INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application contains, as a separate part of disclosure, a sequence listing in computer-readable form (filename: 53908_Seqlisting.txt; Size: 12,788 bytes; Created: Feb. 18, 2019) which is incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to the medical field, particularly relates to an antibody targeted to tissue factor and an antibody-drug conjugate thereof, and preparation method and use thereof.

BACKGROUND ART

Tissue factor (TF) is a 47 kDa transmembrane glycoprotein. Under normal physiological conditions, TF expression is mainly sequestered in the layer of subendothelial cells; once the blood vessels are damaged in an organism, TF will be exposed to the blood stream, and initiate extrinsic coagulation by binding to and activating factor VII.

It has been found in studies that TF is abnormally activated and expressed in many tumor tissues, and plays an important role in the development and progression of tumors. Especially in advanced stage of cancer, most patients are accompanied by spontaneous thrombosis, such as Deep-vein thrombosis (DVT), Disseminated intravascular coagulation (DIC) and Pulmonary embolism (PE) (Thrombosis research, 2013, 131: S59-S62; Journal of Thrombosis and Haemostasis, 2011, 9(s1): 306-315). The abnormal expression of TF in tumor cells is the main cause responsible for these symptoms. Analysis on clinical samples of many tumors shows that the expression level of TF directly affects the deterioration indexes such as metastasis of tumor and occurrence of thrombosis in patients, for example, the percentage of abnormal TF expression was 85.8% in breast cancer, 88.5% in pancreatic cancer, 83.6% in lung cancer, and 91.3% in esophageal cancer, etc. (Blood, 2012, 119: 924-932).

It has been shown in studies that firstly, a TF-FVIIa complex formed by TF and FVII can directly bind to and induce the activation of a transmembrane G protein-coupled receptor, i.e. protease-activated receptor 2 (PAR2). PAR2 is an important signal pathway that regulates inflammatory response. Although there are few studies on PAR2 in the field of tumors, it is conceivable that TF can affect a series of tumor function signals in cells by virtue of PAR2. For example, TF-FVIIa-PAR2 promotes neovascularization, and provides adequate nutrients, energy and a suitable microenvironment for tumor growth by MAPK/ERK phosphorylation and inducing the gene expression of key growth factors, immunoregulators, and chemokines (e.g. VEGF, CSF1/2, IL8, CXCL1, etc.). Secondly, TF can also enhance the migration and adhesion of tumor cells by interaction with Rac1 and β1 family-related integrin, so as to enhance the hematogenous metastasis ability of tumor cells in general. Furthermore, TF-initiated coagulation is also an important cause of tumor thrombosis, and results in deterioration of a plurality of tumors. Meanwhile, TF-induced hypercoagulative state also directly helps tumor cells to evade the immune system in an organism and enhances the interaction between tumor cells and endothelial cells, resulting in an increase in the hematogenous metastasis ability. This is also an important reason why it is difficult to treat cancer.

By utilizing the characteristic that a monoclonal antibody specifically recognizes a specific antigen on the surface of tumor cells, an antibody-drug conjugate (ADC) can accurately deliver an anti-tumor drug (such as a small-molecule chemotherapeutic drug) to tumor target cells and release the drug there, so as to achieve the purpose of accurately killing tumors. ADCs are also regarded as the most potential anti-tumor drugs due to their suitable molecular weights, high stability, strong targeting property, and low toxic side effects. However, with respect to successful development of ADCs, there are also many problems that have to be taken into account and have to be solved, for example, an antibody has to specifically recognize a lesion site, has a low immunosensitization, and can be efficiently and rapidly internalized by a cell; an antibody-drug linker has to be highly stable in blood and can be specifically activated and efficiently release the small-molecule drug in the targeted cell; the conjugated small-molecule drug has a strong ability of killing cells, and so on.

It can be concluded that TF plays an important role in the development and progress of tumor, and antibody-drug conjugates have their unique characteristics and advantages, however, there still lack antibody-drug conjugates highly specific for human TF.

