WO2021068879A1 - 一种靶向功能分子修饰的抗体复合物、组合物及其用途 - Google Patents

一种靶向功能分子修饰的抗体复合物、组合物及其用途 Download PDF

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WO2021068879A1
WO2021068879A1 PCT/CN2020/119919 CN2020119919W WO2021068879A1 WO 2021068879 A1 WO2021068879 A1 WO 2021068879A1 CN 2020119919 W CN2020119919 W CN 2020119919W WO 2021068879 A1 WO2021068879 A1 WO 2021068879A1
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polypeptide
antibody
functional molecule
antibody complex
vap
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French (fr)
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陆伟跃
郭海燕
谢操
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复旦大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/66—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 a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/66—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 a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A61K47/665—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 a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells the pre-targeting system, clearing therapy or rescue therapy involving biotin-(strept) avidin systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to the technical field of pharmacy, and relates to an antibody complex modified by a targeted functional molecule.
  • the complex uses targeted functional molecules to enable the antibody to cross the biological barrier when treating brain diseases, enhance its delivery to the lesion site, and release the antibody in the microenvironment of the lesion to achieve the purpose of enhancing the efficacy of the drug.
  • biomacromolecule drugs are widely used in disease prevention, treatment and diagnosis, and have a good prospect for drug research and development.
  • Antibody drugs as a kind of biomacromolecule drugs, have the advantages of high specificity, effectiveness, and safety in tumors and neurodegenerative diseases (such as Alzheimer’s disease (AD), Parkinson’s disease (PD) ), autoimmune diseases and other diseases occupy an important position in the diagnosis and treatment.
  • AD Alzheimer’s disease
  • PD Parkinson’s disease
  • autoimmune diseases and other diseases occupy an important position in the diagnosis and treatment.
  • PD-1 antibody pembrolizumab and nivolumab suitable for melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, etc.
  • Atezolizumab PD-1 antibody suitable for bladder cancer and non-small cell lung cancer -L1 antibody
  • This type of immune checkpoint antibody binds to the PD-1 on the surface of T lymphocytes or the highly expressed PD-L1 on the surface of tumor cells to prevent the detection of T lymphocytes from tumor cells, thereby preventing tumor cells from immune escape.
  • the therapeutic effect of immune checkpoint antibodies is not optimistic.
  • the inventor of the present application intends to construct an antibody complex to mediate the delivery of the antibody across the blood-brain barrier, blood-tumor barrier, and other biological barriers into the brain by linking targeted functional molecules to the antibody. , And release antibodies at the focus of the disease to improve the therapeutic effect of antibodies on brain diseases and reduce their toxic and side effects.
  • the purpose of the present invention is to construct an antibody complex based on the current state of the art, and specifically relates to an antibody complex modified by a targeted functional molecule.
  • the antibody complex mediates the antibody by connecting the targeted functional molecule to the antibody. It crosses the blood-brain barrier, blood-tumor barrier and other biological barriers and delivers them into the brain, and releases antibodies at the lesion site, improving the therapeutic effect of antibodies on brain diseases and reducing their toxic and side effects.
  • the first aspect of the present invention provides an antibody complex modified by a targeting functional molecule, the complex is an antibody complex formed by a targeting functional molecule and an antibody through covalent and/or non-covalent linkage;
  • the targeted functional molecule is a targeted molecule or a functional molecule composed of a targeted molecule and a specific sensitive structure; and/or
  • the antibodies are anti-tumor antibodies directed against immune checkpoints or related antigens, or antibodies used in the treatment of Alzheimer's disease, or antibodies used in the treatment of Parkinson's disease, or modified forms of the aforementioned antibodies.
  • the targeted functional molecule modified antibody complex wherein the targeted functional molecule is a small molecule or polypeptide molecule that has a blood-brain barrier and/or a blood-brain tumor barrier Or protein molecules.
  • the small molecule that has a blood-brain barrier and/or a blood-brain tumor barrier is selected from para-hydroxybenzoic acid and its derivatives and/or fatty acids;
  • the fatty acid is myristic acid.
  • the targeted functional molecule is a polypeptide molecule and/or protein molecule that has a blood-brain barrier and/or a blood-brain tumor barrier;
  • the polypeptide molecule is selected from one or more of the following: VAP polypeptide, cVAP polypeptide, S VAP polypeptide, D VAP polypeptide, pHA-VAP polypeptide, pHA- S VAP polypeptide, and pHA- D VAP polypeptide, MC- VAP polypeptide, MC- S VAP polypeptide and MC- D VAP polypeptide, D8 polypeptide, D8-VAP polypeptide, D8- S VAP polypeptide and D8-D VAP polypeptide, WSW polypeptide, D WSW polypeptide, WSW-VAP polypeptide, D WSW- S VAP polypeptide and D WSW- D VAP polypeptide, TGN polypeptide, D TGN polypeptide, TGN-VAP polypeptide, D TGN-S VAP polypeptide, and D TGN- D VAP polypeptide; A7R polypeptide, cA7R polypeptide, D A7R polypeptide, D A7
  • the protein molecule is selected from transferrin and/or lactoferrin.
  • the antibody complex modified with targeted functional molecules according to the first aspect of the present invention, wherein the specific sensitive structure is a structural domain or a chemical bond that dissociates in response to the microenvironment of the disease focus;
  • the specific sensitive structure is an enzyme-sensitive polypeptide or a pH-sensitive chemical bond.
  • the enzyme-sensitive polypeptide is a polypeptide substrate of matrix metalloproteinase.
  • the antibody complex modified with targeted functional molecules according to the first aspect of the present invention wherein the antibody is an anti-tumor antibody against immune checkpoints or related antigens, or an antibody used for the treatment of Alzheimer’s disease, Or antibodies used in the treatment of Parkinson's disease, or modified forms of the above antibodies.
  • the anti-tumor antibody is an antibody that acts on PD-1, PD-L1, CTLA-4, LAG-3, and TIM-3 immune checkpoints, or acts on HER-2, VEGFR, EGFR, GD2, PDGF-R ⁇ , gp100, MAGE-1 tumor-associated antigen antibodies.
  • the antibody used for the treatment of Alzheimer's disease is an antibody that acts on A ⁇ and Tau.
  • the antibody used for the treatment of Parkinson's disease is an antibody that acts on leucine-rich repeat sequence kinase 2 (LRRK2), ⁇ -synuclein ( ⁇ -synuclein), DJ-1, RAB8A and RAB10 .
  • LRRK2 leucine-rich repeat sequence kinase 2
  • ⁇ -synuclein ⁇ -synuclein
  • DJ-1 ⁇ -synuclein
  • RAB8A RAB10
  • the antibody modification form is a Fab fragment, a single domain antibody, an Fv fragment, a single chain antibody, a bivalent small molecule antibody, a minibody or a nanobody, which is transformed into an antibody by genetic engineering technology.
  • the target functional molecule modified antibody complex wherein the covalent connection is to directly connect the targeting functional molecule and the antibody through a chemical reaction between the targeting functional molecule and the antibody , Or directly fusion expression of targeted functional molecules with antibodies through genetic engineering.
  • the targeted functional molecule modified antibody complex wherein the non-covalent connection is to indirectly connect the targeted functional molecule to the antibody through the affinity coupling of avidin and biotin. connection.
  • the second aspect of the present invention provides a pharmaceutical composition for treating brain diseases, the pharmaceutical composition comprising:
  • the antibody complex modified by the targeting functional molecule of the first aspect
  • the intracerebral disease is selected from one or more of the following: brain tumor, Alzheimer's disease, Parkinson's disease.
  • the third aspect of the present invention provides a method for treating intracerebral diseases, the method comprising: administering the targeted functional molecule-modified antibody complex of the first aspect or the antibody complex of the second aspect to a subject in need The pharmaceutical composition;
  • the intracerebral disease is selected from one or more of the following: brain tumor, Alzheimer's disease, Parkinson's disease.
  • the fourth aspect of the present invention provides the application of the targeted functional molecule modified antibody complex described in the first aspect in the preparation of drugs for the treatment of brain diseases;
  • the intracerebral disease is selected from one or more of the following: brain tumor, Alzheimer's disease, Parkinson's disease.
  • the targeting functional molecule and the antibody are connected to form an antibody complex modified by the targeting functional molecule.
  • the certain connection mode is covalent connection and/or non-covalent connection.
  • the covalent connection is through the chemical reaction between the targeted functional molecule and the antibody, or the fusion expression of the targeted functional molecule with the antibody through genetic engineering;
  • the non-covalent linkage is through the affinity coupling of avidin and biotin.
  • the linkage method indirectly connects the targeted functional molecule and the antibody.
  • the targeted functional molecules include targeting molecules and specific sensitive structures.
  • the targeting molecule is a molecule that crosses the blood-brain barrier and/or blood-brain tumor barrier.
  • the targeting molecule is a small molecule compound, such as: p-hydroxybenzoic acid (pHA) and its derivatives, and fatty acids such as myristic acid (MC).
