WO2017107914A1 - 一种药物设计方法和获得的药物及其应用 - Google Patents

一种药物设计方法和获得的药物及其应用 Download PDF

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WO2017107914A1
WO2017107914A1 PCT/CN2016/111264 CN2016111264W WO2017107914A1 WO 2017107914 A1 WO2017107914 A1 WO 2017107914A1 CN 2016111264 W CN2016111264 W CN 2016111264W WO 2017107914 A1 WO2017107914 A1 WO 2017107914A1
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drug
antibody
biologically
inert
biologically inert
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PCT/CN2016/111264
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French (fr)
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张美�
季俊虬
高美华
陈军
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合肥立方制药股份有限公司
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Priority to CN201680071364.6A priority Critical patent/CN108430511B/zh
Priority to EP16877724.1A priority patent/EP3395366B1/en
Priority to US16/064,953 priority patent/US20190002516A1/en
Publication of WO2017107914A1 publication Critical patent/WO2017107914A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/54—F(ab')2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif

Definitions

  • the invention relates to the field of drug design and development and the field of pharmaceutical preparations, in particular to the field of drug design and development of diseases related to ED-B, and also relates to medicines and therapeutic applications.
  • Drugs enter organs, tissues, cells, and biological macromolecules and their specific drug binding sites that are active in the human body, which are called target organs, target tissues, target cells, and targets, respectively.
  • the target may be a protein, a nucleic acid or other substance, including a biological macromolecule such as a gene locus, a receptor, an enzyme, an ion channel, or a nucleic acid, and generally has an important physiological or pathological function.
  • the drug can specifically act on the target, but some of the drug targets are distributed on the unintended drug-active organs or tissues, or fall off in the blood, and will compete with the drug to cause some drugs to fail to target the target organs.
  • this will affect the efficacy of the drug, on the one hand consumption of drugs, on the other hand will produce toxic side effects.
  • the target protein that is shed from the tissue is freed in blood or tissue fluid, which affects the pharmacokinetics of the drug.
  • the above characteristics of the target may cause some problems, such as: increased drug dosage, large individual differences, increased drug side effects, narrow therapeutic window, or intractable toxicity.
  • antibody drugs or antibody-related drugs In the development of antibody drugs or antibody-related drugs, the phenomenon of drug off-target and the side effects caused by non-target organ targets are more obvious. Although high hopes for anti-tumor antibody drugs bring hope to cancer patients, the effect is still not satisfactory.
  • antibody drugs developed against human epidermal growth factor receptor 2 (HER2) such as Herceptin, have achieved good results in the treatment of breast cancer, but because of the distribution of HER-2 protein in the transverse tube of the myocardium, long-term use May increase cardiovascular toxicity, cause congestive heart failure, mild or even moderate to severe cardiac dysfunction (Nemeth, BT, et al. (2016). Br J Pharmacol.doi: 10.1111/bph.13643), which is a drug The side effects of the target protein distribution are not high.
  • HER2 human epidermal growth factor receptor 2
  • Additional domain B for fibronectin
  • the developed antibody drugs such as L19-IL2 are not effective by intravenous route (Eigentler, TK, et al., (2011) Clin Cancer Res, 17(24): 7732-42), but pass the tumor lesions locally. The administration was remarkable (Weide, B., et al., (2014) Cancer immunology research 2 (7): 668-678).
  • the present invention provides a kit or kit comprising the biologically inert drug and the biologically active drug of the present invention, wherein
  • the bioactive drug comprises a targeting moiety and a biologically active moiety
  • the bio-inert drug comprises a targeting moiety and a bio-inert moiety, wherein the bio-inert drug and the bioactive drug are capable of targeting the same target, wherein the bio-inert drug and the targeting moiety of the bioactive drug can Same or different.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a biologically inert drug and a biologically active agent according to the invention, and optionally a pharmaceutically acceptable carrier and/or excipient.
  • the targeting moiety is selected from a ligand, receptor, antibody or antibody fragment, such as an intact antibody (eg, a monoclonal antibody, a chimeric antibody, etc.), a CDR region, a variable region, a Fab, a Fab'F(ab)' 2 , single chain Fv (scFv), Fv fragment, antibody light chain, antibody heavy chain, single domain antibody, diabodies and linear antibodies.
  • a ligand, receptor, antibody or antibody fragment such as an intact antibody (eg, a monoclonal antibody, a chimeric antibody, etc.), a CDR region, a variable region, a Fab, a Fab'F(ab)' 2 , single chain Fv (scFv), Fv fragment, antibody light chain, antibody heavy chain, single domain antibody, diabodies and linear antibodies.
  • the kit, kit or pharmaceutical composition is for treating a disease associated with fibronectin ED-B, such as a solid tumor.
  • the targeting moiety is an antibody or fragment thereof that targets fibronectin (FN) extra domain B (Extradomain B, ED-B), such as CGS-1, CGS-2, L19 Or B5 antibody or antibody fragment thereof.
  • FN fibronectin
  • ED-B extra domain B
  • the biologically active moiety is selected from the group consisting of a cytotoxin, a cytokine, a radioisotope, a chemical, a biologically active antibody constant region, and a biologically active cell.
  • the biologically inert moiety is selected from the group consisting of an antibody constant region, albumin, polyethylene glycol, and a nucleic acid aptamer.
  • the biologically active agent is the polypeptide set forth in SEQ ID NO: 6.
  • the biologically inert drug is the polypeptide set forth in SEQ ID NO: 5 or a dimer thereof.
  • the kit or kit comprises a biologically inert drug mixed with the bioactive drug to form a single formulation or composition, or the biologically inert drug and the bioactive drug are separately formulated into separate formulations. Or a composition.
  • the invention provides a biologically inert drug comprising a targeting moiety and a biologically inert moiety, optionally wherein the targeting moiety can be selected from a ligand, a receptor, an antibody, or a fragment thereof.
  • the targeting moiety is an antibody or fragment thereof that targets fibronectin ED-B, such as a CGS-1, CGS-2, L19 or B5 antibody or fragment thereof, eg, SEQ ID NO:3 The polypeptide shown.
  • the biologically inert moiety is selected from the group consisting of an antibody constant region, albumin, polyethylene glycol, and a nucleic acid aptamer, such as the polypeptide set forth in SEQ ID NO:4.
  • the biologically inert drug is the polypeptide set forth in SEQ ID NO: 5 or a dimer thereof.
  • the invention provides a method of treating a disease comprising administering to a subject in need of treatment a biologically inert or biologically active agent of the invention or a pharmaceutical composition of the invention, or using the invention Kit or kit.
  • the bio-inert drug is administered concurrently with the bioactive drug, or sequentially, for example at intervals of 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour. 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours or longer.
  • the method comprises administering a bio-inert drug and then administering the bioactive drug.
  • the disease is a disease associated with fibronectin ED-B, such as a solid tumor.
  • the invention provides the use of a bioinert drug and/or a biologically active agent of the invention in the manufacture of a kit or kit or pharmaceutical composition for treating a disease in a mammalian subject, such as a human.
  • the disease is a disease associated with fibronectin ED-B, For example, solid tumors.
  • the bio-inert drug is mixed with the bioactive drug to form a single formulation or composition, or the bio-inert drug and the bioactive drug are separately formulated into separate formulations or compositions.
  • the invention provides a bioactive drug and/or a biologically inert drug for treating, for example, an ED-B related disease, wherein the bioactive drug comprises a targeting moiety and a organism that specifically binds ED-B An active moiety, the biologically inert drug comprising a targeting moiety and a biologically inert moiety, the treatment comprising administering to the subject having the disease a biologically active drug and a biologically inert drug according to the invention, optionally wherein the biologically inert
  • the drug is administered simultaneously or prior to the bioactive drug, for example, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours. , 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours or longer.
  • the targeting moiety is selected from the group consisting of a ligand, a receptor, an antibody, or a fragment thereof, such as an intact antibody (eg, a monoclonal antibody, a chimeric antibody, etc.), a CDR region, a variable region, a Fab, a Fab'F(ab)' 2 , single chain Fv (scFv), Fv fragment, antibody light chain, antibody heavy chain, single domain antibody, diabodies and linear antibodies.
  • the biologically active moiety is selected from the group consisting of a cytotoxin, a cytokine, a radioisotope, a chemical, a biologically active antibody constant region, and a biologically active cell.
  • the biologically inert moiety is selected from the group consisting of an antibody constant region, albumin, polyethylene glycol, and a nucleic acid aptamer, such as the polypeptide set forth in SEQ ID NO:4.
  • the invention provides the use of a bioinert drug and/or a bioactive drug of the invention in the manufacture of a kit, kit or pharmaceutical composition for use in a disease.
  • the disease is a disease associated with fibronectin ED-B, such as a solid tumor.
  • the bio-inert drug is mixed with the bioactive drug to form a single formulation or composition, or the bio-inert drug and the bioactive drug are separately formulated into separate formulations or compositions.
  • the invention provides a method of drug design comprising:
  • the method is for designing a medicament for treating a disease associated with ED-B, comprising:
  • a targeting moiety that specifically binds ED-B such as an antigen binding portion of an antibody, to a biologically active moiety
  • a targeting moiety that specifically binds ED-B such as an antigen binding portion of an antibody, to a biologically inert moiety.
  • the antigen binding portion is a scFv of a B5 antibody, such as the polypeptide set forth in SEQ ID NO:3.
  • Figure 1 Schematic representation of the B5-Fc fusion protein dimer.
  • Figure 2 Schematic representation of the B5-IL2 fusion protein.
  • Figure 3 Expression of ED-B in the blood of the subject.
  • the invention relates to a drug system development method for improving drug specificity, a corresponding drug and pharmaceutical composition, a kit or a kit, and a related disease treatment method.
  • the invention further improves the specificity of the drug, improves the distribution of the drug in the organ or tissue of the lesion and reduces the distribution in the normal tissue, can reduce the damage to the non-target organ or the non-target tissue, and can also significantly improve the drug. Efficacy while reducing the waste of drugs.
  • the invention achieves precise administration by blocking or eliminating the target of non-administered target tissue.
  • the highly specific drug system, drug and formulation development of the present invention is to design a new basic non-bioactive drug to replace a part of the bioactive drug, distribute it on a non-focal target, reduce the amount of bioactive drug or increase the bioactive drug. The proportion of the distribution in the lesion, thereby reducing the side effects of the drug or improving the efficacy.
  • the highly specific pharmaceutical systems, medicaments, and formulations of the present invention can also be used to target a predominantly or only a diseased site by first administering a biologically inert drug in place of the biologically active drug over the expressed target of the lesion, The bioactive drug is then administered to the new lesion target.
  • the medicament of the present invention may be a nucleic acid drug, a polypeptide drug, a protein drug, or a conjugate of a biopharmaceutical and a chemical drug.
  • the drug enters the organs, tissues, cells and biological macromolecules and their specific drug binding sites that can exert their active functions, which are called target organs, target tissues, target cells, target molecules and targets.
  • the target molecule can be a protein, a nucleic acid or other substance, including a biological macromolecule such as a gene locus, a receptor, an enzyme, an ion channel, a nucleic acid, etc., and generally has an important physiological or pathological function.
  • the "closed or eliminated" drug target on the non-target organ of the present invention may be partially blocked or partially eliminated, and may also have a certain blocking or eliminating effect on the target organ, the target tissue, the lesion and the like. Because the drug target has a higher density or concentration in the lesion than the normal tissue, the drug target distribution in normal tissues is less or difficult to detect, or because the free target in the blood is easier to target than in the tissue. Drug binding, thus giving a bio-inert drug a more pronounced effect on targets in normal tissues or blood than on targets in the lesion.
  • the highly specific pharmaceutical system of the present invention comprises a biologically inert drug and a biologically active drug, wherein the biologically inert drug and the biologically active drug are capable of targeting the same target, for example having the same targeting moiety.
  • the method for developing a highly specific drug system according to the present invention also includes a drug design method, a drug partner design method, a combination method of a drug and a drug partner, and these methods may be used singly or in combination.
  • the biological activity of the present invention refers to a biological effect or a therapeutic effect having biological metabolism or physiological regulation function, such as promoting digestion and absorption, promoting immune regulation, hormone regulation, antibacterial, antiviral, antitumor, cytotoxicity, blood pressure lowering, blood fat lowering Equal activity.
  • the biological inertness of the present invention means that it does not have the above biological activity or has a weak biological activity.
  • the "biologically inert drug” or “biologically inert drug” as used in the present invention has the same meaning, and refers to a substance which is substantially free of biological activity in a living body, especially in a human body, although it can be produced with cells, proteins, etc. in the human body.
  • the combination of effects, but can not significantly affect the body's physiological, biochemical and pathological processes, or the treatment is very weak, mainly used to assist biologically active drugs to improve drug efficacy or reduce side effects.
  • the biologically inert drug is a relatively biologically active drug.
  • the substantially no biological activity mainly means that the biologically inert drug does not have the pharmacological effect of the biologically active drug, or the drug effect is negligible compared with the biologically active drug at the same dose.
  • the biologically inert drug may have other functional or physiological activities.
  • the biologically inert drug may have fluorescence, may block the binding of the target to the biologically active drug, and may bind to other ligands or receptors.
  • the biologically inert drug does not have the biological activity of the corresponding active drug or has no therapeutic effect when used alone.
  • a bio-inert drug is used in conjunction with a bioactive drug and may also be referred to as a drug partner.
  • drug partner refers to an auxiliary drug or an auxiliary molecule which is the same target as the active drug recognition and which is accompanied by the active drug, and refers to a biologically inert drug in the present invention.
  • a biologically active drug recognizes and binds to a specific target, producing a therapeutic effect of the drug.
  • the active portion of the bioactive drug eg, a biologically active molecule
  • can produce a desired therapeutic effect such as a cytotoxin, a cytokine, a radioisotope, a chemical, a biologically active antibody constant region, or a biologically active cell such as T lymphocytes.