CONTENTS OF INVENTION

The objective of the present invention is to provide an antibody-drug conjugate, which specifically targets to human TF, has an activity of inhibiting tumor growth and metastasis, and the like.

In the first aspect, the present invention provides an antibody-drug conjugate comprising:

(a) an antibody moiety; and

(b) a conjugation moiety conjugated to the antibody moiety, wherein the conjugation moiety is selected from the group consisting of: a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof;

wherein, the heavy chain variable region of the antibody comprises the following three complementary determining regions (CDRs):

CDR1 as set forth in SEQ ID NO: 1,

CDR2 as set forth in SEQ ID NO: 2, and

CDR3 as set forth in SEQ ID NO: 3;

wherein any amino acid sequence of said amino acid sequences of the heavy chain variable region further includes a derivative sequence that is optionally resulted from the addition, deletion, modification and/or substitution of at least one amino acid, and can retain a TF-binding activity;

the light chain variable region of the antibody comprises the following three complementary determining regions (CDRs):

CDR1′ as set forth in SEQ ID NO: 4,

CDR2′ as set forth in SEQ ID NO: 5, and

CDR3′ as set forth in SEQ ID NO: 6;

a derivative sequence of any amino acid sequence of said amino acid sequences that is resulted from the addition, deletion, modification and/or substitution of at least one amino acid, and that has a TF-binding affinity.

In another preferred example, the antibody includes an intact antibody or an active fragment thereof.

In another preferred example, the active fragment retains its binding activity for tissue factor.

In another preferred example, an antibody-drug conjugate (ADC) has a formula as follows:
AbLU-D)pwherein:Ab is an anti-TF antibody,LU is a linker;D is a drug;and the subscript p is a value selected from 1-10, preferably from 1-8.

In another preferred example, 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), maleimidopropionyl-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-pyridyldithio)butanoic acid N-hydrosuccinimide ester (SPDB) or N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB).

In another preferred example, LU is SMCC, SPP, SPDB or 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-val-cit-PAB).

In another preferred example, D is selected from the group consisting of: Maytansine derivatives (DM1, DM4), auristatin and dolastatin.

In another preferred example, the D is selected from the group consisting of: Monomethyl auristatin E (MMAE), Monomethylauristatin F (MMAF), Monomethyl Dolastatin 10 (MMAD) derivatives or combinations thereof.

D1DM1D2DM4D3Monomethyl Auristatin E (MMAE)D4Monomethyl Auristatin F (MMAF)D5Monomethyl Dolastatin 10 (MMAD)

In another preferred example, the amino acid residue linked to D is originally present in the antibody (parent antibody) or is introduced exogenously.

In another preferred example, the amino acid residue linked to D is cysteine.

In another preferred example, the cysteine refers to one or more free cysteines introduced into the parent antibody at one or more positions of the light chain according to the Kabat numbering rule and/or at one or more positions of the heavy chain according to the Kabat numbering rule and at one or more positions of the heavy chain according to the EU numbering rule.

In another preferred example, the amino acid residue linked to D is lysine.

In another preferred example, the fragment is selected from: Fab, F(ab′)2, Fv or scFv fragment.

In another preferred example, the antibody is a monoclonal antibody.

In another preferred example, the antibody includes: a double-chain antibody, a single-chain antibody.

In another preferred example, the antibody is recombinant.

In another preferred example, the antibody is generated in a bacterium (e.g.E. coli).

In another preferred example, the antibody is generated in an eukaryotic cell (e.g. CHO cell).

In another preferred example, the antibody is selected from: an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof; more particularly, the antibody is a humanized antibody.

In another preferred example, the antibody has an EC50of 0.005-0.10 nM, more preferably 0.01-0.03 nM or 0.01-0.02 nM, for the affinity for human TF protein.

In another preferred example, the antibody does not bind to wild-type murine TF protein.