  • pHA p-hydroxybenzoic acid
  • MC myristic acid
  • the targeting molecule is a polypeptide molecule or a protein molecule; wherein the polypeptide molecules are: VAP polypeptide, cVAP polypeptide, S VAP polypeptide, D VAP polypeptide, pHA-VAP polypeptide, pHA- S VAP polypeptide, and pHA- D VAP polypeptide , MC-VAP polypeptide, MC- S VAP polypeptide and MC- D VAP polypeptide, D8 polypeptide, D8-VAP polypeptide, D8- S VAP polypeptide and D8-D VAP polypeptide, WSW polypeptide, D WSW polypeptide, WSW-VAP polypeptide, D WSW- S VAP polypeptide and D WSW- D VAP polypeptide, TGN polypeptide, D TGN polypeptide, TGN-VAP polypeptide, D TGN-S VAP polypeptide and D TGN- D VAP polypeptide; A7R polypeptide, cA7R
  • the amino acid sequence of the polypeptide is written in the order of amino-terminal to carboxy-terminal; among them, the VAP polypeptide is the ligand of the GRP78 protein; the D8 polypeptide is the penetrating peptide; the WSW polypeptide is the quorum sensing polypeptide; the TGN polypeptide is the brain obtained by phage display.
  • A7R polypeptide is a ligand for VEGFR2 and NRP-1 receptors
  • RGD polypeptide, RW polypeptide, and mn polypeptide are ligands for integrin
  • T7 polypeptide is a ligand for transferrin receptor
  • RAP12 polypeptide is a low density Lipoprotein receptor related protein-1 is a polypeptide with high binding activity.
  • the specific sensitive structure can dissociate and release antibodies in response to the microenvironment of the disease focus.
  • the specific sensitive structure is an enzyme-sensitive polypeptide or a pH-sensitive chemical bond, and more preferably, the enzyme-sensitive polypeptide is a polypeptide substrate of a matrix metalloproteinase.
  • the antibody is an antibody that acts on immune checkpoints such as PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, or acts on HER-2, VEGFR, EGFR, GD2, PDGF -Anti-tumor antibodies against tumor-related antigens such as Ra, gp100, and MAGE-1; or antibodies used in the treatment of Alzheimer's disease such as A ⁇ and Tau; or acting on leucine-rich repeat sequence kinase 2 (LRRK2), ⁇ -synuclein ( ⁇ -synuclein,) DJ-1, RAB8A, RAB10 and other antibodies used in the treatment of Parkinson’s disease; or a combination of antibody fragments modified by genetic engineering methods of the above antibodies, including Fab fragments, single domain antibodies, Fv fragments, single chain antibodies, bivalent small molecule antibodies, micro antibodies, nano antibodies, etc.
  • immune checkpoints such as PD-1, PD-L1, CTLA-4, LAG-3, TIM-3
  • HER-2
  • the technical scheme adopted by the present invention includes a random site modification method and a site-specific modification method: wherein,
  • Random site modification The free amino group in the antibody is activated to a maleimide group, and then it is covalently linked to a targeted functional molecule with a sulfhydryl group to obtain an antibody complex modified by the targeted functional molecule;
  • Site-specific modification Link the targeted functional molecule to the antibody through affinity coupling, specifically using bioengineering technology to express streptavidin (SA) on the Fc fragment of the antibody, and then combine the SA-antibody with the biological
  • SA streptavidin
  • the present invention provides an antibody complex modified by targeting functional molecules and a preparation method thereof, and has been evaluated for biological activity, in vivo and in vitro targeting, and in vitro and in vivo pharmacodynamics.
  • the results show that the targeted functional molecule modified by the present invention
  • Antibody complexes whose targeted functional molecule modification has little effect on the biological activity of antibodies, can enable antibodies to overcome biological barriers such as the blood-brain barrier, increase the delivery of antibody drugs to brain lesions, and under specific microenvironmental conditions of the lesions The antibody is released from the bottom, which significantly enhances the therapeutic effect of the antibody.
  • the experimental results of the present invention show that the antibody complex modified by the targeted functional molecule has the advantage of crossing the biological barrier, can improve the therapeutic effect of the antibody on brain diseases, has important significance in expanding the scope of clinical application of the antibody, and has a good application prospect .
  • Figure 1 shows the HPLC and mass spectrogram of pHA
  • Chromatographic method chromatographic column (YMC, C18): 150 ⁇ 4.6mm; mobile phase A: water (containing 0.1% trifluoroacetic acid), mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid); elution program: 0- 45min, 5%B-65%B; flow rate: 0.7mL/min; column temperature: 40°C; detection: UV 214nm, retention time: 8.89min, ESI-MS: 415.2Da, which is consistent with the theoretical molecular weight.
  • Figure 2 shows the HPLC and mass spectrogram of mRAP, in which,
  • Chromatographic method chromatographic column (YMC, C18): 150 ⁇ 4.6mm; mobile phase A: water (containing 0.1% trifluoroacetic acid), mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid); elution program: 0- 45min, 5%B-65%B; flow rate: 0.7mL/min; column temperature: 40°C; detection: UV 214nm, retention time: 14.63min, ESI-MS: 2321.8Da, which is consistent with the theoretical molecular weight.
  • Figure 3 shows the HPLC and mass spectrogram of mCDX, in which,
  • Chromatographic method chromatographic column (YMC, C18): 150 ⁇ 4.6mm; mobile phase A: water (containing 0.1% trifluoroacetic acid), mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid); elution program: 0- 45min, 5%B-65%B; flow rate: 0.7mL/min; column temperature: 40°C; detection: UV 214nm, retention time: 17.49min, ESI-MS: 2775.0Da, which is consistent with the theoretical molecular weight.
  • Figure 4 shows the mass spectrometric characterization results of the targeted functional molecule-PDL1 antibody complex
  • the molecular weight of the targeted functional molecule-PDL1 antibody complex was increased compared with the molecular weight of the unmodified antibody, and the average molecular weight increased by about 5-7 targeted functional molecules, confirming the successful preparation of the complex; and mRAP- ⁇ PDL1 and mCDX- ⁇ PDL1 can be After being digested by MMP, the molecular weight is reduced and reduced to the molecular weight of the unmodified antibody, indicating that the enzymatic hydrolysis can remove its targeted functional molecules.
  • Figure 5 shows the gel electrophoresis characterization results of the targeted functional molecule-PDL1 antibody complex
  • Figure 6 shows the functional binding activity characterization of the targeted functional molecule-PDL1 antibody complex
  • Figure A shows the in vitro application of the Elisa method to verify the binding activity of the targeted functional molecule-PDL1 antibody complex and the PDL1 protein.
  • Figure B shows the application of SPR technology to verify the binding activity of the targeted functional molecule-PDL1 antibody complex and the PDL1 protein. The results show , The targeting molecule has little effect on the functional binding activity of the targeting functional molecule-PDL1 antibody complex.
  • Figure 7 shows the binding effect of the targeted functional molecule-PDL1 antibody complex with PDL1 on the surface of tumor cells, where,
  • Figure A is a fluorescent photo showing that the targeted functional molecule-PDL1 antibody complex can block the PDL1 protein on the surface of tumor cells from binding to other PDL1 antibodies.
  • Figure B is a further quantitative verification that the targeted functional molecule-PDL1 antibody complex can block the PDL1 protein.
  • the flow cytometry results showed that the targeted functional molecule-PDL1 antibody complex and the unmodified antibody can also block the binding of PDL1 protein on the surface of tumor cells.
  • Figure 8 shows that the targeted functional molecule-PDL1 antibody complex inhibits the killing effect of PD pathway activation on CD3 + T cells, where,
  • Figure A is a flow chart showing the effect of the targeted functional molecule-PDL1 antibody complex on inhibiting CD3 + T cell apoptosis induced by PD pathway activation
  • the results show that pHA- ⁇ PDL1 can It has the same PD pathway inhibitory ability as ⁇ PDL1, and mRAP- ⁇ PDL1 and mCDX- ⁇ PDL1 can also inhibit T cell apoptosis.
  • Figure 9 shows the evaluation of the in vitro blood-brain barrier ability of the targeted functional molecule-PDL1 antibody complex
  • the targeting molecule can mediate the antibody to cross the blood-brain barrier model barrier in vitro, which is significant compared with the unmodified antibody group difference.
  • Figure 10 shows the in vivo targeting evaluation of the targeted functional molecule-PDL1 antibody complex in normal mice.
  • Figure B shows the distribution of the targeted functional molecule-PDL1 antibody complex in the main organs of normal mice when the tail vein is injected for 8 hours
  • Figure C is the target when the tail vein is injected for 24 hours.
  • Figure D shows that the targeted functional molecule-PDL1 antibody complex is in the main organs of normal mice when injected into the tail vein for 24 hours. The results show that the distribution of the targeted functional molecule-PDL1 antibody complex in the normal brain is more than that of the unmodified antibody, and the PDL1 antibody can cross the complete blood-brain barrier under the mediation of the targeted molecule.
  • Figure 11 shows the targeting evaluation of the targeting functional molecule-PDL1 antibody complex in mice bearing in situ brain tumors, where,
  • Figure B shows the targeted functional molecule injected into the tail vein -PDL1 antibody complex 8h, its distribution in the main organs of mice bearing in situ brain tumor C57BL/6 model mice
  • Figure D shows the main viscera of C57BL/6 model mice bearing in situ brain tumors when the targeted functional molecule-PDL1 antibody complex was injected into the tail vein for 24 hours. The results show that the distribution of targeted functional molecule-PDL1 antibody complexes in brain tumors is greater than that of unmodified antibodies, and that PDL1 antibodies can accumulate more in the brain tumor sites under the mediation of targeting molecules.
  • Figure 12 shows the results of the pharmacokinetic study of the targeted functional molecule-PDL1 antibody complex in normal mice.