  • treatment is a method of obtaining beneficial or desired results, including clinical results.
  • beneficial or desirable clinical outcomes include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, reducing the extent of the disease, and stabilizing the disease (eg, preventing or delaying the progression of the disease), Prevent or delay the spread of disease (such as metastasis), prevent or delay the recurrence of the disease, delay or slow the progression of the disease, improve the disease state, reduce the disease (partially or completely), reduce the dose of one or more other drugs needed to treat the disease, and delay Progression of the disease, increasing or improving quality of life, increasing body weight, and/or prolonging survival.
  • Treatment also culverts Covers reduce the pathological findings of cancer (eg, tumor volume).
  • the present invention relates to a method of drug design and development comprising selecting a targeting moiety that specifically binds to a target according to a target, generating a biologically active drug based on the targeting moiety, and exerting a therapeutic effect based on
  • the targeting moiety or target produces a biologically inert drug that acts as a target shielding or acts to promote target clearance, such as target degradation, target endocytosis, target entry into the digestive system, and the like.
  • the invention achieves precise administration of the lesion by blocking or eliminating non-lesions such as normal tissue or targets in the blood (eg, proteins).
  • non-lesions such as normal tissue or targets in the blood (eg, proteins).
  • methods of shielding and eliminating the target such as a composition or a prescription using a biologically inert drug and a biologically active drug, wherein the biologically inert drug and the biologically active drug are capable of targeting the same target.
  • a biologically active drug recognizes and binds to a specific target to produce a therapeutic effect
  • a biologically inert drug recognizes and binds to a specific target
  • Targets for active drug binding such as, in particular, a target that reduces distribution in a normal tissue that binds to a bioactive drug, as well as a target that reduces distribution in the blood that binds to the bioactive drug.
  • biologically active drugs and biologically inert drugs are used, wherein the biologically active drugs can exert a biological therapeutic effect, and the biologically inert drugs act to block or eliminate targets (such as target proteins) on non-target organs, said elimination targets It can be proteolytic elimination or it can speed up the process of endocytosis of the target by cells.
  • targets such as target proteins
  • the biologically inert drug and the biologically active drug may have the same mother nucleus, may have the same targeting moiety, for example, recognize the same antigenic epitope, may compete for the same target, and may compete for the same target protein or protein complex.
  • the invention provides a method of drug design comprising:
  • the target of the invention may be a biomolecule expressed or present on a particular diseased organ, tissue and/or cell, such as a receptor, ion channel or any other suitable surface molecule.
  • the target can be a specific biomarker for the disease.
  • the target is expressed or present not only on diseased organs, tissues and/or cells, but also on normal organs, tissues and/or cells or in body fluids such as blood.
  • the target of the present invention may include, for example, a receptor tyrosine kinase-like target that promotes neovascularization, such as epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR).
  • EGFR epidermal growth factor receptor
  • VEGFR vascular endothelial growth factor receptor
  • PDGFR platelet-derived growth factor receptor
  • FGFR fibroblast growth factor receptor
  • Highly expressed proteins or tumor markers such as fibronectin (Fn) extra domain A (ED-A), carcinoembryonic antigen (CEA), glycoprotein antigen (eg MUC16/CA125, MUC1/CA) 15-3), epithelial cell adhesion molecule (EpCAM) and carcinoembryonic antigen-associated cell adhesion molecule (CEACAM1).
  • the targets of the invention include, but are not limited to, HER2, ED-B, ED-A, EGFR, VEGFR, PDGFR, FGFR, CEA, glycoprotein antigens (eg, MUC16/CA125, MUC1/CA 15-3) ), EpCAM and CEACAM1.
  • the targeting moiety of the invention may be a molecule or moiety known in the art or recognized in the future that is capable of specifically targeting (eg, recognizing and/or binding) a target of interest, such as an antibody, ligand, receptor, or portion thereof.
  • the targeting moiety is an antibody or a fragment thereof that binds to an antigen, such as an intact antibody (eg, a monoclonal antibody, a chimeric antibody, etc.), a CDR region, a variable region, Fab, Fab', F(ab) ' 2 , single-chain Fv (scFv), Fv fragment, antibody light chain, antibody heavy chain, single domain antibody, diabodies, and linear antibodies.
  • Common targeting moieties are described in Wu, AM and PDSenter. Nat Biotechnol 23(9): 1137-1146 (2005).
  • linear antibody generally refers to an antibody described in Zapata et al., Protein Eng., 8(10): 1057-1062 (1995). These antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that together with the complementary light chain polypeptide form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
  • Papain digestion of the antibodies produces two identical antigen-binding fragments termed "Fab” fragments and the remaining “Fc” fragments.
  • the Fab fragment consists of the entire L chain along the variable region domain (VH) of the H chain and the first constant domain (C H 1) of one heavy chain.
  • VH variable region domain
  • C H 1 first constant domain
  • Each Fab fragment is monovalent with respect to antigen binding, ie, it has a single antigen binding site.
  • Trypsin treatment of the antibody yields a single large F(ab') 2 fragment that roughly corresponds to two disulfide-linked Fab fragments, has bivalent antigen binding activity, and is still capable of cross-linking antigen.
  • Fab 'fragments differ from Fab fragments having additional few residues at the carboxy terminus of the C H 1 domain including one or more cysteines from the antibody hinge region.
  • Fv is the smallest antibody fragment that contains the entire antigen recognition and antigen binding site. This fragment consists of a tightly non-covalently associated dimer of a heavy chain variable region domain and a light chain variable region domain. Folding of these two domains yields six hypervariable loops (each of the three loops from the H and L strands) that contribute to the antigen-bound amino acid residues and confer antigen binding specificity to the antibody. A single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) also has the ability to recognize and bind antigen.
  • Single-chain Fv also abbreviated as “sFv” or “scFv”
  • sFv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for antigen binding.
  • diabodies refers to small antibody fragments prepared by constructing sFv fragments with short linkers (about 5-10 residues) between the V H and V L domains, thereby obtaining a inter-chain V domain and Instead of in-chain pairing, a bivalent fragment, a fragment with two antigen binding sites, is produced.
  • Bispecific diabodies are two "crossover" sFv fragments in heterodimers, V H and V L, wherein the domains of the two antibodies are present on different polypeptide chains. See, for example, EP 404,097, WO 93/11161 and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
  • the targeting moiety of the invention may be covalently linked directly to the biologically active moiety and/or the biologically inert moiety or indirectly via a linker.
  • Methods of attaching a targeting moiety to a biologically active moiety or a biologically inert moiety are known in the art.
  • One skilled in the art can also determine, based on the knowledge in the art, that the targeting moiety is directly covalently linked to a biologically active moiety or a biologically inert moiety or linked via a linker (e.g., the linker set forth in SEQ ID NO: 7 or 8).
  • linker of the invention is a short peptide of no more than 50 amino acids.
  • the bioactive drug is an antibody that is replaced by a portion of the antibody constant region that is capable of producing a biologically active, therapeutic or pharmaceutical effect
  • the active moiety that produces a biological, therapeutic or pharmaceutical effect may be selected from the group consisting of cytotoxins, Cytokines (such as IL-2, TNF ⁇ , IL-12), radioisotopes, chemotherapeutic drugs, and conjugated chemicals.
  • the biologically inert moiety is selected from the group consisting of an antibody constant region such as IgG4 Fc, albumin, polyethylene glycol, and a nucleic acid aptamer.
  • the targeting moiety is an antibody variable region
  • the biologically inert drug is an intact antibody with an antibody constant region
  • the biologically active drug is an antibody in which the constant region of the antibody is replaced with a drug-effect portion.
  • the moiety having a drug effect may be selected from a cytokine (such as IL2, TNF ⁇ , IL12), a radioisotope, and a coupling chemical.
  • the targeting moiety is an antibody variable region
  • the biologically inert portion is the constant region of an IgG4 antibody
  • the biologically active portion is IL-2.
  • the targeting moiety is an antibody variable region
  • the biologically inert portion is an IgG4 antibody constant region
  • the biologically active portion comprises an IgG4 antibody constant region conjugated to a potent anti-microtubule drug DM1 .
  • the invention provides a method of drug design for treating a disease associated with ED-B, comprising:
  • linker eg, the linker set forth in SEQ ID NO: 7 or 8
  • Fibronectin is a multifunctional glycoprotein composed of epithelial cells, endothelial cells, fibroblasts, hepatocytes, decidual cells, and extravillous trophoblasts. Da.
  • the gene of FN is about 75 kb, contains about 50 exons, and has a relative molecular mass of about 250 kDa. It is mainly composed of three kinds of homologous repeating spherical domain units of type I, II and III, and each pair consists of trypsin. Sensitive peptide chain linkage.
  • ED-B Extradomain B
  • ED-B is a complete domain comprising 91 amino acids encoded by a single exon in a type III repeat of FN.
  • FN(B+) FN(B+)
  • FN(B+) FN(B+)
  • FN(B+) is used as a marker for tumor neovascularization (Carnemolla) , Balza et al. (1989). J Cell Biol. 108: 1139-1148.).
  • ED-B refers to a protein comprising an ED-B amino acid sequence, or a homolog thereof, wherein the ED-B amino acid sequence has, for example, the amino acid sequence set forth in SEQ ID NO: 10 or at least 80% with SEQ ID NO: A sequence of 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity.
  • SEQ ID NO: 10 the amino acid sequence set forth in SEQ ID NO: 10 or at least 80% with SEQ ID NO: A sequence of 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity.
  • the present inventors have found in previous studies that ED-B protein is contained in blood and that ED-B protein levels in blood of tissue hyperplasia such as tumor patients are elevated as compared with normal or non-tumor subjects (PCT/CN2015/094727). This patent application is incorporated herein by reference in its entirety.
  • L19-IL2 When administered for the first time, a large amount of ED-B antigen is present in the blood, L19-IL2 will bind to blood ED-B, and the affinity of L19 antibody is high and exists in plasma for a long time, and ED-B bound to L19-IL2 is metabolized with L19-IL2 and cleared from the blood; when re-administered in the same treatment cycle, L19-IL2 binds to it due to a decrease in blood ED-B content. Reduced, free L19-IL2 can quickly enter the solid tumor tissue, so the detected plasma concentration (AUC / D) is lower than the previous dose.
  • the ED-B related diseases of the present invention refer to diseases in which ED-B is present and/or expressed in diseased organs, tissues and/or cells.
  • the ED-B associated disease of the invention is tissue hyperplasia, including, for example, mammary gland hyperplasia, neoplasm, benign tumor, malignancy or cancer, for example selected from various types of squamous cell carcinoma, adenocarcinoma or sarcoma.
  • the tumor of the present invention is a solid tumor selected from, for example, teratomas, upper gastrointestinal cancers such as esophageal cancer, cardiac cancer, laryngeal cancer, gastric cancer, head and neck cancer, liver cancer, biliary tract cancer, gallbladder cancer, colon Cancer, duodenal cancer, lung cancer, bladder cancer, cervical cancer, ovarian cancer, endometrial cancer, breast cancer, melanoma, pancreatic cancer, kidney cancer and prostate cancer.
  • the tumor is a teratoma, nasopharyngeal carcinoma, esophageal cancer, gastric cancer, lung cancer or pancreatic cancer.
  • the tumor is a lymphoma.
  • the ED-B specific antibody comprises, for example, CGS-1, CGS-2 (PCT/GB97/01412; Nissim, Hoogenboom et al. (1994). EMBO J. 13: 692-698), L19 (Pini, Viti et al. (1998). J Biol Chem. 273: 21769-21776), B5 (CN201480001324.5; WO2014/194784).
  • the ED-B specific antibody is an L19 or B5 antibody.
  • the antigen binding portion of the ED-B specific antibody is an intact antibody, CDR region, variable region, Fab, Fab', F(ab)' 2 , single chain Fv (scFv), Fv fragment , antibody light chain, antibody heavy chain, single domain antibody, double antibody and linear antibody.
  • the biologically active moiety is selected from the group consisting of a cytotoxin, a cytokine, a radioisotope, a chemical, a biologically active antibody constant region, and a biologically active cell.
  • the biologically active moiety is a cytokine, such as IL-2, such as the polypeptide set forth in SEQ ID NO:9.
  • the biologically inert moiety is selected from the group consisting of an antibody constant region, albumin, polyethylene glycol, and a nucleic acid aptamer.
  • the biologically inert portion is an antibody constant region, such as an IgG4 antibody constant region (IgG4 Fc), such as the polypeptide set forth in SEQ ID NO:4.
  • the invention also provides a method for developing a biologically inert drug and a corresponding biologically inert drug.
  • the bio-inert drug of the present invention does not have the therapeutic effect of the active drug, or has a therapeutic effect but is different from the indication of the active drug, and more particularly, the inert drug does not have the biological activity of the corresponding active drug.
  • the biologically inert drug may be modified on the active drug to have only the function of recognizing and binding to the target, including but not limited to removing the biologically active structure, inactivating the biologically active site, and replacing the biologically active site of the active drug. Structures that inhibit the action of biological activity, such as by chemical modification of the active site of the drug, or fusion of substantially biologically inactive proteins form steric hindrance.
  • the inert drug (drug partner) of the present invention needs to be used in combination with an active drug.
  • the active drug and drug partner that target the same target can be formulated as a single formulation or composition, or separately as a separate formulation or composition, by administering a drug partner to a specific target to block the target or partially block the target, by The active drug is administered to target unblocked targets or newly generated targets such that the active drug reaches the target of the lesion in a more precise and/or higher ratio.
  • Drug partners are used to increase the safety, efficacy, and/or targeting of existing active drugs.
  • a bio-inert drug can be formulated with a bioactive drug and administered simultaneously; a bio-inert drug can also be an excipient in a pharmaceutical formulation that competes with a bioactive drug for binding to a free target in the blood or other Targets in normal tissues also have the effect of protecting bioactive drugs.
