WO2018036243A1 - 靶向于组织因子的抗体-药物偶联物 - Google Patents

靶向于组织因子的抗体-药物偶联物 Download PDF

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WO2018036243A1
WO2018036243A1 PCT/CN2017/087779 CN2017087779W WO2018036243A1 WO 2018036243 A1 WO2018036243 A1 WO 2018036243A1 CN 2017087779 W CN2017087779 W CN 2017087779W WO 2018036243 A1 WO2018036243 A1 WO 2018036243A1
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antibody
mab
seq
drug conjugate
amino acid
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PCT/CN2017/087779
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French (fr)
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余科
沈竞康
孟韬
马兰萍
张学赛
李晴柔
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复旦大学
中国科学院上海药物研究所
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Priority to EP17842668.0A priority Critical patent/EP3501548A4/en
Priority to JP2019511435A priority patent/JP7020655B2/ja
Priority to US16/326,886 priority patent/US11534495B2/en
Publication of WO2018036243A1 publication Critical patent/WO2018036243A1/zh
Priority to US18/050,240 priority patent/US20230138930A1/en

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    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
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Definitions

  • the present invention relates to the field of medicine, and in particular to an antibody targeted to tissue factor and an antibody-drug conjugate thereof, and a preparation method and use thereof.
  • Tissue factor is a 47 kDa transmembrane glycoprotein. Under normal physiological conditions, TF expression is mainly shielded from the subendothelial cell layer. Once the body's blood vessels are traumatized, TF is exposed to the bloodstream, and the exogenous coagulation reaction is initiated by binding and activating factor VII.
  • TF is abnormally activated and expressed in many tumor tissues and plays an important role in the occurrence and development of tumors.
  • patients are mostly accompanied by spontaneous thrombosis, such as Deep-vein thrombosis (DVT), Disseminated intravascular coagulation (DIC) and Pulmonary embolism (PE).
  • DVT Deep-vein thrombosis
  • DIC Disseminated intravascular coagulation
  • PE Pulmonary embolism
  • TF and FVII form a TF-FVIIa complex that directly binds to and induces activation of the transmembrane G protein-coupled receptor Protease-activated receptor 2 (PAR2).
  • PAR2 is an important signaling pathway regulating inflammatory response.
  • TF-FVIIa-PAR2 phosphorylates through MAPK/ERK, induces the expression of key growth factors, immunoregulators and chemokines (such as VEGF, CSF1/2, IL8, CXCL1, etc.), promotes the formation of new blood vessels, and is a tumor.
  • TF can also enhance the migration and adhesion of tumor cells through interaction with the Rac1 and ⁇ 1 family-associated integrins, thereby enhancing the blood transfer ability of tumor cells as a whole.
  • TF-initiated coagulation is also an important cause of tumor thrombosis, leading to a variety of cancers.
  • the hypertensive state induced by TF directly contributes to the attack of tumor cells from the immune system of the body, and increases the interaction between tumor cells and endothelial cells, resulting in an increase in the ability of tumor cells to metastasize, which is the current cancer. An important reason for difficult treatment.
  • ADC Antibody-drug conjugate
  • ADC which uses monoclonal antibodies to specifically recognize specific antigens on the surface of tumor cells, so as to accurately deliver anti-tumor drugs (such as small molecule chemotherapy drugs) to tumors.
  • the target cells are released and achieve the purpose of accurately killing the tumor.
  • ADC is also considered to be the most potential anti-tumor drug because of its proper molecular weight, high stability, high targeting, and low toxicity.
  • the successful development of ADC also has many problems that must be considered and must be solved. For example, antibodies should specifically identify lesions, have low immunosensitivity, and can efficiently and rapidly undergo endocytosis; antibody-drug linkers are stable in blood. High in sex and capable of being specifically activated and efficiently released in targeted cells Small molecule drugs are released; the coupled small molecule drug cells have strong killing ability.
  • TF plays an important role in the development of tumors, and antibody-drug conjugates have their unique characteristics and advantages. However, there is currently no high specific antibody conjugate for human TF.
  • an antibody-drug conjugate comprising:
  • a coupling moiety coupled to the antibody moiety being selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof;
  • the heavy chain variable region of the antibody comprises the following three complementarity determining region CDRs:
  • amino acid sequence of any one of the above heavy chain variable region amino acid sequences further comprises a derivative sequence which optionally adds, deletes, modifies and/or substitutes at least one amino acid and is capable of retaining TF binding affinity;
  • the light chain variable region of the antibody comprises the following three complementarity determining region CDRs:
  • amino acid sequence of any one of the above light chain variable region amino acid sequences is subjected to addition, deletion, modification and/or substitution of at least one amino acid derivative sequence having TF binding affinity.
  • the antibody comprises an intact antibody or an active fragment thereof.
  • the active fragment retains binding activity bound to tissue factor.
  • the antibody drug conjugate ADC is represented by the following formula:
  • Ab is an anti-TF antibody
  • D is a drug
  • subscript p is a value selected from 1-10, preferably 1-8.
  • LU is selected from the group consisting of 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-val-cit-PAB), 6-maleimidocaproyl-alanine-phenylalanine-p-aminobenzyloxycarbonyl (MC-ala-phe-PAB), maleimido Propionyl-valine-citrulline-p-aminobenzyloxycarbonyl (MP-val-cit-PAB), maleimidopropionyl-alanine-phenylalanine-p-aminobenzyloxycarbonyl (MP) -ala-phe-PAB), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl) Cyclohexane-1-carboxylate (SMCC), 4-(2-pyridyl
  • LU is SMCC, SPP, SPDB or 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-val-cit-PAB).
  • D is selected from the group consisting of: maytansin derivatives (DM1, DM4), auristatin and dolastatin.
  • the D is selected from the group consisting of Monomethyl auristatin E (MMAE), Monomethylauristatin F (MMAF), Monomethyl Dolastatin 10 (MMAD) derivatives, or a combination thereof.
  • MMAE Monomethyl auristatin E
  • MMAF Monomethylauristatin F
  • MMAD Monomethyl Dolastatin 10
  • amino acid residue linked to D is originally present in the antibody (parent antibody) or introduced exogenously.
  • the amino acid residue linked to D is a cysteine amino acid.
  • the cysteine amino acid is in a parent antibody in a light chain according to the Kabat numbering rules
  • One or more free cysteamines introduced at one or more locations and/or at one or more locations of the heavy chain in accordance with the Kabat numbering rules and at one or more locations of the heavy chain in accordance with the EU numbering rules Acid amino acid.
  • the amino acid residue linked to D is lysine.
  • the fragment is selected from the group consisting of a Fab, F(ab')2, Fv or scFv fragment.
  • the antibody is a monoclonal antibody.
  • the antibody comprises: a double-stranded antibody, a single-chain antibody.
  • the antibody is recombinant.
  • the antibody is produced in a bacterium such as E. coli.
  • the antibody is produced in a eukaryotic cell, such as a CHO cell.
  • the antibody is selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof; more specifically, the antibody is a humanized antibody.
  • the affinity of the antibody to human TF protein was EC 50 0.005-0.05nM, or more preferably 0.01-0.03nM 0.01-0.02nM.
  • the antibody does not bind to a wild-type murine TF protein.
  • the antibody has one or more characteristics selected from the group consisting of:
  • the heavy chain variable region sequence of the antibody is selected from the group consisting of SEQ ID NO.: 7, 9, 10, 11, 12, or 13; and/or
  • the light chain variable region sequence of the antibody is selected from the group consisting of SEQ ID NO.: 8, 14, 15, 16, or 17.
  • the antibody is selected from the group consisting of TF-mAb-SC1, TF-mAb-Ch, TF-mAb-H29, TF-mAb-H30, TF-mAb-H31, TF-mAb-H32 , TF-mAb-H33, TF-mAb-H34, TF-mAb-H35, TF-mAb-H36, TF-mAb-H37, TF-mAb-H38, TF-mAb-H39, TF-mAb-H40, TF - mAb-H41, TF-mAb-H42, TF-mAb-H43, TF-mAb-H44, TF-mAb-H45, TF-mAb-H46, TF-mAb-H47, TF-mAb-H48.
  • an antibody-drug conjugate of the first aspect of the invention for (a) preparing a diagnostic reagent; and/or (b) preparing a prophylactic and/or therapeutic TF Drugs related to the disease.
  • the TF-related disease is selected from the group consisting of tumor development, growth and/or metastasis; thrombosis-related diseases; inflammation; metabolic-related diseases;
  • the tumor is a tumor with high expression of TF.
  • the high expression of TF refers to the ratio of the level L1 of the TF transcript and/or protein in the tumor tissue to the level L0 of the transcript and/or protein in the normal tissue, L1/L0 ⁇ 2, Good land ⁇ 3.
  • the tumor is selected from the group consisting of triple negative breast cancer, pancreatic cancer, lung cancer, and malignant glioma.
  • composition comprising:
  • an active ingredient which is an antibody drug conjugate according to the first aspect of the invention, or a combination thereof;
  • the pharmaceutical composition is in the form of a human unit dosage.
  • the pharmaceutical composition is a liquid formulation.
  • the antibody drug conjugate is present in the pharmaceutical composition in an amount of from 0.005 to 50% by weight, preferably from 0.05 to 10% by weight.
  • the medicament further comprises (iii) an additional therapeutic agent.
  • the additional therapeutic agent comprises a chemotherapeutic agent.
  • a method of non-therapeutic inhibition of tumor cells in vitro comprising the step of contacting said tumor cells with an antibody drug conjugate of the first aspect.
  • a method of treating a tumor comprising the step of: administering to a subject in need thereof the antibody-drug conjugate of the first aspect.
  • the subject is a mammal, including a human.
  • the contacting is carried out in an in vitro culture system.
  • a method of slowing tumor growth in a subject comprising the steps of: combining an effective amount of the antibody drug conjugate of the first aspect with one or more treatments selected from the group consisting of : radiation therapy, chemotherapeutic treatment, biological therapy, or a combination thereof.
  • a method of inhibiting cell migration in a subject of treatment comprising the step of: combining an effective amount of the antibody drug conjugate of the first aspect with one or more treatments selected from the group consisting of: Radiation therapy, chemotherapeutic agent therapy, biological therapy, or a combination thereof.
  • a method of inhibiting cell adhesion in a subject of treatment comprising the steps of: combining an effective amount of the antibody drug conjugate of the first aspect with one or more selected from the group consisting of Treatment: radiation therapy, chemotherapeutic treatment, biological therapy, or a combination thereof.
  • a method of making a humanized or chimeric antibody comprising the steps of:
  • a human-mouse chimeric antibody is expressed by transfecting an animal cell.
  • the humanized antibody is expressed by transfecting the animal cell.
  • the antibody is a partially or fully humanized monoclonal antibody.
  • Figure 1 shows that TF-mAb-DM1 significantly inhibits the growth of tumor cells with high expression of TF, and its inhibitory effect is directly proportional to the number of TF molecules on the cell surface.
  • the left panel shows the curve of TF-mAb-DM1 which can inhibit the growth of tumor cells with high expression of TF.
  • the right panel shows the IC 50 value of TF-mAb-DM1 on different cell lines.
  • Figure 2 shows that TF-mAb-MMAE significantly inhibits the growth of tumor cells with high expression of TF, and its inhibition is directly proportional to the number of TF molecules on the cell surface.
  • the left panel shows the curve of TF-mAb-MMAE which can inhibit the growth of tumor cells with high expression of TF.
  • the right panel shows the IC 50 value of TF-mAb-MMAE on different cell lines.
  • Figures 3A and 3B show that TF-mAb-DM1 (see Figure 3A) and TF-mAb-MMAE (see Figure 3B) inhibit the growth of different tumor cells in proportion to the number of molecules on the surface of each cell.
  • the relative molecular number of TF on different cell surfaces was analyzed by CCLE database (Arrays_2013-03-18.tar.gz, Broad-Novartis Cancer Cell line Encyclopedia), and the results showed that TF-mAb-DM1 and TF-mAb-MMAE grew on different cells.
  • the inhibitory effect is proportional to the number of molecules of TF on each cell surface.
  • Figure 4A shows that TF-mAb-DM1 is effective in inhibiting the growth of HCC1806 orthotopic xenografts in a dose-dependent manner. Moreover, compared with the Docetaxel group, the body weight of the TF-mAb-DM1 group did not decrease, indicating that the TF-mAb-DM1 side effect was small.
  • Figure 4B is a graph showing the change in body weight of nude mice.
  • Figure 5A shows that TF-mAb-MMAE is effective at inhibiting the growth of HCC1806 orthotopic xenografts at different doses in a dose-dependent manner.
  • TF-mAb-MMAE almost completely inhibited the growth of HCC1806 orthotopic tumors at a dose of 3.75 mg/kg.
  • the body weight of the TF-mAb-MMAE group did not decrease, indicating that the TF-mAb-MMAE side effects were less.
  • Figure 5B is a graph showing changes in body weight of nude mice.
