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WO2017185662A1 - 多特异性结合偶联物、相关的药物组合物及应用 - Google Patents

多特异性结合偶联物、相关的药物组合物及应用 Download PDF

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Publication number
WO2017185662A1
WO2017185662A1 PCT/CN2016/101984 CN2016101984W WO2017185662A1 WO 2017185662 A1 WO2017185662 A1 WO 2017185662A1 CN 2016101984 W CN2016101984 W CN 2016101984W WO 2017185662 A1 WO2017185662 A1 WO 2017185662A1
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cancer
cell
virus
receptor
antigen
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PCT/CN2016/101984
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English (en)
French (fr)
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于浩洋
李正成
苏静
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深圳市中联生物科技开发有限公司
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Priority to EP23181122.5A priority Critical patent/EP4245775A3/en
Priority to CN201680085024.9A priority patent/CN109195992A/zh
Priority to EP16900170.8A priority patent/EP3450460B1/en
Publication of WO2017185662A1 publication Critical patent/WO2017185662A1/zh
Priority to US16/480,506 priority patent/US20210395387A9/en
Priority to US16/130,031 priority patent/US11000603B2/en
Priority to US17/224,288 priority patent/US20210338836A1/en

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    • 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
    • A61K39/44—Antibodies bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation

Definitions

  • the present invention relates to the field of immunomedicine, and more particularly to multispecific binding conjugates for inducing an immune response to diseases such as cancer or infectious diseases.
  • the invention also relates to related compositions, methods of use and uses of the conjugates.
  • bispecific antibodies can bind to two or more different antigens.
  • a number of recombinant strategies have been developed to synthesize bispecific antibodies, including single-chain variable fragment (scFv)-derived forms, such as diabodies, tandem diabodies, BiTes (bispecific T cell adaptors), and DART (dual affinity) Retargeting) and immunoglobulin G (IgG) based forms such as Triomab, DVD-Ig (double variable domain antibody) and 2-in-1 antibodies.
  • scFv single-chain variable fragment
  • BiTes bispecific T cell adaptors
  • DART dual affinity
  • IgG immunoglobulin G
  • Triomab DVD-Ig (double variable domain antibody) and 2-in-1 antibodies.
  • bispecific antibodies may have poor pharmacokinetic and physical properties (such as immunogenicity) as well as manufacturing difficulties. Therefore, improvements or alternative techniques to such prior art are needed.
  • Multispecific binding conjugates are disclosed herein.
  • the multispecific binding conjugates described herein comprise binding moieties that bind to different cellular receptors, co-receptors, antigens, and/or cellular markers coupled to nanomaterials, such as nanoparticles.
  • the multispecific binding conjugate disclosed herein comprises at least one first binding moiety and at least one second binding moiety conjugated to a nanomaterial, such as a nanoparticle, wherein the first binding moiety binds to a cytotoxic effector cell A receptor, co-receptor, antigen and/or cell marker that binds to a receptor, co-receptor, antigen and/or cellular marker on the target cell.
  • the cytotoxic effector cell can be a cytotoxic cell.
  • the cytotoxic cell can be a white blood cell.
  • the leukocytes may be selected from the group consisting of macrophages, neutrophils, eosinophils, NK cells, B cells, and T cells.
  • the white blood cells can be NK cells.
  • the leukocytes may be NK-like T cells, ie NKT cells.
  • the white blood cells may be NK cells and T cells.
  • the target cell may be a cell to be cleared, and may be a diseased cell.
  • the target cell can be Cancer cells or tumor cells.
  • the target cell can be a leukemia cell.
  • the target cell can be a lymphocyte.
  • the target cell can be a metastatic cell.
  • Target cells can be genetically modified.
  • Target cells can contain genetic mutations. Gene mutations can include oncogene mutations.
  • the gene mutation can be a mutation of a tumor suppressor gene.
  • the gene mutation can be a mutation of the proto-oncogene.
  • the target cell can be an inflammatory cell.
  • the target cell can be an infected cell.
  • the target cell can be a pathogenic cell.
  • the multispecific binding conjugate is preferably bispecific, wherein one binding site is directed to a receptor, co-receptor, antigen and/or cellular marker on leukocytes, and the second binding site binds to the target cell (Can be a receptor, co-receptor, antigen and/or cellular marker on cancer cells or tumor cells or pathogens (ie, microorganisms)).
  • the multispecific binding conjugate may also be trispecific, wherein one binding site (first binding site) targets receptors, co-receptors, antigens and/or cellular markers on leukocytes.
  • the second binding site binds to a receptor, co-receptor, antigen and/or cellular marker on a target cell, which may be a cancer cell or a tumor cell or a pathogen (ie, a microorganism).
  • Exemplary T cell receptors, co-receptors, antigens and/or cell markers are selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD8, CD25, CD28, CD30, CD40, CD40L, CD44, CD45, CD69 and CD90 .
  • Exemplary receptors, co-receptors, antigens and/or cell markers expressed on NK cells are selected from the group consisting of CD2, CD8, CD11b, CD16, CD38, CD56, CD57, ADAM17, KIR, KAR, KLR and CD137.
  • Exemplary monocyte receptors, co-receptors, antigens and/or cell markers are selected from the group consisting of CD74, HLA-DR alpha chain, CD14, CD16, CD64 and CD89.
  • Exemplary neutrophil receptors, co-receptors, antigens and/or cell markers are selected from the group consisting of CEACAM6, CEACAM8, CD16b, CD32a, CD89, CD177, CD11a, CD11b, and SLC44A2.
  • the receptor, co-receptor, antigen and/or cell marker on the leukocyte may also be a checkpoint antigen, which may be selected from the group consisting of LSECtin, CTLA4, PD1, PD-L1, LAG3, B7-H3, B7-H4, KIR and TIM3.
  • the receptor, co-receptor, antigen and/or cell marker on the T cell is CD3, CD4 or CD8, or the receptor, co-receptor, antigen and/or cell marker on the NK cell is CD16 or CD56.
  • the second binding moiety can bind to receptors, co-receptors, antigens and/or cellular markers on diseased cells or pathogens.
  • the second binding portion of the conjugate can target a receptor, co-receptor, antigen, and/or cellular marker on any type of tumor and any type of tumor cell.
  • Exemplary cancer types that can be targeted include acute lymphoblastic leukemia, acute myeloid leukemia, biliary cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, endometrial cancer , esophageal cancer, stomach cancer, head and neck cancer, He Jie King's lymphoma, lung cancer, bone marrow thyroid cancer, non-Hodgkin's lymphoma, multiple myeloma, kidney cancer, ovarian cancer, pancreatic cancer, glioma, melanoma, liver cancer, prostate cancer, bladder cancer, Neuroendocrine cancer, gastrointestinal pancreatic tumor, exocrine pancreatic cancer and Ewing sarcoma.
  • tumor-associated antigens are known to be useful in virtually any type of cancer.
  • the receptor, co-receptor, antigen and/or cell marker on the tumor cell or cancer cell may be selected from the group consisting of: carbonic anhydrase IX, alpha-fetoprotein, ⁇ -actinin-4, A3, A33 antibody specific Sex antigen, AFP, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5 , CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55 , CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83,
  • Receptors, co-receptors, antigens and/or cell markers on the tumor cells or cancer cells are also Selected from cholecystokinin B receptor, gonadotropin releasing hormone receptor, somatostatin receptor 2, avb3 integrin, gastrin releasing peptide receptor, neurokinin 1 receptor, melanocortin 1 cell surface protein of receptor, neurotensin receptor, neuropeptide Y receptor and C-type lectin-like molecule 1.
  • the pathogen may be selected from the group consisting of HIV virus, mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella Pneumophilia), Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans Cryptococcus neoformans), Histoplasma capsulatum, Hemophilis influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa (Pseudomonas aeruginosa), Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type II,
  • the first and/or second binding moiety may be selected from a small molecule, a cell targeting molecule, a ligand, a protein, a peptide, a peptoid, a DNA aptamer, a peptide nucleic acid, a vitamin, a substrate or a substrate analog, an antibody Or a fragment thereof.
  • the binding moiety is preferably a ligand, a receptor, an aptamer, an antibody or a fragment thereof, more preferably an antibody or a fragment thereof.
  • the antibodies used may be murine antibodies, sheep antibodies, etc., chimeric antibodies, humanized and human antibodies or fragments thereof.
  • the antibody may also be a heavy chain antibody (such as a camelid antibody) or a fragment thereof.
  • the antibodies may have various isotypes, preferably human IgGl, IgG2, IgG3 or IgG4, more preferably human IgGl hinge and constant region sequences.
  • the antibody or fragment thereof may be a chimeric human-mouse, humanized (human framework and murine hypervariable (CDR) region) or fully human and variant forms thereof, such as a semi-IgG4 antibody (referred to as a "single antibody" "), as described by van der Neut Kolfschoten et al.
  • the antibody or fragment thereof can be designed or selected to comprise a constant region sequence belonging to a particular allotype, which can reduce immunogenicity when administered to a human subject.
  • the antibody or fragment thereof used may also be a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof.
  • the nanomaterial may be a pharmaceutically acceptable nanomaterial, preferably a biodegradable nanomaterial, such as a nanoparticle. More preferably, the nanomaterial is polylactic acid-glycolic acid, polylactic acid, polycaprolactone, polytetramethylene glycol succinate, polyaniline, polycarbonate, ethylene lactide copolymer or glycolide caprolactone. Any one or a mixture of at least two of the copolymers is most preferably polylactic acid-glycolic acid (PLGA), polylactic acid (PLA) and/or polycaprolactone (PCL).
  • PLGA polylactic acid-glycolic acid
  • PLA polylactic acid
  • PCL polycaprolactone
  • the average particle diameter of the nanoparticles may be, for example, about 10-990 nm, for example, about 900 nm, about 850 nm, about 800 nm, about 750 nm, about 700 nm, about 650 nm, about 600 nm, about 550 nm, about 500 nm, about 450 nm, about 400 nm.
  • the nanoparticles have an average particle diameter in the range of 10 to 500 nm, more preferably in the range of 10 to 300 nm.
  • the present disclosure also provides pharmaceutical compositions comprising the multispecific binding conjugates disclosed herein.
  • the pharmaceutical composition may also comprise leukocytes, preferably NK cells, that bind to the multispecific binding conjugate.
  • the pharmaceutical composition may also contain other therapeutic agents for treating the disease.
  • the present disclosure also provides methods of treating a disease or condition comprising administering the conjugate or pharmaceutical composition to a subject in need thereof.
  • the present disclosure also provides the use of the conjugates and pharmaceutical compositions in the manufacture of a medicament for treating or preventing a disease or condition.
  • the present disclosure also relates to such conjugates and pharmaceutical compositions for treating or preventing a disease or condition.
  • the conjugates and pharmaceutical compositions disclosed herein can be used to treat cancer, such as the cancers disclosed herein.
  • Exemplary cancer types include acute lymphoblastic leukemia, acute myeloid leukemia, biliary cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, endometrial cancer, esophageal cancer, Gastric cancer, head and neck cancer, Hodgkin's lymphoma, lung cancer, bone marrow thyroid cancer, non-Hodgkin's lymphoma, multiple myeloma, kidney cancer, ovarian cancer, pancreatic cancer, glioma, melanoma, liver cancer , prostate cancer, bladder cancer, neuroendocrine cancer, gastrointestinal pancreatic tumor, exocrine pancreatic cancer and Ewing sarcoma.
  • a subject infected with a pathogenic organism such as a bacterium, virus or fungus can be treated using the conjugates or pharmaceutical compositions disclosed herein.
  • fungi that can be treated include Microsporum, Trichophyton, Epidermidis, S. sphaericus, Cryptococcus neoformans, Coccidioides, capsular histoplasma, dermatitis buds or Candida albicans.
  • viruses include human immunodeficiency virus (HIV), herpes virus, cytomegalovirus, rabies virus, influenza virus, human papilloma virus, hepatitis B virus, hepatitis C virus, Sendai virus, feline leukemia virus, Reo virus , poliovirus, human serum parvovirus, simian virus 40, respiratory syncytial virus, mouse mammary tumor virus, varicella-zoster virus, dengue virus, rubella virus, measles virus, adenovirus, human T cell leukemia Toxic leukemia virus, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus or bluetongue virus.
  • HCV human immunodeficiency virus
  • herpes virus cytomegalovirus
  • rabies virus influenza virus
  • human papilloma virus hepatitis
  • Exemplary bacteria include Bacillus anthracis, Streptococcus agalactiae, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcal, Haemophilus influenzae type B, pale Treponema pallidum, Lyme disease spirochete, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis or Mycoplasma.
  • Example 1 is a schematic view showing the assembly of the bispecific antibody of Example 1.
  • Figure 2 shows the killing efficacy of the bispecific binding conjugate anti-CD56-PLGA-anti-MUCl anti-cancer on cancer cells.
  • Figure 3 shows the killing efficacy of the bispecific binding conjugate anti-CD16-PLGA-anti-MUCl on cancer cells.