  • the biologically inert drug can modify, modify or replace the pharmaceutically active structure as described above on the basis of retaining only the drug-targeted binding site, thereby converting the existing active drug into an inert drug, for example, reducing the original activity of the active drug by more than 50%. , 90% or more, 99% or more, 99.9% or more.
  • the inert drug and the active drug may have the same molecular structure, for example, the ratio of the same structure to the molecular structure of the active drug is greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80. %, even greater than 90%.
  • the two are mainly highly similar to the molecular structure of the binding site of the target, for example, the similar proportion of the binding sites is 50%, 60%, 70%, 80%, 90%, 95%, or even 100% identical.
  • the two can not only bind to the same target molecule, but also close to the target site, even highly overlapping, when the inert drug binds to the corresponding target to form steric hindrance, so that the active drug cannot bind to the same target with the inert drug. .
  • a biologically inert drug can recognize and bind to a particular target, can shield the target and/or promote target degradation and/or maintain bioactive drug stability.
  • the inert portion of the biologically inert drug (also referred to as a biologically inert molecule) can shield the target and/or direct degradation of the target, and can be, for example, an antibody constant region, albumin, polyethylene glycol, or a nucleic acid aptamer.
  • Bioly inert drugs can be biopharmaceuticals including, but not limited to, nucleic acid drugs, polypeptide drugs, protein drugs, chemical drugs, and coupling of biopharmaceuticals and chemical drugs. Things.
  • Bio-inert drugs including but not limited to antibodies, antibody recombinant proteins, analogs or modifications of biologically active drugs, are only bioactive drugs that are identical to the target recognition portion of a biologically active drug.
  • the biologically inert drug and the biologically active drug may or may not have the same chemical structure.
  • the biologically inert drug is a nucleic acid aptamer and the biologically active drug is a protein.
  • the biologically inert drug can be based on a constant region fragment of a parental fusion antibody having a targeted specificity, or a domain that fuses human albumin, polyethylene glycol modifications, fusion polypeptide fragments, or native proteins.
  • the invention provides a pharmaceutical partner for an ED-B protein-related drug, such as an antibody to an ED-B protein, in the manufacture of a kit, kit or pharmaceutical composition for treating a tumor in a mammal, such as a human application.
  • the pharmaceutical partner of the present invention is an antibody drug partner that targets fibronectin ED-B, based on a biopharmaceutical with a L19 or B5 antibody as the core, retaining the variable region of the L19 or B5 antibody
  • the antibody constant region crystal fragment (FC) protein is fused to form a drug partner L19- or B5-IgG4Fc, and is administered in combination with a L19 or B5-related drug.
  • the development method of the high specific drug system, the drug and the prescription according to the present invention is not only suitable for the development of anticancer drugs, but also suitable for drug development of autoimmune diseases, and is also suitable for drug development of other diseases.
  • kits, kits and pharmaceutical compositions comprising the corresponding biologically active drugs and/or biologically inert drugs produced by the above methods.
  • the present invention also provides a composition comprising a biologically inert drug and a biologically active drug, wherein the biologically inert drug and the biologically active drug both have the same targeting moiety, wherein the biologically inert drug is composed of the targeting moiety and The biologically inert portion is composed of a covalent bond, and the biologically active drug is composed of a covalent bond of the targeting moiety and the biologically active moiety.
  • the targeting moiety of the bio-inert drug and the bioactive drug is an antibody or antibody fragment.
  • the kit and the kit are used interchangeably and are meant to include a combination of a biologically inert drug and a biologically active drug according to the invention or a pharmaceutical composition according to the invention, or obtained according to the method of the invention.
  • a combination or pharmaceutical composition of a bioactive drug and a biologically inert drug, and optionally a drug-applied tool and/or other desired agent such as a buffer, a reconstituted solvent, or the like.
  • the present invention provides a pharmaceutical system, kit, kit or pharmaceutical composition
  • a pharmaceutical system, kit, kit or pharmaceutical composition comprising the biologically inert drug and biologically active drug of the present invention or a biologically active drug and organism obtained by the method of the present invention
  • the bio-inert drug has the same targeting moiety as the bioactive drug.
  • the bioactive drugs of the present invention recognize and bind to specific targets and produce therapeutic effects.
  • the bioactive drug comprises a targeting moiety and a biologically active moiety, wherein the targeting moiety recognizes and binds to a target site, the biologically active moiety producing a biological activity or therapeutic effect, eg, a specific effect is known Substance, therapeutic effect, indications, etc.
  • the targeting moiety is a molecule, such as an antibody, ligand, receptor, or the like, or a portion thereof that can specifically target a target of interest.
  • the targeting moiety is an intact antibody or fragment or portion thereof, such as a CDR region, a variable region, Fab, Fab ', F ( ab)' 2, single chain Fv (scFv), Fv fragment, Antibody light chain, antibody heavy chain, single domain antibody, double antibody and linear antibody.
  • the biologically active moiety is selected from the group consisting of a cytotoxin, a cytokine (eg, IL-2, TNF ⁇ , IL-12), a radioisotope, a chemotherapeutic drug, a chemical, a biologically active antibody constant region, and a biological Active cells.
  • a cytotoxin e.g, IL-2, TNF ⁇ , IL-12
  • a radioisotope eg., IL-2, TNF ⁇ , IL-12
  • chemotherapeutic drug eg.g, IL-2, TNF ⁇ , IL-12
  • the biologically active moiety is a cytokine, such as the polypeptide set forth in SEQ ID NO:9.
  • the bio-inert drug of the present invention can recognize and bind to a specific target and can shield the target or promote the degradation of the target or promote the endocytosis of the target, promote the target into the digestive system, etc., thereby reducing the binding to the bioactive drug.
  • Targets include reducing targets that are distributed in normal tissues that bind to bioactive drugs, and reducing targets that are distributed in the blood that bind to bioactive drugs.
  • bio-inert drugs block or partially block free protein targets in the blood and protein targets distributed in normal tissues, reducing the binding of bioactive drugs to non-target organs and non-target tissues, so that bioactive drugs can be more Distribution to target organs and target tissues, thereby increasing the effect or therapeutic effect of the bioactive drug at the site of the lesion, reducing the amount of the bioactive drug, reducing the toxic side effects of the drug, improving the safety of the drug;
  • the role of active drugs in stabilizing The bio-inert drug of the present invention itself has no therapeutic effect and needs to be used in combination with a bioactive drug.
  • the bio-inert drug improves health
  • the metabolism and distribution of active drugs is a companion to bioactive drugs, also known as drug partners.
  • the bio-inert drug comprises a targeting moiety and a bio-inert moiety.
  • the targeting moiety recognizes and binds to a target, and the biologically inert moiety can shield the target or direct target degradation.
  • the biologically inert moiety is selected from the group consisting of an antibody constant region, albumin, polyethylene glycol, and a nucleic acid aptamer.
  • the biologically inert portion is an IgG4 antibody constant region, such as the polypeptide set forth in SEQ ID NO:4.
  • the invention provides a pharmaceutical system, kit, kit or pharmaceutical composition for treating a disease associated with ED-B, such as a tumor, comprising:
  • a biologically active agent comprising a biologically active moiety linked to a targeting moiety that targets ED-B, such as directly covalently linked or indirectly linked by a linker (eg, a linker set forth in SEQ ID NO: 7 or 8), and
  • a biologically inert drug comprising a biologically inert moiety linked to a targeting moiety that targets ED-B, for example, directly covalently linked or indirectly via a linker (e.g., a linker set forth in SEQ ID NO: 7 or 8).
  • a linker e.g., a linker set forth in SEQ ID NO: 7 or 8
  • the targeting moiety that targets ED-B is a fragment or portion of an EB-D antibody, such as a CGS-1, CGS-2, L19 antibody or B5 antibody, or a binding antigen thereof, eg, a CDR region, Variable regions, Fab, Fab', F(ab)' 2 , single chain Fv (scFv), Fv fragments, antibody light chains, antibody heavy chains, single domain antibodies, diabodies, and linear antibodies.
  • an EB-D antibody such as a CGS-1, CGS-2, L19 antibody or B5 antibody, or a binding antigen thereof, eg, a CDR region, Variable regions, Fab, Fab', F(ab)' 2 , single chain Fv (scFv), Fv fragments, antibody light chains, antibody heavy chains, single domain antibodies, diabodies, and linear antibodies.
  • the targeting moiety that targets ED-B is a single chain Fv (scFv) of a B5 antibody, wherein the B5 antibody light chain (VL) and heavy chain (HL) are via a suitable linker, eg, (G) 4 S) 3 , the linker shown in SEQ ID NO: 7 or 8.
  • the biologically active moiety is IL-2 or an active fragment thereof, such as the polypeptide set forth in SEQ ID NO:9.
  • the biologically inert moiety is an IgG4 Fc region, such as the polypeptide set forth in SEQ ID NO:4.
  • the bioactive drug is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6.
  • the biologically inert drug is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5 or a dimer thereof.
  • the bioactive drug and the bio-inert drug may each be formulated as separate formulations or compositions, or may be formulated as a single formulation or composition.
  • the content and ratio of the bioactive drug and the bio-inert drug in the formulation or composition can be determined by a person skilled in the art according to actual needs, for example, the molar ratio of the bioactive drug to the bio-inert drug can be 10 : 1 to 1:10, for example, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1: 2. 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.
  • the "formulation" as used in the present invention refers to a medicine which can be supplied to a subject for use in accordance with a certain dosage form requirement for the purpose of adapting to the treatment or prevention of a disease.
  • the preparation may contain the bioactive drug, the biologically inert companion of the present invention, and may also contain other medicinal excipients and tools.
  • auxiliary material refers to a medicinal auxiliary material, which refers to a substance which is used in the preparation of a medicine and a prescription, and which has been evaluated in terms of safety in addition to the active ingredient, and which is included in the pharmaceutical preparation, the purpose thereof In addition to shaping, acting as a carrier or improving stability, it can also have important functions such as solubilization, solubilization, and controlled release.
  • the medicament or pharmaceutical composition of the present invention may be formulated into a dosage form suitable for any suitable administration route such as intravenous, subcutaneous, parenteral, oral, intraperitoneal, etc., such as tablets, powders, solutions and the like.
  • the medicament or pharmaceutical composition of the invention may be formulated in a form suitable for intravenous administration.
  • the invention provides a kit, kit or pharmaceutical composition for treating a tumor in a mammal, such as a human, the kit, kit or pharmaceutical composition comprising an effective amount to reduce ED-B Protein-level drug partners such as ED-B protein antibodies, and antibody drugs that target ED-B protein active drugs such as ED-B protein.
  • reduced level refers to a decrease, such as a decrease of at least 5%, at least 10%, at least 20, from a reference value, such as an object or a mean value observed prior to receiving an inert drug treatment. %, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%.
  • the kit or kit of the invention may also comprise a container, a label and/or instructions.
  • Suitable containers include, for example, bottles, vials, syringes, and the like.
  • the container contains a composition effective to treat the condition and may have a sterile inlet (e.g., the container may be an intravenous solution bag).
  • At least one active agent in the composition is a bioactive drug and/or a biologically inert drug according to the invention, or a pharmaceutical composition according to the invention, or a bioactive drug and/or organism obtained by the method of the invention An inert drug, or a pharmaceutical composition.
  • the label or instruction manual The medicament or composition is used to treat a particular condition, containing information about the indication, usage, dosage, administration, contraindications, and/or precautions of the use of such a therapeutic product.
  • the instructions indicate that the drug or composition is used to treat a disease associated with ED-B, such as a solid tumor associated with ED-B.
  • kit or kit of the present invention may further comprise other containers comprising a pharmaceutically acceptable buffer such as bacteria-inhibiting water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. It may further comprise other materials that meet commercial and user needs, including other buffers, diluents, filters, needles and syringes.
  • a pharmaceutically acceptable buffer such as bacteria-inhibiting water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution.
  • the present invention provides a method of treating a disease comprising administering to a subject in need of treatment a biologically active drug and a biologically inert drug or pharmaceutical composition of the present invention, or a biologically active drug obtained according to the method of the present invention. And a biologically inert drug or pharmaceutical composition.
  • a drug can be first bound to a specific target to block a target or partially block a target, and then another drug can be administered to target a later administered drug to a newly generated target or an unblocked target.
  • the drug to be administered may only have a target blocking effect, so that the drug to be administered later cannot act on the same target; the drug to be administered may also have other biological activities, but has different effects or different effects from the drug to be administered later. Intensive drug activity.
  • the bioactive drug and the biologically inert drug may be administered simultaneously, for example, as a single preparation; or at intervals of, for example, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, 1 Administration of hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours or longer, for example, first administration of bioactive drugs, re-administration A biologically inert drug, or a biologically inert drug, followed by a bioactive drug.
  • the interval may be determined according to technical knowledge or experience known in the art, for example, determining the appropriate interval depending on the pharmacokinetics, half-life, clearance rate, etc. of the administered drug, which are all techniques in the art. Within the technical knowledge and capabilities of the personnel.
  • the drug or pharmaceutical composition can be administered by any suitable route, such as intravenous, subcutaneous, parenteral, oral, intraperitoneal, and the like.
  • the dosage and regimen administered can be determined based on the physician's experience or related manual.
  • the medicament or pharmaceutical composition of the invention may be administered intravenously.
  • the dose and/or ratio of the bioactive drug and the biologically inert drug administered may be According to the doctor's experience or related manuals.
  • the molar ratio of the bioactive drug to the biologically inert drug may range from 10:1 to 1:10, such as 10:1, 9:1, 8:1, 7:1, 6:1, 5:1 , 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1 : 10.
