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WO2009149599A1 - 脂质体药剂及其制备方法和用途 - Google Patents

脂质体药剂及其制备方法和用途 Download PDF

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Publication number
WO2009149599A1
WO2009149599A1 PCT/CN2008/071258 CN2008071258W WO2009149599A1 WO 2009149599 A1 WO2009149599 A1 WO 2009149599A1 CN 2008071258 W CN2008071258 W CN 2008071258W WO 2009149599 A1 WO2009149599 A1 WO 2009149599A1
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WIPO (PCT)
Prior art keywords
liposome
ligand
agent according
hdcfv25
antibody
Prior art date
Application number
PCT/CN2008/071258
Other languages
English (en)
French (fr)
Inventor
刘彦仿
Original Assignee
Liu Yanfang
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Liu Yanfang filed Critical Liu Yanfang
Priority to PCT/CN2008/071258 priority Critical patent/WO2009149599A1/zh
Priority to CN200880129761XA priority patent/CN102056591B/zh
Publication of WO2009149599A1 publication Critical patent/WO2009149599A1/zh
Priority to US12/926,654 priority patent/US20110165068A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6859—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from liver or pancreas cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A61K47/6913—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome the liposome being modified on its surface by an antibody
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/303—Liver or Pancreas
    • 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)

Definitions

  • Liposomal agent preparation method and use thereof
  • the invention belongs to the field of liposome medicaments. More specifically, the present invention relates to a liposome agent having dual targeting effect in which a targeting agent containing an effector molecule in combination with a first ligand is encapsulated in a liposome, and on the surface of the liposome A second ligand is incorporated, wherein the first ligand and the second ligand may be the same or different.
  • the invention further relates to a process for the preparation of said liposome agents and their use in the treatment of diseases, in particular in the treatment of tumors.
  • the invention further relates to a pharmaceutical composition comprising the liposome agent and a pharmaceutically acceptable carrier. Background technique
  • Therapeutic antibodies are tumor-therapeutic antibodies, and the other half are therapeutic antibodies to other diseases. Therapeutic antibodies are an important part of biotech drugs.
  • a monoclonal antibody is used to encapsulate a liposome containing a drug or a toxin, i.e., a once-targeted immunoliposome toxin. Since then, researchers have also tried to cross-link the surface of the liposome with genetically engineered antibodies, especially single-chain antibodies, and internally encapsulated drugs, toxins, cytokines, genes, antisense nucleic acids, etc., and made a targeted immunolipid. Plastid toxin.
  • the liposome-targeting agent complex of the prior art has disadvantages such as insufficient targeting and damage to non-target cells, tissues or organs.
  • a therapeutically active agent that is capable of targeting or treating a target cell, tissue or organ, such as a tumor cell, with greater specificity, such as elimination.
  • the invention provides a dual-targeted liposome agent, which comprises a targeting agent comprising a first ligand and an effector molecule, and a second compound is bounded outside the liposome.
  • the first ligand and the second ligand can specifically bind to a target tissue or target cell of a subject to be treated or treated.
  • the first ligand and the second ligand may be the same or different.
  • the first ligand and/or the second ligand are antibodies, in particular monoclonal antibodies, or antigen-binding fragments thereof, such as Fab fragments, F(ab,) 2 fragments, single chain antibodies, human sources Antibody and the like.
  • the first ligand and the second ligand may be combined with the effector molecule and the liposome surface, respectively, in various suitable manners.
  • the first ligand is covalently or non-covalently bound to the effector molecule, e.g., in the case where both are proteins, the form of the fusion protein can be formed together.
  • the second ligand is conjugated or coupled to the surface of the liposome.
  • Another aspect of the invention relates to a method of preparing the liposome agent having dual targeting.
  • the invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the liposome agent having dual targeting and the use of the agent in the treatment of diseases, especially tumors.
  • the present invention also provides a method of treating a disease comprising administering a liposome agent or a pharmaceutical composition of the present invention to a patient in need thereof.
  • the invention further provides a method of diagnosing a disease comprising administering to a subject a liposomal agent or pharmaceutical composition of the invention.
  • the term "ligand" refers to any molecular entity that can specifically bind to target cells, tissues and/or organs, particularly tumor tissues, ie, should be understood broadly. Although the ligand can be bound to the cell receptor on the surface of the target cell, the present invention is not limited thereto. In one embodiment, the ligand can bind to a target cell or target tissue such as a tumor Related antigens on.
  • the first ligand and the second ligand of the present invention may be the same or different and may be independently selected from any suitable type.
  • One of ordinary skill will be aware of the selection of a suitable first and/or second ligand depending on the particular target tissue to be treated.
  • the first ligand and/or the second ligand are antibodies, in particular monoclonal antibodies, or antigen-binding fragments thereof, such as Fab fragments, F(ab,) 2 fragments, single chain antibodies, human sources Antibody and the like.
  • the first and/or second ligand are independently a monoclonal antibody or antigen-binding fragment thereof directed against an antigen selected from the group consisting of: CD19, CD20, CD22, CD33, epidermal growth Factor receptor (EGFR), MUC-1, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), oncogene c-erbB2 product, ganglioside GD3, GM2, and the like.
  • an antigen selected from the group consisting of: CD19, CD20, CD22, CD33, epidermal growth Factor receptor (EGFR), MUC-1, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), oncogene c-erbB2 product, ganglioside GD3, GM2, and the like.
  • the ligand is an antibody or antibody fragment.
  • Antibody fragments that can be used include, for example, Fab, Fab, F(ab,)2, scFv, and dsFv fragments.
  • the antibody or fragment can be from any species, including mice, rats, rabbits, hamsters, and humans. It is also within the scope of the invention to use chimeric antibody derivatives, i.e., antibody molecules that combine non-human animal variable regions with human constant regions.
  • a chimeric antibody molecule can include a humanized antibody comprising an antigen binding region of an antibody from, for example, a mouse, rat or other species, and a human constant region, which can be prepared by methods conventional in the art.
  • humanized antibodies can be found in EP-B 10 239400.
  • humanized antibodies can be obtained commercially. Chimeric antibodies are expected to be less immunogenic in human subjects than the corresponding non-chimeric antibodies.
  • humanized antibodies can also be further stabilized, for example as described in WO 00/61635.
  • a specific antibody or antibody fragment can also be prepared by screening an expression library encoding an immunoglobulin gene or a portion thereof, which expresses a peptide produced by a nucleic acid molecule encoding the protein in bacteria.
  • a phage expression library can be used to express intact Fab fragments, VH regions and FV regions in bacteria.
  • SCID-hu mice such as mouse models developed by Genpharm, can be used to produce antibodies or fragments thereof.
  • the first ligand and/or the second ligand moiety on the dual targeting liposome of the invention may independently From the source of immunoglobulin.
  • the ligand can be obtained by modifying an immunoglobulin scaffold using standard techniques known in the art.
  • an immunoglobulin domain eg, a heavy chain and/or a light chain variable region
  • ligands can also be formed by chemical reaction or genetic design.
  • a targeting agent can comprise (1) an immunoglobulin-derived polypeptide (eg, an antibody selected from an antibody library) or a variant thereof that specifically binds to a target tissue and/or cell, eg, Tumor cells, and (2) effector molecules such as toxins or variants thereof.
  • an immunoglobulin-derived polypeptide eg, an antibody selected from an antibody library
  • effector molecules such as toxins or variants thereof.
  • immunoglobulin polypeptide ligands can be redesigned to alter their binding characteristics to targets, such as tumor associated molecules, or for example to improve their physical characteristics.
  • the ligand moiety in the dual targeting liposomes of the invention may also independently be based on immunoglobulins.
  • the first ligand and/or the second ligand may be a non-immunoglobulin polypeptide (e.g., Affibody (R)) or a variant thereof that specifically binds to a target tissue such as a tumor cell.
  • the targeting agent may comprise: (1) a non-immunoglobulin polypeptide (eg, Affibody (R)) or a variant thereof that specifically binds to a target tissue such as a tumor cell, and (2) an effector molecule such as a toxin or Variants.
  • non-immunoglobulin ligands can be designed to bind to a target tissue (e.g., a target tumor) related molecule.
  • target tissue e.g., a target tumor
  • non-immunoglobulin polypeptide ligands can be processed to have the desired affinity and designed to withstand a variety of physical conditions, including extreme pH ranges and relatively high temperatures.
  • non-immunoglobulin polypeptides of the invention For use in pharmaceutical compositions, it would be highly advantageous to design the non-immunoglobulin polypeptides of the invention to have relatively long half-lives under physiological conditions (e.g., 37, present by peptidases). In addition, such molecules or variants thereof can exhibit good solubility, small size, proper folding, and can be expressed in a low cost bacterial system that is readily available for production in a commercially reasonable amount.
  • the design of non-immunoglobulin polypeptides is within the abilities of one of ordinary skill in the art. Techniques for designing, producing, and selecting the desired binding partners are generally described, for example, in U.S. Pat. Nos. 5,831,012 and 6,534,628, and are incorporated herein by reference.
  • the first ligand and/or the second ligand of the invention may independently be an epitope binding polypeptide.
  • epitope binding polypeptides include, but are not limited to, ligands containing a fibronectin type III domain.
  • Protein A-based affinity libraries can also be used to identify epitopes Binding polypeptides, such libraries are useful in the present invention for the selection of polypeptides that selectively bind to target tumor cells.
  • the first ligand and/or the second ligand of the invention may also independently be other types of binding molecules.
  • binding molecules are also known in the art and include, but are not limited to, binding molecules based on repeated protein domain assembly.
  • a library of randomly assembled repeat domains can be used to select ligands that selectively bind to target tissues such as tumor cells.
  • the first ligand and/or the second ligand may specifically bind to a tumor tissue to be treated by a tumor marker, such as a carcinoembryonic antigen, a prostate specific antigen, a urethra tumor-associated antigen, Fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialic acid Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, 613 ⁇ 4 8 and 1 55, but not limited to this.
  • a tumor marker such as a carcinoembryonic antigen, a prostate specific antigen, a urethra tumor-associated antigen, Fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialic acid Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, 613 ⁇ 4 8 and 1 55, but not limited to this.
  • the first ligand and/or the second ligand are monoclonal antibodies against liver cancer.
  • the monoclonal antibodies can be prepared by methods well known to those of ordinary skill in the art, such as hybridoma technology.
  • the first ligand and/or the second ligand may independently be YF Liu and CM Hu, Hybridoma 1996; 16(2): 213-215 and YF Liu et al, in Symposium IBS's Seventh Annual International Conference Antibody Engineering, 1997, 2: a monoclonal antibody derived from a HAb25 hybridoma cell disclosed in 171-197 (1), or a derivative of the monoclonal antibody, which has a respective cause of 1-20 Substitutions, insertions, deletions and/or preferably 1 to 15, more preferably 1 to 10, particularly preferably 1 to 8, especially 1 to 5, for example 1, 2, 3 or 4 amino acid residues
  • the heavy and light chains differing from the corresponding sequence of the HAb25 monoclonal antibody were added, but the antigen/target tissue
  • the first ligand and/or the second ligand is a single-chain antibody scFv25 derived from a HAb25 hybridoma cell monoclonal antibody having the amino acid sequence of SEQ ID NO: 1.
  • the coding sequence of the single-chain antibody scFv25 is shown in SEQ ID NO: 2.
  • the first ligand and/or the second ligand may be a variant of the single chain antibody scFv25 having 1-20, preferably 1-15, more preferably 1-10, especially preferably 1- 8,
  • the substitution, insertion, deletion and/or addition of 1-5, for example 1, 2, 3 or 4 amino acid residues differs from the amino acid sequence of SEQ ID NO: 1, but retains the single-chain antibody Antigen/target tissue binding affinity.
  • the single-chain antibody scFv25 see, for example, Reference 2 (YF Liu, CM Hu, P Yang, SM Chen, L Gao, YY Ji, ZN Chen, YF Sui, T Zheng, ZW Sun, MH Zhu, F Ren. Monoclonal antibodies against hepatocellular carcinoma HAb25 and their engineered products. In Symposium IBC's International Conference 1996 2-4 Nor. Antibody Engineering, 1997 2: 171-197, etc.).
  • the first ligand and/or the second ligand may independently be a humanized and/or disulfide-bonded single chain antibody, for example having SEQ ID NO A humanized monoclonal antibody hscFv25 of amino acid sequence of 3 or a humanized disulfide-stabilized single-chain antibody hdcFv25 having the amino acid sequence of SEQ ID NO: 5.
  • the first ligand and/or the second ligand may independently have from 1 to 20, preferably from 1 to 15, more preferably from 1 to 10, particularly preferably from 1 to 8, especially from 1 to 5.
