C 1-6 alkoxy, C 1-6 haloalkyl, C 3-6 cycloalkyl, 6-10 membered aryl and 5-14 membered heteroaryl.
  • Y is a peptide having the following amino acid sequence from N-terminus to C-terminus; Xaa 1 -Xaa 2 -Xaa 3 -Xaa 4 -Xaa 5 ,
  • 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, alanine and valine, preferably valine;
  • Xaa 3 is unsubstituted or substituted lysine
  • Xaa4 is an amino acid selected from leucine, glycine and alanine;
  • Xaa 5 is absent, or is an amino acid selected from glycine and alanine;
  • N-terminal of the amino acid sequence is connected to the Z unit of the linker, and the C-terminal is connected to the M unit or directly connected to the drug D,
  • Xaa 3 is lysine in which the ⁇ -amino group is mono- or di-substituted by C 1 -C 3 alkyl,
  • 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 lysine which is unsubstituted or mono- or di-substituted by C 1 -C 3 alkyl.
  • the ADC conjugates of the invention have a spacer (M) between the releasable peptide-containing unit (Y) and the drug (D).
  • the spacer can be a functional group that facilitates attachment of the peptide-containing unit (Y) to drug D, or can provide an additional structural component to further facilitate the release of drug D from the rest of the conjugate (e.g., a self-degrading group such as p-amino benzyl (PAB) component).
  • PAB p-amino benzyl
  • the M spacer between the Y and Drug D units can be selected from:
  • the wavy line on the left indicates the connection with Y
  • the wavy line on the right indicates the connection with the D unit of the drug.
  • M is a covalent bond
  • Y in formula I and drug D are directly connected, such as through an amide bond.
  • the YM unit in linker L comprises the following structure:
  • the YM unit is connected with the following Z unit:
  • L in formula (I) of the present invention is a linker comprising the following structure:
  • R3 and R4 are each independently selected from methyl, ethyl, propyl.
  • both R3 and R4 are methyl; or both R3 and R4 are ethyl; or both R3 and R4 are propyl.
  • the L-D unit of formula I of the present invention is linked to the antibody by forming a thioether bond with the sulfhydryl group of the free cysteine of the light chain and/or heavy chain of Ab.
  • the present invention provides an ADC conjugate having the following structure, or a pharmaceutically acceptable salt or solvate thereof:
  • q represents an average DAR value of 1-20, such as an average DAR value of about 2, 3, 4, 6 and 8.
  • ADCs according to the present invention have at least one or more of the following advantages:
  • the ADC according to the present invention may also have at least one or more of the following advantages:
  • DAR can be as high as 8.
  • conjugation of the drug-linker to the antibody is accomplished by reaction with amino acid residues of the antibody.
  • a heteroaryl linker L with a leaving group is used to couple a drug D to a cysteine residue of an antibody to prepare a conjugate of formula I of the present invention.
  • interchain disulfide bonds of the antibody are broken and free sulfhydryl groups are exposed for attachment to heteroaryl linkers by controlling the conditions under which the antibody is treated with a reducing agent such as tris(2-hydroxyethyl)phosphine (TCEP). - drug conjugation.
  • TCEP tris(2-hydroxyethyl)phosphine
  • Conjugates prepared by this method may contain zero, one, two, three, four, five, six, seven or eight drugs per antibody molecule.
  • the drug loading of the conjugate is represented by the average DAR, which is the average number of drug molecules per antibody.
  • the average number of drug per antibody of the antibody-drug conjugate composition produced by the preparation can be characterized by conventional means, such as mass spectrometry, ELISA assay and HPLC.
  • the quantitative distribution expressed as q of the antibody-drug conjugate can also be determined.
  • the separation, purification and characterization of homogeneous antibody-drug conjugates in which q is a certain value and antibody-drug conjugates with other drug loadings can be achieved by means such as reversed-phase HPLC or electrophoresis.
  • q represents the number of drug-linker (L-D) moieties coupled to an individual antibody (Ab), and is preferably 1 to 16, 1 to 12, 1 to 10, or An integer from 1 to 8.
  • the individual ADC conjugates may also be referred to as ADC compounds.
  • on the ADC compound according to the invention there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16
  • Each drug linker moiety is coupled to a single antibody.
  • q represents the average DAR of the prepared conjugate composition.
  • q can be in 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 An integer or decimal in the range of 8.
  • q represents an average DAR of about 3.
  • q represents an average DAR of about 6.
  • q represents an average DAR of about 8.
  • the present invention provides a composition, preferably a pharmaceutical composition or pharmaceutical formulation, comprising any ADC described herein, or a pharmaceutically acceptable salt or solvate thereof.
  • the composition further comprises pharmaceutical excipients.
  • a composition eg, a pharmaceutical composition, comprises an ADC of the invention, or a pharmaceutically acceptable salt or solvate thereof, in combination with one or more other therapeutic agents.
  • compositions comprising an ADC of the present invention, or a pharmaceutically acceptable salt or solvate thereof.
  • compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • compositions of the invention can be in a variety of forms. These forms include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (eg, injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories.
  • liquid solutions eg, injectable solutions and infusible solutions
  • powders or suspensions e.g., liposomes and suppositories.
  • liposomes e.g., liposomes and suppositories.
  • a medicament comprising an ADC as described herein may be prepared by admixing an ADC of the invention, or a pharmaceutically acceptable salt or solvate thereof, of the desired degree of purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized in the form of preparations or aqueous solutions.
  • compositions or formulations of the invention may also contain more than one active ingredient as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • active ingredients including chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (eg, immune checkpoint inhibitors or agonists), among others.
  • the active ingredients are suitably present in combination in amounts effective for the intended use.
  • sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody in the form of shaped articles such as films or microcapsules.
  • the present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising the present invention or a pharmaceutically acceptable salt or solvate thereof and one or more other therapeutic agents.
  • Another object of the present invention is to provide a kit of parts comprising the pharmaceutical combination of the present invention, preferably said kit is in the form of pharmaceutical dosage units. Dosage units may thus be presented according to a dosing regimen or interval between drug administrations.
  • 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 pharmaceutically acceptable salt or solvate thereof;
  • the invention provides a combination product wherein the other therapeutic agent is, for example, a therapeutic agent such as an antibody effective to stimulate an immune response thereby further enhancing, stimulating or up-regulating the subject's immune response.
  • the combination is used to prevent or treat TF positive tumors.
  • the present inventors identified a humanized anti-TF monoclonal antibody according to 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, can be quickly and effectively endocytosed by tumor cells, and has little effect on TF-mediated coagulation, so it is not only suitable for use alone or in combination with other anticancer drugs Combinations are used in cancer therapy, and are also suitable as molecular components for forming new anticancer molecules targeting cancer tissues.
  • the present invention also provides a humanized anti-TF monoclonal antibody with various excellent properties, a nucleic acid encoding it, a vector and a host cell comprising the nucleic acid, and an immunoconjugate comprising the antibody , Multispecific antibody, pharmaceutical composition and use.
  • anti-TF antibody of the present invention and its properties are described in detail below. As can be understood by those skilled in the art, unless the context clearly indicates to the contrary, any antibody technical features (including structural features and property features) and any combination thereof mentioned in this section are also applicable as ADCs of the present invention.
  • the present invention provides antibodies or antigen-binding fragments thereof, especially humanized antibodies or antigen-binding fragments thereof, that specifically bind to TF, preferably human TF protein.
  • the invention provides an anti-TF antibody or antigen-binding fragment thereof comprising:
  • the above 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.
  • an anti-TF antibody or antigen-binding fragment thereof comprises 3 complementarity determining regions HCDR of the heavy chain variable region, and 3 complementarity determining regions LCDR of the light chain variable region, wherein:
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:23; or
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:26; or
  • HCDR1 comprises the amino acid sequence of SEQ ID NO:15 or consists of it
  • HCDR2 comprises the amino acid sequence of SEQ ID NO:16 or consists of it
  • HCDR3 comprises the amino acid sequence of SEQ ID NO:17 or consists of it
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:29; or
  • HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 18, and HCDR2 comprises SEQ ID The amino acid sequence of NO:19 or consists of it, HCDR3 comprises the amino acid sequence of SEQ ID NO:20 or consists of it, LCDR1 comprises the amino acid sequence of SEQ ID NO:30 or consists of it, LCDR2 comprises the amino acid sequence of SEQ ID NO:31 sequence or consists of it, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:32.
  • an anti-TF antibody or antigen-binding fragment thereof comprises 3 complementarity determining regions HCDR of the heavy chain variable region, and 3 complementarity determining regions LCDR of the light chain variable region, wherein:
  • HCDR1 comprises the amino acid sequence of SEQ ID NO:33 or consists of it
  • HCDR2 comprises the amino acid sequence of SEQ ID NO:34 or consists of it
  • HCDR3 comprises the amino acid sequence of SEQ ID NO:35 or consists of it
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:47; or
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:50; or
  • HCDR1 comprises the amino acid sequence of SEQ ID NO:39 or consists of it
  • HCDR2 comprises the amino acid sequence of SEQ ID NO:40 or consists of it
  • HCDR3 comprises the amino acid sequence of SEQ ID NO:41 or consists of it
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:53; or
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • an anti-TF antibody or antigen-binding fragment thereof comprises 3 complementarity determining regions HCDR of the heavy chain variable region, and 3 complementarity determining regions LCDR of the light chain variable region, wherein:
  • HCDR1 comprises the amino acid sequence of SEQ ID NO:57 or consists of it
  • HCDR2 comprises the amino acid sequence of SEQ ID NO:58 or consists of it
  • HCDR3 comprises the amino acid sequence of SEQ ID NO:59 or consists of it
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:71; or
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:74; or
  • HCDR1 comprises the amino acid sequence of SEQ ID NO:63 or consists of it
  • HCDR2 comprises the amino acid sequence of SEQ ID NO:64 or consists of it
  • HCDR3 comprises the amino acid sequence of SEQ ID NO:65 or consists of it
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:77; or
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:80.
  • an anti-TF antibody or antigen-binding fragment thereof comprises 3 complementarity determining regions HCDR of the heavy chain variable region, and 3 complementarity determining regions LCDR of the light chain variable region, wherein:
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:95; or
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:98; or
  • HCDR1 comprises the amino acid sequence of SEQ ID NO:87 or consists of it
  • HCDR2 comprises the amino acid sequence of SEQ ID NO:88 or consists of it
  • HCDR3 comprises the amino acid sequence of SEQ ID NO:89 or consists of it
  • 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
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:101; or
  • 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 the amino acid sequence of SEQ ID NO:103 or consists of it
  • LCDR3 comprises the amino acid sequence of SEQ ID NO:104 or consists of it.
  • an anti-TF antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises:
  • 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
  • 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
  • the anti-TF antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region and a light chain variable region, wherein: said light chain variable region comprises:
  • 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
  • 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
  • amino acid sequence shown 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.
  • 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:
  • 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
  • said light chain variable region comprises:
  • 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
  • an anti-TF antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises:
  • said light chain variable region comprises:
  • 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
  • the anti-TF antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO: 1 Amino acid sequence 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 shown in SEQ ID NO: 2, or with it Amino acid sequences having at least 95%, 96%, 97%, 98% or 99% sequence identity.
  • 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.
  • the anti-TF antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO: 3 Amino acid sequence 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 shown in SEQ ID NO: 4, or with it Amino acid sequences having at least 95%, 96%, 97%, 98% or 99% sequence identity.
  • 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.
  • the anti-TF antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO: 5 Amino acid sequence 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 shown in SEQ ID NO: 6, or with it Amino acid sequences having at least 95%, 96%, 97%, 98% or 99% sequence identity.
  • 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.
  • the anti-TF antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO:7 Amino acid sequence or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, and wherein said light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, or with it have at least Amino acid sequences having 95%, 96%, 97%, 98% or 99% sequence identity.
  • 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.
  • an anti-TF antibody or antigen-binding fragment thereof comprises the variable heavy and light chains of any one of the exemplary antibodies 2B12B10-hz1, 30G11B7-hz1, 22F11H5-hz1, and 27H8H3-hz1 of the invention.
  • Region sequences or variants thereof eg, antibodies having the same CDR sequences as one of the exemplary antibodies, and having the same or different framework region sequences.
  • the antibody is a humanized antibody. More preferably, the heavy chain variable regions of the antibody have framework region sequences derived from human germline.
  • An antibody according to the invention may comprise a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region is a heavy chain constant region derived from human immunoglobulin.
  • the light chain constant region is a light chain constant region derived from human immunoglobulin.
  • the heavy chain constant regions comprised in the antibodies of the invention may be of any isotype or subtype, eg, IgG1, IgG2, IgG3 or IgG4 isotype heavy chain constant regions.
  • the invention provides an anti-TF antibody comprising an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, preferably an IgG1 heavy chain constant region, especially a human IgG1 heavy chain constant region, e.g. SEQ ID NO
  • the light chain constant region contained in the antibody of the present invention may be a kappa light chain constant region or a lambda light chain constant region.
  • the invention provides an anti-TF antibody comprising a kappa light chain constant region or a lambda light chain constant region, preferably an antibody of the invention comprising a human kappa light chain constant region, e.g., of SEQ ID NO: 105
  • the heavy chain and/or light chain of an anti-TF antibody or fragment thereof of the invention further comprises a signal peptide sequence.
  • the invention also provides variants of any of the antibodies described herein according to the invention, in particular any of the exemplary antibodies 2B12B10-hz1, 30G11B7-hz1, 22F11H5-hz1 and 27H8H3-hz1 of the invention variant of .
  • the term "variant" in relation to an antibody refers to, compared to a given parental antibody, comprising at least 1, such as 1-30, or 1-20 or 1-10, such as 1 or 2 or 3 or 4 or 5 amino acid substitutions, deletions and/or insertions of the antibody region of interest with amino acid changes, wherein the variant substantially retains at least one biological property (eg, antigen binding ability) of the parent antibody molecule.
  • the antibody region of interest can be the full length of the 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 the specific exemplary antibodies of the invention are contemplated by the invention.
  • antibody variants of the invention preferably retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., substantially the same antigenicity) of the parental antibody prior to the introduction of the alteration or modification. Binding ability and/or receptor endocytic activity, or the degree of influence on blood coagulation is basically the same). It can be understood that the heavy chain variable region or light chain variable region or each CDR region or framework region of the antibody according to the present invention can be changed individually or in combination to generate the antibody variant of the present invention. Furthermore, changes may also be made to the Fc region of the antibodies according to the invention.
  • Alterations in the Fc region may be made alone or in combination with changes in the framework regions and/or CDR regions and/or heavy chain variable regions and/or light chain variable regions.
  • the Fc region can be selectively altered, e.g., to confer one or more altered effector functions of the antibody, e.g., altered serum half-life, complement fixation, Fc receptor binding, and/or antigen dependence Cytotoxicity.
  • antibodies of the invention may be chemically modified (eg, attached to PEG) or their glycosylation pattern altered.
  • antibody variants according to the invention have amino acid changes in the heavy chain sequence and/or the light chain sequence relative to any of the exemplary antibodies of the invention.
  • the variant comprises at least one, two or three, but no more than 20, 10 or 5 amino acid changes in the heavy chain sequence or the light chain sequence or both, compared to the corresponding antibody or an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identity.
  • amino acid changes do not occur in the CDR regions, more preferably not in the variable regions.
  • at least some or all of the amino acid changes comprised in the antibody variant are conservative amino acid substitutions.
  • Constant substitution refers to an amino acid change that results in the substitution of an amino acid for a chemically similar amino acid.
  • Amino acid modifications such as substitutions to achieve conservative 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.
  • the present invention also provides that, with respect to any given antibody according to the present invention (especially the exemplary antibody of the present invention) 2B12B10-hz1, 30G11B7-hz1, 22F11H5-hz1 and 27H8H3-hz1), an anti-tissue factor (TF) antibody or an antigen-binding fragment thereof having one or more of the following properties: (i) combined with the given drug according to the present invention A given antibody binds to the same or overlapping epitope; (ii) competes with a given antibody according to the invention for binding to human tissue factor TF; (iii) inhibits (e.g., competitively inhibits) a given antibody according to the invention from cell surface expression Cellular binding of human tissue factor; and preferably exhibits the same or similar binding affinity and/or specificity as a given antibody according to the invention.
  • TF tissue factor
  • an antibody that "competes for binding to the 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 competition assay and, in turn, gives A given antibody also blocks 50% or more of that antibody's binding to antigen TF in a competition assay.
  • An exemplary competition assay is described in: "Antibodies”, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY).
  • Antibodies that compete for binding may bind to the same epitope region as a given antibody, eg, the same epitope, adjacent epitopes, or overlapping epitopes.
  • an antibody that inhibits (eg, competitively inhibits) the binding of a given antibody to its antigen refers to an antibody that inhibits more than 50%, 60%, 70%, 80%, 90%, or 95% of said given antibody. binding of a given antibody to its antigen; and conversely, said given antibody also inhibits binding of that antibody to its antigen by more than 50%, 60%, 70%, 80%, 90%, or 95%.
  • the binding of an antibody to its antigen can be measured using binding affinity. Methods for determining affinity are known in the art.
  • an antibody showing the same or similar binding affinity and/or specificity as a given antibody refers to an antibody capable of having at least 50%, 60%, 70%, 80%, 90% of the given antibody or greater than 95% binding affinity and/or specificity. This can be determined by any method known in the art for determining binding affinity and/or specificity.
  • antibodies according to the invention are IgG1 antibodies, more particularly IgG1 kappa or IgG1 lambda isotypes. Still preferably, the IgG1 antibody according to the invention has a human IgG1 constant region, more preferably of the human IgG1 kappa isotype.
  • antibodies of the invention have high binding affinity and high binding specificity for TF. Binding EC50 values of antibodies of the invention to TF ectodomain and/or to TF-positive tumor cells can be determined by FACS or ELSA assays, such as those described in the Examples. Alternatively, the binding kinetic parameters of the antibody can be determined by biofilm layer interferometry to characterize the binding affinity and specificity of the antibody. In some embodiments, the anti-TF antibodies of the invention have a KD value of less than 10 ⁇ 7 M, such as a KD value of about 10 ⁇ 8 M or less, as determined by biolayer interferometry ( BIL), preferably, Less than 5 ⁇ 10 -8 M.
  • BIL biolayer interferometry
  • the antibodies of the invention have a K d value of about 10 ⁇ 1 to 10 ⁇ 3 s ⁇ 1 as determined by biolayer interferometry (BIL), for example about 10 ⁇ 2 or about 10 ⁇ 3 s ⁇ 1 A Kd value of 1 .
  • the binding kinetic parameters of the antibody such as KD and Kd values, are determined using the ForteBio assay method described in the Examples; and optionally compared to a reference antibody in the same assay.
  • the antibody has the same or similar, or lesser binding kinetics KD value than a reference antibody.
  • the antibody has a relatively faster off-rate ( Kd value) compared to a reference antibody.
  • anti-TF antibodies of the invention bind strongly to TF-positive tumor cells.
  • the cell-binding affinity of the antibody can be reflected by measuring the cell-binding EC50 value (i.e., the half-maximal effective concentration of cell-binding) and/or the maximum binding amount of the antibody by cell ELISA or flow cytometry assay, and comparing it with a reference antibody .
  • the EC50 value of an antibody of the invention is about 50% to about 160% of the EC50 value of a reference antibody on at least one TF-positive tumor cell, such as NIC-H358 cells or KYSE50 cells, such as about 80%, about 90%, about 100%, about 110%, about 120%, or about 130%.
  • the anti-TF antibodies of the invention exhibit cross-reactivity to human and monkey TF.
  • the KD value of the antibody of the present invention binding to human TF is about the same as the KD value of the antibody binding to monkey TF, for example, the KD ratio between the two is between 1-10, such as 1 -5, more preferably about 1-3.
  • the Kd value of the antibody binding to human TF is about the same as the Kd value of the antibody binding to monkey TF, for example, the ratio of Kd between the two is between 1-10, for example Between 1-5, more preferably about 1-3.
  • antibodies of the invention have TF receptor-mediated endocytic activity. Evaluation of endocytic activity of antibodies can be performed in cell-based assays, such as those described in the Examples. In some embodiments, in an assay based on TF-positive cells, after incubating the antibody to be tested with TF-positive cells (especially TF-positive tumor cells) at 37° C. for a period of time (for example, 2 hours or 4 hours), the relative In the negative control kept at 4°C for the same time, by flow cytometry, the changes in the amount of TF antibody bound to the cell surface were detected by fluorescence to determine the endocytosis rate of the antibody.
  • TF-positive cells especially TF-positive tumor cells
  • an antibody of the invention has a substantially comparable or superior rate of endocytosis compared to a reference antibody.
  • the endocytosis rate of the antibody of the invention is about 50%-200% or higher, for example, about 70% or more, for example about 80% of the endocytosis rate of the reference antibody. % or more, preferably about 100% or more, or about 110% or more, more preferably about 120% or more, such as about 130% or more, about 150% or more, or about 200% or more.
  • the antibody of the invention exhibits more rapid endocytosis, eg, reaches about 30%, 40%, or 50% endocytosis in a shorter time than a reference antibody.
  • the endocytosis rate of the antibody of the invention is, for example, at least 10%, for example, 15- 20%, preferably at least 25%, such as 20-35%, more preferably above 35%, such as about 40% or 45% or above.
  • antibodies of the invention do not substantially affect coagulation, or have reduced coagulation effects.
  • the effect of the antibody on coagulation can be determined by examining the interference of the antibody on coagulation initiated by TF-positive cells in the presence of Ca 2+ ions and human plasma in a cell-based in vitro coagulation assay.
  • an antibody of the invention does not substantially affect coagulation initiated by cell surface TF compared to a negative control with no antibody; or has a reduced coagulation effect compared to a reference antibody. Compared with the negative control without antibody and/or the reference antibody, the change of the coagulation time caused by the antibody can be detected, thereby reflecting the effect of the antibody on coagulation.
  • the change of absorbance at 405 nm over time is estimated to be up to The clotting time (V50) required for the half-maximum OD405 absorbance value to characterize the effect of the antibody on the TF-mediated coagulation process.
  • the concentration of Ca 2+ ions used for detection may be one or more concentrations selected from 1-5 mM, for example, 2 mM and 5 mM.
  • the antibody concentration used for detection may be one or more concentrations selected from 10-50 ⁇ g/ml, for example, 20 ⁇ g/ml and 50 ⁇ g/ml.
  • the effect of the antibody on coagulation is determined in the presence of 5 mM Ca 2+ and 20 ⁇ g/ml antibody. In other embodiments, the effect of the antibody on coagulation is determined in the presence of 2 mM Ca 2+ and 50 ⁇ g/ml antibody.
  • the antibody-induced clotting time (V50) is delayed by no more than 50 seconds under at least one assay condition, e.g., in the presence of 5 mM Ca 2+ and 20 ug/ml antibody, relative to a negative control without antibody, Preferably no more than 40 seconds, no more than 30 seconds, no more than 20 seconds, 10 seconds, or 5 seconds, the antibody is considered to have substantially no or reduced effect on coagulation.
  • the antibody does not cause a delay in clotting time (V50) relative to a reference antibody under at least one assay condition, for example in the presence of 5 mM Ca 2+ and 20 ug/ml antibody, or preferably, (V50) is reduced by about 10% or more, or more preferably about 20% or 30% or 40% or more, and more preferably about 50% or 60% or more, then it can be considered that the antibody does not substantially affect blood coagulation or has a reduced coagulation effects.
  • V50 delay in clotting time
  • the antibody does not cause a delay in clotting time (V50), or preferably, clotting time, relative to a reference antibody, if under at least one assay condition, e.g., in the presence of 2 mM Ca 2+ and 50 ug/ml antibody, A reduction in (V50) of about 10%, 20%, or 30% or more, the antibody is considered to have substantially no effect on coagulation, or to have a reduced effect on coagulation.
  • V50 delay in clotting time
  • the antibodies of the present invention have the ability to block downstream signaling pathways mediated by TF/FVIIa complexes in TF-positive tumor cells.
  • the blocking activity of the anti-TF antibody to the TF downstream signaling pathway can be determined by detecting the release level of IL-8 caused by the activation of the pathway.
  • the antibody of the present invention exhibits an inhibitory effect on the TF signaling pathway.
  • the IC 50 value of the TF signaling pathway blocking activity of the antibody of the present invention is less than 500 ng/ml or 150 ng/ml, and the maximum inhibition rate reaches more than 90%, preferably more than 95%.
  • an antibody of the invention has an IC50 value of TF signaling pathway blocking activity that is comparable ( ⁇ 10%) or less than that of a reference antibody.
  • the anti-TF antibodies or antigen-binding fragments thereof of the present invention also have one or more of the following properties: (vii) good manufacturing performance; (viii) good stability; and (ix) favorable drug kinetic properties.
  • the purified antibody of the present invention can reach a purity of more than 90% as determined by SEC-HPLC; and in HIC In the measurement, it showed good hydrophilicity.
  • the antibodies of the present invention have good stability, including thermal stability and repeated freeze-thaw stability.
  • the SEC-HPLC purity that is, the percentage of antibody monomers determined by SEC-HPCL
  • the SEC-HPLC purity remains at 90%. More than or preferably more than 95%, or reduce no more than 5% or preferably no more than 3%; Above 90%, CE-SDS reduced purity (ie, LC+HC percentage) above 90% or preferably above 95%, or the reduction of the non-reduced purity and reduced purity is no more than 5% or 4%.
  • the SEC purity of the antibody of the present invention is maintained above 95%, or the purity is reduced by no more than 5% or preferably not more than 3%.
  • antibodies of the invention are serum stable.
  • the serum stability of the antibody can be determined by measuring the change in the affinity of the antibody to the TF antigen after incubation in the serum at 37°C for a period of time according to the serum stability determination test in the embodiment. If the change in affinity is not significant, the antibody is considered to be serum stable.
  • antibodies of the invention can be maintained in human serum for at least 14 days, with retention of 90% or more of their antigen binding affinity.
  • the invention provides nucleic acids encoding any of the above anti-TF antibodies or fragments thereof. Also provided are vectors comprising the nucleic acid; and host cells comprising the nucleic acid or the vector.
  • the invention provides nucleic acid molecules comprising a polynucleotide encoding at least one CDR region and typically a heavy chain VH or light chain VL sequence from a TF-binding antibody described above All three CDR regions.
  • the invention provides nucleic acid molecules comprising polynucleotides encoding complete or substantially complete variable region sequences of the heavy and/or light chains of the anti-TF antibodies described above.
  • each antibody or polypeptide amino acid sequence may be encoded by multiple nucleic acid sequences due to codon degeneracy.
  • the invention provides one or more vectors, including cloning vectors and expression vectors, comprising a nucleic acid of the invention.
  • 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 phage, or yeast artificial chromosomes (YACs).
  • the invention provides a host cell comprising a vector of the invention.
  • Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells.
  • antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. After expression in bacteria such as E. coli, antibodies can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • the host cell is a eukaryotic cell.
  • the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the production of antibodies or antigen-binding fragments thereof.
  • Examples of useful mammalian host cell lines include, monkey kidney CV1 line (COS-7) transformed with SV40; 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.
  • COS-7 monkey kidney CV1 line
  • SV40 human embryonic kidney line
  • 293HEK or 293 cells human embryonic kidney line
  • CHO Chinese hamster ovary (CHO) cells, including DHFR-CHO cells
  • myeloma cell lines such as Y0, NS0 and Sp2/0.
  • the invention provides methods of making an anti-TF antibody of the invention.
  • the method of the invention comprises, under conditions suitable for antibody expression, culturing a host cell comprising a nucleic acid encoding an antibody of the invention, and optionally recovering the host cell (or host cell culture medium) from the host cell (or host cell culture medium). said antibody.
  • isolated or artificially synthesized or recombinantly synthesized nucleic acids encoding antibodies can be inserted into one or more vectors for further cloning and/or in host cells or express.
  • the present invention also provides an anti-TF antibody or an antigen-binding fragment thereof having one or more of the following characteristics, and a method for preparing the same:
  • the antibody according to the present invention is especially antibody 2B12B10-hz1, 30G11B7-hz1, 22F11H5-hz1 or 27H8H3-hz1,
  • the preparation method includes: using the antibody according to the present invention as a reference, in a binding affinity determination test, screening antibodies with the above characteristics from the antibody mixture.
  • Antibody mixtures that can be used here include, but are not limited to, antisera from TF immunized animals, or yeast or mammalian display antibody libraries.
  • Anti-TF antibodies provided herein can be identified, screened for, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art.
  • Suitable methods that can be used to determine the binding of antibodies to TF antigens are known in the art.
  • the affinity of an antibody for a TF ectodomain-containing protein (eg, a fusion protein) or TF-positive tumor cells can be readily determined using routine techniques known to those skilled in the art.
  • Such methods include, but are not limited to, assays using ELISA; using a BIAcore 2000 instrument or a ForteBio Instrumental assays; or radioimmunoassays using radiolabeled target antigens; or cell ELISA or FACS techniques or biofilm interferometry techniques described in the examples of this application.
  • the measurement of the affinity parameter is carried out as or substantially as described in the examples.
  • Suitable methods for determining the endocytic activity of antibodies are known in the art.
  • the rate of endocytosis of an antibody after binding to the cell surface can be determined in a cell-based assay, particularly an assay based on TF-positive tumor cells.
  • the endocytic activity of the antibody is carried out as or substantially as described in the Examples.
  • Suitable methods for determining the effect of antibodies on coagulation are known in the art. For example, changes in coagulation parameters such as coagulation time in the presence of the antibody can be measured relative to a control (eg, negative or positive control) to reflect the coagulation effect of the antibody.
  • a control eg, negative or positive control
  • the effect of the antibody on blood coagulation is carried out as described or substantially as described in the examples.
  • the invention provides immunofusions or immunoconjugates produced by fusing or conjugating an antibody of the invention to a heterologous molecule.
  • an antibody of the invention (or an antigen-binding fragment thereof) is linked to a heterologous peptide or polypeptide molecule directly or through an amino acid linker in an immunological fusion.
  • heterologous peptides or polypeptides that may be mentioned include, but are not limited to, proteins or polypeptides that confer another functional activity on the fusion, or tag peptides that facilitate purification or detection of immunofusions.
  • an antibody of the invention (or an antigen-binding fragment thereof) is conjugated to a therapeutic or diagnostic or detectable agent in an immunoconjugate.
  • antibodies of the invention may be conjugated to heterologous molecules in the form of full-length antibodies or antibody fragments.
  • linkers can be used to covalently link different entities of the conjugate. Suitable linkers include chemical linkers or peptide linkers.
  • the linker is a "cleavable linker" which facilitates release of the polypeptide after delivery to the target site. For example, acid labile, peptidase sensitive, photolabile, dimethyl or disulfide containing linkers may be used.
  • suitable therapeutic agents for the conjugate include, but are not limited to, cytotoxins (eg, cytostatic or cytocidal agents), drugs, or radioisotopes.
  • conjugated to diagnostic or detectable agents may be used as part of a clinical assay (e.g., to determine the efficacy of a particular therapy) for monitoring or predicting the onset, development, progression of a disease or condition and/or severity.
  • diagnosis and detection can be accomplished by conjugating the antibody to a detectable agent including, but not limited to, various enzymes such as horseradish peroxidase; prosthetic groups such as streptavidin/biotin; and avidin/biotin; fluorescent substances; luminescent substances; radioactive substances; and positron-emitting metals and non-radioactive paramagnetic metal ions used in various positron emission tomography procedures.
  • the invention provides multispecific (including bispecific) antibody molecules that specifically bind TF.
  • an antibody of the invention (or an antigen-binding fragment thereof) forms a first binding specificity for TF.