  • the invention provides a method of treating a mammal, such as a human, having a disease associated with ED-B, such as a solid tumor, comprising administering to the animal a bioactive drug and a biologically inert drug or combination of drugs of the invention And a bioactive drug and a biologically inert drug or pharmaceutical composition obtained according to the method of the present invention, wherein the bioactive drug comprises a linker to a targeting moiety that targets ED-B (eg, directly covalently linked or through a linker)
  • the biologically inert drug comprises a biologically inert portion linked to a targeting moiety that targets ED-B (eg, directly covalently linked or indirectly through a linker).
  • the targeting moiety that targets ED-B is an EB-D antibody such as a CGS-1, CGS-2, L19 antibody or a B5 antibody or a fragment thereof that binds to an antigen, eg, a CDR region, a variable region Fab, Fab', F(ab)' 2 , single chain Fv (scFv), Fv fragment, antibody light chain, antibody heavy chain, single domain antibody, diabodies and linear antibodies.
  • an antigen eg, a CDR region, a variable region Fab, Fab', F(ab)' 2 , single chain Fv (scFv), Fv fragment, antibody light chain, antibody heavy chain, single domain antibody, diabodies and linear antibodies.
  • the targeting moiety that targets ED-B is a single chain Fv (scFv) of a B5 antibody, wherein the B5 antibody light chain (VL) and heavy chain (HL) are via a suitable linker, eg, (G) 4 S) 3 or SEQ ID NO: 7 or 8.
  • the biologically active moiety is IL-2 or a fragment thereof, as set forth in SEQ ID NO:9.
  • the biologically inert moiety is an IgG4 Fc region, as set forth in SEQ ID NO:4.
  • the bioactive drug is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6.
  • the biologically inert drug is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5 or a dimer thereof.
  • the medicament or pharmaceutical composition of the invention is administered intravenously to the mammal.
  • the biologically inert drug and the biologically active drug are administered simultaneously, for example in the form of a pharmaceutical composition comprising the biologically inert drug and the biologically active drug.
  • the biologically inert drug is administered to the mammal prior to administration of the organism Active drugs, for example, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours or longer.
  • Active drugs for example, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours or longer.
  • the invention provides a design method of the invention drug system, a drug design method and a drug prescription method. Furthermore, the invention relates to the production of pharmaceuticals.
  • the invention is further clarified by the following examples, but any examples or combinations thereof should not be construed as limiting the scope or the embodiments of the invention.
  • N-terminus and C-terminus of this sequence were digested with Nhe I and Not I restriction endonucleases, respectively, and cloned into pCI-neo vector to form pCI-neo-B5-Fc vector and pCI-neo-L19-Fc vector.
  • Vector construction and expression patterns of antibody proteins can be found in (Borsi, Balza et al. (2002). Int J Cancer. 102: 75-85.).
  • the obtained antibody-containing fusion protein-containing vector is utilized by Invitrogen 2000 was transfected into CHO-K1 cells, and the neo gene of the pCI-neo plasmid vector was continuously carried out in a selective medium containing G418 (Davies and Jimenez (1980). Am J Trop Med Hyg. 29: 1089-1092.). After 4 weeks of selective culture, the cloned culture was carried out by limiting dilution method, and the obtained monoclonal cells were further cloned and cultured to obtain a stable strain.
  • the obtained cell line was expressed in suspension with Hyclone CD4 CHO.
  • the antibody protein with Fc tag was purified using Protein A.
  • the purified protein was purified by SDS-PAGE electrophoresis to obtain sample purity, and the sample concentration was measured by an ultraviolet spectrophotometer.
  • the ED-B protein in the blood samples was detected by a sandwich ELISA method using L19-IL2 (see Carnemolla, Borsi et al. (2002). Blood. 99: 1659-1665.) and B5-Fc antibody.
  • the plate was coated at 37 ° C for 2 hours.
  • the plate was washed 3 times with PBST, and HRP-labeled mouse anti-human IgG antibody (Boster BA1070) diluted 5000 with PBST was added, and incubated at 37 ° C for 1 h.
  • the plate was washed 5 times with PBST, 100 ⁇ l/well of TMB coloring solution was added to the sample well, and incubated for 5 minutes at room temperature.
  • the reaction was stopped with 100 ⁇ l/well of 2 mol/L H 2 SO 4 . Double-wavelength detection of OD450nm and OD630nm was selected using a microplate reader. After subtracting the background value of the blank control well, the sample is tested for absorbance.
  • Example 2 Design and expression of biologically inert drugs and biologically active drugs
  • the B5 antibody heavy chain (VH) (SEQ ID NO: 1) and the light chain (VL) (SEQ ID NO: 2) are ligated with a long ligation fragment (SEQ ID NO: 7) to form a single chain antibody B5-ScFv ( SEQ ID NO: 3), the Fc fragment of SEQ ID NO: 4 (SEQ ID NO: 4), and retains the hinge region of the Fc fragment, ie retains cysteine to form a dimer and enhances protein flexibility (B5 For antibodies see patent publication WO 2014/194784).
  • the carboxy terminus of the B5 antibody has three alanine residues and Fc The amino-terminal hinge region is connected in sequence.
  • the protein B5-Fc (SEQ ID NO: 5) was formed after fusion.
  • the B5 antibody heavy chain (VH) (SEQ ID NO: 1) and the light chain (VL) (SEQ ID NO: 2) are ligated with a long ligation fragment (SEQ ID NO: 7) to form a single chain antibody B5-ScFv ( SEQ ID NO: 3), the anterior fusion of human interleukin 2 (IL2) (SEQ ID NO: 9), the carboxy terminus of the B5 antibody linked to IL2 with a long ligation fragment (SEQ ID NO: 8).
  • the DNA fragment encoding the B5-Fc (SEQ ID NO: 5) and B5-IL2 (SEQ ID NO: 6) fusion protein was ligated into the pCI-neo vector, respectively, and the obtained plasmid containing the coding sequence of the antibody fusion protein was obtained.
  • the obtained cell line was accumulatively expressed by Hyclone SFM4CHO-Utility (Thermo Scientific HyClone), and confirmed by ELISA and SDS-PAGE, the target antibody protein was obtained, and the antibody protein yield was about 100 mg/L.