  • Figure 4 shows the killing efficacy of the bispecific binding conjugate anti-CD16-PLGA-anti-CD19 on cancer cells.
  • Figure 5 shows the killing efficacy of the bispecific binding conjugate anti-CD56-PLGA-anti-CD19 on cancer cells.
  • Figure 6 shows the killing efficacy of the bispecific binding conjugate anti-CD16-PLGA-anti-CD20 on cancer cells.
  • Figure 7 shows the killing efficacy of the bispecific binding conjugate and anti-CD56-PLGA-anti-CD20 on cancer cells.
  • Figure 8 shows the ratio of NK (CD3-CD56+) cells and CD3+ T cells in the prepared effector cells.
  • Figure 9 shows the ability of bispecific or trispecific binding conjugates to aid NK in killing cancer cells.
  • Figure 10 shows the efficacy of the prepared bispecific or trispecific binding conjugates against tumors in vivo.
  • a “therapeutic agent” is an atom, molecule or compound that can be used to treat a disease.
  • therapeutic agents include antibodies, antibody fragments, peptides, drugs, toxins, enzymes, nucleases, hormones, immunomodulators, antisense oligonucleotides, small interfering RNAs (siRNAs), chelating agents, boron compounds, photosensitizers, Dyes and radioisotopes.
  • antibody refers to the immunological activity (ie, specificity) of a full-length (ie, naturally occurring or formed by a normal immunoglobulin gene fragment rearrangement process) of an immunoglobulin molecule (eg, an IgG antibody) or an immunoglobulin molecule. Sexually bound) parts, such as antibody fragments.
  • Antibody includes monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, murine antibodies, heavy chain antibodies (such as camelid antibodies), chimeric antibodies, humanized and human antibodies, and the like.
  • a "heavy chain antibody” is an antibody that contains only a heavy chain and does not contain a light chain, such as a camelid antibody, a cartilage fish such as a shark antibody, and the like.
  • an “antibody fragment” is a part of an intact antibody, such as F(ab') 2 , F(ab) 2 , Fab', Fab, Fv, scFv, dAb, and the like. Regardless of the structure, the antibody fragment binds to the same antigen as recognized by the full length antibody.
  • an antibody fragment comprises an isolated fragment consisting of a variable region, such as an "Fv” fragment consisting of a heavy or light chain variable region or a recombinant single sequence in which the light chain and heavy chain variable regions are joined by a peptide linker.
  • Chain polypeptide molecule (“scFv protein").
  • Single-chain antibodies are usually abbreviated as “the scFv", the polypeptide chain comprises V H and V L, composed of domains, said domains interact to form an antigen binding site. And V L, V H domains are usually linked by a peptide having 1-25 amino acid residues.
  • Antibody fragments also include bifunctional antibodies, trifunctional antibodies, and single domain antibodies (dAbs).
  • Antibody fragments also include fragments of heavy chain antibodies, such as single domain antibodies (sdAbs) or fragments comprising variable regions.
  • a “multispecific antibody” is an antibody that can simultaneously bind at least two targets having different structures (eg, two different antigens, two different epitopes on the same antigen).
  • a “bispecific antibody” is an antibody that can simultaneously bind two targets with different structures.
  • the conjugate or composition described herein is said to be administered in a "therapeutically effective amount.” It is physiologically significant if the presence of the agent produces a detectable change in the physiology of the subject. In a particular embodiment, the presence of an antibody formulation is physiologically significant if it causes an anti-cancer response or amelioration of signs and symptoms of an infectious disease state. A physiologically significant effect may also be to elicit a humoral and/or cellular immune response in the subject, thereby causing growth inhibition or death of the target cells.
  • Multispecific binding conjugate means that the conjugate can simultaneously bind at least two targets having different structures (eg, two different antigens, two different epitopes on the same antigen).
  • Targets having different structures eg, two different antigens, two different epitopes on the same antigen.
  • Bispecific binding conjugate means that the conjugate can simultaneously bind two targets having different structures.
  • a first binding moiety comprising a binding leukocyte coupled to a nanomaterial (eg, a nanoparticle) and a receptor, co-receptor, antigen and/or cell marker on a cell associated with or associated with the disease A multispecific binding conjugate of the second binding portion of the substance.
  • the first binding moiety and/or the second binding moiety may have one or more binding specificities, such as one or more monoclonal antibodies or fragments thereof, or a multispecific binding moiety, such as a double Specific antibodies or fragments thereof.
  • Exemplary T cell receptor, co-receptor, antigen and/or cell markers include CD2, CD3, CD4, CD5, CD6, CD8, CD25, CD28, CD30, CD40, CD40L, CD44, CD45, CD69 and CD90.
  • exemplary receptors, co-receptors, antigens, and/or cell markers can be selected from the group consisting of CD8, CD16, CD56, CD57, ADAM17, and CD137; for monocytes, Exemplary receptors, co-receptors, antigens and/or cell markers may be selected from the group consisting of CD74, HLA-DR ⁇ chain, CD14, CD16, CD64 and CD89; and for neutrophils, exemplary receptors, co-receptors
  • the antigen and/or cell markers include CEACAM6, CEACAM8, CD16b, CD32a, CD89, CD177, CD11a, CD11b and SLC44A2.
  • the first binding moiety for NK binds to CD16 or CD56.
  • diseases-associated antigens such as tumor associated antigen (TAA) or antigens expressed by pathogens, are known.
  • TAA tumor associated antigen
  • the TAA is MUC1, CD19, CD20, CD33, CD38, EGFR and HER2.
  • the binding moiety useful in the claimed conjugate is preferably an antibody or fragment thereof.
  • Techniques for preparing monoclonal antibodies that are actually directed against any target antigen are well known in the art.
  • Known and/or commercially available antibodies can also be used.
  • antibodies for the treatment of, for example, cancer include, but are not limited to, LL1 (anti-CD74), LL2 or RFB4 (anti-CD22), veltuzumab (hA20, anti-CD20), rituximab (anti-CD20), Tocilizumab (GA101, anti-CD20), ranibizumab (anti-PD1), nilutuzumab (anti-PD1), MK-3475 (anti-PD1), AMP-224 (anti-PD1), pipizhudan Anti-(PD1), MDX-1105 (anti- PD-L1), MEDI4736 (anti-PD-L1), MPDL3280A (anti-PD-L1), BMS-936559 (anti-PD-L1), ipilimumab (anti-CTLA4), travizumab (anti-CTL4A), RS7 (anti-epithelin-1 (EGP-1, also known as TROP-2)), PAM4 or
  • the receptor, co-receptor, antigen, and/or cellular marker on the target cell can be a cancer cell receptor or a cancer associated antigen, particularly selected from the group consisting of a B cell line antigen (CD19, CD20).
  • VEGF vascular endothelial growth factor
  • VEGFR EGFR
  • CAA carcinoembryonic antigen
  • PlGF placental growth factor
  • axillary protein HER-2/neu
  • EGP-1 EGP-2
  • CD25 CD30 , CD33, CD38, CD40, CD45, CD52, CD74, CD80, CD138, NCA66, CEACAM1, CEACAM6 (carcinoembryonic antigen-associated cell adhesion molecule 6), MUC1, MUC2, MUC3, MUC4, MUC16, IL-6, alpha-fetoprotein (AFP), A3, CA125, colon-specific antigen-p (CSAp), folate receptor, HLA-DR, human chorionic gonadotropin (HCG), Ia, EL-2, insulin-like growth factor (IGF) and IGF receptor, KS-1, Le(y), MAGE, necrotic antigen, PAM-4, prostatic acid phosphatase (PAP), Pr1, prostate cancer, HER
  • MUC1 antigen is highly expressed on the cell surface of various tumors (90% of solid cancers such as liver cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, cervical cancer, kidney cancer, bladder cancer, etc.), making it a potential tumor.
  • a target molecule for targeted therapy is highly expressed on the cell surface of various tumors (90% of solid cancers such as liver cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, cervical cancer, kidney cancer, bladder cancer, etc.
  • B cells express various cell surface molecules during their differentiation and proliferation. Examples include CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85 and CD86 leukocyte surface markers. These markers are generally considered to be therapeutic targets for the treatment of B cell disorders or diseases such as B cell malignancies, autoimmune diseases and transplant rejection. Antibodies that specifically bind to them have been developed, and some have been identified as therapeutic agents for the treatment of diseases and disorders.
  • CD38 and CD138 (Stevenson, Mol Med 2006; 12(11-12): 345-346; Tassone et al, Blood 2004; 104(12): 3688-96), CD74 Suitable targeting (Stein et al, supra), CS1 (Tai et al, Blood 2008; 112(4): 1329-37) and CD40 (Tai et al, 2005; Cancer Res. 65(13): 5988-5906) antibody.
  • CD74 antigen is highly expressed in B cell lymphoma (including multiple myeloma) and leukemia, certain T cell lymphoma, melanoma, colon cancer, lung and kidney cancer, glioblastoma and some other cancers. (Ong et al., Immunology 98: 296-302 (1999)). A review of the use of CD74 antibodies in cancer is contained in Stein et al, Clin Cancer Res. September 15, 2007; 13 (18 Pt 2): 5556s-5563s, which is incorporated herein by reference.
  • Preferred diseases for treatment with anti-CD74 antibodies include, but are not limited to, non-Hodgkin's lymphoma, Hodgkin's disease, melanoma, lung cancer, renal cancer, colon cancer, glioblastoma multiforme, histiocytoma , myeloid leukemia and multiple myeloma.
  • antibodies against pathogens can be used, and such antibodies are known.
  • Commercially available antibodies against a variety of human pathogens eg, KPL, Inc., Gaithersburg, MD
  • the pathogen is selected from the group consisting of HIV virus, Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella legeriae, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae Bacteria, Neisseria meningitidis, pneumococcus, Cryptococcus neoformans, capsular histoplasma, Haemophilus influenzae type B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus, giant cell Virus, herpes simplex virus type I, herpes simplex virus type II, human serum parvovirus, respiratory syncytial virus, varicella-zoster virus,
  • antibodies that may be used include antibodies to infectious disease factors such as bacteria, viruses, mycoplasmas or other pathogens. Many antibodies to such infectious agents are known in the art, and any such known antibodies can be used in the claimed methods and compositions.
  • the following antibodies can be used: antibodies against the gpl20 glycoprotein antigen of human immunodeficiency virus I (HIV-1); antibodies against malaria parasites, such as monoclonal antibodies against sporozoites (circumsporozoite antigens), Antibodies against Schistosoma japonicum surface antigens (Simpson et al, Parasitology, 83: 163-177, 1981; Smith et al, Parasitology, 84: 83-91, 1982; Gryzch et al, J.
  • HIV-1 human immunodeficiency virus I
  • malaria parasites such as monoclonal antibodies against sporozoites (circumsporozoite antigens)
  • Patent 7,910,702 resistant Glucose xylosmannan antibody (Zhong and Priofski, 1998, Clin Diag Lab Immunol 5: 58-64); Candida antibodies (Matthews and Burnie, 2001, Curr Opin Investig Drugs 2: 472-76) and Glycosphingolipid antibodies (Toledo et al, 2010, BMC Microbiol 10:47).
  • Suitable antibodies have been developed for most of the microorganisms (bacteria, viruses, protists, fungi, other parasites) that cause infection in most people, and many have previously been used for in vitro diagnostic purposes. These antibodies, as well as altered antibodies which can be produced by conventional methods, are suitable for use in the present invention.
  • the nanomaterial for coupling the first binding moiety (eg, an antibody or fragment thereof) and the second binding moiety (eg, an antibody or fragment thereof) may be a pharmaceutically acceptable nanomaterial, preferably a biodegradable nanomaterial, more preferably Any one of polylactic acid-glycolic acid, polylactic acid, polycaprolactone, polybutylene glycol succinate, polyaniline, polycarbonate, ethylene lactide copolymer or glycolide caprolactone copolymer Or a mixture of at least two, most preferably polylactic acid-glycolic acid (PLGA), polylactic acid (PLA) and/or polycaprolactone (PCL).
  • PLGA polylactic acid-glycolic acid
  • PLA polylactic acid
  • PCL polycaprolactone
  • the preparation of the multispecific binding conjugate of the present invention is described below by taking, as an example, a bispecific binding conjugate comprising two antibodies conjugated to a nanoparticle.
  • the preparation method comprises the following steps:
  • the nanomaterial obtained in the step (1) is linked to a mixture of the first antibody portion and the second antibody portion.
  • the preparation of the nano material comprises: completely dissolving the nano material with a solvent, stirring, adding water to form a uniform emulsion.
  • the agitation may be carried out at a rotational speed of 500 to 20,000 rpm/min, for example, the rotational speed may be 500 rpm/min, 700 rpm/min, 800 rpm/min, 1000 rpm/min, 1100 rpm/min, 1200 rpm/min, 1300 rpm/min, 1400 rpm/ Min, 1480 rpm/min, 1500 rpm/min, 2000 rpm/min, 2200 rpm/min, 2500 rpm/min, 3000 rpm/min, 3500 rpm/min, 4000 rpm/min, 4200 rpm/min, 4500 rpm/min, 5000 rpm/min, 5500 rpm/min, 6000 rpm/min, 6500 rpm/min, 7000 rpm/min, 7500
  • the nano material is polylactic acid-glycolic acid (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polytetramethylene glycol succinate, polyaniline, polycarbonate, ethylene-propylene cross Any one or a mixture of at least two of an ester copolymer or a glycolide caprolactone copolymer.