  • the multispecific antibody further comprises a second binding specificity, or further comprises second and third binding specificities for two different molecules.
  • the second and third binding specificities can be for example directed to another antigen expressed on the surface of tumor cells, or to an antigen expressed on the surface of T cells.
  • binding specificity is preferably provided by the "binding site” or “antigen binding site” of the antibody (the region of the antibody molecule to which the antigen actually binds). 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).
  • VL antibody light chain variable domain
  • VH antibody heavy chain variable domain
  • compositions comprising an anti-TF antibody or an immunoconjugate/fusion thereof or a multispecific antibody and a composition comprising an anti-TF antibody or an immunoconjugate/fusion thereof or multispecific antibody polynucleotide compositions.
  • compositions may also optionally contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.
  • suitable pharmaceutical excipients such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.
  • anti-TF antibodies, immunoconjugates or multispecific antibodies of the present invention having the desired purity can be prepared by combining with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th Ed., Osol, A. Ed. (1980)) mixing to prepare pharmaceutical formulations comprising the invention.
  • the antibodies of the invention can be the sole active agent, or can be combined with other therapeutic agents.
  • Therapeutic agents that can be combined with the antibodies of the invention include, but are not limited to, therapeutic agents that have beneficial therapeutic effect on the disease and/or condition being treated.
  • the active ingredients may be as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • other pharmaceutical ingredients may provide anticancer activity.
  • the antibodies of the present invention are present in pharmaceutical compositions and pharmaceutical preparations in appropriate combinations with active ingredients in amounts effective for the intended use.
  • the invention also provides a combination product comprising an antibody or antigen-binding fragment thereof, multispecific antibody or immunoconjugate of the invention, and one or more other therapeutic agents (e.g., chemotherapeutics, other Antibodies, cytotoxic agents, antitumor drugs, etc.).
  • therapeutic agents e.g., chemotherapeutics, other Antibodies, cytotoxic agents, antitumor drugs, etc.
  • the components constituting the combination product such as the antibody of the present invention and other therapeutic agents, can be formulated in different formulations, and preferably contained in different containers. According to factors such as the disease to be treated and individual conditions, those skilled in the art can determine the administration method and administration sequence of each component of the combination product. Combinations of the invention may be used in the methods of treatment of the invention.
  • the invention provides a combination product wherein the other therapeutic agent is, for example, a therapeutic agent such as an antibody effective to stimulate an immune response thereby further enhancing, stimulating or up-regulating the subject's immune response.
  • the combination is used to prevent or treat TF positive tumors.
  • the invention provides methods and uses of the TF antibodies of the invention or antigen-binding fragments thereof, e.g., in vivo and in vitro for:
  • the methods and uses of the invention relate to the treatment of disease in an individual subject.
  • the methods and uses of the invention involve detecting the presence of TF in a sample from, eg, a subject.
  • the present invention also provides the use of the anti-TF antibody or antigen-binding fragment thereof of the present invention in the preparation of products (such as pharmaceutical compositions or pharmaceutical products or combination products or detection products) for the above-mentioned purposes.
  • the present invention provides methods and uses for preventing and/or treating TF-positive tumors in a subject using the antibody or antigen-binding fragment of the present invention, comprising administering the antibody or antigen of the present invention in a preventive and/or therapeutically effective amount Combine fragments.
  • the present invention also provides methods and applications for treating or preventing other diseases associated with increased expression of cell membrane TF in subjects using the antibodies or antigen-binding fragments of the present invention, including administering the present invention to subjects in need Invented anti-TF antibodies or antigen-binding fragments thereof.
  • the present invention provides methods and kits for detecting TF in a sample, wherein the method comprises: (a) combining the sample with the present contacting the inventive antibody or antigen-binding fragment thereof or immunoconjugate/fusion; and (b) detecting complex formation between the antibody or antigen-binding fragment or immunoconjugate thereof and the TF protein.
  • the sample is from a cancer patient.
  • the detection can be in vitro or in vivo.
  • 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 complexed with antibody molecules, ELISA assays methods, PCR-techniques (eg, RT-PCR).
  • the biological sample is blood, serum, or other liquid sample of biological origin.
  • a biological sample comprises cells or tissues.
  • the biological sample is from a hyperproliferative or cancerous lesion.
  • the TF to be detected is human TF.
  • anti-TF antibodies are used to select subjects suitable for treatment with anti-TF antibodies.
  • the antibodies of the invention can be used to diagnose cancer or tumors, for example to evaluate (e.g., monitor) the treatment or progression of a disease described herein (e.g., a hyperproliferative or cancerous disease) in a subject, its diagnosis and / or in installments.
  • a disease described herein e.g., a hyperproliferative or cancerous disease
  • Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties that are detected indirectly, such as enzymes or ligands, for example, Through enzymatic reactions or molecular interactions.
  • Exemplary labels include, but are not limited to, radioactive isotopes 32P, 14C, 125I, 3H, and 131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbrella Umbelliferone, luciferase, eg, firefly luciferase and bacterial luciferase (US Patent No.
  • luciferin 2,3-dihydrophthalazinedione, horseradish peroxidase (HR), alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lytic enzyme, carbohydrate oxidase, e.g., 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 tags, phage tags, stable free radicals, and more.
  • HR horseradish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • ⁇ -galactosidase ⁇ -galactosidase
  • glucoamylase lytic
  • the present invention Based on the good targeting of the anti-TF antibody of the present invention or the ADC of the present invention to TF and the above-mentioned excellent properties, the present invention also provides the application and method of the ADC of the present invention and the antibody of the present invention in the treatment and prevention of TF-related diseases.
  • the present invention therefore provides the use of the ADC of the present invention or a pharmaceutically acceptable salt or solvate thereof and the antibody of the present invention or an antigen-binding fragment thereof for preventing and/or treating TF-positive tumors in a subject.
  • the antibody drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof, or the antibody or antigen-binding fragment of the present invention may be administered to a subject as the sole active agent, or may be administered in combination with other therapies or The therapeutic agents are administered to the subject in combination.
  • Said other therapies and therapeutic agents include, for example, drugs that target antigens on the surface of tumor cells to destroy tumors by binding and/or blocking these molecules; drugs that activate the subject's immune system to spontaneously destroy tumors .
  • the present invention also provides a method for preventing or treating TF-positive tumors in a subject, comprising administering the ADC of the present invention or a pharmaceutically acceptable salt or solvate thereof to a subject in need, or to a subject in need A subject in need thereof is administered an anti-TF antibody or antigen-binding fragment thereof of the invention.
  • TF positive tumors suitable for use in the methods and uses of the invention may be selected from the group consisting of: myeloma (e.g. multiple myeloma), acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myelogenous leukemia (AML) and non-Hodgkin's lymphoma tumor, central nervous system tumor, glioma, brain cancer, head and neck cancer, such as head and neck squamous cell carcinoma, gastrointestinal cancer, genitourinary system cancer, lung cancer such as NSCLC, esophageal cancer, gastric cancer, liver and gallbladder cancer, pancreatic cancer, Colorectal cancer, rectal cancer, bladder cancer, kidney cancer, breast cancer, especially triple-negative breast cancer, prostate cancer, endometrial cancer, ovarian cancer, cervical cancer, melanoma, sarcoma, and skin cancer.
  • myeloma e.g. multiple myeloma
  • TF-positive tumors suitable for use in the methods and uses of the present invention may be early, intermediate or advanced or metastatic cancers.
  • TF-positive tumors suitable for use in the methods and uses of the present invention may be tumors that have previously undergone treatment to escape immune evasion.
  • the TF-positive tumors treated according to the methods of the invention are selected from the group consisting of cervical cancer, pancreatic cancer, lung cancer, prostate cancer, bladder cancer, ovarian cancer, breast cancer, colorectal cancer, esophageal cancer, head and neck cancer, gastric cancer, Includes primary or advanced or metastatic cancer.
  • said TF positive tumor is cervical cancer.
  • the TF positive tumor is non-small cell lung cancer.
  • the TF positive tumor is squamous cell carcinoma of the esophagus.
  • said TF positive tumor is breast cancer.
  • TF-positive tumors treated according to the methods of the invention have at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100% of TF positive cells.
  • Tissue factor expression levels on tumor biopsies can be assessed by immunohistochemistry. High percentages of TF-positive cells have been detected on various cancer biopsies, eg, 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%).
  • the methods according to the invention are used to treat tumors with a high percentage of TF-positive cells, for example, tumors with at least 25%, 50%, 75% or 100% of TF-positive cells, such as , tumors with TF positive cells ranging from 25-50% to 75-100%, eg, bladder cancer, lung cancer, pancreatic cancer, prostate cancer, ovarian cancer and cervical cancer.
  • application of the methods of the invention to said cancer induces tumor regression.
  • methods according to the invention may also be used to treat cancers in which the percentage of TF positive cells is below 25% or 20%.
  • application of the methods of the invention to said cancer results in inhibition of tumor growth.
  • the present invention also provides a method for preventing or treating other diseases associated with increased expression of cell membrane TF, comprising administering the anti-TF antibody of the present invention or an antigen-binding fragment thereof to a subject in need, or administering to a subject in need A subject in need thereof is administered the ADC of the present invention or a pharmaceutically acceptable salt or solvate thereof.
  • diseases include, but are not limited to, for example, benign tumors, neurofibromas, hemangiomas; atherosclerosis; vascular diseases, such as retinopathy and macular degeneration; inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, Ankylosing spondylitis; autoimmune inflammation such as multiple sclerosis.
  • the administration of the antibodies or binding fragments thereof according to the invention and the ADCs or pharmaceutically acceptable salts or solvates thereof according to the invention may include 1) therapeutic measures which cure , slow down, alleviate the symptoms of the diagnosed pathological condition or disorder and/or stop the progression of the diagnosed pathological condition or disorder; or 2) preventive or preventive measures, which prevent and/or slow down the progress of the pathological condition or disorder develop.
  • the subject can be an individual already suffering from a disorder, an individual prone to developing a disorder, or an individual in whom a disorder is to be prevented.
  • the invention relates to the treatment of a disease or condition; in other embodiments, the invention relates to the prevention of a disease or condition.
  • Antibodies or binding fragments thereof according to the invention and ADCs or pharmaceutically acceptable salts or solvates thereof according to the invention, and optionally other therapeutic agents in combination therewith, may be administered by any suitable method, including parenteral Administration, intratumoral administration and intranasal administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.
  • Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • suitable doses of the antibodies or binding fragments thereof according to the present invention and the ADCs or pharmaceutically acceptable salts or solvates thereof according to the present invention will depend on the type of disease being treated, the specific type of drug used, the severity and course of the disease, whether the drug is being administered prophylactically or therapeutically, previous therapy, the patient's clinical history and the Antibody response, and the discretion of the attending physician.
  • Antibodies or binding fragments thereof according to the invention and ADCs or pharmaceutically acceptable salts or solvates thereof according to the invention may be administered to a patient in one treatment or over a series of treatments.
  • the subject is receiving or has received other treatments, such as chemotherapy and/or radiation therapy, or other immunotherapy, prior to being treated by the methods of the invention.
  • treatments such as chemotherapy and/or radiation therapy, or other immunotherapy
  • compositions, multispecific antibodies of the invention may be administered in place of the antibodies of the invention or antigen-binding portions thereof or in place of the ADCs of the invention or pharmaceutically acceptable salts or solvates thereof.
  • immunoconjugates/fusions, or combination products in addition to administering an antibody of the invention or an antigen-binding portion thereof or an ADC of the invention or a pharmaceutically acceptable salt or solvate thereof, a composition, multispecific antibody or immunoconjugate of the invention may be further administered. substances/fusions, or combination products.
  • the present invention also provides the use of the ADC of the present invention or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for the aforementioned methods of treatment and prevention.
  • the present invention also provides the use of the antibodies or antigen-binding fragments, compositions, immunoconjugates/fusions, and multispecific antibodies of the present invention in the preparation of medicaments for the aforementioned treatment and prevention methods .
  • the amino acid sequence of human tissue factor refers to P13726 in the Uniprot protein database, and its extracellular segment is aa 33-aa 251; the amino acid sequence of cynomolgus tissue factor refers to A0A2K5VXA0 in the Uniprot protein database, and its extracellular segment is aa 33-aa 252.
  • HEK293-EBNA cells were transiently transfected with PEImax (Polysciences, 24765-1), expressed for 7 days, and then purified by nickel column to obtain human TF-his (h.TF-His) and cynomolgus monkey TF-his (c.TF-His )antigen.
  • PEImax Polysciences, 24765-1
  • the recombinantly expressed and purified TF antigen was used as immunogen or ELISA assay in subsequent examples.
  • Coat ELISA with TF antigen h.TF-His or c.TF-His antigen
  • CBS coating solution 50mM carbonate coating buffer
  • the coating solution was removed the next day, each well was washed once with 300uL PBS, 100uL of 2% BSA (Biotopped, A6020) in PBS was added to each well, and blocked at 37°C for 2 hours.
  • the samples to be tested were serially diluted with 2% BSA in PBS to establish 11 concentration points.
  • TF antigen-positive cells MDA-MB-231 or NCI-H358 cells
  • TF antigen-positive cells MDA-MB-231 or NCI-H358 cells
  • the culture supernatant was removed, each well was washed once with 300uL PBS, and 100uL 4% paraformaldehyde was added to each well to fix at room temperature for 20 minutes. Remove paraformaldehyde, wash each well with 300uL PBS twice, add 100uL 2% BSA in PBS to each well, and block at 37°C for 2 hours.
  • test sample Hybridoma supernatant or purified antibody
  • test sample hybridoma supernatant or purified antibody
  • the liquid was removed, and each well was washed three times with 300uL PBST; 100uL of HRP-conjugated secondary antibody in 2% BSA was added to each well, and incubated at 37°C for 1 hour.
  • HRP-labeled anti-mouse secondary antibody Jackson, 115-035-003
  • humanized antibody use HRP-labeled Anti-human secondary antibody (Jackson, 109-035-088).
  • the recombinantly expressed and purified antibodies were tested for their purity by SEC-HPLC.
  • the detection method is as follows:
  • Instrument parameters sample chamber temperature: 8°C; column temperature: 30°C; flow rate: 0.5ml/min; injection volume: 20ug; detection wavelength: 280nm; isocratic operation: 30min.
  • HIC detection antibody hydrophilicity and hydrophobicity
  • the hydrophilicity and hydrophobicity of the antibody were detected by HIC.
  • the detection method is as follows:
  • Tosoh Tskgel Buty-NPR (2.5), 4.6*100) was used to detect hydrophilicity and hydrophobicity on Agilent HPLC equipment.
  • the mobile phase A was 1.5M (NH 4 ) 2 SO 4
  • the instrument parameters were set as sample chamber temperature: 8°C, column temperature: 30°C, flow rate: 0.5mL/min, detection wavelength: 280nm. Dilute the sample to be tested with mobile phase A to a final concentration of 1mg/mL, and inject 20uL for gradient elution.
  • the elution gradient is as follows:
  • the serum titers of the immunized animals were tested by antigen protein-based ELISA assay using ELISA plates coated with h.TF-His and c.TF-His antigens, respectively, as described in General Methods. Based on the detection results and comprehensively considering the affinity with human and monkey TF, CD1-immunized mice and Balb/c-immunized mice were selected for hybridoma fusion, and the final booster immunization was performed before hybridoma fusion.
  • mice The spleen and lymph nodes of mice were taken to prepare cell suspension, mixed with SP2/0 mouse myeloma cells at a ratio of 1:1, resuspended with cell electrofusion buffer, and electrofusion reaction was performed using BTX-ECM2001 cell electrofusion instrument. After the electrofusion reaction, resuspend with complete fusion medium (RPMI160+15%FBS+1 ⁇ HAT), divide into 96-well cell culture plates according to 20,000-25,000 cells per well, keep at 37°C, 5% CO 2 nourish.
  • complete fusion medium RPMI160+15%FBS+1 ⁇ HAT
  • ELISA re-screening was performed by using ELISA plates coated with human TF-his and cynomolgus monkey TF-his successively; and cell ELISA re-screening was performed using TF-positive tumor cells (MDA-MB-231 or NCI-H358) . Positive mother clones were selected for subcloning.
  • the purified mouse antibody was evaluated by ELISA and cell ELISA by coating human TF-his, cynomolgus monkey TF-his protein and NCI-H358 cells successively.
  • the experimental method is as described in the general method, wherein the murine antibody starts at 10ug/mL, and is serially diluted 3 times to establish 11 concentration points. Based on the assay results, 13 hybridoma monoclonals with high affinity were selected for antibody sequence determination and humanization.
  • the method of CDR grafting was used to humanize the mouse antigen sequence. Firstly, by conventional BLAST method, find the human germline sequence with the highest homology to the original mouse sequence as a template; transplant the CDR of the mouse antibody onto the human template to construct a chimera; analyze the energy of the mouse antibody according to the structure Retain the FR amino acid in its original conformation, and back-mutate the corresponding amino acid in the chimera to the mouse amino acid to maintain the original affinity; calculate and analyze the immunogenicity of the constructed humanized antibody, find highly immunogenic fragments, and carry out Substitution of less immunogenic fragments. The sequences of 17 humanized antibodies were obtained.
  • the weight, weight, and The amino acid sequence of the light variable region was submitted to General Biology for gene synthesis, and after codon optimization, it was constructed on the PTT5 vector. After the plasmid was synthesized, HEK293E cells were transfected by PEImax, expressed for about 7 days, and the supernatant was collected by centrifugation. The supernatant was purified using MabSelectSure ProActive affinity media (GE, Cat. No. 17547401). The expressed reference antibody has 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 the purified antibodies were ultrafiltered into PBS buffer, the concentration was measured, and stored at -20°C.
  • the assay was performed as described in General Methods. In short, the ELISA plates coated with human TF-his protein or cynomolgus monkey TF-his protein were blocked with 2% BSA for 2 hours at 37°C, and then the antibody was added with serial dilutions (10ug/mL starting, 3 times Serial dilution, 11 concentration points), incubated at 37°C for 2 hours. After that, add anti-human secondary antibody (Peroxidase-AffiniPure Goat Anti-Human IgG (H+L), Jackson, 109-035-088), incubate at 37°C for 1 hour, add TMB substrate (Huzhou Yingchuang, TMB-S- 004) color development. After the color reaction was terminated, the absorbance value at 450 nM was read on the computer and the curve was fitted with GraphPad software.
  • anti-human secondary antibody Peroxidase-AffiniPure Goat Anti-Human IgG (H+L), Jackson, 109-03
  • the assay was performed as described in General Methods. In brief, TF antigen-positive NCI-H358 cells were collected by trypsinization, and 5 ⁇ 10 4 cells per well were plated 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 plates were blocked with 2% BSA for 2 hours at 37°C. Add serially diluted antibodies (10ug/mL starting, 3-fold serial dilution, 11 concentration points), and incubate at 37°C for 2 hours.
  • the dynamic affinity of humanized antibodies was determined 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 min. Dilute the antibody to be immobilized to 5 ⁇ g/ml; antigen h.TF-His and c.TF-His start from 75nM, 2-fold dilution, 4 concentration points, set 0 concentration point. Set the program, put the sensor plate and sample plate, start the program, and the sensor is regenerated with 20mM glycine solution (pH 1.7). Use Octet analysis software to analyze the data and export the result graph.
  • Table 5 the six candidate derivatized antibodies have high affinity to human and monkey TF.
  • the endocytic activity of the humanized monoclonal antibody was detected by flow cytometry (FACS).
  • FACS flow cytometry
  • the experimental steps are as follows: NCI-H358 cells were collected by trypsinization and centrifugation, and incubated with the antibody to be detected at a concentration of 10 ⁇ g/mL at 4° C. for 1 hour. Wash 3 times with PBS, resuspend cells with RPMI1640+10% FBS, divide into 3 parts and incubate at 37°C for 0, 2 and 4 hours respectively.
  • the experimental results are shown in Table 6.
  • the endocytosis rate of 2B12B10-hz1, 4F6C3-hz1, 30G11B7-hz1, 22F11H5-hz1, 27B9F2-hz1 was basically equivalent to that of the reference antibody, and the endocytosis rate of 27H8H3-hz1 was better than that of the reference antibody.
  • the binding of FVIIa to tissue factor on the surface of TF-positive tumor cell MDA-MB-231 will activate the downstream signaling pathway of TF, leading to the release of IL-8. Therefore, by detecting the release of IL-8, it can reflect the blocking activity of the anti-TF humanized antibody on the TF signaling pathway.
  • the experimental steps are as follows: FVIIa (40nM, Haematologic Technologies Inc, HCVIIA-0031), serially diluted anti-TF humanized antibody (starting at 60 ⁇ g/mL, 3-fold serial dilution, 11 concentration points) and MDA-MB-231 Cells (5 ⁇ 10 4 cells/well, Nanjing Kebai, CBP60382) co-incubated for 5 hours, the supernatant was collected, and the release of IL-8 was detected by IL-8 detection kit (MABTECH, 3114-1H-20).
  • IL-8 detection steps are as follows: the night before the experiment, coat the capture antibody MT8H6 in PBS at 200ng/well in a 96-well plate, overnight at 4°C; remove the coating solution, wash once with 300 ⁇ L PBST, and wash all 96 wells Add 100 ⁇ L of 2% BSA in PBS to each well of the plate, block at 37°C for 2 hours; directly add 50 ⁇ L of cell culture supernatant, and incubate at 37°C for 2 hours; remove the supernatant, wash each well with 300uL PBST three times, and add 100uL ( 1 ug/ml) MT8F19-biotin secondary antibody (in 2% BSA), incubate at 37°C for 1 hour; remove the supernatant, wash four times with 300 ⁇ L PBST per well, add 100 ⁇ L peroxidase-conjugated streptavidin per well Heparin (1:10000 dilution), incubate at 37°C for 1
  • the Tm value of the humanized antibody was determined by DSF to reflect the thermal stability of the antibody.
  • the experimental steps are as follows: dilute the antibody sample to be tested to 1 mg/mL with PBS; dilute the dye SYPRO Orange dye (Thermo#56651) to 40 times with ddH 2 O; reaction system: sample 12.5 ⁇ L + 40X dye 2.5 ⁇ L + ddH 2 O 5 ⁇ L; sealed film, centrifuged briefly; detected by Q-PCR, Q-PCR parameter setting: Target (ROX), program (25°C, 3min; 1% rate, 95°C; 95°C, 2min).
  • the anti-TF humanized antibody was diluted to 20 ⁇ g/mL with human serum, placed at 37°C for 14 days, and then the change of affinity was detected by ELISA based on the antigen protein.
  • the experimental procedure of the ELISA method is as described in the general method. The experimental results are shown in Figures 5A and 5B, there is no significant change in the affinity of 22F11H5-hz1 and 27H8H3-hz1 after being placed in human serum for 14 days.
  • the sample to be tested was injected into male rats (Chengdu Dashuo) through the tail vein, 3 rats in each group, and the administration dose was 10 mg/kg. Serum samples were collected 30min, 1h, 2h, 6h, 24h, 48h, 96h, 168h, 336h after administration. Dilute the corresponding antibody with 2% BSA containing negative rat serum, starting at 1 ⁇ g/mL, 2-fold serial dilution, 11 concentration points, and use this as the standard curve sample. Plasma drug concentrations were detected by antigenic protein-based ELISA as described in General Methods. Experimental results show that the half-life of the humanized antibodies 22F11H5-hz1 and 27H8H3-hz1 in rats is about 120 hours or more.
  • Embodiment 1 Preparation of antibody-drug conjugate (ADC)
  • Step 1 (S)-7-ethyl-7-hydroxy-14-(3-chloropropyl)-10,13-dihydro-11H-[1,3]dioxole[4, Synthesis of 5-g]pyrano[3',4':6,7]indoxazino[1,2-b]quinoline-8,11(7H)-dione (A1B)
  • the reaction solution was stirred at 0° C. for 5 minutes, then warmed to room temperature, and stirred for 3 hours.
  • the reaction solution was diluted with water (50 mL), extracted with ethyl acetate (80 mL ⁇ 2), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product.
  • the crude product was further purified with a C18 column (acetonitrile/0.05% formic acid in water: 5%-60%) to obtain the target compound A1B (yellow solid, 400 mg, yield: 67%).
  • Step 2 (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxole[4, Synthesis of 5-g]pyrano[3',4':6,7]indoxazino[1,2-b]quinoline-8,11(7H)-dione (A1)
  • Example 1.1.2 (S)-2-amino-N-((3-(7-ethyl-7-hydroxyl-8,11-dioxo-7,8,11,13-tetrahydro-10H -[1,3]dioxole[4,5-g]pyrano[3',4':6,7]indoleazino[1,2-b]quinoline-14- base) propoxy) methyl) acetamide (B1) synthesis
  • Step 2 B1B (240 mg) was dissolved in DMF (5 ml), piperidine (1 ml) was added, the compound was stirred for 20 minutes, the solution was decompressed to remove low boiling point components, and the residue was directly used in the next step of synthesis. A small amount of crude product was purified by reverse phase chromatography (acetonitrile/0.05% FA in water: 5% to 50%) to obtain the target compound B1.
  • 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)
  • HOBT 1-hydroxybenzotriazole
  • EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • the reaction solution was directly purified by C18 column reverse phase (acetonitrile and 0.05% aqueous formic acid system) to obtain the target compound N 6 ,N 6 -dimethyl-N 2 -((6-(2-(methylsulfonyl)pyrimidine- 5-yl)hex-5-ynoyl)-L-valine)-L-lysine (C1, white solid, 327 mg).
  • 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)
  • Example 1.1.5 N 6 , N 6 -di-n-propyl- N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valline acid)-L-lysine (C3)
  • the reaction solution was directly purified by C18 column reverse phase (acetonitrile and 0.05% aqueous formic acid 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, yield 28%), as a pale yellow solid.
  • Example 1.2.1 N-((11S,14S)-11-(4-(di-n-propylamino)butyl)-1-((S)-7-ethyl-7-hydroxyl-8,11 -dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxaleno[4,5-g]pyrano[3',4':6,7 ]indoleazino[1,2-b]quinolin-14-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazadeca Hexa-14-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (DL-01)
  • Example 1.2.2 N-((11S,14S)-11-(4-(diethylamino)butyl)-1-((S)-7-ethyl-7-hydroxyl-8,11- Dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxole[4,5-g]pyrano[3',4':6,7] Indolazino[1,2-b]quinolin-14-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadecano Alk-14-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (DL-02)
  • Example 1.2.3 N-((11S,14S)-11-(4-(dimethylamino)butyl)-1-((S)-7-ethyl-7-hydroxyl-8,11- Dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxole[4,5-g]pyrano[3',4':6,7] Indolazino[1,2-b]quinolin-14-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadecano Alk-14-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (DL-03)
  • Ab is an anti-TF antibody.
  • the ultrafiltration tube replaced the sample into a 20 mM histidine buffer with a pH of 6.0 to remove low-molecular substances, and finally concentrated the sample to obtain a solution TF-ADC (DAR3) containing an ADC composition of an anti-TF antibody.
  • the DAR value was determined to be 3.0 by mass spectrometry.
  • Example 1.4 Determination of the DAR value of the sample after coupling by mass spectrometry
  • Chromatographic column PLRP-S, 2.1*50mm, 5 ⁇ m;
  • Sample treatment Take 50 ⁇ g of samples, add 2 ⁇ l of 1M DTT, add ultrapure water to 50 ⁇ l to dilute to a concentration of about 1.0 mg/ml, mix well, and restore at room temperature for 30 minutes.
  • Mass spectrometry conditions Gas temp: 320°C; Drying Gas: Nitrogen; Nebulizer: 35psi; Sheath Gas Temp: 350°C; sheath Gas Flow: 11l/min; m/z 500 ⁇ 3000
  • mAb represents unconjugated monoclonal antibody
  • LC represents antibody light chain
  • HC represents antibody heavy chain
  • DAR1 represents a conjugate containing light chain or heavy chain coupled with a toxin molecule
  • DAR2 represents light chain Or a conjugate of two toxin molecules coupled to a heavy chain
  • DAR3 represents a conjugate comprising three toxin molecules coupled to a light chain or a heavy chain; wherein, the theoretical molecular weight of the monoclonal antibody is calculated based on the G0F glycoform.
  • mAb, LC, HC, DAR1, DAR2, and DAR3 are as described above.
  • the test results showed that on TF-ADC (DAR3), the light chain of the antibody was coupled to 0-1 toxin molecule (LC, DAR1 ratios were 53.8%, 46.2%), and the heavy chain was coupled to 0-3 toxin molecules (mAb, DAR1 , DAR2, and DAR3 were 30.6%, 42.4%, 20.2%, and 6.8%, respectively), so the coupling ratio (average DAR value) of the TF-ADC (DAR3) sample was calculated to be 3.0.
  • Sample treatment Take 50 ⁇ g of samples, add 2 ⁇ l of 1M DTT, add ultrapure water to 50 ⁇ l to dilute to a concentration of about 1.0 mg/ml, mix well, and restore at room temperature for 30 minutes.
  • Mass spectrometry conditions Gas1:45; Gas2:45; CUR: 30; TEM: 450; ISVF: 5000; DP: 120; CE: 12; mass range: 600-4000
  • the test results showed that on TF-ADC (DAR8), the light chain of the antibody was coupled to 0 to 1 toxin molecule (LC, DAR1 ratios were 0%, 100.0%), and the heavy chain was coupled to 0 to 3 toxin molecules (mAb, DAR1 , DAR2, and DAR3 are respectively 0%, 0%, 0%, and 100.0%), thus calculating the coupling ratio (average DAR value) of the TF-ADC (DAR8) sample to be 8.0.
  • Embodiment 2 anti-TF antibody ADC evaluation
  • Collect TF-positive tumor cells KYSE520 cells by trypsinization, resuspend the cells with RPMI1640+2% FBS, and plate 2000 cells per well; use RPMI1640+2%FBS to serially dilute the ADC to be tested, starting at 50 ⁇ g/mL, 3-fold dilution, 11 concentration points; add diluted ADC to the plate and incubate at 37°C for 120 hours; after incubation, add CCK8 reagent, 20 ⁇ L/well, react for 2-4 hours, read at 450nm on a microplate reader and import it into Graphpad Prism for curve fitting combine.
  • TGI (%) (1-T/C) ⁇ 100%
  • T and C are respectively the treatment group and the control group at a specific time Relative tumor volume of each point
  • Photos of tumor volume and tumor weight at the end point of the test are respectively the treatment group and the control group at a specific time Relative tumor volume of each point
  • Photos of tumor volume and tumor weight at the end point of the test are respectively the treatment group and the control group at a specific time Relative tumor volume of each point.