  • Example 3 Animal modeling, grouping and administration methods
  • B5- prepared in Example 1 in a solution Embodiment
  • the Fc and B5-IL2 fusion proteins were each formulated into a 0.4 mg/ml solution.
  • mice Healthy Balb/c nu nude mice were housed in the SPF barrier system to 8 weeks of age, and subcutaneously inoculated mouse teratoma cells (F9) in the back of the mouse (Shanghai Fuxiang Biotechnology Co., Ltd.) , 2.5 ⁇ 10 6 cells per injection point. Mice with a tumor volume of approximately 60 mm 3 were selected on day 6 of the inoculation for group experiments. According to 8 groups per group, groups A, B, C, D, and E were administered by tail vein injection on the 6th, 7th, and 8th day after inoculation, among which:
  • Group A was a negative control group, and saline was administered twice a day at intervals of 6 hours, 50 ⁇ l/piece each time.
  • Group B was a positive control group.
  • B5-IL2 solution and normal saline were administered once a day.
  • Physiological saline was administered first, and B5-IL2 solution was administered at intervals of 6 hours, respectively, 50 ⁇ l/mouse.
  • Group C was given B5-Fc solution daily, and 6 hours later, it was administered to B5-IL2 solution, respectively, at 50 ⁇ l/head.
  • Group D was administered with an equal volume mixture of B5-IL2 solution and B5-Fc solution once a day, 100 ⁇ l/head.
  • the E group was the control group, and the B5-Fc solution and the physiological saline were administered once a day, and the B5-Fc solution was administered first, and physiological saline was administered at intervals of 6 hours, respectively, being 50 ⁇ l/head.
  • Group A and Group E were negative control groups, and the tumor volume was significantly larger than that of group B, C, and D, and the difference was statistically significant.
  • the tumor volume of group C or group D was significantly smaller than that of group B, and the difference was statistically significant. Therefore, the treatment effect of group C and group D was better than that of group B; there was no significant difference between group C and group D. There was no significant difference between group A and group E.

Abstract

本发明提供了药物体系设计方法，包括选择特异性结合感兴趣靶的靶向部分；将靶向部分与生物活性部分连接；和/或将靶向部分与生物惰性部分连接。本发明还提供了试剂盒、药盒或药物组合物，包含包括靶向部分和生物惰性部分的生物惰性药物和包括靶向部分和生物活性部分的生物活性药物，其中所述生物惰性药物与所述生物活性药物能够靶向相同靶。本发明还提供了使用所述药物或药物组合物治疗疾病例如与ED-B相关的疾病的方法。

Description

一种药物设计方法和获得的药物及其应用 发明领域
本发明涉及药物设计开发领域和药物制剂领域，特别涉及与ED-B相关疾病的药物设计开发领域，还涉及药物以及治疗应用。
发明背景
药物进入人体内能够发挥活性作用的器官、组织、细胞和生物大分子及其特定的药物结合位点，分别称为靶器官、靶组织、靶细胞和靶点。靶点可以为蛋白质、核酸或其他物质，包括基因位点、受体、酶、离子通道、核酸等生物大分子，一般具有重要的生理或病理功能。药物能够特异性的作用于靶点，但是部分药物靶点分布在非预期的药物作用器官或组织上，或者脱落在血液中，会与药物竞争性结合致使部分药物不能与靶器官上的靶点结合，这会影响药效的发挥，一方面消耗药物，另一方面会产生毒副作用。另外，从组织中脱落的靶点蛋白游离在血液或组织液中，会影响药物的代谢动力学。靶点的上述特点会引起一些列的问题，比如：给药量增大、个体差异大、药物毒副作用增大、治疗窗较窄的药物会无效或产生难以耐受的毒性等。
在抗体药物或抗体相关药物开发过程中，药物脱靶现象以及非靶器官靶点带来的副作用更加明显。寄予厚望的抗肿瘤抗体药物虽然给肿瘤患者带来希望，但效果仍不理想。如针对人类表皮生长因子受体2(HER2)开发的抗体药物，如赫赛汀，在乳腺癌治疗中取得了良好的效果，但由于HER-2蛋白在心肌横小管上有分布，长期使用有可能增加心血管毒性，引起充血性心衰，轻度甚至中至重度的心功能减退(Nemeth，B.T.，et al.(2016).Br J Pharmacol.doi：10.1111/bph.13643)，这是药物靶点蛋白分布的器官特异性不高产生的副作用。而靶向HER2的抗体偶联化药(ADC)药物，如T-DM1(曲妥珠单抗偶联美登木素DM1)，携带高效的细胞毒类药物，能抑制微管蛋白聚合和微管动力学，但同样由于HER2在心脏组织的分布会导致心脏毒性，并且T-DM1在非病灶部位的代谢也会产生肝脏毒性等(Yan，H.，et al.(2016).Mol Cancer Ther 15(3)：480-490)。针对纤维连接蛋白的额外结构域B(ED-B) 开发的抗体药物，如L19-IL2，通过静脉给药途径药效不明显(Eigentler，T.K.，et al.，(2011)Clin Cancer Res，17(24)：7732-42)，而通过肿瘤病灶局部给药则效果显著(Weide，B.，et al.，(2014)Cancer immunology research2(7)：668-678)。
发明概述
在一个方面，本发明提供了一种试剂盒或药盒，其包含本发明所述的生物惰性药物和生物活性药物，其中
-所述生物活性药物包括靶向部分和生物活性部分；
-所述生物惰性药物包括靶向部分和生物惰性部分，其中所述生物惰性药物与所述生物活性药物能够靶向相同靶，其中所述生物惰性药物与所述生物活性药物的靶向部分可以相同或不同。
在一个方面，本发明提供了一种药物组合物，其包含本发明所述的生物惰性药物和生物活性药物以及任选存在的药学可接受的载体和/或赋形剂。
在一个实施方案中，所述靶向部分选自配体、受体、抗体或抗体片段，例如完整抗体(如单克隆抗体、嵌合抗体等)、CDR区、可变区、Fab、Fab′、F(ab)′2、单链Fv(scFv)、Fv片段、抗体轻链、抗体重链、单域抗体、双抗体及线性抗体。
在一个实施方案中，所述试剂盒、药盒或药物组合物用于治疗与纤维连接蛋白ED-B相关的疾病，例如实体肿瘤。
在一个实施方案中，所述靶向部分为靶向纤维连接蛋白(Fibronectin，FN)额外结构域B(Extradomain B，ED-B)的抗体或其片段，例如CGS-1、CGS-2、L19或B5抗体或其抗体片段。
在一个实施方案中，所述生物活性部分选自细胞毒素、细胞因子、放射性同位素、化学药物、具有生物活性的抗体恒定区和具有生物活性的细胞。
在一个实施方案中，所述生物惰性部分选自抗体恒定区、白蛋白、聚乙二醇和核酸适配体。
在一个实施方案中，所述生物活性药物是SEQ ID NO：6所示的多肽。
在一个实施方案中，所述生物惰性药物是SEQ ID NO：5所示的多肽或其二聚体。
在一个实施方案中，所述试剂盒或药盒包含的生物惰性药物与所述生物活性药物混合制成单一制剂或组合物，或者所述生物惰性药物与所述生物活性药物分别制成独立制剂或组合物。
在一个方面，本发明提供了一种生物惰性药物，其包括靶向部分和生物惰性部分，任选其中所述靶向部分可选自配体、受体、抗体或其片段。
在一个实施方案中，所述靶向部分为靶向纤维连接蛋白ED-B的抗体或其片段，例如CGS-1、CGS-2、L19或B5抗体或其片段，例如SEQ ID NO：3所示的多肽。
在一个实施方案中，所述生物惰性部分选自抗体恒定区、白蛋白、聚乙二醇和核酸适配体，例如SEQ ID NO：4所示的多肽。
在一个实施方案中，所述生物惰性药物是SEQ ID NO：5所示的多肽或其二聚体。
在一个方面，本发明提供了一种治疗疾病的方法，包括给予需要治疗的对象施用本发明所述的生物惰性药物和生物活性药物或本发明所述的药物组合物、或使用本发明所述的试剂盒或药盒。
在一个实施方案中，所述生物惰性药物与生物活性药物同时施用，或者顺序施用，例如间隔5分钟、10分钟、15分钟、20分钟、25分钟、30分钟、40分钟、50分钟、1小时、2小时、3小时、4小时、5小时、6小时、7小时、8小时、9小时、10小时、11小时、12小时或更长时间。
在一个实施方案中，所述方法包括施用生物惰性药物，然后施用生物活性药物。
在一个实施方案中，所述疾病是与纤维连接蛋白ED-B相关的疾病，例如实体肿瘤。
在一个方面，本发明提供了本发明所述的生物惰性药物和/或生物活性药物在制备用于治疗哺乳动物对象例如人的疾病的试剂盒或药盒或药物组合物中的应用。
在一个实施方案中，所述疾病是与纤维连接蛋白ED-B相关的疾病， 例如实体肿瘤。
在一个实施方案中，所述生物惰性药物与所述生物活性药物混合制成单一制剂或组合物，或者所述生物惰性药物与所述生物活性药物分别制成独立制剂或组合物。
在一个方面，本发明提供了一种生物活性药物和/或生物惰性药物用于治疗例如与ED-B有关的疾病，其中所述生物活性药物包括特异性结合ED-B的靶向部分和生物活性部分，所述生物惰性药物包括靶向部分和生物惰性部分，所述治疗包括给予患有所述疾病的对象施用本发明所述的生物活性药物和生物惰性药物，任选其中所述生物惰性药物同时或先于生物活性药物施用，例如间隔5分钟、10分钟、15分钟、20分钟、25分钟、30分钟、40分钟、50分钟、1小时、2小时、3小时、4小时、5小时、6小时、7小时、8小时、9小时、10小时、11小时、12小时或更长时间。
在一个实施方案中，所述靶向部分选自配体、受体、抗体或其片段，例如完整抗体(如单克隆抗体、嵌合抗体等)、CDR区、可变区、Fab、Fab′、F(ab)′2、单链Fv(scFv)、Fv片段、抗体轻链、抗体重链、单域抗体、双抗体及线性抗体。
在一个实施方案中，所述所述生物活性部分选自选自细胞毒素、细胞因子、放射性同位素、化学药物、具有生物活性的抗体恒定区和具有生物活性的细胞。
在一个实施方案中，所述生物惰性部分选自抗体恒定区、白蛋白、聚乙二醇和核酸适配体，例如SEQ ID NO：4所示的多肽。
在一个方面，本发明提供了本发明所述的生物惰性药物和/或生物活性药物在制备用于疾病的试剂盒、药盒或药物组合物中的应用。
在一个实施方案中，所述疾病是与纤维连接蛋白ED-B相关的疾病，例如实体肿瘤。
在一个实施方案中，所述生物惰性药物与所述生物活性药物混合制成单一制剂或组合物，或者所述生物惰性药物与所述生物活性药物分别制成独立制剂或组合物。
在一个方面，本发明提供了一种药物设计方法，包括：
-选择特异性结合感兴趣靶的靶向部分；
-将靶向部分与生物活性部分连接，直接连接或通过接头连接，获得生物活性药物；和/或
-将靶向部分与生物惰性部分连接，直接连接或通过接头连接，获得生物惰性药物。
在一个实施方案中，所述方法用于设计治疗与ED-B相关的疾病的药物，包括：
-将特异性结合ED-B的靶向部分例如抗体的抗原结合部分与生物活性部分连接；和/或
-将特异性结合ED-B的靶向部分例如抗体的抗原结合部分与生物惰性部分连接。
在一个实施方案中，所述抗原结合部分是B5抗体的scFv，例如SEQ ID NO：3所示的多肽。
附图简述
图1：B5-Fc融合蛋白二聚体示意图。
图2：B5-IL2融合蛋白示意图。
图3：ED-B在对象血液中的表达。
发明详述
除非特别指出，本文使用的科学和技术术语应具有本领域技术人员通常已知的含义。另外除非特别需要，则单数术语应包括复数，复数术语应包括单数。前述技术和方法通常根据本领域熟知的及在本说明书引用的参考文献所述的常规方法进行。见例如并入作参考的Sambrook et al.Molecular Cloning：A Laboratory Manual(3rd ed.，Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y(2001))所述。本文引用的所有参考文献，包括专利、专利申请、文章、教科书等及其中引用的参考文献在此均以其全部并入本文作参考。
本发明涉及一种提高药物特异性的药物体系开发方法、相应的药物和药物组合物、试剂盒或药盒以及相关疾病治疗方法。本发明进一步提高药物的特异性，提高药物在病灶器官或组织的分布而降低在正常组织的分布，可以降低对非靶器官或非靶组织的伤害，也可以显著提高药物 疗效，同时减少药物的浪费。
在一个方面，本发明通过封闭或消除非给药目标组织的靶点实现精准给药。本发明的高特异性药物体系、药物和配方开发是设计一种新的基本无生物活性药物代替一部分有生物活性药物，分布在非病灶靶点上，减少生物活性药物的用量或者提高生物活性药物在病灶部位的分布比例，从而降低药物毒副作用或提高药效。在另一方面，本发明的高特异性药物体系、药物和配方也可用于靶主要或仅存在于患病部位，通过先给予生物惰性药物代替生物活性药物分布在已表达的病灶靶点上，再给予生物活性药物分布于新生的病灶靶点。