  • Figure 6 shows that TF-mAb-MMAE is also effective in inhibiting the growth of HCC1806 orthotopic xenografts at lower doses, and this experiment showed that the minimum effective dose was 0.7 mg/kg.
  • Figure 7A shows that TF-mAb-MMAE is effective in inhibiting the growth of BxPC-3 subcutaneous xenografts in a dose-dependent manner. Moreover, compared with the Docetaxel group, the body weight of the TF-mAb-MMAE group did not decrease, indicating that the TF-mAb-MMAE side effects were less.
  • Figure 7B is a graph showing the change in body weight of nude mice.
  • Figure 8 shows the results of molecular sieve high performance liquid chromatography of TF-mAb-H39-MMAE.
  • Figure 9 shows the results of hydrophobic chromatography of TF-mAb-H39-MMAE.
  • Figure 10 is a mass spectrometric characterization result of TF-mAb-H39-MMAE.
  • Figure 11 shows the results of molecular sieve high performance liquid chromatography of TF-mAb-H44-MMAE.
  • Figure 12 shows the results of hydrophobic chromatography of TF-mAb-H44-MMAE.
  • Figure 13 is a mass spectrometric characterization result of TF-mAb-H44-MMAE.
  • TF-mAb-H39-MMAE can significantly inhibit the growth of tumor cells TF expression and TF is proportional to the number of cell surface molecules, the right table corresponding IC 50 values.
  • Figure 15 shows TF-mAb-H44-MMAE can significantly inhibit the growth of tumor cells TF expression and TF is proportional to the number of cell surface molecules, the right table corresponding IC 50 values.
  • Figure 16A and Figure 16B show the relative molecular number of TF on different cell surfaces analyzed by the CCLE database (Arrays_2013-03-18.tar.gz, Broad-Novartis Cancer Cell line Encyclopedia), and the results show that TTF-mAb-H39-MMAE (see Figure The killing effect of 16A) and TF-mAb-H44-MMAE (see Figure 16B) on different cells is directly proportional to the number of molecules of TF on each cell surface.
  • Figure 17 shows that TF-mAb-H44-MMAE can effectively inhibit the growth of HCC1806 orthotopic xenografts, and completely inhibit the growth of HCC1806 tumors at a dose of 3 mg/kg.
  • Figure 18 shows that TF-mAb-H39-MMAE is effective in inhibiting the growth of HCC1806 orthotopic xenografts with a minimum effective dose of 1 mg/kg.
  • Figure 19TF-mAb-H44-MMAE can effectively inhibit the growth of BxPC-3 subcutaneous xenografts, and can completely inhibit the growth of BxPC-3 subcutaneous xenografts at a dose of 1 mg/kg.
  • Figure 20TF-mAb-H39-MMAE is effective in inhibiting the growth of BxPC-3 subcutaneous xenografts with a minimum effective dose of 0.3 mg/kg.
  • the inventors have unexpectedly obtained an anti-TF monoclonal antibody TF-mAb-SC1 through extensive screening, and the experimental results show that the monoclonal antibody against the TF protein is an IgG2b type antibody.
  • the highly specific antibody capable of binding TF antigen with high affinity ELISA which measured about 0.019 nM EC 50
  • said antibody has significant anti-tumor activity, but does not have visible mammals
  • the toxic side effects The chimeric antibody, the humanized antibody, and the corresponding ADC obtained based on the TF-mAb-SC1 also have excellent characteristics.
  • the present invention has been completed on this basis.
  • antibody drug conjugate As used herein, the terms “antibody drug conjugate”, “antibody conjugate”, “antibody drug conjugate”, “antibody-drug conjugate” “immunoconjugate” are used interchangeably and mean (a a conjugate of an antibody or active fragment thereof with (b) a drug.
  • antibody drug conjugate of the invention is used interchangeably to refer to an antibody of the invention having a tissue factor or A conjugate of the active fragment with the drug.
  • antibody or "immunoglobulin” is a heterotetramer of about 150,000 daltons having the same structural features.
  • a glycoprotein consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds.
  • Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions.
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain .
  • Particular amino acid residues form an interface between the variable regions of the light and heavy chains.
  • variable means that certain portions of the variable regions of an antibody differ in sequence, which form the binding and specificity of various specific antibodies for their particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the variable region of the light and heavy chains called the complementarity determining region (CDR) or hypervariable region. The more conserved portion of the variable region is referred to as the framework region (FR).
  • the variable regions of the native heavy and light chains each comprise four FR regions which are substantially in a beta-sheet configuration and are joined by three CDRs forming a linker, in some cases forming a partial beta sheet structure.
  • the CDRs in each chain are closely joined together by the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH Publ. No. 91-3242, Vol. I, pp. 647-669). (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as antibody-dependent cytotoxicity of the participating antibodies.
  • the "light chain" of a vertebrate antibody can be classified into one of two distinct classes (called kappa and lambda) depending on the amino acid sequence of its constant region.
  • Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy chain constant regions corresponding to different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
  • variable regions which are divided into four framework regions (FR), four
  • FR framework regions
  • the amino acid sequence of FR is relatively conservative and is not directly involved in the binding reaction.
  • CDRs form a cyclic structure in which the ⁇ -sheets formed by the FRs are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • the amino acid sequence of the same type of antibody can be compared to determine which amino acids constitute the FR or CDR regions.
  • the present invention encompasses not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
  • antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be obtained by standard DNA recombination techniques, all of which are useful antibodies.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as a variable region having a monoclonal antibody from a murine, and a chimeric antibody from a constant region of a human immunoglobulin (see, e.g., U.S. Patent 4,816,567 and U.S. Patent No. 4,816,397, incorporated herein by reference in its entirety herein.
  • a humanized antibody refers to an antibody molecule derived from a non-human species having one or more complementarity determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Patent 5,585,089, This article is hereby incorporated by reference in its entirety.
  • CDRs complementarity determining regions
  • These chimeric and humanized monoclonal antibodies can be prepared using recombinant DNA techniques well known in the art.
  • the antibody may be monospecific, bispecific, trispecific, or more multiple specificity.
  • the antibody of the present invention further includes a conservative variant thereof, which means that there are up to 10, preferably up to 8, more preferably up to 5, optimally compared to the amino acid sequence of the antibody of the present invention. Up to 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.
  • the present invention provides a highly specific and high affinity antibody against TF comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region (VH) amino acid sequence, the light chain comprising a light chain variable Region (VL) amino acid sequence.
  • VH heavy chain variable region
  • VL light chain variable Region
  • the respective CDRs of the heavy chain variable region (VH) amino acid sequence and the light chain variable region (VL) amino acid sequence are selected from the group consisting of:
  • the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence preferably has a homology of at least 80%, preferably at least 85%, more preferably at least 90. %, optimally at least 95% of the amino acid sequence.
  • the antibody has an activity of inhibiting a TF-related signaling pathway; has anticoagulant activity; has anti-FXa production activity, or a combination thereof.
  • the invention provides an anti-TF antibody having: a heavy chain variable region of the invention; and/or a light chain variable region of the invention;
  • the heavy chain variable region of the antibody comprises the following three complementarity determining region CDRs:
  • any one of the above amino acid sequences further comprises a derivative sequence which optionally adds, deletes, modifies and/or substitutes at least one amino acid and is capable of retaining TF binding affinity;
  • the light chain variable region of the antibody comprises the following three complementarity determining region CDRs:
  • Any one of the above amino acid sequences is subjected to addition, deletion, modification and/or substitution of at least one amino acid derivative sequence having TF binding affinity.
  • the heavy chain variable region sequence of the antibody is selected from the group consisting of SEQ ID NO.: 7, 9, 10, 11, 12, or 13; and/or the light chain variable region of the antibody The sequence is selected from the group consisting of SEQ ID NO.: 8, 14, 15, 16, or 17.
  • the antibody is selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof.
  • the number of amino acids added, deleted, modified and/or substituted does not exceed 40% of the total amino acid number of the initial amino acid sequence.
  • the number of amino acids added, deleted, modified and/or substituted is from 1 to 7.
  • the at least one amino acid sequence added, deleted, modified and/or substituted is an amino acid sequence having a homology of at least 80%.
  • the at least one amino acid added, deleted, modified, and/or substituted has any one or more of an activity of inhibiting TF-related signaling pathway, anticoagulant activity, and anti-FXa production activity. .
  • the antibody of the present invention may be a double-stranded or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a humanized antibody, more preferably a humanized antibody, a human-animal chimeric antibody, more preferably a whole Humanized antibody.
  • the antibody derivative of the present invention may be a single chain antibody, and/or an antibody fragment such as Fab, Fab', (Fab') 2 or other known antibody derivatives in the field, and IgA, IgD, IgE. Any one or more of IgG and IgM antibodies or antibodies of other subtypes.
  • the animal is preferably a mammal, such as a mouse.
  • the antibody of the invention may be a chimeric antibody, a humanized antibody, a CDR grafted and/or a modified antibody that targets human TF.
  • any one or more of the above SEQ ID No.: 1 - SEQ ID No.: 3, or they are added, deleted, modified and/or substituted for at least one amino acid A sequence having TF binding affinity, located in the CDR region of the heavy chain variable region (VH).
  • any one or more of the above SEQ ID No.: 4-SEQ ID No.: 6, or they are added, deleted, modified and/or substituted for at least one amino acid A sequence having TF binding affinity, located in the CDR region of the light chain variable region (VL).
  • VH CDR1, CDR2, CDR3 are each independently selected from any one or more of SEQ ID No.: 1 - SEQ ID No.: 3, or they are added, A sequence having TF binding affinity that lacks, modifies, and/or substituted at least one amino acid;
  • VL CDR1, CDR2, CDR3 are each independently selected from any one or more of SEQ ID No.: 4-SEQ ID No.: Or a sequence having TF binding affinity for addition, deletion, modification and/or substitution of at least one amino acid.
  • the number of amino acids added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1-33% of the total amino acid number of the initial amino acid sequence. More preferably, it is 5-30%, more preferably 10-25%, and still more preferably 15-20%.
  • the number of amino acids added, deleted, modified and/or substituted may be 1-7, more preferably 1-5, more preferably 1-3, more preferably It is 1-2.
  • the antibody that targets TF is TF-mAb-SC1 (formerly known as TF-mAb).
  • the heavy chain variable region (VH) amino acid sequence of the antibody TF-mAb-SC1 is the amino acid sequence set forth in SEQ ID NO.: 7.
  • the light chain variable region (V-Kappa) amino acid sequence of the antibody TF-mAb-SC1 is the amino acid sequence set forth in SEQ ID NO.: 8.
  • sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique such as PCR amplification or genomic library screening.
  • the coding sequences of the light and heavy chains can also be fused together to form a single chain antibody.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Preferred animal cells include, but are not limited to, CHO-S, HEK-293 cells.
  • the resulting host cells are cultured under conditions suitable for expression of the antibody of the invention.
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc.
  • the antibodies of the present invention are purified by conventional separation and purification means well known to those skilled in the art.
  • the resulting monoclonal antibodies can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • the binding affinity of a monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson et al, Anal. Biochem., 107: 220 (1980).
  • the antibodies of the invention can be expressed intracellularly, or on the cell membrane, or secreted extracellularly.
  • the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • Drugs that can be used to form the ADC of the invention include, but are not limited to, cytotoxic agents.
  • cytotoxic agent refers to a substance that inhibits or prevents the expression of cells, the function of cells, and/or the destruction of cells.
  • the term includes radioisotopes, chemotherapeutic agents, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
  • cytotoxic agents include, but are not limited to, auristatins (eg, auristatin E, auristatin F, MMAE, and MMAF), chlortetracycline, etometanol, ricin, ricin A-chain, Butatin, doxymethine, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide , vincristine, vinblastine, colchicine, dihydroxy anthrax dione, actinomycin, diphtheria toxin, pseudomonas exotoxin (PE) A, PE40, acacia toxin, abrin toxin A chain , lotus root toxin A chain, alpha-tripococcus, white toxin, mittollin, retstrictocin, phenolic acid, a
  • Preferred small molecule drugs are compounds having high cytotoxicity, preferably monomethylaustatatin, calicheamicin, maytansinoids, or combinations thereof; more preferably selected from: monomethyl au Statin -E (MMAE), monomethyl auristatin-D (MMAD), monomethyl auristatin-F (MMAF), or a combination thereof.
  • MMAE monomethyl au Statin -E
  • MMAD monomethyl auristatin-D
  • MMAF monomethyl auristatin-F
  • ADC Antibody-drug conjugate
  • the invention also provides an antibody-drug conjugate (ADC) based on an antibody of the invention.
  • ADC antibody-drug conjugate
  • the antibody-conjugated drug comprises the antibody, and an effector molecule, which is coupled to the effector molecule, and is preferably chemically coupled.
  • the effector molecule is preferably a therapeutically active drug.
  • the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug or a radionuclide.
  • the antibody of the present invention and the effector molecule may be coupled by a coupling agent.
  • the coupling agent may be any one or a combination of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond.