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Abstract

本发明涉及抗体和抗体-药物偶联物，更具体地涉及靶向组织因子的抗体和抗体-药物偶联物(ADC)以及含有所述抗体或ADC的组合物及其治疗应用。

Description

一种抗体及其药物偶联物和用途 技术领域
本发明涉及抗体以及抗体-药物偶联物，更具体地涉及靶向组织因子(Tissue Factor，TF)的抗体及抗体-药物偶联物(ADC)以及含有所述抗体或ADC的组合物及其治疗应用。
背景技术
组织因子(Tissue Factor，TF)又称促凝血酶原激酶(thromboplastin)、CD142、或凝血因子3。作为一种跨膜糖蛋白，TF由胞外域、跨膜区和胞内域组成。
TF是外源凝血事件启动的必需分子，以功能形式表达在细胞表面。作为血浆中凝血因子VII(FVII，一种丝氨酸蛋白酶)的高亲和力受体，TF在与VII的活化形式VIIa形成复合体后，引发催化事件，通过特定的有限蛋白裂解，激活因子IX或X，启动凝血蛋白酶级联。在正常生理条件下，TF位于血管壁外膜细胞、包绕血管的成纤维细胞等上，而在血管中膜或内膜层却很稀少。只有当血管壁的完整性遭到破坏时TF才暴露于循环血液，通过激活凝固级联反应发挥止血作用。在该过程中，TF依靠其与细胞膜的紧密结合发挥“锚”作用，使生理性凝血局限于损伤部位，而不从血液凝固的起始部位向远处播散。
与正常组织和细胞上有限的表达相反，已经证实，TF在多种恶性肿瘤上过表达，包括宫颈癌、胰腺癌、肺癌、前列腺癌、膀胱癌、卵巢癌、乳腺癌、结直肠癌等。因此，TF可以作为抗体类药物和ADC(抗体-药物偶联物)药物开发的靶点。Seagen Inc.(SGEN)/Genmab A/S(GMAB)公司开发的靶向TF的ADC药物Tivdak(Tisotumab Vedotin)，已于2021年9月获批用于宫颈癌适应症，并且在卵巢癌等其它适应症中的应用也已经处于临床II期。
然而，目前开发的许多抗TF抗体或抗体类药物，尽管在癌症治疗上表现出一定的功效，但同时也已经检测到对凝血的干扰或抑制，和由此引发的相关副反应(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.)。
因此，本领域仍然存在着开发新的TF抗体以及包含其的新ADC药物的需求，所述的TF抗体应具有特异性靶向肿瘤细胞表面TF的能力，但同时对正常组织中TF介导的凝血反应具有最小影响，以便为癌症患者提供更广更优的用药选择。
发明概述
为满足上述的本领域需要，本发明人经过深入研究，提供了新的TF抗体及由其构成的抗体-药物偶联物(ADC)。如实施例所示，本发明的抗TF抗体不仅对TF阳性肿瘤细胞展示出高结合亲和力和高特异性，能够快速有效地被肿瘤细胞内吞，且对TF介导的凝血影响小。进一步地，如实施例所示，由本发明TF抗体构成的ADC不仅具有良好的物理性质，无明显的聚集现象，且在动物模型中展示了显著的抑瘤生长活性和良好的动物用药耐受性。
因此，在第一方面，本发明提供了具有下式(I)的抗体-药物偶联物(ADC)或其可药用盐或溶剂化物：
Ab-[L-D]q      (I)
其中，
Ab表示抗TF抗体，
L表示连接体，
D表示细胞毒性或细胞抑制性药物，例如，拓扑异构酶I抑制剂，且
q＝1至20，例如，q＝1-10、1-8、3-8、4-8、或6-8，
其中，所述Ab包含：
-SEQ ID NO:1的重链可变区(VH)序列的三个CDR和SEQ ID NO:2的轻链可变区(VL)序列的三个CDR，
-SEQ ID NO:3的重链可变区(VH)序列的三个CDR和SEQ ID NO:4的轻链可变区(VL)序列的三个CDR，
-SEQ ID NO:5的重链可变区(VH)序列的三个CDR和SEQ ID NO:6的轻链可变区(VL)序列的三个CDR，或者
-SEQ ID NO:7的重链可变区(VH)序列的三个CDR和SEQ ID NO:8的轻链可变区(VL)序列的三个CDR，
其中优选地，所述CDR根据Chothia、AbM、Kabat、IMGT或其任意组合进行定义，
更优选地，所述Ab包含SEQ ID NO:7的重链可变区(VH)序列的三个CDR和SEQ ID NO:8的轻链可变区(VL)序列的三个CDR。
在第二方面，本发明提供了包含本发明ADC或其可药用盐或溶剂化物的组合物。
在第三方面，本发明提供了本发明ADC或其可药用盐或溶剂化物及其组合物在治疗或预防TF阳性肿瘤中的用途，以及在制备用于所述治疗或预防的药物中的用途。
在第四方面，本发明提供了抗TF抗体、及其药物组合物和用途。
在下面的附图和具体实施方案中进一步说明本发明。然而，这些附图和具体实施方案不应被认为限制本发明的范围，并且本领域技术人员容易想到的改变将包括在本发明的精神和所附权利要求的保护范围内。
附图说明
图1A-1D显示，通过基于抗原蛋白的ELSIA测定，检测本发明示例性抗体与人TF或猴TF的结合亲和力。BM为参照抗体。
图2A-2D显示，通过流式细胞术，检测本发明示例性抗体与不同的TF阳性肿瘤细胞(NCI-H358和KYSE520)的细胞结合亲和力。BM为参照抗体。
图3A-3C显示，通过IL-8释放抑制实验，检测本发明人源化抗体对TF/FVIIa复合物介导的下游信号通路的阻断活性。
图4A-4B显示，与参照抗体BM相比，本发明人源化抗体对凝血的影响。图4A：2mM Ca2+,50μg/mL抗体浓度；图4B：5mM Ca2+,20μg/mL抗体浓度。
图5A-5B显示，在人血清中37℃孵育一段时间后，本发明人源化抗体的血清稳定性检测结果。
图6A-6B显示，在将抗TF人源化抗体与毒素-Linker(B81，即，DL-01分子)偶联前后，通过ELISA测定的抗体亲和力变化。
图7A-7B显示，本发明ADC的体外杀伤活性检测结果。
图8显示，本发明ADC，22F11H5-hz1和30G11B7在偶联药物-连接体(B81)之后，在小鼠体内的抑瘤效果。
图9显示，包含本发明抗体22F11H5-hz1和30G11B7的ADC偶联物在施用后对小鼠体重的影响。
图10显示，本发明ADC，2B12B10-hz1和27H8H3-hz1在偶联B81之后，在小鼠体内的抑瘤效果。
图11显示，包含本发明抗体2B12B10-hz1和27H8H3-hz1的ADC偶联物在施用后对小鼠体重的影响。
发明详述
定义
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。
当在本文中使用商品名时，除非上下文另有指出，否则该商品名包括商品名产品的产品配方、通用名药物和活性药物成分。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
用于连接两个或两个以上可选项的术语“和/或”，应理解为意指可选项中的任一项或可选项中的任意两项或多项的组合。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
在本文中，术语“组织因子”或“TF”可互换使用，并且除非另有说明，否则包括人组织因子的任何变体，包括序列变体，尤其是天然存在的变体、等位基因变体，以及翻译后修饰变体和构象变体，并涵盖其物种同源物。此外，应理解，该术语不仅涵盖由细胞天然或重组表达的TF或在天然或重组细胞上表达的TF，也涵盖重组表达的包含TF胞外域的融合蛋白。组织因子的一个例子是包含UniProtKB-P13726下的氨基酸序列的人TF蛋白，或包含所述蛋白胞外域的重组蛋白。组织因子的另一个例子是包含UniProtKB-A0A2K5VXA0下的氨基酸序列的猴TF蛋白，或包含所述蛋白胞外域的重组蛋白。在本文中，在不特别指明的情况下，术语“组织因子”或“TF”是指来源于人的组织因子。
在本文中，术语“TF阳性”细胞是指，呈TF细胞表面表达阳性的细胞，例如癌细胞、经改造的癌细胞或经改造的非肿瘤细胞。可以通过本领域已知用于确定细胞表面抗原表达水平的任何常规方法，例如，FACS检测方法或免疫荧光染色方法，确定细胞表面的TF表达水平。TF在多种肿瘤细胞上具有显著高于正常组织/细胞上的表达水平，例如，MDA-MB-231(乳腺癌；>大约350,000个TF分子/细胞)，BxPC-3(胰腺癌；>大约350,000个TF分子/细胞)。优选地，在本文中，TF阳性细胞是TF阳性肿瘤细胞。
在本文中，术语“抗体”是指，至少包含轻链或重链免疫球蛋白可变区的多肽，所述免疫球蛋白可变区特异性识别并结合抗原。该术语涵盖各种抗体结构，包括、但不限于单克隆抗体、多克隆抗体、单链抗体或多链抗体、单特异性或多特异性抗体(例如双特异性抗体)、嵌合抗体或人源化抗体、全长抗体和抗体片段，只要它们呈现期望的抗原结合活性即可。
在本文中，“全抗体”(在本文中可与“全长抗体”、“完全抗体”和“完整抗体”互换使用)是指，包含至少两条重链(H)和两条轻链(L)的免疫球蛋白分子。每条重链由重链可变区(本文中缩写为VH)和重链恒定区组成。每条轻链由轻链可变区(本文中缩写为VL)和轻链恒定区组成。可变区是抗体的重链或轻链中参与抗体与其抗原结合的结构域。恒定区不直接参与抗体与抗原的结合，但是显示出多种效应子功能。抗体的轻链可以基于其恒定区的氨基酸序列归为两种类型(称为kappa(κ)和lambda(λ))。抗体的重链可以基于其恒定区的氨基酸序列而划分为主要5种不同的类型：IgA、IgD、IgE、IgG和IgM，并且这些类型中的几种 可以进一步划分成亚类，如，IgG1、IgG2、IgG3和IgG4、IgA1以及IgA2。
在本文中，术语抗体的“抗体片段”和“抗原结合片段”可互换使用，是指并非完整抗体的分子，其包含完整抗体中用于结合该完整抗体所结合的抗原的部分。如本领域技术人员理解的，为实现抗原结合目的，抗体片段通常包含来自“互补决定区”或“CDR”的氨基酸残基。可以通过重组DNA技术、或通过酶或化学切割完整的抗体制备抗体片段。抗体片段的例子包括但不限于，Fab、scFab、二硫键连接的scFab、Fab’、F(ab’)2、Fab’-SH、Fv、scFv、二硫键连接的scFv、线性抗体、双链抗体(diabody)、三链抗体(triabody)、四链抗体(tetrabody)、微型抗体(minibody)。在根据本发明的一些实施方案中，抗体片段包含用于在轻链和重链之间或重链与重链之间形成链间二硫键的半胱氨酸残基部分，例如，IgG1抗体Fab区和/或铰链区的半胱氨酸残基，以提供可用于巯基偶联化学的氨基酸残基位点。在根据本发明的另一些实施方案中，抗体片段包含引入Fc区的半胱氨酸残基，以提供可用于巯基偶联化学的氨基酸残基位点。
在本文中，术语“免疫球蛋白”指具有天然存在抗体的结构的蛋白质。例如，IgG类免疫球蛋白是由二硫键键合的两条轻链和两条重链组成的约150,000道尔顿的异四聚体蛋白。从N端至C端，每条免疫球蛋白重链具有一个重链可变区(VH)，也称作重链可变结构域，随后是三个重链恒定结构域(CH1、CH2和CH3)。从N端至C端，每条免疫球蛋白轻链具有一个轻链可变区(VL)，也称作轻链可变结构域，随后是一个轻链恒定结构域(CL)。相应地，在本文中，提及一个抗体是IgG抗体，是指该抗体是具有IgG类免疫球蛋白结构的异四聚体蛋白。在IgG抗体中，通常重链的VH-CH1与轻链的VL-CL配对形成特异性结合抗原的Fab片段。因此，一个IgG抗体基本上由借助免疫球蛋白铰链区连接的两个Fab分子和两个二聚化的Fc区组成。IgG免疫球蛋白可以基于重链恒定区的序列，划分成亚类，例如γ1(IgG1)、γ2(IgG2)、γ3(IgG3)、和γ4(IgG4)。IgG免疫球蛋白的轻链也可以基于其恒定结构域的氨基酸序列而划分成两种类型，称作κ和λ。在一些实施方案中，根据本发明的抗体是IgG抗体，例如IgG1,IgG2,IgG3或IgG4抗体。在另一些实施方案中，根据本发明的抗体是IgGκ或IgGλ抗体，例如，IgG1κ或IgG1λ抗体。
在本文中，术语“互补决定区”或“CDR区”或“CDR”或“高变区”可互换使用，是指抗体可变结构域中在序列上高度可变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。在本文中，抗体重链和轻链的CDR从N-端开始顺序编号，通常称作CDR1、CDR2和CDR3。位于抗体重链可变结构域内的CDR也称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR则称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，可以采用本领域公知的多种方案确定其CDR序列。例如，可以在http://www.abysis.org/abysis/，获得给定的轻链可变区或重链可变区中CDR的注释，包括基于Kabat、AbM、Chothia、Contact、IMGT定义的CDR序列。此外，CDR也可以基于与参考CDR序列具有相同的Kabat编号位置而确定。除非另有说明，否则在本发明中，当提及抗体可变区中的残基位置(包括重链可变区残基和轻链可变区残基)时，是指根据Kabat编号系统(Kabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991))的编号位置。
在本文中，“可变区”或“可变结构域”是抗体的重链或轻链中参与抗体与其抗原的结合的结构域。重链可变区(VH)和轻链可变区(VL)可以进一步再划分为高变区(HVR，又称作互补决定区(CDR))，其间插有较保守的区域(即，构架区(FR))。每个VH和VL由三个CDR和4个FR组成，从氨基端到羧基端以如下顺序排列：FR1,CDR1,FR2,CDR2,FR3,CDR3,FR4。在一些方面，可以对抗体的两个可变区之一或两者(即VH和/或VL)中的一个或多个残基进行修饰，例如，对一个或多个CDR区和/或对一个或多个构架区进行残基修改，尤其是保守残基取代，以获得仍基本上保持改变之前的该亲本抗体的至少一个生物学特性(例如，抗原结合能力)的抗体变体。在再一些方面，可以通过CDR移植，修饰抗体可变区。由于CDR序列负责大多数抗体-抗原相互作用，故可以构建模拟已知抗体的性质的重组抗体变体。在此类抗体变体中，来自已知抗体的CDR序列被移植到具有不同性质的不同抗体的构架区上。可以在体外或体内测定试验中评估突变和/修饰后的抗体或包含其的ADC偶联物的性质，例如靶抗原结合性质或其它期望的功能性质，例如内吞活性、凝血影响、药代动力学、和体内肿瘤杀伤活性。
在本文中，术语“嵌合抗体”是指可变区序列源自一个物种、恒定区序列源自另一物种的抗体，例如， 其中可变区序列源自小鼠抗体、恒定区序列源自人抗体的抗体。
术语“人源化抗体”是指将源自非人哺乳动物物种例如小鼠种系的CDR序列嫁接到人构架序列上的抗体。可以在人构架序列内进行额外的构架区修饰，和/或在CDR序列中进行额外的氨基酸修饰，例如以进行抗体的亲和力成熟。在本文中，在一些实施方案中，本发明的人源化抗体具有“来源于”特定的人种系序列的构架区序列。在此，“来源于”是指，所述抗体构架区的氨基酸序列与由所述人种系免疫球蛋白基因编码的相应构架区氨基酸序列具有至少90％，更优选至少95％，甚至更优选至少96％、97％、98％或99％同一性，并且所述抗体保持抗原结合活性。
在本文中，术语“分离的”抗体是已经与它的天然环境中的组分分离的抗体。在一些实施方案中，抗体被纯化至大于90％、95％或99％纯度，所述纯度可以通过例如电泳(例如，SDS-PAGE、等电聚焦(IEF)、毛细管电泳)或色谱(例如，离子交换或反相HPLC)确定。
在本文中，术语“表位”是指抗体所结合的抗原区域。表位可以由连续的氨基酸形成或者由通过蛋白的三级折叠而并置的非连续氨基酸形成。在本发明中，优选地，根据本发明的抗体结合人TF的天然表位，更优选地，结合细胞表面表达的人TF胞外域的天然表位。
在本文中，术语“亲和力”(affinity)或“结合亲和力”指，反映结合对子的成员之间相互作用的固有结合亲和力。亲和力可以由本领域已知的常见方法测量。用于测量亲和力的一个具体方法是本文中实施例描述的基于抗原蛋白或细胞的ELISA测定法，另一具体方法是本文中实施例描述的流式细胞测定法。也可以使用基于生物膜层光学干涉(BLI)技术，进行抗体的动态亲和力评价。
术语“结合”或“特异性结合”，在涉及抗体与关联抗原(在本文中，TF抗原)结合的上下文中，用于指具有大约10-6M或更小的KD值亲和力的结合，例如，KD值为大约10-7M或更小，或大约10-8M或更小。与非特异性抗原(例如，无关抗原，如BSA)的结合KD值相比，抗体与其关联抗原的结合KD值，优选低至少100倍或例如至少1000倍。KD值的测量是本领域已知的，例如基于生物膜层光学干涉(BLI)技术。可以在例如ForteBio仪器中，使用抗体为配体，使用抗原(例如，包含TF胞外域的融合蛋白，如TF-His)为分析物，进行所述测定。
术语“KD”(M)在本文中指，特定抗体-抗原相互作用的解离平衡常数。亲和力(affinity)与KD值成反相关，即，亲和力越高，KD值越小；反之，亲和力越低，KD值越高。一般，KD值取决于相互作用的抗体-抗原对之间的解离速率常数(Kd或Kdis,sec-1)和结合速率常数(Ka,M-1x sec-1)。
如本领域技术人员理解，特异性结合人TF的抗体，可以与来自其它物种的TF蛋白具有交叉反应性。在本文中，术语“交叉反应”是指抗体结合来自不同物种的TF的能力。例如，在一些实施方案中，特异于人TF的根据本发明的抗体，还可结合来自其它物种的TF(例如，食蟹猴TF)。测定交叉反应性的方法包括实施例中所述的方法以及本领域已知的标准测定法，例如通过使用流式细胞术或细胞ELISA技术测定。抗体的物种交叉反应性在一些情况下是有利的。例如，当目标抗体对临床前实验动物，例如灵长类动物具有物种交叉反应性时，将促进目标抗体在人体治疗或诊断应用之前的临床前安全性和功效评价。
在本文中，术语“同种型”是指由抗体重链恒定区确定的抗体类型。例如，根据本发明的抗体可以是IgA(例如IgA1或IgA2)、IgG1、IgG2(例如IgG2a或IgG2b)、IgG3、IgG4、IgE、IgM和IgD抗体，并具有所述免疫球蛋白类型的重链恒定区。本发明的抗体也可以是具有人IgG1恒定区的IgG1抗体。此外，本发明不仅考虑采用天然序列恒定区的抗体，也考虑包含变体序列恒定区的抗体。
在本文中，术语“天然序列Fc区”涵盖天然存在的各种免疫球蛋白Fc区序列，例如各种Ig亚型以及其同种异型的Fc区序列(Gestur Vidarsson等,IgG subclasses and allotypes:from structure to effector functions,20October 2014,doi:10.3389/fimmu.2014.00520.)。在一些实施方案中，人IgG重链Fc区具有自Cys226或自Pro230延伸至重链羧基端的氨基酸序列。然而，Fc区的C端末端赖氨酸(Lys447)可以存在或不存在。在再一些实施方案中，人IgG重链Fc区在N端带有天然免疫球蛋白的铰链序列或部分铰链序列，例如根据EU编号，E216到T225的序列或D221到T225的序列。
在本文中，术语“变体序列Fc区”是指，相对于天然序列Fc区多肽包含修饰的Fc区多肽。所述的修饰可以是氨基酸残基的添加，缺失或取代。取代可以包括天然存在的氨基酸和非天然存在的氨基酸。修饰的目的可以是旨在改变由Fc区与其受体的结合及其由此引发的效应子功能。
术语“效应子功能”是指，可归因于抗体的Fc-区的那些生物活性，其随抗体类别而改变。已经已知IgG Fc区可以介导几种重要的效应子功能，例如细胞因子诱导、ADCC、吞噬作用、补体依赖性细胞毒性(CDC)、以及抗体和抗原-抗体复合物的半衰期/清除速率。在一些情况下，取决于治疗目的，这些效应子功能对于治疗性抗体是理想的，但在其他情况下可能是不必要的。因此，在一个实施方案中，本发明提供具有引起效应子功能例如ADCC或CDC的Fc区的抗体，从而在携带TF抗原的肿瘤细胞中诱导肿瘤细胞凋亡、细胞裂解、和/或抑制携带抗原TF的肿瘤细胞的增殖、播散和/或转移。在另一些实施方案中，本发明提供具有改变的效应子功能的Fc区的抗体。可以通过对抗体的Fc区进行序列改变，以改变效应子功能。或者，可制备Fc区具有改变类型的糖基化的抗体，例如具有减小量的岩藻糖基残基的低或无岩藻糖化抗体或具有增加的等分GlcNac结构的抗体。这类改变的糖基化模式已显示可增加抗体的ADCC能力。也可以考虑在与Fc区连接的寡糖中具有至少一个半乳糖残基的抗体变体，这样的抗体变体可具有提高的CDC功能。Fc区糖基化模式的改变可通过改变Fc区的氨基酸序列以便产生或移除一或多个糖基化位点而方便地实现。
在本文中，术语“受体介导的内吞”是指，由配体与细胞表面上的相应受体结合所触发的、配体/受体复合物被内化并递送到细胞溶质中或转移至合适的细胞内区室的过程。在一些实施方案中，本发明的抗体在与细胞表面表达的TF结合后引发由TF受体介导的内吞。在本文中，可以通过例如实施例中所述的方法测定内吞率，表征抗体所具有的受体介导的内吞活性。在一些实施方案中，具有受体介导的内吞活性的本发明抗体可以用作在本发明的ADC中运载抗肿瘤药物进入癌细胞中的工具。
在本文中，“序列同一性”是指在比较窗中以逐个核苷酸或逐个氨基酸为基础的序列相同的程度。可以通过以下方式计算“序列同一性百分比”：将两条最佳比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同一性百分比。为了确定序列同一性百分比而进行的最佳比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。
在本发明中，就抗体序列而言，氨基酸序列同一性百分比通过将候选抗体序列与给定抗体序列最佳比对后，在一个优选方案中按照Kabat编号规则进行最佳比对后，予以确定。在本文中，在不指定比较窗(即待比较的目标抗体区域)的情况下，将适用于在给定抗体序列的全长上进行比对。
在本文中，除非另有说明，否则参照抗体(BM)是指，采用来自于专利CN 103119065 B中公布的Tisotumab抗体部分的重链和轻链可变区氨基酸序列(SEQ ID NOs:107和108)构建的抗TF抗体。在涉及与参照抗体比较的上下文中，所述参照抗体将与待比较的抗体在可变区之外的部分具有相同的抗体结构，例如在两者均具有重链和轻链恒定区结构时，具有相同的重链和轻链恒定区序列。
在本文中，术语“卤素”通常是指氟、氯、溴、碘，例如可以是氟、氯。
本文所用的术语“烷基”是指由碳原子和氢原子组成的直链或支链的饱和烃基团。具体地，烷基具有1-10个碳原子，例如1至8个、1至6个、1至5个、1至4个、1至3个或1至2个碳原子。例如，如本文中所使用，术语“C1-C6烷基”指具有1至6个碳原子的直链或支链的饱和烃基团，其实例例如甲基、乙基、丙基(包括正丙基和异丙基)、丁基(包括正丁基、异丁基、仲丁基或叔丁基)、戊基(包括正戊基、异戊基、新戊基)、己基(包括正己基、2-甲基戊基、3-甲基戊基、3,3-二甲基丁基、2,2-二甲基丁基、1,1-二甲基丁基、1,2-二甲基丁基、1,3-二甲基丁基、2,3-二甲基丁基、2-乙基丁基)，等。
本文所用的术语“烯基”是指由碳原子和氢原子组成的包含至少一个双键的直链或支链的不饱和烃基团。 具体地，烯基具有2-8个，例如2至6个、2至5个、2至4个或2至3个碳原子。例如，如本文中所使用，术语“C2-C6烯基”指具有2至6个碳原子的直链或支链的烯基，例如乙烯基、丙烯基、烯丙基、1-丁烯基、2-丁烯基、1,3-丁二烯基、1-戊烯基、2-戊烯基、3-戊烯基、1,3-戊二烯基、1,4-戊二烯基、1-己烯基、2-己烯基、3-己烯基、1,4-己二烯基等。
本文所用的术语“炔基”是指由碳原子和氢原子组成的包含至少一个叁键的直链或支链的不饱和烃基团。具体地，炔基具有2-8个，例如2至6个、2至5个、2至4个或2至3个碳原子。例如，如本文中所使用，术语“C2-C6炔基”指具有2至6个碳原子的直链或支链的炔基，例如乙炔基、丙炔基、炔丙基、1-丁炔基、2-丁炔基、1-戊炔基、2-戊炔基、3-戊炔基、4-甲基-1-戊炔基、1-己炔基、2-己炔基、3-己炔基、5-甲基-2-己炔基、等。
本文所用的术语“亚烷基”是指，从直链或支链的饱和烷烃的相同或两个不同碳原子上移去两个氢原子得到的二价基团。具体地，亚烷基具有1-10个碳原子，例如1至6个、1至5个、1至4个、1至3个或1至2个碳原子。例如，如本文中所使用，术语“C1-C6亚烷基”指具有1至6个碳原子的直链或支链的亚烷基，包括，但不限于，亚甲基、亚乙基、亚丙基、亚丁基，等。
本文所用的术语“亚烯基”是指，从包含至少一个双键的直链或支链的不饱和烯烃的相同或两个不同碳原子上移去两个氢原子得到的二价基团。具体地，亚烯基具有2-8个，例如2至6个、2至5个、2至4个或2至3个碳原子。例如，如本文中所使用，术语“C2-C6亚烯基”指具有2至6个碳原子的直链或支链的亚烯基，例如亚乙烯基、亚丙烯基、亚烯丙基、亚丁烯基、亚戊烯基、和亚己烯基。
本文所用的术语“亚炔基”是指，从包含至少一个叁键的直链或支链的不饱和炔烃的相同或两个不同碳原子上移去两个氢原子得到的二价基团。具体地，亚炔基具有2-8个，例如2至6个、2至5个、2至4个或2至3个碳原子。例如，如本文中所使用，术语“C2-C6亚炔基”指具有2至6个碳原子的直链或支链的亚炔基，例如亚乙炔基、亚丙炔基、亚炔丙基、亚丁炔基、亚戊炔基和亚己炔基。
本文所用的术语“环烷基”是指具有指定环原子数的单环、稠合多环、桥接多环或螺环非芳族单价烃环结构，其可以是饱和或不饱和的，例如包含1个或多个双键。环烷基基团在环中可包含3个或更多个例如3-18、3-10、或3-8个碳原子，例如C3-10环烷基、C3-8环烷基、C3-6环烷基、C5-6环烷基。环烷基基团的实例包括但不限于环丙基、环丁基、环戊基、环己基、环庚基。
本文所用的术语“杂环”或“杂环基”是指，具有1-4个独立地选自N、O或S的杂原子环成员的5-20元(例如5-14元、5-8元、5-6元)的芳族或非芳族单环、二环、或多环环系。杂环中的一个或多个N、C或S原子可被氧化。优选地，杂环是5-10元环系，为单环或稠合双环。代表性实例包括但不限于吡咯烷、氮杂环丁烷、哌啶、吗啉、四氢呋喃、四氢吡喃、苯并呋喃、苯并噻吩、吲哚、苯并吡唑、吡咯、噻吩(噻吩)、呋喃、噻唑、咪唑、吡唑、嘧啶、吡啶、吡嗪、哒嗪、异噻唑和异噁唑。应该理解，该术语包含本文所定义的杂芳基。
术语"芳基"指在环部分具有6-20例如6-12个碳原子的单环或多环芳香烃基团。优选地，芳基是(C6-C10)芳基。非限制性示例包括苯基、联苯基、萘基或四氢萘基，它们各自可以任选被1-4个取代基取代，所述取代基例如烷基、三氟甲基、环烷基、卤素、羟基、烷氧基、酰基、烷基-C(O)-O-、芳基-O-、杂芳基-O-、氨基、巯基、烷基-S-、芳基-S-、硝基、氰基、羧基、烷基-O-C(O)-、氨基甲酰基、烷基-S(O)-、磺酰基、磺酰氨基、杂环基等。
术语"杂芳基"指含有选自N、O或S的1-4个杂原子的5-20元(例如5-14元、5-8元、5-6元)的芳族单环状或多环状环系，其可以是取代的或未取代的。优选地，杂芳基是5-10元环系，为单环或稠合双环。代表性的杂芳基基团包括2-或3-噻吩基、2-或3-呋喃基、2-或3-吡咯基、2-、4-或5-咪唑基、3-、4-或5-吡唑基、2-、4-或5-噻唑基、3-、4-或5-异噻唑基、2-、4-或5-噁唑基、3-、4-或5-异噁唑基、3-或5-1,2,4-三唑基、4-或5-1,2,3-三唑基、四唑基、2-、3-或4-吡啶基、3-或4-哒嗪基、3-、4-或5-吡嗪基、2-吡嗪基、2-、4-或5-嘧啶基。
术语“杂烷基”是指完全饱和或含1至3个不饱和度、由所示数量的碳原子和一至十个、优选一至三个选自O、N、Si和S的杂原子组成的稳定的直链或支链烃，其中的氮和硫原子可任选被氧化并且氮杂原子可任选被季铵化。杂原子O、N、Si和S可位于杂烷基基团的任何内部位置处或位于杂烷基基团与分子的其余部分连接的位置处。杂烷基的代表性实例包括–CH2-CH2-O-CH3、-CH2-CH2-NH-CH3、-CH2-CH2-N(CH3)-CH3、-CH2-S-CH2-CH3、-CH2-CH2-S(O)-CH3、-NH-CH2-CH2-NH-C(O)-CH2-CH3、-CH2-CH2-S(O)2-CH3、-CH＝CH-O-CH3、-Si(CH3)3、-CH2-CH＝N-O-CH3和–CH＝CH-N(CH3)-CH3。至多两个杂原子可以是连续的，如例如-CH2-NH-OCH3和–CH2-O-Si(CH3)3。通常，C1至C4杂烷基或亚杂烷基具有1至4个碳原子和1或2个杂原子，C1至C3杂烷基或亚杂烷基具有1至3个碳原子和1或2个杂原子。在一些方面，杂烷基和亚杂烷基是饱和的。
除非另外指出，否则本文定义各个基团时所使用的术语“被取代的”，是指相应基团可以被例如但不限于以下的基团取代：烷基、烯基、炔基、环烷基、芳基、杂芳基、杂环基、卤素、氰基、硝基、叠氮基、羧基、羟基、巯基、氨基、单或二烷基氨基、单或二环烷基氨基、单或二芳基氨基、单或二杂环基氨基、单或二杂芳基氨基、烷基-或环烷基-或杂环基-或杂芳基-或芳基-氧基、烷基-或环烷基-或杂环基-或杂芳基-或芳基-硫基、烷基-或环烷基-或杂环基-或杂芳基-或芳基-酰基、烷基-或环烷基-或杂环基-或杂芳基-或芳基-酰氨基、烷基-或环烷基-或杂环基-或杂芳基-或芳基-酰氧基、烷基-或环烷基-或杂环基-或杂芳基-或芳基-磺酰基、烷基-或环烷基-或杂环基-或杂芳基-或芳基-磺酰氧基、烷基-或环烷基-或杂环基-或杂芳基-或芳基-磺酰氨基、或上述任选取代的氨基-甲酰基，以及其各自被其余可选取代基进一步取代的基团，其中的各类基团如本文所定义。取代基的实例包括但不限于一个或多个独立地选自以下的基团：卤素、OH、SH、CN、NH2、NHCH3、N(CH3)2、NO2、N3、C(O)CH3、COOH、C(O)-氨基、OCOCH3、甲基、乙基、丙基、异-丙基、丁基、异丁基、仲丁基、叔丁基、环丙基、甲氧基、乙氧基、丙氧基、氧代基、三氟甲基、二氟甲基、磺酰基氨基、甲磺酰基氨基、SO、SO2、苯基、哌啶基、哌嗪基和嘧啶基。
本文所用的术语“取代”或“取代的”是指所指定的原子上的一个或多个(例如1、2、3或4个)氢被所指定的基团代替，条件是未超出所指定原子在当前情况下的正常价键并且形成稳定的化合物，取代基和变量的组合仅当这种组合形成稳定的化合物时才是允许的。
在本文中，术语“连接体”是指在药物-抗体偶联物中将药物与抗体相连的双官能部分。本发明的连接体可以具有多个组分(例如，在一些实施方式中具有负责与抗体偶联的连接基；可降解的肽单元；以及任选地间隔基)。
在本文中，术语“PEG单元”是指包含重复的乙烯氧基亚单元(PEG或PEG亚单元)的有机部分，其可以是多分散的、单分散的或离散的(即，具有离散数目的乙烯-氧基亚单元)。多分散PEG为大小和分子量的非均匀混合物，而单分散PEG通常是从非均匀混合物纯化的并因此具有单一的链长和分子量。优选的PEG单元包含离散的PEG，其是以逐步的方式而非经由聚合过程合成的化合物。离散的PEG提供具有限定和指定链长的单一分子。
术语“药学上可接受的盐”表示，能保持本发明的ADC偶联物的生物学效应和性能的盐，并且该盐在生物学上或其它方面不是不被期望的。本发明的ADC偶联物可以以它们的药学上可接受的盐形式存在，包括酸加成盐和碱加成盐。在本发明中，药学上可接受的无毒的酸加成盐表示本发明中的ADC偶联物与有机或无机酸形成的盐，有机或无机酸包括但不限于盐酸、硫酸、氢溴酸、氢碘酸、磷酸、硝酸、高氯酸、乙酸、草酸、马来酸、富马酸、酒石酸、苯磺酸、甲磺酸、水杨酸、琥珀酸、柠檬酸、乳酸、丙酸、苯甲酸、对甲苯磺酸、苹果酸等。药学上可接受的无毒的碱加成盐表示本发明中的ADC偶联物与有机或无机碱所形成的盐，包括但不限于碱金属盐，例如锂、钠或钾盐；碱土金属盐，例如钙或镁盐；有机碱盐，例如通过与含N基团的有机碱形成的铵盐。
术语“溶剂化物”表示一个或多个溶剂分子与本发明中的ADC偶联物所形成的缔合物。形成溶剂化物的溶剂包括但不限于水、甲醇、乙醇、异丙醇、乙酸乙酯、四氢呋喃、N,N-二甲基甲酰胺、二甲亚砜等。
在根据上下文无矛盾的情况下，本文中“药学上可接受的”和“药用”可互换使用。
术语“药物:抗体比”或“DAR”是指，在ADC偶联物中，偶联于本文所述的Ab部分上的药物部分(D)与Ab部分的比例。在本文所述的一些实施方案中，DAR可由式I中的q确定，例如DAR可以为1至20，例如2-18、4-16、5-12、6-10、2-8、3-8、2-6、4-6、6-10，例如2、3、4、5、6、7、8、9、10、11、12、13、14或15。DAR也可被计算为产品中分子群体的平均DAR，即通过检测方法(例如通过常规方法如质谱法、ELISA测定、电泳和/或HPLC)测得的产品中偶联于本文所述的Ab部分的小分子药物部分(D)与Ab部分的总体比例，此DAR在文中称为平均DAR。在一些实施方案中，本发明偶联物的平均DAR值是1至20，例如2-18、4-16、5-12、6-10、2-8、3-8、2-6、4-6、6-10，例如1.0-8.0，2.0-6.0，例如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或10.0，以及以这些数值中的两个作为端点的范围。
本文所述的术语“药物”涵盖在预防或治疗肿瘤，例如癌症中有效的任何物质，包括化疗剂、细胞因子、血管生成抑制剂、细胞毒性剂、其它抗体、小分子药物或免疫调节剂(例如免疫抑制剂)。
术语“细胞毒性剂”用在本发明中指抑制或防止细胞功能和/或引起细胞死亡或破坏的物质。细胞毒性剂的实例包含但不限于喜树碱类药物、奥瑞他汀、金霉素、美登木素生物碱、蓖麻毒素、蓖麻毒素A链、卡贝塔汀(combrestatin)、倍癌霉素、海兔毒素、多柔比星、道诺霉素、他克唑、顺铂、cc1065、溴化乙锭、丝裂霉素、依托泊苷、替诺泊苷(tenoposide)、长春新碱、长春碱、秋水仙碱、二羟基蒽二酮、放线菌素、白喉毒素、假单胞菌外毒素(PE)A、PE40、相思豆毒素、相思豆毒素A链、蒴莲根毒素A链、α-帚曲霉素(sarcin)、白树毒素、丝林霉素(mitogellin)、局限曲菌素(retstrictocin)、酚霉素、伊诺霉素、麻疯树毒素(curicin)、巴豆毒素、卡奇霉素、肥皂草(Sapaonariaofficinalis)抑制剂和糖皮质激素和其它化学治疗剂，以及放射性同位素，例如At211、I131、I125、Y90、Re186、Re188、Sm153、Bi212或213、P32，以及Lu的放射性同位素，包含Lu177。
术语“小分子药物”是指低分子量的能够调节生物过程的有机化合物。“小分子”被定义为分子量小于10kD、通常小于2kD和优选小于1kD的分子。小分子包括但不限于无机分子、有机分子、含无机组分的有机分子、含放射性原子的分子、合成分子、肽模拟物和抗体模拟物。作为治疗剂，小分子可以比大分子更能透过细胞、对降解更不易感和更不易于引发免疫应答。
术语“药物组合物”指这样的组合物，其允许包含在其中的活性成分以生物学活性有效的形式存在，并且不包含对施用所述组合物的受试者具有不可接受的毒性的另外的成分。
术语“药用辅料”指与活性物质一起施用的稀释剂、佐剂(例如弗氏佐剂(完全和不完全的))、赋形剂、载体或稳定剂等。