本发明所述的药物可以是核酸类药物，多肽类药物，蛋白类药物，或者是生物药物与化学药物的偶联物。药物进入人体内能够发挥活性作用的器官、组织、细胞和生物大分子及其特定的药物结合位点，分别称为靶器官、靶组织、靶细胞、靶分子和靶点。靶分子可以为蛋白质、核酸或其他物质，包括基因位点、受体、酶、离子通道、核酸等生物大分子，一般具有重要的生理或病理功能。
本发明所述“封闭或消除”非靶器官上的药物靶点，可以是部分封闭或部分消除，也可能会对靶器官、靶组织、病灶等部位产生一定的封闭或消除作用。由于药物靶点在病灶部位的分布密度或浓度较正常组织高，而正常组织上药物靶点分布较少或很难检测到，或者由于血液中游离的靶点比组织中的靶点更容易与药物结合，因此给予生物惰性药物对正常组织或血液中靶点的作用比对病灶中的靶点更显著。
本发明所述的高特异性药物体系包括生物惰性药物和生物活性药物，其中所述生物惰性药物与所述生物活性药物能够靶向同一个靶点，例如具有相同的靶向部分。本发明所涉及的高特异性药物体系开发方法，也包含药物设计方法，药物伴侣的设计方法，药物及药物伴侣的组合使用方法，这些方法可单独使用，也可组合使用。
本发明所述生物活性是指具有生物代谢或生理调节功能的生物效应或治疗作用，如促进消化吸收，促进免疫调节、激素调节、抗菌、抗病毒、抗肿瘤、细胞毒性、降血压、降血脂等活性。
本发明所述的“生物活性药物”或“具有生物活性的药物”具有相同的含义，是指在生物体内尤其是指在人体内具有生物活性的物质，能够 与人体内细胞、蛋白等相互作用并能影响机体生理、生化和病理过程的、用于预防、诊断、治疗疾病。
本发明所述生物惰性，指不具有上述生物活性或具有微弱的生物活性。本发明所述的“生物惰性药物”或“具有生物惰性的药物”具有相同含义，是指在生物体内尤其是指在人体内基本无生物活性的物质，虽然能够与人体内细胞、蛋白等产生结合作用，但不能显著影响机体生理、生化和病理过程，或治疗作用很弱，主要用于辅助生物活性药物提高药效或降低毒副作用等目的。所述的生物惰性药物也可以产生一定的生物效应，比如具有较弱的ADCC活性或CDC活性，但生物惰性药物与生物活性药物具有不同的药物活性，或生物惰性药物的药物活性显著低于生物活性药物。
生物惰性药物是相对生物活性药物而言，基本无生物活性主要指生物惰性药物不具备生物活性药物的药效，或者在同等给药剂量下药效较生物活性药物可忽略不计。但不排除生物惰性药物可以有其他功能或生理活性，例如生物惰性药物可以有荧光，可以阻断靶点与生物活性药物的结合，可以与其他配体或受体结合。特别地，生物惰性药物不具有对应活性药物的生物活性或单独使用不具有治疗作用。在本发明中，生物惰性药物伴随生物活性药物使用，也可称为药物伴侣。本发明所述的“药物伴侣”是指与活性药物识别相同的靶点并伴随活性药物使用的辅助药物或辅助分子，在本发明中是指生物惰性药物。
具有生物活性的药物可识别特定靶点并与之结合，产生药物的治疗作用。生物活性药物的活性部分(例如为生物活性分子)可产生希望的治疗作用，可以是例如细胞毒素，细胞因子，放射性同位素，化学药物，具有生物活性的抗体恒定区，或具有生物活性的细胞如T淋巴细胞。
如本文使用，“治疗”是获得有益的或希望的结果包括临床结果的方法。对于本发明而言，有益的或希望的临床结果包括但不限于如下的一或多种：缓解疾病所导致的一或多个症状，减轻疾病程度，稳定疾病(例如预防或延缓疾病恶化)，预防或延缓疾病传播(例如转移)，预防或延缓疾病复发，延缓或减慢疾病进展，改善疾病状态，疾病(部分或全部)减轻，减少治疗疾病需要的一或多种其它药物的剂量，延缓疾病进展，增加或改善生活质量，增加体重，和/或延长存活。“治疗”还涵 盖减少癌症的病理学结果(例如肿瘤体积)。
在一个方面，本发明涉及一种药物设计和开发方法，包括根据靶点选择特异性结合该靶点的靶向部分，基于该靶向部分产生具有生物活性的药物，发挥药物治疗作用，同时基于该靶向部分或靶点产生具有生物惰性的药物，发挥靶点屏蔽作用或者发挥或促进靶点清除，如靶点降解、靶点被细胞内吞、靶点进入消化系统等。
在一个实施方案中，本发明通过封闭或消除非病灶如正常组织或血液中的靶点(例如蛋白)实现对病灶的精准给药。屏蔽和消除靶的方法有多种，例如使用具有生物惰性的药物和具有生物活性的药物的组合物或药方，其中具有生物惰性的药物与具有生物活性的药物能够靶向同一个靶点。具有生物活性的药物可识别特定靶点并与之结合，产生治疗作用，而具有生物惰性的药物可识别特定靶点并与之结合，能屏蔽靶点或引导靶点降解，从而减少可与生物活性药物结合的靶点，例如尤其是减少在正常组织中分布的可与生物活性药物结合的靶点，也包括减少在血液中分布的可与生物活性药物结合的靶点。
或者，使用生物活性药物和生物惰性药物，其中生物活性药物可发挥生物治疗作用，而生物惰性药物发挥封闭或消除非靶器官上的靶点(例如靶蛋白)的作用，所述的消除靶点可以是蛋白水解消除，也可以是加快靶点被细胞内吞的过程。
在本发明中，生物惰性药物与生物活性药物可以具有同一母核，可以具有相同靶向部分例如识别同一抗原表位，可以竞争同一靶点，可以竞争同一靶蛋白或蛋白复合物。
在一个实施方案中，本发明提供了药物设计方法，包括：
-选择特异性结合感兴趣靶的靶向部分；
-将靶向部分与生物活性部分连接，例如直接共价连接或通过接头间接连接，获得生物活性药物；和/或
-将靶向部分与生物惰性部分连接，例如直接共价连接或通过接头间接连接，获得生物惰性药物。
本发明所述靶可以是在特定的患病器官、组织和/或细胞上表达或存在的生物分子，例如受体、离子通道或其它任何适合的表面分子。在一个实施方案中，所述靶可以是疾病的特异性生物标记物。在一个实施 方案中，所述靶不仅在患病器官、组织和/或细胞上表达或存在，也在正常器官、组织和/或细胞上表达或存在或者在体液例如血液中存在。在一个实施方案中，本发明所述靶可包括例如具有促进新生血管生成作用的受体酪氨酸激酶类靶点，如表皮生长因子受体(EGFR)、血管内皮细胞生长因子受体(VEGFR)、血小板衍生生长因子受体(PDGFR)、成纤维细胞生长因子受体(FGFR)等，其在部分正常组织或血管壁中也有表达；以及包括例如在肿瘤或炎症组织细胞膜或细胞外基质中特异性高表达的蛋白或肿瘤标志物，如纤维连接蛋白(fibronectin，Fn)的额外结构域A(ED-A)、癌胚抗原(CEA)、糖蛋白抗原(如MUC16/CA125、MUC1/CA 15-3)、上皮细胞粘附分子(EpCAM)和癌胚抗原相关细胞黏附分子(CEACAM1)等。
在一个实施方案中，本发明所述靶包括但不限于HER2、ED-B、ED-A、EGFR、VEGFR、PDGFR、FGFR、CEA、糖蛋白抗原(如MUC16/CA125、MUC1/CA 15-3)、EpCAM和CEACAM1。
本发明所述靶向部分可以是本领域已知的或将来鉴别的能够特异性靶向(例如识别和/或结合)感兴趣靶的分子或部分，例如抗体、配体、受体或其部分。在一个实施方案中，靶向部分是抗体或其可以结合抗原的片段，例如完整抗体(如单克隆抗体、嵌合抗体等)、CDR区、可变区、Fab、Fab′、F(ab)′2、单链Fv(scFv)、Fv片段、抗体轻链、抗体重链、单域抗体、双抗体及线性抗体。常见的靶向部分如Wu，A.M.and P.D.Senter.Nat Biotechnol 23(9)：1137-1146(2005)中所述。
用语“线性抗体”通常是指在Zapata et al.，Protein Eng.，8(10)：1057-1062(1995)中描述的抗体。这些抗体包含一对串联的Fd节段(VH-CH1-VH-CH1)，其与互补的轻链多肽一起形成一对抗原结合区。线性抗体可以是双特异性或单特异性的。
木瓜蛋白酶消化抗体产生称作“Fab”片段的两个相同的抗原结合片段和剩余的“Fc”片段。Fab片段由沿着H链的可变区结构域(VH)的整个L链及一条重链的第一个恒定结构域(CH1)组成。每个Fab片段关于抗原结合均是单价的，即其具有单一抗原结合位点。对抗体进行胰蛋白酶处理产生单一的大F(ab′)2片段，其大致相应于两个二硫键连接的Fab片段，具有二价抗原结合活性，仍能交联抗原。Fab’片段与Fab片 段不同，在CH1结构域的羧基末端具有另外几个残基，包括来自抗体铰链区的一或多个半胱氨酸。
“Fv”’是最小抗体片段，其含有完整的抗原识别和抗原结合位点。这个片段由一个重链可变区结构域和一个轻链可变区结构域的紧密非共价缔合的二聚体组成。这两个结构域的折叠产生6个超变环(各来自H和L链的3个环)，贡献抗原结合的氨基酸残基并赋予抗体的抗原结合特异性。单一的可变结构域(或者包含仅三个特异于抗原的CDR的一半Fv)也具有识别和结合抗原的能力。
“单链Fv”也缩写为“sFv”或“scFv”，是包含连接为单一多肽链的VH和VL抗体结构域的抗体片段。优选地，sFv多肽进一步包含在VH与VL结构域之间的多肽接头，其使得sFv形成用于抗原结合所需的结构。关于sFv的综述，见The Pharmacology of Monoclonal Antibodies，vol.113，Rosenburg and Moore eds.，Springer-Verlag，New York，pp.269-315(1994)。
术语“双抗体”是指小抗体片段，通过在VH与VL结构域之间用短接头(大约5-10个残基)构建sFv片段而制备，由此获得V结构域的链间而不是链内配对，产生二价片段，即具有两个抗原结合位点的片段。双特异性双抗体是两个“交叉”sFv片段的异源二聚体，其中两个抗体的VH和VL结构域存在于不同的多肽链上。参见例如EP 404,097、WO93/11161及Hollinger et al.，Proc.Natl.Acad.Sci.USA，90：6444-6448(1993)。
本发明所述靶向部分可与生物活性部分和/或生物惰性部分直接共价连接或通过接头间接连接。本领域已知将靶向部分与生物活性部分或生物惰性部分进行连接的方法。本领域技术人员根据本领域技术知识也可以确定将所述靶向部分与生物活性部分或生物惰性部分直接共价连接或通过接头(例如SEQ ID NO：7或8所示的接头)连接，以实现靶向部分的靶向功能(例如特异性结合感兴趣的靶)和/或生物活性部分的治疗功能，例如杀伤癌细胞，或生物惰性部分的功能，例如屏蔽靶点或降解靶点。所述接头可以有多种设计方式，常见的短肽接头参见Chen，X.，et al.Adv Drug Deliv Rev 65(10)：1357-1369(2013)。在一个实施方案中，本发明所述接头是不超过50个氨基酸的短肽。在一个实施方案中， 本发明所述接头是GS接头例如(GGGGS)n(其中n＝1、2、3、4、5、6、7、8、9、10)，(Gly)n接头(其中n＝1、2、3、4、5、6、7、8、9、10)例如(Gly)6或(Gly)8，(EAAAK)n接头(其中n＝1、2或3)，[A(EAAAK)nA]m接头(其中n＝2、3、4或5，m＝1或2)，(Ala-Pro)n接头(其中n＝5-17)，或(XP)n接头(其中X表示任意氨基酸，优选Ala、Lys或Glu；n＝1、2、3、4、5、6、7、8、9、10)。
在一个实施方案中，生物活性药物是抗体恒定区置换为能够产生生物活性、治疗作用或药物效应的部分的抗体，所述产生生物活性、治疗作用或药物效应的活性部分可以选自细胞毒素、细胞因子(如IL-2、TNFα、IL-12)、放射性同位素、化疗药物和偶联化学药物。
在一个实施方案中，所述生物惰性部分选自抗体恒定区例如IgG4Fc、白蛋白、聚乙二醇和核酸适配体。
在一个实施方案中，所述靶向部分是抗体可变区，生物惰性药物是带有抗体恒定区的完整抗体，而生物活性药物是抗体的恒定区置换为具有药物效应的部分的抗体，所述具有药物效应的部分可以选自细胞因子(如IL2、TNFα、IL12)、放射性同位素和偶联化学药物。
在一个实施方案中，所述靶向部分是抗体可变区，所述生物惰性部分是IgG4抗体的恒定区，而生物活性部分是IL-2。
在一个实施方案中，所述靶向部分是抗体可变区，所述生物惰性部分是IgG4抗体恒定区，而所述生物活性部分包含偶联了强效抗微管药物DM1的IgG4抗体恒定区。
在一个实施方案中，本发明提供了一种用于治疗与ED-B相关的疾病的药物设计方法，包括：
-将ED-B特异性抗体的抗原结合部分与生物活性部分连接，例如直接共价连接或通过接头(例如SEQ ID NO：7或8所示的接头)间接连接；和/或
-将ED-B特异性抗体的抗原结合部分与生物惰性部分连接，例如直接共价连接或通过接头(例如SEQ ID NO：7或8所示的接头)间接连接。
纤维连接蛋白(fibronectin，FN)是一种多功能的糖蛋白，由上皮细胞、内皮细胞、成纤维细胞、肝细胞、蜕膜细胞及绒毛外滋养细胞等表 达。FN的基因约有75kb，约含50个外显子，相对分子质量约250kDa，主要由I，II，III型三类同源重复的球形结构域单元构成，各结构域之间由对胰蛋白酶敏感的肽链连接。ED-B(Extradomain B)是包含在FN的III型重复序列中，由单个外显子编码的包含91个氨基酸的完整结构域。几乎所有的人类实体肿瘤，其原发病灶及转移位点都能检测到含有ED-B的FN(FN(B+))的高表达，因此FN(B+)被作为肿瘤新生血管的标志物(Carnemolla，Balza et al.(1989).J Cell Biol.108：1139-1148.)。
“ED-B”是指含有ED-B氨基酸序列的蛋白或其同源物，其中ED-B氨基酸序列例如具有SEQ ID NO：10所示氨基酸序列或与SEQ ID NO：10具有至少80％、85％、90％、95％、96％、97％、98％、99％或更高序列相同性的序列。本发明人在之前的研究中发现血液中含有ED-B蛋白并且在组织增生例如肿瘤患者的血液中ED-B蛋白水平与正常人或者未患肿瘤的对象相比升高(PCT/CN2015/094727，该专利申请以其全文并入本文参考)。
L19-IL2药物I期临床药物代谢实验数据表明：1)在同一个治疗周期内连续给药时，在相同给药剂量下血液中的L19-IL2浓度(单位剂量的浓度-时间曲线下面积，AUC/D)会逐次降低；2)在相同给药次数时，其血液中的L19-IL2浓度(单位剂量的浓度-时间曲线下面积，AUC/D)会随给药剂量的增加而降低(Johannsen，Spitaleri et al.(2010).Eur J Cancer.46：2926-2935)。不受任何理论约束，推测：1)当首次给药时，血液中大量存在ED-B抗原，L19-IL2会与血液ED-B结合，L19抗体的亲和力较高而长期存在于血浆中，并且与L19-IL2结合的ED-B会与L19-IL2一起代谢而从血液中被清除；当同一治疗周期内再次给药时，由于血液ED-B含量降低，从而使与之结合的L19-IL2减少，游离的L19-IL2则可以快速进入实体肿瘤组织，因此检测到的血药浓度(AUC/D)较前一次给药时低。当给药剂量较低时，L19-IL2与血液中ED-B充分结合而游离L19-IL2较少，但随着L19-IL2剂量的加大，使之与ED-B的结合接近饱和或过饱和，则血液中游离的L19-IL2增加，游离的L19-IL2更容易进入实体肿瘤组织中，因此检测到的血液L19-IL2浓度(AUC/D)随剂量增加而降低。因此可通过给予较大剂量的生物惰性的L19抗体先与血液中的ED-B过饱和结合，然后给予含有L19抗体 识别结构的生物活性药物。