  • PLGA polylactic acid-glycolic acid
  • PLA polylactic acid
  • PCL polycaprolactone
  • polytetramethylene glycol succinate polyaniline
  • polycarbonate polycarbonate
  • the solvent is any one of acetone, methyl ethyl ketone, methanol, ethanol or isopropanol Or a mixture of at least two.
  • the collecting of the nanomaterial comprises: collecting the prepared nanomaterial by centrifugation, then resuspending it with deionized water, and repeating the operation of washing the nanomaterial twice.
  • the centrifugation can be carried out at a rotational speed of 8000-15000 rpm/min, for example, the rotational speed can be 8000 rpm/min, 9000 rpm/min, 10000 rpm/min, 11000 rpm/min, 12000 rpm/min, 13000 rpm/min, 14000 rpm/min, 14500 rpm/min. , 14800 rpm / min, 15000 rpm / min. Higher speeds can be used if necessary.
  • Nanomaterials can be collected or further purified by other methods. The nanoparticles may have an average particle diameter as described above
  • the activation of the nanomaterial comprises: using 1-10 mg/mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDS) and N-hydroxysuccinyl
  • EDS 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • NHS N-hydroxysuccinyl
  • the amine (NHS) mixed solvent activates the nanomaterial at room temperature for 0.5-5 h.
  • the joining comprises: collecting the activated nanomaterial by centrifugation, and then washing the nanomaterial once with the linking reaction solution. Adding the first antibody portion and the second antibody portion to be ligated in equal amounts to the ligation reaction solution, and then resuspending the nano material with the ligation reaction solution containing the first antibody portion and the second antibody portion, and performing the reaction at room temperature 0.5-5h. After the reaction, the nanomaterials were collected by centrifugation, and the nanomaterials were washed twice with Dunsen's phosphate buffer solution, and then resuspended in Dun's phosphate buffer solution (D-PBS) and stored at 4 ° C until use. Other methods of activation of the nanomaterial can be employed.
  • the preparation method of the multi-specific binding conjugate of the present invention specifically includes the following steps:
  • the antibody portion and the second antibody portion are mixed in equal amounts to the ligation reaction solution, and then the nanomaterial is resuspended with the ligation reaction solution containing the first antibody portion and the second antibody portion, and the reaction is allowed to react at room temperature for 0.5-5 h, and the reaction is centrifuged at the end of the reaction.
  • the nanomaterials were collected, and the nanomaterials were washed twice with Dunsen's phosphate buffer solution, and then resuspended in Dun's phosphate buffer solution and stored at 4 ° C until use.
  • the invention also relates to a pharmaceutical composition comprising the multispecific binding conjugate.
  • the pharmaceutical composition may also comprise leukocytes, such as T cells, NK cells.
  • compositions herein may be formulated using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate the passage of the active agent into the pharmaceutically acceptable formulation. Proper formulation depends on the route of administration chosen. For example, an overview of pharmaceutical compositions can be found, for example, in Ansel et al, PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th edition (Lea & Febiger 1990); and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th edition (Mack Publishing) Company 1990) and its revised edition.
  • the pharmaceutical compositions disclosed herein may further comprise one or more diluents, excipients or carriers that are pharmaceutically acceptable.
  • the pharmaceutical composition may comprise a pharmaceutical or pharmaceutical agent, a carrier, an adjuvant such as a preservative, a stabilizer, a wetting or emulsifying agent, a dissolution promoter, a salt for regulating the osmotic pressure, and/or a buffer.
  • an adjuvant such as a preservative, a stabilizer, a wetting or emulsifying agent, a dissolution promoter, a salt for regulating the osmotic pressure, and/or a buffer.
  • compositions may also contain other therapeutically valuable substances, such as one or more other therapeutic agents for treating the disease.
  • other therapeutic agents may be other agents for treating cancer, such as cyclophosphamide, etoposide, carmustine, vincristine, etc., and may also be used to treat cancer antibodies, antibody-drug conjugates. (ADC), interferon and/or checkpoint inhibitor antibodies.
  • ADC antibody-drug conjugates.
  • Various embodiments are directed to methods of treating a disease or condition (eg, cancer) in a subject, such as a mammal, including a human, domesticated or companion pet, such as a dog and a cat, comprising administering to the subject a therapeutically effective amount of the text.
  • a disease or condition eg, cancer
  • a subject such as a mammal, including a human, domesticated or companion pet, such as a dog and a cat
  • administering comprising administering to the subject a therapeutically effective amount of the text.
  • Said conjugate or pharmaceutical composition comprising administering to the subject a therapeutically effective amount of the text.
  • the conjugates or pharmaceutical compositions described herein may be formulated, administered subcutaneously or even by other parenteral routes, such as intravenously, intramuscularly, intraperitoneally or intravascularly, to a mammal via, for example, bolus injection or continuous infusion.
  • parenteral routes such as intravenously, intramuscularly, intraperitoneally or intravascularly
  • the conjugate or pharmaceutical composition is infused for a period of less than about 4 hours, and more preferably for a period of less than about 3 hours.
  • the first bolus can be infused over 30 minutes, preferably even 15 minutes, and the remainder is infused over the next 2-3 hours.
  • the injectable preparation may be presented in unit dosage form, for example, in ampoules or in multi-dose containers with the addition of a preservative.
  • compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulations such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in the form of a powder which is reconstituted with a suitable vehicle (for example, sterile pyrogen-free water) before use.
  • Controlled release formulations can be prepared by using a polymer to complex or adsorb the agent to be administered.
  • biocompatible polymers include poly(ethylene-vinyl acetate) copolymer matrices and polyanhydride copolymer matrices of stearic acid dimers and sebacic acid. Sherwood et al, Bio/Technology 10: 1446 (1992). The rate of release from such a matrix depends on the molecular weight of the therapeutic agent, the amount of agent within the matrix, and the size of the dispersed particles. Saltzman et al, Biophys. J.
  • the dosage of the conjugate administered to a human will vary depending on factors such as the age, weight, height, sex, general medical condition, and past medical history of the patient. It may be desirable to provide the recipient with a conjugate dose as a single intravenous infusion in the range of about 0.1 [mu]g/kg to 25 mg/kg (to bind the first binding moiety and the second binding moiety in the multispecific binding conjugate) The total amount), but lower or higher doses can also be administered, as the case may be. For example, for a 70 kg patient, a dose of 0.1 ⁇ g/kg to 20 mg/kg is 0.7 ⁇ g to 1400 mg.
  • the dose can be repeated as needed, for example, once a week for 4-10 weeks, once a week for 8 weeks or once a week for 4 weeks. In maintenance therapy, it can also be administered at a lower frequency, if desired, such as every other week for several months, or monthly or quarterly for many months. It is also possible to administer 2, 3, 4, 5 or 6 times continuously for each course of treatment, for example, about 10 days, 15 days, 20 days, 25 days, 30 days, 35 days, 40 days after continuous administration. 45 days, 50, 55 days or 60 days or more for the next course of treatment.
  • the conjugate can be combined with effector cells such as leukocytes (such as T cells, NK cells) Administration, for example, intravenous reinfusion.
  • effector cells such as leukocytes (such as T cells, NK cells) Administration
  • the conjugate and effector cells can also be administered at a lower frequency, such as every few weeks or weeks, for several months, or monthly or quarterly for many months. It is also possible to administer 2, 3, 4, 5 or 6 times continuously for each course of treatment, for example, about 10 days, 15 days, 20 days, 25 days, 30 days, 35 days, 40 days after continuous administration. 45 days, 50, 55 days or 60 days or more for the next course of treatment.
  • the dosage of other therapeutic agents for human administration will vary depending on factors such as the age, weight, height, sex, general medical condition, and past medical history of the patient. It may be desirable to provide the recipient with a conjugate dose as a single intravenous infusion in the range of about 1 mg/kg to 25 mg/kg, but lower or higher doses may also be administered, as appropriate. For example, for a 70 kg patient, a dose of 1-20 mg/kg is 70-1,400 mg. The dose can be repeated as needed, for example, once a week for 4-10 weeks, once a week for 8 weeks or once a week for 4 weeks. In maintenance therapy, it can also be administered at a lower frequency, if desired, such as every other week for several months, or monthly or quarterly for many months.
  • Other therapeutic agents can be administered in one dose every 2 weeks or 3 weeks, for a total of at least 3 doses repeated.
  • the combination can be administered twice a week for 4 to 6 weeks. If so the dose was reduced to about 200-300mg / m 2 (per dose for patients 1.7m 340mg, for a 70kg patient is or 4.9mg / kg), it may be administered once or twice a week even for 4 to 10 weeks.
  • the time course of administration can be reduced, i.e., every 2 or 3 weeks for 2-3 months. However, it has been determined that even higher doses can be administered even by slow iv infusion, such as 20 mg/kg once a week or once every 2-3 weeks, for repeated dosing cycles.
  • the time course of administration can optionally be repeated at other intervals, and the dosage can be administered by various parenteral routes with appropriate adjustments in dosage and time course.
  • the conjugates or pharmaceutical compositions described herein are useful in the treatment of cancer therapies.
  • cancer include, but are not limited to, carcinoma, lymphoma, glioblastoma, melanoma, sarcoma and leukemia, myeloma or lymphoid malignancies.
  • squamous cell carcinoma eg, epithelial squamous cell carcinoma
  • Ewing's sarcoma e.g., Ewing's sarcoma
  • Weems's tumor astrocytoma
  • lung cancer including small cell lung cancer, Non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma
  • peritoneal cancer hepatocellular carcinoma
  • gastric cancer or stomach cancer including gastrointestinal cancer
  • pancreatic cancer polymorphism Glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrine tumor, medullary thyroid carcinoma, thyroid differentiation cancer, breast cancer, ovarian cancer, colon cancer, rectal cancer Endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or kidney cancer, prostate cancer, vaginal cancer, anal cancer, penile cancer, and head and neck cancer.
  • squamous cell carcinoma
  • cancer includes primary malignant cells or tumors (eg, cells that have not migrated to a site other than the original malignant disease or tumor site in the subject) and secondary malignant cells or tumors (eg, by metastasis, ie, A malignant cell or tumor cell migrates to a tumor produced by a secondary site different from the original tumor site).
  • primary malignant cells or tumors eg, cells that have not migrated to a site other than the original malignant disease or tumor site in the subject
  • secondary malignant cells or tumors eg, by metastasis, ie, A malignant cell or tumor cell migrates to a tumor produced by a secondary site different from the original tumor site.
  • Cancers that benefit from the therapeutic methods of the invention are directed to cells that express, overexpress, or aberrantly express IGF-IR.
  • cancer or malignant diseases include, but are not limited to, acute childhood lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adrenocortical carcinoma, adult (primary) hepatocytes Cancer, adult (primary) liver cancer, adult acute lymphocytic leukemia, adult acute myeloid leukemia, adult Hodgkin's lymphoma, adult lymphocytic lymphoma, adult non-Hodgkin's lymphoma, adult primary Liver cancer, adult soft tissue sarcoma, AIDS-related lymphoma, AIDS-related malignant disease, anal cancer, astrocytoma, cholangiocarcinoma, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, renal pelvis And ureteral cancer, central nervous system (primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, cerebral a
  • conjugates, compositions, and methods described and claimed herein can be used to treat malignant or pre-malignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those described above. Indicating such use for conditions that are known or suspected to progress in advance into a tumor or cancer, especially in the case of non-neoplastic cell growth that has occurred from hyperplasia, metaplasia or, most particularly, dysplasia (on this) For a review of abnormal growth conditions, see Robbins and Angell, BASIC PATHOLOGY, 2nd ed., WBSaunders Co., Philadelphia, pp. 68-79 (1976).
  • Dysplasia is often a precursor to cancer and is mainly found in the epithelium. It is the most disordered form of non-neoplastic cell growth and involves the loss of individual cell identity and cell structure orientation. In the presence of chronic irritation or inflammation, dysplasia characteristically occurs.
  • Dysplastic disorders that can be treated include, but are not limited to, non-performing ectodermal dysplasia, anterior dysplasia, asphyxiating thoracic dysplasia, atrial-finger dysplasia, bronchopulmonary dysplasia, brain dysplasia, Cervical dysplasia, cartilage ectodermal dysplasia, clavicular skull dysplasia, congenital ectodermal dysplasia, skull dysplasia, craniofacial dysplasia, dysplasia of the skull, dentine dysplasia, poor backbone structure, external Germ dysplasia, enamel dysplasia, cerebral ocular dysplasia, unilateral epiphyseal dysplasia, multiple epiphyseal dysplasia, punctate epiphyseal dysplasia, epithelial dysplasia, facial genital dysplasia, familial jaw fibrosis Poor, familial white wrinkle dysplasia, fibromuscular dysp
  • pre-neoplastic conditions include, but are not limited to, benign abnormal proliferative disorders (eg, benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps or adenomas and esophageal dysplasia), leukoplakia, keratosis, Bowen's Disease, farmer's skin, solar cheilitis and solar keratosis.