术语“药物组合”是指非固定组合产品或固定组合产品，包括但不限于药盒、药物组合物。术语“非固定组合”意指两种或更多种活性成分(例如，(i)本发明的ADC分子或本发明的抗体、以及(ii)其他治疗剂)以分开的实体被同时地或依次地，不受特定时间限制或以相同或不同的时间间隔，施用于患者，其中这类施用在患者体内提供预防或治疗有效水平的所述两种或更多种活性成分。术语“固定组合”意指两种或更多种活性成分以单个实体的形式被同时施用于患者。在涉及药物组合的一些情况下，优选对两种或更多种活性成分的施用剂量和/或时间间隔进行选择，从而使各成分的联合使用能够在治疗疾病或病症时产生大于单独使用任何一种成分所能达到的效果。包含在药物组合中的各成分可以各自呈单独的制剂形式，并且其制剂形式可以相同也可以不同。在一些实施方案中，药物组合中使用的本发明的ADC分子或本发明的抗体和其他治疗剂以不超过它们单独使用时的水平施用。
术语“组合疗法”或“治疗组合”是指施用两种或更多种治疗剂或治疗方式(例如放射疗法或手术)以治疗本文所述疾病。这种施用包括以基本上同时的方式共同施用这些治疗剂，例如施用具有固定比例的两种或多种活性成分的单一胶囊。或者，这种施用包括对于在多种制剂(例如片剂、胶囊、粉末和液体)中的 或在分开的容器中的两种或多种活性成分的共同施用。粉末和/或液体可以在施用前重构或稀释至所需剂量。此外，这种施用还包括以大致相同的时间或在不同的时间以顺序的方式使用每种类型的治疗剂。在任一情况下，治疗方案将提供所述的治疗组合在治疗本文所述的病症或病状中的有益作用。
在本文中，术语“个体”、“受试者”或“患者”可互换地使用，是指哺乳动物。哺乳动物包括但不限于驯化动物(例如，奶牛、绵羊、猫、犬和马)、灵长类(例如，人和非人灵长类如猴)、兔和啮齿类(例如，小鼠和大鼠)。优选地，受试者是人。
术语“肿瘤”和“癌症”在本文中可互换地使用，是指哺乳动物中特征通常为细胞生长不受调节的生理疾患。该术语涵盖肿瘤的原发性形式和转移性形式。该术语也涵盖所有赘生性(neoplastic)的细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性的细胞和组织。
术语“抗肿瘤作用”指可以通过多种手段展示的抗肿瘤生物学效果，包括但不限于例如，肿瘤体积减少、肿瘤细胞数目减少、肿瘤细胞增殖减少或肿瘤细胞存活减少。
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性。
用于本文时，“预防”包括对疾病或病症或特定的疾病或病症的症状的发生或发展的抑制。通常，在癌症的背景中，术语“预防”是指在癌症的病征或症状发生前，特别是在具有癌症风险的受试者中，的药物施用。在一些实施方式中，具有癌症家族病史的受试者是预防性方案的候选者。
用于本文时，术语“有效量”指药物(例如，本发明的抗体药物偶联物或可药用盐或溶剂化物、或本发明的抗体或其抗原结合片段、或其组合物或药物组合)的这样的量，所述的量以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。
用于本文时，术语“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。治疗有效量也是这样的一个量，其中在将所述量施用于受试者时，由所述施用带来的任何有毒或有害作用将不及其带来的治疗有益作用。相对于未治疗的个体，“治疗有效量”优选地对可度量参数(例如肿瘤体积)实现至少约30％、甚至更优选地至少约40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％甚至100％的改善。
用于本文时，术语“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在受试者中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量将小于治疗有效量。
本发明的各方面将在下面各小节中进一步详述。
本发明的抗体-药物偶联物
I.抗体-药物偶联物
在一个方面，本发明提供了具有下式(I)的抗体-药物偶联物(ADC)或其可药用盐或溶剂化物：
Ab-[L-D]q     (I)
其中，
Ab表示抗TF抗体，
L表示连接体，
D表示细胞毒性或细胞抑制性药物，例如，拓扑异构酶I抑制剂，且
q表示1至20的整数或小数，例如，q＝1-10、1-8、3-8、4-8、或6-8。
以下就本发明ADC偶联物的组件及由其组成的本发明ADC偶联物分别进行详述描述。本领域技术人员可以理解，除非上下文有明确相反指示，这些组件的任何技术特征的任何组合均在本发明考虑范畴之中。并且，本领域技术人员可以理解，除非上下文有明确相反指示，本发明的ADC偶联物可以包含任何这样 的组合特征。
抗体Ab单元
在一些方面，本发明提供了抗体-药物偶联物(ADC)，其包含特异性结合TF的本发明抗体作为本发明式I偶联物的Ab单元。用作Ab单元的本发明抗TF抗体可以是全长抗体或是抗体片段。在一些实施方案中，本发明式(I)中的Ab是包含重链恒定区和轻链恒定区的全长抗体。在一些实施方案中，本发明式(I)中的Ab具有由两条轻链和两条重链形成的四聚体结构。在再一些实施方案中，本发明式(I)中的Ab是IgG抗体，尤其是IgG1抗体。在进一步的实施方案中，本发明式(I)中的Ab是人源化抗体。
抗体性质
用于本发明ADC中的本发明抗TF抗体具有以下一个或多个特性：
(i)以高亲和力与人TF胞外域结合；
(ii)以高亲和力与TF阳性肿瘤细胞结合；
(iii)具有与食蟹猴TF的交叉反应性；
(iv)具有TF受体介导的内吞活性；
(v)阻断TF阳性细胞中由TF/FVIIa复合物介导的下游信号通路；和
(vi)基本不影响由细胞表面TF起始的凝血或具有降低的凝血影响。
优选地，所述抗TF抗体与抗体-药物偶联物能够以高亲和力与TF阳性肿瘤细胞结合，且具有基本不影响由细胞表面TF起始的凝血或具有降低的凝血影响。
关于用于本发明ADC中的本发明抗TF抗体的性质，也参见下文“本发明的抗TF抗体”部分提供的详细描述。
抗体CDR区
CDR是抗体中主要负责与抗原表位结合的区域。在一些实施方案中，用于本发明ADC中的抗TF抗体包含如下CDR序列：
(i)如SEQ ID NO:1所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:2所示的轻链可变区的LCDR1、2和3序列，或者
(ii)如SEQ ID NO:3所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:4所示的轻链可变区的LCDR1、2和3序列，或者
(iii)如SEQ ID NO:5所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:6所示的轻链可变区的LCDR1、2和3序列，或者
(iv)如SEQ ID NO:7所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:8所示的轻链可变区的LCDR1、2和3序列。
优选地，所述CDR根据Chothia、AbM、Kabat、IMGT或其任意组合进行定义。再优选地，所述CDR根据Kabat或IMGT或其组合进行定义，更优选地，所述CDR根据IMGT定义。但应当明了，所述的CDR也可以采用本领域已知的任何其他方式定义。
此外，本领域知晓，尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat,Chothia,AbM和Contact方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。这样的最小结合单位可以是CDR的一个子部分。而CDR序列其余部分的残基，正如本领域技术人员明了，可以通过抗体的结构和蛋白折叠予以确定。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而其余CDR残基可以被替代。
在一些实施方案中，优选地，用于本发明ADC中的抗TF抗体包含重链可变区的3个互补决定区HCDR，以及轻链可变区的3个互补决定区LCDR，其中：
(i)根据Chothia定义，HCDR1包含SEQ ID NO:9的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:10的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:11的氨基酸序列或由其组成，LCDR1包含SEQ  ID NO:21的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:22的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:23的氨基酸序列或由其组成；或者
(ii)根据AbM定义，HCDR1包含SEQ ID NO:12的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:13的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:14的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:24的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:25的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:26的氨基酸序列或由其组成；或者
(iii)根据Kabat定义，HCDR1包含SEQ ID NO:15的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:16的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:17的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:27的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:28的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:29的氨基酸序列或由其组成；或者
(iv)根据IMGT定义，HCDR1包含SEQ ID NO:18的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:19的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:20的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:30的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:31的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:32的氨基酸序列或由其组成。
在一些实施方案中，优选地，用于本发明ADC中的抗TF抗体包含重链可变区的3个互补决定区HCDR，以及轻链可变区的3个互补决定区LCDR，其中：
(i)根据Chothia定义，HCDR1包含SEQ ID NO:33的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:34的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:35的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:45的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:46的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:47的氨基酸序列或由其组成；或者
(ii)根据AbM定义，HCDR1包含SEQ ID NO:36的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:37的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:38的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:48的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:49的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:50的氨基酸序列或由其组成；或者
(iii)根据Kabat定义，HCDR1包含SEQ ID NO:39的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:40的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:41的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:51的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:52的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:53的氨基酸序列或由其组成；或者
(iv)根据IMGT定义，HCDR1包含SEQ ID NO:42的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:43的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:44的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:54的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:55的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:56的氨基酸序列或由其组成。
在一些实施方案中，优选地，用于本发明ADC中的抗TF抗体包含重链可变区的3个互补决定区HCDR，以及轻链可变区的3个互补决定区LCDR，其中：
(i)根据Chothia定义，HCDR1包含SEQ ID NO:57的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:58的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:59的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:69的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:70的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:71的氨基酸序列或由其组成；或者
(ii)根据AbM定义，HCDR1包含SEQ ID NO:60的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:61的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:62的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:72的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:73的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:74的氨基酸序列或由其组成；或者
(iii)根据Kabat定义，HCDR1包含SEQ ID NO:63的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:64的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:65的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:75的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:76的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:77的氨基酸序列或由其组成；或者
(iv)根据IMGT定义，HCDR1包含SEQ ID NO:66的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:67的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:68的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:78的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:79的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:80的氨基酸序列或由其组成。
在一些实施方案中，优选地，用于本发明ADC中的抗TF抗体包含重链可变区的3个互补决定区HCDR，以及轻链可变区的3个互补决定区LCDR，其中：
(i)根据Chothia定义，HCDR1包含SEQ ID NO:81的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:82的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:83的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:93的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:94的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:95的氨基酸序列或由其组成；或者
(ii)根据AbM定义，HCDR1包含SEQ ID NO:84的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:85的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:86的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:96的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:97的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:98的氨基酸序列或由其组成；或者
(iii)根据Kabat定义，HCDR1包含SEQ ID NO:87的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:88的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:89的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:99的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:100的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:101的氨基酸序列或由其组成；或者
(iv)根据IMGT定义，HCDR1包含SEQ ID NO:90的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:91的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:92的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:102的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:103的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:104的氨基酸序列或由其组成。
抗体可变区
“可变区”或“可变结构域”是抗体的重链或轻链中参与抗体与其抗原的结合的结构域。在一些实施方案中，用于本发明ADC中的本发明抗TF抗体包含：本发明示例性抗体2B12B10-hz1、30G11B7-hz1、22F11H5-hz1和27H8H3-hz1之任一的重链和轻链可变区序列或其变体，例如，具有与所述示例性抗体之一相同的CDR序列，并具有相同或不同的构架区序列的抗体或其片段，例如人源化抗体。对于构架区的替代，可以使用序列相似性检索工具，例如Gapped BLAST，比较亲本抗体蛋白序列与数据库中的蛋白序列；选择与待进行改变的亲本抗体的构架序列具有高度结构相似性，例如，具有序列同一性至少80％，85％，90％，或95％、96％、97％、98％、99％以上的数据库构架序列，用于构架区替代。在一些情况下，在构架区替代后，可以基于需要，对替代的构架序列进行1至几个残基突变，例如回复突变，并在体外或体内测定试验 中评估突变后的抗体的抗原结合性质或其它功能性质的保持或改善情况。
在一个实施方案中，用于本发明ADC中的本发明抗TF抗体包含重链可变区和轻链可变区，其中：所述重链可变区包含：
(i)SEQ ID NO:1所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
(ii)SEQ ID NO:3所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
(iii)SEQ ID NO:5所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，
(iv)SEQ ID NO:7所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
在另一个实施方案中，用于本发明ADC中的抗TF抗体包含重链可变区和轻链可变区，其中：所述述轻链可变区包含：
(i)SEQ ID NO:2所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
(ii)SEQ ID NO:4所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
(iii)SEQ ID NO:6所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，
(iv)SEQ ID NO:8所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
在一些优选的实施方案中，所述抗TF抗体包含重链可变区和轻链可变区，其中所述重链可变区包含SEQ ID NO:1所述的氨基酸序列或与其具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列，且其中所述轻链可变区包含SEQ ID NO:2所示的氨基酸序列、或与其具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列。优选地，所述抗体或抗原结合片段包含SEQ ID No:1的重链可变区和SEQ ID No:2的轻链可变区，或由其组成。
在一些优选的实施方案中，所述抗TF抗体包含重链可变区和轻链可变区，其中所述重链可变区包含SEQ ID NO:3所述的氨基酸序列或与其具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列，且其中所述轻链可变区包含SEQ ID NO:4所示的氨基酸序列、或与其具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列。优选地，所述抗体或抗原结合片段包含SEQ ID No:3的重链可变区和SEQ ID No:4的轻链可变区，或由其组成。
在一些优选的实施方案中，所述抗TF抗体包含重链可变区和轻链可变区，其中所述重链可变区包含SEQ ID NO:5所述的氨基酸序列或与其具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列，且其中所述轻链可变区包含SEQ ID NO:6所示的氨基酸序列、或与其具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列。优选地，所述抗体或抗原结合片段包含SEQ ID No:5的重链可变区和SEQ ID No:6的轻链可变区，或由其组成。
在一些优选的实施方案中，所述抗TF抗体包含重链可变区和轻链可变区，其中所述重链可变区包含SEQ ID NO:7所述的氨基酸序列或与其具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列，且其中所述轻链可变区包含SEQ ID NO:8所示的氨基酸序列、或与其具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列。优选地，所述抗体或抗原结合片段包含SEQ ID No:7的重链可变区和SEQ  ID No:8的轻链可变区，或由其组成。
抗体重链和轻链
用于本发明ADC中作为Ab单元的本发明抗TF抗体，在一些情况下，可以包含重链恒定区和/或轻链恒定区。优选地，所述重链恒定区为来源于人免疫球蛋白的重链恒定区。优选地，所述轻链恒定区为来源于人免疫球蛋白的轻链恒定区。在一些实施方案中，包含在所述抗TF抗体中的重链恒定区可以为任何同种型或亚型，例如IgG1,IgG2，IgG3或IgG4同种型的重链恒定区。在一些实施方案中，所述抗TF抗体优选包含IgG1重链恒定区，尤其是人IgG1重链恒定区，例如SEQ ID NO:106的氨基酸序列或与其具有至少90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。在另一些实施方案中，包含在所述抗TF抗体中的轻链恒定区可以为κ轻链恒定区或λ轻链恒定区。在一些实施方案中，所述抗TF抗体包含来源于人的κ轻链恒定区或λ轻链恒定区，优选地人κ轻链恒定区，例如，SEQ ID NO:105的氨基酸序列或与其具有至少90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
本发明也考虑上述任何抗体实施方案的变体。可用作根据本发明的Ab单元的抗体变体优选保持引入改变前的亲本抗体的至少60％,70％,80％,90％,或100％的生物学活性(例如抗原结合能力)。更优选地，所述改变不导致抗体变体丧失对抗原的结合，但任选地赋予诸如提高的抗原亲和力等性质。可以理解的，抗体的重链可变区或轻链可变区、或各CDR区或构架区可以单独改变或组合改变。此外，也可以对抗体的Fc区进行改变。Fc区的改变可以单独进行，或与构架区和/或CDR区的改变相组合。可以改变Fc区，例如，以改变抗体的一种或多种效应子功能，例如血清半衰期，补体固定、Fc受体结合、和/或抗原依赖性细胞毒性。此外，还可以对本发明抗体进行化学修饰(例如，与PEG连接)或改变其糖基化模式。
为形成ADC偶联物，根据本发明的抗体可以利用其上的一些天然连接位点与毒素-连接体偶联。这样的天然连接位点有半胱氨酸的巯基和赖氨酸的氨基。通常，应用抗体的链间二硫桥可以实现较为确定的药物-抗体比(DAR)。因此，在一个实施方案中，在还原抗体的链间二硫键后，通过巯基化学，将毒素偶联到本发明抗体上，形成式(I)的ADC偶联物。此外，也可以考虑在抗体中引入人工连接位点，实现更为定点的偶联。
药物D单元
抗体-药物偶联物的药物D单元在本文中也称作ADC药物的有效载荷(payload)。可用于本发明ADC中的药物D并无特别限制，可以为任何对细胞具有毒性或抑制性的药物或其前药。本领域技术人员可以根据所需的ADC药物起效机制和细胞杀伤效果，选择合适的药物分子作为ADC的有效载荷。
在一些实施方案中，药物D是细胞毒性剂。在本领域中，已经报道了多种不同机制的适用作有效载荷的细胞毒性剂，包括，但不限于，
(1)微管抑制/破坏剂：例如，但不限于，auristatin类(例如，MMAE或MMAF)，美登素衍生物(例如DM2，DM4)，微管溶素(Tubulysins)，隐粘菌素(Cryptomycins)，抗有丝分裂EG5抑制剂(例如，纺锤体驱动蛋白KSP抑制剂)；
(2)DNA损伤剂：例如，但不限于，吡咯并苯并二氮杂卓类(Pyrrolobenzodiazepines)(例如，吡咯并[2,1-c][1,4]苯并二氮杂卓(PBD))，杜卡霉素,Indolinobenzodiazepine；Duocarmycins；Calicheamicin类；
(3)拓扑异构酶抑制剂：例如，但不限于，喜树碱类(例如，依喜替康及其衍生物Dxd)；
(4)其他：凋亡诱导剂(Bcl-xL抑制剂)，thailanstatin及其类似物，鹅膏毒素，烟酰胺磷酸核糖基转移酶(NAMPT)抑制剂，卡马霉素。
本发明式(I)中的药物D可以是选自上述的任何化合物。在一些实施方案中，药物D为选自美登素衍生物、卡里奇霉素衍生物、奥瑞他汀衍生物的抗肿瘤生长抑制剂。在一个实施方案中，药物D为微管蛋白抑制剂/稳定性破坏剂，例如长春花生物碱类、长春新碱、紫杉醇和多西他赛。在一个实施方案中，药物D为DNA合成抑制剂，例如，甲氨蝶呤、5-氟尿嘧啶、阿糖胞苷、吉西他滨、巯基嘌呤、喷司他丁、氟达拉滨、克拉屈滨。在一个实施方案中，药物D为DNA拓扑异构酶抑制剂，例如，拓扑异构酶I抑制剂(例 如，喜树碱类)和拓扑异构酶II抑制剂(例如，放线菌素D、阿霉素、米托蒽醌)。
在一些优选的实施方案中，根据本发明的ADC的药物D为拓扑异构酶I抑制剂。拓扑异构酶I抑制剂的一个典型例子是喜树碱类药物，例如，但不限于,喜树碱(CPT)、羟基喜树碱、9-氨基喜树碱、exatecan,topotecan,belotecan,irinotecan，SN-38，和FL118，及其衍生物。参见例如Vesela Kostova等,The Chemistry Behind ADCs，Pharmaceuticals 2021,14,442.https://doi.org/10.3390/ph14050442；和WO2019/195665，所述文献在此并入本文作为参考。
在本发明的一些实施方案中，本发明ADC的药物D为选自以下的喜树碱类药物：
-喜树碱及其衍生物，例如，
其中RA选自例如：氢、任选被取代的烷基，其中取代基包括但不限于羟基、氨基，其中氨基部分或羟基部分可以是取代或未取代的，例如被烷基、烷基酰基、或烷基磺酰基取代；在一个实施方案中，所述药物D为
-10,11-methylenedioxy CPT(也简称为MDCPT或FL118)及其衍生物，例如，
其中RB选自例如：氢、环烷基、苯基、任选被取代的烷基，其中取代基包括但不限于卤素、羟基、任选被取代的烷氧基、环烷基、杂环烷基、苯基、氨基，其中氨基部分可以任选被取代；在一个实施方案中，所述药物D为
-10-hydroxy CPT(也简称为HCPT)及其衍生物，例如，
其中，RC选自例如任选被取代的烷基和环烷基；R’C选自例如H、烷酰基或任选取代的杂环烷基酰基；在一个实施方案中，所述药物D为7-乙基-10-羟基CPT(SN-38)或其前药伊立替康(irinotecan,CPT-11)，或所述药物D为拓扑替康
-Exatecan及其衍生物，例如，
其中，RD选自例如H、任选被取代的烷基、任选被取代的烷基-C(＝O)-，所述取代基为例如-OH或取代或未取代的氨基；在一个实施方案中，所述药物D为:
Exatecan、Dxd(1)或Dxd(2)；在另一个实施方案中，所述药物D为：
F118((20S)-10,11-methylenedioxy喜树碱)通过高通量筛选化合物文库获得。与其他喜树碱衍生物相比，FL118已经在许多不同的癌症类型中被证实具有高得多的体内和体外抗癌活性。除抑制拓扑异构酶I外，FL118也可以选择性抑制抗凋亡蛋白，例如survivin,XIAP,cIAP2和Mcl-1的基因启动子活性和内源性表达。参见Xiang Ling等,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。尽管FL118的单独应用由于其极差的水溶性和毒性副作用而受到严重阻碍，但如实施例所示，由该化合物与本发明抗体和连接体组合形成的偶联物，不仅有效地发挥了该化合物的肿瘤杀死效果，且克服了该化合物的缺陷，所形成的偶联物的聚集度小并具有良好的动物耐受性。
因此，在本发明的一些优选实施方案中，本发明ADC的药物D为FL118衍生物，尤其是7位取代的FL118。
在一些实施方案中，本发明的式(I)中的药物D为包含以下式D结构的喜树碱类药物：
其中，
Rx、Ry各自独立地选自H、卤素、-OH、C1-C6烷基，或者Rx和Ry与各自连接的碳原子一起形成具有1或2个选自N、S和O的5-6元杂环；
Ra选自H、卤素、-OH、任选取代的C1-C8烷基或C3-C8炔基或C3-C8烯基、任选取代的C1-C8烷氧基、任选取代的C3-C8环烷基、任选取代的苯基、任选取代的杂环烷基、任选取代的杂芳基，
优选地，其中，Ra选自：
氢；
C3-C8环烷基；
苯基；
任选被选自以下的取代基取代的C1-C8烷基或C3-C8炔基或C3-C8烯基：卤素、羟基、任选被NH2、NH(C1-C4烷基)和N(C1-C4烷基)2取代的C1-C4烷氧基、C3-C8环烷基、杂环烷基、苯基、和NR1R2，
其中R1和R2彼此独立地选自
氢；
任选地被选自以下的取代基取代的C1-C8烷基：羟基、氨基、被一个或两个C1-C4烷基取代的氨基、被一个或两个C1-C4羟烷基取代的氨基、被(C1-C4羟烷基)和(C1-C4烷基)取代的氨基；
被1或2个C3-C10环烷基、C3-C10杂环烷基、苯基或杂芳基取代的C1-C4烷基；
C3-C10环烷基；
C3-C10杂环烷基
C2-C6杂烷基；
杂芳基；
任选卤代的苯基；
任选被羟基或氨基取代的C1-C8烷基-C(＝O)-；
或者，R1和R2与各自连接的氮原子组合形成具有0至3个取代基的5-、6-或7-元杂环，所述取代基选自卤素、C1-C4烷基、OH、C1-C4烷氧基、NH2、NH(C1-C4烷基)和N(C1-C4烷基)2，
其中环烷基、杂环烷基、苯基、杂芳基在每次出现时各自独立地任选被0-3个选自以下的取代基取代：OH、C1-C4烷基、C1-C4烷氧基、NH2、NH(C1-C4烷基)和N(C1-C4烷基)2取代。