  • Substitution, insertion, deletion and/or addition of 1, 2, 3 or 4 amino acid residues for example, differs from SEQ ID NO: 3 or 5, but retains the single-chain antibody hscFv25 or hdcFv25 Antigen/target tissue binding affinity.
  • drug may be used interchangeably with the term “effector molecule” or "biological or therapeutically active agent”, meaning that it is capable of exerting physiological effects on a biological system such as a prokaryotic or eukaryotic cell in vivo or in vitro (for example) Any agent that treats or prevents the effect) Examples include, but are not limited to, chemotherapeutic agents, toxins, radiotherapeutic agents, radiation sensitizers, gene therapy vectors, antisense nucleic acid constructs, transcription factor decoys, imaging agents, diagnostic agents, agents known to interact with intracellular proteins , vaccines, polypeptides and polynucleotides such as inhibitory RNA, antisense RNA, genes and the like.
  • an "effector molecule” is any molecule that exhibits a predetermined function, either directly or indirectly, within a target cell, such as a therapeutically active agent.
  • the effector molecule is a therapeutically active agent, which may be any suitable active agent capable of treating or preventing a subject or disease to be treated, such as a drug such as doxorubicin, a cytotoxic agent such as PE38, a cytokine such as TNF, radioisotope such as 131 1, therapeutic protein, and the like.
  • the therapeutically active agent is a cytotoxic agent, including, for example, bacterial and phytotoxins.
  • cytotoxic agents are Pokeweed antiviral protein, saponin, leucovorin, ricin, abrin, Pseudomonas exotoxin, diphtheria toxin alpha- ochratoxin, bouganin, xenogenic It is not limited to this, but it is limited to scorpion venom protein, local avermectin, Shiga toxin and its variants.
  • the cytotoxin is an active fragment of human perforin.
  • perforin for the use of perforin in targeting agents, see, for example, the description of CN98113025.9.
  • the therapeutically active agent is a toxin having DNA damaging effects.
  • the toxins include, but are not limited to, enediynes (eg, calicheamicin and esperamicin) and non-diacetylene small molecule agents such as bleomycin, methidiumpropyl-EDT A-Fe(II))schreib
  • Other toxins useful in the present invention include daunorubicin, doxorubicin, statin, cisplatin, mitomycin C, ecteinascidins, and bleomycin /Pilomycin and the like.
  • the therapeutically active agent is a toxin having microtubule disruption.
  • toxins include, but are not limited to, rhizoxin/maytansine, paclitaxel, vincristine and vinblastine, colchicine, and the like.
  • the therapeutically active agent can also be of other types well known to those of ordinary skill in the art. There are numerous documents available in the art regarding the types of therapeutically active agents available. for reference.
  • the effector molecule is RNase, which is an enzyme that decomposes RNA, is non-toxic to normal cells, and is toxic to tumor cells.
  • the effector molecule is a diagnostic agent, such as a label.
  • the label includes, for example, a fluorescent label, an enzyme label, a radioactive label, a nuclear magnetic resonance activity label, a luminescent label or a chromophore label.
  • the diagnostic agent can be used for diagnostic purposes after being targeted by the first ligand to the target tissue, including, for example, by imaging or the like.
  • the targeting agent of the present invention comprises: (1) a first ligand that specifically binds to a target cell; and (2) an effector molecule capable of exerting a desired function such as cytotoxicity on the cell.
  • Effector molecules such as therapeutically active agents can be combined with the first ligand in any of a variety of suitable manners, such as in a conjugated or unconjugated manner.
  • the first ligand can be combined with an effector molecule by chemical or recombinant means. Chemical methods for preparing fusions or conjugates are well known in the art and can be used to prepare the targeted agents of the present invention.
  • the method used to conjugate the first ligand to the effector molecule must be capable of linking the first ligand to the effector molecule without interfering with the ability of the ligand to bind to a target molecule, such as a target molecule on a tumor cell.
  • one or more effector molecules may be coupled to the first ligand by, for example, covalent bonding, affinity binding, intercalation, coordination binding, chelation or complexation, and the like. Conjugation. Where covalent bonding can be achieved by direct condensation of existing side chains or by incorporation of external bridging molecules. Many divalent or multivalent agents can be used to couple protein molecules to other proteins, peptides, or amino functions, and the like, including, but not limited to, carbodiimides, diisocyanates, glutaraldehyde, and the like.
  • the antibody can be first derivatized and then the effector molecule linked to the derivative product.
  • Suitable crosslinking agents in this regard include, for example, SPDP (N-succinimide-3-(2-pyridyldithio)propionate) and SMPT (4-succinimidyl-oxocarbonyl-indenyl- (2-pyridinium fluorenyl) fluorene.
  • SPDP N-succinimide-3-(2-pyridyldithio)propionate
  • SMPT 4-succinimidyl-oxocarbonyl-indenyl- (2-pyridinium fluorenyl) fluorene.
  • both the first ligand and the effector molecule are proteins that can be conjugated using techniques well known in the art.
  • crosslinkers are known to be used to conjugate two proteins.
  • the crosslinker is typically selected based on the first antibody or ligand and the reactive functional group inserted or available for use on the effector molecule.
  • a photoactivatable crosslinking agent can be used. In some cases, it may be desirable to have a spacer between the first ligand and the effector molecule.
  • Crosslinkers known in the art are bis-functional reagents: glutaraldehyde, dinonyl adipate and bis(diazabenzidine), and diisofunctional reagents: m-maleimidobenzoquinone Acyl-N-hydroxysuccinimide and sulfo-maleimidobenzoyl-N-hydroxysuccinimide.
  • Fusions of the first ligand protein to the effector molecule protein can also be made by recombinant DNA techniques.
  • a DNA sequence encoding the first ligand protein can be fused with a DNA sequence encoding the effector molecule to obtain a chimeric DNA molecule.
  • the chimeric DNA sequence is then transfected into a host cell expressing the first ligand-effector fusion protein and the fusion protein is recovered and purified from the culture using techniques known in the art.
  • the effector molecule is a radionuclide. It can generally be coupled to the first ligand of the invention by chelating.
  • a bifunctional chelating agent is typically used to link the isotope to the first ligand.
  • the chelating agent is first attached to the first ligand and then the chelating agent-first ligand complex is contacted with the metal radionuclide.
  • a number of bifunctional chelating agents are known which can be used for this purpose, including, for example, the diethylenetriaminepentaacetic acid (DTPA) series of amino acids, which are described in U.S. Patent Nos.
  • the targeting agent is encapsulated in a liposome to facilitate its delivery.
  • a liposome Any suitable liposome can be adapted for use in the present invention.
  • Liposomes are understood to mean such structures which enclose an aqueous interior by a membrane containing lipids. Unless otherwise stated, such a structure may have one or more layers of lipid membranes, although liposomes typically contain only one membrane. Liposomes of such monolayer membranes are referred to herein as “single layer” and multilamellar liposomes are referred to as "multilayered".
  • the liposome may be of any suitable size, for example, 1 ⁇ -50 ⁇ , preferably lOnm- ⁇ , more preferably 30 ⁇ -1 ⁇ , especially 50-500nm.
  • the liposome is a nanoliposome.
  • the liposome used in the present invention is preferably formed of a lipid which, when combined, forms a relatively stable blister.
  • lipids are known in the art for forming such liposomes. Preferred species include, but are not limited to, neutral and negatively charged phospholipids or sphingolipids and guanidines Alcohols, such as cholesterol.
  • the choice of lipids usually takes into account the size of the liposomes and the stability of the liposomes in the bloodstream.
  • the liposome is composed of sphingomyelin and cholesterol, wherein the ratio of sphingomyelin to cholesterol may vary, usually from 75:25 to 30:50 mol/mol%, preferably from about 70:30 to about 40,45 mol. /mol%. Or the ratio between phospholipid (SPC) and cholesterol (Choi) is 10: 1-1: 1 , more preferably 8: 1-2: 1 , particularly preferably 6: 1-3: 1 , for example 5 by weight : 1-4: 1.
  • the liposome of the present invention may further comprise other lipids, for example, to prevent oxidation of the lipid or to attach a ligand to the surface of the liposome, such as cholesterol polyethylene glycol ester, which is incorporated in a ratio of phospholipid The number may be from 1 to 15%, preferably from 3 to 10%, more preferably from 5 to 8%, for example 6%.
  • lipids for example, to prevent oxidation of the lipid or to attach a ligand to the surface of the liposome, such as cholesterol polyethylene glycol ester, which is incorporated in a ratio of phospholipid The number may be from 1 to 15%, preferably from 3 to 10%, more preferably from 5 to 8%, for example 6%.
  • This type of liposome is described in detail in U.S. Patent 5,814,335. The disclosure of this document is hereby incorporated by reference in its entirety.
  • Liposomes can be prepared in a variety of ways, and such methods are taught in the prior art, including, for example, U.S. Patent Nos. 4,235,871 and 4,501, 728 each incorporated herein by reference.
  • the step of producing a liposome generally comprises: mixing the lipid component in an organic solvent, drying and reconstituting the liposome in an aqueous solvent, and determining the size of the liposome and the like.
  • the introduction of the targeting agent into the liposome can be carried out in a passive or active manner. Passive introduction usually requires the addition of a drug to the buffer during the reconstitution step. This allows the drug to be trapped inside the liposome, where the drug will remain if the drug is insoluble in the lipid and the bleb remains intact.
  • Passive introduction usually requires the addition of a drug to the buffer during the reconstitution step. This allows the drug to be trapped inside the liposome, where the drug will remain if the drug is insoluble in the lipid and the bleb remains intact.
  • Active introduction involves a variety of methods that can achieve 100% encapsulation efficiency by using a transmembrane pH or ion gradient.
  • One of ordinary skill in the art is familiar with such introduction methods, which involve the establishment of certain forms of gradients by which the lipophilic component is fed into the interior of the liposome.
  • the second ligand can be bound or coupled to its surface by a variety of suitable methods. Such methods are well known to those of ordinary skill in the art.
  • the second ligand can be attached directly to the liposome or via a linker.
  • Linkers useful in the present invention are well known to those of ordinary skill in the art and include, for example, carbodiimides, glutaraldehyde. Wait.
  • the dual targeting liposome agents of the invention can be administered alone.
  • the dual targeting liposomal agents of the invention can be administered in combination with other drugs or bioactive agents or therapeutic regimens.
  • other drugs or bioactive agents include, but are not limited to, antioxidants, free radical scavengers, peptides, growth factors, antibiotics, bacterial inhibitors, immunosuppressants, anticoagulants, buffers, anti-inflammatory agents, Antipyretics, analgesics, steroids and corticosteroids.
  • the treatment may also include surgery and/or chemotherapy.
  • dual-targeted liposomes can be administered in combination with radiation therapy and Platinol, fluorouracil (5-FU, Adrucil), Paraplatin and/or Taxol (Taxol).
  • the dual targeting liposome agents of the invention are used in combination with conventional radiation therapy.
  • a lower dose of radiation therapy or a lower frequency of radiation treatment can be utilized, thereby enabling, for example, a reduction in side effects associated with radiation therapy.
  • the dual-targeted liposome agent of the invention can be administered in combination with one or more cytokines including, but not limited to, lymphokines, tumor necrosis factor, tumor necrosis factor-like cells Factor, lymphotoxin, interferon, macrophage inflammatory protein, monocyte-granulocyte colony-stimulating factor, interleukin (including, but not limited to, interleukin-1, interleukin-2, interleukin-6, interleukin-12, interleukin- 15 and interleukin-18) and variants thereof, including pharmaceutically acceptable salts thereof.
  • cytokines including, but not limited to, lymphokines, tumor necrosis factor, tumor necrosis factor-like cells Factor, lymphotoxin, interferon, macrophage inflammatory protein, monocyte-granulocyte colony-stimulating factor, interleukin (including, but not limited to, interleukin-1, interleukin-2, interleukin-6, interleukin-12, interleukin- 15
  • the dual-targeted liposome agents of the invention can be used in combination with a cancer vaccine, including, but not limited to, autologous cells or tissues, non-autologous cells or tissues, carcinoembryonic antigens, abortions Protein, human chorionic gonadotropin, live BCG vaccine, melanocyte lineage protein, and mutant tumor-specific antigen.
  • a cancer vaccine including, but not limited to, autologous cells or tissues, non-autologous cells or tissues, carcinoembryonic antigens, abortions Protein, human chorionic gonadotropin, live BCG vaccine, melanocyte lineage protein, and mutant tumor-specific antigen.
  • the dual targeting liposome agents of the invention can be used in combination with hormonal therapies.
  • Hormone therapy includes, but is not limited to, hormone agonists, hormone antagonists (such as flutamide, tamoxifen, leuprolide acetate (LUPRON)) and steroids (eg dexamethasone, retinoid) Retinoid, betamethasone.