  • the non-selective coupling agent refers to a compound that forms a covalent bond between an effector molecule and an antibody, such as glutaraldehyde or the like.
  • the coupling agent using a carboxyl group may be any one or more of an cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acyl hydrazine coupling agent (coupling site is an acylhydrazine).
  • Certain residues on the antibody are used to link to a variety of functional groups, including imaging agents (such as chromophores and fluorophores), diagnostic reagents (such as MRI contrast agents and radioisotopes). , stabilizers (such as ethylene glycol polymers) and therapeutic agents.
  • imaging agents such as chromophores and fluorophores
  • diagnostic reagents such as MRI contrast agents and radioisotopes
  • stabilizers such as ethylene glycol polymers
  • therapeutic agents such as ethylene glycol polymers
  • the antibody can be conjugated to a functional agent to form a conjugate of the antibody-functional agent.
  • Functional agents eg, drugs, detection reagents, stabilizers
  • the functional agent can be attached to the antibody either directly or indirectly via a linker.
  • Typical coupling methods suitable for use in the present invention include both K-Lock and C-Lock coupling methods.
  • K lysine
  • C cysteine in the antibody sequence
  • Antibodies can be coupled to drugs to form antibody drug conjugates (ADCs).
  • ADC antibody drug conjugates
  • the ADC comprises a linker between the drug and the antibody.
  • the linker can be a degradable or non-degradable linker.
  • Degradable linkers are typically susceptible to degradation under the intracellular environment, such as degradation of the linker at the target site, thereby releasing the drug from the antibody.
  • Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (eg, lysosomal proteases or endosomal proteases), or sugar linkers, for example, which can be glucuronide Enzymatically degraded glucuronide-containing linker.
  • Peptidyl linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine or valine-alanine.
  • Other suitable degradable linkers include, for example, pH sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as barium splices) and linkers that degrade under reducing conditions (e.g., disulfide bond linkers).
  • Non-degradable linkers typically release the drug under conditions in which the antibody is hydrolyzed by a protease.
  • the linker Prior to attachment to an antibody, the linker has an reactive reactive group capable of reacting with certain amino acid residues, and attachment is achieved by reactive reactive groups.
  • Sulfhydryl-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (eg, iodine, bromine or chlorinated); haloesters (eg, iodine, bromine or chlorinated) Halogenated methyl ketone (eg iodine, bromine or chlorinated), benzyl halide (eg iodine, bromine or chlorinated); vinyl sulfone, pyridyl disulfide; mercury derivative such as 3,6- Di-(mercurymethyl)dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate.
  • the linker can include, for example, a maleimide attached to the antibody via
  • the drug can be any cytotoxic, cytostatic or immunosuppressive drug.
  • the linker binds the antibody to the drug, and the drug has a functional group that can bond to the linker.
  • the drug may have an amino group, a carboxyl group, a thiol group, a hydroxyl group, or a ketone group which may be bonded to a linker.
  • the drug is directly attached to the linker, the drug has a reactive group that is reactive prior to attachment to the antibody.
  • Particularly useful classes of drugs include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapeutic sensitizers, topoisomerase inhibition Agent, vinca alkaloids, etc.
  • cytotoxic drugs include, for example, DNA minor groove binding reagents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs including, for example, auristatin, camptothecin (camptothecins), docamycin/duocarmycins, etoposides, maytansines and maytansinoids (eg DM1 and DM4), taxanes ( Taxanes), benzodiazepines or benzodiazepine containing drugs (eg pyrrolo[1,4]benzodiazepines (PBDs), porphyrin benzodiazepines Classes (indolinobenzodiazepines) and oxazolidinobenzodiazepines and vinca alkaloids.
  • typical cytotoxic drugs including, for example, auristatin, camptothecin (camptothecins), docamycin/duocarmycins, etoposides, maytansines and maytansinoids (eg DM1 and DM4), tax
  • the drug-linker can be used to form an ADC in a simple step.
  • the bifunctional linker compound can be used to form an ADC in a two or more step process. For example, a cysteine residue is reacted with a reactive moiety of the linker in a first step, and in a subsequent step, a functional group on the linker reacts with the drug to form an ADC.
  • a functional group on the linker is selected to facilitate specific reaction with a suitable reactive group on the drug moiety.
  • a portion based on an azide compound can be used to specifically react with a reactive alkynyl group on a drug moiety.
  • the drug is covalently bound to the linker by a 1,3-dipolar cycloaddition between the azide and the alkynyl group.
  • Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for amines) And alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols).
  • the invention also provides a method of making an ADC, which can further comprise: binding the antibody to a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
  • the methods of the invention comprise: binding an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the invention further comprise: binding the antibody linker conjugate to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via a linker.
  • the antibody drug conjugate ADC is represented by the following formula:
  • Ab is an antibody
  • D is a drug
  • subscript p is a value selected from 1-10, preferably 1-8.
  • the invention also provides the use of an antibody of the invention, for example for the preparation of a diagnostic preparation, or for the preparation of a medicament for the prevention and/or treatment of a TF-related disease.
  • TF-related diseases include tumorigenesis, growth and/or metastasis, thrombosis-related diseases, inflammation, metabolic-related diseases, and the like.
  • the tumor includes (but is not limited to): breast cancer (such as triple negative breast cancer), pancreatic cancer, lung cancer, glioblastoma, gastric cancer, liver cancer, esophageal cancer, kidney cancer, colorectal cancer, bladder cancer, prostate cancer , endometrial cancer, ovarian cancer, cervical cancer, leukemia, bone marrow cancer, angiosarcoma, etc.; especially triple-negative breast cancer, pancreatic cancer, malignant glioma and lung cancer, more preferably triple-negative breast cancer and/or pancreatic cancer .
  • breast cancer such as triple negative breast cancer
  • pancreatic cancer lung cancer
  • glioblastoma gastric cancer
  • liver cancer liver cancer
  • esophageal cancer kidney cancer
  • colorectal cancer bladder cancer
  • prostate cancer endometrial cancer
  • ovarian cancer cervical cancer
  • leukemia bone marrow cancer
  • angiosarcoma etc.
  • triple-negative breast cancer pancreatic cancer
  • thrombosis-related diseases include, but are not limited to, atherosclerosis, acute coronary syndrome, acute myocardial infarction, stroke, hypertension, deep vein thrombosis, pulmonary embolism, renal embolism and arterial surgery, adjacent to the coronary artery Thrombosis caused by road transplantation.
  • the inflammation includes (but is not limited to): rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, gout, Lytle syndrome, psoriasis arthropathy, infectious arthritis, tuberculous arthritis, viral joints Inflammation, fungal arthritis, glomerulonephritis, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, acute lung injury, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis.
  • the metabolic related diseases include, but are not limited to, diabetes, foodborne obesity, and fat inflammation.
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition comprising the above antibody or active fragment thereof or a fusion protein thereof or an ADC thereof, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used for binding to a TF protein molecule, and thus can be used for the prevention and treatment of diseases such as tumors.
  • other therapeutic agents can also be used simultaneously, for example, various cytokines such as TNF, IFN, IL-2, etc.; various tumor chemotherapy drugs, such as 5-FU, methotrexate and the like, which affect nucleic acid biosynthesis; nitrogen mustard, An alkylating agent such as cyclophosphamide; Doxorubicin, actinomycin D and other drugs that interfere with the transcription process to prevent RNA synthesis; vincristine, camptothecin.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001 to 99% by weight, preferably 0.01 to 90% by weight, more preferably 0.1 to 80% by weight) of the above-mentioned monoclonal antibody (or a conjugate thereof) of the present invention and pharmacy An acceptable carrier or excipient.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably prepared under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram body weight to about 5 milligrams per kilogram body weight per day.
  • the polypeptides of the invention may also be used with other therapeutic agents.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, Preferably, the dosage is from about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the antibody of the present invention has excellent biological activity and specificity and has a high affinity (the EC 50 can be as high as about 0.01 to 0.03 nM by ELISA). In addition, it has a good binding affinity to cell surface TF and can be used as an antibody targeting TF.
  • the humanized antibody of the present invention has not only activity comparable to that of a murine antibody but also lower immunogenicity.
  • the antibodies and ADCs of the present invention not only have significant therapeutic effects in a plurality of tumor models, but are also applicable to other diseases associated with high expression of TF.
  • Step 1 preparation of hybridoma cells:
  • TF extracellular domain protein was 100 ⁇ g/head to prepare immune spleen cells; timely preparation of murine myeloma cells (SP2/0) and feeder cells for fusion.
  • the spleen cells and SP2/0 cells are fused by PEG-mediated fusion, and PEG is removed.
  • the HAT complete medium containing the feeder cells was resuspended, inoculated into a 96-well plate, and subjected to positive well screening by ELISA.
  • the cells of the positive wells were cloned and cultured by limiting dilution method.
  • the cells with high titer, good morphology and monoclonal growth were screened by ELSIA or immunofluorescence to continue subcloning screening until the positive clones were screened for three consecutive times. At 100%, the cell strain can be expanded and constructed.
  • Step 2 Preparation of human TF murine monoclonal antibody ascites:
  • the hybridoma cells screened in step 1 were expanded and cultured, and the mice were intraperitoneally injected with pristane (0.5 mL/mouse) to provide a favorable environment for the growth of hybridoma cells. After 7-10 days, each intraperitoneal injection was 10 ⁇ . From the 7th day, the hybridoma cells of 10 6 were observed daily for the ascites production and mental state of the mice, and ascites was taken and centrifuged to remove the oil at -80 ° C for purification.
  • pristane 0.5 mL/mouse
  • step 2 The ascites frozen in step 2 was thawed on ice, filtered through a 0.45 ⁇ m filter, dialyzed against PBS overnight at 4 ° C, and finally purified by FPLC technique and concentrated to the desired concentration by ultrafiltration, and dispensed, Store at -80 °C for later use.
  • Step 4 Determination of the biological activity and targeting specificity of a monoclonal antibody directed against human TF mice:
  • TF-mAb-SC1 is for humans.
  • TF has a higher affinity than the other five antibodies.
  • TF extracellular domain protein was coated with ELISA plate, 0.05 ⁇ g/well, and subjected to ELISA.
  • the results showed that TF-mAb-SC1 had strong affinity for TF extracellular domain protein; cell binding affinity experiments showed that TF-mAb-SC1 has a very high binding affinity for TF-negative breast cancer cells MDA-MB-231 and pancreatic cancer cells BxPC-3 cells with high expression of TF; and TF-mAb-SC1 can significantly inhibit TF-PAR2 downstream MAPK/ The phosphorylation level of ERK is dose dependent.
  • TF-mAb-SC1 Based on the fact that TF-mAb-SC1 exhibits very high specificity, very high affinity, and significant inhibition of the phosphorylation level of MAPK/ERK, it was selected for sequencing and subsequent studies.
  • the CDR amino acid sequence of the heavy chain variable region is:
  • SEQ ID No.: 1 SYWMN;
  • SEQ ID No.: 2 MIYPADSETRLNQKFKD;
  • SEQ ID No.: 3 EDYGSSDY.
  • VH amino acid sequence is set forth in SEQ ID NO.: 7.
  • the CDR amino acid sequence of the light chain variable region is:
  • SEQ ID No.: 4 SASSSVSYMN;
  • SEQ ID No.: 5 GISNLAS
  • SEQ ID No.: 6 QQKSSFPWT.
  • VL amino acid sequence is shown in SEQ ID NO.: 8:
  • a human-mouse chimeric antibody was constructed based on the obtained highly active and specific murine TF-mAb-SC1.
  • Primers were designed to introduce EcoR I and Nhe I in the heavy chain variable region, and to introduce the Age I and BsiW I restriction endonuclease sites in the light chain variable region, and then the above-mentioned antibody heavy and light chains were obtained.
  • the variable region sequences were cloned into vectors containing the human IgG1 heavy chain constant region and the Kappa chain constant region, respectively, and the chimeric constructs were constructed using transfection techniques and mammalian expression systems (CHO-S or HEK-293 cells). The antibody was expressed and purified, and the obtained human-mouse chimeric antibody was designated as TF-mAb-Ch.
  • the CDR region of the murine antibody TF-mAb-SC1 is then transplanted onto the selected humanized template, the CDR region of the human template is replaced, and then recombined with the IgG1 constant region, and based on the three-dimensional structure of the murine antibody, Residues with embedded residues, direct interactions with CDR regions, and residues that have important effects on the conformation of VL and VH are subjected to back mutation to obtain variable regions of 5 humanized heavy chains (SEQ ID NO.
  • the TF-mAb-SC1 stock solution was replaced with a G25 desalting column into a reaction buffer (50 mM potassium phosphate/50 mM NaCl/2 mM EDTA, pH 7.5) to a final concentration of 7.8 mg/ml; then 9 eq of 11 mg/ml was added.
  • MCC-DM1 dissolved in DMA, DMA content in the reaction system was less than 5%
  • the supernatant was purified by Q column and purified by cation column to remove excess small molecules.
  • it was replaced with 10 mM PBS solution by G25 desalting column or ultrafiltration, and stored at -80 ° C until use.