在一些优选实施方案中，Rx和Ry各自独立地为H。在另一些优选实施方案中，Rx为F且Ry为甲基。在再一些优选实施方案中，Rx和Ry与各自连接的碳原子一起形成含有2个O的5元杂环。
在一些实施方案中，本发明的式(I)中的药物D为包含以下式Da或式Db结构的喜树碱类药物：
尤其是式(Da)的喜树碱类药物，
其中，Ra选自：
氢；
C3-C8环烷基；
苯基；
任选被选自以下的取代基取代的C1-C8烷基、C3-C8炔基或C3-C8烯基：卤素、羟基、任选被NH2、NH(C1-C4烷基)和N(C1-C4烷基)2取代的C1-C4烷氧基、C3-C8环烷基、杂环烷基、苯基、和NR1R2，
其中R1和R2彼此独立地选自
氢；
任选地被选自以下的取代基取代的C1-C8烷基：羟基、氨基、被一个或两个C1-C4烷基取代的氨基、被一个或两个C1-C4羟烷基取代的氨基、被(C1-C4羟烷基)和(C1-C4烷基)取代的氨基；
被1或2个C3-C10环烷基、C3-C10杂环烷基、苯基或杂芳基取代的C1-C4烷基；
C3-C10环烷基；
C3-C10杂环烷基
C2-C6杂烷基；
杂芳基；
任选卤代的苯基；
任选被羟基或氨基取代的C1-C8烷基-C(＝O)-；
或者，R1和R2与各自连接的氮原子组合形成具有0至3个取代基的5-、6-或7-元杂环，所述取代基选自卤素、C1-C4烷基、OH、C1-C4烷氧基、NH2、NH(C1-C4烷基)和N(C1-C4烷基)2，
其中环烷基、杂环烷基、苯基、杂芳基在每次出现时各自独立地任选被0-3个选自以下的取代基取代：OH、C1-C4烷基、C1-C4烷氧基、NH2、NH(C1-C4烷基)和N(C1-C4烷基)2取代。
在一些实施方案中，Ra是氢。
在一些实施方案中，Ra是C3-C8环烷基(例如，环丙基，环庚基，环戊基)。
在一些实施方案中，Ra是苯基。在另一些实施方案中，Ra是被NH2或NH(C1-C4烷基)取代的苯基。
在一些实施方案中，Ra是C1-C6烷基，例如，甲基，乙基，丙基，2-甲基丙基，丁基，异丁基，2,2-二甲基-丙基，2,2-二甲基-丁基，正己基，正戊基，3-乙基-戊基。
在一些实施方案中，Ra是C1-C4烷基，其任选地被羟基或C1-C4烷氧基取代，其中烷氧基任选地进一步被-NH2、-NH(C1-C4烷基)和-N(C1-C4烷基)2取代。
在一些实施方案中，Ra是C2-C4烯基或C2-C4炔基，其任选地被羟基或氨基取代。
在一些实施方案中，Ra是被具有1或2个选自N、S和O的5-6元杂环烷基取代的C1-C4烷基，其中杂环烷基部分任选地被C1-C4烷基进一步取代，优选地，所述杂环烷基为哌嗪基或吗啉基。
在一些实施方案中，Ra是被-NR1R2取代的C1-C4烷基，尤其是-亚甲基-NR1R2，其中R1和R2彼此独立地选自：
氢；
-C1-C4烷基；
被以下取代基取代的-C1-C4烷基：
-OH；-NH2；-NH(C1-C4烷基)；-N(C1-C4烷基)2；-苯基；-亚苯基-NH2；-亚苯基-C1-C4烷氧基；-二苯基；-C3-C10环烷基；-C3-C10亚环烷基-C1-C4烷基；-C3-C10杂环烷基，其中所述杂环烷基具有1或2个选自N、S和O的杂原子，优选所述杂环烷基为哌啶基；
苯基；卤代的苯基；
C3-C10环烷基；
-C(＝O)-C1-C4烷基-OH；-C(＝O)-C1-C4烷基-NH2。
在一些实施方案中，Ra是被-NR1R2取代的C1-C4烷基，尤其是-亚甲基-NR1R2，其中R1和R2与各自连接的氮原子组合形成5-、6-或7-元杂环烷基环。在一些方面，R1和R2与各自连接的氮原子组合形成6-元环。在一些方面，所述6-元环为吗啉基或哌嗪基。
在一些优选实施方案中，Ra选自：-C1-C4亚烷基-OH、-C1-C4亚烷基-O-C1-C4亚烷基-NH2、-C1-C4亚烷基-NH2、-C1-C4亚烷基-NH(C1-C4羟烷基)、-C1-C4亚烷基-N(C1-C4羟烷基)2、-C1-C4亚烷基-N(C1-C4氨烷基)(C1-C4羟烷基)、-C1-C4亚烷基-NH(C1-C4氨烷基)、-C1-C4亚烷基-NH-C(＝O)-C1-C4亚烷基-OH；-C1-C4亚烷基-NH-C(＝O)-C1-C4亚烷基-NH2、-C1-C4亚烷基-N(C1-C4烷基)(C(＝O)-C1-C4亚烷基-OH)；-C1-C4亚烷基-N(C1-C4烷基)(C(＝O)-C1-C4亚烷基-NH2)。
在一些再优选的实施方案中，Ra为-C1-C4亚烷基-NH-C(＝O)-C1-C4亚烷基-OH；-C1-C4亚烷基-NH-C(＝O)-C1-C4亚烷基-NH2。
在一些再优选的实施方案中，Ra为-C2-C4亚烯基-NH2或-C2-C4亚烯基-OH，例如，-CH＝CH-CH2-NH2或-CH＝CH-CH2-OH。
在一些再优选的实施方案中，Ra为-C1-C4亚烷基-OH、-C1-C4亚烷基-O-C1-C4亚烷基-NH2、或-C1-C4亚烷基-NH2。
在一些更优选的实施方案中，Ra为-C1-C4亚烷基-OH。在一些方面，Ra为羟甲基。在一些方面，Ra为羟乙基。在一些方面，Ra为羟丙基。
在另一些更优选的实施方案中，Ra为-C1-C4亚烷基-NH2。在一些方面，Ra为氨基甲基。在一些方面，Ra为氨基乙基。在一些方面，Ra为氨基丙基。
优选地，药物D通过其上的游离羟基或氨基基团，与连接体L连接，以偶联到抗体上。更优选地，式D或式Da或Db的药物D通过Ra上的羟基或氨基基团，与连接体L连接，以偶联到抗体上。优选地，在与连接体连接后Ra具有选自以下的结构：
其中，n为1-4的整数，优选n＝2或3，其中R’为H或C1-C4烷基，优选为H，其中，左侧波浪线表示与连接体L连接的位置，右侧星号表示与喜树碱母核连接的位置；
更优选地，与连接体连接后的Ra具有选自以下的结构：
最优选地，与连接体连接后的Ra具有选自以下的结构：
其中，左侧波浪线表示与连接体L连接的位置，右侧星号表示与喜树碱母核连接的位置。
在一些优选的实施方案中，本发明的式(I)中的L-D单元包含以下结构：
更优选地，式(I)中的L-D单元包含以下结构：
连接体L单元
适用于本发明的连接体L可以是能够实现将本发明抗体与药物偶联的任何连接体。适宜地，连接体的加入应保证本发明的ADC在循环系统中的充分稳定性，同时可以在靶位点(例如肿瘤细胞或肿瘤环境)中提供快速和有效的毒性药物活性形式的释放。
在一些实施方案中，本发明式(I)中的连接体是不可降解型连接体。不可降解型连接体的例子包括，但不限于，N-琥珀酰亚胺基-4-(N-马来酰亚胺甲基)环己烷-1-羧酸盐(SMCC)。通常，包含此类连接体的ADC必须被细胞内化，由细胞内溶酶体蛋白酶降解ADC的抗体部分，释放药物活性分子。
在再一些实施方案中，本发明式(I)中的连接体是可降解型连接体。包含此类连接体的ADC的药物释放由连接体中的切割位点的性质触发。因此，可以根据靶治疗位点(例如，肿瘤细胞溶酶体和/或肿瘤环境)的特点，设计此类连接体的切割位点。在大多数情况下，可降解连接体可以由偶联部分、可降解部分、和任选地间隔基部分组成。偶联部分负责抗体与连接体-药物的连接，可以根据期望采用的抗体偶联化学来进行选择。可降解部分在基于酶的释放机制下将包含可被酶识别的肽或肽类似物，例如，可被蛋白水解酶降解的Val-Ala,Val-Cit,Phe-Lys,Gly-Phe-Leu-Gly,Ala-Leu-Ala-Leu,Gly-Gly-Phe-Gly,环丁基-Ala,环丁基-Cit等寡肽或寡肽类似物。在一些情况下，可以通过在连接体中邻近酶切割位点的肽残基位置引入修饰，来改善ADC的性质，例如，ADC在血液循环中的稳定性和/或ADC在靶位点的药效。在一些情况下，也可以根据需要，在连接体的可降解部分与药物D之间引入间隔基，来促进药物活性分子自偶联物的其余部分的释放(尤其是，无痕释放)，例如，在酸性介质中可自发消除的对氨基苄基氨基甲酸酯(PABA)或氨基甲基(-NHCH2-)间隔基。此外，在药物疏水性高时，在一些情况下，可以考虑(但不是必需)加入诸如PEG单元，来改善ADC的性质，例如减少沉淀和聚集。可适用于本发明的连接体包括但不限于WO2022/170971中公开的连接体(该文献特此并入作为参考)。
在一些实施方案中，本发明式(I)中的连接体L具有以下式(II)的结构：
-Z-Y-M-     (II)，
其中
Z为与Ab连接的连接基，
Y是2-5个氨基酸的肽，优选二肽、三肽或四肽，
M不存在，或是用于与药物D连接的间隔基。
Z连接基
通常，抗体半胱氨酸的硫基，以二硫键的形式存在。打开抗体的二硫键，可以提供自由巯基作为偶联位点。通过与抗体巯基进行偶联形成ADC的一种方式是，使抗体上的自由巯基与杂环类(例如马来酰亚胺类)连接体发生Michael加成反应。另一种方式是，使包含离去基团取代的杂芳环的连接体与抗体分子中的自由巯基进行亲核取代反应，来获得抗体-药物偶联物。两种方式均适用于本发明的ADC偶联物。因此，根据本发明的连接体在一些方面可以包含杂环类(例如马来酰亚胺类)或杂芳环类(例如嘧啶类)的Z连接基； 并且，在一些情况中，优选包含杂芳环类(例如嘧啶类)连接基，以提供在血液循环中具有更高稳定性的偶联物。
在一些实施方案中，式(II)中的Z具有以下结构：
-Z1-Z2-Z3-Z4-，
其中Z1为Ab中的硫原子，
Z2为5-10元杂环基，优选地含1或2个选自N,S和O的杂原子；
Z3选自键、-C(＝O)-、-C1-C10亚烷基-C(＝O)-、-C3-C10亚炔基-C(＝O)-、-C3-C10亚烯基-C(＝O)-、-C1-C10亚杂烷基-C(＝O)-、-C3-C8亚环烷基-C(＝O)-、-O-C1-C8亚烷基-C(＝O)-、-亚芳基-C(＝O)-、-C1-C10亚烷基-亚芳基-C(＝O)-、-亚芳基-C1-C10亚烷基-C(＝O)-、-C1-C10亚烷基-C3-C8亚环烷基-C(＝O)-、-C3-C8亚环烷基-C1-C10亚烷基-C(＝O)-、-C3-C8亚杂环基-C(＝O)-、-C1-C10亚烷基-C3-C8亚杂环基-C(＝O)-、-C3-C8亚杂环基-C1-C10亚烷基-C(＝O)-、-亚杂芳基-C(＝O)-、-C1-C10亚烷基-亚杂芳基-C(＝O)-、-亚杂芳基-C1-C10亚烷基-C(＝O)-、，
Z4是键或是下式表示的PEG单元，
其中，R5选自C1-4亚烷基、-NH-、-NH-C1-4亚烷基-杂芳基-，其中杂芳基为5元或6元的含氮杂芳基，优选三唑基；R6为-C(＝O)-、-C1-4亚烷基、-C1-4亚烷基-C(＝O)-、-NH-C(＝O)-(CH2OCH2)-C(＝O)-、-C1-4亚烷基-NH-C(＝O)-(CH2OCH2)-C(＝O)-，其中m为2-12的整数，例如m＝2,4,6,或8。
在一个实施方案中，Z2为5-10元杂芳基。在一个实施方案中，Z2选自任选被一个或多个独立地选自氢、卤素、硝基、C1-6烷基和卤代C1-6烷基取代的嘧啶、噻唑、苯丙噻唑、噁唑、喹唑啉和吡咯并[2,3d]嘧啶。在一些实施方案中，Z2为选自如下的杂芳基，
其中，Z2通过与杂原子相邻的碳原子与Z1连接。在一个优选实施方案中，Z2为亚嘧啶基，优选更优选其中左侧波浪线表示与Z1连接的位置；右侧波浪线表示与Z3连接的位置。
在一些实施方案中，Z2为亚马来酰亚胺基，其中左侧波浪线表示与Z1连接的位置；右侧波浪线表示与Z3连接的位置。
在一些实施方案中，Z3为-C3-C8亚杂环基-C(＝O)-、-C1-C10亚烷基-C3-C8亚杂环基-C(＝O)-、-C3-C8亚杂环基-C1-C10亚烷基-C(＝O)-，优选地，其中所述亚杂环基中的杂环是杂芳环，例如三唑、吡唑、噻唑、噁唑、异噁唑、或哒嗪的杂芳环基。在一些实施方案中，Z3为-亚杂芳基-C(＝O)-、-C1-C10亚烷基-亚杂芳基-C(＝O)-、-亚杂芳基-C1-C10亚烷基-C(＝O)-。
在一些实施方案中，Z3为-亚杂芳基-C1-C10亚烷基-C(＝O)-，尤其是，
其中n’为1-6，优选地，Z3为更优选
其中左侧波浪线表示与Z2连接的位置，右侧波浪线表示与Z4连接的位置，且其中优选地，Z2为亚嘧啶基。
在一些实施方案中，Z3为亚芳基-或亚杂芳基-C(＝O)-,尤其是，
其中Z3通过-C-(＝O)-与Z4连接，且其中优选地，Z2为亚嘧啶基。
在另一些实施方案中，Z3为-(C≡C)-C1-5亚烷基-C(＝O)-,-(CH＝CH)-C1-5亚烷基-C(＝O)-,-C1-6亚烷基-C(＝O)-，或-C3-8亚环烷基-C(＝O)-，其中Z3通过-C-(＝O)-与Z4连接。
在一些优选实施方案中，Z2为亚嘧啶基，Z3为-C(＝O)-。
在一些优选实施方案中，Z2为亚嘧啶基，Z3为-(C≡C)-C1-5亚烷基-C(＝O)-或-(CH＝CH)-C1-5亚烷基-C(＝O)-，尤其是-(C≡C)-C1-5亚烷基-C(＝O)-。
在一些优选实施方案中，Z2为亚马来酰亚胺基，Z3为-C1-6亚烷基-C(＝O)-，或-C3-8亚环烷基-C(＝O)-。
在一些实施方案中，Z4为键，Z3与式(II)中的Y直接连接。
在一些实施方案中，Z4为包含2-12个PEG的单元。在一些实施方案中，Z4为
其中m＝1-8,例如，2,3,4,5,6,7或8。
在一些实施方案中，Z4选自：
其中左侧波浪线表示与Z3连接的位置；右侧波浪线表示与式II中的Y连接的位置。
在一些实施方案中，本发明式(I)中的Z具有以下结构：
其中，x1＝1-8，例如,x1＝3。
在一些实施方案中，本发明式(I)中的Z具有以下结构：
其中，x2＝1-6。
在一些优选的实施方案中，本发明式(I)中的Z具有以下结构：
其中S为Ab中的硫原子，且Rb为未取代的或取代的-C3-10炔基-C(＝O)-或-C3-10烯基-C(＝O)-或-亚杂芳基-C1-C10亚烷基-C(＝O)-，
优选地，Rb是
其中，左侧波浪线表示与嘧啶基连接的位置；右侧波浪线表示与Y连接的位置；
更优选地，Z具有结构：
在一些实施方案中，本发明式(I)中的Z具有以下结构：
其中RE是氢、C1-6烷基、C1-6氨基烷基、C1-6卤代烷基、C1-6羟基烷基，其中y＝0-4,例如,0,2,或5，其中烷基、氨基和羟基部分任选地被取代；在一些实施方案中，RE是任选被 取代的氨基烷基，例如被-C1-4亚烷基-NH2、-C1-4亚烷基NHRF和-C1-4亚烷基N(RF)2所取代，其中每个RF独立地选自C1-6烷基和C1-6卤代烷基，或者两个RF基团与它们所附连的氮组合形成氮杂环丁烷基、吡咯烷基或哌啶基基团。
在一些实施方案中，本发明式(I)中的Z具有以下结构：
Y含肽单元
出于限制或最小化ADC的非靶向毒性，同时保证毒素分子在靶肿瘤位置释放的目的，在一些方面，有利的是，设计抗体-药物偶联物，以使其在高选择性地靶向肿瘤细胞的同时，能够被肿瘤细胞或环境中的蛋白水解酶(尤其是，相对于血液，在肿瘤细胞和/或肿瘤环境中具有显著更高活性的酶)降解。为此，可以在此类ADC偶联物的连接体中包含可以被此类蛋白水解酶识别和切割的寡肽或寡肽类似物。
在一个实施方案中，因此，作为本发明连接体一部分的Y单元是含肽的可降解部分，例如，含有两个或更多个(例如,2-12，例如2,3,4,5,或6个)连续或不连续的氨基酸的可降解肽接头。含肽单元的每个氨基酸可以彼此独立地选自天然氨基酸或非天然氨基酸，例如选自：丙氨酸、精氨酸、天冬氨酸、天冬酰胺、组氨酸、甘氨酸、谷氨酸、谷氨酰胺、苯丙氨酸、赖氨酸、取代的赖氨酸、亮氨酸、丝氨酸、酪氨酸、苏氨酸、异亮氨酸、脯氨酸、色氨酸、缬氨酸、半胱氨酸、甲硫氨酸、硒代半胱氨酸、鸟氨酸、β-丙氨酸、瓜氨酸、及其衍生物。在一些实施方式中，每个氨基酸独立地选自丙氨酸、精氨酸、天冬氨酸、天冬酰胺、组氨酸、甘氨酸、谷氨酸、谷氨酰胺、苯丙氨酸、赖氨酸、亮氨酸、丝氨酸、酪氨酸、苏氨酸、异亮氨酸、脯氨酸、色氨酸、缬氨酸、半胱氨酸、甲硫氨酸及其衍生物。在一些实施方式中，每个氨基酸独立地选自丙氨酸、精氨酸、天冬氨酸、天冬酰胺、组氨酸、甘氨酸、谷氨酸、谷氨酰胺、苯丙氨酸、赖氨酸、亮氨酸、丝氨酸、酪氨酸、苏氨酸、异亮氨酸、脯氨酸、色氨酸和缬氨酸、N-甲基甘氨酸、β-丙氨酸、及其衍生物。在一些方面，ADC在内化进入肿瘤细胞中后，Y单元中的酰胺键将被肿瘤细胞溶酶体中的酶识别并降解，释放出药物部分D。在另一些方面，Y单元中的酰胺键可以被肿瘤环境中的酶识别并降解，释放出药物部分D。通过设计偏向肿瘤分布的抗体-药物偶联物，具有此类Y含肽单元的连接体将促进毒素分子在肿瘤细胞和环境中的释放。
在一些实施方案中，Y为包含选自以下的单元：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,环丁基-Ala,环丁基-Cit。在一些实施方案中，Y为包含选自以下的单元：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,Ala-Ala-Asn。
在一些优选的实施方案中，Y为包含取代的赖氨酸的二肽、三肽、四肽或五肽。在一个实施方案中，所述取代的赖氨酸为：
其中，R3和R4彼此独立地选自：H，C1-6烷基，-CO-NH2,-CONH(C1-6烷基)，和-CONH(C1-6烷基)2，其中所述烷基任选地被选自以下的基团取代：卤素、C1-6烷氧基、C1-6卤代烷基、C3-6环烷基、6-10元芳基和5-14元杂芳基。
在一些优选实施方案中，Y为从N端到C端具有下式氨基酸序列的肽；
Xaa1-Xaa2-Xaa3-Xaa4-Xaa5，
其中Xaa1不存在，或为选自缬氨酸、甘氨酸、丙氨酸和谷氨酸的氨基酸；
Xaa2为选自苯丙氨酸、亮氨酸、丙氨酸和缬氨酸的氨基酸，优选缬氨酸；
Xaa3为未取代的或取代的赖氨酸；
Xaa4为选自亮氨酸、甘氨酸和丙氨酸的氨基酸；
Xaa5不存在，或为选自甘氨酸和丙氨酸的氨基酸；
其中，所述氨基酸序列的N端与连接体的Z单元连接，且C端与M单元连接或与药物D直接连接，
优选地，Xaa3为ε-氨基被C1-C3烷基单取代或二取代的赖氨酸，
再优选地，Y为选自以下的肽：Phe-Lys-Gly、Leu-lys-Gly、Gly-Val-Lys-Gly、Val-Lys-Gly-Gly、Val-Lys-Gly、Val-Lys-Ala、Val-Lys-Leu，其中Lys残基为未取代的或被C1-C3烷基单取代或二取代的赖氨酸。
M间隔基
在一些实施方式中，本发明的ADC偶联物在可释放的含肽单元(Y)与药物(D)之间具有间隔基(M)。间隔基可以是促进含肽单元(Y)与药物D连接的官能团，或者可以提供附加的结构组分以进一步促进药物D从偶联物的其余部分释放(例如，自消解基团，如对氨基苄基(PAB)组分)。
在一些实施方案中，连接Y和药物D单元之间的M间隔基可以选自：
且优选为
其中左侧波浪线表示与Y连接，右侧波浪线表示与药物D单元连接。
在另一些实施方案中，M为共价键，式I中的Y和药物D之间直接连接，如通过酰胺键连接。
在一个优选实施方案中，连接体L中的Y-M单元包含以下结构：
且优选地，所述Y-M单元与如下Z单元连接：
示例性连接体L
在一些优选的实施方案中，本发明式(I)中的L为包含如下结构的连接体：
其中，R3和R4各自独立地选自甲基、乙基、丙基。
在一些更优选的实施方案中，R3和R4均为甲基；或R3和R4均为乙基；或R3和R4均为丙基。
在一些实施方案中，本发明式I的L-D单元通过与Ab的轻链和/或重链的自由半胱氨酸的巯基形成硫醚键，从而与抗体连接。
示例性ADC偶联物
在一些实施方案中，本发明提供了具有以下结构的ADC偶联物或其可药用盐或溶剂化物：

其中，q表示1-20的平均DAR值，例如大约2、3、4、6和8的平均DAR值。
在一些实施方案中，根据本发明的ADC具有至少一项或多项以下优点：
-相对于ADC的血液循环中分布，显著地促进ADC向肿瘤环境的分布和富集；
-有效地限制了有效载荷在血浆中的过早释放，具有较高的血液循环稳定性，
-能在肿瘤环境中提供有效的毒性药物活性形式释放；
-赋予高效的动物体内肿瘤杀伤效果；和
-良好的动物耐受性。
在再一些实施方案中，根据本发明的ADC还可以具有至少一项或多项以下优点：
-毒素-连接体的加入不诱导聚集，并可以实现高的载药量，DAR可以高达8；和
-可接受的PK特性。
II.抗体-药物偶联物制备
抗体-药物偶联物的生成可以通过本领域的技术人员已知的任何技术来完成。在一些方面，通过与抗体的氨基酸残基反应来完成药物-连接体与抗体的偶联。在一些实施方案中，应用带离去基团的杂芳基连接体L，将药物D偶联到抗体的半胱氨酸残基上，以制备本发明式I的偶联物。在一些实施方案中，可以通过控制以还原剂例如三(2-羟乙基)膦(TCEP)处理抗体的条件，破坏抗体的链间二硫键并暴露自由巯基用于与杂芳基连接体-药物偶联。对于IgG1型抗体，至多可以还原四个链接二硫键，从而生成至多8个反应性巯基基团用于偶联。通过此方法制备的偶联物可以在每个抗体分子中含有零个、一个、两个、三个、四个、五个、六个、七个或八个药物。
在制备的偶联物是具有不同药物偶联位点和/或个数的偶联物的组合物时，偶联物的载药量由平均DAR表示，其为平均每抗体的药物分子数。可以通过常规手段，例如质谱、ELISA测定和HPLC，表征制备产生的抗体-药物偶联物组合物的平均每抗体的药物数。在另一些实施方案中，也可以确定抗体-药物偶联物以q表示的定量分布。可以通过如反相HPLC或电泳等手段实现其中q为某一值的同质抗体-药物偶联物与具有其它载药量的抗体-药物偶联物的分离、纯化和表征。
因此，在本发明的一个方面，q表示偶联在一个单独的抗体(Ab)上的药物-连接体(L-D)部分的数目，并且优选为1至16、1至12、1至10、或1至8的整数。在此情况下，单独的ADC偶联物也可称为ADC化合物。在任何本文的实施方式中，在根据本发明的ADC化合物上，可以有1、2、3、4、5、6、7、8、9、10、11、12、13、14、15或16个药物连接体部分偶联到一个单独的抗体上。
在本发明的另一个方面，q表示制备的偶联物组合物的平均DAR。在此情况下中，q可以为在1至约16、1至约12、1至约10、或1至约8、2至约16、2至约12、2至约10、或2至约8的范围内的整数或小数。在一些方面，q表示约3的平均DAR。在一些方面，q表示约6的平均DAR。在一些方面，q表示约8的平均DAR。
III.药物组合物
在一些实施方案中，本发明提供包含本文所述的任何ADC或其可药用盐或溶剂化物的组合物，优选地组合物为药物组合物或药物制剂。在一个实施方案中，所述组合物还包含药用辅料。在一个实施方案中，组合物，例如，药物组合物，包含本发明的ADC或其可药用盐或溶剂化物以及一种或多种其它治疗剂的组合。
本发明还包括包含本发明的ADC或其可药用盐或溶剂化物的组合物(包括药物组合物)。这些组合物还可以包含合适的药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。
如本文所用，“药用载体”包括生理上相容的任何和全部溶剂、分散介质、等渗剂和吸收延迟剂等。
对于药用辅料的使用及其用途，亦参见“Handbook of Pharmaceutical Excipients”,第八版,R.C.Rowe,P.J.Seskey和S.C.Owen,Pharmaceutical Press,London,Chicago。
本发明的组合物可以处于多种形式。这些形式例如包括液体、半固体和固体剂型，如液态溶液剂(例如，可注射用溶液剂和可输注溶液剂)、散剂或混悬剂、脂质体剂和栓剂。优选的形式取决于预期的施用模式和治疗用途。
可以通过将具有所需纯度的本发明的ADC或其可药用盐或溶剂化物与一种或多种任选的药用辅料混合来制备包含本文所述的ADC的药物，优选地以冻干制剂或水溶液的形式。
本发明的药物组合物或制剂还可以包含超过一种活性成分，所述活性成分是被治疗的特定适应证所需的，优选具有不会不利地彼此影响的互补活性的那些活性成分。例如，理想的是还提供其它治疗剂，包括化疗剂、血管生成抑制剂、细胞因子、细胞毒性剂、其它抗体、小分子药物或免疫调节剂(例如免疫检查点抑制剂或激动剂)等。所述活性成分以对于目的用途有效的量合适地组合存在。
可制备持续释放制剂。持续释放制剂的合适实例包括含有抗体的固体疏水聚合物的半渗透基质，所述基质呈成形物品，例如薄膜或微囊形式。
IV.药物组合和药盒
在一些实施方案中，本发明还提供了药物组合或药物组合产品，其包含本发明的或其可药用盐或溶剂化物以及一种或多种其它治疗剂。
本发明的另一个目的是提供一种成套药盒，其包含本发明的药物组合，优选地所述药盒为药物剂量单元形式。由此可以依据给药方案或药物施用间隔提供剂量单元。
在一个实施方案中，本发明的成套药盒在同一包装内包含：
-含有包含本发明的ADC或其可药用盐或溶剂化物的药物组合物的第一容器；
-含有包含其它治疗剂的药物组合物的第二容器。
根据待治疗的疾病以及个体状况等因素，本领域技术人员可以确定组合成品之各组分的施用方式和施用顺序。本发明的组合产品可用于本发明的治疗方法中。在一些实施方案中，本发明提供组合产品，其中所述其它治疗剂为例如有效刺激免疫反应从而进一步增强、刺激或上调受试者的免疫反应的治疗剂如抗体。在一些实施方案中，所述组合产品用于预防或治疗TF阳性肿瘤。
本发明的抗TF抗体
通过筛选具有序列独特性和高抗原结合特异性的抗体序列；并进一步通过对所获抗体的性质，包括亲和力、内吞活性、信号通路阻断、凝血影响、稳定性、药代动力学等进行评价，本发明人鉴定了具有优良性质的根据本发明的人源化抗TF单克隆抗体。本发明的抗体不仅对TF阳性肿瘤细胞展示出高结合亲和力和高特异性，能够快速有效地被肿瘤细胞内吞，且对TF介导的凝血影响小，因此不仅适宜单独或与其他抗癌药物组合用于癌症治疗、也适宜作为分子组件用于形成靶向癌症组织的新抗癌分子。
因此，在一些方面，本发明也提供了具有多种优良性质的人源化抗TF单克隆抗体、其编码核酸、包含所述核酸的载体和宿主细胞、以及包含所述抗体的免疫缀合物、多特异性抗体、药物组合物和用途。
I.本发明抗体及其性质
以下就本发明的抗TF抗体及其性质进行详述描述。如本领域技术人员可以理解，除非上下文有明确相反指示，否则在此部分中述及的任何抗体技术特征(包括，结构特征和性质特征)及其任何组合，也同样适用于作为本发明ADC偶联物的Ab单元的本发明抗体。
本发明的抗TF抗体
本发明提供特异性地结合TF，优选人TF蛋白质的抗体或其抗原结合片段，尤其是人源化抗体或其抗原结合片段。
在一些实施方案中，本发明提供抗TF抗体或其抗原结合片段，其包含：
(i)如SEQ ID NO:1所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:2所示的轻链可变区的LCDR1、2和3序列，或者
(ii)如SEQ ID NO:3所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:4所示的轻链可变区的LCDR1、2和3序列，或者
(iii)如SEQ ID NO:5所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:6所示的轻链可变区的LCDR1、2和3序列，或者
(iv)如SEQ ID NO:7所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:8所示的轻链可变区的LCDR1、2和3序列，或者
(v)(i)-(iv)任一项的CDR组合的变体，优选地，所述变体在6个CDR区上共包含1-10个氨基酸改变(包括但不限于，插入、缺失和/或取代,优选氨基酸取代，优选保守取代)，且优选地重链CDR3保持不变。
优选地，上述CDR根据Kabat或IMGT或其组合进行定义，更优选地，根据IMGT定义。但也可以采用本领域已知的任何其他方式定义所述的CDR。
在一些实施方案中，根据本发明的抗TF抗体或其抗原结合片段包含重链可变区的3个互补决定区HCDR，以及轻链可变区的3个互补决定区LCDR，其中：
(i)根据Chothia定义，HCDR1包含SEQ ID NO:9的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:10的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:11的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:21的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:22的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:23的氨基酸序列或由其组成；或者
(ii)根据AbM定义，HCDR1包含SEQ ID NO:12的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:13的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:14的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:24的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:25的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:26的氨基酸序列或由其组成；或者
(iii)根据Kabat定义，HCDR1包含SEQ ID NO:15的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:16的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:17的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:27的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:28的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:29的氨基酸序列或由其组成；或者
(iv)根据IMGT定义，HCDR1包含SEQ ID NO:18的氨基酸序列或由其组成，HCDR2包含SEQ ID  NO:19的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:20的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:30的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:31的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:32的氨基酸序列或由其组成。
在一些实施方案中，根据本发明的抗TF抗体或其抗原结合片段包含重链可变区的3个互补决定区HCDR，以及轻链可变区的3个互补决定区LCDR，其中：
(i)根据Chothia定义，HCDR1包含SEQ ID NO:33的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:34的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:35的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:45的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:46的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:47的氨基酸序列或由其组成；或者
(ii)根据AbM定义，HCDR1包含SEQ ID NO:36的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:37的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:38的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:48的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:49的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:50的氨基酸序列或由其组成；或者
(iii)根据Kabat定义，HCDR1包含SEQ ID NO:39的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:40的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:41的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:51的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:52的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:53的氨基酸序列或由其组成；或者
(iv)根据IMGT定义，HCDR1包含SEQ ID NO:42的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:43的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:44的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:54的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:55的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:56的氨基酸序列或由其组成。
在一些实施方案中，根据本发明的抗TF抗体或其抗原结合片段包含重链可变区的3个互补决定区HCDR，以及轻链可变区的3个互补决定区LCDR，其中：
(i)根据Chothia定义，HCDR1包含SEQ ID NO:57的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:58的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:59的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:69的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:70的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:71的氨基酸序列或由其组成；或者