  • hormone agonists such as flutamide, tamoxifen, leuprolide acetate (LUPRON)
  • steroids eg dexamethasone, retinoid Retinoid, betamethasone.
  • Testosterone is the same as testosterone and progestin.
  • the dual targeting liposome agents of the invention can be used in combination with gene therapy protocols to treat or prevent diseases such as cancer.
  • the dual targeting liposome agent of the invention may be included in a pharmaceutical composition or drug.
  • Pharmaceutical compositions suitable for direct administration include lyophilized powders or sterile injectable solutions or suspensions, aqueous or non-aqueous, which may further comprise an antioxidant, a buffer, a bacteriostatic agent, and such compositions and intended recipients
  • the blood is basically isotonic solute.
  • Other components that may be present in such compositions include, for example, water, alcohols, polyols, glycerin, and vegetable oils.
  • the injectable solutions and suspensions are prepared from sterile powders, granules and tablets.
  • the dual-targeted liposome agent of the present invention can be provided as, for example, a lyophilized powder, but is not limited thereto.
  • the lyophilized powder may be reconstituted with sterile water or saline prior to administration to the patient for ease of use.
  • compositions of the invention may comprise a pharmaceutically acceptable carrier.
  • suitable pharmaceutically acceptable carriers include substantially chemically inert, non-toxic compositions which do not interfere with the effectiveness of the biological activity of the pharmaceutical composition.
  • suitable pharmaceutical carriers include, but are not limited to, water, saline solution, glycerol solution, ethanol, N-(l(2,3-dioleyloxy)propyl)indole, indole, indole-trimethylammonium chloride (DOTMA), dioleoylphosphatidylethanolamine (DOPE) and liposomes.
  • DOTMA dioleoylphosphatidylethanolamine
  • liposomes Such compositions should contain a therapeutically effective amount of the dual targeting liposomal agent and a suitable amount of carrier to provide a form for administration to a patient.
  • the pharmaceutical composition comprises a dual targeting liposome agent of the invention and one or more additional therapeutic agents, such as a cancer therapeutic, optionally in a pharmaceutically acceptable carrier.
  • compositions of this invention may be administered in a variety of suitable manners including, but not limited to, intra-arterial, intramuscular, intravenous, intranasal, and oral routes.
  • the pharmaceutical compositions of the invention may be administered topically to the area in need of treatment.
  • Such topical administration can be accomplished, for example, by local infusion, injection, or by catheter during a surgical procedure.
  • the pharmaceutical composition is administered directly to a target tissue or target cell region, such as a tumor region, for example by topical infusion, topical application during a surgical procedure (eg, in combination with a wound dressing after surgery) Use), injection, catheterization, suppository or implant.
  • a target tissue or target cell region such as a tumor region
  • the implant can be a porous, non-porous or gelatin sample Material, including film or fiber.
  • Suppositories usually contain from 0.5 to 10% by weight of active ingredient.
  • the controlled release system can be placed adjacent to the target tissue or target cell.
  • a controlled dose can be delivered directly to the target tissue or target cell using a micropump, thereby finely adjusting the timed release and concentration of the pharmaceutical composition.
  • the invention also provides a kit comprising an effective amount of a dual-targeted liposome agent of the invention, and optionally one or more active agents, such as a therapeutically active or diagnostic agent, Contains instructions for its use.
  • the target cells, tissues, and organs to be treated or diagnosed may be, for example, prostate tissue, ovarian tissue, colon tissue, epithelial tissue, blood cells, lung tissue, liver tissue, pancreatic tissue, etc., the condition or condition thereof, or
  • the tumors in the corresponding tissue cells are of course not limited to these.
  • One of ordinary skill can select the appropriate first ligand and second ligand depending on the type of tissue or condition to be treated or diagnosed.
  • the condition or condition is a hyperproliferative disease, such as a tumor, including, for example, bladder cancer, colon cancer, liver cancer, lung cancer, gastric cancer, prostate cancer, breast cancer, brain tumor, skin cancer, etc.; or an artery Sclerosis, etc.
  • the dual targeting liposome agent and/or other therapeutically active agent is delivered to the patient by direct administration.
  • the dual-targeted liposome agents and/or other therapeutically active agents of the present invention can be introduced into a target tissue, such as a tumor, by introducing, for example, one or more direct injections into a target tissue, such as a tumor, or into a dual target.
  • the liposome drug reservoir is introduced into a target tissue, such as a tumor, into a sustained release device and/or directly to a target tissue such as a tumor.
  • the mode of administration to "intratumor” also includes introduction of a dual-targeted liposome agent of the present invention and/or other cancer therapeutic agent into a tumor region or a blood vessel or lymphatic vessel that substantially directly flows into the tumor region.
  • the pharmaceutical composition is administered in an amount sufficient to achieve a therapeutic endpoint, and if necessary, a pharmaceutically acceptable carrier can be included therein.
  • the dual targeting liposome agent of the invention can be administered by intratumoral means, while any other cancer therapeutic agent is delivered to the patient by other routes of administration (e.g., intravenously).
  • the dual-targeted liposome agent of the present invention and one or more of the cancer therapeutic agents can be delivered intratumorally, while other cancer therapeutic agents can be passed Other routes of administration (e.g., intravenous or oral) are administered.
  • the invention also provides a method of treating a disease comprising administering a liposomal agent or pharmaceutical composition of the invention to a patient in need thereof. In one embodiment, the method further comprises administering to the patient other therapies, such as hormone therapy, radiation therapy, and the like, sequentially or simultaneously.
  • the invention further provides a method of diagnosing a disease comprising administering to a subject a liposomal agent or pharmaceutical composition of the invention.
  • the method further comprises the step of imaging the subject.
  • Figure 1 shows the amino acid sequence of the anti-hepatocarcinoma genetically engineered single-chain antibody scFv25 and its coding sequence (SEQ ID NO: 1&2);
  • Figure 2 shows the single-chain antibody HdcFv25 obtained after humanization and disulfide stabilization of scFv25 The coding sequence;
  • Figures 3A and 3B list the amino acid sequences of the humanized and disulfide stabilized single chain antibody hdcFv25 and the corresponding coding sequences.
  • the nucleotide sequence encoding the single-chain antibody scFv25 was prepared by chemical synthesis with reference to the sequence shown in SEQ ID NO: 2, cloned into pUC19 plasmid (Novagen) into pUC19mscFv25 and expressed. See Reference 2 (YF Liu, CM Hu, P Yang, SM Chen, L Gao, YY Ji, ZN Chen, YF Sui, T Zheng, ZW Sun, MH Zhu, F Ren. Monoclonal antibodies against hepatocellular carcinoma HAb25 and their engineered Products. In Symposium IBC's International Conference 1996 2-4 Nor. Antibody Engineering, 1997; 2: 171-197).
  • the experimental results show that the murine single-chain antibody mscFv25 (also referred to in the present invention)
  • the single-chain antibody scFv25 increased the imaging ability of human liver cancer transplanted in mice by 84% (tumor/liver ratio 9.6).
  • the amino acid sequence of the single-chain antibody scFv25 is shown in SEQ ID NO: 1, and its coding nucleotide sequence is shown in SEQ ID NO: 2, see also FIG.
  • the resulting expression vector is pUC19hscFv25 0 wherein the humanized single chain antibody scFv25 referred hscFv25, the amino acid sequence shown in SEQ ID NO: 3.
  • primer 5 CCGCTCGACCTGGAGACGGTGACCAGGATGCCCAGCCCCA 3
  • SEQ ID NO: 6 to transform V H position 105 of the amino acid is cys
  • the V L at position 43 can also transformed into cys
  • prepared humanized A disulfide-stabilized single-chain antibody coding sequence is shown in SEQ ID NO: 4.
  • the humanized disulfide stabilized single chain antibody is referred to as hdcFv25 and the amino acid sequence thereof is set forth in SEQ ID NO: 5.
  • the resulting coding sequence was cloned between the Ncol and Notl sites in the expression vector pET15 bs to obtain the expression vector pET15b-hdcFv25, which was transformed into BL21 (DE3) to express the hdcFv25 protein.
  • hdcFv25 BACK 5 ATAGTTTAGCGGCCGCTTTGATCTCGACCTGGTCCC3' (SEQ ID NO: 7) and FOR: 5' CGGAATTCATGACCCAGACTCCACTC 3, (SEQ ID NO: 8) as primers, PCR amplification of hdcFv25, via EcoR I, Not I double digestion, recover DNA.
  • the pTIH was digested with EcoRI and Hindlll, and the above-mentioned double-digested hdcFv25 was inserted into EcoRI and NotI to form a highly efficient expression vector pTIH-hdcFv25.
  • P1 5' AAGCGGCCGCCTCAGAACTGGGCAACATT 3' (SEQ ID NO: 9 , wherein a Notl cleavage site is introduced, see the underlined portion);
  • the cDNA was synthesized by RT-PCR.
  • the above-mentioned PI and P2 primers were used to amplify the bullfrog-RNase (RC-RNase RNse) gene.
  • the pUCm-T vector (purchased from Shanghai Boya Biotechnology Co., Ltd.) was cloned to obtain a pUCm-RNase recombinant plasmid.
  • the plasmid was digested with Notl and Hind III and analyzed by DNA sequencing. The gene sequence was identical to the GeneBank report, indicating that the bullfrog RNase gene was successfully cloned.
  • RNase expression The pUCm-RNase recombinant plasmid was used as a template, and the gene fragment encoding the RNase mature protein was amplified by PCR from P2 and P3 primers, and then inserted into the prokaryotic expression by the same digestion and digestion after digestion with BamHI and Hindlll.
  • the vectors pRSET-A and pET32a(+) are prokaryotic expression vectors for the RNase fusion protein. After the enzyme digestion and DNA sequence determination were confirmed, the above two recombinant prokaryotic expression vectors were transformed into E. coli and induced by IPIG, and the expressed product was subjected to SDS-PAGE electrophoresis and thin layer scanning of the target protein.
  • the expressed products mainly existed in the form of inclusion bodies. After the inclusion bodies were isolated and purified, M- Further purification by NTA agarose affinity chromatography (see Fu Yong, Liu Y rotating, Su Qin et al, Humanized anti-hepatocarcinoma single-chain antibody and bovine ribonucleic acid fusion gene construction and expression biotechnology communication,
  • p4 5 AAGCGGCCGCTCAGAACTGGGCAACATT 3, (SEQ ID NO: 12, introduction of Notl site) and p5: 5, AAGCGGCCGCTTAATGATGATGAT GATGATGACGCGGTTCCAGCGGATACGGCACCGGCGCACCA GGACATCGTCCTATTCCAGC 3, (SEQ ID NO: 13 , introduction of Notl position
  • 6xHis and E-tag genes were used as primers, PCR amplification, and the product was digested with Notl to RNase.
  • hdcFv25 was also digested with Notl1, and the hHdcFv25-RNase fusion protein gene was ligated into the expression vector pTIH to form the expression plasmid pTIH-hdcFv25-RNase ⁇ , and the competent cells were transformed after sequencing.
  • Bacterial E Coli cultured, lysed bacteria to isolate fusion proteins.
  • hdcFv25 was amplified using the above expression vector pET15b-hdcFv25 as a template, wherein hdcFv25 BACK: 5'ATAGTTTAGCGGCCGCTTTGATCTCGA CCTGGTCCC3' (SEQ ID NO: 14) and FOR: 5'CGGAATTC ATGACCCAGACTCC ACTC 3' (SEQ ID NO: 15) were used as primers. .
  • the PCR amplification product hdcFv25 was digested with EcoRI and Notl to recover DNA.
  • PE38 was amplified using primers pCS18dPE38 with PE BACK: 5' GGAAGCTTTTAATGA TGATGATGATGATGCTTCAGGTCCTCGCGCGGCGG 3' (SEQ ID NO: 16) and PE FOR: 5'ATAGTTTAGCGGCCGCTCAGGAG GGCGGCAGCCTGGCCGCG3' (SEQ ID NO: 17).
  • PE BACK 5' GGAAGCTTTTAATGA TGATGATGATGATGCTTCAGGTCCTCGCGCGGCGG 3'
  • Hind III and Not l were digested to recover DNA.
  • the above pTIH expression vector was double-digested with EcoR I and Hind III. Receive DNA.
  • pTIH-hdcFv25-PE38 0 which is a highly efficient soluble expression vector, transformed into competent E. coli cells, and in a three-resistant LB medium containing 200 mg/L ampicillin , cultured in 12.5mg/L tetracycline and 15mg/L kanamycin, induced by IPTG O.lmM, 30" culture for 4 hours, the expression mainly occurred in the supernatant. The expression amount accounted for 21% of the total soluble protein.
  • the expressed product was subjected to SDS-PAGE electrophoresis. The results showed that a protein band appeared at 66KD and was identified by Western blot with His antibody.
  • mTNF-a was PCR-amplified.