  • the obtained antibody drug conjugate was named TF-mAb. -DM1.
  • the TF-mAb-SC1 stock solution was replaced with a G25 desalting column into a reaction buffer (50 mM potassium phosphate/50 mM NaCl/2 mM EDTA, pH 6.5) to a final concentration of 10 mg/ml; then 8 eq of SMCC (dissolved in DMSO) was added. The reaction was carried out at 10-12 ° C for 3 hours, and excess SMCC was removed through a G25 desalting column. Then, 12 eq of DM1 was added to P-MCC, reacted at 25 ° C for 18 hours, and finally replaced with 10 mM PBS solution by G25 desalting column, and stored at -80 ° C until use.
  • the obtained antibody drug conjugate was named TF-mAb-DM1. .
  • Example 6 In vitro antitumor activity of TF-mAb-ADCs against triple negative breast cancer cells and pancreatic cancer with high expression of TF
  • the cell lines used in this example were purchased from the American Type Culture Collection (ATCC) or the Chinese Academy of Sciences Cell Bank and cultured according to the corresponding instructions, including: MCF7, MDA453, T47D, A549, U87MG, H1975, MDA-MB- 231, BxPC-3, HCC1806, Hs578T.
  • the cells in the logarithmic growth phase were inoculated into 96-well cell culture plates at a density of 1,000-3,000 cells per well (depending on the growth rate of different cells), 150 ⁇ L/well, 37 ° C, 5% CO. 2
  • different concentrations of TF-mAb-ADCs ie TF-mAb-DM1 and TF-mAb-MMAE
  • 3 replicate wells were set for each drug concentration, and the corresponding vehicle control and blank control wells were set.
  • TF-mAb-DM1 and TF-mAb-MMAE could inhibit the growth of tumor cells with high expression of TF in vitro, and the inhibition was proportional to the number of TF molecules on the cell surface.
  • the left panel shows the curve of TF-mAb-DM1 which can inhibit the growth of tumor cells with high expression of TF.
  • the right panel shows the IC 50 value of TF-mAb-DM1 on different cell lines. 2
  • the left is TF-mAb-MMAE can better inhibit the expression profile of tumor cell growth of TF
  • Example 7 In vivo antitumor activity of TF-mAb-ADCs against triple negative breast cancer and pancreatic cancer models with high expression of TF
  • the HCC1806 and BxPC-3 cells in the logarithmic growth phase were inoculated subcutaneously into the 6-week-old Balb/c female nude mouse or the back of the pad at a density of 3 ⁇ 10 6 and 10 ⁇ 10 6 per 200 ⁇ L of serum-free medium, respectively.
  • Balb/c nude mice were purchased from Shanghai Xipuer-Beikai Experimental Animal Co., Ltd.). After the tumors were grown to 100-200 mm 3 , the animals were randomly divided into 8 tumors in each group.
  • TF-mAb-DM1 was administered once a week at 3.75 mg/kg and 15 mg/kg, TF-mAb-MMAE at doses of 0.7 mg/kg, 2 mg/kg, 3.75 mg/kg, 7 mg/kg and 15 mg/kg.
  • the tail vein was administered, and normal mouse IgG (IgG) and docetaxel (Dacetaxel) were used as negative and positive control drugs, respectively.
  • Tumor volume and nude mouse weight were measured 2-3 times per week and recorded to plot tumor growth curves.
  • the tumor volume (V) is calculated as:
  • V 1/2 ⁇ a ⁇ b 2
  • a and b represent the length and width of the tumor, respectively.
  • Figure 4A shows that TF-mAb-DM1 inhibits the growth curve of HCC1806 orthotopic xenografts in a dose-dependent manner.
  • Figure 4B is a graph showing changes in body weight of nude mice.
  • Figure 5A and Figure 6 show that TF-mAb-MMAE inhibits the growth curve of HCC1806 orthotopic tumors at different doses, and Figure 5B shows the changes in body weight of nude mice. From the results, it was found that TF-mAb-MMAE can effectively inhibit the growth of HCC1806 tumor at a dose of 0.7 mg/kg, and the growth of HCC1806 tumor can be almost completely inhibited at a dose of 3.75 mg/kg.
  • TF-mAb-MMAE was effective in inhibiting the growth of BxPC-3 subcutaneous xenografts in a dose-dependent manner. Moreover, compared with the Docetaxel group, the body weight of the TF-mAb-MMAE group did not decrease, indicating that the TF-mAb-MMAE side effects were less.
  • Figure 7B is a graph showing changes in body weight of nude mice.
  • Example 4-7 was repeated except that TF-mAb-SC1 was replaced with the following humanized antibody: TF-mAb-H29, TF-mAb-H30, TF-mAb-H31, TF-mAb-H32, TF-mAb -H33, TF-mAb-H34, TF-mAb-H35, TF-mAb-H36, TF-mAb-H37, TF-mAb-H38, TF-mAb-H39, TF-mAb-H40, TF-mAb-H41 TF-mAb-H42, TF-mAb-H43, TF-mAb-H44, TF-mAb-H45, TF-mAb-H46, TF-mAb-H47, TF-mAb-H48.
  • humanized antibody TF-mAb-H29, TF-mAb-H30, TF-mAb-H31, TF-mAb-H32, TF-mAb -H33
  • conjugates of these humanized antibodies with MMAE were separately prepared, wherein the TF-mAb-H39-MMAE and TF-mAb-H44-MMAE were characterized as follows.
  • Figure 8 is a molecular sieve high performance liquid chromatography of TF-mAb-H39-MMAE;
  • Figure 9 is a hydrophobic chromatography chromatogram of TF-mAb-H39-MMAE;
  • Figure 10 is a mass spectrum of TF-mAb-H39-MMAE.
  • Figure 11 is a molecular sieve high performance liquid chromatography of TF-mAb-H44-MMAE;
  • Figure 12 is a hydrophobic chromatography chromatogram of TF-mAb-H44-MMAE;
  • Figure 13 is a mass spectrum of TF-mAb-H44-MMAE.
  • test results indicate that ADCs based on these humanized antibodies also have high affinity (compared to TF-mAb-MMAE, preferably the relative affinity of the ADC of the humanized antibody is between 60-140%), high cytotoxicity , and low toxic side effects, and showed significant anti-tumor effects.
  • TF-mAb-H39-MMAE significantly inhibited the growth of TF-expressing tumor cells and was proportional to the number of TF molecules on the cell surface.
  • the right panel is TF-mAb-H39-MMAE. IC 50 values on the different cell lines.
  • TF-mAb-H44-MMAE significantly inhibited the growth of TF-expressing tumor cells and was directly proportional to the number of TF molecules on the cell surface.
  • the right panel is TF-mAb-H44-MMAE in different cell lines. IC 50 value on.
  • TF-mAb-H44-MMAE was effective in inhibiting the growth of HCC1806 tumors in a dose-dependent manner, and completely inhibited HCC1806 tumor growth at a dose of 3 mg/kg.
  • the minimum effective dose (MED) was 1 mg/kg.