  • the results show that pHA- ⁇ PDL1, mRAP- ⁇ PDL1 and unmodified There is no significant difference in the pharmacokinetic behavior of ⁇ PDL1, and the blood clearance of mCDX- ⁇ PDL1 is faster.
  • Figure 13 shows the pharmacodynamic results of the targeted functional molecule-PDL1 antibody complex on the C57BL/6 model of tumor in situ bearing GL261, where,
  • Figures B and C are The ki67 and tunnel immunohistochemical sample photos of tumor-bearing C57BL/6 mice and their quantitative results indicate that there are fewer malignant cells and more apoptosis in the targeted functional molecule-PDL1 antibody complex group. The results are consistent.
  • Figure 14 shows the immunomodulatory effect of the targeted functional molecule-PDL1 antibody complex on glioma in situ, where,
  • Figure GI is the immune effect of the cervical lymph nodes of tumor-bearing mice
  • Figure JL is the immune effect of the spleen of tumor-bearing mice Immune effect.
  • the immunosuppressive cells of the targeted functional molecule-PDL1 antibody complex group are significantly reduced, and the effector cells are significantly increased, which can improve the immunosuppressive environment of the tumor and the body.
  • Figure 15 shows the toxic and side effects of the targeted functional molecule-PDL1 antibody complex, where,
  • Figure B and Figure C show the blood levels of inflammatory factors after administration.
  • the results show that the liver function indicators of the targeted functional molecule-PDL1 antibody complex are similar to those of the PDL1 antibody complex.
  • the PBS group is close, and the unmodified antibody group is more toxic to the liver.
  • the level of inflammatory factors in the blood further confirms that the targeted functional molecule-PDL1 antibody complex delivery system has lower toxic and side effects.
  • Synthesis and characterization of pHA targeting molecules Use solid-phase synthesis technology to prepare peptides, first weigh MBHA resin (substitution degree is 0.54mmol/g), add N,N-dimethylformamide for swelling, and add 20% piperidine -DMF solution to remove the Fmoc protective group on the resin, after washing, add 8.8 times the amount of Fmoc-amino acid (dissolved in the DMF solution of HBTU/HOBT/DIEA), and place it in an air shaker (37°C, 180rpm) ) Shake for 45 minutes.
  • mRAP was synthesized according to the above method, and its amino acid sequence was CGPLGIAGQEAKIEKHNHYQK. The HPLC and ESI-MS characterization results are shown in Figure 2.
  • mCDX was synthesized according to the above method, and its amino acid sequence was greirtgraerwsekfGPLGIAGQC (lower case D configuration amino acid, upper case L configuration amino acid), and the HPLC and ESI-MS characterization results are shown in Figure 3.
  • ⁇ PDL1 In the ⁇ PDL1 solution, add Sulfo-SMCC (1 mg/mL), react at room temperature for 30 minutes, and remove the excess Sulfo-SMCC from the desalting column. Add 108 ⁇ L of pHA PBS solution (2mg/mL), mix well, put it at 4°C for 12h, dialyze in pure water twice (30min each time) in a 14K Da dialysis bag, collect the liquid in the dialysis bag to obtain pHA modification PDL1 antibody complex (pHA- ⁇ PDL1). Prepare mRAP- ⁇ PDL1 and mCDX- ⁇ PDL1 in the same way.
  • the above products were characterized by MALDI-TOF and gel electrophoresis.
  • the results of MALDI-TOF are shown in Figure 4.
  • the gel electrophoresis method is as follows: prepare a 12% separation gel and take 0.35 mg/mL pHA- ⁇ PDL1 and mRAP respectively. - ⁇ PDL1, mCDX- ⁇ PDL1, mRAP- ⁇ PDL1+MMP and
  • SPR surface plasmon resonance
  • PD pathway blocking is the use of fluorescein-labeled PDL1 antibody (PE- ⁇ PDL1) to compete for the binding of the PDL1 antibody complex modified by the targeted functional molecule to the PDL1 protein, and to investigate the binding of the targeted functional molecule-PDL1 antibody complex to the PDL1 protein Blocking ability.
  • Digest and disperse GL261 cells inoculate a laser confocal cell culture dish at a density of 5 ⁇ 10 3 cells per well, add cell culture medium containing IFN- ⁇ to each well, and incubate in a cell incubator for 48 hours to increase the surface PDL1 protein expression.
  • GL261 cells were stimulated with IFN- ⁇ for 48h to make the surface PDL1 protein highly expressed.
  • GL261 cells with high PDL1 protein expression were digested with trypsin, and the culture medium neutralized the pancreas. After enzyme, centrifugation and redispersion in PBS buffer.
  • IgG, ⁇ PDL1, pHA- ⁇ PDL1, mRAP- ⁇ PDL1, mCDX- ⁇ PDL1 were added to the tumor cells with high expression of PDL1, and incubated for 5 minutes, then PE- ⁇ PDL1 was added to incubate for 15 minutes, washed with PBS and resuspended to 200 ⁇ L In the PBS buffer, the positive cells were counted with a flow cytometer, and the biological activity of the modified ⁇ PDL1 was investigated. The result is shown in FIG. 7.
  • mice 10 6 cells GL261, 5 days after tumor inoculation, the mice spleens taken, ground into a single cell, beads sorted CD3 + T cells, cultured coated with anti-CD3 antibody On the U-shaped plate, add anti-CD28 antibody for co-stimulation and culture for 48 hours.
  • the GL261 cells were stimulated with IFN- ⁇ for 48h to make the surface PDL1 protein highly expressed.
  • Tumor cells with high expression of PDL1 protein were inoculated into 96-well plates (5 ⁇ 10 3 cells/well), with or without equimolar amounts of IgG, ⁇ PDL1, pHA- ⁇ PDL1, mRAP- ⁇ PDL1, mCDX- ⁇ PDL1.
  • the stimulated CD3 + T cells (105 cells/well) were cultured for 24 hours. After staining with the apoptosis kit, flow cytometer detection, the results are shown in Figure 8.
  • TEER transmembrane resistance
  • 60 ⁇ L ⁇ PDL1 antibody solution (0.5mg / mL), was added to the radiation dose of 125 I 0.78mCi, 20 ⁇ L PBS, the reaction in Iodogen tube (80 g) in 5min (about 34-37 °C).
  • 125 I-labeled ⁇ PDL1 was purified by Sephadex G-25 cartridge. The measured radiation dose was 660 ⁇ Ci, the labeling rate was about 81%, and the specific activity was 21.06 ⁇ Ci/ ⁇ g.
  • 125 I-labeled ⁇ PDL1 was used to prepare 125 I-labeled pHA- ⁇ PDL1, 125 I-labeled mRAP- ⁇ PDL1, and 125 I-labeled mCDX- ⁇ PDL1.
  • mice Twelve normal mice were selected, three in each group, the groups were ⁇ PDL1, pHA- ⁇ PDL1, mRAP- ⁇ PDL1, mCDX- ⁇ PDL1, and the radiation dose administered to each mouse was 15 ⁇ Ci.
  • the brain was taken at 8h and 24h, weighed, and the radiation dose was measured to investigate the distribution of the complex in the brain and main tissues of normal mice. The results are shown in Figure 10.
  • a male C57BL/6 weighing about 20g was selected and positioned on the stereotaxic device to the striatum area 1.8 mm to the right of the bregma, 0.6 mm to the front, and 3 mm in depth, and injected into 1 ⁇ 10 6 in logarithmic growth.
  • Stage GL261 cells (dispersed in 5 ⁇ L PBS buffer) were raised in SPF environment after tumor inoculation, and the survival status of model mice was observed regularly.
  • a total of 12 mice inoculated with glioma in situ were divided into 4 groups, the same as the above groups.
  • mice 16 male C57BL/6 mice were randomly divided into 4 groups with 4 mice in each group.
  • the tail vein was injected with ⁇ PDL1, pHA- ⁇ PDL1, mRAP- ⁇ PDL1, mCDX- ⁇ PDL1, and the dose was equivalent to containing 5mg/kg of ⁇ PDL1 in 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h blood was taken from the orbit, the PDL1 antibody content was determined by ELISA method, the drug-time curve was drawn and the pharmacokinetic parameters were calculated. The results are shown in Figure 12.
  • Example 7 Pharmacodynamic evaluation of the mouse model of tumor in situ by the target functional molecule-PDL1 antibody complex
  • Example 8 The immunomodulatory effect of targeting functional molecule-PDL1 antibody complex on in situ glioma
  • mice Twelve male C57BL/6 mice were inoculated with orthotopic glioma cells GL261 (1 ⁇ 10 6 /mouse), and they were randomly divided into 3 groups with 4 mice in each group. The tumor was inoculated on day 0. On the 3rd, 5th, 7th and 9th days, PBS, ⁇ PDL1, and pHA- ⁇ PDL1 were injected into the tail vein at a dose equivalent to 5mg/kg of ⁇ PDL1. On the 10th day, brain tumors, cervical lymph nodes, and spleen were taken and processed into a single cell suspension.