(ii)根据AbM定义，HCDR1包含SEQ ID NO:60的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:61的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:62的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:72的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:73的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:74的氨基酸序列或由其组成；或者
(iii)根据Kabat定义，HCDR1包含SEQ ID NO:63的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:64的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:65的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:75的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:76的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:77的氨基酸序列或由其组成；或者
(iv)根据IMGT定义，HCDR1包含SEQ ID NO:66的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:67的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:68的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:78的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:79的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:80的氨基酸序列或由其组成。
在一些实施方案中，根据本发明的抗TF抗体或其抗原结合片段包含重链可变区的3个互补决定区HCDR，以及轻链可变区的3个互补决定区LCDR，其中：
(i)根据Chothia定义，HCDR1包含SEQ ID NO:81的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:82的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:83的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:93的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:94的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:95的氨基酸序列或由其组成；或者
(ii)根据AbM定义，HCDR1包含SEQ ID NO:84的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:85的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:86的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:96的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:97的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:98的氨基酸序列或由其组成；或者
(iii)根据Kabat定义，HCDR1包含SEQ ID NO:87的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:88的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:89的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:99的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:100的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:101的氨基酸序列或由其组成；或者
(iv)根据IMGT定义，HCDR1包含SEQ ID NO:90的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:91的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:92的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:102的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:103的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:104的氨基酸序列或由其组成。
在一些实施方案中，根据本发明的抗TF抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：所述重链可变区包含：
(i)SEQ ID NO:1所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
(ii)SEQ ID NO:3所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
(iii)SEQ ID NO:5所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，
(iv)SEQ ID NO:7所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
在一些实施方案中，根据本发明的抗TF抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：所述述轻链可变区包含：
(i)SEQ ID NO:2所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
(ii)SEQ ID NO:4所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
(iii)SEQ ID NO:6所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，
(iv)SEQ ID NO:8所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
在一些实施方案中，根据本发明的抗TF抗体或其抗原结合片段包含重链可变区和轻链可变区，其中： 所述重链可变区包含：
(i)SEQ ID NO:1所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
(ii)SEQ ID NO:3所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，
和/或其中所述轻链可变区包含：
(i)SEQ ID NO:2所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
(ii)SEQ ID NO:4所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
在一些实施方案中，根据本发明的抗TF抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：所述重链可变区包含：
(i)SEQ ID NO:5所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
(ii)SEQ ID NO:7所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，
和/或其中所述轻链可变区包含：
(i)SEQ ID NO:6所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
(ii)SEQ ID NO:8所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
在一些优选的实施方案中，根据本发明的抗TF抗体或其抗原结合片段包含重链可变区和轻链可变区，其中所述重链可变区包含SEQ ID NO:1所述的氨基酸序列或与其具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列，且其中所述轻链可变区包含SEQ ID NO:2所示的氨基酸序列、或与其具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列。优选地，所述抗体或抗原结合片段包含SEQ ID No:1的重链可变区和SEQ ID No:2的轻链可变区。
在一些优选的实施方案中，根据本发明的抗TF抗体或其抗原结合片段包含重链可变区和轻链可变区，其中所述重链可变区包含SEQ ID NO:3所述的氨基酸序列或与其具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列，且其中所述轻链可变区包含SEQ ID NO:4所示的氨基酸序列、或与其具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列。优选地，所述抗体或抗原结合片段包含SEQ ID No:3的重链可变区和SEQ ID No:4的轻链可变区。
在一些优选的实施方案中，根据本发明的抗TF抗体或其抗原结合片段包含重链可变区和轻链可变区，其中所述重链可变区包含SEQ ID NO:5所述的氨基酸序列或与其具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列，且其中所述轻链可变区包含SEQ ID NO:6所示的氨基酸序列、或与其具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列。优选地，所述抗体或抗原结合片段包含SEQ ID No:5的重链可变区和SEQ ID No:6的轻链可变区。
在一些优选的实施方案中，根据本发明的抗TF抗体或其抗原结合片段包含重链可变区和轻链可变区，其中所述重链可变区包含SEQ ID NO:7所述的氨基酸序列或与其具有至少95％、96％、97％、98％或99％序列同一性的氨基酸序列，且其中所述轻链可变区包含SEQ ID NO:8所示的氨基酸序列、或与其具有至少 95％、96％、97％、98％或99％序列同一性的氨基酸序列。优选地，所述抗体或抗原结合片段包含SEQ ID No:7的重链可变区和SEQ ID No:8的轻链可变区。
在一些实施方案中，根据本发明的抗TF抗体或其抗原结合片段包含本发明示例性抗体2B12B10-hz1、30G11B7-hz1、22F11H5-hz1和27H8H3-hz1之任一的重链和轻链可变区序列或其变体，例如，具有与所述示例性抗体之一相同的CDR序列，并具有相同或不同的构架区序列的抗体。优选地，所述抗体为人源化抗体。更优选地，所述抗体的重链可变区具有来源于人种系的构架区序列。
根据本发明的抗体可以包含重链恒定区和/或轻链恒定区。优选地，所述重链恒定区为来源于人免疫球蛋白的重链恒定区。优选地，所述轻链恒定区为来源于人免疫球蛋白的轻链恒定区。包含在本发明抗体中的重链恒定区可以为任何同种型或亚型，例如IgG1,IgG2，IgG3或IgG4同种型的重链恒定区。在一些实施方案中，因此，本发明提供了抗TF抗体，其包含IgG1,IgG2，IgG3或IgG4重链恒定区，优选地IgG1重链恒定区，尤其是人IgG1重链恒定区，例如SEQ ID NO:106的氨基酸序列或与其具有至少90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。包含在本发明抗体中的轻链恒定区可以为κ轻链恒定区或λ轻链恒定区。在一些实施方案中，因此，本发明提供了包含κ轻链恒定区或λ轻链恒定区的抗TF抗体，优选地本发明抗体包含人κ轻链恒定区，例如，SEQ ID NO:105的氨基酸序列或与其具有至少90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。在一些实施方案中，本发明抗TF抗体或其片段的重链和/或轻链还包含信号肽序列。
在一些方面，本发明也提供在本文中所述及的根据本发明的任何抗体的变体，尤其是本发明示例性抗体2B12B10-hz1、30G11B7-hz1、22F11H5-hz1和27H8H3-hz1之任一的变体。
在本文中，与抗体相关的术语“变体”指，与给定的亲本抗体相比，包含已经通过至少1个，例如1-30，或1-20或1-10个，例如1或2或3或4或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的目标抗体区域的抗体，其中变体基本上保持亲本抗体分子的至少一个生物学特性(例如，抗原结合能力)。目标抗体区域可以是抗体全长、或重链可变区或轻链可变区或其组合、或(一个或多个)重链CDR区或(一个或多个)轻链CDR区或其组合。本发明的具体示例性抗体的此类变体，在本发明的考虑中。
在一些实施方案中，本发明的抗体变体优选保持引入改变或修饰前的亲本抗体的至少60％,70％,80％,90％,或100％的生物学活性(例如，基本相同的抗原结合能力和/或受体内吞活性，或对凝血的影响程度基本相同)。可以理解的，根据本发明的抗体的重链可变区或轻链可变区或各个CDR区或构架区，均可以单独改变或组合改变，以产生本发明的抗体变体。此外，也可以对根据本发明的抗体的Fc区进行改变。Fc区的改变可以单独进行，或与构架区和/或CDR区和/或重链可变区和/或轻链可变区上的改变相组合。可以根据抗体分子的具体应用，选择性改变Fc区，以例如赋予抗体的一种或多种改变的效应子功能，例如改变的血清半衰期，补体固定、Fc受体结合、和/或抗原依赖性细胞毒性。此外，还可以对本发明抗体进行化学修饰(例如，与PEG连接)或改变其糖基化模式。
在一些实施方案中，相对于本发明示例性抗体之任一，根据本发明的抗体变体在重链序列和/或轻链序列上具有氨基酸改变。优选地，所述变体在重链序列或轻链序列或两者上，与对应的抗体相比，包含至少一个，两个或三个，但不超过20个，10个或5个氨基酸改变的氨基酸序列，或具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列。优选地，氨基酸改变不发生在CDR区，更优选地不发生在可变区。在一些实施方案中，包含在抗体变体中的至少一些或全部氨基酸改变为保守氨基酸取代。“保守性取代”是指导致某个氨基酸置换为化学上相似的氨基酸的氨基酸改变。可以通过本领域已知的标准方法，例如定点诱变和PCR介导的诱变，将氨基酸修饰如取代引入本发明的抗体中，以实现保守性取代。
在再一些方面，本发明也提供，相对于前述根据本发明的任何给定抗体(尤其是本发明示例性抗体 2B12B10-hz1、30G11B7-hz1、22F11H5-hz1和27H8H3-hz1之任一)，具有以下一个或多个特性的抗组织因子(TF)抗体或其抗原结合片段：(i)与根据本发明的给定抗体结合相同或重叠的表位；(ii)与根据本发明的给定抗体竞争结合人组织因子TF；(iii)抑制(例如，竞争性抑制)根据本发明的给定抗体与细胞表面表达人组织因子的细胞的结合；且优选地，与根据本发明的给定抗体显示相同或相似的结合亲和力和/或特异性。
在本文中，与给定抗体“竞争结合TF抗原”的抗体是指这样的抗体，所述抗体在竞争检验中阻断给定抗体与抗原TF结合的50％或更多，并且反过来，给定抗体在竞争检验中也阻断该抗体与抗原TF结合的50％或更多。示例性竞争检验描述于：“Antibodies”,Harlow and Lane(Cold Spring Harbor Press,Cold Spring Harbor,NY)。竞争结合的抗体可以与给定抗体结合相同的表位区，例如相同表位、相邻表位或重叠表位。
在本文中，抑制(例如竞争性抑制)给定抗体与其抗原的结合的抗体是指这样的抗体，其抑制50％、60％、70％、80％、90％或95％以上的所述给定抗体与其抗原的结合；并且反过来，所述给定抗体也抑制50％、60％、70％、80％、90％或95％以上的该抗体与其抗原的结合。抗体与其抗原的结合可以使用结合亲和力衡量。测定亲和力的方法是本领域已知的。
在本文中，与给定抗体显示相同或相似的结合亲和力和/或特异性的抗体是指这样的抗体，其能够具有给定抗体的至少50％、60％、70％、80％、90％或95％以上的结合亲和力和/或特异性。这可以通过本领域已知的任何测定结合亲和力和/或特异性的方法进行测定。
在一些实施方案中，根据本发明的抗体是IgG1抗体，更特别地IgG1κ或IgG1λ同种型。再优选地，根据本发明的IgG1抗体具有人IgG1恒定区，更优选地具有人IgG1κ同种型。
本发明抗TF抗体的特性
本发明抗TF抗体具有以下一个或多个特性：
(i)以高亲和力与人TF胞外域结合；
(ii)以高亲和力与TF阳性肿瘤细胞结合；
(iii)具有与食蟹猴TF的交叉反应性；
(iv)具有TF受体介导的内吞活性；
(v)阻断TF阳性细胞中由TF/FVIIa复合物介导的下游信号通路；和
(vi)基本不影响由细胞表面TF起始的凝血或具有降低的凝血影响。
在一些方面，本发明的抗体对TF具有高结合亲和力和高结合特异性。可以通过FACS或ELSA测定(例如实施例中所述的测定)，确定本发明抗体对TF胞外域和/或对TF阳性肿瘤细胞的结合EC50值。或者，可以通过生物膜层干涉技术测定抗体的结合动力学参数，以表征抗体的结合亲和力和特异性。在一些实施方案中，通过生物膜层干涉技术(BIL)测定，本发明抗TF抗体具有小于10-7M的KD值，例如大约10- 8M或更小的KD值，优选地，小于5×10-8M。在另一些实施方案中，通过生物膜层干涉技术(BIL)测定，本发明抗体具有大约10-1至10-3s-1的Kd值，例如大约10-2或大约10-3s-1的Kd值。优选地，采用实施例描述的ForteBio测定方法，确定抗体的结合动力学参数，例如KD值和Kd值；并任选地，与在同一测定中的参照抗体进行比较。在一些实施方案中，抗体与参照抗体相比具有相同或相似，或更小的结合动力学KD值。在另一些实施方案中，抗体与参照抗体相比具有相对较快的解离速率(Kd值)。之前的研究已经显示，在一些情况下，具有较快的解离速率的抗体可以更为有效地浸润肿瘤，以利于抗体在实体肿瘤中的分布。(参见，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)
在一些实施方案中，本发明抗TF抗体与TF阳性肿瘤细胞强结合。可以通过细胞ELISA或流式细胞术测定，测定抗体的细胞结合EC50值(即，细胞结合的半最大有效浓度)和/或最大结合量，并与参照抗体进行比较，来反映抗体的细胞结合亲和力。在一些实施方案中，在至少一种TF阳性肿瘤细胞，例如NIC-H358细胞或KYSE50细胞上，本发明抗体的EC50值为参照抗体的EC50值的大约50％至大约160％，例如大约80％、大约90％、大约100％、大约110％、大约120％或大约130％。
在一些实施方案中，本发明抗TF抗体表现出对人和猴TF的交叉反应性。在一些实施方案中，本发明抗体与人TF结合的KD值，与所述抗体与猴TF结合的KD值大约相当，例如，两者之KD比值在1-10之间，例如1-5之间，更优选地大约1-3之间。在再一些实施方案中，所述抗体与人TF结合的Kd值，与所述抗体与猴TF结合的Kd值大约相当，例如，两者的Kd比值在1-10之间，例如1-5之间，更优选地大约1-3之间。
在一些实施方案中，本发明抗体具有TF受体介导的内吞活性。可以在基于细胞的测定试验中，如在实施例所述的测定试验中，进行抗体的内吞活性评价。在一些实施方案中，在基于TF阳性细胞的测定试验中，将待测抗体与TF阳性细胞(尤其是TF阳性肿瘤细胞)在37℃孵育一段时间(例如,2小时或4小时)后，相对于保持在4℃相同时间的阴性对照，通过流式细胞术，荧光检测细胞表面结合的TF抗体量的变化，以确定抗体的内吞率。在一些实施方案中，与参照抗体相比，本发明抗体具有基本相当或更优的内吞率。在另一些实施方案中，在等同的测定条件下，本发明抗体的内吞率是参考抗体的内吞率的大约50％-200％或更高，例如，大约70％或以上，例如大约80％或以上，优选地大约100％或以上,或大约110％或以上，更优选地大约120％或以上，例如大约130％或以上，大约150％或以上，或大约200％或以上。在再一些实施方案中，与参照抗体相比，本发明抗体表现出更快速的内吞，例如，在更短的时间达到大约30％、40％或50％内吞。在再一些实施方案中，在如实施例中所述的内吞活性评价测定试验中，本发明抗体的内吞率为，例如，在37℃2小时孵育后，至少10％，例如，15-20％，优选地，至少25％，例如20-35％，再优选地35％以上，例如大约40％或45％或以上。
在一些实施方案中，本发明抗体基本上不影响凝血，或具有降低的凝血影响。可以通过基于细胞的体外凝血测定试验，检测在Ca2+离子和人血浆存在下，抗体对TF阳性细胞起始的凝血的干扰作用，确定抗体对凝血的影响。在一些实施方案中，相比于无抗体的阴性对照，本发明抗体基本不影响由细胞表面TF起始的凝血；或相比于参照抗体，具有降低的凝血影响。可以相对于无抗体的阴性对照和/或参照抗体，检测抗体引起的凝血时间的变化，由此反映抗体对凝血的影响。优选地，按照实施例所述凝血测定试验，检测在包含TF阳性细胞以及高或低浓度的游离Ca2+离子和待测抗体的溶液中加入人血浆后，405nm吸光度随时间的变化，估计达半最大OD405吸光值所需的凝血时间(V50)，以表征抗体对TF介导的凝血过程的影响。用于检测的Ca2+离子浓度可以为选自1-5mM的一个或多个浓度，例如，2mM和5mM。用于检测的抗体浓度可以为选自10-50μg/ml的一个或多个浓度，例如，20μg/ml和50μg/ml。在一些实施方案中，在5mM Ca2+和20μg/ml抗体存在下测定抗体对凝血的影响。在另一些实施方案中，在2mM Ca2+和50μg/ml抗体存在下测定抗体对凝血的影响。在一些实施方案中，如果在至少一个测定条件下，例如在5mM Ca2+和20ug/ml抗体存在下，相对于无抗体的阴性对照，抗体引起的凝血时间(V50)延迟不超过50秒，优选地不超过40秒、不超过30秒、不超过20秒、10秒、或5秒，则可以认为所述抗体基本不影响凝血，或具有降低的凝血影响。在另一些实施方案中，如果在至少一个测定条件下，例如在5mM Ca2+和20ug/ml抗体存在下，相对于参照抗体，抗体不引起凝血时间(V50)延迟，或优选地，凝血时间(V50)减少大约10％或以上，或更优选地大约20％或30％或40％或以上，再优选大约50％或60％或以上，则可以认为所述抗体基本不影响凝血或具有降低的凝血影响。在再一些实施方案中，如果在至少一个测定条件下，例如在2mM Ca2+和50ug/ml抗体存在下，相对于参照抗体，抗体不引起凝血时间(V50)延迟，或优选地，凝血时间(V50)减少大约10％、20％、或30％或以上，则可以认为所述抗体基本不影响凝血，或具有降低的凝血影响。
在再一些实施方案中，本发明抗体具有阻断TF阳性肿瘤细胞中由TF/FVIIa复合物介导的下游信号通路的能力。可以通过检测由该通路激活导致的IL-8释放水平，确定抗TF抗体对该TF下游信号通路的阻断活性。在一些实施方案中，按照实施例所述的信号通路阻断活性评价试验，本发明的抗体对TF信号通路显示抑制作用。在一些实施方案中，本发明抗体的TF信号通路阻断活性IC50值小于500ng/ml或150ng/ml，且最大抑制率达到90％以上，优选地95％以上。在一些实施方案中，本发明抗体的TF信号通路阻断活性IC50值，与参照抗体相当(±10％)或更小。
在再一些实施方案中，本发明的抗TF抗体或其抗原结合片段还具有以下一个或多个特性：(vii)良好的生产性能；(viii)良好的稳定性；和(ix)有利的药代动力学性质。
在一些实施方案中，在采用蛋白A亲和层析从重组哺乳动物细胞产生的抗体产物中一步纯化后，根据SEC-HPLC测定，纯化的本发明抗体可以达到90％以上的纯度；且在HIC测定中，表现出良好的亲水性。
在再一些实施方案中，本发明抗体具有良好的稳定性，包括热稳定性和反复冻融稳定性。在一些实施方案中，按实施例的加速稳定性测定，在37℃放置3-14天后，本发明抗体的SEC-HPLC纯度(即，SEC-HPCL测定的抗体单体百分数)均保持在90％以上或优选地95％以上，或降低不超过5％或优选地不超过3％；在另一些实施方案中，CE-SDS非还原纯度(即，主峰百分数)均保持在85％以上或优选地90％以上，CE-SDS还原纯度(即，LC+HC百分数)在90％或优选地95％以上，或所述非还原纯度和还原纯度的降低均不超过5％或4％。在再一些实施方案中，按实施例的反复冻融测定，在反复冻融4-8次后，本发明抗体的SEC纯度均保持在95％以上，或纯度降低不超过5％或优选地不超过3％。
在再一些实施方案中，本发明抗体具有血清稳定性。可以按实施例的血清稳定性测定试验，测定抗体在血清中37℃孵育一段时间后对TF抗原的亲和力变化，确定抗体的血清稳定性。如果所述亲和力的变化不显著，则可以认为抗体具有血清稳定性。在一些实施方案中，本发明抗体可以在人血清中保持至少14天，其抗原结合亲和力保留90％或以上。
II.多核苷酸、载体和宿主
再一方面，本发明提供编码以上任何抗TF抗体或其片段的核酸。还提供包含所述核酸的载体；和包含所述核酸或所述载体的宿主细胞。
在一些实施方案中，本发明提供包含如下多核苷酸的核酸分子，所述多核苷酸编码来自上文所述的结合TF的抗体的重链VH或轻链VL序列的至少一个CDR区和通常全部三个CDR区。在一些进一步的实施方案中，本发明提供包含编码上文所述的抗TF抗体的重链和/或轻链的完整或基本上完整可变区序列的多核苷酸的核酸分子。如本领域技术人员明了的，因为密码子简并性，每一个抗体或多肽氨基酸序列可以由多种核酸序列编码。
在一个实施方案中，本发明提供包含本发明核酸的一个或多个载体，包括克隆载体和表达载体。在一个实施方案中，载体是表达载体，例如真核表达载体。可用于本发明的载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。
在一个实施方案中，本发明提供包含本发明载体的宿主细胞。用于克隆或表达编码抗体的载体的适当宿主细胞包括原核或真核细胞。例如，抗体可在细菌中产生，特别当不需要糖基化和Fc效应子功能时。在细菌例如大肠杆菌中表达后，抗体可以从可溶级分中的细菌细胞糊状物分离，并且可以进一步纯化。在再一个实施方案中，宿主细胞是真核细胞。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞。有用的哺乳动物宿主细胞系的实例包括，用SV40转化的猴肾CV1系(COS-7)；人胚肾系(293HEK或293细胞)；中国仓鼠卵巢(CHO)细胞，包括DHFR-CHO细胞；以及骨髓瘤细胞系如Y0，NS0和Sp2/0。
III.抗体的制备
在再一方面，本发明提供了制备本发明抗TF抗体的方法。
在一个实施方案中，本发明的方法包括，在适合抗体表达的条件下，培养包含编码本发明抗体的核酸的宿主细胞，和任选地从所述宿主细胞(或宿主细胞培养基)回收所述抗体。为了重组产生抗TF抗体，可以将分离的或人工合成的或重组合成的编码抗体(例如上文所描述的抗体)的核酸，插入一个或多个载体，用于在宿主细胞中进一步克隆和/或表达。
在再一个实施方案中，本发明体也提供具有以下一个或多个特性的抗TF抗体或其抗原结合片段、及其制备方法：
(i)与根据本发明的任一抗体结合相同或重叠的表位；
(ii)与根据本发明的任一抗体竞争结合人TF；
(iii)抑制(例如，竞争性抑制)根据本发明的任一抗体与细胞表面表达人TF的细胞的结合；
其中，所述根据本发明的抗体尤其是抗体2B12B10-hz1、30G11B7-hz1、22F11H5-hz1或27H8H3-hz1，
优选地，所述制备方法包括：利用根据本发明的抗体作为参照，在结合亲和力测定试验中，从抗体混合物中筛选具有上述特性的抗体。在此可以使用的抗体混合物包括，但不限于，来自TF免疫动物的抗血清、或为酵母或哺乳动物展示抗体文库。
IV.测定法
可以通过本领域中已知的多种测定法对本文中提供的抗TF抗体进行鉴定，筛选，或表征其物理/化学特性和/或生物学活性。
可用于确定抗体与TF抗原的结合的适宜方法是本领域已知的。例如，可以使用本领域技术人员已知的常规技术，容易地确定抗体对包含TF胞外域的蛋白(例如融合蛋白)或TF阳性肿瘤细胞的亲和力。所述方法包括但不限于，采用ELISA的测定试验；使用BIAcore 2000仪器或ForteBio仪器的测定方法；或通过使用经放射性标记的靶抗原的放射免疫测定试验；或本申请实施例中描述的细胞ELISA或FACS技术或生物膜层干涉技术。在本文中，优选地，亲和力参数的测量按照或基本上按照实施例中描述的方式进行。
适用于测定抗体的内吞活性的方法是本领域已知的。例如，可以在基于细胞的测定中，尤其是基于TF阳性肿瘤细胞的测定中，确定抗体在与所述细胞表面结合后的内吞率。在本文中，优选地，抗体的内吞活性按照或基本上按照实施例中描述的方式进行。
适用于测定抗体对凝血的影响的方法是本领域已知的。例如，可以相对于对照(例如阴性对照或阳性对照)，测定在抗体存在时凝血参数例如凝血时间的变化，来反映抗体的凝血影响。在本文中，优选地，抗体对凝血的影响，按照或基本上按照实施例中描述的方式进行。
V.免疫缀合物和免疫融合物
再一方面，本发明提供通过将本发明抗体融合或缀合于异源分子而产生的免疫融合物或免疫缀合物。
在一个实施方案中，在免疫融合物中，本发明的抗体(或其抗原结合片段)与异源肽或多肽分子直接或通过氨基酸接头连接。可以提及的异源肽或多肽包括但不限于，赋予该融合另一功能活性的蛋白或多肽、或利于免疫融合物纯化或检测的标签肽。
在一个实施方案中，在免疫缀合物中，本发明的抗体(或其抗原结合片段)与治疗剂或诊断剂或可检测剂缀合。在一些实施方案中，本发明抗体可以以全长抗体或抗体片段的形式与异源分子缀合。在缀合物中，可以使用接头来共价连接缀合物的不同实体。适宜的接头包括化学接头或肽接头。有利地的是，接头是利于多肽在递送至靶位点后释放的“可裂解接头”。例如，可以使用酸不稳定性接头、肽酶敏感性接头、光不稳定性接头、二甲基接头或含二硫化物的接头。
在与治疗剂缀合的实施方案中，适用于缀合物的治疗剂包括但不限于细胞毒素(例如细胞生长抑制剂或细胞杀伤剂)，药物或放射性同位素。
在与诊断剂或可检测剂缀合的实施方案中，这类缀合物可以作为临床检验方法的部分(如确定特定疗法的效力)，用于监测或预测疾病或病症的发作、形成、进展和/或严重性。可以通过将抗体与可检测剂偶联实现这类诊断和检测，所述可检测剂包括但不限于多种酶，例如辣根过氧化物酶；辅基，例如链霉亲和素/生物素和抗生物素蛋白/生物素；荧光物质；发光物质；放射性物质；和用于各种正电子发射成像术中的正电子发射金属和非放射性顺磁金属离子。
VI.多特异性抗体
在再一方面，本发明提供特异性地结合TF的多特异性(包括双特异性)抗体分子。在一个实施方案 中，在多特异性抗体中，本发明的抗体(或其抗原结合片段)形成针对TF的第一结合特异性。在再一实施方案中，所述多特异性抗体还包含第二结合特异性、或还包含针对两种不同分子的第二和第三结合特异性。所述的第二、第三结合特异性例如可以针对肿瘤细胞表面上表达的另一抗原、或针对T细胞表面表达的抗原。
在此类多特异性分子中，结合特异性优选由抗体的“结合位点”或“抗原结合位点”(抗体分子中与抗原实际结合的区域)提供。更优选地，抗原结合位点由抗体轻链可变结构域(VL)和抗体重链可变结构域(VH)组成的VH/VL对构成。
VII.药物组合物和药物制剂
本发明还提供，包含抗TF抗体或其免疫缀合物/融合物或多特异性抗体的组合物(包括药物组合物或药物制剂)和包含编码抗TF抗体或其免疫缀合物/融合物或多特异性抗体的多核苷酸的组合物。这些组合物还可以任选地包含合适的药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。对于赋形剂的使用及其用途，可参见“Handbook of Pharmaceutical Excipients”,第五版,R.C.Rowe,P.J.Seskey和S.C.Owen,Pharmaceutical Press,London,Chicago。在一些实施方案中，可以通过将具有所需纯度的本发明的抗TF抗体、免疫缀合物或多特异性抗体，与一种或多种任选的药用辅料(Remington′s Pharmaceutical Sciences，第16版，Osol，A.编(1980))混合，来制备包含本发明的药物制剂。
在本发明的药物组合物和药物制剂中，本发明的抗体可以是唯一的活性剂，或可以与其它治疗剂联合。可以与本发明抗体联合的治疗剂包括但不限于对于待治疗的疾病和/或病症具有有益治疗功效的治疗剂。例如，所述活性成分可以是被治疗的特定适应症所需的，优选具有不会不利地影响彼此的互补活性的那些活性成分。例如，可以提供抗癌活性的其它药物成分。本发明抗体与活性成分以对于目的用途有效的量合适地组合存在于药物组合物和药物制剂中。
VIII.组合产品
在再一方面，本发明还提供了组合产品，其包含本发明的抗体或其抗原结合片段、多特异性抗体或免疫缀合物，以及一种或多种其它治疗剂(例如化疗剂、其他抗体、细胞毒性剂、抗肿瘤药等)。构成组合产品的各组分，例如本发明抗体与其他治疗剂，可以分别配制在不同的制剂中，并优选地容纳在不同的容器中。根据待治疗的疾病以及个体状况等因素，本领域技术人员可以确定组合成品之各组分的施用方式和施用顺序。本发明的组合产品可用于本发明的治疗方法中。在一些实施方案中，本发明提供组合产品，其中所述其它治疗剂为例如有效刺激免疫反应从而进一步增强、刺激或上调受试者的免疫反应的治疗剂如抗体。在一些实施方案中，所述组合产品用于预防或治疗TF阳性肿瘤。
IX.方法和用途
在一方面，本发明提供应用本发明TF抗体或其抗原结合片段的方法和用途，例如，体内和体外用于：
(1)靶向性结合TF阳性肿瘤细胞；和/或
(2)阻断TF阳性肿瘤细胞中由TF/VIIa介导的下游信号通路，以及
(3)抑制和/或杀伤TF阳性肿瘤细胞。
在一些实施方案中，本发明的方法和用途涉及在受试者个体的疾病治疗。在另一些实施方案中，本发明的方法和用途涉及在来自例如受试者的样品中检测TF的存在。在再一些实施方案中，本发明也提供本发明抗TF抗体或其抗原结合片段在制备用于上述用途的产品(例如药物组合物或药物产品或组合产品或检测产品)中的用途。
在一个方面，本发明提供了应用本发明抗体或抗原结合片段在受试者中预防和/或治疗TF阳性肿瘤的方法和用途，包括以预防和/或治疗有效量施用本发明的抗体或抗原结合片段。在另一方面，本发明也提供了应用本发明抗体或抗原结合片段在受试者中治疗或预防与细胞膜TF表达上升相关的其他疾病的方法和用途，包括向有需要的受试者施用本发明的抗TF抗体或其抗原结合片段。