  • TF-mAb-H39-MMAE was effective in inhibiting the growth of HCC1806 tumors with a minimum effective dose (MED) of 1 mg/kg.
  • MED minimum effective dose
  • TF-mAb-H44-MMAE significantly inhibited BxPC-3 tumor growth in a dose-dependent manner and completely inhibited BxPC-3 tumor growth at a dose of 1 mg/kg.
  • TF-mAb-H39-MMAE significantly inhibited BxPC-3 tumor growth with a minimum effective dose (MED) of 0.3 mg/kg.
  • MED minimum effective dose

Abstract

靶向于组织因子的抗体-药物偶联物以及制备该抗体-药物偶联物（ADC）的方法。该ADC能够高特异性地结合TF抗原，其具有很高的亲和力、很低的免疫原性，以及很高的细胞毒性，并且具有显著的抗肿瘤作用。

Description

靶向于组织因子的抗体-药物偶联物 技术领域
本发明涉及医药领域，具体地涉及一种靶向于组织因子的抗体及其抗体-药物偶联物、以及制法和用途。
背景技术
组织因子(Tissue factor，TF)是一个47kDa的跨膜糖蛋白。正常生理状态下TF表达主要屏蔽于血管内皮下细胞层，一旦机体血管受到创伤，TF暴露于血流，通过结合并激活VII因子从而启动外源性凝血反应。
研究发现，TF在众多肿瘤组织中异常激活表达，在肿瘤的发生和发展过程中起着重要作用。特别是在癌症晚期，病人大多伴随自发性血栓，如深度静脉血栓(Deep-vein thrombosis，DVT)、弥漫性血管内凝血(Disseminated intravascular coagulation，DIC)和肺栓塞(Pulmonary embolism，PE)等(Thrombosis research,2013,131:S59-S62；Journal of Thrombosis and Haemostasis,2011,9(s1):306-315)。TF在肿瘤细胞中的异常表达则是这些症状发生的主要诱因。对众多肿瘤临床样本分析表明，TF的表达水平直接影响肿瘤的转移、病人血栓的发生等恶化指标，如在乳腺癌中TF异常表达率为85.8％，在胰腺癌中为88.5％，在肺癌中为83.6％，食道癌中为91.3％等(Blood,2012,119:924-932)。
研究表明，首先，TF与FVII形成TF-FVIIa复合物,能直接结合并诱导跨膜G蛋白偶联受体Protease-activated receptor 2(PAR2)的活化。PAR2是调控炎症反应的重要信号通路，虽然对PAR2在肿瘤领域的研究目前还比较少，但可以想象，TF通过PAR2能影响细胞内一系列肿瘤功能信号。如TF-FVIIa-PAR2通过MAPK/ERK磷酸化，诱导关键生长因子、免疫调节因子和趋化因子的基因表达(如VEGF、CSF1/2、IL8、CXCL1等)，促进新生血管的形成，为肿瘤的生长提供了充足的养分、能量和适宜的微环境。其次，TF还可以通过与Rac1、β1家族相关整合素的相互作用，以提高肿瘤细胞的迁移性和粘附性，从而在整体上增强肿瘤细胞的血行转移能力。再次，TF起始的凝血作用也是肿瘤性血栓发生的重要诱因，导致多种癌症恶化。同时，TF诱导的高凝状态又直接有助于肿瘤细胞逃离机体免疫系统的攻击，并增加了肿瘤细胞与内皮细胞的相互作用，导致肿瘤细胞的血行转移能力的提高，这也正是当前癌症难治疗的重要原因。
抗体-药物偶联物(antibody-drug conjugate，ADC)，是利用单克隆抗体特异性识别肿瘤细胞表面特定抗原的特点，从而实现精准地将抗肿瘤药物(如小分子化疗药物等)递送到肿瘤靶细胞并释放，达到精准杀伤肿瘤的目的。ADC也因为其分子量大小合适，稳定性高，靶向性强，毒副作用小被认为是最具潜力的抗肿瘤药物。但成功开发ADC也存在诸多必须考虑且必须解决的问题，如抗体要特异性的识别病变部位，免疫致敏性低，能够高效迅速的发生细胞内吞作用；抗体-药物接头，在血液中稳定性要高并能在靶向细胞中特异的被激活并高效释 放小分子药物；所偶联的小分子药物细胞杀伤能力要强等。
可见，TF在肿瘤发生发展过程中起着重要作用，而抗体-药物偶联物因其独具的特点和优势，然而，目前尚缺乏高特异性的针对人TF的抗体药物偶联物。
发明内容
本发明目的就是提供了一种特异性针对人TF的抗体-药物偶联物，它具有特异性靶向于人TF、具有抑制肿瘤生长和转移活性等特性。
在本发明第一方面，提供了一种抗体-药物偶联物，所述抗体药物偶联物含有：
(a)抗体部分；和
(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合；
其中，所述抗体的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO.:1所示的CDR1，
SEQ ID NO.:2所示的CDR2，和
SEQ ID NO.:3所示的CDR3；
其中，上述重链可变区氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留TF结合亲和力的衍生序列；
所述抗体的轻链可变区包括以下三个的互补决定区CDR：
SEQ ID NO.:4所示的CDR1'，
SEQ ID NO.:5所示的CDR2'，和
SEQ ID NO.:6所示的CDR3'；
其中，上述轻链可变区氨基酸序列中任意一种氨基酸序列经过添加、缺失、修饰和/或取代至少一个氨基酸的具有TF结合亲和力的衍生序列。
在另一优选例中，所述的抗体包括完整抗体或其活性片段。
在另一优选例中，所述的活性片段保留了结合于组织因子的结合活性。
在另一优选例中，抗体药物偶联物ADC如下分子式所示：
Figure PCTCN2017087779-appb-000001
其中：
Ab是抗TF的抗体，
LU是接头；
D是药物；
而且下标p是选自1-10，较佳地1-8的值。
在另一优选例中，LU选自下组：6-马来酰亚氨基己酰基-缬氨酸-瓜氨酸-对氨基苄氧羰 基(MC-val-cit-PAB)、6-马来酰亚氨基己酰基-丙氨酸-苯丙氨酸-对氨基苄氧羰基(MC-ala-phe-PAB)、马来酰亚氨基丙酰基-缬氨酸-瓜氨酸-对氨基苄氧羰基(MP-val-cit-PAB)、马来酰亚氨基丙酰基-丙氨酸-苯丙氨酸-对氨基苄氧羰基(MP-ala-phe-PAB)、N-琥珀酰亚氨基4-(2-吡啶基硫基)戊酸酯(SPP)、N-琥珀酰亚氨基4-(N-马来酰亚氨基甲基)环己烷-1-羧酸酯(SMCC)、4-(2-吡啶基二硫代)丁酸-N-羟基琥珀酰亚胺酯(SPDB)或N-琥珀酰亚氨基(4-碘-乙酰基)氨基苯甲酸酯(SIAB)。
在另一优选例中，其中LU是SMCC、SPP、SPDB或6-马来酰亚氨基己酰基-缬氨酸-瓜氨酸-对氨基苄氧羰基(MC-val-cit-PAB)。
在另一优选例中，其中D选自下组：美登素衍生物(DM1,DM4)，auristatin和多拉司他汀。
在另一优选例中，所述的D选自下组：Monomethyl auristatin E(MMAE)，Monomethylauristatin F(MMAF)，Monomethyl Dolastatin 10(MMAD)类衍生物或其组合。
Figure PCTCN2017087779-appb-000002
在另一优选例中，所述的与D相连的氨基酸残基是原本存在于抗体(亲本抗体)或外源引入的。
在另一优选例中，所述的与D相连的氨基酸残基为半胱氨酸氨基酸。
在另一优选例中，所述半胱氨酸氨基酸是在亲本抗体中在依照Kabat编号规则的轻链的 一个或多个位置处和/或在依照Kabat编号规则的重链的一个或多个位置处和在依照EU编号规则的重链的一个或多个位置处所引入的一个或多个游离半胱氨酸氨基酸。
在另一优选例中，所述的与D相连的氨基酸残基为赖氨酸。
在另一优选例中，所述片段选自下组：Fab、F(ab′)2、Fv或scFv片段。
在另一优选例中，所述的抗体是单克隆抗体。
在另一优选例中，所述的抗体包括：双链抗体、单链抗体。
在另一优选例中，所述抗体是重组的。
在另一优选例中，所述抗体是在细菌(如大肠杆菌)中生成的。
在另一优选例中，所述抗体是在真核细胞(如CHO细胞)中生成的。
在另一优选例中，述抗体选自：动物源抗体、嵌合抗体、人源化抗体、或其组合；更加地，所述抗体是人源化抗体。
在另一优选例中，所述抗体对人TF蛋白的亲和力的EC50为0.005-0.05nM，更佳地0.01-0.03nM或0.01-0.02nM。
在另一优选例中，所述抗体不结合于野生型的鼠TF蛋白。
在另一优选例中，所述抗体具有选自下组的一个或多个特性：
(a)抑制肿瘤细胞迁移或转移；
(b)抑制肿瘤生长。
在另一优选例中，所述抗体的重链可变区序列选自下组：SEQ ID NO.:7、9、10、11、12、或13；和/或
所述的抗体的轻链可变区序列选自下组：SEQ ID NO.:8、14、15、16、或17。
在另一优选例中，所述抗体选自下组：TF-mAb-SC1、TF-mAb-Ch、TF-mAb-H29、TF-mAb-H30、TF-mAb-H31、TF-mAb-H32、TF-mAb-H33、TF-mAb-H34、TF-mAb-H35、TF-mAb-H36、TF-mAb-H37、TF-mAb-H38、TF-mAb-H39、TF-mAb-H40、TF-mAb-H41、TF-mAb-H42、TF-mAb-H43、TF-mAb-H44、TF-mAb-H45、TF-mAb-H46、TF-mAb-H47、TF-mAb-H48。
在本发明的第二方面，提供了本发明第一方面的抗体-药物偶联物的应用，所述抗体用于(a)制备诊断试剂；和/或(b)制备预防和/或治疗TF相关的疾病的药物。
在另一优选例中，所述TF相关的疾病选自下组：肿瘤的发生、生长和/或转移；血栓类相关疾病；炎症；代谢相关疾病；或其组合。
在另一优选例中，所述肿瘤为TF高表达的肿瘤。
在另一优选例中，所述的TF高表达指肿瘤组织中TF转录本和/或蛋白的水平L1与正常组织中转录本和/或蛋白的水平L0之比，L1/L0≥2，较佳地≥3。
在另一优选例中，所述的肿瘤选自下组：三阴性乳腺癌、胰腺癌、肺癌和恶性胶质瘤。
在本发明的第三方面，提供了一种药物组合物，它含有：
(i)活性成分，所述活性成分为如本发明第一方面所述的抗体药物偶联物或其组合；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为人用单位剂量形式。
在另一优选例中，所述药物组合物为液态制剂。
在另一优选例中，所述药物组合物中，所述抗体药物偶联物的含量为0.005-50wt％，较佳地0.05-10wt％。
在另一优选例中，所述的药物还包括(iii)额外的治疗剂。
在另一优选例中，所述的额外治疗剂包括化疗剂。
在本发明的第四方面，提供了一种体外非治疗性抑制肿瘤细胞的方法，包括步骤：将所述肿瘤细胞与第一方面的抗体药物偶联物接触。
在本发明的第五方面，提供了一种治疗肿瘤的方法，包括步骤：给需要的对象使用第一方面的抗体-药物偶联物。
在另一优选例中，所述对象为哺乳动物，包括人。
在另一优选例中，所述的接触是在体外培养体系中进行。
在本发明的第六方面，提供了一种减缓治疗对象中肿瘤生长的方法，包括步骤：联用有效量的第一方面的抗体药物偶联物与一种或多种选自下组的治疗：辐射治疗、化疗剂治疗、生物治疗、或其组合。
在本发明的第七方面，提供了一种抑制治疗对象中细胞迁移的方法，包括步骤：联用有效量第一方面的抗体药物偶联物与一种或多种选自下组的治疗：辐射治疗、化疗剂治疗、生物治疗、或其组合。
在本发明的第八方面，提供了一种抑制治疗对象中细胞粘附的方法，包括步骤：联用有效量的第一方面的抗体药物偶联物与一种或多种选自下组的治疗：辐射治疗、化疗剂治疗、生物治疗、或其组合。
在本发明的第九方面，提供了一种制备人源化或嵌合抗体的方法，包括步骤：
将本发明的鼠源抗体可变区的核苷酸序列克隆入含有人抗体恒定区的表达载体后，通过转染动物细胞表达人-鼠嵌合抗体。
将本发明的含人源FR区的抗体可变区的核苷酸序列克隆入含有人抗体恒定区的表达载体后，通过转染动物细胞表达人源化抗体。
在另一优选例中，所述的抗体是部分或全人源化的单克隆抗体。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了TF-mAb-DM1能显著的抑制TF高表达的肿瘤细胞的生长，并且其抑制作 用与细胞表面的TF分子数成正比。左图为TF-mAb-DM1能较好抑制TF高表达的肿瘤细胞生长的曲线，右表为TF-mAb-DM1在不同细胞株上的IC50值。
图2显示了TF-mAb-MMAE能显著的抑制TF高表达的肿瘤细胞的生长，并且其抑制作用与细胞表面的TF分子数成正比。左图为TF-mAb-MMAE能较好抑制TF高表达的肿瘤细胞生长的曲线，右表为TF-mAb-MMAE在不同细胞株上的IC50值。
图3A和图3B显示了TF-mAb-DM1(见图3A)和TF-mAb-MMAE(见图3B)对不同肿瘤细胞生长的抑制作用与各细胞表面TF的分子数成正比。通过CCLE数据库(Arrays_2013-03-18.tar.gz，Broad-Novartis Cancer Cell line Encyclopedia)分析不同细胞表面TF的相对分子数，结果表明TF-mAb-DM1和TF-mAb-MMAE对不同细胞生长的抑制作用与各细胞表面TF的分子数成正比。
图4A显示了TF-mAb–DM1能有效的抑制HCC1806原位移植瘤的生长，并呈一定的剂量依赖性。而且与Docetaxel组相比，TF-mAb-DM1组小鼠体重没有下降，说明TF-mAb-DM1毒副作用较小。图4B为裸鼠的体重变化曲线。
图5A显示了TF-mAb-MMAE在不同的剂量下均能有效的抑制HCC1806原位移植瘤的生长，并呈一定的剂量依赖性。尤其是TF-mAb-MMAE在3.75mg/kg的剂量，几乎可以完全抑制HCC1806原位移植瘤的生长。而且与Docetaxel组相比，TF-mAb-MMAE组小鼠体重没有下降，说明TF-mAb-MMAE毒副作用较小。图5B为裸鼠的体重变化曲线。
图6显示了TF-mAb-MMAE在更低的剂量下也能有效的抑制HCC1806原位移植瘤的生长，且本实验显示，最低有效剂量为0.7mg/kg。