  • the amplified product was digested with Sail and Xhol and cloned into the hscFv25 3 end of the same digested pGEX4T-1 vector. It was ligated into pGEX4T-l-scFv25-m-TNFa by DNA ligase to transform E. Coli JM109 competent. Monoclones were picked at 37 ° C overnight and transferred to Amp/LB medium for further culture. Induction by IPTG, the cells were collected by centrifugation, and the bacteria were lysed by adding lysozyme and sodium deoxycholate.
  • HdcFv25-RNase using a membrane sonication: liposome-coated targeting agent, 2 mg/ml of HdcFv25-RC-RNase was prepared using buffer pH 7.0 of 10 mM Tris-HCl, 20 mM NaCl. Add a certain amount of drug: fat ratio (1:15) into the dry film bottle, mix the rotating vibration quickly, and ultrasonically use the ultrasonic generator. Filter, 220 whole tablets and store at 4 G C. A fully expanded Sephadex G50 column (1.6 cm X 30 cm) was used to load 1 ml of liposomes at a flow rate of 0.5 ml/min. The absorbance was measured in a 2 ml tube at 280 nm to distinguish between free HdcFv25-RNase and total HdcFv25-RNase, and the encapsulation efficiency (E%) was determined:
  • the RNase was encapsulated in a similar manner to form RNase-Lp liposomes.
  • the double-targeted liposome hdcFv25-PE38-lp-hdcFv25 and the control hdcFv25-PE38 and PE38-lp were also encapsulated.
  • Example 7 Crosslinking on the surface of liposomes HdcFv25
  • the antibody can be labeled with a radioisotope such as 131 1 or 125 1 . Labeling is carried out using the chloramine T method, in which I is covalently bonded to the antibody (see Reference 10: Ya-You Ji, Yan-Fang Liu, Zhi-Nan Chen. Radioimmunodection and Autoradioigraphic Localization of Monoclonal Antibody against Human Hepatocellular Carcinoma In Xenografts. Cancer 1992; (8): 2055-2059).
  • the MTT method is based on the metabolic reduction of dimethylthiazol diphenyl tetrazolium bromide (MTT).
  • MTT dimethylthiazol diphenyl tetrazolium bromide
  • NAADP-related dehydrogenases are present in living cells, which can reduce yellow MTT to blue-violet substances and are markers of living cells. In the dead cells, the enzyme disappeared and the MTT could not be restored, still yellow. Therefore, the optical density is measured at a wavelength of 550 nm by a microplate reader to indicate the cell death rate, and LD50 is a dose of poison that 50% of the tumor cells are inhibited.
  • the anti-tumor cell activity test of the dual-targeted liposome hdcFv25-RNase-lp-hdcFv25 in the present invention was carried out by using the MTT assay and the cultured hepatoma cell line (SMMC-7721) as the target tumor cell, and the examples were as follows.
  • the RNase-lp-hdcFv25 liposome constructed in 5 was used as a control.
  • the results are shown in Table 2.
  • Table 2 Comparison of hdcFv25-RNase-lp-hdcFv25 and hdcFv25-RNase, RNase-lp MTT LD50 reduction Formulation MTT LD50 MW MTT LD50
  • Hp-hdcFv25 Example 7 Construction of dual-targeted liposomes HdcFv25-PE38-lp-HdcFv25 and control HdcFv25-PE38 and PE38-lp and anti-tumor cell activity assay
  • the dual-targeted liposome HdcFv25-PE38-lp-HdcFv25 and the control HdcFv25-PE38 and PE38-lp were constructed as described in Examples 4 and 5, and tested as described in Example 6, and the results are shown in Table 3.
  • the dose of LD 50 was significantly reduced, indicating that its ability to kill tumor cells was significantly enhanced.
  • the dual-targeted liposome agent of the present invention exerts enhanced targeted anti-tumor activity by surface cross-linking of HdcFv25 liposomes and their encapsulated HdcFv25-toxin (eg RNase) targets liver cancer cells. If the liposome is destroyed during transport, the release of HdcFv25-toxin still has a targeting effect, which is the second targeting effect, thereby enhancing the killing effect on tumor cells.

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Description

脂质体药剂及其制备方法和用途 技术领域
本发明属于脂质体药剂领域。 更具体地， 本发明涉及一种具有双 靶向作用的脂质体药剂， 其在脂质体中包裹有含有效应分子与第一配 体相联合的靶向药剂， 并且在脂质体表面上结合有第二配体， 其中所 述第一配体与第二配体可相同或不同。 本发明还涉及所述脂质体药剂 的制备方法及其在疾病治疗、 特别是肿瘤治疗中的用途。 本发明另外 涉及含有所述脂质体药剂以及药学上可接受的载体的药物组合物。 背景技术
由于传统的手术治疗、化疗、放疗对于肿瘤的治疗作用十分有限， 世界各国在药物开发上都投入巨资， 以求得新的进展。 随着生物技术 的发展， 治疗性抗体及肿瘤治疗性抗体的研发取得了突破性进展， 已 经成为生物技术类药物中重要的增长点和竟相研发的焦点。 在美国
FDA批准的治疗性抗体中， 一半为肿瘤治疗性抗体， 另一半为其它疾 病的治疗性抗体。 治疗性抗体是生物技术药物重要的组成部分之一。
单克隆抗体问世后， 人们把希望寄托于单克隆抗体。 现有技术中 早已报道过应用单克隆抗体包裹含有药物或毒素等的脂质体， 即一次 靶向的免疫脂质体毒素。 此后， 科研人员还尝试用基因工程抗体特别 是单链抗体交联脂质体表层， 内部分别包裹药物、 毒素、 细胞因子、 基因、 反义核酸等， 制成的同样是一次靶向性免疫脂质体毒素。
但是， 现有技术中的脂质体靶向药剂复合物存在着靶向性不足、 会对非靶细胞、 组织或器官造成损害作用等缺点。 因此， 现有技术中 迫切需要一种能够以更高特异性靶向于靶细胞、 组织或器官如肿瘤细 胞进行治疗或处理如消除的治疗活性剂。 发明内容
本发明中出乎意料地发现， 通过将效应分子如治疗活性剂与对靶 组织有靶向作用的两种配体相组合， 结合脂质体技术， 能够制备有非 常高的靶组织特异性的双靶向脂质体。
本发明一方面提供了一种具有双靶向作用的脂质体药剂， 其内包 裹有包含第一配体和效应分子相联合的靶向药剂， 并在该脂质体外面 结合了第二配体， 该第一配体和第二配体可特异性结合待处理或治疗 的受试者靶组织或靶细胞。 所述第一配体与所述第二配体可以相同或 不同。 优选地， 所述第一配体和 /或第二配体是抗体， 尤其是单克隆抗 体， 或者其抗原结合性片段， 例如 Fab片段、 F(ab，)2片段、单链抗体、 人源化抗体等。
在本发明中， 第一配体和第二配体可以以各种合适的方式分别与 效应分子和脂质体表面相结合。 在一个实施方案中， 所述第一配体与 所述效应分子共价或非共价结合， 例如在二者均为蛋白质的情形下可 以一起形成融合蛋白的形式。 在另一个实施方案中， 所述第二配体与 脂质体表面相缀合或偶联。
本发明另一方面涉及所述具有双靶向作用的脂质体药剂的制备方 法。
本发明还涉及含有所述具有双靶向作用的脂质体药剂的药物组合 物以及该药剂在疾病、 尤其是肿瘤治疗中的用途。
本发明还提供了一种治疗疾病的方法， 其中包括对有此需要的患 者施用本发明的脂质体药剂或者药物组合物。
本发明另外提供了一种诊断疾病的方法， 包括对受试者施用本发 明的脂质体药剂或者药物组合物。
具体实施方式
在本发明中， 术语 "配体" 指可以与靶细胞、 组织和 /或器官、 尤 其是肿瘤组织特异性结合的任何分子实体， 即应当从广义上理解。 虽 然配体可以通过靶细胞表面的细胞受体与之结合， 但本发明并不局限 于此。 在一个实施方案中， 所述配体可结合靶细胞或靶组织例如肿瘤 上的相关抗原。
本发明的第一配体和第二配体可以相同或不同， 并可独立地选自 任何合适的类型。 普通技术人员知晓根据具体的待治疗靶组织而选择 合适的第一和 /或第二配体。 优选地， 所述第一配体和 /或第二配体是 抗体， 尤其是单克隆抗体， 或者其抗原结合性片段， 例如 Fab片段、 F(ab，)2片段、 单链抗体、 人源化抗体等。 例如， 在一个实施方案中， 所述第一和 /或第二配体独立地是针对选自下组中的抗原的单克隆抗 体或者其抗原结合片段： CD19、 CD20、 CD22、 CD33、 表皮生长因 子受体 (EGFR)、 MUC-1、 前列腺特异性抗原（PSA)、 前列腺特异性膜 抗原（PSMA)、 癌基因 c-erbB2产物、 神经节苷酯 GD3、 GM2等。 普 通技术人员显然可以认识到， 本发明并不局限于上述具体列举的那些 类型。