本发明所述与ED-B相关的疾病是指在患病的器官、组织和/或细胞存在和/或表达ED-B的疾病。
在一个实施方案中，本发明所述与ED-B相关的疾病是组织增生，包括例如乳腺增生，赘生物，良性肿瘤，恶性肿瘤或癌症例如选自各类鳞癌、腺癌或肉瘤。在一个实施方案中，本发明所述肿瘤是实体肿瘤，选自例如畸胎瘤，上消化道癌症例如食管癌、贲门癌、喉癌、胃癌，头颈癌，肝癌，胆道癌，胆囊癌，结肠癌，十二指肠癌，肺癌，膀胱癌，子宫颈癌，卵巢癌，子宫内膜癌，乳腺癌，黑色素瘤，胰腺癌，肾癌和前列腺癌。在一个实施方案中，所述肿瘤是畸胎瘤、鼻咽癌、食管癌、胃癌、肺癌或胰腺癌。在一个实施方案中，所述肿瘤是淋巴瘤。
在一个实施方案中，所述ED-B特异性抗体包括例如CGS-1、CGS-2(PCT/GB97/01412；Nissim，Hoogenboom et al.(1994).EMBO J.13：692-698)、L19(Pini，Viti et al.(1998).J Biol Chem.273：21769-21776)、B5(CN201480001324.5；WO2014/194784)。在一个实施方案中，所述ED-B特异性抗体是L19或B5抗体。在一个实施方案中，所述ED-B特异性抗体的抗原结合部分是完整抗体、CDR区、可变区、Fab、Fab′、F(ab)′2、单链Fv(scFv)、Fv片段、抗体轻链、抗体重链、单域抗体、双抗体及线性抗体。
在一个实施方案中，所述生物活性部分选自细胞毒素、细胞因子、放射性同位素、化学药物、具有生物活性的抗体恒定区和具有生物活性的细胞。在一个实施方案中，所述生物活性部分是细胞因子，例如IL-2，如SEQ ID NO：9所示的多肽。
在一个实施方案中，所述生物惰性部分选自抗体恒定区、白蛋白、聚乙二醇和核酸适配体。在一个实施方案中，所述生物惰性部分是抗体恒定区，例如IgG4抗体恒定区(IgG4Fc)，如SEQ ID NO：4所示的多肽。
本发明还提供了一种生物惰性药物的开发方法以及相应的生物惰性药物。本发明所述生物惰性药物不具有活性药物的治疗作用，或者具有治疗作用但与所述活性药物针对不同的适应症，更特别地，惰性药物不具有对应活性药物的生物活性。
生物惰性药物可以是在活性药物上修改，使其仅具备识别并结合靶点的作用，包括但不限于去除生物活性作用的结构、使生物活性作用部位突变失活、置换活性药物的生物活性部位、增加抑制生物活性作用的结构如通过化学修饰屏蔽药物的活性部位、或者融合基本无生物活性的蛋白形成空间位阻。
本发明所述的惰性药物(药物伴侣)需要与活性药物联合使用。靶向同一靶点的活性药物和药物伴侣可以制成单一制剂或组合物、或分别制成独立制剂或组合物，通过给予药物伴侣与特定靶点结合以封闭靶点或部分封闭靶点，通过给予活性药物靶向未被封闭的靶点或新产生的靶点，从而活性药物以更精准和/或更高比例到达病灶靶点。药物伴侣用于提高既有活性药物的安全性、有效性和/或靶向性。例如，生物惰性药物可与生物活性药物制成制剂，同时给药；生物惰性药物也可以成为药物配方中的一种辅料，其作用是与生物活性药物竞争结合血液中的游离的靶点或者其他正常组织中的靶点，同时也具有保护生物活性药物稳定的作用。
生物惰性药物可以在仅保留药物靶向结合部位的基础上如上所述被修改、修饰或置换药物活性结构，使既有活性药物变成惰性药物，例如使活性药物的原有活性下降50％以上、90％以上、99％以上、99.9％以上。惰性药物与活性药物可以有相同的分子结构，例如相同结构占所述活性药物分子结构的比例大于5％、10％、20％、30％、40％、50％、60％、70％、80％、甚至大于90％。特别地，二者主要是与靶点结合部位的分子结构高度相似，例如结合部位的相似比例达到50％、60％、70％、80％、90％、95％、甚至100％相同性。二者不仅能够结合到同一靶点分子，而且结合在靶点上的部位接近，甚至高度重叠，当惰性药物与相应靶点结合后形成空间位阻，使活性药物不能与惰性药物结合同一靶点。
具有生物惰性的药物可识别特定靶点并与之结合，能屏蔽靶点和/或促进靶点降解和/或维持生物活性药物稳定。生物惰性药物的惰性部分(也可称为生物惰性分子)可屏蔽靶点和/或引导靶点降解，可以是例如抗体恒定区，白蛋白，聚乙二醇，或核酸适配体。
具有生物惰性的药物可以为生物药物包括但不限于核酸类药物、多肽类药物、蛋白类药物，化学药物以及生物药物与化学药物的偶联化合 物。具有生物惰性的药物，包括但不限于抗体，抗体重组蛋白，具有生物活性的药物的类似物或修饰物，仅与具有生物活性的药物的靶点识别部分相同的生物惰性药物。
具有生物惰性的药物与具有生物活性的药物也可以不具有同一化学结构，也可以不具有同一母核。在一个实施方案中，生物惰性药物是核酸适配体，而生物活性药物是蛋白质。在一个实施方案中，生物惰性药物可基于具有靶向特异性的母核融合抗体的恒定区片段，或融合人白蛋白、聚乙二醇修饰、融合多肽片段或天然蛋白的结构域。
在一个实施方案中，本发明提供了ED-B蛋白相关药物的药物伴侣，例如ED-B蛋白的抗体在制备用于治疗哺乳动物例如人中肿瘤的试剂盒、药盒或药物组合物中的应用。在一个实施方案中，本发明涉及的药物伴侣是靶向纤维连接蛋白ED-B的抗体药物伴侣，基于以L19或B5抗体为母核的生物药物，在保留L19或B5抗体可变区的基础上，融合抗体恒定区结晶片段(FC)蛋白，形成药物伴侣L19-或B5-IgG4Fc，并与L19或B5相关药物配伍给药。
本发明所涉及的高特异性药物体系、药物和药方的开发方法，不仅适合抗癌药物的开发，也适合自身免疫疾病的药物开发，也适合其他疾病的药物开发。
本发明还提供了通过上述方法产生的包含相应生物活性药物和/或生物惰性药物的试剂盒、药盒和药物组合物。
在一个方面，本发明还提供了一种包含生物惰性药物和生物活性药物的组合物，其中生物惰性药物与生物活性药物二者均具有相同的靶向部分，其中生物惰性药物由靶向部分和生物惰性部分经共价键结合组成，生物活性药物由靶向部分和生物活性部分经共价键结合组成。在一个实施方案中，所述生物惰性药物与生物活性药物的靶向部分为抗体或抗体片段。
在本发明中，试剂盒和药盒可互换使用，指包括本发明所述的生物惰性药物和生物活性药物的组合或本发明所述的药物组合物、或根据本发明所述方法获得的生物活性药物和生物惰性药物的组合或药物组合物、以及任选存在的施用药物的工具和/或其它所需试剂例如缓冲液、复性溶剂等的生产物品。
在一个方面，本发明提供了一种药物体系、试剂盒、药盒或药物组合物，其包含本发明所述的生物惰性药物和生物活性药物或通过本发明的方法获得的生物活性药物和生物惰性药物以及任选存在的施用工具、给药说明、药学可接受的载体和/或赋形剂，其中所述生物惰性药物与所述生物活性药物能够靶向相同靶点。在一个实施方案中，所述生物惰性药物与所述生物活性药物具有相同的靶向部分。
本发明所述的生物活性药物可识别并结合特定靶点以及产生治疗作用。在一个实施方案中，所述生物活性药物包括靶向部分和生物活性部分，其中靶向部分可识别并结合靶点，所述生物活性部分产生生物活性或治疗作用，例如已知有特定的作用靶点、治疗效果、适应症等的物质。
在一个实施方案中，所述靶向部分是可以特异性靶向感兴趣靶的分子例如抗体、配体、受体等或其部分。在一个实施方案中，所述靶向部分是完整抗体或其一部分或片段，例如CDR区、可变区、Fab、Fab′、F(ab)′2、单链Fv(scFv)、Fv片段、抗体轻链、抗体重链、单域抗体、双抗体及线性抗体。
在一个实施方案中，所述生物活性部分选自细胞毒素、细胞因子(如IL-2、TNFα、IL-12)、放射性同位素、化疗药物、化学药物、具有生物活性的抗体恒定区和具有生物活性的细胞。在一个特定的实施方案中，所述生物活性部分是细胞因子，例如SEQ ID NO：9所示的多肽。
本发明所述的生物惰性药物可识别并结合特定靶点以及能屏蔽靶点或促进靶点降解或促进靶点被细胞内吞、促进靶点进入消化系统等，从而减少可与生物活性药物结合的靶点，包括减少在正常组织中分布的可与生物活性药物结合的靶点，和减少在血液中分布的可与生物活性药物结合的靶点。特别地，生物惰性药物将血液中游离的蛋白靶点和正常组织中分布的蛋白靶点封闭或部分封闭，减少生物活性药物在非靶器官和非靶组织的结合，使生物活性药物可以更多的分布到靶器官和靶组织中，从而提高生物活性药物在病灶部位的效应或治疗效果，减少所述生物活性药物的用量，以减少药物的毒副作用，提高药物安全性；同时也具有保护生物活性药物稳定的作用。本发明的生物惰性药物本身不具有治疗作用，需要伴随生物活性药物组合使用。所述生物惰性药物改善生 物活性药物的代谢与分布，是生物活性药物的伴侣，也可称为药物伴侣。
在一个实施方案中，所述生物惰性药物包括靶向部分和生物惰性部分。所述靶向部分可识别并结合靶点，生物惰性部分能屏蔽靶点或引导靶点降解。在一个实施方案中，所述生物惰性部分选自抗体恒定区、白蛋白、聚乙二醇和核酸适配体。在一个实施方案中，所述生物惰性部分是IgG4抗体恒定区，例如SEQ ID NO：4所示的多肽。
在一个实施方案中，本发明提供了一种用于治疗与ED-B相关的疾病例如肿瘤的药物体系、试剂盒、药盒或药物组合物，其包含：
-生物活性药物，其包含与靶向ED-B的靶向部分连接的生物活性部分，例如直接共价连接或通过接头(例如SEQ ID NO：7或8所示的接头)间接连接，和
-生物惰性药物，其包含与靶向ED-B的靶向部分连接的生物惰性部分，例如直接共价连接或通过接头(例如SEQ ID NO：7或8所示的接头)间接连接。
在一个实施方案中，所述靶向ED-B的靶向部分是EB-D抗体如CGS-1、CGS-2、L19抗体或B5抗体或其结合抗原的片段或部分，例如CDR区、可变区、Fab、Fab′、F(ab)′2、单链Fv(scFv)、Fv片段、抗体轻链、抗体重链、单域抗体、双抗体及线性抗体。
在一个实施方案中，所述靶向ED-B的靶向部分是B5抗体的单链Fv(scFv)，其中所述B5抗体轻链(VL)和重链(HL)通过适当接头例如(G4S)3、SEQ ID NO：7或8所示的接头连接。
在一个实施方案中，所述生物活性部分是IL-2或其活性片段，如SEQ ID NO：9所示的多肽。
在一个实施方案中，所述生物惰性部分是IgG4Fc区，如SEQ ID NO：4所示的多肽。
在一个实施方案中，所述生物活性药物是包含SEQ ID NO：6所示氨基酸序列的多肽。
在一个实施方案中，所述生物惰性药物是包含SEQ ID NO：5所示氨基酸序列的多肽或其二聚体。
在一个实施方案中，所述生物活性药物和生物惰性药物可分别配制为独立制剂或组合物，或者混合配制为单一制剂或组合物。在单一制剂 或组合物的情况中，所述生物活性药物和生物惰性药物在制剂或组合物中的含量以及比例可以由本领域技术人员根据实际需要确定，例如生物活性药物和生物惰性药物的摩尔比例可以为10∶1至1∶10，例如10∶1、9∶1、8∶1、7∶1、6∶1、5∶1、4∶1、3∶1、2∶1、1∶1、1∶2、1∶3、1∶4、1∶5、1∶6、1∶7、1∶8、1∶9或1∶10。
本发明所述的“制剂”是指为适应治疗或预防疾病的需要，按照一定的剂型要求所制成的可提供给用药对象使用的药品。所述的制剂中可以含有本发明所述生物活性药物、生物惰性伴侣，同时也可以含有其他药用辅料和工具。
本发明所述的“辅料”即药用辅料，是指生产药品和调配处方时使用的除活性成分以外，在安全性方面已进行了合理的评估，且包含在药物制剂中的物质，其目的除赋形、充当载体或提高稳定性外，还可以具有增溶、助溶、缓控释等重要功能。
本发明的药物或药物组合物可以配制为适合任何合适给予途径例如静脉内、皮下、胃肠外、口服、腹膜内等的剂型，例如片剂、粉剂、溶液等。在一个实施方案中，本发明所述药物或药物组合物可以配制为适合用于静脉内施用的形式。
在一个实施方案中，本发明提供了用于治疗哺乳动物例如人中肿瘤的试剂盒、药盒或药物组合物，所述试剂盒、药盒或药物组合物包括有效量的能降低ED-B蛋白水平的药物伴侣例如ED-B蛋白抗体，和靶向ED-B蛋白的活性药物例如ED-B蛋白的抗体药物。
本文所述“降低水平”是指与参照值例如在未接受治疗的对象或者在接受惰性药物治疗前观察到的中间值或者平均值相比降低，例如降低至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％或100％。
本发明所述试剂盒或药盒也可包含容器、标签和/或者说明书。合适的容器包括例如瓶、小瓶、注射器等。通常，容器含有有效治疗病症的组合物及可具有无菌入口(例如容器可以是静脉内注射溶液袋)。组合物中至少一种活性剂是本发明所述的生物活性药物和/或生物惰性药物、或本发明所述的药物组合物、或通过本发明所述方法获得的生物活性药物和/或生物惰性药物、或药物组合物。所述标签或说明书指示所 述药物或组合物用于治疗特定病症，含有关于使用这种治疗产品的适应症、用法、剂量、给药、禁忌症和/或注意事项的信息。在一个实施方案中，所述说明书指出所述药物或组合物用于治疗与ED-B相关的疾病，例如与ED-B相关的实体肿瘤。
此外，本发明所述试剂盒或药盒可进一步包含其它容器，其包含药物可接受的缓冲液，如抑制细菌的注射用水(BWFI)、磷酸盐缓冲盐水、Ringer′s溶液和葡萄糖溶液。其可进一步包含满足商业和用户需要的其它材料，包括其它缓冲液、稀释剂、滤器、针头和注射器。
在一个方面，本发明提供了一种疾病治疗方法，包括给予需要治疗的对象施用本发明所述的生物活性药物和生物惰性药物或药物组合物、或根据本发明所述方法获得的生物活性药物和生物惰性药物或药物组合物。
例如，可以先给予一种药物与特定靶点结合封闭靶点或部分封闭靶点，然后再给予另一种药物，使后给予的药物能靶向新产生的靶点或未被封闭的靶点。先给予的药物可以仅具备靶点封闭作用，使后给予的药物不能作用与同一个靶点；先给予的药物也可以具有其他生物活性，但与后给予的药物具有不同的药效或具有不同强度的药物活性。
所述生物活性药物和生物惰性药物可以同时给予，例如以单一制剂形式给予；或者间隔一定时间例如5分钟、10分钟、15分钟、20分钟、25分钟、30分钟、40分钟、50分钟、1小时、2小时、3小时、4小时、5小时、6小时、7小时、8小时、9小时、10小时、11小时、12小时或更长时间先后给予，例如先给予生物活性药物、再给予生物惰性药物，或者先给予生物惰性药物、再给予生物活性药物。
在先后给药的情况中，间隔时间可以根据本领域已知的技术知识或经验确定，例如根据给予的药物的药物动力学、半衰期、清除速率等确定合适的间隔时间，这都在本领域技术人员的技术知识和能力范围内。
所述药物或药物组合物可以通过任何合适的途径给予，例如静脉内、皮下、胃肠外、口服、腹膜内等。给予的剂量和方案可以根据医师的经验或者相关手册确定。在一个实施方案中，本发明所述药物或药物组合物可以通过静脉内施用。
给予的生物活性药物和生物惰性药物的剂量和/或比例可以可以根 据医师的经验或者相关手册确定。在一个实施方案中，生物活性药物和生物惰性药物的摩尔比例可以为10∶1至1∶10，例如10∶1、9∶1、8∶1、7∶1、6∶1、5∶1、4∶1、3∶1、2∶1、1∶1、1∶2、1∶3、1∶4、1∶5、1∶6、1∶7、1∶8、1∶9或1∶10。