在再一方面，本发明提供了检测样品中TF的方法和试剂盒，其中所述方法包括：(a)将所述样品与本 发明抗体或其抗原结合片段或免疫缀合物/融合物接触；和(b)检测所述抗体或其抗原结合片段或免疫缀合物和TF蛋白之间复合物的形成。在一些实施方案中，样品来自癌症患者。所述检测可以是体外的或体内的。
术语“检测”用于本文中时，包括定量或定性检测，示例性的检测方法可以涉及免疫组织化学、免疫细胞化学、流式细胞术(例如，FACS)、抗体分子复合的磁珠、ELISA测定法、PCR-技术(例如，RT-PCR)。在某些实施方案中，生物样品是血、血清或生物来源的其他液体样品。在某些实施方案中，生物样品包含细胞或组织。在一些实施方案中，生物样品来自过度增生性或癌性病灶。在某些实施方案中，待检测的TF是人TF。
在一个实施方案中，抗TF抗体被用于选择适合利用抗TF抗体治疗的受试者。在再一个实施方案中，可以使用本发明抗体诊断癌症或肿瘤，例如评价(例如监测)受试者中本文所述疾病(例如，过度增生性或癌性疾病)的治疗或进展、其诊断和/或分期。
在某些实施方案中，提供标记的抗TF抗体。标记包括但不限于，被直接检测的标记或部分(如荧光标记、发色团标记、电子致密标记、化学发光标记和放射性标记)，以及被间接检测的部分，如酶或配体，例如，通过酶促反应或分子相互作用。示例性标记包括但不限于，放射性同位素32P、14C、125I、3H和131I，荧光团如稀土螯合物或荧光素及其衍生物，罗丹明及其衍生物，丹酰(dansyl)，伞形酮(umbelliferone)，荧光素酶(luceriferase)，例如，萤火虫荧光素酶和细菌荧光素酶(美国专利号4,737,456)，荧光素，2，3-二氢酞嗪二酮，辣根过氧化物酶(HR)，碱性磷酸酶，β-半乳糖苷酶，葡糖淀粉酶，溶解酶，糖类氧化酶，例如，葡萄糖氧化酶，半乳糖氧化酶，和葡萄糖-6-磷酸脱氢酶，杂环氧化酶如尿酸酶和黄嘌呤氧化酶，以及利用过氧化氢氧化染料前体的酶如HR，乳过氧化物酶，或微过氧化物酶(microperoxidase)，生物素/亲和素，自旋标记，噬菌体标记，稳定的自由基，等等。
本发明的治疗方法和用途
基于本发明抗TF抗体或本发明ADC对于TF的良好靶向性以及上述优良性质，本发明也提供了本发明ADC和本发明抗体在TF相关疾病治疗和预防中的应用和方法。
TF在多种来源的癌组织细胞表面过表达，因此是适用于开发癌症免疫疗法的合适靶点。在一个方面，因此，本发明提供了，本发明ADC或其可药用盐或溶剂化物以及本发明抗体或其抗原结合片段用于在受试者中预防和/或治疗TF阳性肿瘤的应用。在所述应用中，本发明的抗体药物偶联物或其可药用盐或溶剂化物、或本发明的抗体或抗原结合片段可以作为唯一活性剂施用于受试者，或可以与其它疗法或治疗剂联合施用于受试者。所述的其它疗法和治疗剂包括，例如，靶向肿瘤细胞表面的抗原，通过结合和/或阻断这些分子而消灭肿瘤的药物；激活受试者的免疫系统，促使其自发消灭肿瘤的药物。
在再一个方面，本发明也提供了在受试者中预防或治疗TF阳性肿瘤的方法，包括向有需要的受试者施用本发明的ADC或其可药用盐或溶剂化物、或向有需要的受试者施用本发明的抗TF抗体或其抗原结合片段。
适用于本发明方法和应用的TF阳性肿瘤可以选自：骨髓瘤(例如多发性骨髓瘤)、急性成淋巴细胞白血病、慢性成淋巴细胞白血病、急性髓性白血病(AML)和非霍奇金淋巴瘤、中枢神经系统肿瘤、神经胶质瘤、脑癌、头颈癌，如头颈鳞状细胞癌、胃肠道癌症、泌尿生殖系统癌症、肺癌如NSCLC、食道癌、胃癌、肝胆癌、胰腺癌、结直肠癌、直肠癌、膀胱癌、肾癌、乳腺癌，尤其是三阴性乳腺癌、前列腺癌、子宫内膜癌、卵巢癌、宫颈癌、黑素瘤、肉瘤、和皮肤癌。
适用于本发明方法和应用的TF阳性肿瘤可以是处于早期、中期或晚期或是转移性的癌。此外，适用于本发明方法和应用的TF阳性肿瘤可以是先前接受过治疗而发生免疫逃逸的肿瘤。
在一些实施方案中，根据本发明方法治疗的TF阳性肿瘤选自：宫颈癌、胰腺癌、肺癌、前列腺癌、膀胱癌、卵巢癌、乳腺癌、结直肠癌、食道癌、头颈癌、胃癌，包括原发性或晚期或转移性癌症。在再一个实施方案中，所述TF阳性肿瘤是宫颈癌。在另一个实施方案中，所述TF阳性肿瘤是非小细胞肺癌。在再一实施方案中，所述TF阳性肿瘤是食道鳞状细胞癌。在再一实施方案中，所述TF阳性肿瘤是乳腺癌。
在一些实施方案中，根据本发明方法治疗的TF阳性肿瘤具有至少10％、20％、30％、40％、50％、60％、70％、75％、80％、85％、90％、95％、100％的TF阳性细胞。可以通过免疫组织化学，评估肿瘤活检物上的组织因子表达水平。已经在多种癌症活检物上检测到高的TF阳性细胞百分比，例如，宫颈癌(100％)、非小细胞肺癌(34-88％)、子宫内膜癌(14-100％)、前列腺癌(47-75％)、卵巢癌(75-100％)、食道癌(43-91％)和膀胱癌(78％)。
优选地，在一些实施方案中，根据本发明的方法用于治疗具有高百分比的TF阳性细胞的肿瘤，例如，具有至少25％、50％、75％或100％的TF阳性细胞的肿瘤，例如，具有范围在25-50％至75-100％的TF阳性细胞的肿瘤，例如，膀胱癌、肺癌、胰腺癌、前列腺癌、卵巢癌和宫颈癌。在一些实施方案中，本发明方法在所述癌症中的应用，诱导肿瘤消退。
在再一些实施方案中，根据本发明的方法也可以用于治疗TF阳性细胞百分比低于25％或20％的癌症。在一些实施方案中，本发明方法在所述癌症中的应用，导致肿瘤生长抑制。
在再一方面，本发明也提供了用于预防或治疗与细胞膜TF表达上升相关的其他疾病的方法，包括向有需要的受试者施用本发明的抗TF抗体或其抗原结合片段、或向有需要的受试者施用本发明的ADC或其可药用盐或溶剂化物。所述疾病包括但不限于，例如，良性肿瘤、神经纤维瘤、血管瘤；动脉粥样硬化；血管疾病，例如，视网膜病和黄斑变性；炎症疾病，例如类风湿性关节炎、骨关节炎、强直性脊柱炎；自身免疫炎症例如多发性硬化。
在本发明方法的上述任何一个实施方案中，根据本发明的抗体或其结合片段和根据本发明的ADC或其可药用盐或溶剂化物的施用，可以包括1)治疗性措施，该措施治愈、减缓、减轻经诊断的病理状况或疾患的症状及/或停止该经诊断的病理状况或疾患的进展；或2)预防性或防范性措施，该措施预防及/或减缓病理状况或疾患的发展。因此，在本发明的方法中，受试者可以是，已罹患疾患的个体、易于罹患疾患的个体，或欲预防疾患的个体。所述个体将受益于所述的治疗性措施或预防性措施，并相比于未接受所述处理的个体，表现出在疾病、病症、病状、和/或症状的发生、复发或发展上的减轻或改善。在一些实施方案中，本发明涉及疾病或病症的治疗；在另一些实施方案中，本发明涉及疾病或病症的预防。
根据本发明的抗体或其结合片段和根据本发明的ADC或其可药用盐或溶剂化物，以及任选地与之联用的其他治疗剂，可以通过任何合适的方法给药，包括肠胃外给药，肿瘤内给药和鼻内给药。肠胃外输注包括肌内、静脉内、动脉内、腹膜内或皮下给药。本文中涵盖各种用药时程，包括，但不限于，单次给药或在多个时间点多次给药、推注给药及脉冲输注。
为了预防或治疗疾病，根据本发明的抗体或其结合片段和根据本发明的ADC或其可药用盐或溶剂化物的合适剂量(当单独或与一种或多种其他的治疗剂组合使用时)，将取决于待治疗疾病的类型、所用药物的具体类型、疾病的严重性和进程、所述药物是以预防目的施用还是以治疗目的施用、以前的治疗、患者的临床病史和对所述抗体的应答，和主治医师的判断力。根据本发明的抗体或其结合片段和根据本发明的ADC或其可药用盐或溶剂化物，可以以一次治疗或经过一系列治疗施用于患者。
在一些实施方案中，在接受本发明方法治疗之前，所述受试者正在接受或已经接受过其它治疗，例如化疗治疗和/或放射疗法、或其他免疫疗法。
在上述本发明方法和应用的任何实施方案中，可以替代本发明抗体或其抗原结合部分或替代本发明的ADC或其可药用盐或溶剂化物，施用本发明的组合物、多特异性抗体或免疫缀合物/融合物、或组合产品。或者，在这些方法中，除了施用本发明抗体或其抗原结合部分或本发明的ADC或其可药用盐或溶剂化物，还可以进一步施用本发明的组合物、多特异性抗体或免疫缀合物/融合物、或组合产品。
在一些实施方案中，本发明也提供本发明的ADC或其可药用盐或溶剂化物在制备用于前述治疗和预防方法的药物中的用途。在另一些实施方案中，本发明也提供了，本发明抗体或抗原结合片段、组合物、免疫缀合物/融合物、多特异性抗体在制备用于前述治疗和预防方法的药物中的用途。
上文以及整个本申请中所描述的任何或所有特征可以在本发明的各种实施方案中组合。以下实施例进一步说明本发明，然而，应理解实施例以举例说明为目的，不应理解为构成任何限制。
实施例
实施例I抗TF人源化抗体的制备和表征
一般方法
细胞因子抗原重组表达
人组织因子的氨基酸序列参考Uniprot蛋白数据库中的P13726，其胞外段为aa 33-aa 251；食蟹猴组织因子的氨基酸序列参考Uniprot蛋白数据库中的A0A2K5VXA0，其胞外段为aa 33-aa 252。
以上胞外段氨基酸序列经由安徽通用公司进行密码子优化，合成于表达载体pTT5-His上。通过PEImax(Polysciences，24765-1)瞬时转染HEK293-EBNA细胞，表达7天后，通过镍柱纯化，获得人TF-his(h.TF-His)和食蟹猴TF-his(c.TF-His)抗原。该重组表达并纯化的TF抗原在后续实施例中用作免疫原或用于ELISA测定。
基于抗原蛋白的ELISA
用50mM碳酸盐包被缓冲液(CBS包被液)中的TF抗原(h.TF-His或c.TF-His抗原)，以包被浓度1ug/mL，体积100uL，4℃包被ELISA板过夜。第二天去除包被液，每孔用300uL PBS洗一次，每孔加入100uL在PBS中的2％BSA(Biotopped，A6020)，37℃封闭2小时。用在PBS中的2％BSA梯度稀释待测样品，建立11个浓度点。取梯度稀释的样品100uL加入板中，37℃孵育2小时；去除上清，每孔用300uL PBST洗三次，每孔加入100uL在2％BSA中的HRP缀合的二抗，37℃孵育1小时。对于来自杂交瘤的鼠源抗体，采用HRP标记的抗小鼠二抗(Jackson，115-035-003)；对于人源化抗体，采用HRP标记的抗人二抗(Jackson，109-035-088)。在孵育结束后，去除二抗，每孔用300uL PBST洗五次，随后每孔加入100uL TMB底物(湖州英创，TMB-S-004)，显色5-20分钟；每孔加入50uL 2N HCl终止反应，酶标仪(厂家：MD，型号：SpectraMax)读取OD450nM读数，导入到GraphPad软件进行拟合作图，确定样品对TF抗原蛋白的结合。
基于细胞的ELISA
实验前一天将TF抗原阳性细胞(MDA-MB-231或NCI-H358细胞)铺至96孔板中，每孔5×104细胞。实验当天去除培养上清，每孔用300uL PBS洗一次，每孔加入100uL 4％多聚甲醛室温固定20分钟。去除多聚甲醛，每孔用300uL PBS洗两次，每孔加入100uL在PBS中的2％BSA，37℃封闭2小时。封闭完成后，每孔加入30uL待测样品(杂交瘤上清或纯化抗体)，37℃孵育2小时。去除液体，每孔用300uL PBST洗三次；每孔加入100uL在2％BSA中的HRP缀合的二抗，37℃孵育1小时。在待测样品为杂交瘤或鼠源抗体的情况下，采用HRP标记的抗小鼠二抗(Jackson，115-035-003)；在待测样品为人源化抗体的情况下，采用HRP标记的抗人二抗(Jackson，109-035-088)。去除二抗，每孔用300uL PBST洗五次，随后每孔加入100uL TMB底物(湖州英创，TMB-S-004)，显色5-20分钟；每孔加入50uL 2N HCl终止反应，酶标仪(厂家：MD，型号：SpectraMax)读取OD450nm数值，确定样品对TF阳性细胞的结合。
SEC-HPLC检测抗体纯度
经重组表达和纯化的抗体，采用SEC-HPLC检测抗体纯度。检测方法如下进行：
仪器：Waters Alliance e2695HPLC；
色谱柱：Thermo MabPac SEC-1，5um，7.8*300mm；
流动相：61mmol/L Na2HPO4，39mmol/L NaH2PO4，200mmol/L NaCl，5％IPA；
仪器参数：样品室温度：8℃；柱温：30℃；流速：0.5ml/min；进样量：20ug；检测波长：280nm；等度运行：30min。
HIC检测抗体亲疏水性
经重组表达和纯化的抗体，采用HIC检测抗体亲疏水性。检测方法如下进行：
利用东曹公司的疏水色谱柱(TOSOH Tskgel Buty-NPR(2.5)，4.6*100)，在安捷伦HPLC设备上进行亲疏水性检测，流动相A为1.5M(NH4)2SO4，流动相B为25mM Na2HPO4(pH＝7.0)+25％IPA。仪器参数设置为样品室温度：8℃，柱温：30℃，流速：0.5mL/min，检测波长：280nm。将待测样品用流动相A稀释至终浓度1mg/mL，进样20uL进行梯度洗脱。洗脱梯度如下：
实施例1：抗人TF鼠源单克隆抗体的制备
选用Balb/C、昆明(KM)以及CD01 3个品系各3只5-6周龄雌性小鼠。采用人TF-his蛋白和食蟹猴TF-his蛋白进行免疫。免疫方案如表1所示。
表1.小鼠免疫方案

*:CFA:弗氏完全佐剂；IFA：弗氏不完全佐剂；CpG：CpG寡脱氧核苷酸佐剂。
在2次加强免疫过后，通过基于抗原蛋白的ELISA测定，按照一般方法中所述，采用分别包被h.TF-His和c.TF-His抗原的ELISA板，检测免疫动物的血清效价。基于检测结果，综合考虑与人和猴TF的亲和力，选取CD1免疫小鼠和Balb/c免疫小鼠用于杂交瘤融合，并在杂交瘤融合前，进行最后的加强免疫。
取小鼠脾脏、淋巴结制备细胞悬液，与SP2/0小鼠骨髓瘤细胞1：1比例进行混合，采用细胞电融合缓冲液进行重悬，使用BTX-ECM2001细胞电融合仪进行电融合反应。电融合反应后，用完全融合培养基(RPMI160+15％FBS+1×HAT)重悬，按每孔2万-2.5万个细胞分至96孔细胞培养板中，37℃、5％CO2培养。
培养7天后，先后通过使用包被人TF-his和食蟹猴TF-his的ELISA板，进行ELISA复筛；并采用TF阳性肿瘤细胞(MDA-MB-231或NCI-H358)进行细胞ELISA复筛。挑选出阳性母克隆用于亚克隆。
每个母克隆取75个细胞分至96孔板中，进行亚克隆培养。之后，取亚克隆上清，先后通过包被人TF-his、食蟹猴TF-his、大鼠TF-his蛋白及NCI-H358细胞，进行ELISA和细胞ELISA筛选。基于筛选结果，选取共119个阳性克隆，采用无血清培养基进行放大培养。收集培养的杂交瘤单克隆的上清，通过抗体纯化介质ProA(GE,Mabselect XL)填料进行纯化，获得鼠源抗体。
对纯化的鼠源抗体，先后通过包被人TF-his、食蟹猴TF-his蛋白及NCI-H358细胞，进行ELISA和细胞ELISA评价。实验方法如一般方法中所述，其中，鼠源抗体按照10ug/mL起始，3倍梯度稀释，建立11个浓度点。基于测定结果，选取亲和力高的13个杂交瘤单克隆，用于抗体序列确定和人源化。
实施例2抗TF鼠源单克隆抗体序列确定和人源化
收集约1×105个候选杂交瘤细胞，通过Trizol(Invitrogen，15596026)提取RNA，然后使用PrimeScript RT reagent试剂盒(TAKARA，RR047A)通过PolyA进行反转录获得cDNA。分别于重链、轻链上游设计上 游引物，并于重链CH1区域、轻链CL区域设计下游引物。通过PCR扩增产物后，采用琼脂糖胶回收试剂盒进行片段回收；送样北京擎科生物技术有限公司进行测序，获得鼠源抗体序列。
采用CDR grafting的方法，进行鼠源抗原序列的人源化。首先通过常规BLAST方法，找到同原始鼠源序列同源性最高的人germline序列，作为模板；将鼠源抗体的CDR移植到人源模板上，构建成嵌合体；根据结构分析鼠源抗体中能保留其原始构象的FR氨基酸，将嵌合体中相应的氨基酸回复突变为鼠源氨基酸以保持原有亲和力；将构建的人源化抗体进行计算及免疫原性分析，找到高免疫原性片段，进行低免疫原性片段的替换。获得17种人源化抗体的序列。
实施例3人源化抗体表达和纯化
将上述各人源化抗体以及参照抗体(Benchmark，简称BM，为Genmab公司的Tisotumab，序列来自于专利CN 103119065B的SEQ ID NO:5(VH)和SEQ ID NO:45(VL))的重、轻可变区氨基酸序列提交给通用生物进行基因合成，进行密码子优化后，构建到PTT5载体上。质粒合成好后，通过PEImax转染HEK293E细胞，表达7天左右，离心收集上清。上清采用MabSelectSure ProActive亲和填料(GE，货号17547401)进行纯化。表达的参照抗体与人源化抗体具有相同的人IgG1重链恒定区(SEQ ID NO:106)和人Kappa轻链恒定区(SEQ ID NO:105)。纯化好的抗体全部超滤到PBS缓冲液中，测定浓度，-20℃保存。
实施例4人源化抗体的ELISA亲和力评价
基于抗原蛋白的ELISA检测
如一般方法中所述进行所述检测。简言之，将分别包被有人TF-his蛋白或食蟹猴TF-his蛋白的ELISA板，采用2％BSA在37℃封闭2小时，加入梯度稀释的抗体(10ug/mL起始，3倍梯度稀释，11个浓度点)，37℃孵育2小时。之后，加入抗人二抗(Peroxidase-AffiniPure Goat Anti-Human IgG(H+L)，Jackson，109-035-088)，37℃孵育1小时，加入TMB底物(湖州英创，TMB-S-004)显色。终止显色反应后上机读取450nM吸光值并用GraphPad软件拟合曲线。
基于细胞的ELISA检测：
如一般方法中所述进行所述检测。简言之，胰酶消化收集TF抗原阳性细胞NCI-H358细胞，每孔5×104个细胞铺至96孔板中过夜，之后4％多聚甲醛固定细胞。去除多聚甲醛并用PBS洗涤细胞后，使用2％BSA在37℃封闭板2小时。加入梯度稀释的抗体(10ug/mL起始，3倍梯度稀释，11个浓度点)，37℃孵育2小时。之后，加入抗人二抗(Peroxidase-AffiniPure Goat Anti-Human IgG(H+L)，Jackson，109-035-088)，37℃孵育1小时，加入TMB底物(湖州英创，TMB-S-004)显色，终止显色反应后上机读取450nM吸光值并用GraphPad软件拟合曲线。
实验结果如表2以及图1所示。选取与人/食蟹猴TF-his以及TF阳性细胞显示出良好的结合活性的人源化抗体，2B12B10-hz1、4F6C3-hz1、30G11B7-hz1、22F11H5-hz1、27B9F2-hz1及27H8H3-hz1，进行后续评价。
表2.人源化抗体ELISA检测结果

实施例5人源化抗体质量鉴定
上述经重组表达和蛋白A亲和纯化的人源化抗体，按照一般方法中所述，通过SEC-HPLC鉴定抗体的纯度；并通过HIC检测抗体的亲疏水性。SEC及HIC检测结果如表3所示。SEC结果表明选取的6种人源化抗体纯度较高，均＞90％；HIC结果表明选取的6种人源化抗体与疏水色谱柱子结合较弱，保留时间较短，亲水性都较好。
表3.人源化抗体质量鉴定
实施例6人源化抗体的流式亲和力评价
胰酶消化离心收集TF中低表达细胞系NCI-H358(来源：南京科佰，CBP60136)及TF高表达细胞系KYSE520细胞(来源：南京科佰，CBP60658)，预冷PBS洗三次，1％BSA重悬细胞，每孔3×105个细胞铺至96孔尖底板中。加入梯度稀释的抗体，4℃孵育1小时，预冷PBS洗三次后，每孔加入1μL抗人荧光二抗(APC anti-human IgG Fc Antibody，Biolegend，410712)，4℃孵育0.5小时。预冷PBS洗三次后重悬细胞，流式细胞仪(Sony，LE-SA3800GA)检测，将数据导入GraphPad软件拟合曲线。
实验结果如表4和图2所示，6个候选人源化抗体与肿瘤细胞均有较强结合。
表4.人源化抗体流式检测结果
实施例7人源化抗体的动态亲和力评价
通过ForteBio测定人源化抗体的动态亲和力。实验步骤如下：取出ProA传感器(18-5010，Octet)，用PBST稀释液(PH7.4)将传感器预湿10min。将需要固定化的抗体稀释至5μg/ml；抗原h.TF-His和c.TF-His从75nM起始，2倍稀释，4个浓度点，设置0浓度点。设置程序，放入传感器板和样品板，开始程序，传感器用20mM甘氨酸溶液(pH 1.7)再生。使用Octet分析软件分析数据，导出结果图。实验结果如表5所示，6个候选人源化抗体与人、猴TF均有较高亲和力。
表5.人源化抗体动态亲和力检测结果
实施例8人源化抗体的内吞活性评价
通过流式细胞技术(FACS)，检测人源化单克隆抗体的内吞活性。实验步骤如下：胰酶消化离心收集NCI-H358细胞，用浓度为10μg/mL的待检测抗体4℃孵育1小时。PBS洗3次，用RPMI1640+10％FBS重悬细胞，分成3份分别在37℃孵育0、2、4小时。PBS洗三次后加入1μL抗人荧光二抗(APC anti-human IgG Fc Antibody，Biolegend，410712)，4℃孵育0.5小时，PBS洗三次后重悬细胞上机。使用同型对照阴性抗体HLE IgG1作为阴性对照。按照以下公式计算内吞率：内吞率(％)＝【1-(该时间点检测样品平均荧光值-该时间点阴性对照样品平均荧光值)/(0小时检测样品平均荧光值-0小时阴性对照样品平均荧光值)】×100。
实验结果如表6所示，2B12B10-hz1、4F6C3-hz1、30G11B7-hz1、22F11H5-hz1、27B9F2-hz1内吞率与参照抗体基本相当，27H8H3-hz1内吞率优于参照抗体。
表6.人源化抗体内吞检测结果
实施例9人源化抗体的信号通路阻断活性评价
FVIIa与TF阳性肿瘤细胞MDA-MB-231表面的组织因子结合，将激活TF下游信号通路，导致IL-8的释放。因此通过检测IL-8的释放，能够反映抗TF人源化抗体对该TF信号通路的阻断活性。
实验步骤如下：将FVIIa(40nM，Haematologic Technologies Inc，HCVIIA-0031)、梯度稀释的抗TF人源化抗体(60μg/mL起始，3倍梯度稀释，11个浓度点)与MDA-MB-231细胞(5×104细胞/孔，南京 科佰，CBP60382)共孵育5小时后，收集上清，通过IL-8检测试剂盒(MABTECH，3114-1H-20)检测IL-8的释放。IL-8检测步骤如下：实验前一晚，将在PBS中的捕获抗体MT8H6按200ng/孔包被在96孔板中，4℃过夜；去除包被液，300μL PBST洗1遍，所有96孔板每孔加入100μL在PBS中的2％BSA，37℃封闭2小时；直接加50μL细胞培养上清，37℃孵育2小时；去除上清，每孔用300uL PBST洗三次，每孔加入100uL(1ug/ml)MT8F19-biotin二抗(在2％BSA中)，37℃孵育1小时；去除上清，每孔用300μL PBST洗四次，每孔加入100μL过氧化物酶缀合的链霉亲和素(1：10000稀释)，37℃孵育1小时；去除上清，每孔用300μL PBST洗五次，随后每孔加入100μL TMB底物，显色5-20分钟；每孔加入50μL 2N HCl终止反应，酶标仪读取OD450mm读数。将结果导入GraphPad作图并计算抑制率和IC50。
实验结果如表7和图3所示，除27B9F2-hz1外，其余5个人源化抗体均对TF信号通路有较强抑制作用。
表7.人源化抗体IL-8释放阻断活性检测结果
实施例10评价人源化抗体对凝血的影响
用含有CaCl2的HEPES溶液稀释抗TF人源化抗体，随后与50000个TF阳性细胞MDA-MB-231细胞在37℃孵育30分钟，然后加入50μL人血浆，迅速混匀，立刻连续读取405nm吸光值，以计算对细胞表面TF起始凝血的抗凝作用。将结果导入GraphPad作图。共进行两次实验，第一次实验CaCl2浓度为2mM，抗体终浓度为50μg/mL；第二次实验CaCl2浓度为5mM，抗体终浓度为20μg/mL。实验结果如图4(图4A:2mM Ca2+,50μg/mL抗体浓度；图4B：5mM Ca2+,20μg/mL抗体浓度)和下表8所示。综合两次实验结果，在测试的抗体中，27H8H3-hz1和2B12B10-hz1对人凝血影响最小，且优于参照抗体。
表8：人源化抗体对凝血时间V50的影响

*：在2mM Ca2+和50μg/mL抗体4F6C3-hz1存在下,在2000秒内基本上无凝血发生。
实施例11人源化抗体的稳定性评价
人源化抗体的加速稳定性评价
将抗TF人源化抗体样品超滤置换至PBS(PH＝7.4)中，浓度5mg/mL,过滤除菌，分别在37℃放置0、3、7、14天及反复冻融4次、8次。随后，按照一般方法中所述，检测样品的SEC-HPLC及CE-SDS纯度。
实验结果显示，在加速及反复冻融情况下，进行检测的22F11H5-hz1与27H8H3-hz1人源化抗体拥有良好的稳定性。在加速稳定性及反复冻融试验后，两种人源化抗体的SEC纯度均保持在95％以上；CE-SDS非还原纯度均保持在87％以上，CE-SDS还原纯度在95％以上。
人源化抗体Tm值测定
采用DSF测定人源化抗体Tm值，以反映抗体的热稳定性。实验步骤如下：将待测抗体样品用PBS稀释到1mg/mL；将染料SYPRO Orange dye(Thermo#56651)用ddH2O稀释至40倍；反应体系：样品12.5μL+40X染料2.5μL+ddH2O 5μL；封膜，瞬时离心；通过Q-PCR检测，Q-PCR参数设置：Target(ROX)，程序(25℃，3min；1％速率，95℃；95℃，2min)。
结果显示，5个候选人源化抗体2B12B10-hz1、4F6C3-hz1、30G11B7-hz1、22F11H5-hz1、27H8H3-hz1均有较高的Tm值(均高于70℃)，表明其拥有较好的热稳定性。
实施例12人源化抗体的血清稳定性评价
用人血清将抗TF人源化抗体稀释至20μg/mL，在37℃放置14天，随后通过基于抗原蛋白的ELISA检测亲和力的变化。ELISA方法的实验步骤如一般方法中所述。实验结果如图5A和5B显示，22F11H5-hz1与27H8H3-hz1在人血清中放置14天后亲和力没有明显变化。
实施例13人源化抗体大鼠体内PK检测
将待检测样品通过尾静脉注射给予雄性大鼠(成都达硕)，每组3只，给药剂量为10mg/kg。给药后采集30min、1h、2h、6h、24h、48h、96h、168h、336h血清样品。用含阴性大鼠血清的2％BSA稀释相应抗体，1μg/mL起始，2倍梯度稀释，11个浓度点，以此作为标准曲线样品。通过基于抗原蛋白的ELISA检测血药浓度，ELISA方法如一般方法中所述。实验结果显示，人源化抗体22F11H5-hz1和27H8H3-hz1在大鼠体内的半衰期为大约120小时以上。
根据前述抗体表征，确定4种候选人源化抗体，2B12B10-hz1、30G11B7-hz1、22F11H5-hz1和27H8H3-hz1，用于后续的ADC构建和性能表征。序列表中显示了所选4种抗体的序列。
实施例II抗体偶联药物(ADC)的制备和表征
实施例1抗体偶联药物(ADC)的制备
实施例1.1：在“药物-连接体化合物”合成中使用的中间体的合成
实施例1.1.1：(S)-7-乙基-7-羟基-14-(3-羟基丙基)-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1)的合成
步骤一：(S)-7-乙基-7-羟基-14-(3-氯丙基)-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1B)的合成
冰浴条件下，向化合物(S)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1A，Cas No.：151636-76-9，500mg)的75％硫酸溶液(5mL)中，加入七水合硫酸亚铁(570mg七水合硫酸亚铁溶于1mL水中)和4,4-二甲氧基氯丁烷(3.89g)，反应液搅拌三分钟后滴加双氧水(29％，2.5mL)。反应液在0℃下搅拌反应5分钟后升至室温，并搅拌反应3小时。反应液加入水(50mL)稀释，乙酸乙酯(80mL×2)萃取，有机相用饱和食盐水洗涤，无水硫酸钠干燥，浓缩得到粗产品。粗产品用C18柱(乙腈/0.05％甲酸的水溶液：5％-60％)进一步纯化，得到目标化合物A1B(黄色固体，400mg，收率：67％)。
LCMS(ESI)[M+H]+：468.9；
1H NMR(400MHz,DMSO-d6)δ7.65(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＝5.9Hz,2H),3.22(s,2H),1.98(d,J＝6.7Hz,4H),0.88(t,J＝7.2Hz,3H)。
步骤二：(S)-7-乙基-7-羟基-14-(3-羟基丙基)-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1)的合成
将化合物(S)-7-乙基-7-羟基-14-(3-氯丙基)-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(100mg，0.213mmol)溶于10％硫酸(5mL)溶液中，反应在110℃下反应48小时。向反应液中加入饱和碳酸氢钠(30mL)溶液，用二氯甲烷(10mL×5)萃取，无水硫酸钠干燥，抽滤，减压浓缩得到粗产品。经高效液相制备纯化(乙腈/水含0.05％甲酸)，得到(S)-7-乙基-7-羟基-14-(3-羟基丙基)-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1，1.78mg)。
LCMS(ESI)[M+H]+：451.0；
1H NMR(400MHz,DMSO-d6)δ7.63(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)。
实施例1.1.2：(S)-2-氨基-N-((3-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)丙氧基)甲基)乙酰胺(B1)的合成
步骤一：化合物(B1A)(Cas No.：1599440-06-8，368mg)、化合物(A1)(440mg)和对甲苯磺酸吡啶盐(PPTS)(25mg)在二氯甲烷(20ml)中回流20小时，然后用碳酸氢钠水溶液和盐酸水溶液分别洗涤，减压除去有机溶剂，得到粗产物。粗产品经柱层析分离纯化(二氯甲烷:甲醇＝10/1)，得到目标化合物B1B(240mg)。
LCMS(ESI)[M+H]+：759.5。
步骤二：B1B(240mg)溶于DMF(5ml)，加入哌啶(1ml)，化合物搅拌20分钟，溶液减压除去低沸点组分，残留物直接用于下步合成。少量粗产物经反相色谱(乙腈/0.05％FA的水溶液:5％到50％)纯化后得目标化合物B1。
ESI-MS(m/z):537.4[M+H]+；
1H NMR(400MHz,DMSO-d6)δ9.13(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)。
实施例1.1.3：N6,N6-二甲基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(C1)
将6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酸(268mg，Cas No.2356229-58-6)、化合物C1A(328mg)、三乙胺(322mg)溶于N,N-二甲基甲酰胺(5mL)。然后再加入1-羟基苯并三唑(HOBT，162mg)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI，229mg),反应液室温搅拌16小时。反应液直接经过C18柱反相(乙腈和0.05％的甲酸水溶液体系)提纯，得到目标化合物N6,N6-二甲基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(C1，白色固体，327mg)。
LCMS(ESI)[M+H]+：524.4。
1H NMR(400MHz,)δ9.13(s,2H),7.95(t,J＝8.8Hz,2H),4.21(dd,J＝8.8,6.9Hz,1H),4.08–4.03(m,1H),3.41(s,3H),2.55(t,J＝7.0Hz,2H),2.42–2.32(m,4H),2.27(s,6H),1.98(dd,J＝13.6,6.8Hz,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＝12.8,6.8Hz,6H)。
实施例1.1.4：N6,N6-二乙基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(C2)
步骤一：
将化合物C2A(5.0g,12.05mmol，Cas：1872-06-8)溶在二氯甲烷(100mL)中，向反应液中加入乙醛(3.2g,72.3mmol)，室温下搅拌反应10分钟，再向反应液中加入三乙酰氧基硼氢化钠(12.8g,60.25mmol)，反应在室温下搅拌反应1小时。LCMS显示反应完全。向反应液中加入氯化铵饱和水溶液搅拌一个小时，旋干，过滤后滤液用C18柱反向分离纯化(乙腈比0.05％的甲酸水溶液:5％到55％)，得到目标化合物C2B(4.57g,收率82.0％)为白色固体。
LCMS(ESI)[M+H]+＝436.4；
1H NMR(400MHz,DMSO-d6)δ7.70(d,J＝7.0Hz,1H),7.41(d,J＝9.0Hz,1H),7.38–7.26(m,5H),5.08–4.99(m,2H),4.00(dd,J＝12.6,6.5Hz,1H),3.86(dd,J＝8.6,6.8Hz,1H),2.74(dd,J＝14.0,6.9Hz,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＝7.1Hz,6H),0.87–0.81(m,6H)。
步骤二：
室温下，将化合物C2B(1.6g，3.68mmol)溶于甲醇(80mL)中，然后向反应液中加入Pd/C(0.16g)，氢气下室温下搅拌反应12小时。LCMS显示反应完成。将反应液过滤，滤液减压浓缩得到目标化合物C2C(900mg,收率82％)，为米白色固体。
1H NMR(400MHz,DMSO-d6)δ8.04(br s,1H),4.02–3.99(m,1H),3.10(d,J＝4.5Hz,1H),2.65(q,J＝7.1Hz,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＝7.1Hz,6H),0.89(d,J＝6.9Hz,3H),0.79(d,J＝6.8Hz,3H)。
步骤三：