在一个优选实施方案中， 所述配体是抗体或抗体片段。 可以使用 的抗体片段包括例如 Fab、 Fab，、 F(ab，)2、 scFv和 dsFv片段。 所述 抗体或片段可以来自任何物种， 包括小鼠、 大鼠、 兔、 仓鼠和人。 在 本发明范围内还可以使用嵌合抗体衍生物， 即将非人动物可变区与人 恒定区相组合的抗体分子。 嵌合抗体分子可以包括人源化抗体， 其中 包含来自例如小鼠、 大鼠或者其它物种的抗体的抗原结合区以及人恒 定区， 其可通过本领域内常规的方法进行制备。 例如， 人源化抗体的 制备可参阅 EP-B 10 239400。 另外， 还可通过商业途径获得人源化抗 体。 预计嵌合抗体在人受试者中的免疫原性将比相应的非嵌合抗体更 低。 为了改善其性质， 还可以对人源化抗体进一步稳定化， 例如参照 WO 00/61635中所述进行。
还可以通过对编码免疫球蛋白基因或者其部分的表达文库进行 筛选来制备特异性抗体或抗体片段， 该表达文库在细菌中表达由编码 所述蛋白的核酸分子所产生的肽。 例如， 可以使用噬菌体表达文库在 细菌中表达完整的 Fab片段、 VH区和 FV区。或者，可以使用 SCID-hu 小鼠， 例如 Genpharm开发的小鼠模型， 来生产抗体或者其片段。
本发明双靶向脂质体上的第一配体和 /或第二配体部分独立地可 以来源于免疫球蛋白。 例如， 可以通过釆用本领域中已知的标准技术 修饰免疫球蛋白支架而获得所述配体。 在另一个非限制性的实施方案 中，可以将免疫球蛋白结构域 （例如重链和 /或轻链可变区）与非免疫球 蛋白支架相连接。 此外， 还可以通过化学反应或遗传设计形成配体。 因此， 在非限制性的实例中， 靶向药剂可以包含 (1) 来源于免疫球蛋 白的多肽 （例如选自抗体文库的抗体)或者其变体， 其特异性结合靶组 织和 /或细胞如肿瘤细胞， 和 (2)效应分子如毒素或者其变体。可以重新 设计这样的免疫球蛋白多肽配体， 以改变它们对靶标例如肿瘤相关分 子的结合特征， 或者例如改善它们的物理特征。
本发明双靶向脂质体中的配体部分独立地也可以无需基于免疫 球蛋白。 例如， 在本发明中， 第一配体和 /或第二配体可以是特异性结 合靶组织如肿瘤细胞的非免疫球蛋白多肽 (例如 Affibody(R))或者其变 体。 相应地， 所述靶向药剂可以包含： （1) 特异性结合靶组织如肿瘤 细胞的非免疫球蛋白多肽 (例如 Affibody(R))或者其变体， 和 (2) 效应 分子如毒素或者其变体。 可将这样的非免疫球蛋白配体设计成结合靶 组织 (如靶肿瘤)相关分子。 此外， 可将非免疫球蛋白多肽配体加工成 具有所需的亲和力， 以及设计成可以耐受多种物理条件， 包括极端 pH 范围和相对高温度。
为了应用于药物组合物， 将本发明的非免疫球蛋白多肽设计成在 生理条件下 (例如 37 ， 由肽酶存在)具有相对较长的半寿期将是非常 有利的。 此外， 这样的分子或其变体可以显示出良好的溶解性、 小尺 寸、适当折叠， 并且可以在很容易利用的低成本细菌体系中进行表达， 从而以商业上合理的量加以生产。 非免疫球蛋白多肽的设计在本领域 普通技术人员的能力范围内。 有关设计、 生产和选择所需的结合配偶 体的技术可一般性参阅例如 U.S. Pat. Nos. 5,831,012和 6,534,628, 并 且可作适应性修改。
在又一个实施方案中，本发明的第一配体和 /或第二配体可以独立 地是表位结合多肽。 表位结合多肽的实例包括、 但不限于含有纤连蛋 白 III型结构域的配体。 还可釆用基于蛋白 A的亲和文库来鉴定表位 结合多肽， 这样的文库在本发明中可用于选择出与靶肿瘤细胞选择性 结合的多肽。
本发明的第一配体和 /或第二配体也可以独立地是其它类型的结 合分子。 这样的结合分子在本领域中也是已知的， 包括、 但不限于基 于重复蛋白质结构域装配的结合分子。 按照本发明， 随机装配的重复 结构域的文库可以用于选择出选择性结合靶组织如肿瘤细胞的配体。
优选地，所述第一配体和 /或第二配体可通过肿瘤标记与待治疗的 肿瘤组织特异性结合， 所述肿瘤标记包括例如癌胚抗原、 前列腺特异 性抗原、尿道肿瘤相关抗原、胎抗原、酪氨酸酶 (p97)、 gp68、 TAG-72、 HMFG、 唾液酸 Lewis抗原、 MucA、 MucB、 PLAP、 雌激素受体、 层粘连蛋白受体、 61¾ 8和 1 55 , 但不限于此。
更优选地， 所述第一配体和 /或第二配体是针对肝癌的单克隆抗 体。 所述单克隆抗体可通过本领域普通技术人员熟知的方法制备， 例 如杂交瘤技术。 在一个实施方案中， 所述第一配体和 /或第二配体可以 独立地是 YF Liu和 CM Hu, Hybridoma 1996; 16(2): 213-215和 YF Liu et al , in Symposium IBS's Seventh Annual International Conference Antibody Engineering, 1997, 2: 171-197(1)中公开的来源 于 HAb25杂交瘤细胞的单克隆抗体，或者该单克隆抗体的衍生物，所 述衍生物具有分别各因 1-20个、 优选 1-15个、 更优选 1-10个、 尤其 优选 1-8个、 特别是 1-5个、 例如 1、 2、 3或 4个氨基酸残基的取代、 插入、 缺失和 /或添加而与 HAb25单克隆抗体相应序列有所不同的重 链和轻链， 但保留了 HAb25杂交瘤细胞单克隆抗体的抗原 /靶组织结 合亲和力。 测定抗原 /靶组织 -抗体间结合亲和力的方法是本领域普通 技术人员众所周知的。
进一步优选地， 所述第一配体和 /或第二配体是来源于 HAb25杂 交瘤细胞单克隆抗体的单链抗体 scFv25 , 其具有 SEQ ID NO: 1所示 的氨基酸序列。 单链抗体 scFv25的编码序列示于 SEQ ID NO: 2中。 或者， 所述第一配体和 /或第二配体可以是单链抗体 scFv25 的变体， 其具有因 1-20个、 优选 1-15个、 更优选 1-10个、 尤其优选 1-8个、 特别是 1-5个、 例如 1、 2、 3或 4个氨基酸残基的取代、 插入、 缺失 和 /或添加而与 SEQ ID NO: 1有所不同的氨基酸序列， 但保留了该单 链抗体的抗原 /靶组织结合亲和力。 有关单链抗体 scFv25 的制备可参 见例如参考文献 2(YF Liu, CM Hu, P Yang, SM Chen, L Gao, YY Ji， ZN Chen, YF Sui, T Zheng, ZW Sun, MH Zhu, F Ren. Monoclonal antibodies against hepatocellular carcinoma HAb25 and their engineered products. In Symposium IBC's International Conference 1996 2-4 Nor. Antibody Engineering, 1997 2: 171-197等）。
普通技术人员知晓， 还可以对单链抗体进行人源化和二硫键稳定 化， 以改善单链抗体的性质， 更有利于临床应用。 因此， 在本发明的 一个优选实施方案中， 所述第一配体和 /或第二配体独立地可以是经过 人源化和 /或二硫键化的单链抗体， 例如具有 SEQ ID NO: 3所示氨基 酸序列的人源化单克隆抗体 hscFv25或者具有 SEQ ID NO: 5所示氨 基酸序列的人源化二硫键稳定化的单链抗体 hdcFv25。 有关人源化抗 体 hscFv25的制备可参阅参考文献 4(袁清安、 俞炜源和黄翠芬， 肝癌 特异性鼠源及人源化单链抗体基因的构建及在大肠杆菌中的表达， 生 物工程学报， 200 4 ,16(1),86 ~ 90) , 而人源化二硫键稳定化单链抗体 hdcFv25的制备可参阅参考文献 5(孙志伟、 俞炜源、 吕国华等， 二硫 键稳定的抗肝癌人源化单链抗体-突变体人 TNF a融合基因在 CHO细 胞中的表达 军事医学科学院院刊， 2001年 12月第 25卷第 4期）。 这 些文献的公开内容均通过参考并入本文中。 或者， 所述第一配体和 / 或第二配体可以独立地具有因 1-20个、优选 1-15个、更优选 1-10个、 尤其优选 1-8个、 特别是 1-5个、 例如 1、 2、 3或 4个氨基酸残基的 取代、 插入、 缺失和 /或添加而与 SEQ ID NO: 3或 5有所不同的氨基 酸序列，但保留了单链抗体 hscFv25或 hdcFv25的抗原 /靶组织结合亲 和力。 在本发明中， 术语 "药物" 可以与术语 "效应分子" 或 "生物或 治疗活性剂" 互换使用， 指的是能够对生物体系例如原核或真核细胞 在体内或体外发挥生理效应 （例如治疗或预防效果） 的任何药剂， 其 实例包括、 但不限于化疗剂、 毒素、 放疗剂、 辐射致敏剂、 基因治疗 载体、 反义核酸构建体、 转录因子诱饵、 成像剂、 诊断剂、 已知与胞 内蛋白发生相互作用的药剂、疫苗、多肽和多核苷酸例如抑制性 RNA、 反义 RNA、 基因等。 在一个实施方案中， "效应分子" 是任何能在靶细胞内直接或间 接发挥预定功能的分子， 如治疗活性剂。 在一个实施方案中， 所述效 应分子是治疗活性剂， 其可以是能够对待治疗目标或疾病进行治疗或 预防的任何合适活性剂， 例如药物如阿霉素、 细胞毒性剂如 PE38、 细 胞因子如 TNF、 放射性同位素如1311、 治疗性蛋白等。
在一个非限制性的实施方案中， 所述治疗活性剂是细胞毒性剂， 包括例如细菌和植物毒素。细胞毒性剂的实例有美洲商陆抗病毒蛋白、 皂苷、 白树毒蛋白、 蓖麻毒蛋白、 相思豆毒蛋白、 假单胞菌外毒素 、 白喉毒素 α -帚曲霉素、 bouganin、 异株泻才艮毒蛋白、 局限曲菌素、 志 贺菌毒素及其变体等， 但并不局限于此。
在另一个的实施方案中， 所述细胞毒素是人穿孔素 (perforin)的活 性片段。 有关穿孔素在靶向药剂中的应用可参见例如 CN98113025.9 的记载。
在另一个非限制性的实施方案中， 所述治疗活性剂是具有 DNA 破坏作用的毒素。 所述毒素包括、但不限于烯二炔类 (enediynes, 例如 加里刹霉素（calicheamicin)和埃斯波霉素 (esperamicin)) 和非烯二炔 类小分子药剂例如博来霉素， methidiumpropyl-EDT A-Fe(II))„本发明 中可用的其它毒素包括柔红霉素、 多柔比星、 司他霉素 、 顺铂、 丝 裂霉素 C、 海鞘素 (ecteinascidins)以及博来霉素 /培洛霉素等。
在又一个非限制性的实施方案中， 所述治疗活性剂是具有微管蛋 白破坏作用的毒素。所述毒素包括、但不限于根霉素 (rhizoxin)/美登素 (maytansine)、 紫杉醇 (paclitaxel)、 长春新械 (vincristine)和长春械 (vinblastine)、 秋水仙械 (colchicine)等。
在本发明中， 治疗活性剂还可以是本领域普通技术人员熟知的其 它类型。 有关可用的治疗活性剂的类型， 在本领域中有众多的文献可 供参考。
在又一个实施方案中， 所述效应分子是 RNase, 其是能分解 RNA 的酶， 对正常细胞无毒， 而对肿瘤细胞有毒。
在进一步的实施方案中， 所述效应分子是诊断剂， 例如标记物。 所述标记物包括如荧光标记、 酶标记、 放射性标记、 核磁共振活性标 记、 发光标记或生色团标记等。 所述诊断剂在被第一配体靶向到目标 组织后可以实现诊断目的， 包括例如通过成像等方法进行。
如前所述， 本发明的靶向药剂包含： （1) 与靶细胞特异性结合的 第一配体； 和 (2) 对该细胞能发挥所需功能例如细胞毒性的效应分子。 效应分子如治疗活性剂可以以多种合适方式中的任一种与第一配体相 联合， 例如缀合或非缀合的方式。 例如， 所述第一配体可通过化学或 重组方式与效应分子结合。 制备融合体或缀合物的化学方法在本领域 中是众所周知的， 它们均可以用来制备本发明的靶向药剂。 用来缀合 第一配体与效应分子的方法必须能够将该第一配体与效应分子相连接 而不干扰该配体与靶组织如肿瘤细胞上靶分子结合的能力。
按照本发明， 在一个实施方案中， 可以有一个或多个效应分子通 过例如共价结合、 亲和结合、 嵌入、 配位结合、 螯合或络合等而与第 一配体相偶联 /缀合。其中共价结合可通过现有侧链的直接缩合或者通 过掺入外部桥连分子来实现。 许多二价或多价药剂可用于使蛋白质分 子偶联其它蛋白质、 肽、 或氨基功能等， 包括例如碳二亚胺、 二异氰 酸酯、 戊二醛等， 但并不局限于此。 在一些实施方案中， 可以首先衍生所述抗体， 然后将效应分子连 接到衍生产物上。 这方面的合适交联剂包括例如 SPDP(N-琥珀酰亚胺 -3-(2-吡啶二硫基）丙酸酯）和 SMPT(4-琥珀酰亚胺基-氧代羰基 -曱基 -(2-吡啶二巯基）曱苯)。 在另一些实施方案中， 所述第一配体与效应分子均是蛋白质， 它 们能够利用本领域中众所周知的技术缀合起来。 已知有数百种交联剂 可用来将两种蛋白质缀合起来。 通常根据第一抗体或配基与效应分子 上插入的或可供利用的活性官能团来选择交联剂。 此外， 如果没有活 性官能团， 则可以使用可光活化交联剂。 在某些情形下， 可能比较希 望在第一配体与效应分子之间存在间隔基。 本领域中已知的交联剂有 双同官能团试剂： 戊二醛、 己二酸二曱酯和双 (二氮杂联苯胺）， 以及 双异官能团试剂：间马来酰亚胺基苯曱酰基 -N-羟基琥珀酰亚胺以及磺 基间马来酰亚胺基苯曱酰基 -N-羟基琥珀酰亚胺。