  • mice Twelve male C57BL/6 mice were inoculated with orthotopic glioma cells GL261 (1 ⁇ 10 6 /mouse), and they were randomly divided into 3 groups with 4 mice in each group. The tumor was inoculated on day 0. On the 3rd, 5th, 7th and 9th days, PBS, ⁇ PDL1 and pHA- ⁇ PDL1 were injected into the tail vein at a dose of 5 mg/kg equivalent to ⁇ PDL1. Detect and record the weight of each mouse. On the 10th day, the blood was collected and centrifuged at 4000 rpm/min for 10 minutes. The plasma was collected to determine the TNF- ⁇ and IFN- ⁇ factors in the blood by ELISA; and the ALT and AST liver function indexes were tested to evaluate its safety. The result is shown in Figure 15.

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Abstract

本发明属于药学技术领域，涉及一种靶向功能分子修饰的抗体复合物，本发明将靶向功能分子与抗体通过一定的方式连接形成复合物，用于改善抗体在治疗脑部疾病时，如脑肿瘤(血-脑屏障和血-脑肿瘤屏障)、阿尔兹海默症和帕金森病(血-脑屏障)由于无法跨越相关生物屏障而影响其疗效的问题，试验结果显示：本发明的靶向功能分子修饰的抗体复合物不影响抗体的活性，能克服生物屏障的限制，促使抗体跨生物屏障进入病灶部位，并在病灶部位微环境响应下释放抗体，显著改善抗体对脑部疾病的治疗效果，具有良好的临床应用前景。

Description

一种靶向功能分子修饰的抗体复合物、组合物及其用途 技术领域
本发明属药学技术领域，涉及一种靶向功能分子修饰的抗体复合物。该复合物利用靶向功能分子使抗体在治疗脑部疾病时可跨越生物屏障，增强其向病灶部位的递送，并可在病灶的微环境中释放抗体，达到增强药效的目的。
背景技术
随着生物医学技术的迅猛发展，生物大分子药物被广泛应用于疾病的预防、治疗和诊断，具有良好的药物研究开发前景。抗体类药物作为一种生物大分子药物，以其高特异性、有效性、和安全性的优势，在肿瘤、神经退行性疾病(如阿尔兹海默症(AD)、帕金森病(PD))、自身免疫病等多种疾病的诊断和治疗中占据重要地位。例如在抗肿瘤领域，FDA批准的适用于黑色素瘤、非小细胞肺癌、肾细胞癌、膀胱癌等的pembrolizumab和nivolumab(PD-1抗体)，适用于膀胱癌与非小细胞肺癌的Atezolizumab(PD-L1抗体)都具有非常好的抗肿瘤效果。这类免疫检查点抗体通过结合T淋巴细胞表面的PD-1或肿瘤细胞表面高表达的PD-L1，阻止T淋巴细胞与肿瘤细胞的检测点识别，从而避免肿瘤细胞发生免疫逃逸。但是，对于同样高表达PD-L1的脑肿瘤，免疫检查点抗体的治疗效果并不乐观，临床试验结果显示，免疫检查点抗体对脑肿瘤的治疗响应率低，Atezolizumab(PD-L1抗体)的治疗并未显著提高生存期。这是由于脑肿瘤存在生物屏障(包括血-脑屏障(BBB)、血-脑肿瘤屏障(BBTB))，而抗体作为生物大分子药物，其分子尺度大，难以跨越生物屏障进入脑肿瘤部位，因此免疫检查点抗体在用于脑肿瘤治疗时达不到预期的效果。除了脑肿瘤外，其它脑部疾病如神经退行性疾病同样存在生物屏障的问题，这导致相关疾病的抗体药物由于没法跨屏障而无效。
基于现有技术现状，本申请的发明人拟构建一种抗体复合物，通过靶向功能分子与抗体连接的方式，介导抗体跨越血-脑屏障、血-肿瘤屏障等生物屏障递送入脑内，并在病灶部位释放抗体，提高抗体对脑内疾病的治疗效果并降低其毒副作用。
发明内容
本发明的目的是基于现有技术现状，构建一种抗体复合物，具体涉及一种靶向功能分子修饰的抗体复合物，该抗体复合物通过靶向功能分子与抗体连接的方式，介导抗体跨越血-脑屏障、血-肿瘤屏障等生物屏障递送入脑内，并在病灶部位释放抗体，提高抗体对脑内疾病的治疗效果并降低其毒副作用。
本发明的第一方面提供了一种靶向功能分子修饰的抗体复合物，所述复合物为由靶向 功能分子与抗体通过共价连接和/或非共价连接方式形成抗体复合物；
优选地，所述的靶向功能分子为靶向分子或靶向分子和特定敏感结构组成的功能分子；和/或
所述的抗体是针对免疫检查点或相关抗原的抗肿瘤抗体，或用于阿尔兹海默症治疗的抗体，或用于帕金森病治疗的抗体，或上述抗体的改造形式。
根据本发明第一方面所述的靶向功能分子修饰的抗体复合物，其中，所述的靶向功能分子为具有跨血-脑屏障和/或跨血-脑肿瘤屏障的小分子、多肽分子或蛋白分子。
优选地，所述具有跨血-脑屏障和/或跨血-脑肿瘤屏障的小分子选自对羟基苯甲酸及其衍生物和/或脂肪酸；
更优选地，所述脂肪酸为肉豆蔻酸。
优选地，所述的靶向功能分子为具有跨血-脑屏障和/或跨血-脑肿瘤屏障的多肽分子和/或蛋白分子；
更优选地，所述多肽分子选自以下一种或多种：VAP多肽、cVAP多肽、 SVAP多肽、 DVAP多肽，pHA-VAP多肽、pHA- SVAP多肽及pHA- DVAP多肽，MC-VAP多肽、MC- SVAP多肽及MC- DVAP多肽，D8多肽、D8-VAP多肽、D8- SVAP多肽及D8- DVAP多肽，WSW多肽、 DWSW多肽、WSW-VAP多肽、 DWSW- SVAP多肽及 DWSW- DVAP多肽，TGN多肽、 DTGN多肽、TGN-VAP多肽、 DTGN- SVAP多肽及 DTGN- DVAP多肽；A7R多肽、cA7R多肽、 DA7R多肽，pHA-A7R多肽及pHA- DA7R多肽，MC-A7R多肽及MC- DA7R多肽，D8-A7R多肽及D8- DA7R，WSW-A7R多肽及 DWSW- DA7R多肽，TGN-A7R多肽及 DTGN- DA7R多肽；RGD多肽、Stapled-RGD多肽，pHA-RGD多肽，MC-RGD多肽，D8-RGD多肽，WSW-RGD多肽，TGN-RGD多肽；RW多肽、mn多肽，pHA-RW多肽及pHA-mn多肽，MC-RW多肽及MC-mn多肽，D8-RW多肽及D8-mn多肽，WSW-RW多肽及 DWSW-mn多肽，TGN-RW多肽及 DTGN-mn多肽；T7多肽及 DT7多肽；RAP12多肽及 DRAP12多肽；和/或
所述蛋白分子选自转铁蛋白和/或乳铁蛋白。
根据本发明第一方面所述的靶向功能分子修饰的抗体复合物，其中，所述的特定敏感结构为响应疾病病灶微环境发生解离的结构域或化学键；
优选地，所述的特定敏感结构为酶敏感多肽或pH敏感化学键。
更优选地，所述的酶敏感多肽为基质金属蛋白酶的多肽底物。
根据本发明第一方面所述的靶向功能分子修饰的抗体复合物，其中，所述的抗体是针对免疫检查点或相关抗原的抗肿瘤抗体，或用于阿尔兹海默症治疗的抗体，或用于帕金森病治疗的抗体，或上述抗体的改造形式。
优选地，所述的抗肿瘤抗体为作用于PD-1、PD-L1、CTLA-4、LAG-3、TIM-3免疫检查点的抗体，或作用于HER-2、VEGFR、EGFR、GD2、PDGF-Rα、gp100、MAGE-1肿瘤相关抗原的抗体。
优选地，所述的用于阿尔兹海默症治疗的抗体为作用于Aβ、Tau的抗体。
优选地，所述的用于帕金森病治疗的抗体为作用于富亮氨酸重复序列激酶2 (LRRK2)、α-突触核蛋白(α-synuclein)、DJ-1、RAB8A和RAB10的抗体。
优选地，所述的抗体改造形式为利用基因工程技术将抗体改造为的Fab片段、单域抗体、Fv片段、单链抗体、双价小分子抗体、微抗体或纳米抗体。
根据本发明第一方面所述的靶向功能分子修饰的抗体复合物，其中，所述的共价连接是通过靶向功能分子与抗体之间的化学反应方式将靶向功能分子与抗体直接连接，或通过基因工程直接将靶向功能分子与抗体融合表达。
根据本发明第一方面所述的靶向功能分子修饰的抗体复合物，其中，所述的非共价连接是通过亲合素与生物素的亲和偶联方式将靶向功能分子与抗体间接连接。
本发明的第二方面提供了一种用于治疗脑内疾病的药物组合物，所述药物组合物包括：
第一方面所述的靶向功能分子修饰的抗体复合物；和
药学上可接受的辅料；
优选地，所述脑内疾病选自以下一种或多种：脑肿瘤、阿尔兹海默症、帕金森病。
本发明的第三方面提供了一种脑内疾病的治疗方法，所述方法包括：对有需要的受试者给予第一方面所述的靶向功能分子修饰的抗体复合物或第二方面所述的药物组合物；
优选地，所述脑内疾病选自以下一种或多种：脑肿瘤、阿尔兹海默症、帕金森病。
本发明的第四方面提供了第一方面所述的靶向功能分子修饰的抗体复合物在制备用于治疗脑内疾病的药物中的应用；