图7A显示TF-mAb-MMAE能有效的抑制BxPC-3皮下移植瘤生长，并呈一定的剂量依赖性。而且与Docetaxel组相比，TF-mAb-MMAE组小鼠体重没有下降，说明TF-mAb-MMAE毒副作用较小。图7B为裸鼠的体重变化曲线。
图8显示了TF-mAb-H39-MMAE的分子筛高效液相色谱结果。
图9为TF-mAb-H39-MMAE的疏水层析色谱结果。
图10为TF-mAb-H39-MMAE的质谱表征结果。
图11显示了TF-mAb-H44-MMAE的分子筛高效液相色谱结果。
图12为TF-mAb-H44-MMAE的疏水层析色谱结果。
图13为TF-mAb-H44-MMAE的质谱表征结果。
图14显示TF-mAb-H39-MMAE能显著的抑制TF高表达肿瘤细胞的生长，并与细胞表面的TF分子数成正比,右表为相应的IC50值。
图15显示TF-mAb-H44-MMAE能显著的抑制TF高表达肿瘤细胞的生长，并与细胞表面 的TF分子数成正比，右表为相应的IC50值。
图16A和图16B显示通过CCLE数据库(Arrays_2013-03-18.tar.gz,Broad-Novartis Cancer Cell line Encyclopedia)分析不同细胞表面TF的相对分子数，结果表明TTF-mAb-H39-MMAE(见图16A)和TF-mAb-H44-MMAE(见图16B)对不同细胞的杀伤作用与各细胞表面TF的分子数成正比。
图17显示TF-mAb-H44-MMAE能有效的抑制HCC1806原位移植瘤的生长，并且在3mg/kg剂量下即可完全抑制HCC1806肿瘤的生长。
图18显示TF-mAb-H39-MMAE能有效的抑制HCC1806原位移植瘤的生长，且其最低有效剂量为1mg/kg。
图19TF-mAb-H44-MMAE能有效的抑制BxPC-3皮下移植瘤生长，并且在1mg/kg剂量下即可完全抑制BxPC-3皮下移植瘤生长。
图20TF-mAb-H39-MMAE能有效的抑制BxPC-3皮下移植瘤生长，且其最低有效剂量为0.3mg/kg。
具体实施方式
本发明人通过广泛而深入的研究，经过大量筛选，意外地获得一种抗TF单克隆抗体TF-mAb-SC1，实验结果表明，该针对TF蛋白的单克隆抗体为IgG2b型抗体。所述的抗体能够高特异性地结合TF抗原，其具有很高的亲和力(ELISA测定其EC50约为0.019nM)，并且所述的抗体具有显著的抗肿瘤活性，而对于哺乳动物本身没有可见的毒副作用。基于该TF-mAb-SC1而获得的嵌合抗体、人源化抗体以及相应的ADC也具有优异的特性。在此基础上完成了本发明。
术语
如本文所用，术语“抗体药物偶联体”、“抗体偶联物”、“抗体药物偶联物”、“抗体-药物偶联物”“免疫偶联物”可互换使用，指(a)抗体或其活性片段与(b)药物形成的偶联物。
如本文所用，术语“本发明的抗体药物偶联体”、“本发明的抗体与药物偶联物”或“本发明的ADC”可互换使用，指具有针对组织因子的本发明抗体或其活性片段与药物形成的偶联物。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
抗体
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚 糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
脊椎动物抗体(免疫球蛋白)的“轻链”可根据其恒定区的氨基酸序列归为明显不同的两类(称为κ和λ)中的一类。根据其重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类。主要有5类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，其中一些还可进一步分成亚类(同种型)，如IgG1、IgG2、IgG3、IgG4、IgA和IgA2。对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
一般，抗体的抗原结合特性可由位于重链和轻链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
在本发明中，抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分，可以通过标准的DNA重组技术获得，它们都是有用的抗体。嵌合抗体是一个分子，其中不同的部分来自不同的动物种，例如具有来自鼠的单克隆抗体的可变区，和来自人免疫球蛋白的恒定区的嵌合抗体(见例如美国专利4,816,567和美国专利4,816,397,在此通过引用方式整体引入本文)。人源化的抗体是指来源于非人物种的抗体分子，具有一个或多个来源于非人物种的互补决定区(CDRs)和来源于人免疫球蛋白分子的框架区域(见美国专利5,585,089，在此通过引用方式整体引入本文)。这些嵌合和人源化的单克隆抗体可以采用本领域熟知的DNA重组技术制备。
在本发明中，抗体可以是单特异性、双特异性、三特异性、或者更多的多重特异性。
在本发明中，本发明的抗体还包括其保守性变异体，指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明的抗TF的抗体
本发明提供一种针对TF的高特异性和高亲和力的抗体，其包括重链和轻链，所述重链含有重链可变区(VH)氨基酸序列，所述轻链含有轻链可变区(VL)氨基酸序列。
优选地，重链可变区(VH)氨基酸序列和轻链可变区(VL)氨基酸序列的各自CDR选自下组：
a1)SEQ ID No.:1；
a2)SEQ ID No.:2；
a3)SEQ ID No.:3；
a4)SEQ ID No.:4；
a5)SEQ ID No.:5；
a6)SEQ ID No.:6；
a7)上述氨基酸序列中任意一种氨基酸序列经过添加、缺失、修饰和/或取代至少一个氨基酸的具有TF结合亲和力的序列。
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列优选为同源性为至少80％，较佳地至少85％，更佳地至少为90％，最佳地至少95％的氨基酸序列。
优选地，所述的抗体具有抑制TF相关信号通路的活性；具有抗凝血活性；具有抗FXa生成活性、或其组合。
典型地，本发明提供了一种抗TF的抗体，所述抗体具有：本发明的重链可变区；和/或本发明的轻链可变区；
其中，所述抗体的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO.:1所示的CDR1，
SEQ ID NO.:2所示的CDR2，和
SEQ ID NO.:3所示的CDR3；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留TF结合亲和力的衍生序列；
所述抗体的轻链可变区包括以下三个的互补决定区CDR：
SEQ ID NO.:4所示的CDR1'，
SEQ ID NO.:5所示的CDR2'，和
SEQ ID NO.:6所示的CDR3'；
上述氨基酸序列中任意一种氨基酸序列经过添加、缺失、修饰和/或取代至少一个氨基酸的具有TF结合亲和力的衍生序列。
较佳地，所述抗体的重链可变区序列选自下组：SEQ ID NO.:7、9、10、11、12、或13；和/或所述的抗体的轻链可变区序列选自下组：SEQ ID NO.:8、14、15、16、或17。
本发明中，所述抗体选自：动物源抗体、嵌合抗体、人源化抗体、或其组合。在另一优选例中，所述添加、缺失、修饰和/或取代的氨基酸数量，不超过初始氨基酸序列总氨基酸数量的40％。
在另一优选例中，所述添加、缺失、修饰和/或取代的氨基酸数量为1-7个。
在另一优选例中，所述经过添加、缺失、修饰和/或取代的至少一个氨基酸序列为同源性为至少80％的氨基酸序列。
在另一优选例中，，所述经过添加、缺失、修饰和/或取代的至少一个氨基酸具有抑制TF相关信号通路的活性、抗凝血活性、抗FXa生成活性中的任意一种或几种。
本发明所述抗体可以是双链或单链抗体，并且可以是选自动物源抗体、嵌合抗体、人源化抗体，更优选为人源化抗体、人-动物嵌合抗体，更优选为全人源化抗体。
本发明所述抗体衍生物可以是单链抗体、和/或抗体片段，如：Fab、Fab'、(Fab')2或该领域内其他已知的抗体衍生物等，以及IgA、IgD、IgE、IgG以及IgM抗体或其他亚型的抗体中的任意一种或几种。
其中，所述动物优选为哺乳动物，如鼠。
本发明抗体可以是靶向人TF的嵌合抗体、人源化抗体、CDR嫁接和/或修饰的抗体。
在本发明的一种优选实施例中，上述SEQ ID No.:1-SEQ ID No.:3中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有TF结合亲和力的序列，位于重链可变区(VH)的CDR区。
在本发明的一种优选实施例中，上述SEQ ID No.:4-SEQ ID No.:6中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有TF结合亲和力的序列，位于轻链可变区(VL)的CDR区。
在本发明的一种更优选实施例中，VH CDR1、CDR2、CDR3分别独立地选自SEQ ID No.:1-SEQ ID No.:3中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有TF结合亲和力的序列；VL CDR1、CDR2、CDR3分别独立地选自SEQ ID No.:4-SEQ ID No.:6中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有TF结合亲和力的序列。
本发明上述内容中，所述添加、缺失、修饰和/或取代的氨基酸数量，优选为不超过初始氨基酸序列总氨基酸数量的40％，更优选为不超过35％，更优选为1-33％，更优选为5-30％，更优选为10-25％，更优选为15-20％。
本发明上述内容中，更优选地，所述添加、缺失、修饰和/或取代的氨基酸数量，可以是1-7个，更优选为1-5个，更优选为1-3个，更优选为1-2个。
在另一优选例中，所述靶向TF的抗体为TF-mAb-SC1(原名称为TF-mAb)。
在另一优选例中，所述抗体TF-mAb-SC1的重链可变区(VH)氨基酸序列为如SEQ ID NO.:7所示的氨基酸序列。
在另一优选例中，所述抗体TF-mAb-SC1的轻链可变区(V-Kappa)氨基酸序列为如SEQ ID NO.:8所示的氨基酸序列。
抗体的制备
本发明抗体或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码所述的本发明的抗体(或其片段，或其衍生 物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。优选的动物细胞包括(但并不限于)：CHO-S、HEK-293细胞。
通常，在适合本发明抗体表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本发明的抗体。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。单克隆抗体的结合亲和力例如可用Munson等,Anal.Biochem.,107:220(1980)的Scatchard分析来测定。
本发明的抗体可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超声处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
细胞毒剂
可用于构成本发明ADC的药物包括但并不限于：细胞毒剂。
术语“细胞毒剂”是指抑制或阻止细胞表达活性、细胞功能和/或造成细胞破坏的物质。该术语包括放射性同位素、化学治疗剂以及毒素，如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，包括其片段和/或变体。细胞毒剂的例子包括但不限于：耳他汀类(例如，耳他汀E、耳他汀F、MMAE和MMAF)、金霉素、类美坦西醇、篦麻毒素、篦麻毒素A-链、考布他汀、多卡米星、多拉司他汀、阿霉素、柔红霉素、紫杉醇、顺铂、cc1065、溴化乙锭、丝裂霉素、依托泊甙、替诺泊甙(tenoposide)、长春新碱、长春碱、秋水仙素、二羟基炭疽菌素二酮、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-八叠球菌、白树毒素、迈托毒素(mitogellin)、局限曲菌素(retstrictocin)、酚霉素、依诺霉素、麻疯树毒蛋白(curicin)、巴豆毒素、卡奇霉素、肥皂草(Sapaonaria officinalis)抑制剂以及糖皮质激素和其它化学治疗剂，以及放射性同位素，如At211、I131、I125、Y90、Re186、Re188、Sm153、Bi212或213、P32和包括Lu177在内的Lu的放射性同位素。抗体也可与能够将前药转化成其活性形式的抗癌前药活化酶偶联。
优选的小分子药物为具有高细胞毒性的化合物，优选单甲基澳瑞他汀(monomethylauristatin)、加利车霉素、美登素类、或其组合；更佳地选自：单甲基澳瑞他汀 -E(MMAE)、单甲基澳瑞他汀-D(MMAD)、单甲基澳瑞他汀-F(MMAF)、或其组合。
抗体-药物偶联物(ADC)
本发明还提供了基于本发明抗体的抗体偶联药物(antibody-drug conjugate，ADC)。
典型地，所述抗体偶联药物包括所述抗体、以及效应分子，所述抗体与所述效应分子偶联，并优选为化学偶联。其中，所述效应分子优选为具有治疗活性的药物。此外，所述效应分子可以是毒蛋白、化疗药物、小分子药物或放射性核素中的一种或多种。
本发明抗体与所述效应分子之间可以是通过偶联剂进行偶联。所述偶联剂的例子可以是非选择性偶联剂、利用羧基的偶联剂、肽链、利用二硫键的偶联剂中的任意一种或几种。所述非选择性偶联剂是指使效应分子和抗体形成共价键连接的化合物，如戊二醛等。所述利用羧基的偶联剂可以是顺乌头酸酐类偶联剂(如顺乌头酸酐)、酰基腙类偶联剂(偶联位点为酰基腙)中的任意一种或几种。
抗体上某些残基(如Cys或Lys等)用于与多种功能基团相连，其中包括成像试剂(例如发色基团和荧光基团)，诊断试剂(例如MRI对比剂和放射性同位素)，稳定剂(例如乙二醇聚合物)和治疗剂。抗体可以被偶联到功能剂以形成抗体-功能剂的偶联物。功能剂(例如药物，检测试剂，稳定剂)被偶联(共价连接)至抗体上。功能剂可以直接地、或者是通过接头间接地连接于抗体。