第一配体蛋白质与效应分子蛋白质的融合体也可通过重组 DNA 技术制备。 在这种情形下， 可将编码该第一配体蛋白质的 DNA序列 与编码所述效应分子的 DNA序列融合在一起， 得到嵌合 DNA分子。 然后将该嵌合 DNA序列转染到表达第一配体 -效应分子融合蛋白质的 宿主细胞中， 并使用本领域中已知的技术从培养物中回收和纯化融合 蛋白质。
在又一个实施方案中， 所述效应分子是放射性核素。 其一般可通 过螯合偶联到本发明的第一配体上。 例如， 在金属放射性核素的情况 下， 双功能螯合剂通常用于连接所述同位素与所述第一配体。 通常， 螯合剂首先附着在第一配体上，然后将螯合剂-第一配体复合物与金属 放射性核素接触。 已知有许多可用于此目的的双功能螯合剂， 包括例 如二乙三胺五乙酸 (DTPA)系列氨基酸， 其描述于美国专利 5,124,471、 5,286,850和 5,434,287, 其通过参考并入本文。 在本发明中， 所述靶向药剂被包封在脂质体中， 以便于其递送。 任何合适的脂质体在本发明中均可适用。 脂质体应理解为指由含有脂 类的膜包裹含水的内部的这种结构。 除非另有说明， 这种结构可以有 一层或多层脂类的膜， 尽管脂质体通常只含有一层膜。 这种单层膜的 脂质体在本文中被称为 "单层的" ， 而多层脂质体被称为 "多层的" 。 在本发明中， 脂质体可以是任何合适的大小， 例如 1ηιη-50μιη, 优选 lOnm-ΙΟμιη, 更优选 30ηιη-1μιη, 尤其是 50-500nm。 在一个实施方案 中， 所述脂质体是纳米脂质体。
用在本发明中的脂质体最好由脂类形成， 当结合时可以形成相对 稳定的嚢泡。 本领域中已知有许多种脂类可用来形成此类脂质体。 优 选的种类包括、 但不限于中性和带负电荷的磷脂类或神经鞘脂类和甾 醇类， 比如胆固醇。 对脂类的选择通常要考虑脂质体的尺寸和脂质体 在血流中的稳定性。
在本发明中， 优选脂质体由鞘磷脂和胆固醇构成， 其中鞘磷脂和 胆固醇的比例可以改变， 通常是在 75: 25到 30: 50mol/mol% , 优选 约 70: 30-约 40、 45mol/mol%。 或者以重量计， 磷脂（SPC)与胆固醇 (Choi)之间的比例为 10: 1-1: 1 , 更优选为 8: 1-2: 1 , 尤其优选 6: 1-3: 1 , 例如 5: 1-4: 1。 如果必要的话， 本发明的脂质体中还可以包 含其它的脂类， 比如为防止脂类氧化或在脂质体表面附着配体， 例如 胆固醇聚乙二醇酯， 其掺入比例以磷脂摩尔数计可以为 1-15 % , 优选 3-10 % , 更优选 5-8 % , 例如 6 %。 美国专利 5814335中对这种类型的 脂质体有详细的描述。 该文献的公开内容在此全文并入本文中作为参 考。
脂质体可釆用多种方法制备， 这样的方法在现有技术中有众多的 教导， 包括例如美国专利 4235871和 4501728等， 在此将它们全文引 入本文作为参考。 制造脂质体的步骤通常包括： 将脂类组分混合在有 机溶剂中， 干燥并在含水溶剂中重建脂质体， 以及确定脂质体的尺寸 等。
靶向药剂在脂质体中的引入可釆用被动或主动的方式进行。 被动 的引入通常需要在重建步骤时在緩冲液中添加药物。 这使得药物被截 留在脂质体内部， 如果药物不溶于脂类并且嚢泡保持完整的话药物将 保留在这里。 这样的方法公开在例如 WO 95/08986中， 其全文并入本 文中作为参考。
主动引入包括多种方法， 通过使用跨膜 pH或离子梯度可以使包 裹效率达到 100 %。 普通技术人员熟知这样的引入方法， 这些方法都 包括建立某些形式的梯度， 借助梯度将亲脂性组分送入脂质体内部。
在脂质体形成后， 可以通过各种合适的方法将第二配体结合或偶 联在其表面上。 这样的方法对于本领域普通技术人员来说是熟知的。 该第二配体可与脂质体直接连接， 或者借助接头连接。 在本发明中可 用的接头是本领域普通技术人员熟知的， 包括例如碳二亚胺、 戊二醛 等。
本发明的双靶向脂质体药剂可以单独施用。 或者， 本发明的双靶 向脂质体药剂可以与其它药物或生物活性剂或者治疗方案组合施用。 所述其它药物或生物活性剂的实例包括、 但不限于抗氧化剂、 自由基 清除剂、 肽、 生长因子、 抗生素、 细菌抑制剂、 免疫抑制剂、 抗凝血 剂、 緩冲剂、 抗炎剂、 退热剂、 止痛药、 类固醇和皮质类固醇等。 该 治疗也可包括外科手术和 /或化疗。 例如， 可以将双靶向脂质体与放疗 和顺铂 （Platinol)、 氟尿嘧啶 (5-FU, Adrucil). 碳铂 (Paraplatin)和 /或 紫杉醇 (Taxol)组合施用。
在一个实施方案中， 本发明的双靶向脂质体药剂与常规的放疗组 合使用。 在这种组合使用时， 可以利用更低剂量的放疗或频率更低的 放疗处理， 从而能够例如降低与放疗有关的副作用。
在另一个实施方案中， 本发明的双靶向脂质体药剂可以与一种或 多种细胞因子组合施用， 所述细胞因子包括、 但不限于淋巴因子、 肿 瘤坏死因子、 肿瘤坏死因子样细胞因子、 淋巴毒素、 干扰素、 巨噬细 胞炎性蛋白、 单核细胞-粒细胞集落刺激因子、 白介素（包括、 但不限 于白介素 -1、白介素 -2、白介素 -6、白介素 -12、白介素 -15和白介素 -18) 及其变体， 包括其药学可接受的盐。
在又一个实施方案中， 本发明的双靶向脂质体药剂可以与癌症疫 苗组合使用， 所述癌症疫苗包括、 但不限于自体细胞或组织、 非自体 细胞或组织、 癌胚抗原、 曱胎蛋白、 人绒毛膜促性腺激素、 BCG活疫 苗、 黑素细胞谱系蛋白以及突变的肿瘤特异性抗原。
在仍然又一个实施方案中， 可以将本发明的双靶向脂质体药剂与 激素疗法组合使用。 激素疗法包括、 但不限于激素激动剂、 激素拮抗 剂 （例如氟他胺（flutamide)、 他莫西芬、 乙酸亮丙立德（leuprolide acetate, LUPRON))和类固醇 (例如地塞米松、 类视色素 (retinoid)、 倍 他米松 (betamethasone). 皮质醇 (Cortisol)、 可的松 (cortisone)、 泼尼 松 (prednisone)、 脱氢睾 δ同 (dehydrotestosterone)、 糖皮质类固醇 (glucocorticoid)^盐皮质类固醇 (mineralocorticoid)、雄激素 (estrogen), 睾 S同 (testosterone)、 孕 δ同 (progestin)。
在更进一步的一个实施方案中， 本发明的双靶向脂质体药剂可以 与基因疗法方案组合使用， 以治疗或预防疾病例如癌症。
本发明的双靶向脂质体药剂可以包含在药物组合物或药物中。 适 于直接给药的药物组合物包括冻干粉或者含水或不含水的无菌可注射 溶液或悬液， 其中可以进一步包含抗氧化剂、 緩冲剂、 抑菌剂以及使 该组合物与预定接受者的血液基本等渗的溶质。 这样的组合物中可以 存在的其它组分包括例如水、 醇、 多元醇、 甘油和植物油。 现用注射 溶液和悬液可从无菌粉末、 颗粒和片剂制备。 本发明的双靶向脂质体 药剂可以作为例如冻干粉来提供， 但不限于此。 可以在向患者给药前 将冻干粉用无菌水或盐水来重溶， 以便于使用。
本发明的药物组合物可以包含药学可接受的载体。 合适的药学可 接受载体包括基本上化学惰性的无毒组合物， 其不干扰该药物组合物 生物活性的有效性。 合适药物载体的实例包括、 但不限于水、 盐水溶 液、 甘油溶液、 乙醇、 N-(l(2，3-二油酰氧基)丙基) Ν,Ν,Ν-三曱基氯化铵 (DOTMA), 二油酰基磷脂酰乙醇胺 （DOPE)以及脂质体。 这样的组合 物应当包含治疗有效量的所述双靶向性脂质体药剂以及合适量的载 体， 以便提供直接对患者施用的形式。
在另一个实施方案中， 所述药物组合物包含本发明的双靶向性脂 质体药剂以及一种或多种额外的治疗剂例如癌症治疗剂， 任选地处于 药学可接受载体中。
本发明的药物组合物可以多种合适的方式给药， 包括、 但不限于 动脉内、 肌内、 静脉内、 鼻内和口服途径。 在一个具体的实施方案中， 本发明的药物组合物可以向需要治疗的区域局部给药。 这种局部给药 可通过例如外科手术期间的局部输注、 注射或者通过导管来实现。
在本发明的一些实施方案中， 将所述药物组合物直接施用到靶组 织或靶细胞区域例如肿瘤区域， 例如通过在外科手术期间局部输注、 局部施用 （例如在外科手术后与创伤敷料组合使用）、注射、导管方式、 栓剂方式或者植入物方式。 植入物可以是多孔的、 无孔的或明胶样材 料， 包括膜或者纤维。 栓剂通常含有 0.5-10wt%的活性成分。
在另一些实施方案中， 可以将控释体系放置在靶组织或靶细胞附 近。 例如， 可以釆用微型泵将受控剂量直接投递到靶组织或靶细胞附 近， 由此精细地调节药物组合物的定时释放和浓度。
本发明还提供了一种试剂盒， 其中包含有效量的本发明双靶向脂 质体药剂， 以及任选地与之相组合的一种或多种活性剂例如治疗活性 剂或诊断剂， 还包含其使用说明。
在本发明中， 待治疗或诊断的靶细胞、 组织、 器官可以是例如前 列腺组织、 卵巢组织、 结肠组织、 上皮组织、 血液细胞、 肺组织、 肝 脏组织、 胰组织等， 其病症或状况， 或者相应组织细胞内的肿瘤， 当 然并不局限于这些。 普通技术人员可以根据待治疗或诊断的组织或病 症的不同类型而选择适用的第一配体和第二配体。在一个实施方案中， 所述病症或状况是过度增殖疾病， 如肿瘤， 包括例如膀胱癌、 结肠癌、 肝癌、 肺癌、 胃癌、 前列腺癌、 乳腺癌、 脑部肿瘤、 皮肤癌等； 或者 是动脉硬化症等。
根据本发明的一个方面， 所述双靶向脂质体药剂和 /或其它治疗活 性剂通过直接施用而递送给患者。 因此， 本发明的双靶向脂质体药剂 和 /或其它治疗活性剂可通过例如向靶组织如肿瘤内一次或多次直接 注射、 持续或间断地向靶组织如肿瘤内灌注、 导入双靶向脂质体药剂 储库、 向靶组织如肿瘤内导入緩释装置和 /或直接向靶组织如肿瘤应 用。 在本发明中， 向 "肿瘤内" 施用的给药方式也包括向肿瘤区域或 者基本上直接流入肿瘤区域的血管或淋巴管内导入本发明的双靶向脂 质体药剂和 /或其它癌症治疗剂。 在每种情形下， 药物组合物以至少足 以实现治疗终点的量施用， 并且必要时，其中可包含药学可接受载体。
可以预见到， 可以通过肿瘤内方式施用本发明的双靶向脂质体药 剂， 而通过其它施用途径 (例如静脉内）将任何其它癌症治疗剂递送给 患者。 另外， 若旨在向患者递送多种癌症治疗剂， 则可以通过肿瘤内 方式递送本发明的双靶向脂质体药剂以及一种或多种所述癌症治疗 剂， 而其它癌症治疗剂可通过其它给药途径 (例如静脉内或口服)施用。 本发明还提供了一种治疗疾病的方法， 其中包括对有此需要的患 者施用本发明的脂质体药剂或者药物组合物。 在一个实施方案中， 所 述方法还包括依次或同时向患者施用其他的疗法， 例如激素疗法、 放 射性疗法等。
本发明进一步提供了一种诊断疾病的方法， 包括对受试者施用本 发明的脂质体药剂或者药物组合物。 优选地， 所述方法还包括对受试 者成像的步骤。 附图说明
图 1列出了抗肝癌基因工程单链抗体 scFv25的氨基酸序列及其 编码序列（SEQ ID NO: 1&2); 图 2 列出了 scFv25 经人源化及二硫键稳定化后所得单链抗体 HdcFv25的编码序列； 以及 图 3A和 3B列出了人源化及二硫键稳定化单链抗体 hdcFv25的氨 基酸序列以及相应的编码序列。
具体实施方式
通过下述示例性实施例可更好地理解本发明。 本文中列出的各个 实施例仅是举例说明， 而并非意图限定本发明的范围。
实施例 1单链抗体 scFv25的编码核苷酸序列的制备
参考 SEQ ID NO: 2所示序列， 通过化学合成制备编码单链抗体 scFv25的核苷酸序列，将其克隆到 pUC19质粒 (Novagen公司）中成为 pUC19mscFv25并进行表达。参阅参考文献 2(YF Liu, CM Hu, P Yang, SM Chen, L Gao, YY Ji， ZN Chen, YF Sui, T Zheng, ZW Sun, MH Zhu, F Ren. Monoclonal antibodies against hepatocellular carcinoma HAb25 and their engineered products. In Symposium IBC's International Conference 1996 2-4 Nor. Antibody Engineering, 1997; 2: 171-197)。 实验结果表明， 鼠源单链抗体 mscFv25(在本发明中也称 为单链抗体 scFv25)把棵鼠中移植的人肝癌的显像能力提高了 84% (瘤 /肝比值为 9.6)。单链抗体 scFv25的氨基酸序列如 SEQ ID ΝΟ:1所示， 其编码核苷酸序列如 SEQ ID NO: 2所示， 也可参见图 1。