优选地，所述脑内疾病选自以下一种或多种：脑肿瘤、阿尔兹海默症、帕金森病。
本发明将靶向功能分子与抗体通过一定的连接方式形成靶向功能分子修饰的抗体复合物。
本发明中，所述的一定的连接方式为共价连接和/或非共价连接。
其中，共价连接是通过靶向功能分子与抗体之间的化学反应连接，或通过基因工程将靶向功能分子与抗体融合表达；非共价连接是通过亲和素与生物素的亲和偶联方式将靶向功能分子与抗体间接连接。
本发明中，所述的靶向功能分子包括靶向分子和特定敏感结构。
本发明中，所述的靶向分子为具有跨血-脑屏障和/或血-脑肿瘤屏障的分子。
优选地，所述的靶向分子为小分子化合物，如：对羟基苯甲酸(pHA)及其衍生物，脂肪酸如肉豆蔻酸(MC)。
优选地，所述靶向分子为多肽分子或蛋白分子；其中多肽分子为：VAP多肽、cVAP多肽、 SVAP多肽、 DVAP多肽，pHA-VAP多肽、pHA- SVAP多肽及pHA- DVAP多肽，MC-VAP多肽、MC- SVAP多肽及MC- DVAP多肽，D8多肽、D8-VAP多肽、D8- SVAP多肽及D8- DVAP多肽，WSW多肽、 DWSW多肽、WSW-VAP多肽、 DWSW- SVAP多肽及 DWSW- DVAP多肽，TGN多肽、 DTGN多肽、TGN-VAP多肽、 DTGN- SVAP多肽及 DTGN- DVAP多肽；A7R多肽、cA7R多肽、 DA7R多肽，pHA-A7R多肽及pHA- DA7R多肽，MC-A7R多肽及MC- DA7R多肽，D8-A7R多肽及D8- DA7R多肽，WSW-A7R多肽及 DWSW- DA7R多肽，TGN-A7R多肽及 DTGN- DA7R多肽；RGD多肽、Stapled-RGD多肽，pHA-RGD多肽，MC-RGD多肽，D8-RGD多肽，WSW-RGD多肽，TGN-RGD多肽；RW多肽、mn多肽，pHA-RW多肽及pHA-mn多肽，MC-RW多肽及MC-mn多肽，D8-RW多肽及D8-mn多肽，WSW-RW多肽及 DWSW-mn多肽，TGN-RW多肽及 DTGN-mn多肽；T7多肽及 DT7多肽；RAP12多肽及 DRAP12多肽；其中蛋白分子为转铁蛋白和乳铁蛋白等，本发明上述多肽分子或蛋白分子中可以一种或任意种组合使用。
本发明中，多肽的氨基酸序列书写顺序为氨基端向羧基端；其中，VAP多肽为GRP78蛋白的配体；D8多肽为穿膜肽；WSW多肽为群体感应多肽；TGN多肽为噬菌体展示获得的脑靶向多肽；A7R多肽为VEGFR2和NRP-1受体的配体；RGD多肽、RW多肽、mn多肽为整合素的配体；T7多肽为转铁蛋白受体的配体；RAP12多肽为低密度脂蛋白受体相关蛋白‐1的高结合活性多肽。
表1
Figure PCTCN2020119919-appb-000001
Figure PCTCN2020119919-appb-000002
Figure PCTCN2020119919-appb-000003
Figure PCTCN2020119919-appb-000004
本发明中，所述的特定敏感结构可响应疾病病灶部位微环境，解离释放抗体。优选地，所述的特定敏感结构为酶敏感多肽或pH敏感化学键，更优选地，所述的酶敏感多肽为基质金属蛋白酶的多肽底物。
本发明中，所述的抗体为作用于PD-1、PD-L1、CTLA-4、LAG-3、TIM-3等免疫检查点的抗体或作用于HER-2、VEGFR、EGFR、GD2、PDGF-Rα、gp100、MAGE-1等肿瘤相关抗原的抗肿瘤抗体；或作用于Aβ、Tau等用于阿尔兹海默症治疗的抗体；或作用于富亮氨酸重复序列激酶2(LRRK2)、α-突触核蛋白(α-synuclein、)DJ-1、RAB8A和RAB10等用于帕金森病治疗的抗体；或以上抗体通过基因工程手段改造的抗体片段组合，包括Fab片段、单域抗体、Fv片段、单链抗体、双价小分子抗体、微抗体、纳米抗体等。
为实现上述目的，本发明采用的技术方案中包括随机位点修饰方法和定点位点修饰方法：其中，
随机位点修饰：将抗体中游离氨基活化为马来酰亚胺基团，再将其与带有巯基的靶向功能分子共价连接，得到靶向功能分子修饰的抗体复合物；
定点位点修饰：通过亲和偶联作用将靶向功能分子与抗体连接，具体为利用生物工程技术将链霉亲和素(SA)表达于抗体的Fc片段上，然后将SA-抗体与生物素化靶向功能分子混合，得到靶向功能分子修饰的抗体复合物。
本发明提供了一种靶向功能分子修饰的抗体复合物及其制备方法，并进行了生物活性、体内外靶向性及体内外药效评价，结果表明，本发明的靶向功能分子修饰的抗体复合物，其靶向 功能分子的修饰对抗体的生物活性影响较小，能使抗体克服血-脑屏障等生物屏障，能增加对脑内病灶的抗体药物递送，并在病灶特定微环境条件下释放出抗体，显著增强了抗体的治疗效果。
本发明实验结果表明，所述的靶向功能分子修饰的抗体复合物具有跨越生物屏障的优势，可提高抗体对脑部疾病的治疗效果，对拓展抗体的临床应用范围有重要意义，应用前景好。
附图的简要说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1示出了pHA的HPLC与质谱图，其中
色谱方法：色谱柱(YMC，C18)：150×4.6mm；流动相A：水(含0.1％三氟乙酸)，流动相B：乙腈(含0.1％三氟乙酸)；洗脱程序：0-45min，5％B-65％B；流速：0.7mL/min；柱温：40℃；检测：UV 214nm，保留时间：8.89min，ESI-MS：415.2Da，与理论分子量相符合。
图2示出了mRAP的HPLC与质谱图，其中，
色谱方法：色谱柱(YMC，C18)：150×4.6mm；流动相A：水(含0.1％三氟乙酸)，流动相B：乙腈(含0.1％三氟乙酸)；洗脱程序：0-45min，5％B-65％B；流速：0.7mL/min；柱温：40℃；检测：UV 214nm，保留时间：14.63min，ESI-MS：2321.8Da，与理论分子量相符合。
图3示出了mCDX的HPLC与质谱图，其中，
色谱方法：色谱柱(YMC，C18)：150×4.6mm；流动相A：水(含0.1％三氟乙酸)，流动相B：乙腈(含0.1％三氟乙酸)；洗脱程序：0-45min，5％B-65％B；流速：0.7mL/min；柱温：40℃；检测：UV 214nm，保留时间：17.49min，ESI-MS：2775.0Da，与理论分子量相符合。
图4示出了靶向功能分子-PDL1抗体复合物的质谱表征结果，
其中显示，靶向功能分子-PDL1抗体复合物的分子量较未修饰抗体分子量增加，平均分子量增加约5-7个靶向功能分子，证实了复合物成功制备；且mRAP-αPDL1与mCDX-αPDL1可被MMP酶解后，分子量降低，还原为未修饰抗体分子量，表明酶解作用能脱去其靶向功能分子。
图5示出了靶向功能分子-PDL1抗体复合物的凝胶电泳表征结果，
其中显示，靶向功能分子-PDL1抗体复合物的轻链与重链的分子量均较未修饰抗体分子量增加，表明靶向功能分子在抗体的轻链与重链上均有修饰，进一步证实复合物成功制备；而且mRAP-αPDL1与mCDX-αPDL1可被MMP酶解后，分子量降低，表明酶解作用能脱去其靶向功能分子。
图6示出了靶向功能分子-PDL1抗体复合物的功能性结合活性表征，其中，
图A为在体外应用Elisa法验证靶向功能分子-PDL1抗体复合物与PDL1蛋白的结合活性，图B为应用SPR技术验证靶向功能分子-PDL1抗体复合物与PDL1蛋白的结合活性，结果显示，靶向分子对于靶向功能分子-PDL1抗体复合物的功能性结合活性的影响较小。
图7示出了靶向功能分子-PDL1抗体复合物与肿瘤细胞表面PDL1结合作用，其中，
图A为靶向功能分子-PDL1抗体复合物能阻断肿瘤细胞表面的PDL1蛋白与其他PDL1抗体结合的荧光照片，图B为进一步定量验证靶向功能分子-PDL1抗体复合物对PDL1蛋白阻断作用的流式定量结果，结果显示，靶向功能分子-PDL1抗体复合物与未修饰抗体同样能阻断肿瘤细胞表面的PDL1蛋白结合。
图8示出了靶向功能分子-PDL1抗体复合物抑制PD通路激活对CD3 +T细胞的杀伤作用，其中，
图A为靶向功能分子-PDL1抗体复合物对抑制PD通路激活诱发的CD3 +T细胞凋亡作用的流式图，图B为其定量结果(n＝3)，结果显示，pHA-αPDL1能与αPDL1具有相当的PD通路抑制能力，mRAP-αPDL1与mCDX-αPDL1也能抑制T细胞凋亡。
图9示出了靶向功能分子-PDL1抗体复合物的体外跨血-脑屏障能力评价，
显示了靶向功能分子-PDL1抗体复合物体外跨血-脑屏障评价(n＝3)，靶向分子能介导抗体跨过体外血-脑屏障模型屏障，与未修饰抗体组比较具有显著性差异。
图10示出了靶向功能分子-PDL1抗体复合物的正常小鼠体内靶向性评价，其中，图A为尾静脉注射8h时，靶向功能分子-PDL1抗体复合物在正常C57BL/6小鼠的脑内分布(n＝3)，图B为尾静脉注射8h时，靶向功能分子-PDL1抗体复合物在正常小鼠的主要脏器的分布，图C为尾静脉注射24h时，靶向功能分子-PDL1抗体复合物在正常C57BL/6小鼠的脑内分布(n＝3)，图D为尾静脉注射24h时，靶向功能分子-PDL1抗体复合物在正常小鼠的主要脏器的分布，结果显示，靶向功能分子-PDL1抗体复合物在正常脑内的分布比未修饰抗体增多，PDL1抗体能在靶向分子的介导下跨越完整的血脑屏障。