典型的适用于本发明的偶联方式，包括K-Lock和C-Lock两种偶联方式。在K-Lock偶联方式中，药物分子偶联于抗体序列中赖氨酸(K)残基，在C-Lock偶联方式中，药物分子偶联于抗体序列中的半胱氨酸(C)残基。
抗体可以偶联药物从而形成抗体药物偶联物(ADCs)。典型地，ADC包含位于药物和抗体之间的接头。接头可以是可降解的或者是不可降解的接头。可降解的接头典型地在细胞内环境下容易降解，例如在目标位点处接头发生降解，从而使药物从抗体上释放出来。合适的可降解的接头包括，例如酶降解的接头，其中包括可以被细胞内蛋白酶(例如溶酶体蛋白酶或者内体蛋白酶)降解的含有肽基的接头，或者糖接头例如，可以被葡糖苷酸酶降解的含葡糖苷酸的接头。肽基接头可以包括，例如二肽，例如缬氨酸-瓜氨酸，苯丙氨酸-赖氨酸或者缬氨酸-丙氨酸。其它合适的可降解的接头包括，例如，pH敏感接头(例如pH小于5.5时水解的接头，例如腙接头)和在还原条件下会降解的接头(例如二硫键接头)。不可降解的接头典型地在抗体被蛋白酶水解的条件下释放药物。
连接到抗体之前，接头具有能够和某些氨基酸残基反应的活性反应基团，连接通过活性反应基团实现。巯基特异性的活性反应基团是优选的，并包括：例如马来酰亚胺类化合物，卤代酰胺(例如碘、溴或氯代的)；卤代酯(例如碘、溴或氯代的)；卤代甲基酮(例如碘、溴或氯代)，苄基卤代物(例如碘、溴或氯代的)；乙烯基砜，吡啶基二硫化物；汞衍生物例如3,6-二-(汞甲基)二氧六环，而对离子是醋酸根、氯离子或者硝酸根；和聚亚甲基二甲基硫醚硫代磺酸盐。接头可以包括，例如，通过硫代丁二酰亚胺连接到抗体上的马来酰亚胺。
药物可以是任何细胞毒性，抑制细胞生长或者免疫抑制的药物。在实施方式中，接头连接抗体和药物，而药物具有可以和接头成键的功能性基团。例如，药物可以具有可以和连接物成键的氨基，羧基，巯基，羟基，或者酮基。在药物直接连接到接头的情况下，药物在连接到抗体之前，具有反应的活性基团。
特别有用的药物类别包括，例如，抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱等。特别有用的细胞毒性药物类的例子包括，例如，DNA小沟结合试剂、DNA烷基化试剂、和微管蛋白抑制剂、典型的细胞毒性药物包括、例如奥瑞他汀(auristatins)、喜树碱(camptothecins)、多卡霉素/倍癌霉素(duocarmycins)、依托泊甙(etoposides)、美登木素(maytansines)和美登素类化合物(maytansinoids)(例如DM1和DM4)、紫杉烷(taxanes)、苯二氮卓类(benzodiazepines)或者含有苯二氮卓的药物(benzodiazepine containing drugs)(例如吡咯并[1,4]苯二氮卓类(PBDs)，吲哚啉苯并二氮卓类(indolinobenzodiazepines)和噁唑烷并苯并二氮卓类(oxazolidinobenzodiazepines))和长春花生物碱(vinca alkaloids)。
在本发明中，药物-接头可以用于在一个简单步骤中形成ADC。在其它实施方式中，双功能连接物化合物可以用于在两步或多步方法中形成ADC。例如，半胱氨酸残基在第一步骤中与接头的反应活性部分反应，并且在随后的步骤中，接头上的功能性基团与药物反应，从而形成ADC。
通常，选择接头上功能性基团，以利于特异性地与药物部分上的合适的反应活性基团进行反应。作为非限制性的例子，基于叠氮化合物的部分可以用于特异性地与药物部分上的反应性炔基基团反应。药物通过叠氮和炔基之间的1,3-偶极环加成，从而共价结合于接头。其它的有用的功能性基团包括，例如酮类和醛类(适合与酰肼类和烷氧基胺反应)，膦(适合与叠氮反应)；异氰酸酯和异硫氰酸酯(适合与胺类和醇类反应)；和活化的酯类，例如N-羟基琥珀酰亚胺酯(适合与胺类和醇类反应)。这些和其它的连接策略，例如在《生物偶联技术》，第二版(Elsevier)中所描述的，是本领域技术人员所熟知的。本领域技术人员能够理解，对于药物部分和接头的选择性反应，当选择了一个互补对的反应活性功能基团时，该互补对的每一个成员既可以用于接头，也可以用于药物。
本发明还提供了制备ADC的方法，可进一步地包括：将抗体与药物-接头化合物，在足以形成抗体偶联物(ADC)的条件下进行结合。
在某些实施方式中，本发明方法包括：在足以形成抗体-接头偶联物的条件下，将抗体与双功能接头化合物进行结合。在这些实施方式中，本发明方法还进一步地包括：在足以将药物部分通过接头共价连接到抗体的条件下，将抗体接头偶联物与药物部分进行结合。
在一些实施方式中，抗体药物偶联物ADC如下分子式所示：
Figure PCTCN2017087779-appb-000003
其中：
Ab是抗体，
LU是接头；
D是药物；
而且下标p是选自1-10，较佳地1-8的值。
应用
本发明还提供了本发明抗体的用途，例如用于制备诊断制剂、或制备用于预防和/或治疗TF相关的疾病的药物。所述TF相关的疾病包括肿瘤发生、生长和/或转移、血栓类相关疾病、炎症、代谢相关疾病等。
本发明抗体、ADC或CAR-T等的用途，包括(但并不限于)：
(i)诊断、预防和/或治疗肿瘤发生、生长和/或转移，尤其是TF高表达的肿瘤。所述肿瘤包括(但并不限于)：乳腺癌(如三阴性乳腺癌)、胰腺癌、肺癌、恶性胶质瘤、胃癌、肝癌、食道癌、肾癌、结直肠癌、膀胱癌、前列腺癌、子宫内膜癌、卵巢癌、宫颈癌、白血病、骨髓癌、血管肉瘤等；尤其是三阴性乳腺癌、胰腺癌、恶性胶质瘤和肺癌，更优选为三阴性乳腺癌和/或胰腺癌。
(ii)诊断、预防和/或治疗血栓类相关疾病。所述血栓类相关疾病包括(但并不限于)：动脉粥样硬化、急性冠状动脉综合症、急性心肌梗塞、中风、高血压、深静脉血栓、肺栓塞、肾栓塞及动脉手术、冠状动脉旁路移植引起的血栓等。
(iii)诊断、预防和/或治疗炎症。所述炎症包括(但并不限于)：风湿性关节炎、骨关节炎、强直性脊柱炎、痛风、莱特尔综合征、牛皮癣性关节病、感染性关节炎、结核性关节炎、病毒性关节炎、真菌性关节炎、肾小球性肾炎、全身性红斑狼疮、克罗恩病、溃疡性结肠炎、急性肺损伤、慢性阻塞性肺疾病、特发性肺纤维化。
(iv)诊断、预防和/或治代谢相关疾病。所述代谢相关疾病包括(但并不限于)：糖尿病、食源性肥胖和脂肪炎症等。
药物组合物
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白或其ADC，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明的药物组合物可直接用于结合TF蛋白分子，因而可用于预防和治疗肿瘤等疾病。此外，还可同时使用其他治疗剂，例如，各种细胞因子，如TNF、IFN、IL-2等；各种肿瘤化疗药物，如5-FU、氨甲喋呤等影响核酸生物合成的药物；氮芥、环磷酰胺等烷化剂类药物； 阿霉素、放线菌素D等干扰转录过程阻止RNA合成的药物；长春新碱、喜树碱类。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单克隆抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约20毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明的主要优点包括：
(a)本发明抗体具有优异的生物活性和特异性，并具有很高的亲和力(ELISA测定其EC50可高达为约0.01-0.03nM)。此外，对细胞表面TF具有良好的结合亲合力，可用做靶向TF的抗体。
(b)本发明的人源化抗体不仅具有与鼠源抗体相当的活性，而且具有更低的免疫原性。
(c)本发明抗体和ADC均具有显著的抗肿瘤活性，而对于哺乳动物本身没有可见的毒副作用。
(d)本发明抗体和ADC不仅在多个肿瘤模型中有显著的治疗效应，而且还适用于其他与TF高表达有关的疾病。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。细胞株为常规的市售产品或购自ATCC，质粒均为市售产品。
实施例1靶向人TF单克隆抗体的发现和制备
步骤①，杂交瘤细胞的制备：
首先将人TF蛋白的胞外区部分(UniProtKB/Swiss-Prot:P13726.1，从第34位到第251位氨基酸)免疫8周龄Balb/c雌性小鼠，TF胞外区蛋白的用量为100μg/只，以制备免疫脾细胞；适时的制备鼠骨髓瘤细胞(SP2/0)和饲养细胞以备融合之需。
待上述三种细胞准备完毕，通过PEG介导融合免疫脾细胞和SP2/0细胞，去除PEG，用 含有饲养细胞的HAT完全培养基重悬，接种到96孔板中培养，通过ELISA法进行阳性孔筛选。最后再对阳性孔的细胞通过有限稀释法进行克隆化培养，通过ELSIA或免疫荧光法筛选效价高、形态好、呈单克隆生长的细胞继续进行亚克隆筛选，直到连续三次筛选阳性克隆率全为100％，即可对该细胞株进行扩大培养和建库。
步骤②，靶向人TF鼠源单克隆抗体腹水的制备：
将步骤①中筛选出来的杂交瘤细胞扩大培养，小鼠适应性培养后腹腔注射降植烷(0.5mL/只)以为杂交瘤细胞生长提供有利环境，7-10d后，每只腹腔注射10×106的杂交瘤细胞，自第7天起，每天观察小鼠腹水产生状况和精神状态，采取腹水，离心去除油脂-80℃冻存，以备纯化。
步骤③，靶向人TF鼠源单克隆抗体的纯化：
将步骤②中冻存的腹水于冰上融化，经0.45μm滤膜过滤后，用PBS于4℃透析过夜，最后通过FPLC技术对该抗体进行纯化并超滤浓缩至所需浓度，分装、-80℃冻存备用。
步骤④，靶向人TF鼠源单克隆抗体的生物活性和靶向特异性确定：
经过初步筛选，选定约30个杂交瘤细胞进行二次有限稀释克隆筛选，然后在其中选定6个抗体，进行大量表达纯化，并在10μg/mL的浓度下利用流式细胞仪测定各个抗体对人乳腺癌细胞MDA-MB-231，人胰腺癌细胞BxPC-3和鼠黑色素瘤细胞B16-F10的亲和力。
结果表明测试的抗体可以特异性的靶向结合人源TF(MDA-MB-231和BxPC-3细胞)而不靶向结合鼠源TF(B16-F10细胞)，其中TF-mAb-SC1对人TF的亲和力比其他5个抗体都要高。
随后，将TF胞外区蛋白包被ELISA板，0.05μg/孔，进行ELISA实验，结果表明，TF-mAb-SC1对TF胞外区蛋白有很强的亲和性；细胞结合亲和力实验表明，TF-mAb-SC1对TF高表达的三阴性乳腺癌细胞MDA-MB-231、胰腺癌细胞BxPC-3细胞有非常高的结合亲和力；而且TF-mAb-SC1可以显著抑制TF-PAR2下游MAPK/ERK的磷酸化水平并呈一定的剂量依赖性。
基于TF-mAb-SC1表现出非常高的特异性、非常高的亲和力以及对MAPK/ERK的磷酸化水平的显著抑制作用，故被选定用于测序以及后续研究。
采用常规测序，并通过Kabat数据库分析，得到以下序列信息：
重链可变区的CDR氨基酸序列为：
SEQ ID No.:1：SYWMN；
SEQ ID No.:2：MIYPADSETRLNQKFKD；
SEQ ID No.:3：EDYGSSDY。
完整的VH氨基酸序列如SEQ ID NO.:7所示。
Figure PCTCN2017087779-appb-000004
轻链可变区的CDR氨基酸序列为：
SEQ ID No.:4：SASSSVSYMN；
SEQ ID No.:5：GISNLAS；
SEQ ID No.:6：QQKSSFPWT。
完整的VL氨基酸序列如SEQ ID NO.:8所示：
Figure PCTCN2017087779-appb-000005
实施例2人-鼠嵌合抗体的制备
在已获得的活性高、特异性强的鼠源TF-mAb-SC1的基础上，构建人-鼠嵌合抗体。
设计引物在重链可变区引入EcoR I和Nhe I，在轻链可变区引入Age I和BsiW I限制性内切酶酶切位点，然后将上述所获得抗体重链和轻链的可变区序列分别克隆入含有人IgG1重链恒定区和Kappa链恒定区的载体，经鉴定无误后，利用转染技术和哺乳动物表达系统(CHO-S或HEK-293细胞)将构建的嵌合型抗体表达、纯化，所获得的人-鼠嵌合型抗体，命名为TF-mAb-Ch。
实施例3TF-mAb-SC1的人源化及活性测定
参照TF-mAb-SC1的抗体重链可变区序列(SEQ ID NO.:7)和轻链可变区序列(SEQ ID NO.:8)，在Germline数据库中选取与其非CDR区匹配最好的人源化模板。然后将鼠源抗体TF-mAb-SC1的CDR区移植到所选择的人源化模板上，替换人源模板的CDR区，再与IgG1恒定区重组，同时以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变，得到5个人源化重链的可变区(SEQ ID NO.:9，SEQ ID NO.:10，SEQ ID NO.:11，SEQ ID NO.:12，SEQ ID NO.:13)及4个人源化轻链的可变区(SEQ ID NO.:14，SEQ ID NO.:15，SEQ ID NO.:16，SEQ ID NO.:17)。
表B
Figure PCTCN2017087779-appb-000006
Figure PCTCN2017087779-appb-000007
基于所述改造的VH和VL，分别组合表达这些人源化的重链及轻链，最终共得到的20个人源化抗体，即TF-mAb-H29至TF-mAb-H48。各抗体相应的重链和轻链组合如下表所示：
表C
序列编号 SEQ ID NO.:9 SEQ ID NO.:10 SEQ ID NO.:11 SEQ ID NO.:12 SEQ ID NO.:13
SEQ ID NO.:14 TF-mAb-H29 TF-mAb-H30 TF-mAb-H31 TF-mAb-H32 TF-mAb-H33
SEQ ID NO.:15 TF-mAb-H34 TF-mAb-H35 TF-mAb-H36 TF-mAb-H37 TF-mAb-H38
SEQ ID NO.:16 TF-mAb-H39 TF-mAb-H40 TF-mAb-H41 TF-mAb-H42 TF-mAb-H43
SEQ ID NO.:17 TF-mAb-H44 TF-mAb-H45 TF-mAb-H46 TF-mAb-H47 TF-mAb-H48
首先通过ELISA结合实验测定这20个人源化抗体对TF胞外区蛋白的亲和活性(实验方法参照实施例1，步骤④)，结果如表1所示。
表1人源化抗体对TF胞外区蛋白的结合亲和力