按照参考文献 4(袁清安、 俞炜源、 黄翠芬， 肝癌特异性鼠源及人 源化单链抗体基因的构建既在大肠杆菌中的表达， 生物工程学报， 2004,16,86 ~ 90)中所述将单链抗体 scFv25 人源化， 所得表达载体为 pUC19hscFv250 其中所述人源化单链抗体 scFv25被称为 hscFv25 , 其氨基酸序列示于 SEQ ID NO: 3中。 进一步地， 以 pUC19hscFv25 为 模 版 ， 用 引 物 5， CCGCTCGACCTGGAGACGGTGACCAGGATGCCCAGCCCCA 3， (SEQ ID NO:6)将 VH第 105位的氨基酸改造为 cys, 将 VL第 43位也 改造成 cys ,制得人源化二硫键稳定化单链抗体编码序列，示于 SEQ ID NO: 4中。 该人源化二硫键稳定化单链抗体被称为 hdcFv25 , 其氨基 酸序列列于 SEQ ID NO: 5中。 将所制得的编码序列克隆到表达载体 pET15 bs 中 的 Ncol 和 Notl 位点之间 ， 制得表达载体 pET15b-hdcFv25 , 将之转化到 BL21(DE3)中， 从而表达 hdcFv25蛋 白。
釆用 Invitrogen公司的高效原核表达载体 pET22b(+)实现可溶性 表达。 基本操作参阅参考文献 6(赵君， 孙志伟， 刘彦仿等 二硫键稳 定的抗肝癌单链抗体 -PE38融合基因的构建、表达于能检测 细胞与分 子免疫学杂志， 2003;19(6) , 585 ~ 587)进行。
用以上表达载体 pET15b-hdcFv25为模版，以 hdcFv25 BACK: 5， ATAGTTTAGCGGCCGCTTTGATCTCGACCTGGTCCC3' (SEQ ID NO: 7)和 FOR: 5' CGGAATTCATGACCCAGACTCCACTC 3， (SEQ ID NO: 8)为引物， PCR扩增 hdcFv25 , 经 EcoR I、 Not I双酶 切， 回收 DNA。 再对 pTIH进行 EcoRI及 Hindlll双酶切， 插入上述 经 EcoRI、NotI双酶切的 hdcFv25 ,构成高效表达载体 pTIH-hdcFv25。 实施例 2 RNase表达载体的构建及其表达 根据文献 Huang HC et al: Biochem 1998 273(11): 6395-7014中公 开的牛蛙 RNase全长 cDNA序列， 应用计算机引物设计软件 Primer premier 5.0和 oligo软件优化设计引物如下：
P1: 5' AAGCGGCCGCCTCAGAACTGGGCAACATT 3'(SEQ ID NO: 9 , 其中引入 Notl酶切位点， 见划线部分)；
P2: 5， CCAAGCTTTGACAGCATGAAAACTAACTAAG 3， (SEQ IDNO: 10, 其中引入 Hind III酶切位点， 见划线部分)；
P3: 5， AAGGATCCCAGAACTGGGCAAC 3'(SEQ ID NO: 11， 其中引入 Bam HI酶切位点， 见划线部分）。
从雌性牛蛙肝脏釆用异¾氰酸胍 /酚 /氯方法分离提出总 RNA, 应 用 RT-PCR 的方法合成 cDNA 以上述 PI、 P2 引物扩增牛蛙 -RNase(RC-RNase简称 RNse)基因， 再克隆入 pUCm-T载体 (购自上 海博亚生物技术有限公司）， 得到 pUCm-RNase重组质粒。 对该质粒 经 Notl和 Hind III双酶切鉴定， 并进行 DNA序列测定分析。 其基因 序列与 GeneBank报道完全一致，表明成功的克隆了牛蛙 RNase基因。
关于 RNase的表达： 以 pUCm-RNase重组质粒为模板， 从 P2、 P3引物釆用 PCR扩增编码 RNase成熟蛋白的基因片段， 经 BamHI 和 Hindlll 后消化分别将其插入经同样酶切消化的原核表达载体 pRSET-A和 pET32a(+)中，成为 RNase融合蛋白原核表达载体。经酶 切鉴定和 DNA序列测定验证正确后， 将上述两种重组原核表达载体 转化大肠杆菌， 用 IPIG诱导表达， 表达产物经 SDS-PAGE电泳和薄 层扫描目的蛋白。 结果表明， 两种载体在诱导下， 表达蛋白的分子量 分别为 16KD和 31KD, 表达量分别到达 12.5%和 34% , 表达产物主 要以包涵体的形式存在， 将包涵体分离纯化后， 经 M-NTA agarose 亲合层析进一步纯化 (参阅付勇， 刘彦仿， 苏勤等人， 人源化抗肝癌单 链抗体与牛蛙核糖核酸融合基因的构建及表达 生物技术通讯，
Vol.14, No.5 Sep, 2003,353 ~ 356)。 实施例 3 构建靶向药剂 HdcFv25-RNase 融合蛋白的表达载体 pTIH-hdcFv25-RNase
以上述克隆载体 pUCm-RNase 为模板， 以 p4： 5， AAGCGGCCGCTCAGAACTGGGCAACATT 3， (SEQ ID NO: 12 ,引 入 Notl 位点）及 p5： 5， AAGCGGCCGCTTAATGATGATGAT GATGATGACGCGGTTCCAGCGGATACGGCACCGGCGCACCA GGACATCGTCCTATTCCAGC 3， (SEQ ID NO: 13 ,引入 Notl位点、 6xHis 和 E-tag基因）为引物， PCR扩增，产物经 Notl单酶切为 RNase。
以上述 pTIH-hdcFv25同样经 Notl单酶切得 hdcFv25 , 经聚合酶 连接为 hHdcFv25-RNase融合蛋白基因，并克隆在表达载体 pTIH中， 成为表达质粒 pTIH-hdcFv25-RNase<, 测序正确后转化感受态细菌 E Coli, 培养， 裂解细菌分离融合蛋白。 电泳并进行 Western blot分析 鉴定，结果表明表达 HdcFv25-RNase融合蛋白（参阅参考文献 7:付勇， 刘彦仿， 苏勤等人， 人源化抗肝癌单链抗体与牛蛙核糖核酸酶融合基 因的构建及表达， 生物技术通讯， Vol.14 , No.5 Sep,2003,353 ~ 356)o 实施例 4 hdcFv25-PE38和 hscFv25-mTNF-a融合蛋白的构建、表 达和纯化
1. hdcFv25-PE38的构建、 表达与纯化
用以上表达载体 pET15b-hdcFv25为模板扩增 hdcFv25 ,其中釆 用 hdcFv25 BACK: 5'ATAGTTTAGCGGCCGCTTTGATCTCGA CCTGGTCCC3' (SEQ ID NO: 14)和 FOR: 5'CGGAATTC ATGACCCAGACTCC ACTC 3' (SEQ ID NO: 15)为引物。 将 PCR 扩增产物 hdcFv25 经 EcoRI、 Notl 双酶切， 回收 DNA。 以 pCS18dPE38 为模版， 以 PE BACK: 5' GGAAGCTTTTAATGA TGATGATGATGATGCTTCAGGTCCTCGCGCGGCGG 3' (SEQ ID NO: 16)及 PE FOR: 5'ATAGTTTAGCGGCCGCTCAGGAG GGCGGCAGCCTGGCCGCG3' (SEQ ID NO: 17)为引物扩增 PE38。 扩增产物经电泳鉴定大小后， 进行 Hind III、 Not l双酶切， 回收 DNA。 对上述 pTIH表达载体经 EcoR I、 Hind III双酶切后回 收 DNA。
对以上三种酶切片段加聚合酶连接成 pTIH-hdcFv25-PE380 这 是一种高效可溶性表达载体， 转化感受态大肠杆菌细胞， 并在三抗性 LB培养液 (其中含有 200mg/L氨苄青霉素、 12.5mg/L四环素和 15mg/L 卡那霉素）中培养， 经 IPTG O.lmM, 30 " 培养 4小时诱导表达， 表达 主要发生在上清中。 表达量占总可溶性蛋白的 21% , 对表达产物进行 SDS-PAGE电泳。 结果显示， 表达产物在 66KD处出现一蛋白条带， 以 His抗体进行 Western blot鉴定。 参阅参考文献 6: 赵君， 孙志伟， 刘彦仿等， 二硫键稳定的抗肝癌单链抗体 -PE38 融合基因的构建、 表 达与检测， 细胞与分子免疫学杂志， 2003;19(6), 585 - 587 中所述对 表达上清进行 Ni-NTA agarone亲合柱层析纯化。
2. hscFv25-mTNF-a的构建与表达
参照参考文献 3: 刘彦仿、 张静、 胡川闽等， 抗肝癌单链抗体 (鼠 及人源化)融合突变型 TNF- a的构建、 表达及其作用研究， 细胞与分 子免疫学杂志， 2000;16(5) , 372 中所述， 以 pUC18-mTNF-a为模版 (mTNF-a为突变型 TNF-a), 利用计算机 Oligo软件设计的上、 下游 引物 BACK 5， ACGCGTCGACCGCAAACGTAAGCCTGTA 3'(SEQ ID NO: 18) , FOR: 5'ACTCTGAGTCAGAAGGCAATGAT CCCAAAGT 3， (SEQ ID NO: 19 , 其中引入 Sail. Xhol酶切位点）， PCR扩增 mTNF-a。 扩增产物经 Sail及 Xhol双酶切后克隆到经相同 酶切的 pGEX4T-l载体中的 hscFv25 3， 端。 经 DNA连接酶连接成 pGEX4T-l-scFv25-m-TNFa, 转化 E. Coli JM109感受态。 挑单克隆 37° C过夜， 转接于 Amp/LB培养液中继续培养。 加加 IPTG诱导， 离心收集菌体， 加溶菌酶再加脱氧胆酸钠裂解细菌， 离心 12000r/min lOmin后沉淀用 0.5%TritonX-100，洗涤 3次， 离心， 沉淀为包涵体。 包涵体经过变性和复性后在 GST亲合层析柱纯化。 用 Werstern blot 方法釆用 GST抗体 (购自 Pharmacia公司）鉴定目的蛋白。表达产物加 入 0.2%凝血酶消化， 切去 GST, 上 GST亲合层析柱进一步纯化， 获 得 scFv25-mTNF-a的目的蛋白， 经 western blot进一步确认产物正 确 。 实施例 5 融合蛋白的体外毒性测试
釆用 MTT测试上述融合蛋白的活性， 结果显示， 它们在体外均 有很好的杀伤作用。用这些融合蛋白每天尾静脉注入一次，每次 0.3ml 含 20-30ug, 共 14天， 对荷肝癌棵鼠进行实验治疗。 结果表明， 抑制 率均在 75— 79%范围。比阿霉素（临床常用治疗肝癌药物）的效果为高， 见下表 1。 表 1 融合蛋白治疗棵鼠荷人肝癌的实验治疗效果
Figure imgf000020_0001
CR 完全緩解； PR 不完全緩解； NR 无緩解 实施例 6 将靶向药剂 HdcFv25-RNase融合蛋白和 RNase自身包 封在纳米脂质体中
此实施例参照参考文献 8(张桂红、 刘彦仿等人， 抗肝癌单链抗体 免疫脂质体的制备及其体外抑瘤实验， 医学研究生学报.2006:19(1): 3-5)和参考文献 9(HAIYANG HU,DAWEI CHEN,YANFANG LIU, et al. Target Ability and Therapy Efficacy of Immunoliposomes Using a Humanized Antihepatoma Disulfide-Stabilized Fv Fragment on Tumor Cells . J Pharm Sci. 2006 Jan; 95(1): 192-9)中所述进行， 简述 如下：
1. 制备空间稳定脂质体
将大豆卵磷脂（SPC)100 mg， 胆固醇 (Chol)25 mg， 两者比是 4: 1(W/W)选用分子量 2000 的胆固醇琥珀酸酯（Chol-PEG-OH) , PEG-Chol用量按磷脂的 6 %，溶于氯仿， 使用旋转薄膜蒸发仪， 在真 空减压下除去氯仿， 于瓶壁中形成均勾一致的干膜。
2. 使用薄膜超声法制备 HdcFv25-RNase: 脂质体包裹靶向药剂， 用緩冲液 PH7.0 的 10 mM Tris-HCl、 20 mM NaCl, 制备 2mg/ml 的 HdcFv25-RC-RNase。 按一定的药：脂比例（1:15)加入干膜瓶中， 快 速混合旋转震荡， 用超声波发生器超声。 过滤， 220整粒后 4GC保存。 用充分膨胀的 Sephadex G50柱 (1.6cmX30cm)， 取脂质体 1 ml 上样， 流速 0.5 ml/min。 用 2ml管， 紫外 280nm 处测定吸光度， 以区分游 离 HdcFv25-RNase及总 HdcFv25-RNase， 测包封率 (E%):
C游离量
E(%)=(1- ) X 100%
C总量 完成脂质体包裹 HdcFv25-RC-RNase 的工作， 使之成为 HdcFv25-RNase-Lp。