  • benign abnormal proliferative disorders eg, benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps or adenomas and esophageal dysplasia
  • leukoplakia keratosis
  • Bowen's Disease farmer's skin
  • solar cheilitis solar cheilitis
  • the method of the invention is used to inhibit the growth, progression and/or metastasis of cancer, particularly the cancers listed above.
  • hyperproliferative diseases, disorders, and/or conditions include, but are not limited to, progression and/or metastasis of malignant diseases and related conditions, such as leukemia (including acute leukemia (eg, acute lymphocytic leukemia, acute myeloid leukemia (including bone marrow formation) Cytoplasmic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia) and chronic leukemia (eg, chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia), true red blood cells Hyperplasia, lymphoma (eg, Jay's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, including but not limited to sarcoma and Carcinoma, such as fibrosarcoma, mucinous sarcoma, liposarcoma, chondrosarcom
  • kits/kit containing a component suitable for treating or diagnosing a diseased tissue of a patient.
  • An exemplary kit/kit can contain one or more conjugates, leukocytes, and/or other therapeutic agents as described herein.
  • a device capable of delivering the kit components by some other route may be included.
  • One type of device for applications such as parenteral delivery is a syringe for injecting a composition into a subject.
  • An inhalation device can also be used.
  • the therapeutic agent can be provided in the form of a prefilled syringe or automatic injection pen containing a sterile liquid formulation or a lyophilized formulation.
  • the kit/kit components can be packaged together or separated into two or more containers.
  • the container can be a vial containing a sterile lyophilized formulation of a composition suitable for reconstitution.
  • the kit/kit may also contain one or more buffers suitable for reconstituting and/or diluting other reagents.
  • Other containers that may be used include, but are not limited to, bags, trays, boxes, tubes, and the like.
  • the kit/kit components can be aseptically packaged and maintained in a container. Another component that can be included is the instructions for the user of the drug delivery kit/kit.
  • a bispecific binding conjugate anti-CD56-PLGA-anti-CD20 with anti-CD56 antibody and anti-CD20 antibody conjugated to different nanoparticles was prepared.
  • the specific preparation method of the bispecific binding conjugate anti-CD56-PLGA-anti-CD20 is as follows:
  • PLGA was completely dissolved in acetone to a concentration of 5 mg/mL, and a solution of PLGA and acetone was magnetically stirred at 1000 rpm/min in a volume ratio of acetone to deionized water of 1:4. Add deionized water to form a uniform emulsion, and then continue to stir until the acetone is evaporated;
  • the anti-CD56 monoclonal antibody (mAb) to be ligated and the anti-CD20 mAb are mixed in an equal amount to the ligation reaction solution, and then the anti-CD56 mAb is contained and The anti-CD20 mAb was ligated to the nanomaterial and the ligation reaction was carried out for 0.5 h at room temperature.
  • the nanomaterials were collected by centrifugation, and the nanomaterials were washed twice with Dunsen's phosphate buffer solution, and then resuspended in Dun's phosphate buffer solution and stored at 4 ° C for use.
  • the specific preparation method of the bispecific binding conjugate anti-CD56-PLA-anti-CD20 is as follows:
  • PLA was completely dissolved in methanol to a concentration of 15 mg/mL, and a solution of PLA and methanol was magnetically stirred at 500 rpm/min in a volume ratio of methanol to deionized water of 1:4. Adding deionized water to form a uniform emulsion, and then continuing to stir until the methanol is evaporated;
  • the specific preparation method of the bispecific binding conjugate anti-CD56-PCL-anti-CD20 is as follows:
  • PCL nanoparticles completely dissolve PCL with isopropyl alcohol to a concentration of 30 mg/mL, and according to the volume ratio of isopropyl alcohol and deionized water of 1:4, PCL with magnetic stirring at 1500 rpm/min. A solution of isopropanol is added to the deionized water to form a uniform emulsion, and then stirring is continued until the isopropanol is volatilized;
  • the nanomaterials were collected by centrifugation, and the nanomaterials were washed twice with Dunsen's phosphate buffer solution, and then resuspended in Dun's phosphate buffer solution and stored at 4 ° C for use.
  • bispecific binding conjugates - anti-CD16-PLGA-anti-MUCl, anti-CD56-PLGA-anti-MUCl, anti-CD16-PLGA-anti-CD19, anti-CD56- were prepared in a manner similar to that described in Example 1.
  • PLGA-anti-CD19, anti-CD16-PLGA-anti-CD20 and anti-CD56-PLGA-anti-CD20 were prepared in a manner similar to that described in Example 1.
  • the antibodies used therein are all commercially available monoclonal antibodies.
  • NK cells were adjusted with cytokine-free X-vivo 15 medium (purchased from lonza) Peripheral blood mononuclear cells from healthy blood donors were induced to amplify, so that the number of NK cells in a volume of 100 ⁇ l was 4 times that of the target cells (the effective target ratio was 4:1).
  • Killing rate [1 - (experimental group - effect control group) / (target control group - blank control group)] ⁇ 100%
  • the blank control indicates the addition of the medium
  • the target control group indicates the addition of the target cells + medium
  • the effect control group indicates the addition of the effector cells + medium
  • the experimental group indicates the effector cells + target cells + medium + bispecific binding conjugate .
  • HKC human embryonic kidney epithelial cells
  • A549 human non-small cell lung cancer cells
  • HepG2 human liver cancer cells
  • HT29 human colon cancer cells
  • AGS human gastric adenocarcinoma cells
  • Hela human cervical cancer cells
  • 5637 human bladder Cancer cells
  • OS-RC-2 human kidney cancer cells.
  • Anti-CD56-PLA-anti-CD19 and anti-CD56-PCL-anti-CD19 showed a killing rate of 83% or more for black Burkitt lymphoma cells and human B lymphoma cells (specific data not shown).
  • Anti-CD16-PLA-anti-CD20 and anti-CD16-PCL-anti-CD20 showed a killing rate of 83% or more for black Burkitt lymphoma cells and human B lymphoma cells (specific data not shown).
  • Anti-CD16-PLA-anti-MUCl and anti-CD16-PCL-anti-MUC1 showed greater than 95% killing rate for human non-small cell lung cancer cells; more than 92% killing for human bladder cancer cells and human breast cancer cells Rate; nearly 85% killing rate for human cervical cancer cells and human gastric adenocarcinoma cells; more than 80% killing rate for human colon cancer cells, human kidney cancer cells and human liver cancer cells.
  • Anti-CD3 and/or anti-CD16 and anti-MUC1 bispecific or trispecific binding conjugates were prepared according to the methods of the previous examples.
  • the ability of the bispecific or trispecific binding conjugate to assist NK killing of A549 cells was evaluated. Specifically, 50 ml of peripheral blood of healthy people was collected, monocytes (PBMC) were isolated, and then NK cytokines were added for induction and expansion culture. The percentage of NK cells was measured by cell culture until day 21. The test results were as follows: wherein the ratio of NK cells was 86.9%, and the ratio of CD3+ T cells was 10.2% (see Fig. 8). In a 96-well plate, A549 cells as target cells were cultured at 5000 cells/well for 12 hours, and then the original medium was discarded.
  • the NK cell density was adjusted using the cytokine-free X-vivo 15 medium to make the number of NK cells in a volume of 100 ⁇ l 4 times that of the target cells (effective target ratio of 4:1).
  • 100 ⁇ l of NK cell suspension was added to the cancer cell culture plate, and 10 ⁇ l of the prepared bispecific binding conjugate (0.2 mg of the bispecific binding conjugate preparation and 0.2 ⁇ g of the monoclonal antibody-containing component) was added.
  • CCK-8 reagent CCK-8 reagent
  • the killing rate of NK cells against cancer cells was calculated according to the following formula.
  • Killing rate [1 - (experimental group - effect control group) / (target control group - blank control group)] ⁇ 100%
  • NK, CD3 mAb + NK, CD16 mAb + NK, MUC1 single Anti-NK showed a certain effect.
  • the statistical results showed that the tumor inhibition rates were 26.1%, 33.02%, 26.22%, and 37.51%, respectively, and there was no significant difference between the groups.
  • the tumor inhibition rates of CD3-MUC1, CD16-MUC1, CD3/CD16-MUC1 helper NK cells were 71.02%, 81.84%, and 90.91%, respectively, which showed good efficacy, among which CD3/CD16-MUC1>CD16-MUC1> CD3-MUC1 is also closely related to the cellular components in NK cells.
  • NK generally expresses CD16, and the proportion of NK cells is much larger than that of CD3+ T cells. Therefore, it is appropriate to use CD16 as the recognition site of NK. Because of the current in vitro induced NK cells, the NK ratio is about 86.9%, and some other effector cells, including CD3+ T cells, are 10.2%, so CD16 and CD3 are simultaneously connected, and the antitumor effect is better.
  • Example 4 The efficacy of the bispecific or trispecific binding conjugates described in Example 4 against tumors in vivo was evaluated. Specifically, in healthy Balb-c nude mice (clean grade, female, four weeks old, body weight 18-22g, purchased from Beijing Vital Lihua Experimental Animal Technology Co., Ltd.), human lung adenocarcinoma A549 was inoculated under the armpit, inoculation 3 After 4 weeks, 90 nude mice with a tumor diameter of about 0.5 x 0.5 cm were randomly divided into 8 groups for experiments (grouping is shown below).
  • each experimental group was injected with 20 ⁇ l of antibody or bound conjugate preparation each time, and the preparation contained 12.5 ⁇ g of each monoclonal antibody, that is, each of the monoclonal antibody groups.
  • Animals were injected with 0.25 ⁇ g of antibody, the total amount of antibody injected per animal in the bispecific binding conjugate group was 0.50 ⁇ g, and the total amount of antibody injected per animal in the trispecific binding conjugate group was 0.75 ⁇ g.
  • the control group was injected with the same volume of normal saline.
  • the volume of the recorded tumor was measured from the first day.
  • the therapeutic effect of the bispecific/trispecific binding conjugate on the tumor was calculated.
  • Figure 10 shows the statistical changes in tumor volume.
  • NK CD3 monoclonal antibody + NK
  • CD16 monoclonal antibody + NK CD16 monoclonal antibody + NK
  • MUC1 monoclonal antibody + NK showed a certain effect
  • the current statistical results show that compared with the control group, the tumor inhibition rate was 39.24% 36.19%, 33.49%, 37.01%, there was no significant difference between the groups.
  • the tumor inhibition rates of CD3-MUC1, CD16-MUC1, CD3/CD16-MUC1 and NK cells were 81.22%, 88.24%, and 95.15%, respectively, and all showed good results, among which CD3/CD16-MUC1>CD16-MUC1>CD3 -MUC1 is also closely related to the cellular components in NK cells.
  • NK generally expresses CD16, and its proportion is much larger than that of CD3+ T cells. Therefore, it is appropriate to use CD16 as the recognition site of NK. Because of the current in vitro induced NK cells, the NK ratio is about 86.9%, and some other effector cells, including CD3+ T cells, are 10.2%, so CD16 and CD3 are simultaneously connected, and the antitumor effect is better.