图11示出了靶向功能分子-PDL1抗体复合物在荷原位脑肿瘤小鼠体内靶向性评价，其中，
图A为尾静脉注射靶向功能分子-PDL1抗体复合物8h时，其在荷原位脑肿瘤C57BL/6模型鼠的脑内分布(n＝3)，图B为尾静脉注射靶向功能分子-PDL1抗体复合物8h时，其在荷原位脑肿瘤C57BL/6模型鼠的主要脏器的分布，图C为尾静脉注射靶向功能分子-PDL1抗体复合物24h时，其在荷原位脑肿瘤C57BL/6模型鼠的脑内分布(n＝3)，图D为尾静脉注射靶向功能分子-PDL1抗体复合物24h时，其在荷原位脑肿瘤C57BL/6模型鼠的主要脏器的分布，结果显示，靶向功能分子-PDL1抗体复合物在脑肿瘤内的分布比未修饰抗增多，PDL1抗体能够在靶向分子的介导下更多地蓄积到脑肿瘤部位。
图12示出了靶向功能分子-PDL1抗体复合物在正常小鼠的药动学研究结果，
显示了靶向功能分子-PDL1抗体复合物经尾静脉注射后在正常小鼠体内的药动学曲线及药动学参数(n＝4)，结果表明，pHA-αPDL1、mRAP-αPDL1与未修饰αPDL1药动学行为无明显差异，mCDX-αPDL1血液清除较快。
图13示出了靶向功能分子-PDL1抗体复合物对荷GL261原位瘤的C57BL/6模型的药效结果，其中，
图A为荷瘤C57BL/6小鼠的生存曲线(n＝8)，表明与未修饰抗体组及PBS组相比，靶向功能分子-PDL1抗体复合物显著延长生存期；图B、C为荷瘤C57BL/6小鼠的脑肿瘤切片ki67和tunnel免疫组化示例照片及其定量结果，说明靶向功能分子-PDL1抗体复合物组的恶性细胞较少，且细胞凋亡较多，与药效结果一致。
图14示出了靶向功能分子-PDL1抗体复合物对原位脑胶质瘤的免疫调节效应，其中，
图A-F荷原位脑胶质瘤模型小鼠的脑肿瘤部位的免疫效应(n＝3)，图G-I为荷瘤小鼠的颈部淋巴结的免疫效应，图J-L为荷瘤小鼠的脾脏的免疫效应，与未修饰抗体及PBS组相比，靶向功能分子-PDL1抗体复合物组的免疫抑制细胞明显减少，效应细胞明显增多，可改善肿瘤及机体的免疫抑制环境。
图15示出了靶向功能分子-PDL1抗体复合物的毒副作用，其中，
图A为给药后血液的ATL生化指标(n＝3)，图B与图C为给药后血液中炎症因子的含量，结果显示，靶向功能分子-PDL1抗体复合物的肝脏功能指标与PBS组接近，未修饰抗体组则肝脏毒性较大，同时血液中炎症因子水平进一步确证了靶向功能分子-PDL1抗体复合物递送系统具有更低的毒副作用。
实施发明的最佳方式
下面通过具体的实施例进一步说明本发明，但是，应当理解为，这些实施例仅仅是用于更详细具体地说明之用，而不应理解为用于以任何形式限制本发明。
本部分对本发明试验中所使用到的材料以及试验方法进行一般性的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的，但是本发明仍然在此作尽可能详细描述。本领域技术人员清楚，在上下文中，如果未特别说明，本发明所用材料和操作方法是本领域公知的。
实施例1靶向分子的合成与表征
pHA靶向分子的合成与表征：采用固相合成技术制备多肽，首先称取MBHA树脂(取代度为0.54mmol/g)，加入N,N-二甲基甲酰胺溶胀后，加入20％哌啶-DMF溶液以脱去树脂上的Fmoc保护基，洗净后，加入8.8倍量的Fmoc-氨基酸(溶解于HBTU/HOBT/DIEA的DMF溶液中)，置于空气摇床中(37℃，180rpm)振摇45min。反应结束后，新鲜DMF洗涤并抽干树脂，加入20％哌啶-DMF溶液以脱去α-氨基上的Fmoc保护基。按照序列(pHA序列：p-Hydroxybenzoic acid-SSC)重复上述操作。待所有氨基酸缩合完毕，将树脂洗净抽干，加入多肽切割试剂(TFA/TIPS/H2O＝95/2.5/2.5,v/v)搅拌2h后，旋蒸除去TFA，经冰乙醚沉淀后过滤收集得到多肽粗品。用制备液相纯化，冻干，HPLC和ESI-MS对pHA多肽分子进行表征，结果如图1所示。
mRAP靶向分子的合成与表征：按上述方法合成mRAP，其氨基酸序列为CGPLGIAGQEAKIEKHNHYQK，HPLC和ESI-MS表征结果如图2所示。
mCDX靶向分子的合成与表征：按上述方法合成mCDX，其氨基酸序列为greirtgraerwsekfGPLGIAGQC(小写为D构型氨基酸，大写为L构型氨基酸)，HPLC和ESI-MS表征结果如图3所示。
实施例2靶向功能分子-PDL1抗体复合物的制备与表征
靶向功能分子-PDL1抗体复合物的制备：
在αPDL1溶液中，加入Sulfo-SMCC(1mg/mL)，室温反应30min，脱盐柱除去过量的Sulfo-SMCC。加入pHA的PBS溶液(2mg/mL)108μL，混匀，置于4℃反应12h后，于14K Da透析袋内，纯水透析2次(每次30min)，收集透析袋内液 体，得pHA修饰的PDL1抗体复合物(pHA-αPDL1)。同法制备mRAP-αPDL1与mCDX-αPDL1。
MMP酶解验证：
MMP-2加入1μL APMA(1M)用反应试液稀释至100μL，37℃孵育1h。激活后，取50μL酶与50μL的mRAP-αPDL1与mCDX-αPDL1混匀，37℃孵育2h后，透析。
靶向功能分子-PDL1抗体复合物的表征：
上述产物分别用MALDI-TOF与凝胶电泳进行表征，MALDI-TOF结果如图4所示，其中凝胶电泳方法为：配制12％的分离胶，分别取0.35mg/mL的pHA-αPDL1、mRAP-αPDL1、mCDX-αPDL1、mRAP-αPDL1+MMP与
mCDX-αPDL1+MMP溶液60μL，加入0.6μL的DTT(1M)溶液，DTT终浓度为10mM，4℃放置过夜后，加入15μL上样缓冲液，100℃水浴煮沸5min，冷却至室温，上样20μL。以80V电压浓缩样品，以120V分离样品，用纯水洗3次，每次5min；SimpleBlue染色，室温轻摇1h；纯水洗1h，共洗2次，拍照，结果如图5所示。
实施例3靶向功能分子-PDL1抗体复合物的功能性结合活性表征
ELISA结合活性：
用鼠源的PD-L1蛋白100μL/well包被96孔ELISA板，室温放置2小时，洗板2次，加入室温下BSA封闭1h，加入系列浓度梯度的pHA-αPDL1、mRAP-αPDL1、mCDX-αPDL1或未修饰的αPDL1(600ng/mL，400ng/mL，200ng/mL，100ng/mL，50ng/mL，25ng/mL，10ng/mL，5ng/mL，2ng/mL)，置于37℃恒温摇床孵育1h，洗板后，加入anti-rat Ig(H+L)/HRP孵育1h，加入100μL显色液室温放置10min，加入100μL 1M硫酸终止反应，酶标仪测定吸光度值，检测波长450nm。结果见附图6(A)。
SPR结合活性：
利用表面等离子共振(SPR)技术，考察pHA-αPDL1、mRAP-αPDL1、mCDX-αPDL1与PDL1蛋白的结合活性。通过biacore系统进行预结合分析，选取CM5芯片。将重组鼠PDL1蛋白偶联至CM5芯片上，RU值达到目标值。将未修饰的αPDL1、pHA-αPDL1、mRAP-αPDL1、mCDX-αPDL1分别配置成浓度为0.78125、1.5625、3.125、6.25、12.5、25和50nM的样品溶液。按浓度从低到高依次进样，用Biacore T200 Evaluation software软件分析靶向功能分子-PDL1抗体复合物与PDL1蛋白的结合活性，并分别计算其K D值。结果见附图6(B-E)。
PD通路阻断：
PD通路阻断是利用有荧光素标记的PDL1抗体(PE-αPDL1)竞争靶向功能分子修饰的PDL1抗体复合物与PDL1蛋白的结合，考察靶向功能分子-PDL1抗体复合物与PDL1蛋白的结合阻断能力。将GL261细胞消化分散，以每孔5×10 3个细胞的密度接种于激光共聚焦细胞培养皿，每孔加入含有IFN-γ的细胞培养液，于细胞培养箱培养48h,使表面PDL1蛋白高表达。弃去培养液，PBS洗涤，4％多聚甲醛固定15min, DAPI染核10min，PBS再次洗涤后，同上述方法加样孵育，用激光共聚焦显微镜观测多肽修饰抗体对细胞表面PDL1蛋白的阻断作用。结果如附图7。
进一步进行复合物对PD通路的阻断作用的定量考察，将GL261细胞用IFN-γ刺激48h，使表面PDL1蛋白高表达，取PDL1蛋白高表达的GL261细胞用胰酶消化，培养液中和胰酶后，离心，在PBS缓冲液中重新分散。在4℃下，将IgG、αPDL1、pHA-αPDL1、mRAP-αPDL1、mCDX-αPDL1分别加至高表达PDL1的肿瘤细胞中，共孵育5min，再加入PE-αPDL1共孵育15min，PBS洗涤并重悬至200μL PBS缓冲液中，用流式细胞计数仪对阳性细胞计数，考察修饰后的αPDL1的生物活性，结果如图7所示。
实施例4体外CD3 +T细胞杀伤实验
小鼠CD3 +T细胞的提取：