以类似的方法包封 RNase, 形成 RNase-Lp脂质体。 同样包封双 靶向脂质体 hdcFv25-PE38-lp-hdcFv25 以及对照 hdcFv25-PE38 和 PE38-lp。 实施例 7 在脂质体表面交联 HdcFv25
取包裹 hdcFv25-RNase 的脂质体 1 ml, 分别加入 100 μ ΐ 0.5 mol/L EDC (乙基（3-二 曱胺基）丙基）碳二亚胺盐酸盐， ethyl-(3-dimethylaminopropyl) Cardiimide-HCl) 和 100 μ 1 0.5 mol/L S-NHS (N-羟基硫代琥珀酰亚胺， N-hydroxysulfosuccinimide), 室温 下反应 30 min.， 按 hdcFv25/磷脂为 1: 500， 加入反应液， 4 "C过夜， 使 hdcFv25 通过以上试剂交联到脂质体表面的 PEG 分子上， 成为 hdcFv25-RNase-lp-hdcFv25。
类似地在包裹 RC-RNase 的脂质体表面交联 hdcFv25 , 形成 RNase-lp- hdcFv25对照脂质体。
为方便测试， 可以将抗体标记上放射性同位素， 如 131 1或 1251。 标记釆用氯胺 T法进行，其中 I与抗体发生共价键结合 (参阅参考文献 10: Ya-You Ji, Yan-Fang Liu, Zhi-Nan Chen。 Radioimmunodection and Autoradioigraphic Localization of Monoclonal Antibody Against Human Hepatocellular Carcinoma in Xenografts. Cancer 1992; (8): 2055-2059)。 至于抗体与药物如阿霉素的结合， 则是通过醛类与 NH2 发生共价结合 (参阅参考文献 8: 张桂红、 刘彦仿等， 抗肝癌单链抗体 免疫脂质体的制备及其体外抑瘤实验， 医学研究生学报， 2006:19(1): 3-5和参考文献 9: HAIYANG HU, DAWEI CHEN,YANFANG LIU, et al, Target Ability and Therapy Efficacy of Immunoliposomes Using a Humanized Antihepatoma Disulfide-Stabilized Fv Fragment on Tumor Cells . J Pharm Sci. 2006 Jan; 95(1): 192-9(8， 9)。 实施例 8 本发明药物对培养肝癌细胞的毒性测试
MTT 法是以代谢还原溴化二曱塞噻唑二苯四氮唑溴化物 (dimethylthiazol diphenyl tetrazolium bromide, MTT)为基 。 活细胞中存在 NAADP相关的脱氢酶， 可将黄色的 MTT还原为蓝紫 色的物质，是活细胞的标记。而在死细胞中此酶消失，不能还原 MTT, 仍为黄色。 因此， 用酶标仪在 550nm波长处测光密度， 以指示细胞死 亡率的情况， LD50是指 50%的瘤细胞被抑制的毒物剂量。
利用 MTT测定法， 以培养的肝癌细胞株 (SMMC— 7721)作为靶 肿瘤细胞， 对本发明中的双靶向脂质体 hdcFv25-RNase-lp- hdcFv25 进行了 抗肿瘤细胞活性测试， 并以 实施例 5 中构建的 RNase-lp-hdcFv25脂质体作对照。 结果见表 2中。 表 2 hdcFv25-RNase-lp-hdcFv25与 hdcFv25-RNase、 RNase-lp MTT LD50减量的比较 剂 型 MTT LD50 MW MTT LD50
以重量计 以摩尔浓度计
( μ g/mL) ( μ Μ)
PBS
hdcFv25 26000
RNase 53 14000 3.78
hdcFv25-RNase 53 40000 1.33
RNase-lip- 38 40000 0.95
hdcFv25- hdcFv25-Rnase 30 66000 0.45
Hp-hdcFv25 实施例 7 双靶向脂质体 HdcFv25-PE38-lp-HdcFv25 和对照 HdcFv25-PE38 和 PE38-lp的构建以及抗肿瘤细胞活性测试
如实施例 4 和 5 所述构建双靶向脂质体 HdcFv25-PE38- lp-HdcFv25以及对照 HdcFv25-PE38 和 PE38-lp, 并如实施例 6所述 进行测试， 结果如表 3所示。
表 3· HdcFv25-PE38-lp-HdcFv25与 HdcFv25-PE38、 PE38-lp MTT LD50减量的比较
Figure imgf000023_0001
L D 50的剂量明显减少， 说明其杀伤瘤细胞的能力明显增强。 不局限于某种特定的理论， 推测本发明的双靶向脂质体药剂是通 过如下方式发挥增强的靶向抗肿瘤活性，即表面交联的 HdcFv25将脂 质体及其包裹的 HdcFv25-毒素（例如 RNase)靶向肝癌细胞。 在运送过 程中若脂质体破坏， 释放出 HdcFv25-毒素仍然有靶向作用， 这是第二 次靶向作用， 由此增强了对肿瘤细胞的杀伤作用。
本发明所提及的所有文献都全文并入本申请中作为参考， 就如同 每一篇文献被单独引用作为参考那样。 此外， 还应当理解， 在阅读了 本发明的上述教导内容之后， 本领域普通技术人员可以对本发明作各 种改动或修改。 这些等同形式同样落入本申请所附权利要求书所限定 的保护范围之内。
参考文献
1. YF Liu, CM Hu. A group of monoclonal antibodies to hepatocellular carcinoma HAb25, HAb27-30. Hybridoma 1996; 16(2): 213-215
2. YF Liu, CM Hu, P Yang, SM Chen, L Gao, YY Ji， ZN Chen,
YF Sui, T Zheng, ZW Sun, MH Zhu, F Ren. Monoclonal antibodies against hepatocellular carcinoma HAb25 and their engineered products. In Symposium IBC's International Conference 1996 2-4 Nor. Antibody Engineering, 1997; 2: 171-197
3、 刘彦仿， 张静， 胡川闽等 抗肝癌单链抗体 (鼠及人源化)融合 突变型 TNF- α的构建、 表达及其作用研究 细胞与分子免疫学杂志， 2000;16(5) , 372
4、 袁清安， 俞炜源， 黄翠芬 肝癌特异性鼠源及人源化单链抗体 基因的构建既在大肠杆菌中的表达 生物工程学报， 200 4 ,16(1),86 - 90
5、 孙志伟， 俞炜源， 吕国华等 二硫键稳定的抗肝癌人源化单链 抗体-突变体人 TNF a融合基因在 CHO细胞中的表达 军事医学科学 院院刊， 2001年 12月第 25卷 第 4期，
6、赵君，孙志伟，刘彦仿等 二硫键稳定的抗肝癌单链抗体 -PE38 融合基因的构建、表达于能检测 细胞与分子免疫学杂志， 2003;19(6),
585 ~ 587
7、 付勇， 刘彦仿， 苏勤等 人源化抗肝癌单链抗体与牛蛙核糖核 酸融合基因的构建及表达 生物技术通讯， Vol.14 , No.5 Sep, 2003,353 ~ 356
8、 张桂红，刘彦仿等 抗肝癌单链抗体免疫脂质体的制备及其体 外抑瘤实验 医学研究生学报.2006:19(1): 3-5
9、 HAIYANG HU,DAWEI CHEN,YANFANG LIU, et alTarget Ability and Therapy Efficacy of Immunoliposomes Using a Humanized Antihepatoma Disulfide-Stabilized Fv Fragment on Tumor Cells . J Pharm Sci. 2006 Jan; 95(1): 192-9.
10 . Ya-You Ji, Yan-Fang Liu , Zhi-Nan Chen 。
Radioimmunodection and Autoradioigraphic Localization of Monoclonal Antibody Against Human Hepatocellular Carcinoma in Xenografts. Cancer 1992; (8): 2055-2059

Claims

权 利 要 求
1. 一种脂质体药剂， 其在脂质体内包含含有效应分子与第一配体 相联合的靶向药剂， 并在该脂质体表面上结合有第二配体， 该第一配 体和第二配体可特异性结合待处理或治疗的受试者靶组织或靶细胞。
2. 根据权利要求 1的脂质体药剂， 其中所述第一配体和第二配体 是相同的。
3. 根据权利要求 1的脂质体药剂， 其中所述第一配体和第二配体 是不同的。
4. 根据权利要求 1-3中任一项的脂质体药剂， 其中所述第一配体 和 /或第二配体单独或者同时是免疫球蛋白， 优选单克隆抗体， 更优选 所述抗体是针对肿瘤抗原的特异性抗体。
5. 根据权利要求 4的脂质体药剂， 其中所述第一配体和第二配体 各自独立地是单克隆抗体， 其 Fab或 F(ab，)2片段， 或者基因工程化 单链抗体 scFv, 或者其人源化抗体。
6. 根据权利要求 5的脂质体药剂， 其中所述抗体还进行了二硫键 稳定化。
7. 根据权利要求 5的脂质体药剂，其中所述第一配体和 /或第二配 体独立地选自： 具有 SEQ ID NO: 1 所示氨基酸序列的单链抗体 scFv25、 具有 SEQ ID NO: 3 所示氨基酸序列的人源化单链抗体 hscFv25和具有 SEQ ID NO: 5所示氨基酸序列的人源化二硫键稳定化 单链抗体 HdcFv25, 或者其因 1-20个、优选 1-15个、 更优选 1-10个、 尤其优选 1-8个、 特别是 1-5个、 例如 1、 2、 3或 4个氨基酸残基的 取代、 插入、 缺失和 /或添加而与 SEQ ID NO: 1、 3或 5所示序列有所 不同的变体。
8. 根据权利要求 7的脂质体药剂， 其中所述第一配体和第二配体 之一或二者均为人源化二硫键稳定化抗肝癌单链抗体 HdcFv250
9. 根据权利要求 1的脂质体药剂， 其中所述效应分子选自下述组 中： 毒素、 药物、 酶、 细胞因子、 放射性同位素或化疗药物或抑癌基 因等。
10. 根据权利要求 9的脂质体药剂，其中所述效应分子是生物毒素， 优选其选自细菌来源的白喉毒素、 假单胞菌外毒素、 植物来源的蓖麻毒 素、 相思豆毒素和其它来源的核糖核酸酶、 磷脂酶 C、 补体等， 更优选 为 RNA酶或者 PE38。
11. 根据权利要求 10的脂质体药剂，其中所述靶向药剂是 HdcFv25 与 RNA酶或者 PE38通过或不通过连接肽融合形成的融合蛋白。
12. 权利要求 1的脂质体药剂， 其中所述第二配体与脂质体直接连 接， 或通过脂质体表面上掺入的化学基团如 PEG连接。
13. 根据权利要求 1的脂质体药剂， 其中所述脂质体由磷脂 (SPC) 与胆固醇 (Choi)构成， 优选二者的重量之比为 10: 1-1 : 1 , 更优选为
8: 1-2: 1 , 尤其优选 6: 1-3: 1 , 例如 5: 1-4: 1。
14. 根据权利要求 13的脂质体药剂， 其中所述脂质体内还可选地 掺入了胆固醇衍生物。
15. 根据权利要求 14的脂质体药剂， 其中所述脂质体内掺入了胆 固醇聚乙二醇酯，其掺入比例以磷脂摩尔数计为 1-15 % ,优选 3-10 % , 更优选 5-8 % , 例如 6 %。
16. 根据权利要求 1 的脂质体药剂， 其中所述脂质体是纳米脂质 体。
17. 一种制备权利要求 1-16 中任一项所述脂质体药剂的方法， 其 中包括如下步骤：
(1) 制备空间稳定脂质体；
(2) 用脂质体包裹含有效应分子与第一配体相联合的靶向药剂； 和
(3) 在脂质体表面上交联第二配体。
18. 根据权利要求 17的方法， 其中步骤 (1)是如下进行的： 将磷脂 与胆固醇按合适的重量百分比以及任选地胆固醇聚乙二醇酯溶于氯仿 中， 然后除去氯仿、 优选通过减压的方法除去氯仿， 形成均勾一致的 脂质体干膜。
19. 一种药物组合物， 其中包含权利要求 1-16 中任一项的脂质体 药剂以及任选地药学上可接受的载体。
20. 一种治疗疾病的方法，其中包括对有此需要的患者施用权利要 求 1-16中任一项的脂质体药剂或者权利要求 19的药物组合物。
21. 根据权利要求 20的方法， 其中还包括依次或同时向患者施用 其他的疗法， 例如激素疗法、 放射性疗法等。
22. 一种诊断疾病的方法， 包括对受试者施用权利要求 1-16 中任 一项的脂质体药剂或者权利要求 19的药物组合物。
23. 根据权利要求 22的方法， 其中还包括对受试者成像的步骤。
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