将化合物6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酸(268mg,1mmol)溶于DMF(8mL)中，依次加入HATU(380mg,1mmol)和三乙胺(322mg,2.5mmol)，室温搅拌20分钟后加入化合物C2C(301mg,1mmol)，室温继续搅拌30分钟。LCMS检测反应完成后，反应液直接经过C18柱反相(乙腈和0.05％的甲酸水溶液体系)提纯得到目标化合物C2(280mg，收率51％)，为白色固体。
LCMS(ESI)[M+H]+＝552.3；
1H NMR(400MHz,DMSO-d6)δ9.13(s,2H),7.95(d,J＝8.9Hz,1H),7.86(d,J＝7.2Hz,1H),4.18(dd,J＝8.8,6.8Hz,1H),4.02(dd,J＝12.8,7.2Hz,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＝7.2Hz,6H),0.87–0.82(m,6H)。
实施例1.1.5：N6,N6-二正丙基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(C3)
步骤一：
将化合物C2A(5.0g，12mmol)溶在二氯甲烷(100mL)中，向反应液中加入正丙醛(4.2g，72.3mmol)，室温下搅拌反应10分钟，再向反应液中加入三乙酰氧基硼氢化钠(12.8g，60.25mmol)，反应在室温下搅拌反应1小时。LCMS显示反应完全。向反应液中加入氯化铵饱和水溶液搅拌一个小时，旋干，过滤后滤液用C18柱反向分离纯化(乙腈比0.05％的甲酸水溶液:5％到55％)得到目标化合物C3A(4.57g,收率82.0％)，为白色固体。
LCMS(ESI)[M+H]+＝464.0；
1H NMR(400MHz,DMSO-d6)δ7.81(d,J＝7.3Hz,1H),7.38–7.28(m,5H),5.04(d,J＝1.7Hz,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)。
步骤二：
室温下，将化合物C3A(2.0g，4.32mmol)溶于甲醇(80mL)中，然后向反应液中加入Pd/C(0.16g)，氢气下室温下搅拌反应12小时。LCMS显示反应完成。将反应液过滤，滤液减压浓缩得到目标化合物C3B(1.2g,收率85.5％)，为白色固体。
步骤三：
将化合物6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酸(100mg,0.373mmol)，溶于N,N-二甲基甲酰胺(1mL),然后加入HATU(142mg,0.373mmol)和N,N-二异丙基乙胺(120mg,0.93mmol)，该体系搅拌30分钟，然后加入化合物C3B(122mg，0.371mmol)，反应液室温搅拌1小时。LCMS检测反应完成后，反应液直接经过C18柱反相(乙腈和0.05％的甲酸水溶液体系)提纯得到目标化合物N6,N6-二正丙基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(C3，50mg，收率28％)，为淡黄色固体。
LCMS(ESI)[M+H]+＝580.0；
1H NMR(400MHz,DMSO-d6)δ8.24(s,2H),7.98–7.93(m,2H),4.24–4.16(m,1H),4.10(d,J＝5.2Hz,1H),3.41(s,3H),2.79–2.64(m,6H),2.55(t,J＝7.1Hz,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)。
实施例1.2药物-连接体化合物的合成
实施例1.2.1：N-((11S,14S)-11-(4-(二正丙基氨基)丁基)-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环烯烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-01)
将化合物C3(43mg,0.074mmol)、化合物B1(40mg，0.075mmol)，溶于N,N-二甲基甲酰胺(1mL)，然后依次加入HBTU(28mg,0.075mmol)和N,N-二异丙基乙胺(24mg,0.187mmol)，反应液室温搅拌1小时。LCMS检测反应完成后，反应液直接经制备色谱(0.01％三氟乙酸水溶液,乙腈)纯化得到目标化合物(DL-01，8.5mg,收率10％)，为黄色固体。
LCMS(ESI)[M+H]+＝1098.6；
1H NMR(400MHz,DMSO-d6)δ9.10(s,2H),9.03(s,1H),8.64(t,J＝6.4Hz,1H),8.19(t,J＝5.9Hz,1H),8.08(d,J＝7.4Hz,1H),7.92(d,J＝8.5Hz,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＝5.5Hz,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)。
实施例1.2.2：N-((11S,14S)-11-(4-(二乙基氨基)丁基)-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-02)
将化合物C2(40mg,0.075mmol)、化合物B1(41mg,0.075mmol)溶于DMF(1mL)，然后再加入HOBt(15.2mg,0.113mmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(21.5mg,0.113mmol)，然后再加入三乙胺(23mg,0.225mmol)，加完后反应液室温搅拌16小时。LCMS检测反应完成后，将反应液浓缩得粗产品。粗产品经制备色谱(0.01％TFA水溶液,乙腈)纯化得到目标化合物(DL-02,16mg,收率20％)，为黄色固体。
LCMS(ESI)[M+H]+＝1070.6；
1H NMR(400MHz,DMSO-d6)δ9.13–9.08(m,2H),9.01(s,1H),8.64(t,J＝6.5Hz,1H),8.20(t,J＝5.7Hz,1H),8.09(d,J＝7.4Hz,1H),7.92(d,J＝8.4Hz,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＝6.5Hz,1H),4.20–4.10(m,1H),3.74(d, J＝5.5Hz,2H),3.50(t,J＝5.8Hz,2H),3.41(s,3H),3.14–3.07(m,6H),2.98(s,2H),2.55(d,J＝7.3Hz,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＝7.2Hz,6H),0.91–0.78(m,9H)。
实施例1.2.3：N-((11S,14S)-11-(4-(二甲基氨基)丁基)-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-03)
将化合物B1(100mg,0.186mmol)和化合物N6,N6-二甲基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(C1，100mg，0.191mmol)溶于DMF(2mL)。然后再加入1-羟基苯并三唑(38mg,0.280mmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(53mg,0.280mmol),然后再加入三乙胺(56mg,0.559mmol)，加完后反应液室温搅拌16小时。LCMS检测反应完成后，将反应液经制备色谱(0.01％TFA水溶液,乙腈)纯化得到目标化合物(DL-03，20mg,收率10％)，为黄色固体。
LCMS(ESI)[M+H]+＝1042.5；
1H NMR(400MHz,DMSO-d6)δ9.28(s,1H,TFA),9.10(s,2H),8.64(br s,1H),8.19(br s,1H),8.09(d,J＝6.4Hz,1H),7.92(d,J＝7.8Hz,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)。
实施例1.3抗TF抗体-药物偶联物的制备
Ab为抗TF抗体。
1.3.1 TF-ADC(DAR8)样品制备
将根据实施例I制备的抗TF抗体30mg，用稀释液(20mM PB，pH 7.5)稀释，加入终浓度为1mM的依地酸钠溶液，混匀；加入抗体6.0倍当量的TCEP溶液，混匀，室温放置60分钟；向上述溶液体系加入抗体15倍当量的溶解于二甲基亚砜中的DL-01，混匀，室温静置1小时，得到偶联后样品，反应结束后使用30KDa的超滤管将样品置换到pH为6.0的20mM组氨酸缓冲液中并去除低分子物质，最后将样品浓缩以获得含有抗TF抗体的ADC组合物的溶液TF-ADC(DAR8)。利用实施例1.4的质谱法测定DAR值 为8.0。
1.3.2 TF-ADC(DAR3)样品制备
将根据实施例I制备的抗TF抗体30mg，用稀释液(20mM PB，pH 7.5)稀释，加入终浓度为1mM的依地酸钠溶液，混匀；加入抗体6.0倍当量的TCEP溶液，混匀，室温放置60分钟；向上述溶液体系加入抗体4.0倍当量的溶解于二甲基亚砜中的DL-01，混匀，室温静置1小时，得到偶联后样品，反应结束后使用30KDa的超滤管将样品置换到pH为6.0的20mM组氨酸缓冲液中并去除低分子物质，最后将样品浓缩以获得含有抗TF抗体的ADC组合物的溶液TF-ADC(DAR3)。质谱法测定DAR值为3.0。
实施例1.4采用质谱法进行偶联后样品的DAR值测定
1.4.1对TF-ADC(DAR3)进行LC-MS分子量和DAR值分析
色谱条件：
色谱柱：PLRP-S，2.1*50mm,5μm；
流动相A：0.1％FA/H2O；流动相B：0.1％FA/ACN
柱温：30℃
样品室温度：8℃
流速：0.6ml/min
进样量：2μl
样品处理：分别取样品50μg，加入1M DTT 2μl，加超纯水至50μl以稀释至约1.0mg/ml浓度，混匀，室温还原30min。
LC/MS型号：Agilent 1290-6545XT Q-TOF
质谱条件：Gas temp：320℃；Drying Gas：Nitrogen；Nebulizer：35psi；Sheath Gas Temp：350℃；sheath Gas Flow：11l/min；m/z 500～3000
结果如下表9所示：
表9：理论分子量及实测分子量
表9中，mAb表示未偶联的单克隆抗体；LC代表抗体轻链；HC代表抗体重链；DAR1代表包含轻链或重链偶联1个毒素分子的偶联物；DAR2代表包含轻链或重链偶联2个毒素分子的偶联物；DAR3代表包含轻链或重链偶联3个毒素分子的偶联物；其中，单抗理论分子量以G0F糖型计算。下文中mAb、LC、HC、DAR1、DAR2、DAR3如上说明。
检测结果显示，TF-ADC(DAR3)上抗体轻链偶联0～1个毒素分子(LC，DAR1比例分别为53.8％，46.2％)、重链偶联0～3个毒素分子(mAb、DAR1、DAR2、DAR3的比例分别为30.6％，42.4％，20.2％，6.8％)，由此计算TF-ADC(DAR3)样品的偶联比(平均DAR值)为3.0。
1.4.2对TF-ADC(DAR8)进行LC-MS分子量和DAR值分析
色谱条件：
色谱柱：Xbridge Protein BEH SEC(2.5μm,4.6*150mm)
流动相A：0.1％FA/H2O；流动相B：0.1％FA/ACN
柱温：30℃
样品室温度：8℃
流速：0.3ml/min
进样量：1μl
样品处理：分别取样品50μg，加入1M DTT 2μl，加超纯水至50μl以稀释至约1.0mg/ml浓度，混匀，室温还原30min。
LC/MS型号：UPLC(AB SCIEX)，高分辨质谱仪(AB SCIEX)
质谱条件：Gas1:45；Gas2:45；CUR：30；TEM：450；ISVF：5000；DP：120；CE：12；质量数范围：600-4000
结果如下表10示：
表10理论分子量及实测分子量
检测结果显示，TF-ADC(DAR8)上抗体轻链偶联0～1个毒素分子(LC，DAR1比例分别为0％，100.0％)、重链偶联0～3个毒素分子(mAb、DAR1、DAR2、DAR3的比例分别为0％，0％，0％，100.0％)，由此计算TF-ADC(DAR8)样品的偶联比(平均DAR值)为8.0。
实施例2抗TF抗体ADC评价
实施例2.1抗TF ADC亲和力检测
根据前述实施例中用于制备ADC样品TF-ADC(DAR8)的方法，制备本发明4种候选抗体(2B12B10-hz1，30G11B7-hz1，22F11H5-hz1,27H8H3hz1)以及参照抗体(BM)分别与毒素-Linker(B81，即，DL-01分子)的ADC偶联物。
在将抗TF人源化抗体与毒素-Linker(B81，即，DL-01分子)偶联前后，按照与实施例I中所述相似的方法，通过基于抗原蛋白的ELISA测定抗体亲和力的变化。
实验结果如表11和图6A和6B所示，4种人源化抗体在偶联B81后，亲和力没有明显变化。
表11.人源化抗体ADC偶联前后亲和力检测
实施例2.2 ADC体外杀伤活性检测
胰酶消化收集TF阳性肿瘤细胞KYSE520细胞，用RPMI1640+2％FBS重悬细胞，每孔铺2000个细胞；用RPMI1640+2％FBS梯度稀释待测ADC，50μg/mL起始，3倍稀释，11个浓度点；将稀释后的ADC加入板中，37℃孵育120小时；孵育完成后，加入CCK8试剂，20μL/孔，反应2-4小时，酶标仪450nm读数并导入Graphpad Prism进行曲线拟合。
实验结果如图7A和7B所示，2B12B10-hz1、30G11B7-hz1、22F11H5-hz1及27H8H3-hz1偶联B81之后，均能够有效杀伤KYSE520细胞，且杀伤IC50值与参照抗体偶联的ADC基本相当。
实施例2.3 ADC体内药效
为验证抗TF人源化ADC药物的体内药效，评价受试药物在KYSE520皮下异种移植雌性Balb/c Nude小鼠模型中的抗肿瘤作用，购买5-6周龄的雌性Balb/c Nude小鼠。将生长至对数生长期的KYSE520细胞采用胰酶消化及PBS重悬，每只小鼠皮下接种5*106细胞。待肿瘤生长至250mm3大小，采用静脉给药，每周一次，每次1mg/kg或3mg/kg，给药待测ADC，具体方案如下表13和14所示。本实验的主要观察指标为：1)TGI(％)，计算公式为：TGI(％)＝(1-T/C)×100％(T和C分别为治疗组和对照组在某一特定时间点的相对肿瘤体积)2)试验终点肿瘤体积大小照片、肿瘤重量。3)ADC药物对小鼠体重的影响。共开展了2次实验。
第一次实验的给药方案和结果如表12和图8所示，22F11H5-hz1和30G11B7偶联B81之后，在小鼠体内均有良好的抑瘤效果，3mg/kg剂量下TGI与BM-B81相当，1mg/kg剂量下两者均优于BM-B81。小鼠体重变化情况如图9所示，表明整个给药过程中2个ADC对小鼠体重无影响。
表12.抗TF ADC体内药效
第二次实验的实验方案和结果如表13和图10所示，2B12B10-hz1和27H8H3-hz1偶联B81之后，在小鼠体内均有良好的抑瘤效果，1mg/kg剂量下两者均优于BM-B81。小鼠体重变化情况如图11所示，表明2个ADC对小鼠体重无影响。
表13.抗TF ADC体内药效
序列表：




Claims (27)

  1. 具有下式(I)的抗体-药物偶联物(ADC)或其可药用盐或溶剂化物：
    Ab-[L-D]q        (I)
    其中，
    Ab表示抗TF抗体，
    L表示连接体，
    D表示细胞毒性或细胞抑制性药物，例如，拓扑异构酶I抑制剂，且
    q＝1-20，例如，q＝1-10、1-8、3-8、4-8、或6-8，
    其中，所述Ab包含：
    -SEQ ID NO:1的重链可变区(VH)序列的三个CDR和SEQ ID NO:2的轻链可变区(VL)序列的三个CDR，
    -SEQ ID NO:3的重链可变区(VH)序列的三个CDR和SEQ ID NO:4的轻链可变区(VL)序列的三个CDR，
    -SEQ ID NO:5的重链可变区(VH)序列的三个CDR和SEQ ID NO:6的轻链可变区(VL)序列的三个CDR，或者
    -SEQ ID NO:7的重链可变区(VH)序列的三个CDR和SEQ ID NO:8的轻链可变区(VL)序列的三个CDR，
    其中，所述CDR根据Chothia、AbM、Kabat、IMGT或其任意组合进行定义。
  2. 根据权利要求1的抗体-药物偶联物或其可药用盐或溶剂化物，其中，所述Ab包含3个重链互补决定区(HCDR)以及3个轻链互补决定区(LCDR)，其中：
    (i)根据IMGT定义，HCDR1包含SEQ ID NO:18的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:19的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:20的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:30的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:31的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:32的氨基酸序列或由其组成；或
    (ii)根据IMGT定义，HCDR1包含SEQ ID NO:42的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:43的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:44的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:54的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:55的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:56的氨基酸序列或由其组成；或
    (iii)根据IMGT定义，HCDR1包含SEQ ID NO:66的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:67的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:68的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:78的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:79的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:80的氨基酸序列或由其组成；或
    (iv)根据IMGT定义，HCDR1包含SEQ ID NO:90的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:91的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:92的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:102的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:103的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:104的氨基酸序列或由其组成。
  3. 根据权利要求1-2任一项的抗体-药物偶联物或其可药用盐或溶剂化物，其中，所述Ab包含：
    -包含SEQ ID NO:1或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列的重链可变区；和包含SEQ ID NO:2或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列的轻链可变区，或
    -包含SEQ ID NO:3或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性 的氨基酸序列的重链可变区；和包含SEQ ID NO:4或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列的轻链可变区，或
    -包含SEQ ID NO:5或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列的重链可变区；和包含SEQ ID NO:6或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列的轻链可变区，或
    -包含SEQ ID NO:7或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列的重链可变区；和包含SEQ ID NO:8或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列的轻链可变区，
    优选地，所述Ab为IgG1抗体。
  4. 根据权利要求1-3任一项的抗体-药物偶联物或其可药用盐或溶剂化物，其中，D表示喜树碱类药物，优选地，FL118或其衍生物，例如，包含以下结构的喜树碱类药物：
    其中，Ra选自：
    氢；
    C3-C8环烷基；
    苯基；
    任选被选自以下的取代基取代的C1-C8烷基：卤素、羟基、任选地被NH2、NH(C1-C4烷基)和N(C1-C4烷基)2取代的C1-C4烷氧基、C3-C8环烷基、杂环烷基、苯基、NR1R2，
    其中R1和R2彼此独立地选自
    氢；
    任选地被选自以下的取代基取代的C1-C8烷基：羟基、氨基、被一个或两个C1-C4烷基取代的氨基、被一个或两个C1-C4羟烷基取代的氨基、被(C1-C4羟烷基)和(C1-C4烷基)取代的氨基；
    被1或2个C3-C10环烷基、C3-C10杂环烷基、苯基或杂芳基取代的C1-C4烷基；
    C3-C10环烷基；
    C3-C10杂环烷基
    C2-C6杂烷基；
    杂芳基；
    任选卤代的苯基；
    任选被羟基或氨基取代的C1-C8烷基-C(＝O)-；
    或者，R1和R2与各自连接的氮原子组合形成具有0至3个取代基的5-、6-或7-元杂环，所述取代基选自卤素、C1-C4烷基、OH、C1-C4烷氧基、NH2、NH(C1-C4烷基)和N(C1-C4烷基)2，
    其中环烷基、杂环烷基、苯基、杂芳基在每次出现时各自独立地任选被0-3个选自以下的取代基取代：OH、C1-C4烷基、C1-C4烷氧基、NH2、NH(C1-C4烷基)和N(C1-C4烷基)2取代，
    优选地，Ra为-C1-C4烷基-OH、-C1-C4烷基-O-C1-C4烷基-NH2、或-C1-C4烷基-NH2，
    其中，优选地，所述药物D单元通过其上存在的羟基或氨基基团，与连接体L单元连接。
  5. 根据权利要求1-3任一项的抗体-药物偶联物或其可药用盐或溶剂化物，其中，式(I)中的L-D单元包含以下结构：
    优选地，式(I)中的L-D单元包含以下结构：
  6. 根据权利要求1-5任一项的抗体-药物偶联物或其可药用盐或溶剂化物，其中，L为含肽连接体，并包含下式(II)的结构：
    -Z-Y-M-，    (II)
    其中
    Z为与Ab连接的连接基，
    Y是2-5个氨基酸的肽，优选二肽、三肽或四肽，
    M不存在，或是用于与药物D连接的间隔基。
  7. 根据权利要求6的抗体-药物偶联物或其可药用盐或溶剂化物，其中，Y为从N端到C端具有下式氨基酸序列的肽；
    Xaa1-Xaa2-Xaa3-Xaa4-Xaa5，
    其中Xaa1不存在，或为选自缬氨酸、甘氨酸、丙氨酸和谷氨酸的氨基酸；
    Xaa2为选自苯丙氨酸、亮氨酸、和缬氨酸的氨基酸，优选缬氨酸；
    Xaa3为未取代的或取代的赖氨酸；
    Xaa4为选自亮氨酸、甘氨酸和丙氨酸的氨基酸；
    Xaa5不存在，或为选自甘氨酸和丙氨酸的氨基酸；
    其中，所述氨基酸序列的N端与Z连接，且C端与M连接(如果M存在的话)或直接与药物D连接，
    优选地，Xaa3为ε-氨基被C1-C3烷基单取代或二取代的赖氨酸，
    再优选地，Y为选自以下的肽：Phe-Lys-Gly、Leu-lys-Gly、Gly-Val-Lys-Gly、Val-Lys-Gly-Gly、Val-Lys-Gly、Val-Lys-Ala、Val-Lys-Leu，其中Lys残基为未取代的或被C1-C3烷基单取代或二取代的赖氨酸。
  8. 根据权利要求6-7任一项的抗体-药物偶联物或其可药用盐或溶剂化物，其中，Y为
    其中，R3和R4各自独立地选自甲基、乙基和丙基，
    优选地，R3和R4相同，更优选地，R3和R4各自独立地为丙基，
    其中，左侧波浪线表示与Z连接的位置；右侧波浪线表示与M连接的位置。
  9. 根据权利要求6-8的抗体-药物偶联物或其可药用盐或溶剂化物，其中，Z具有以下结构：
    -Z1-Z2-Z3-Z4-，
    其中Z1为Ab中的硫原子，
    Z2为5-10元杂环基，优选地含1或2个选自N,S和O的杂原子；
    Z3选自键、-C(＝O)-、-C1-C10亚烷基-C(＝O)-、-C3-C10亚炔基-C(＝O)-、-C3-C10亚烯基-C(＝O)-、-C1-C10亚杂烷基-C(＝O)-、-C3-C8亚环烷基-C(＝O)-、-O-C1-C8亚烷基-C(＝O)-、-亚芳基-C(＝O)-、-C1-C10亚烷基-亚芳基-C(＝O)-、-亚芳基-C1-C10亚烷基-C(＝O)-、-C1-C10亚烷基-C3-C8亚环烷基-C(＝O)-、-C3-C8亚环烷基-C1-C10亚烷基-C(＝O)-、-C3-C8亚杂环基-C(＝O)-、-C1-C10亚烷基-C3-C8亚杂环基-C(＝O)-、-C3-C8亚杂环基-C1-C10亚烷基-C(＝O)-，
    Z4是键或是下式表示的PEG单元，
    其中，R5选自C1-4亚烷基、-NH-、-NH-C1-4亚烷基-杂芳基-，其中杂芳基
    为5元或6元的含氮杂芳基，优选三唑基；R6为-C(＝O)-、C1-4亚烷基、C1-4亚烷基-C(＝O)-、-NH-C(＝O)-(CH2OCH2)-C(＝O)-、C1-4亚烷基-NH-C(＝O)-(CH2OCH2)-C(＝O)-，其中m为2-12的整数，例如m＝2,4,6,或8，
    优选地，Z具有以下结构：
    其中,Rb是亚炔基-C(＝O)-或亚烯基-C-(＝O)-、
    优选地，Rb是
    其中，左侧波浪线表示与抗体(Ab)连接的位置；右侧波浪线表示与Y连接的位置；
    更优选地，Z具有结构：
  10. 根据权利要求6-9的抗体-药物偶联物或其可药用盐或溶剂化物，其中，M不存在、或是氨基-C1- C3亚烷基，例如，-NH-CH2-，或氨基-苯基-C1-C3亚烷基-O-C(＝O)-，优选地M为：
    其中左侧波浪线表示与Y连接，右侧波浪线表示与药物D单元连接。
  11. 根据权利要求6-10的抗体-药物偶联物或其可药用盐或溶剂化物，其中，L为包含如下结构的连接体：
    其中，R3和R4各自独立地选自甲基、乙基、丙基。
  12. 根据权利要求11的抗体-药物偶联物或其可药用盐或溶剂化物，其中，R3和R4均为甲基；或R3和R4均为乙基；或R3和R4均为丙基。
  13. 权利要求1的抗体-药物偶联物或其可药用盐或溶剂化物，其中，所述式I的L-D单元通过与Ab的轻链和/或重链的半胱氨酸的巯基形成硫醚键，从而与抗体连接。
  14. 根据权利要求1的抗体-药物偶联物或其可药用盐或溶剂化物，其中，所述ADC选自：

    其中，q为1-8的平均DAR值，优选地，q为大约2、大约3、大约4、大约5、大约6，大约7或大约8，
    优选地，所述Ab为IgG1全长抗体，且包含SEQ ID NO:7的重链可变区(VH)序列的三个CDR和SEQ ID NO:8的轻链可变区(VL)序列的三个CDR；或更优选地包含SEQ ID NO:7的重链可变区和包含SEQ ID NO:8的轻链可变区。
  15. 药物组合物，其包含前述权利要求1-14任一项的抗体-药物偶联物或其可药用盐或溶剂化物以及任选地药用辅料。
  16. 结合组织因子(TF)的抗体或其抗原结合片段，其包含
    (i)如SEQ ID NO:1所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:2所示的轻链可变区的LCDR1、2和3序列，或者
    (ii)如SEQ ID NO:3所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:4所示的轻链可变区的LCDR1、2和3序列，或者
    (iii)如SEQ ID NO:5所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:6所示的轻链可变区的LCDR1、2和3序列，或者
    (iv)如SEQ ID NO:7所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:8所示的轻链可变区的LCDR1、2和3序列，
    优选地，其中所述CDR根据Chothia、Abm、Kabat或IMGT或其组合进行定义。
  17. 权利要求16的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
    (i)重链可变区包含如SEQ ID NO:1所示的氨基酸序列或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的氨基酸序列，以及轻链可变区包含如SEQ ID NO:2所示的氨基酸序列或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的氨基酸序列，或者
    (ii)重链可变区包含如SEQ ID NO:3所示的氨基酸序列或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的氨基酸序列，以及轻链可变区包含如SEQ ID NO:4所示的氨基酸序列或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的氨基酸序列，或者
    (iii)重链可变区包含如SEQ ID NO:5所示的氨基酸序列或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的氨基酸序列，以及轻链可变区包含如SEQ ID NO:6所示的氨基酸序列或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的氨基酸序列，或者
    (iv)重链可变区包含如SEQ ID NO:7所示的氨基酸序列或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的氨基酸序列，以及轻链可变区包含如SEQ ID NO:8所示的氨基酸序列或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的氨基酸序列；
    优选地，
    其中所述抗体或其抗原结合片段包含选自以下的重链可变区和轻链可变区：
    (i)包含SEQ ID NO:1所示的氨基酸序列的重链可变区，和包含SEQ ID NO:2所示的氨基酸序列的轻链可变区，或者
    (ii)包含SEQ ID NO:3所示的氨基酸序列的重链可变区，和包含SEQ ID NO:4所示的氨基酸序列的轻链可变区，或者
    (iii)包含SEQ ID NO:5所示的氨基酸序列的重链可变区，和包含SEQ ID NO：6所示的氨基酸序列的轻链可变区，或者，
    (iv)包含SEQ ID NO:7所示的氨基酸序列的重链可变区，和包含SEQ ID NO：8所示的氨基酸序列的轻链可变区。
  18. 权利要求16-17任一项的抗体或其抗原结合片段，其中
    所述抗体包含Fc区，优选地人Fc区；或者
    所述抗体是IgG1,IgG2,IgG3或IgG4抗体，且优选地具有人IgG1重链恒定区；或者
    所述抗原结合片段是选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体、scFv、scFab、二硫键连接的scFv,二硫键连接的scFab、(Fab’)2片段或线性抗体。
  19. 权利要求16-18任一项的抗体或其抗原结合片段，其中所述抗体是鼠源抗体、或嵌合抗体、或人源化抗体，优选地为人源化抗体。
  20. 一种分离的核酸，其编码权利要求16-19任一项的抗TF抗体或其抗原结合片段。
  21. 一种载体，其包含权利要求20的核酸，优选地所述载体是表达载体。
  22. 一种宿主细胞，其包含权利要求20的核酸或权利要求21的载体，优选地，所述宿主细胞是哺乳动物细胞。
  23. 包含权利要求16-19中任一项的抗体或其抗原结合片段的免疫缀合物或免疫融合物或多特异性抗体。
  24. 药物组合物，其包含前述权利要求16-19任一项的抗体或其抗原结合片段或权利要求23的免疫缀合物或免疫融合物或多特异性抗体，以及任选地药用辅料。
  25. 权利要求16-19任一项的抗体或其抗原结合片段或权利要求23的免疫缀合物或免疫融合物或多特异性抗体的用途，用于：在体内或在体外，
    (1)靶向性结合TF阳性肿瘤细胞；
    (2)阻断TF阳性肿瘤细胞中由TF/VIIa介导的下游信号通路；或
    (3)抑制和/或杀伤TF阳性肿瘤细胞，
    或用于制备在前述任一项中使用的药物。
  26. 在受试者中预防或治疗TF阳性肿瘤的方法，所述方法包括向所述受试者施用有效量的前述权利要求1-14任一项的抗体-药物偶联物或其可药用盐或溶剂化物、或权利要求16-19任一项的抗TF抗体或其抗原结合片段、或权利要求23的免疫缀合物或免疫融合物或多特异性抗体、或权利要求15或24的药物组合物，优选地，所述肿瘤是宫颈癌、胰腺癌、肺癌、前列腺癌、膀胱癌、卵巢癌、乳腺癌、结直肠癌、食道癌、头颈癌、胃癌，包括原发性或晚期或转移性癌症。
  27. 检测样品中TF的方法，所述方法包括
    (a)将样品与前述权利要求16-19中任一项的抗体或其抗原结合片段接触；和
    (b)检测所述抗体或其抗原结合片段和TF间的复合物的形成；任选地，抗体是被可检测地标记的。
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