在一个方面，本发明提供了治疗患有与ED-B相关的疾病例如实体肿瘤的哺乳动物例如人的方法，包括给所述动物施用本发明所述的生物活性药物和生物惰性药物或药物组合物、或根据本发明所述方法获得的生物活性药物和生物惰性药物或药物组合物，其中所述生物活性药物包含与靶向ED-B的靶向部分连接(例如直接共价连接或通过接头间接连接)的生物活性部分，生物惰性药物包含与靶向ED-B的靶向部分连接(例如直接共价连接或通过接头间接连接)的生物惰性部分。
在一个实施方案中，所述靶向ED-B的靶向部分是EB-D抗体如CGS-1、CGS-2、L19抗体或B5抗体或其结合抗原的片段，例如CDR区、可变区、Fab、Fab′、F(ab)′2、单链Fv(scFv)、Fv片段、抗体轻链、抗体重链、单域抗体、双抗体及线性抗体。
在一个实施方案中，所述靶向ED-B的靶向部分是B5抗体的单链Fv(scFv)，其中所述B5抗体轻链(VL)和重链(HL)通过适当接头例如(G4S)3或SEQ ID NO：7或8所示。
在一个实施方案中，所述生物活性部分是IL-2或其片段，如SEQ ID NO：9所示。
在一个实施方案中，所述生物惰性部分是IgG4Fc区，如SEQ ID NO：4所示。
在一个实施方案中，所述生物活性药物是包含SEQ ID NO：6所示氨基酸序列的多肽。
在一个实施方案中，所述生物惰性药物是包含SEQ ID NO：5所示氨基酸序列的多肽或其二聚体。
在一个实施方案中，给所述哺乳动物静脉内施用本发明的药物或药物组合物。在一个实施方案中，同时给予所述生物惰性药物和生物活性药物，例如以包含所述生物惰性药物和生物活性药物的药物组合物的形式。
在一个实施方案中，先给哺乳动物施用生物惰性药物，再施用生物 活性药物，例如间隔5分钟、10分钟、15分钟、20分钟、25分钟、30分钟、40分钟、50分钟、1小时、2小时、3小时、4小时、5小时、6小时、7小时、8小时、9小时、10小时、11小时、12小时或更长时间。
实施例
本发明给出发明药物体系设计方法、药物设计方法和药物组方方法。此外，本发明涉及药物的生产方式。本发明通过下述实施例进一步阐明，但任何实施例或其组合不应当理解为对本发明的范围或实施方式的限制。
实施例1：检测血液中的ED-B蛋白
(A)ED-B蛋白抗体表达和纯化
分别合成B5、L19的单链抗体(scFv)结构的DNA序列(参见专利申请号CN201480001324.5，Pini，Viti et al.(1998).J Biol Chem.273：21769-21776和Borsi，Balza et al.(2002).Int J Cancer.102：75-85)。在抗体的DNA序列5’端添加一个编码信号肽的DNA序列和在3’端添加一个编码IgG1Fc标签的DNA序列(Cao，Cao et al.(2009).Appl Biochem Biotechnol.157：562-574)。该序列N端和C端分别通过Nhe I和Not I限制性内切酶酶切并克隆至pCI-neo载体中，形成pCI-neo-B5-Fc载体、pCI-neo-L19-Fc载体。抗体蛋白的载体构建和表达方式可参见(Borsi，Balza et al.(2002).Int J Cancer.102：75-85.)。
将获得的含抗体融合蛋白的载体利用Invitrogen的
Figure PCTCN2016111264-appb-000001
2000转染至CHO-K1细胞，借助pCI-neo质粒载体的neo基因在含G418的选择性培养基(Davies and Jimenez(1980).Am J Trop Med Hyg.29：1089-1092.)中进行连续4周的选择培养后，经有限稀释法进行克隆化培养，将获得的单克隆细胞继续克隆化培养以获得稳定株。
获得的细胞株用Hyclone CD4CHO悬浮表达。带Fc标签的抗体蛋白用Protein A纯化。纯化获得的蛋白通过SDS-PAGE电泳检测纯化获得样本纯度，通过紫外分光光度计检测样本浓度。
(B)ELISA检测血液中的ED-B
取健康人血液和患者(鼻咽癌、食管癌、胃癌、肺癌、胰腺癌、乳腺增生、良性肿瘤)血液经枸橼酸钠抗凝剂处理后于4℃、3000g离心2 分钟后，弃沉淀，重复一次。
采用L19-IL2(参见Carnemolla，Borsi et al.(2002).Blood.99：1659-1665.)和B5-Fc抗体通过夹心ELISA方法检测血液样本中的ED-B蛋白。
简而言之，将抗体L19-IL2用pH＝9.6的碳酸盐缓冲液(Na2CO31.59g/L，NaHCO32.93g/L)稀释至浓度为2ng/μl，按100μl/孔在37℃包被酶标板2小时。用PBST(NaCl 8g/L，KCl 0.2g/L，Na2HPO41.42g/L，KH2PO40.27g/L，pH＝7.4，0.5‰Tween-20)洗涤包被后的酶标板3次，以含4％BSA的PBS溶液(NaCl 8g/L，KCl 0.2g/L，Na2HPO41.42g/L，KH2PO40.27g/L，pH＝7.4)在37℃封闭酶标板1小时。用PBST洗涤酶标板3次。
PBS(NaCl 8g/L，KCl 0.2g/L，Na2HPO41.42g/L，KH2PO40.27g/L，pH＝7.4)稀释50倍的血清样品按100μl/孔加入到酶标板样品孔中。同时以BSA为阴性对照。将酶标板置于37℃孵育1h。以PBST洗涤酶标板3次，以PBST将B5-Fc抗体稀释至2ng/μl，按100μl/孔加入到酶标板样品孔中，37℃孵育1h。以PBST洗涤酶标板3次，加入用PBST稀释5000的HRP标记小鼠抗人IgG抗体(Boster BA1070)，37℃孵育1h。PBST洗涤酶标板5次，向样品孔中加入100μl/孔的TMB显色液，室温孵育5分钟。用100μl/孔的2mol/L的H2SO4终止反应。用酶标仪选择OD450nm和OD630nm双波长检测。扣除空白对照孔的本底值，即为样品检测吸光值。
结果示于图3，显示实体肿瘤患者血液中ED-B水平显著升高，与健康人相比差异具有显著统计学意义(Mann-Whitney test)。
实施例2：生物惰性药物与生物活性药物的设计与表达
1)生物惰性药物B5-Fc融合蛋白(SEQ ID NO：5)的设计与表达
B5抗体重链(VH)(SEQ ID NO：1)与轻链(VL)(SEQ ID NO：2)之间用长连接片段(SEQ ID NO：7)连接，形成单链抗体B5-ScFv(SEQ ID NO：3)，后端融合抗体IgG4的Fc片段(SEQ ID NO：4)，并保留Fc片段的铰链区，即保留半胱氨酸以形成二聚体，并提高蛋白的柔性(B5抗体见专利公开WO2014/194784)。B5抗体的羧基端以三个丙氨酸残基与Fc 的氨基端铰链区序列连接。融合后形成蛋白B5-Fc(SEQ ID NO：5)。
2)生物活性药物B5-IL2融合蛋白(SEQ ID NO：6)的设计与表达
B5抗体重链(VH)(SEQ ID NO：1)与轻链(VL)(SEQ ID NO：2)之间用长连接片段(SEQ ID NO：7)连接，形成单链抗体B5-ScFv(SEQ ID NO：3)，后端融合人白介素2(IL2)(SEQ ID NO：9)，B5抗体的羧基端以长连接片段(SEQ ID NO：8)与IL2连接。融合后形成的蛋白为B5-IL2(SEQ ID NO：6)。
3)B5-Fc与B5-IL2融合蛋白表达
将编码B5-Fc(SEQ ID NO：5)与B5-IL2(SEQ ID NO：6)融合蛋白的DNA片段分别连接至pCI-neo载体中，将获得的含所述抗体融合蛋白编码序列的质粒大量提取后，利用
Figure PCTCN2016111264-appb-000002
2000(Invitrogen)将质粒DNA转染至CHO-K1细胞(购自中国典型培养物保藏中心)，借助pCI-neo质粒载体的neo基因在含G418的选择性DMEM/F12培养基(Thermo Scientific HyClone)中进行连续4-8周的选择培养后，经有限稀释法进行克隆化培养，将获得的单克隆细胞继续克隆化培养以获得稳定株。
获得的细胞株经Hyclone SFM4CHO-Utility(Thermo Scientific HyClone)驯化后悬浮表达，经ELISA和SDS-PAGE实验验证，获得了目标抗体蛋白，抗体蛋白产量约为100mg/L。
实施例3：动物造模、分组和给药方法
溶液配制：在PBS(NaCl 8g/L，KCl 0.2g/L，Na2HPO41.42g/L，KH2PO40.27g/L，pH＝7.4)溶液中将实施例1中制备的B5-Fc与B5-IL2融合蛋白分别配制成0.4mg/ml的溶液。
小鼠移植瘤模型建立：健康Balb/c nu裸鼠在SPF级屏障系统中饲养至8周龄，在小鼠背部皮下接种小鼠畸胎瘤细胞(F9)(上海复祥生物科技有限公司)，每个注射点2.5×106个细胞。接种第6天选择肿瘤体积约60mm3的小鼠进行分组实验。按8只每组分组，设A、B、C、D、E组，在接种后第6天、7天、8天分别通过尾静脉注射给药，其中：
A组为阴性对照组，每天给予生理盐水两次，间隔6小时，每次50μl/只。
B组为阳性对照组，每天给予B5-IL2溶液和生理盐水各一次，先给予生理盐水，间隔6小时再给予B5-IL2溶液，分别为50μl/只。
C组每天先给予B5-Fc溶液，6小时后给药予B5-IL2溶液，分别为50μl/只。
D组每天给予B5-IL2溶液与B5-Fc溶液制成的等体积混合溶液一次，100μl/只。
E组为对照组，每天给予B5-Fc溶液和生理盐水各一次，先给予B5-Fc溶液，间隔6小时再给予生理盐水，分别为50μl/只。
最后一次给药后第6天，即小鼠皮下接种造模后第14天测量体积。结果示于下表1：A组、E组为阴性对照组，肿瘤体积显著大于B组、C组、D组的肿瘤体积，差异有统计学意义。C组或D组的肿瘤体积显著小于B组肿瘤体积，差异有统计学意义，因此C组和D组的治疗效果优于B组；C组和D组之间无显著差异。A组和E组之间无显著差异。
表1
Figure PCTCN2016111264-appb-000003

Claims (20)

  1. 一种试剂盒，其包含生物惰性药物和生物活性药物，其中
    -所述生物活性药物包括靶向部分和生物活性部分，
    -所述生物惰性药物包括靶向部分和生物惰性部分，
    所述生物惰性药物与所述生物活性药物能够靶向相同靶，任选所述生物惰性药物与所述生物活性药物具有相同的靶向部分。
  2. 一种药物组合物，其包含生物惰性药物和生物活性药物以及任选存在的药学可接受的载体和/或赋形剂，其中
    -所述生物活性药物包括靶向部分和生物活性部分，
    -所述生物惰性药物包括靶向部分和生物惰性部分，
    所述生物惰性药物与所述生物活性药物能够靶向相同靶，任选所述生物惰性药物与所述生物活性药物具有相同的靶向部分。
  3. 根据权利要求1所述的试剂盒或权利要求2所述的药物组合物，其中所述靶向部分选自配体、受体、抗体或其结合靶的片段，例如完整抗体、抗体CDR区、抗体可变区、Fab片段、Fab′片段、F(ab)′2片段、单链Fv(scFv)、Fv片段、抗体轻链、抗体重链、单域抗体、双抗体及线性抗体。
  4. 根据权利要求1-3任一项所述的试剂盒或药物组合物，其中所述生物惰性部分选自抗体恒定区、白蛋白、聚乙二醇和核酸适配体，例如IgG4 Fc区。
  5. 根据权利要求1-4任一项所述的试剂盒或药物组合物，其中所述生物活性部分选自细胞毒素、细胞因子如TNF-alpha、IL-2或IL-12、放射性同位素、化学药物、具有生物活性的抗体恒定区和具有生物活性的细胞。
  6. 根据权利要求1-5任一项所述的试剂盒或药物组合物，其中所述靶向部分靶向纤维连接蛋白额外结构域B(ED-B)，例如选自CGS-1、 CGS-2、L19或B5抗体或其结合ED-B的片段，例如完整抗体、抗体CDR区、抗体可变区、Fab片段、Fab′片段、F(ab)′2片段、单链Fv(scFv)、Fv片段、抗体轻链、抗体重链、单域抗体、双抗体及线性抗体。
  7. 根据权利要求1-6任一项所述的试剂盒或药物组合物，其中所述生物惰性部分是抗体恒定区，例如SEQ ID NO：4所示的肽。
  8. 根据权利要求1-7任一项所述的试剂盒或药物组合物，其中所述生物活性部分是细胞因子IL-2，例如SEQ ID NO：9所示的肽。
  9. 根据权利要求1-8任一项所述的试剂盒或药物组合物，其中所述生物惰性部分是SEQ ID NO：4所示的多肽，或者所述生物活性部分是SEQ ID NO：9所示的多肽。
  10. 根据权利要求1-9任一项所述的试剂盒或药物组合物，其中所述生物惰性药物是SEQ ID NO：5所示的多肽或其二聚体，或者所述生物活性药物是SEQ ID NO：6所示的多肽。
  11. 根据权利要求1-10任一项所述的试剂盒，其中所述生物惰性药物与所述生物活性药物混合制成单一制剂或组合物，或者所述生物惰性药物与所述生物活性药物分别制成独立制剂或组合物。
  12. 一种生物惰性药物，其包括靶向感兴趣靶的靶向部分和生物惰性部分。
  13. 根据权利要求12所述的生物惰性药物，其中所述靶向部分选自配体、受体、抗体或抗体片段，例如完整抗体、抗体CDR区、抗体可变区、Fab片段、Fab′片段、F(ab)′2片段、单链Fv(scFv)、Fv片段、抗体轻链、抗体重链、单域抗体、双抗体及线性抗体。
  14. 根据权利要求12或13所述的生物惰性药物，其中所述靶向部分靶向纤维连接蛋白额外结构域B，例如选自CGS-1、CGS-2、L19或B5抗体或其结合ED-B的片段，例如抗体CDR区、抗体可变区、Fab片段、Fab′片段、F(ab)′2片段、单链Fv(scFv)、Fv片段、抗体轻链、抗体重链、单域抗体、双抗体及线性抗体，如SEQ ID NO：3所示的肽。
  15. 根据权利要求12-14任一项所述的生物惰性药物，其中所述生物惰性部分选自抗体恒定区、白蛋白、聚乙二醇和核酸适配体，例如IgG4Fc区，如SEQ ID NO：4所示的肽。
  16. 根据权利要求12-15任一项所述的生物惰性药物，其中所述生物惰性药物是SEQ ID NO：5所示的多肽或其二聚体。
  17. 一种治疗疾病的方法，包括给予需要治疗的对象例如哺乳动物施用权利要求1-11任一项中所限定的生物惰性药物和生物活性药物、或权利要求2-10任一项所述的药物组合物，或使用权利要求1-11任一项的试剂盒。
  18. 根据权利要求17所述的方法，其中所述生物惰性药物与生物活性药物同时施用，或者顺序施用，例如间隔5分钟、10分钟、15分钟、20分钟、25分钟、30分钟、40分钟、50分钟、1小时、2小时、3小时、4小时、5小时、6小时、7小时、8小时、9小时、10小时、11小时、12小时或更长时间，例如先施用生物惰性药物，然后施用生物活性药物。
  19. 根据权利要求17或18所述的方法，其中所述疾病是与纤维连接蛋白ED-B相关的疾病，例如实体肿瘤。
  20. 权利要求17-19任一项的方法，其中所述对象是人。
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