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Abstract

本发明提供了多特异性结合偶联物、其相关的组合物和用途。所述偶联物包含通过纳米材料偶联的针对两种或更多种不同受体、共受体、抗原或细胞标志物的结合部分，可用于调节免疫应答，治疗或预防如癌症、自身免疫病、病原体感染或炎性疾病等疾病或病症。

Description

多特异性结合偶联物、相关的药物组合物及应用 技术领域
本发明涉及免疫医学领域，尤其涉及用于诱导对诸如癌症或传染病等疾病的免疫应答的多特异性结合偶联物。本发明还涉及所述偶联物的相关组合物和使用方法及用途。
发明背景
一些抗体偶联物，诸如多特异性抗体(如双特异性抗体)，可与两种或更多种不同抗原结合。已开发了许多重组体策略来合成双特异性抗体，包括单链可变片段(scFv)衍生的形式，诸如双抗体、串联双抗体、BiTes(双特异性T细胞衔接体)和DART(双亲和力再靶向)以及基于免疫球蛋白G(IgG)的形式如Triomab、DVD-Ig(双可变域抗体)和二合一抗体。然而，双特异性抗体可能具有较差的药代动力学和物理性质(诸如免疫原性)以及和制造上的困难。因此，需要对这样的现有技术的改善或替代技术。
发明内容
本文公开了多特异性结合偶联物。本文所述的多特异性结合偶联物包含偶联于纳米材料(如纳米微粒)上的结合不同细胞上受体、共受体、抗原和/或细胞标志物的结合部分。
本文公开的多特异性结合偶联物包含偶联于纳米材料(如纳米微粒)上的至少一种第一结合部分和至少一种第二结合部分，其中第一结合部分结合细胞毒性效应细胞上受体、共受体、抗原和/或细胞标志物，所述第二结合部分结合靶细胞上受体、共受体、抗原和/或细胞标志物。
所述细胞毒性效应细胞可以是细胞毒性细胞。所述细胞毒性细胞可以是白细胞。所述白细胞可以选自巨噬细胞、嗜中性粒细胞、嗜酸性粒细胞、NK细胞、B细胞和T细胞。白细胞可以是NK细胞。所述白细胞可以是NK样T细胞，即NKT细胞。所述白细胞可以是NK细胞和T细胞。
靶细胞可以是有待清除的细胞,可以是患病细胞。例如，靶细胞可以是 癌细胞或肿瘤细胞。靶细胞可以是白血病细胞。靶细胞可以是淋巴细胞。靶细胞可以是转移细胞。靶细胞可进行遗传修饰。靶细胞可包含基因突变。基因突变可包含癌基因突变。基因突变可以是肿瘤抑制基因的突变。基因突变可以是原癌基因的突变。靶细胞可以是炎症细胞。靶细胞可以是受感染细胞。靶细胞可以是病原细胞。
所述多特异性结合偶联物优选是双特异性的，其中一个结合位点针对白细胞上的受体、共受体、抗原和/或细胞标志物，且第二结合位点结合于靶细胞(可以是癌细胞或肿瘤细胞或病原体(即微生物))上的受体、共受体、抗原和/或细胞标志物。所述多特异性结合偶联物也可以是三特异性的，其中一个结合位点(第一结合位点)针对白细胞上的受体、共受体、抗原和/或细胞标志物，而所述第二结合位点结合于靶细胞(可以是癌细胞或肿瘤细胞或病原体(即微生物))上的受体、共受体、抗原和/或细胞标志物。
示例性T细胞上受体、共受体、抗原和/或细胞标志物选自CD2、CD3、CD4、CD5、CD6、CD8、CD25、CD28、CD30、CD40、CD40L、CD44、CD45、CD69和CD90。NK细胞上表达的示例性受体、共受体、抗原和/或细胞标志物选自CD2、CD8、CD11b、CD16、CD38、CD56、CD57、ADAM17、KIR、KAR、KLR和CD137。示例性单核细胞上受体、共受体、抗原和/或细胞标志物选自CD74、HLA-DRα链、CD14、CD16、CD64和CD89。示例性嗜中性粒细胞上受体、共受体、抗原和/或细胞标志物选自CEACAM6、CEACAM8、CD16b、CD32a、CD89、CD177、CD11a、CD11b和SLC44A2。白细胞上受体、共受体、抗原和/或细胞标志物还可以是检查点抗原，可以选自LSECtin、CTLA4、PD1、PD-L1、LAG3、B7-H3、B7-H4、KIR和TIM3。优选地，T细胞上受体、共受体、抗原和/或细胞标志物是CD3、CD4或CD8，或NK细胞上受体、共受体、抗原和/或细胞标志物是CD16或CD56。
所述第二结合部分可以结合患病细胞或病原体上的受体、共受体、抗原和/或细胞标志物。在某些实施方案中，所述偶联物的第二结合部分可以靶向任何类型的肿瘤和任何类型的肿瘤细胞上的受体、共受体、抗原和/或细胞标志物。可以靶向的示例性癌症类型包括急性淋巴母细胞性白血病、急性髓性白血病、胆癌、乳腺癌、子宫颈癌、慢性淋巴细胞性白血病、慢性髓性白血病、结肠直肠癌、子宫内膜癌、食道癌、胃癌、头颈癌、何杰 金氏淋巴瘤、肺癌、骨髓甲状腺癌、非何杰金氏淋巴瘤、多发性骨髓瘤、肾癌、卵巢癌、胰腺癌、神经胶质瘤、黑素瘤、肝癌、前列腺癌、膀胱癌、神经内分泌癌、胃肠胰肿瘤、外分泌胰腺癌和尤因肉瘤。然而，本领域普通技术人员应认识到，已知肿瘤相关抗原实际上可用于任何类型的癌症。
所述肿瘤细胞或癌细胞上的受体、共受体、抗原和/或细胞标志物可以选自：碳酸酐酶IX、甲胎蛋白、α-辅肌动蛋白-4、A3、A33抗体特异性抗原、AFP、ART-4、B7、Ba 733、BAGE、BrE3-抗原、CA125、CAMEL、CAP-1、CASP-8/m、、CCCL19、CCCL21、CD1、CD1a、CD2、CD3、CD4、CD5、CD8、CD11A、CD14、CD15、CD16、CD18、CD19、CD20、CD21、CD22、CD23、CD25、CD29、CD30、CD32b、CD33、CD37、CD38、CD40、CD40L、CD45、CD46、CD52、CD54、CD55、CD59、CD64、CD66a-e、CD67、CD70、CD70L、CD74、CD79a、CD79b、CD80、CD83、CD95、CD126、CD132、CD133、CD138、CD147、CD154、CDC27、CDK-4/m、CDKN2A、CXCR4、CXCR7、CXCL12、HIF-1α、结肠特异性抗原-p(CSAp)、CEA(CEACAM5)、CEACAM6、c-met、DAM、EGFR、EGFRvIII、EGP-1、EGP-2、ELF2-M、Ep-CAM、Flt-1、Flt-3、叶酸受体、G250抗原、GAGE、gp100、GROB、HLA-DR、HM1.24、人绒毛膜促性腺激素(HCG)和其亚单位、HER2/neu、HMGB-1、低氧可诱导因子(HIF-1)、HSP70-2M、HST-2、Ia、IGF-1R、IFN-γ、IFN-α、IFN-β、IL-2、IL-4R、IL-6R、IL-13R、IL-15R、IL-17R、IL-18R、IL-6、IL-8、IL-12、IL-15、IL-17、IL-18、IL-23、IL-25、胰岛素样生长因子-1(IGF-1)、KC4-抗原、KS-1-抗原、KS1-4、Le-Y、LDR/FUT、巨噬细胞迁移抑制因子(MIF)、MAGE、MAGE-3、MART-1、MART-2、NY-ESO-1、TRAG-3、mCRP、MCP-1、MIP-1A、MIP-1B、MIF、MUC1、MUC2、MUC3、MUC4、MUC5ac、MUC13、MUC16、MUM-1/2、MUM-3、NCA66、NCA95、NCA90、胰腺癌粘蛋白、胎盘生长因子、p53、PLAGL2、前列腺酸性磷酸酶、PSA、PRAME、PSMA、PlGF、ILGF、ILGF-1R、IL-6、IL-25、RS5、RANTES、T101、SAGE、S100、存活蛋白、存活蛋白-2B、TAC、TAG-72、腱生蛋白、TRAIL受体、TNF-α、Tn抗原、汤姆森-弗里德里希抗原、肿瘤坏死抗原、TROP-2、VEGFR、ED-B纤连蛋白、WT-1、17-1A-抗原、补体因子C3、C3a、C3b、C5a、C5、血管生成标记物、bcl-2、bcl-6、Kras、cMET、致癌基因产物。
所述肿瘤细胞或癌细胞上的受体、共受体、抗原和/或细胞标志物也可 以选自胆囊收缩素B受体、促性腺激素释放激素受体、促生长素抑制素受体2、avb3整联蛋白、胃泌素释放肽受体、神经激肽1受体、黑皮质素1受体、神经降压肽受体、神经肽Y受体和C型凝集素样分子1的细胞表面蛋白。
所述病原体可以选自HIV病毒、结核分枝杆菌(mycobacterium tuberculosis)、无乳链球菌(Streptococcus agalactiae)、抗甲氧西林性金黄色葡萄球菌(meticillin-resistant Staphylococcus aureus)、嗜肺军团菌(Legionella pneumophilia)、酿脓链球菌(Streptococcus pyogenes)、大肠杆菌(Escherichia coli)、淋病奈瑟氏菌(Neisseria gonorrhoeae)、脑膜炎奈瑟氏菌(Neisseria meningitidis)、肺炎球菌(Pneumococcus)、新型隐球菌(Cryptococcus neoformans)、荚膜组织胞浆菌(Histoplasma capsulatum)、B型流感嗜血杆菌(Hemophilis influenzae B)、苍白密螺旋体(Treponema pallidum)、莱姆病螺旋体(Lyme disease spirochetes)、铜绿假单胞菌(Pseudomonas aeruginosa)、麻风分枝杆菌(Mycobacterium leprae)、流产布鲁氏菌(Brucella abortus)、狂犬病病毒、流感病毒、巨细胞病毒、I型单纯疱疹病毒、II型单纯疱疹病毒、人血清细小样病毒、呼吸道合胞病毒、水痘-带状疱疹病毒、乙型肝炎病毒、丙型肝炎病毒、麻疹病毒、腺病毒、人T细胞白血病病毒、埃-巴二氏病毒、鼠白血病病毒、腮腺炎病毒、水泡性口膜炎病毒、辛德毕斯病毒、淋巴细胞性脉络丛脑膜炎病毒、疣病毒、蓝舌病病毒、仙台病毒、猫白血病毒、呼肠孤病毒、脊髓灰质炎病毒、猿猴病毒40、小鼠乳腺瘤病毒、登革热病毒、风疹病毒、西尼罗河病毒、恶性疟原虫(Plasmodium falciparum)、间日疟原虫(Plasmodium vivax)、刚地弓形虫(Toxoplasma gondii)、让氏锥虫(Trypanosoma rangeli)、克氏锥虫(Trypanosoma cruzi)、罗德西亚锥虫(Trypanosoma rhodesiensei)、布氏锥虫(Trypanosoma brucei)、曼森氏裂体吸虫(Schistosoma mansoni)、日本裂体吸虫(Schistosoma japonicum)、牛巴贝虫(Babesia bovis)、艾美耳球虫(Elmeria tenella)、旋盘尾丝虫(Onchocerca volvulus)、热带利什曼虫(Leishmania tropica)、旋毛虫(Trichinella spiralis)、小泰勒虫(Theileria parva)、泡状带绦虫(Taenia hydatigena)、羊绦虫(Taenia ovis)、牛肉绦虫(Taenia  saginata)、细粒棘球绦虫(Echinococcus granulosus)、科氏中殖孔绦虫(Mesocestoides corti)、关节炎支原体(Mycoplasma arthritidis)、鼻支原体(M.hyorhinis)、口腔支原体(M.orale)、精氨酸支原体(M.arginini)、拉氏无胆甾原体(Acholeplasma laidlawii)、唾液支原体(M.salivarium)和肺炎支原体(M.pneumoniae)。
所述第一和/或第二结合部分可选自小分子、细胞靶向分子、配体、蛋白质、肽、类肽、DNA适体、肽核酸、维生素、底物或底物类似物、抗体或其片段。所述结合部分优选为配体、受体、适体、抗体或其片段，更优选为抗体或其片段。