将C57BL/6小鼠腹部皮下接种10 6个GL261细胞，接种肿瘤第5天后，取小鼠脾脏，研磨成单细胞，磁珠分选出CD3 +T细胞，培养于包被有anti-CD3 antibody的U型板上，再加入anti-CD28 antibody共刺激，培养48h。
T细胞介导的细胞杀伤实验：
将GL261细胞用IFN-γ刺激48h，使表面PDL1蛋白高表达。将高表达PDL1蛋白的肿瘤细胞接种于96孔板内(5×10 3个/孔)，加入或不加等摩尔量的IgG、αPDL1、pHA-αPDL1、mRAP-αPDL1、mCDX-αPDL1。再加刺激后的CD3 +T细胞(105个/孔)共培养24h。用凋亡试剂盒染色后，流式细胞计数仪检测，结果如图8所示。
实施例5靶向功能分子-PDL1抗体复合物的靶向性评价
体外跨血-脑屏障能力：
4周龄SD大鼠断头后取脑，于冰冷的D-Hanks溶液中迅速分离得到大脑皮层，除去脑膜和脑部大血管后剪碎，加入胶原酶和DNA酶，消化，离心，将底部微血管转移至培液中，接种于预先铺有鼠尾胶原的24孔transwell中，将transwell移入二氧化碳培养箱中，37℃，5％CO 2及饱和湿度条件下培养24h使微血管段贴壁后，换成含有嘌呤霉素的内皮专用培养液继续培养72h后，再换成不含嘌呤霉素的内皮专用培养液培养72h，测得跨膜电阻(TEER)超过250Ω·cm 2，表明体外血-脑屏障建模成功。
将αPDL1、pHA-αPDL1、mRAP-αPDL1、mCDX-αPDL1分别以BCA法定量并用PBS稀释至相同浓度(1.0mg/mL)，取10μL加入transwell上室每孔，再补加90μL含有培养因子的EBM-2培养液，下室加入600μL的EBM-2培养液。在0.5h和1h收集60μL下室溶液，并补液60μL。CD274包被的ELISA板测定收集样品中的抗体含量，结果如图9所示。
体内脑靶向性验证：
60μLαPDL1抗体溶液(0.5mg/mL)，加放射剂量为0.78mCi的 125I，20μL PBS，于Iodogen管(80μg)中反应5min(约34-37℃)。转移后经2次水洗，Sephadex G-25小柱纯化得 125I标记的αPDL1，测得其放射剂量为660μCi，标记率约81％，比活度 为21.06μCi/μg。用 125I标记的αPDL1再分别制备 125I标记的pHA-αPDL1、 125I标记的mRAP-αPDL1、 125I标记的mCDX-αPDL1。
选取12只正常小鼠，每组三只，组别为αPDL1、pHA-αPDL1、mRAP-αPDL1、mCDX-αPDL1，每只小鼠给药的放射剂量为15μCi。在8h、24h取脑，称重，测定放射量，以考察复合物在正常小鼠体内的脑及主要组织分布情况，结果如图10所示。
同时，选取体重约20g的雄性C57BL/6，于脑立体定位仪定位至前囟向右1.8mm,向前0.6mm,纵深3mm的纹状体区域，注射入1×10 6个处于对数生长期的悬浮GL261细胞(分散于5μL PBS缓冲液)，接种肿瘤后饲养于SPF环境，定期观察模型鼠生存状况。取接种原位脑胶质瘤的小鼠，共12只，分为4组，同上述组别。在接种肿瘤后第7天，同法进行放射性组织分布实验，每只小鼠给药的放射剂量为15μCi。在8h、24h取脑瘤，称重，测定放射量，结果如图11所示。
实施例6靶向功能分子-PDL1抗体复合物在正常小鼠中的药动学评价
将16只雄性C57BL/6小鼠，随机分成4组每组4只，尾静脉注射αPDL1、pHA-αPDL1、mRAP-αPDL1、mCDX-αPDL1，剂量为相当于含有5mg/kg的αPDL1，于5min、15min、30min、1h、2h、4h、6h、8h眼眶取血，用ELISA法测定PDL1抗体含量，绘制药时曲线并计算药动学参数，结果如图12所示。
实施例7靶向功能分子-PDL1抗体复合物对的原位瘤小鼠模型药效学评价
将33只雄性C57BL/6小鼠接种原位脑胶质瘤细胞GL261(1×10 6个/只)，
将其随机分成3组，接种肿瘤为第0天。分别于第3天、第5天、第7天、第9天，尾静脉注射PBS、αPDL1、pHA-αPDL1，剂量为相当于含有5mg/kg的αPDL1，观察并记录死亡时间，绘制生存曲线。最后一次给药结束后24h，即第10天，每组取3只小鼠的脑组织进行石蜡包埋，切片后进行Ki67、TUNEL免疫组化染色，考察各给药组肿瘤增殖能力及凋亡情况，结果如图13所示。
实施例8靶向功能分子-PDL1抗体复合物对原位脑胶质瘤的免疫调节效应
将12只雄性C57BL/6小鼠接种原位脑胶质瘤细胞GL261(1×10 6个/只)，将其随机分成3组每组4只，接种肿瘤为第0天。分别于第3天、第5天、第7天、第9天，尾静脉注射PBS、αPDL1、pHA-αPDL1，剂量相当于αPDL1的5mg/kg。于第10天取脑肿瘤、颈部淋巴结、脾脏，处理为单细胞悬液，加入流式细胞表面抗体先用CD8、CD4、PD-1、CD62L、CD44、CD25荧光标记抗体染色，而后破细胞核膜染Foxp3；另一部分细胞加入Brefeldin A培养箱孵育6小时，经细胞表面染色后，破细胞膜染胞内抗体IFN-γ、TNF-α。确定其中CD8 +T细胞、CD4 +T细胞、IFN-γ +T细胞、TNF-α +、PD-1 +T细胞及T reg细胞数目，及其比例变化，结果如图14所示。
实施例9靶向功能分子-PDL1抗体复合物的毒副作用
将12只雄性C57BL/6小鼠接种原位脑胶质瘤细胞GL261(1×10 6个/只)，将其 随机分成3组每组4只，接种肿瘤为第0天。分别于第3天、第5天、第7天、第9天，尾静脉注射PBS、αPDL1、pHA-αPDL1，剂量为相当于αPDL1的5mg/kg。检测并记录每只小鼠体重。于第10天取血液，4000rpm/min离心10分钟，取血浆用ELISA法测定血液中TNF-α、IFN-γ因子；并检测ALT、AST肝功指标，评价其安全性。结果如图15所示。
尽管本发明已进行了一定程度的描述，明显地，在不脱离本发明的精神和范围的条件下，可进行各个条件的适当变化。可以理解，本发明不限于所述实施方案，而归于权利要求的范围，其包括所述每个因素的等同替换。

Claims (15)

  1. 一种靶向功能分子修饰的抗体复合物，其特征在于，所述复合物为由靶向功能分子与抗体通过共价连接和/或非共价连接方式形成抗体复合物；
    优选地，所述的靶向功能分子为靶向分子或靶向分子和特定敏感结构组成的功能分子；和/或所述的抗体是针对免疫检查点或相关抗原的抗肿瘤抗体，或用于阿尔兹海默症治疗的抗体，或用于帕金森病治疗的抗体，或上述抗体的改造形式。
  2. 按权利要求1所述的靶向功能分子修饰的抗体复合物，其特征在于，所述的靶向功能分子为具有跨血-脑屏障和/或跨血-脑肿瘤屏障的小分子、多肽分子或蛋白分子。
  3. 根据权利要求2所述的靶向功能分子修饰的抗体复合物，其特征在于，所述具有跨血-脑屏障和/或跨血-脑肿瘤屏障的小分子选自对羟基苯甲酸及其衍生物和/或脂肪酸；
    优选地，所述脂肪酸为肉豆蔻酸。
  4. 根据权利要求2所述的靶向功能分子修饰的抗体复合物，其特征在于，所述的靶向功能分子为具有跨血-脑屏障和/或跨血-脑肿瘤屏障的多肽分子和/或蛋白分子；
    优选地，所述多肽分子选自以下一种或多种：VAP多肽、cVAP多肽、 SVAP多肽、 DVAP多肽，pHA-VAP多肽、pHA- SVAP多肽及pHA- DVAP多肽，MC-VAP多肽、MC- SVAP多肽及MC- DVAP多肽，D8多肽、D8-VAP多肽、D8- SVAP多肽及D8- DVAP多肽，WSW多肽、 DWSW多肽、WSW-VAP多肽、 DWSW- SVAP多肽及 DWSW- DVAP多肽，TGN多肽、 DTGN多肽、TGN-VAP多肽、 DTGN- SVAP多肽及 DTGN- DVAP多肽；A7R多肽、cA7R多肽、 DA7R多肽，pHA-A7R多肽及pHA- DA7R多肽，MC-A7R多肽及MC- DA7R多肽，D8-A7R多肽及D8- DA7R，WSW-A7R多肽及 DWSW- DA7R多肽，TGN-A7R多肽及 DTGN- DA7R多肽；RGD多肽、Stapled-RGD多肽，pHA-RGD多肽，MC-RGD多肽，D8-RGD多肽，WSW-RGD多肽，TGN-RGD多肽；RW多肽、mn多肽，pHA-RW多肽及pHA-mn多肽，MC-RW多肽及MC-mn多肽，D8-RW多肽及D8-mn多肽，WSW-RW多肽及 DWSW-mn多肽，TGN-RW多肽及 DTGN-mn多肽；T7多肽及 DT7多肽；RAP12多肽及 DRAP12多肽；和/或
    所述蛋白分子选自转铁蛋白和/或乳铁蛋白。
  5. 按权利要求1所述的靶向功能分子修饰的抗体复合物，其特征在于，所述的特定敏感结构为响应疾病病灶微环境发生解离的结构域或化学键；
    优选地，所述的特定敏感结构为酶敏感多肽或pH敏感化学键。
  6. 按权利要求5所述的靶向功能分子修饰的抗体复合物，其特征在于，所述的酶敏感多肽为基质金属蛋白酶的多肽底物。
  7. 按权利要求1所述的靶向功能分子修饰的抗体复合物，其特征在于，所述的抗肿瘤抗体为作用于PD-1、PD-L1、CTLA-4、LAG-3、TIM-3免疫检查点的抗体，或作用于HER-2、VEGFR、EGFR、GD2、PDGF-Rα、gp100、MAGE-1肿瘤相关抗原的抗体。