In another preferred example, the antibody has one or more characteristics selected from the group consisting of:

(a) inhibiting migration or metastasis of tumor cells; and

(b) inhibiting tumor growth;

In another preferred example, the sequence of the heavy chain variable region of the antibody is selected from the group consisting of: SEQ ID NO: 7, 9, 10, 11, 12, or 13; and/or the sequence of the light chain variable region of the antibody is selected from the group consisting of: SEQ ID NO: 8, 14, 15, 16, or 17.

In another preferred example, 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, and TF-mAb-H48.

In the second aspect, the present invention provides use of the antibody-drug conjugate according to the first aspect of the present invention, for (a) manufacture of a diagnostic agent; and/or (b) manufacture of a medicament for preventing and/or treating a TF-related disease.

In another preferred example, the TF-related disease is selected from: tumorigenesis, tumor growth and/or metastasis, a thrombosis-related diseases, inflammation, a metabolism-related disease, or a combination thereof.

In another preferred example, the tumor is a tumor with high TF expression.

In another preferred example, the expression “high TF expression” means that when the TF transcript and/or protein level L1 in a tumor tissue is compared with the transcript and/or protein level L0 in a normal tissue, L1/L0≥2, preferably ≥3.

In another preferred example, the tumor is selected from the group consisting of: triple negative breast cancer, pancreatic cancer, lung cancer and malignant glioma.

In the third aspect, the present invention provides a pharmaceutical composition, comprising:

(i) an active ingredient that is the antibody-drug conjugate according to the first aspect or a combination thereof; and

(ii) a pharmaceutically acceptable carrier.

In another preferred example, the pharmaceutical composition is an unit dosage form for human.

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

In another preferred example, in the pharmaceutical composition, the antibody-drug conjugate is in an amount of 0.005-50 wt %, preferably 0.05-10 wt %.

In another preferred example, the pharmaceutical composition further comprises (iii) an additional therapeutic agent.

In another preferred example, the additional therapeutic agent includes a chemotherapeutic agent.

In the fourth aspect, the present invention provides a method for non-therapeutically inhibiting tumor cells in vitro, comprising the step of: contacting the tumor cells with the antibody-drug conjugate according to the first aspect.

In the fifth aspect, the present invention provides a method for treating a tumor, comprising the step of: administering to a subject in need thereof the antibody-drug conjugate according to the first aspect.

In another preferred example, the subject is a mammal, including human.

In another preferred example, the contacting is carried out in an in vitro culture system.

In the sixth aspect, the present invention provides a method for slowing tumor growth in a subject, comprising the step of: using an effective amount of the antibody-drug conjugate according to the first aspect in combination with one or more therapies selected from: radiation therapy, chemotherapy, biological therapy, or a combination thereof.

In the seventh aspect, the present invention provides a method for inhibiting cell migration in a subject, comprising the step of: using an effective amount of the antibody-drug conjugate according to the first aspect in combination with one or more therapies selected from: radiation therapy, chemotherapy, biological therapy, or a combination thereof.

In the eighth aspect, the present invention provides a method for inhibiting cell adhesion in a subject, comprising the step of: using an effective amount of the antibody-drug conjugate according to the first aspect in combination with one or more therapies selected from: radiation therapy, chemotherapy, biological therapy, or a combination thereof.

In the ninth aspect, the present invention provides a method for preparing a humanized or chimeric antibody, comprising the steps of:

after cloning the nucleotide sequence of a murine antibody variable region according to the present invention into an expression vector containing a human antibody constant region, expressing the human-mouse chimeric antibody by transfecting animal cells;

after cloning the nucleotide sequence of an antibody variable region comprising human FR region according to the present invention into an expression vector comprising a human antibody constant region, expressing the humanized antibody by transfecting animal cells.

In another preferred example, the antibody is a partially or fully humanized monoclonal antibody.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (e.g. in Examples) may be combined with each other so as to constitute a new or preferred technical solution. For the purpose of space saving, these combinations are no longer described here.