所使用的抗体可以是鼠类抗体、羊抗体等、嵌合抗体、人源化和人抗体或其片段。所述抗体也可以是重链抗体(如骆驼抗体)或其片段。抗体可以具有各种同型，优选人IgG1、IgG2、IgG3或IgG4，更优选地包含人IgG1铰链和恒定区序列。所述抗体或其片段可以是嵌合人类-小鼠、人源化(人类架构和鼠类高变(CDR)区)或完全人类的以及其变异形式，诸如半IgG4抗体(称为“单抗体”)，如van der Neut Kolfschoten等所描述(Science 2007；317:1554-1557)。更优选地，抗体或其片段可以经过设计或选择而包含属于特定同种异型的恒定区序列，这在施用于人类受试者时可以降低免疫原性。所使用的抗体或其片段也可以是多特异性抗体(例如双特异性抗体)或其片段。
所述纳米材料可以是可药用的纳米材料，优选为生物可降解纳米材料，例如为纳米微粒。所述纳米材料更优选为聚乳酸-羟基乙酸、聚乳酸、聚己内酯、聚丁二醇丁二酸酯、聚苯胺、聚碳酸酯、乙丙交酯共聚物或乙交酯己内酯共聚物中的任意一种或至少两种的混合物，最优选为聚乳酸-羟基乙酸(PLGA)、聚乳酸(PLA)和/或聚己内酯(PCL)。所述纳米微粒的平均粒径可以例如为大约10-990nm，例如为约900nm、约850nm、约800nm、约750nm、约700nm、约650nm、约600nm、约550nm、约500nm、约450nm、约400nm、约350nm、约300nm、约250nm、约200nm、约150nm、约100nm或小于100nm，例如大小为约90nm、约80nm、约70nm、约60nm、约50nm、约40nm、约30nm、约20nm/或约10nm。优选纳米颗粒的平均粒径在10-500nm范围内，更优选在10-300nm范围内。
本公开还提供药物组合物，其包含本文所公开的多特异性结合偶联物。所述药物组合物还可以包含与所述多特异性结合偶联物相结合的白细胞，优选NK细胞。所述药物组合物还可以含有治疗疾病的其他治疗剂。
本公开也提供疾病或病症的治疗方法，其包括给有需要的受治疗者施用所述偶联物或药物组合物。
本公开还提供所述偶联物和药物组合物在制备用治疗或预防疾病或病症的药物中的用途。
本公开也涉及用于治疗或预防疾病或病症的所述偶联物和药物组合物。
在某些实施方案中，本文公开的偶联物和药物组合物可以用于治疗癌症，例如本文公开的癌症。示例性癌症类型包括急性淋巴母细胞性白血病、急性髓性白血病、胆癌、乳腺癌、子宫颈癌、慢性淋巴细胞性白血病、慢性髓性白血病、结肠直肠癌、子宫内膜癌、食道癌、胃癌、头颈癌、何杰金氏淋巴瘤、肺癌、骨髓甲状腺癌、非何杰金氏淋巴瘤、多发性骨髓瘤、肾癌、卵巢癌、胰腺癌、神经胶质瘤、黑素瘤、肝癌、前列腺癌、膀胱癌、神经内分泌癌、胃肠胰肿瘤、外分泌胰腺癌和尤因肉瘤。在其它实施方案中，可以使用本文公开的偶联物或药物组合物治疗感染了诸如细菌、病毒或真菌等致病生物体的受试者。可以治疗的示例性真菌包括小孢霉、发癣菌、表皮癣菌、申克孢子丝菌、新型隐球菌、粗球孢子菌、荚膜组织胞浆菌、皮炎芽生菌或白色念珠菌。示例性病毒包括人免疫缺陷病毒(HIV)、疱疹病毒、巨细胞病毒、狂犬病病毒、流感病毒、人类乳头状瘤病毒、乙型肝炎病毒、丙型肝炎病毒、仙台病毒、猫白血病毒、Reo病毒、脊髓灰质炎病毒、人血清细小样病毒、猿猴病毒40、呼吸道合胞病毒、小鼠乳腺瘤病毒、水痘-带状疱疹病毒、登革热病毒、风疹病毒、麻疹病毒、腺病毒、人类T细胞白血病毒白血病病毒、埃-巴二氏病毒、鼠类白血病毒白血病病毒、腮腺炎病毒、水泡性口膜炎病毒、辛德毕斯病毒、淋巴细胞性脉络丛脑膜炎病毒或蓝舌病病毒。示例性细菌包括炭疽杆菌、无乳链球菌、嗜肺军团菌、酿脓链球菌、大肠杆菌、淋病奈瑟氏菌、脑膜炎奈瑟氏球菌、肺炎球菌属、B型流感嗜血杆菌、苍白密螺旋体、莱姆病螺旋体、铜绿假单胞菌、麻风分枝杆菌、流产布鲁氏菌、结核分枝杆菌或支原体。
附图简述
图1是实施例1的双特异性抗体的组装示意图。
图2显示了双特异性结合偶联物抗CD56-PLGA-抗MUC1抗对癌细胞的杀伤效力。
图3显示了双特异性结合偶联物抗CD16-PLGA-抗MUC1对癌细胞的杀伤效力。
图4显示了双特异性结合偶联物抗CD16-PLGA-抗CD19对癌细胞的杀伤效力。
图5显示了双特异性结合偶联物抗CD56-PLGA-抗CD19对癌细胞的杀伤效力。
图6显示了双特异性结合偶联物抗CD16-PLGA-抗CD20对癌细胞的杀伤效力。
图7显示了双特异性结合偶联物和抗CD56-PLGA-抗CD20对癌细胞的杀伤效力。
图8显示所制备的效应细胞中NK(CD3-CD56+)细胞及CD3+T细胞的比例。
图9显示双特异性或三特异性结合偶联物辅助NK杀伤癌细胞的能力。
图10显示所制备的双特异性或三特异性结合偶联物于体内抗肿瘤的效力。
具体实施方案
在描述本发明方法和组合物之前，应当理解，本发明并不局限于所述的特定方法或组合物，因此当然可能会有所不同。还应当理解的是，本文所使用的术语仅用于描述特定的实施方案，并非限制性的。描述实施例是为本领域的普通技术人员提供如何制造和使用本发明的完整的公开内容和描述，并非旨在限制发明人视为其发明的范围，也并非旨在表示下面的实验是进行的全部或仅有的实验。已经努力确保所用数字(例如量、温度等)的准确度，但一些实验误差和偏差应该予以考虑。
除非另有定义，否则本文使用的所有技术和科学术语具有如由本发明所属领域的普通技术人员通常理解的相同的含义。现在描述一些潜在的和优选的方法和材料，尽管类似或等同于本文描述的任何方法和材料可以用 于实施或测试本发明。本文提及的所有出版物在此通过引用并入，以公开和描述与所引用的出版物有关的方法和/或材料。可以理解的是，存在矛盾的情况下，以本公开内容取代所引用的出版物中的任何公开内容。
如在阅读本公开内容时对本领域技术人员显而易见的，每个本文所述和说明的单独的实施方案具有分立的组件和特征，其可容易地与其他若干实施方案中的特征分离或结合，而不脱离本发明的范围或实质。可以按所列举事件的顺序或逻辑上可能的任何其他顺序实施任何列举的方法。
除非另外说明，否则本文和所附权利要求中所用的“一个(种)”意指“一个(种)或多个(种)”。
当提供数值范围时，应当理解，该范围的上限和下限之间的每个中间值(至下限单位的十分之一)也被具体地公开，除非上下文另有明确说明。在所述范围中的任何所述值或中间值之间的各个较小的范围以及在所述范围内的其他所述值或中间值包含在本发明之内。这些较小范围的上限和下限可独立地包含在该范围内或排除在该范围之外，并且服从于所述范围中任何所特定地排除的，其中上下限中任一个或两个或无一个包含在所述较小范围内的每个范围也包含在本发明内。当所述范围包含一个或两个界限，除去任一或两个那些包含在内的界限的范围也包含在本发明中。
“治疗剂”是可用于治疗疾病的原子、分子或化合物。治疗剂的实例包括抗体、抗体片段、肽、药物、毒素、酶、核酸酶、激素、免疫调节剂、反义寡核苷酸、小干扰RNA(siRNA)、螯合剂、硼化合物、光敏剂、染料和放射性同位素。
本文所使用的“抗体”是指全长(即天然存在的或由正常免疫球蛋白基因片段重排过程形成的)免疫球蛋白分子(例如IgG抗体)或免疫球蛋白分子的免疫活性(即特异性结合)部分，如抗体片段。“抗体”包括单克隆抗体、多克隆抗体、双特异性抗体、多特异性抗体、鼠类抗体、重链抗体(如骆驼抗体)、嵌合抗体、人源化和人抗体等。“重链抗体”是仅含有重链而不含轻链的抗体，例如骆驼抗体、软骨鱼如鲨鱼抗体等等。
“抗体片段”是完整抗体的一部分，诸如F(ab')2、F(ab)2、Fab'、Fab、Fv、scFv、dAb等等。无论结构如何，抗体片段结合与全长抗体所识别的相同抗原。举例来说，抗体片段包括由可变区组成的经分离片段，诸如由 重链或轻链可变区组成的“Fv”片段或轻链与重链可变区通过肽连接子连接的重组单链多肽分子(“scFv蛋白”)。“单链抗体”通常缩写为“scFv”，由包含VH和VL结构域的多肽链组成，所述结构域相互作用以形成抗原结合位点。VH和VL结构域通常通过具有1至25个氨基酸残基的肽连接。抗体片段还包括双功能抗体、三功能抗体和单结构域抗体(dAb)。“抗体片段”也包括重链抗体的片段，如单域抗体(sdAb)或包含可变区的片段。
“多特异性抗体”是可以同时结合至少两个具有不同结构的靶标(例如两个不同的抗原、同一抗原上的两个不同的表位)的抗体。“双特异性抗体”是可以同时结合两个具有不同结构的靶标的抗体。
如果施用量具有生理学显著性，则称本文中所描述的偶联物或组合物是以“治疗有效量”施用。如果药剂的存在在受治疗者的生理学方面产生可检测的变化，则它具有生理学显著性。在特定实施方案中，若抗体制剂的存在引起抗癌反应或缓解传染病状态的体征和症状，则其具有生理学显著性。生理学上显著的效应还可以是在受治疗者中引起体液和/或细胞免疫应答，从而引起靶细胞的生长抑制或死亡。
多特异性(如双特异性结合)偶联物
“多特异性结合偶联物”是指所述偶联物可以同时结合至少两个具有不同结构的靶标(例如两个不同的抗原、同一抗原上的两个不同的表位)。“双特异性结合偶联物”是指所述偶联物可以同时结合两个具有不同结构的靶标。
各种实施方案涉及包含偶联于纳米材料(如纳米微粒)上的结合白细胞的第一结合部分和结合疾病相关的或有待清除的细胞上的受体、共受体、抗原和/或细胞标志物的第二结合部分的多特异性结合偶联物。所述第一结合部分和/或第二结合部分可以具有一种或多种结合特异性，例如可以为一种或多种单克隆抗体或其片段，也可以为多特异性结合部分，如双特异性抗体或其片段。
示例性T细胞上受体、共受体、抗原和/或细胞标志物包括CD2、CD3、CD4、CD5、CD6、CD8、CD25、CD28、CD30、CD40、CD40L、CD44、CD45、CD69和CD90。对于NK细胞，其它示例性受体、共受体、抗原和/或细胞标志物可以选自CD8、CD16、CD56、CD57、ADAM17和CD137；对于单核细胞，示 例性受体、共受体、抗原和/或细胞标志物可以选自CD74、HLA-DRα链、CD14、CD16、CD64和CD89；而对于嗜中性粒细胞，示例性受体、共受体、抗原和/或细胞标志物包括CEACAM6、CEACAM8、CD16b、CD32a、CD89、CD177、CD11a、CD11b和SLC44A2。在优选的实施方案中，针对NK的第一结合部分结合于CD16或CD56。如以下所论述，疾病相关抗原，诸如肿瘤相关抗原(TAA)或病原体表达的抗原的许多实例是已知的。优选TAA是MUC1、CD19、CD20、CD33、CD38、EGFR和HER2。
可用于要求保护的偶联物中的结合部分优选为抗体或其片段。用于制备实际上针对任何靶抗原的单克隆抗体的技术在本领域中是熟知的。也可以使用已知的和/或市售的抗体。例如，可以使用选自以下的抗体：hR1(抗IGF-1R)、hPAM4(抗粘蛋白)、KC4(抗粘蛋白)、hA20(抗CD20)、hA19(抗CD19)、hIMMU31(抗AFP)、hLL1(抗CD74)、hLL2(抗CD22)、RFB4(抗CD22)、hMu-9(抗CSAp)、hL243(抗HLA-DR)、hMN-14(抗CEACAM5)、hMN-15(抗CEACAM6)、hRS7(抗TROP-2)、hMN-3(抗CEACAM6)、CC49(抗TAG-72)、J591(抗PSMA)、D2/B(抗PSMA)、G250(抗碳酸酐酶IX)、英夫利昔单抗(抗TNF-α)、聚乙二醇化赛妥珠单抗(certolizumab pegol)(抗TNF-α)、阿达木单抗(抗TNF-α)、阿仑单抗(抗CD52)、贝伐单抗(抗VEGF)、西妥昔单抗(抗EGFR)、吉姆单抗(抗CD33)、替伊莫单抗(抗CD20)、帕尼单抗(panitumumab)(抗EGFR)、利妥昔单抗(抗CD20)、托西莫单抗(抗CD20)、GA101(抗CD20)、曲妥单抗(抗HER2/neu)、托珠单抗(tocilizumab)(抗IL-6受体)、巴利昔单抗(抗CD25)、达克珠单抗(抗CD25)、依法利珠单抗(efalizumab)(抗CD11a)、莫罗单抗(muromonab)-CD3(抗CD3受体)、那他珠单抗(抗α4整合素)、BWA-3(抗组蛋白H2A/H4)、LG2-1(抗组蛋白H3)、MRA12(抗组蛋白H1)、PR1-1(抗组蛋白H2B)、LG11-2(抗组蛋白H2B)、LG2-2(抗组蛋白H2B)、P4/D10(抗gp120)和奥玛珠单抗(omalizumab)(抗IgE)。