  8. 按权利要求1所述的靶向功能分子修饰的抗体复合物，其特征在于，所述的用于阿尔兹海默症治疗的抗体为作用于Aβ、Tau的抗体。
  9. 按权利要求1所述的靶向功能分子修饰的抗体复合物，其特征在于，所述的用于帕金森病PD治疗的抗体为作用于富亮氨酸重复序列激酶2(LRRK2)、α-突触核蛋白(α-synuclein)、DJ-1、RAB8A和RAB10的抗体。
  10. 按权利要求1所述的靶向功能分子修饰的抗体复合物，其特征在于，所述的抗体改造形式为利用基因工程技术将抗体改造为的Fab片段、单域抗体、Fv片段、单链抗体、双价小分子抗体、微抗体或纳米抗体。
  11. 按权利要求1所述的靶向功能分子修饰的抗体复合物，其特征在于，所述的共价连接是通过靶向功能分子与抗体之间的化学反应方式将靶向功能分子与抗体直接连接，或通过基因工程直接将靶向功能分子与抗体融合表达。
  12. 按权利要求1所述的靶向功能分子修饰的抗体复合物，其特征在于，所述的非共价连接是通过亲合素与生物素的亲和偶联方式将靶向功能分子与抗体间接连接。
  13. 一种用于治疗脑内疾病的药物组合物，其特征在于，所述药物组合物包括：
    权利要求1至12中任一项所述的靶向功能分子修饰的抗体复合物；和
    药学上可接受的辅料；
    优选地，所述脑内疾病选自以下一种或多种：脑肿瘤、阿尔兹海默症、帕金森病。
  14. 一种脑内疾病的治疗方法，其特征在于，所述方法包括：对有需要的受试者给予权利要求1至12中任一项所述的靶向功能分子修饰的抗体复合物或权利要求13所述的药物组合物；
    优选地，所述脑内疾病选自以下一种或多种：脑肿瘤、阿尔兹海默症、帕金森病。
  15. 权利要求1至12中任一项所述的靶向功能分子修饰的抗体复合物在制备用于治疗脑内疾病的药物中的应用；
    优选地，所述脑内疾病选自以下一种或多种：脑肿瘤、阿尔兹海默症、帕金森病。
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Publication number Priority date Publication date Assignee Title
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107029239A (zh) * 2016-02-03 2017-08-11 复旦大学 一种多功能靶向分子及其用途
WO2018177420A1 (zh) * 2017-03-31 2018-10-04 复旦大学 一种生物膜包载药物纳米晶体的制备方法及其用途

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104174024B (zh) * 2013-05-21 2017-12-01 复旦大学 一种肉豆蔻酸介导的脑靶向聚合物胶束递药系统及其制备方法和应用
CN106333926A (zh) * 2015-07-10 2017-01-18 复旦大学 一种稳定性多肽介导跨屏障膜的脑部肿瘤多重靶向递药系统
WO2018119001A1 (en) * 2016-12-19 2018-06-28 Fred Hutchinson Cancer Research Center Peptide-antibody compositions and methods of use thereof
CN109384850A (zh) * 2017-08-11 2019-02-26 复旦大学 全过程靶向多肽及其在构建肿瘤靶向诊治递药系统中的应用
CN109422801B (zh) * 2017-08-31 2022-07-08 复旦大学 多功能靶向多肽rap及其在制备肿瘤靶向递送系统中的用途
US20200354458A1 (en) * 2017-11-20 2020-11-12 Taizhou Mabtech Pharmaceutical Co., Ltd. Bifunctional Fusion Protein Targeting CD47 and PD-L1

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107029239A (zh) * 2016-02-03 2017-08-11 复旦大学 一种多功能靶向分子及其用途
WO2018177420A1 (zh) * 2017-03-31 2018-10-04 复旦大学 一种生物膜包载药物纳米晶体的制备方法及其用途

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ASTRID J. SCHRAA, ROBBERT J. KOK, SANDER M. BOTTER, SEBO WITHOFF, DIRK K.F. MEIJER, LOU F.M.H. DE LEIJ, GRIETJE MOLEMA: "RGD-modified anti-CD3 antibodies redirect cytolytic capacity of cytotoxic T lymphocytes toward alphavbeta3-expressing endothelial cells", INTERNATIONAL JOURNAL OF CANCER, JOHN WILEY & SONS, INC., US, vol. 112, no. 2, 1 November 2004 (2004-11-01), US, pages 279 - 285, XP002683865, ISSN: 0020-7136, DOI: 10.1002/ijc.20413 *
BELHADJ ZAKIA; YING MAN; CAO XIE; HU XUEFENG; ZHAN CHANGYOU; WEI XIAOLI; GAO JIE; WANG XIAOYI; YAN ZHIQIANG; LU WEIYUE: "Design of Y-shaped targeting material for liposome-based multifunctional glioblastoma-targeted drug delivery", JOURNAL OF CONTROLLED RELEASE, ELSEVIER, AMSTERDAM, NL, vol. 255, 6 April 2017 (2017-04-06), AMSTERDAM, NL, pages 132 - 141, XP085032972, ISSN: 0168-3659, DOI: 10.1016/j.jconrel.2017.04.006 *
GUO HAIYAN; WANG RUIFENG; WANG DONGLI; WANG SONGLI; ZHOU JIANFEN; CHAI ZHILAN; YAO SHENGYU; LI JINYANG; LU LINWEI; LIU YU; XIE CAO: "Deliver anti-PD-L1 into brain by p-hydroxybenzoic acid to enhance immunotherapeutic effect for glioblastoma", JOURNAL OF CONTROLLED RELEASE, ELSEVIER, AMSTERDAM, NL, vol. 320, 7 January 2020 (2020-01-07), AMSTERDAM, NL, pages 63 - 72, XP086115470, ISSN: 0168-3659, DOI: 10.1016/j.jconrel.2020.01.005 *
NOUNOU MOHAMED ISMAIL; ADKINS CHRIS E.; RUBINCHIK EVELINA; TERRELL-HALL TORI B.; AFROZ MOHAMED; VITALIS TIM; GABATHULER REINHARD; : "Anti-cancer Antibody Trastuzumab-Melanotransferrin Conjugate (BT2111) for the Treatment of Metastatic HER2+ Breast Cancer Tumors in the Brain: anIn-VivoStudy", PHARMACEUTICAL RESEARCH, SPRINGER NEW YORK LLC, US, vol. 33, no. 12, 15 August 2016 (2016-08-15), US, pages 2930 - 2942, XP036089064, ISSN: 0724-8741, DOI: 10.1007/s11095-016-2015-0 *

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