抗体 EC50(nM) 抗体 EC50(nM)
TF-mAb-Ch 0.0100 TF-mAb-H39 0.0125
TF-mAb-H29 0.0178 TF-mAb-H40 0.0131
TF-mAb-H30 0.0147 TF-mAb-H41 0.0134
TF-mAb-H31 0.0145 TF-mAb-H42 0.0128
TF-mAb-H32 0.0168 TF-mAb-H43 0.0116
TF-mAb-H33 0.0189 TF-mAb-H44 0.0120
TF-mAb-H34 0.0154 TF-mAb-H45 0.0138
TF-mAb-H35 0.0105 TF-mAb-H46 0.0119
TF-mAb-H36 0.0234 TF-mAb-H47 0.0130
TF-mAb-H37 0.0173 TF-mAb-H48 0.0153
TF-mAb-H38 0.0178 IgG(阴性对照) ＞6.67
通过流式细胞仪测定这20个人源化抗体分别在10μg/mL和1μg/mL浓度下，对1×105个MDA-MB-231细胞的结合亲和力，结果如表2所示。
表2人源化抗体对MDA-MB-231细胞的结合亲和力
Figure PCTCN2017087779-appb-000008
实施例4抗体药物偶联物，TF-mAb-MMAE的制备
在TF-mAb-SC1原液中加入PBS/D(pH＝7.4)缓冲液使其浓度在20mg/ml，然后用2.6eq的TCEP于25℃还原2小时，取出后置于冰上冷却，未纯化直接加入6eq的MMAE，0℃反应1小时，加Cyst终止反应。采用G25脱盐柱除去过量的小分子，并置换至10mM PBS溶液中(pH 7.4)，-80℃保存备用，所得的抗体药物偶联物命名为TF-mAb-MMAE。
实施例5抗体药物偶联物，TF-mAb-DM1的制备
①一步法的制备
首先利用G25脱盐柱将TF-mAb-SC1原液置换至反应缓冲液(50mM磷酸钾盐/50mM NaCl/2mM EDTA，pH7.5)中，终浓度7.8mg/ml；然后加入9eq的11mg/ml的MCC-DM1(溶解在DMA中，反应体系中DMA含量小于5％)，室温反应6小时。离心，上清经Q柱，阳离子柱纯化去除过量的小分子，最后经G25脱盐柱或超滤置换至10mM PBS溶液中，-80℃保存备用,所得的抗体药物偶联物命名为TF-mAb-DM1。
②二步法的制备
利用G25脱盐柱将TF-mAb-SC1原液置换至反应缓冲液(50mM磷酸钾盐/50mM NaCl/2mM EDTA，pH6.5)中，终浓度10mg/ml；然后加入8eq的SMCC(溶解在DMSO中)，10-12℃反应3小时，过G25脱盐柱去除过量的SMCC。再于P-MCC中加入12eq的DM1，于25℃反应18小时，最后经G25脱盐柱置换至10mM PBS溶液中，-80℃保存备用,所得的抗体药物偶联物命名为TF-mAb-DM1。
实施例6TF-mAb-ADCs针对TF高表达的三阴性乳腺癌细胞、胰腺癌的体外抗肿瘤活性
本实例所使用细胞系购自于美国典型培养物保藏中心(ATCC)或中国科学院细胞库,并按照相应的说明进行培养，包括：MCF7，MDA453，T47D，A549，U87MG，H1975，MDA-MB-231，BxPC-3，HCC1806，Hs578T。
将上述处于对数生长期的细胞，分别以每孔1,000-3,000个细胞的密度(依不同细胞的生长速率而定)接种至96孔细胞培养板中，150μL/孔，37℃，5％CO2培养约16h后，分别加入不同浓度的TF-mAb-ADCs(即TF-mAb-DM1和TF-mAb-MMAE)，每个药物浓度设置3个复孔，及相应的溶媒对照和空白对照孔，作用4天后，倾去培养液，加入MTS反应液(购自Promega,cat#G3581)，100μL/孔，于37℃反应至预期颜色深浅，测定每组的细胞活力(OD490nm)，并按照以下公式计算细胞存活率：
存活率＝(OD给药-OD空白)/(OD对照-OD空白)×100％。
通过GraphPad Prism 5软件分析上述数据,并分别计算TF-mAb-DM1和TF-mAb-MMAE在不同细胞株上的IC50值。
实验结果表明，在体外，TF-mAb-DM1和TF-mAb-MMAE均能较好抑制TF高表达的肿 瘤细胞的生长，并且其抑制作用与细胞表面的TF分子数成正比。如图1，左图为TF-mAb-DM1能较好抑制TF高表达的肿瘤细胞生长的曲线，右表为TF-mAb-DM1在不同细胞株上的IC50值。如图2，左图为TF-mAb-MMAE能较好抑制TF高表达的肿瘤细胞生长的曲线，右表为TF-mAb-MMAE在不同细胞株上的IC50值。
通过CCLE(Broad-Novartis Cancer Cell line Encyclopedia)数据库分析不同细胞表面TF的相对分子数，结果表明TF-mAb-DM1和TF-mAb-MMAE对不同细胞生长的抑制作用与各细胞表面TF的分子数成正比，分别如图3A和图3B。
实施例7TF-mAb-ADCs针对TF高表达的三阴性乳腺癌、胰腺癌模型的体内抗肿瘤活性
将处于对数生长期的HCC1806和BxPC-3细胞分别按照每200μL无血清培养基含3×106和10×106的密度接种到6周龄Balb/c雌性裸鼠乳或垫背部皮下(Balb/c裸鼠购自上海西普尔-必凯实验动物有限公司)，待肿瘤生长至100-200mm3后，将动物随机分组，每组8个肿瘤。将TF-mAb-DM1按照3.75mg/kg和15mg/kg，TF-mAb-MMAE按照0.7mg/kg，2mg/kg，3.75mg/kg，7mg/kg和15mg/kg的剂量，每周一次通过尾静脉给药，正常小鼠IgG(IgG)和多烯紫杉醇(Docetaxel)分别作为阴性和阳性对照药物。每周测量2-3次肿瘤体积及裸鼠重量并记录以绘制肿瘤生长曲线。肿瘤体积(V)计算公式为：
V＝1/2×a×b2其中a、b分别表示肿瘤的长、宽。
如图4所示，图4A为TF-mAb-DM1抑制HCC1806原位移植瘤生长曲线，并呈一定的剂量依赖性。图4B为裸鼠体重变化曲线。
图5A和图6为TF-mAb-MMAE在不同剂量下抑制HCC1806原位移植瘤生长曲线,图5B为裸鼠体重变化曲线。由结果可知TF-mAb-MMAE在0.7mg/kg剂量下即可有效抑制HCC1806肿瘤生长，而在3.75mg/kg的剂量，几乎可以完全抑制HCC1806肿瘤的生长。
如图7A所示，TF-mAb-MMAE能有效的抑制BxPC-3皮下移植瘤生长，并呈一定的剂量依赖性。而且与Docetaxel组相比，TF-mAb-MMAE组小鼠体重没有下降，说明TF-mAb-MMAE毒副作用较小。图7B为裸鼠体重变化曲线。
实施例8人源化抗体的ADC
重复实施例4-7，不同点在于用如下人源化抗体替换TF-mAb-SC1：TF-mAb-H29、TF-mAb-H30、TF-mAb-H31、TF-mAb-H32、TF-mAb-H33、TF-mAb-H34、TF-mAb-H35、TF-mAb-H36、TF-mAb-H37、TF-mAb-H38、TF-mAb-H39、TF-mAb-H40、TF-mAb-H41、TF-mAb-H42、TF-mAb-H43、TF-mAb-H44、TF-mAb-H45、TF-mAb-H46、TF-mAb-H47、TF-mAb-H48。
然后，分别制得这些人源化抗体与MMAE的偶联物，其中TF-mAb-H39-MMAE和TF-mAb-H44-MMAE表征结果如下。
图8为TF-mAb-H39-MMAE的分子筛高效液相色谱；图9为TF-mAb-H39-MMAE的疏水层析色谱；图10为TF-mAb-H39-MMAE的质谱图。图11为TF-mAb-H44-MMAE的分子筛高效液相色谱；图12为TF-mAb-H44-MMAE的疏水层析色谱；图13为TF-mAb-H44-MMAE的质谱图。
上述实验结果表明，TF-mAb-H39-MMAE和TF-mAb-H44-MMAE的药物-抗体偶联比(DAR)主要为2和4，平均DAR约为4。
测试结果表明，基于这些人源化抗体的ADC同样具有高亲和力(与TF-mAb-MMAE相比，较佳地人源化抗体的ADC的相对亲和力为60-140％之间)、高细胞毒性，和低的毒副作用，并表现出显著的抗肿瘤作用。
两种优选的人源化抗体TF-mAb-H39-MMAE和TF-mAb-H44-MMAE的体外、体内抗肿瘤活性结果分别如图14-20所示。
如图14所示，实验结果表明TF-mAb-H39-MMAE能显著的抑制TF高表达肿瘤细胞的生长，并与细胞表面的TF分子数成正比，右表为TF-mAb-H39-MMAE在不同细胞株上的IC50值。
如图15所示为TF-mAb-H44-MMAE能显著的抑制TF高表达肿瘤细胞的生长，并与细胞表面的TF分子数成正比，右表为TF-mAb-H44-MMAE在不同细胞株上的IC50值。
通过CCLE数据库分析不同细胞表面TF的相对分子数，结果表明TF-mAb-H39-MMAE和TF-mAb-H44-MMAE对不同细胞的杀伤作用与各细胞表面TF的分子数成正比，分别如图16A和图16B。
如图17所示，TF-mAb-H44-MMAE能有效的抑制HCC1806肿瘤的生长，且呈剂量依赖性，并且在3mg/kg剂量下即可完全抑制HCC1806肿瘤生长，最低有效剂量(MED)为1mg/kg。
如图18所示，TF-mAb-H39-MMAE能有效的抑制HCC1806肿瘤的生长，且最低有效剂量(MED)为1mg/kg。
如图19所示，TF-mAb-H44-MMAE能显著的抑制BxPC-3肿瘤生长，且呈剂量依赖性，并且在1mg/kg剂量下即可完全抑制BxPC-3肿瘤生长。
如图20所示TF-mAb-H39-MMAE能显著的抑制BxPC-3肿瘤生长，且最低有效剂量(MED)为0.3mg/kg。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种抗体-药物偶联物，其特征在于，所述抗体药物偶联物含有：
    (a)抗体部分；和
    (b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合；
    其中，所述抗体的重链可变区包括以下三个互补决定区CDR：
    (H1)SEQ ID NO.:1所示的CDR1，
    (H2)SEQ ID NO.:2所示的CDR2，和
    (H3)SEQ ID NO.:3所示的CDR3；
    其中，上述重链可变区氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留TF结合亲和力的衍生序列；和/或
    所述抗体的轻链可变区包括以下三个互补决定区CDR：
    (L1)SEQ ID NO.:4所示的CDR1'，
    (L2)SEQ ID NO.:5所示的CDR2'，和
    (L3)SEQ ID NO.:6所示的CDR3'；
    其中，上述轻链可变区氨基酸序列中任意一种氨基酸序列经过添加、缺失、修饰和/或取代至少一个氨基酸的具有TF结合亲和力的衍生序列。
  2. 如权利要求1所述的抗体-药物偶联物，其特征在于，所述抗体药物偶联物ADC如下分子式所示：
    Figure PCTCN2017087779-appb-100001
    其中：
    Ab是抗TF的抗体，
    LU是接头；
    D是药物；
    而且下标p是选自1-10，较佳地1-8的值。
  3. 如权利要求2所述的抗体-药物偶联物，其特征在于，LU选自下组：6-马来酰亚氨基己酰基-缬氨酸-瓜氨酸-对氨基苄氧羰基(MC-val-cit-PAB)、6-马来酰亚氨基己酰基-丙氨酸-苯丙氨酸-对氨基苄氧羰基(MC-ala-phe-PAB)、马来酰亚氨基丙酰基-缬氨酸-瓜氨酸-对氨基苄氧羰基(MP-val-cit-PAB)、马来酰亚氨基丙酰基-丙氨酸-苯丙氨酸-对氨基苄氧羰基(MP-ala-phe-PAB)、N-琥珀酰亚氨基4-(2-吡啶基硫基)戊酸酯(SPP)、N-琥珀酰亚氨基4-(N-马来酰亚氨基甲基)环己烷-1-羧酸酯(SMCC)、4-(2-吡啶基二硫代)丁酸-N-羟基琥珀酰亚胺酯(SPDB)或N-琥珀酰亚氨基(4-碘-乙酰基)氨基苯甲酸酯(SIAB)。
  4. 如权利要求2所述的抗体-药物偶联物，其特征在于，其中D选自下组：
    (i)美登素衍生物(DM1,DM4)，auristatin和多拉司他汀。
    (ii)Monomethyl auristatin E(MMAE)，Monomethylauristatin F(MMAF)，Monomethyl Dolastatin 10(MMAD)类衍生物或其组合。
  5. 如权利要求1所述的抗体-药物偶联物，其特征在于，所述抗体选自：动物源抗体、嵌合抗体、人源化抗体、或其组合。
  6. 如权利要求1所述的抗体-药物偶联物，其特征在于，所述抗体的重链可变区序列选自下组：SEQ ID NO.:7、9、10、11、12、或13；和/或
    所述的抗体的轻链可变区序列选自下组：SEQ ID NO.:8、14、15、16、或17。
  7. 如权利要求1所述的抗体-药物偶联物，其特征在于，所述抗体选自下组：TF-mAb-SC1、TF-mAb-Ch、TF-mAb-H29、TF-mAb-H30、TF-mAb-H31、TF-mAb-H32、TF-mAb-H33、TF-mAb-H34、TF-mAb-H35、TF-mAb-H36、TF-mAb-H37、TF-mAb-H38、TF-mAb-H39、TF-mAb-H40、TF-mAb-H41、TF-mAb-H42、TF-mAb-H43、TF-mAb-H44、TF-mAb-H45、TF-mAb-H46、TF-mAb-H47、TF-mAb-H48。
  8. 一种权利要求1所述的抗体-药物偶联物的应用，其特征在于，所述抗体用于(i)制备诊断试剂；和/或(ii)制备预防和/或治疗TF相关的疾病的药物。
  9. 根据权利要求8所述的应用，其特征在于，所述TF相关的疾病选自下组：肿瘤的发生、生长和/或转移。
  10. 一种药物组合物，其特征在于，它含有：
    (i)活性成分，所述活性成分为如权利要求1所述的抗体药物偶联物或其组合；以及
    (ii)药学上可接受的载体。
PCT/CN2017/087779 2016-08-22 2017-06-09 靶向于组织因子的抗体-药物偶联物 WO2018036243A1 (zh)

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