用于治疗例如癌症的示例性抗体包括但不限于LL1(抗CD74)、LL2或RFB4(抗CD22)、维妥珠单抗(hA20、抗CD20)、利妥昔单抗(抗CD20)、奥妥珠单抗(GA101、抗CD20)、兰利珠单抗(抗PD1)、尼鲁单抗(抗PD1)、MK-3475(抗PD1)、AMP-224(抗PD1)、皮地珠单抗(抗PD1)、MDX-1105(抗 PD-L1)、MEDI4736(抗PD-L1)、MPDL3280A(抗PD-L1)、BMS-936559(抗PD-L1)、伊匹单抗(抗CTLA4)、曲维珠单抗(抗CTL4A)、RS7(抗上皮糖蛋白-1(EGP-1，也称为TROP-2))、PAM4或KC4(均为抗粘蛋白)、MN-14(抗癌胚抗原(CEA，也称为CD66e或CEACAM5)、MN-15或MN-3(抗CEACAM6)、Mu-9(抗结肠特异性抗原-p)、Immu 31(抗甲胎蛋白)、R1(抗IGF-1R)、A19(抗CD19)、TAG-72(例如CC49)、Tn、J591或HuJ591(抗PSMA(前列腺特异性膜抗原))、AB-PG1-XG1-026(抗PSMA二聚体)、D2/B(抗PSMA)、G250(抗碳酸酐酶IX MAb)、L243(抗HLA-DR)、阿仑单抗(抗CD52)、贝伐单抗(抗VEGF)、西妥昔单抗(抗EGFR)、吉姆单抗(抗CD33)、替伊莫单抗(抗CD20)；帕尼单抗(抗EGFR)；托西莫单抗(抗CD20)；PAM4(又名克里伏单抗，抗粘蛋白)、BWA-3(抗组蛋白H2A/H4)、LG2-1(抗组蛋白H3)、MRA12(抗组蛋白H1)、PR1-1(抗组蛋白H2B)、LG11-2(抗组蛋白H2B)、LG2-2(抗组蛋白H2B)和曲妥单抗(抗ErbB2)。此类抗体在本领域中是已知的。
在某些实施方案中，靶细胞上受体、共受体、抗原和/或细胞标志物可以是癌细胞受体或癌症相关抗原，特别选自以下的抗原：B细胞系抗原(CD19、CD20、CD21、CD22、CD23等)、VEGF、VEGFR、EGFR、癌胚抗原(CEA)、胎盘生长因子(PlGF)、腱生蛋白、HER-2/neu、EGP-1、EGP-2、CD25、CD30、CD33、CD38、CD40、CD45、CD52、CD74、CD80、CD138、NCA66、CEACAM1、CEACAM6(癌胚抗原相关细胞粘连分子6)、MUC1、MUC2、MUC3、MUC4、MUC16、IL-6、甲胎蛋白(AFP)、A3、CA125、结肠特异性抗原-p(CSAp)、叶酸受体、HLA-DR、人绒膜促性腺激素(HCG)、Ia、EL-2、胰岛素样生长因子(IGF)和IGF受体、KS-1、Le(y)、MAGE、坏死抗原、PAM-4、前列腺酸性磷酸酶(PAP)、Pr1、前列腺特异性抗原(PSA)、前列腺特异性膜抗原(PSMA)、S100、T101、TAC、TAG72、TRAIL受体和碳酸酐酶IX。
MUC1抗原在多种肿瘤(肝癌、肺癌、乳腺癌、食管癌、胃癌、结直肠癌、宫颈癌、肾癌、膀胱癌等90％的实体癌)细胞表面高度异常表达，使其成为潜在的肿瘤靶向治疗的靶分子。
B细胞在其分化和增殖期间表达各种细胞表面分子。实例包括CD10、CD19、CD20、CD21、CD22、CD23、CD24、CD37、CD53、CD72、CD74、CD75、 CD77、CD79a、CD79b、CD80、CD81、CD82、CD83、CD84、CD85和CD86白细胞表面标记物。这些标记物通常被认为是治疗B细胞失调或疾病如B细胞恶性肿瘤、自身免疫病和移植排斥的治疗靶点。开发了与它们特异性结合的抗体，一些经测定成为治疗疾病和失调的治疗剂。
对于多发性骨髓瘤疗法，已经描述了针对例如CD38和CD138(Stevenson,Mol Med 2006；12(11-12):345-346；Tassone等,Blood 2004；104(12):3688-96)、CD74(Stein等,出处同上)、CS1(Tai等,Blood 2008；112(4):1329-37)和CD40(Tai等,2005；Cancer Res.65(13):5898-5906)的合适的靶向抗体。
CD74抗原是高度表达于B细胞淋巴瘤(包括多发性骨髓瘤)和白血病、某些T细胞淋巴瘤、黑素瘤、结肠癌、肺癌和肾癌、成胶质细胞瘤和某些其它癌症上(Ong等,Immunology 98:296-302(1999))。CD74抗体用于癌症中的用途的综述包含于Stein等,Clin Cancer Res.2007年9月15日；13(18 Pt 2):5556s-5563s中，该文献通过引用并入本文中。优选用抗CD74抗体治疗的疾病包括但不限于非何杰金氏淋巴瘤、何杰金氏病、黑素瘤、肺癌、肾癌、结肠癌、多形性成胶质细胞瘤、组织细胞瘤、骨髓性白血病和多发性骨髓瘤。
在另一个优选的实施方案中，可以使用针对病原体的抗体，这些抗体是已知的。可利用针对多种人类病原体的市售抗体(例如KPL,Inc.，Gaithersburg，MD)，包括针对金黄色葡萄球菌(目录号011-90-05)、无乳链球菌(目录号011-90-08)、酿脓链球菌(目录号01-90-07)、幽门螺旋杆菌(目录号01-93-94)、伯氏疏螺旋体(目录号05-97-91)、大肠杆菌(目录号01-95-91；01-95-96)、军团菌属(目录号01-90-03)、李斯特菌属(目录号01-90-90)、霍乱弧菌(目录号01-90-50)、志贺氏菌属(目录号16-90-01)和弯曲杆菌属(目录号01-92-93)的抗体。
在一优选的实施方案中，病原体选自HIV病毒、结核分支杆菌、无乳链球菌、耐甲氧西林金黄色葡萄球菌、嗜肺性军团病菌、酿脓链球菌、大肠杆菌、淋病奈瑟氏菌、脑膜炎奈瑟氏菌、肺炎球菌、新型隐球菌、荚膜组织胞浆菌、B型流感嗜血杆菌、苍白密螺旋体、莱姆病螺旋体、铜绿假单胞菌、麻风分枝杆菌、流产布鲁氏菌、狂犬病病毒、流感病毒、巨细胞 病毒、I型单纯疱疹病毒、II型单纯疱疹病毒、人血清细小样病毒、呼吸道合胞病毒、水痘-带状疱疹病毒、乙型肝炎病毒、丙型肝炎病毒、麻疹病毒、腺病毒、人T细胞白血病病毒、埃-巴二氏病毒、鼠类白血病病毒、腮腺炎病毒、水泡性口膜炎病毒、辛德比斯病毒、淋巴细胞性脉络丛脑膜炎病毒、疣病毒、蓝舌病病毒、仙台病毒、猫白血病病毒、呼肠孤病毒、脊髓灰质炎病毒、猿猴病毒40、鼠乳房肿瘤病毒、登革热病毒、风疹病毒、西尼罗河病毒、恶性疟原虫、间日疟原虫、刚地弓形虫、让氏锥虫、克氏锥虫、罗德西亚锥虫、布氏锥虫、曼氏裂体吸虫、日本裂体吸虫、牛巴贝虫、艾美耳球虫、旋盘尾丝虫、热带利什曼虫、旋毛虫、小泰勒虫、泡状带绦虫、羊绦虫、牛肉绦虫、细粒棘球绦虫、科氏中殖孔绦虫、关节炎支原体、鼻支原体、口腔支原体、精氨酸支原体、拉氏无胆甾原体、唾液支原体和肺炎支原体，如美国专利号6,440,416中所公开，该专利的实施例部分以引用的方式并入本文中。
可使用的其它抗体包括针对诸如细菌、病毒、支原体或其它病原体的感染性疾病因素的抗体。许多针对此类感染性因素的抗体在本领域中是已知的，并且任何此类已知抗体都可以用于要求保护的方法和组合物中。举例来说，可以使用以下抗体：针对人免疫缺陷病毒I(HIV-1)的gpl20糖蛋白抗原的抗体；针对疟疾寄生虫的抗体，诸如针对子孢子(环子孢子抗原)的单克隆抗体，针对血吸虫童虫表面抗原的抗体(Simpson等,Parasitology,83:163-177,1981；Smith等,Parasitology,84:83-91,1982；Gryzch等,J.Immunol.,129:2739-2743,1982；Zodda等,J.Immunol.129:2326-2328,1982；Dissous等,J.Immunol.,129:2232-2234,1982)；抗真菌抗体，诸如抗核盘菌抗体(美国专利7,910,702)，抗葡萄糖醒酸木糖甘露聚糖抗体(Zhong和Priofski,1998,Clin Diag Lab Immunol5:58-64)；抗假丝酵母抗体(Matthews和Burnie,2001,Curr Opin Investig Drugs 2:472-76)和抗鞘糖脂抗体(Toledo等,2010,BMC Microbiol 10:47)。
已经开发了针对大部分造成大多数人感染的微生物(细菌、病毒、原生生物、真菌、其它寄生虫)的合适的抗体，且许多抗体先前已用于体外诊断目的。这些抗体以及可以通过常规方法产生的改变的抗体适用于本发明。
用于偶联第一结合部分(例如抗体或其片段)和第二结合部分(例如抗体或其片段)的纳米材料可以是可药用的纳米材料，优选为生物可降解纳米材料，更优选为聚乳酸-羟基乙酸、聚乳酸、聚己内酯、聚丁二醇丁二酸酯、聚苯胺、聚碳酸酯、乙丙交酯共聚物或乙交酯己内酯共聚物中的任意一种或至少两种的混合物，最优选为聚乳酸-羟基乙酸(PLGA)、聚乳酸(PLA)和/或聚己内酯(PCL)。这些纳米材料及其制备方法是本领域已知的，例如可以采用下文描述的方法制备所述纳米材料。
本发明偶联物的制备
以下以包含偶联于纳米微粒的两种抗体的双特异性结合偶联物为例，描述本发明的多特异性结合偶联物的制备方法。所述制备方法包括以下步骤：
(1)纳米材料的制备、收集和活化；
(2)将步骤(1)得到的纳米材料与第一抗体部分和第二抗体部分的混合物进行连接。
在步骤(1)中，所述纳米材料的制备包括：利用溶剂将纳米材料完全溶解，搅拌，加水，形成均匀的乳浊液。其中所述搅拌可以在500-20000rpm/min的转速下进行，例如转速可以是500rpm/min、700rpm/min、800rpm/min、1000rpm/min、1100rpm/min、1200rpm/min、1300rpm/min、1400rpm/min、1480rpm/min、1500rpm/min、2000rpm/min、2200rpm/min、2500rpm/min、3000rpm/min、3500rpm/min、4000rpm/min、4200rpm/min、4500rpm/min、5000rpm/min、5500rpm/min、6000rpm/min、6500rpm/min、7000rpm/min、7500rpm/min、8000rpm/min、8500rpm/min、9000rpm/min、9500rpm/min、10000rpm/min、11000rpm/min、12000rpm/min、13000rpm/min、14000rpm/min、15000rpm/min、16000rpm/min、17000rpm/min、18000rpm/min、19000rpm/min或20000rpm/min。必要时，可以采用更高的转速。
优选地，所述纳米材料为聚乳酸-羟基乙酸(PLGA)、聚乳酸(PLA)、聚己内酯(PCL)、聚丁二醇丁二酸酯、聚苯胺、聚碳酸酯、乙丙交酯共聚物或乙交酯己内酯共聚物中的任意一种或至少两种的混合物。
优选地，所述溶剂为丙酮、丁酮、甲醇、乙醇或异丙醇中的任意一种 或至少两种的混合物。
优选地，所述纳米材料的收集包括：通过离心收集制备的纳米材料，然后用去离子水重悬，重复操作2次洗涤纳米材料。所述离心可以在8000-15000rpm/min的转速下进行，例如转速可以是8000rpm/min、9000rpm/min、10000rpm/min、11000rpm/min、12000rpm/min、13000rpm/min、14000rpm/min、14500rpm/min、14800rpm/min、15000rpm/min。必要时，可以采用更高的转速。可以采用其他方法收集或进一步纯化纳米材料(纳米微粒)。纳米微粒可以具有如上所述的平均粒径
优选地，所述纳米材料的活化包括：用1-10mg/mL的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDS)和N-羟基琥珀酰亚胺(NHS)混合溶剂于室温活化纳米材料0.5-5h。
本发明的步骤(2)中，所述连接包括：活化后的纳米材料通过离心收集，然后用连接反应液洗涤纳米材料1次。将需要连接的第一抗体部分和第二抗体部分等量混合加入到连接反应液，然后用含有所述第一抗体部分和第二抗体部分的连接反应液重悬纳米材料，于室温进行接反应0.5-5h。反应结束后离心收集纳米材料，用杜氏磷酸盐缓冲溶液洗涤纳米材料2次，然后再重悬到杜氏磷酸盐缓冲溶液(D-PBS)中放4℃保存备用。可以采用其他方法进行纳米材料的活化。
本发明多特异性结合偶联物的制备方法例如具体包括以下步骤：
(1)纳米材料的制备：利用丙酮将纳米材料完全溶解至浓度为5-30mg/mL，按照丙酮和去离子水1:4的体积比，在500-1500rpm/min磁力搅拌的状态下将纳米材料与丙酮的溶液加入去离子水中，形成均匀的乳浊液，然后继续搅拌至丙酮挥发；
(2)纳米材料的收集：8000-15000rpm/min离心收集制备的纳米材料，然后用去离子水重悬，重复操作2次洗涤纳米材料；
(3)纳米材料的活化：利用1-10mg/mL的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺混合溶剂室温活化纳米材料0.5-5h；
(4)纳米材料连接抗体：将活化后的纳米材料离心收集，然后用0.1M、pH＝8.0的杜氏磷酸盐缓冲溶液洗涤纳米材料1次，将需要连接的第一 抗体部分和第二抗体部分等量混合加入到连接反应液，然后用含有所述第一抗体部分和第二抗体部分的连接反应液重悬纳米材料，室温连接反应0.5-5h，反应结束后离心收集纳米材料，用杜氏磷酸盐缓冲溶液洗涤纳米材料2次，然后再重悬到杜氏磷酸盐缓冲溶液中放4℃保存备用。
药物组合物
本发明还涉及包含所述多特异性结合偶联物的药物组合物。所述药物组合物还可以包含白细胞，例如T细胞、NK细胞。
本文的药物组合物可使用一种或多种生理上可接受的载体进行配制，该载体包含促进活性剂进入药学上使用的制剂的过程的赋形剂和助剂。适当的配制依赖于所选的施用途径。例如，药物组合物的概述可以在例如以下文献中找到：Ansel等,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS,第5版(Lea&Febiger 1990)；和Gennaro(编),REMINGTON’S PHARMACEUTICAL SCIENCES,第18版(Mack Publishing Company 1990)和其修订版。本文公开的药物组合物可进一步包含药学上可接受的一种或多种稀释剂、赋形剂或载体。所述药物组合物可包含医药或药物剂、载体、佐剂诸如防腐剂、稳定剂、润湿剂或乳化剂、溶解促进剂、用于调节渗透压的盐和/或缓冲剂。
此外，该药物组合物还可以含有在治疗上有价值的其他物质,诸如一种或多种治疗疾病的其他治疗剂。例如，其他治疗剂可以是用于治疗癌症的其他药剂，例如环磷酰胺、依托泊苷、卡莫司汀、长春新碱等，也可以也用于治疗癌症的抗体、抗体-药物缀合物(ADC)、干扰素和/或检查点抑制子抗体。
治疗性治疗方法
各种实施方案涉及治疗受治疗者(诸如哺乳动物，包括人、驯养或伴侣宠物，诸如犬和猫)的疾病或病症(如癌症)的方法，其包括向受治疗者施用治疗有效量的本文所述的偶联物或药物组合物。
本文所述的偶联物或药物组合物可以经配制，经皮下或甚至通过其它肠胃外途径，诸如经静脉内、肌肉内、腹膜内或血管内施用于哺乳动物，经由例如推注或连续输注用于静脉内施用。优选地，偶联物或药物组合物输注持续少于约4个小时的时间，且更优选地持续少于约3个小时的时间。 举例来说，第一次推注可在30分钟，优选地甚至15分钟内输注，且其余部分在接下来的2-3小时内输注。注射制剂可以呈单位剂型提供，例如，在安瓿或多剂量容器中，其中添加防腐剂。组合物可采用诸如于油性或水性媒剂中的混悬液、溶液或乳液的形式，并且可含有配制剂，诸如悬浮剂、稳定剂和/或分散剂。替代地，活性成分可呈在使用前用合适的媒剂(例如无菌无热原质水)复原的粉末形式。
可以采用额外药学方法来控制治疗偶联物、药物组合物和/或其他治疗剂的作用持续时间。可以通过使用聚合物以复合或吸附欲施用的药剂来制备控制释放制剂。举例来说，生物相容性聚合物包括聚(乙烯-乙酸乙烯酯)共聚物基质和硬脂酸二聚体与癸二酸的聚酐共聚物基质。Sherwood等,Bio/Technology 10:1446(1992)。从此类基质释放的速率取决于治疗剂的分子量、基质内的药剂的量，和分散粒子的大小。Saltzman等,Biophys.J.55:163(1989)；Sherwood等,同上。其它固体剂型描述于以下文献中：Ansel等,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS,第5版(Lea&Febiger 1990)；以及Gennaro(编),REMINGTON’SPHARMACEUTICAL SCIENCES,第18版(Mack Publishing Company 1990)和其修订版。
更一般来说，所述的偶联物用于人类的施用剂量将取决于诸如患者年龄、体重、身高、性别、一般医学病状和既往病史的因素而变化。可能需要给接受者提供在约0.1μg/kg-25mg/kg范围内的作为单次静脉输注的偶联物剂量(以多特异性结合偶联物中的第一结合部分和第二结合部分的总量计)，但也可以施用更低或更高的剂量，视情况决定。举例来说，对于70kg患者，0.1μg/kg-20mg/kg的剂量是0.7μg-1400mg。需要时剂量可以重复，例如每周一次持续4-10周、每周一次持续8周或每周一次持续4周。在维持疗法中，需要时其还可以较低频率给予，诸如每隔一周一次持续若干个月，或者每月或每季度一次持续许多个月。也可以每个疗程连续施用2次、3次、4次、5次或6次，在连续施用后间隔例如约10天、15天、20天、25天、30天、35天、40天、45天、50、55天或60天或更长时间再进行下一个疗程。
所述的偶联物可以与效应细胞如白细胞(诸如T细胞、NK细胞)一起 施用，例如一起静脉回输。当偶联物与效应细胞一起施用时，例如每周一次持续4-10周、每周一次持续8周或每周一次持续4周。在维持疗法中，需要时偶联物与效应细胞还可以较低频率给予，诸如每隔一周或数周一次持续若干个月，或者每月或每季度一次持续许多个月。也可以每个疗程连续施用2次、3次、4次、5次或6次，在连续施用后间隔例如约10天、15天、20天、25天、30天、35天、40天、45天、50、55天或60天或更长时间再进行下一个疗程。
一般来说，其他治疗剂用于人类的施用剂量将取决于诸如患者年龄、体重、身高、性别、一般医学病状和既往病史的因素而变化。可能需要给接受者提供在约1mg/kg至25mg/kg范围内的作为单次静脉输注的偶联物剂量，但也可以施用更低或更高的剂量，视情况决定。举例来说，对于70kg患者，1-20mg/kg的剂量是70-1,400mg。需要时剂量可以重复，例如每周一次持续4-10周、每周一次持续8周或每周一次持续4周。在维持疗法中，需要时其还可以较低频率给予，诸如每隔一周一次持续若干个月，或者每月或每季度一次持续许多个月。
其他治疗剂可以按照每2周或3周一个剂量施用，总共重复至少3个剂量。或者，所述组合可以每周施用2次，持续4至6周。如果使剂量降至约200-300mg/m2(对于1.7m患者是每剂340mg，或对于70kg患者是4.9mg/kg)，则其可每周施用一次或甚至两次持续4至10周。替代地，给药时程可减少，即，每2或3周一次持续2-3个月。然而已经确定，甚至可通过缓慢i.v.输注施用甚至更高的剂量，诸如20mg/kg每周一次或每2-3周一次，用于重复给药循环。给药时程可任选地以其它间隔重复，且剂量可在对剂量和时程进行适当调节的情况下通过各种胃肠外途径给予。
在优选的实施方案中，本文所述的偶联物或药物组合物可用于治疗癌症疗法。癌症的实例包括但不限于癌瘤、淋巴瘤、成胶质细胞瘤、黑素瘤、肉瘤和白血病、骨髓瘤或淋巴恶性疾病。此类癌症的更特定实例如下所述且包括：鳞状细胞癌(例如上皮鳞状细胞癌)、尤因氏肉瘤、韦尔姆斯氏肿瘤、星形细胞瘤、肺癌(包括小细胞肺癌、非小细胞肺癌、肺腺癌和肺鳞状癌)、腹膜癌、肝细胞癌、胃癌或胃部癌症(包括胃肠癌)、胰腺癌、多形性 成胶质细胞瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、肝细胞瘤、肝细胞癌瘤、神经内分泌肿瘤、甲状腺髓样癌、甲状腺分化癌、乳腺癌、卵巢癌、结肠癌、直肠癌、子宫内膜癌或子宫癌、唾液腺癌、肾脏癌或肾癌、前列腺癌、阴门癌、肛门癌、阴茎癌以及头颈癌。术语“癌症”包括原发性恶性细胞或肿瘤(例如细胞未迁移至受治疗者体内除原始恶性病或肿瘤部位以外的部位的肿瘤)和继发性恶性细胞或肿瘤(例如由转移，即，恶性细胞或肿瘤细胞迁移至与原始肿瘤部位不同的次级部位而产生的肿瘤)。得益于本发明的治疗方法的癌症涉及表达、过度表达或异常表达IGF-1R的细胞。
癌症或恶性病的其它实例包括但不限于：急性儿童成淋巴细胞性白血病、急性成淋巴细胞性白血病、急性淋巴细胞性白血病、急性骨髓性白血病、肾上腺皮质癌、成人(原发性)肝细胞癌、成人(原发性)肝癌、成人急性淋巴细胞性白血病、成人急性骨髓性白血病、成人何杰金氏淋巴瘤、成人淋巴细胞性淋巴瘤、成人非何杰金氏淋巴瘤、成人原发性肝癌、成人软组织肉瘤、AIDS相关性淋巴瘤、AIDS相关性恶性病、肛门癌、星形细胞瘤、胆管癌、膀胱癌、骨癌、脑干神经胶质瘤、脑瘤、乳腺癌、肾盂和输尿管癌、中枢神经系统(原发性)淋巴瘤、中枢神经系统淋巴瘤、小脑星形细胞瘤、大脑星形细胞瘤、宫颈癌、儿童(原发性)肝细胞癌、儿童(原发性)肝癌、儿童急性成淋巴细胞性白血病、儿童急性骨髓性白血病、儿童脑干神经胶质瘤、儿童小脑星形细胞瘤、儿童大脑星形细胞瘤、儿童颅外胚细胞瘤、儿童何杰金氏病、儿童何杰金氏淋巴瘤、儿童下丘脑和视通路神经胶质瘤、儿童成淋巴细胞性白血病、儿童成神经管细胞瘤、儿童非何杰金氏淋巴瘤、儿童松果体和幕上原始神经外胚层瘤、儿童原发性肝癌、儿童横纹肌肉瘤、儿童软组织肉瘤、儿童视通路和下丘脑神经胶质瘤、慢性淋巴细胞性白血病、慢性髓细胞性白血病、结肠癌、皮肤T细胞淋巴瘤、内分泌胰岛细胞癌、子宫内膜癌、室管膜瘤、上皮癌、食道癌、尤因氏肉瘤和相关肿瘤、外分泌胰腺癌、颅外胚细胞瘤、性腺外胚细胞瘤、肝外胆管癌、眼癌、女性乳癌、高歇氏病、胆囊癌、胃部癌、胃肠道良性肿瘤、胃肠道肿瘤、胚细胞瘤、妊娠性滋养层细胞瘤、毛细胞白血病、头颈癌、肝细胞癌、何杰金氏淋巴瘤、高丙种球蛋白血症、下咽癌、肠癌、眼内黑素瘤、胰岛细胞癌、胰岛细胞胰腺癌、卡波济氏肉瘤、肾癌、喉癌、唇口腔癌、 肝癌、肺癌、淋巴增生性病症、巨球蛋白血症、男性乳腺癌、恶性间皮瘤、恶性胸腺瘤、成神经管细胞瘤、黑素瘤、间皮瘤、转移性原发灶隐匿性鳞状颈癌、转移性原发性鳞状颈癌、转移性鳞状颈癌、多发性骨髓瘤、多发性骨髓瘤/浆细胞赘瘤、骨髓发育不良综合症、髓细胞性白血病、骨髓性白血病、骨髓增生性病症、鼻腔和副鼻窦癌、鼻咽癌、成神经细胞瘤、非何杰金氏淋巴瘤、非黑素瘤皮肤癌、非小细胞肺癌、转移性原发灶隐匿性鳞状颈癌、口咽癌、骨肉瘤/恶性纤维肉瘤、骨肉瘤/恶性纤维组织细胞瘤、骨肉瘤/骨骼的恶性纤维组织细胞瘤、卵巢上皮癌、卵巢胚细胞瘤、卵巢低恶性潜能肿瘤、胰腺癌、病变蛋白血症、真性红细胞增多症、副甲状腺癌、阴茎癌、嗜铬细胞瘤、垂体肿瘤、原发性中枢神经系统淋巴瘤、原发性肝癌、前列腺癌、直肠癌、肾细胞癌、肾盂和输尿管癌、成视网膜细胞瘤、横纹肌肉瘤、唾液腺癌、肉状瘤病肉瘤、塞扎里综合症、皮肤癌、小细胞肺癌、小肠癌、软组织肉瘤、鳞状颈癌、胃癌、幕上原始神经外胚层和松果体瘤、T细胞淋巴瘤、睾丸癌、胸腺瘤、甲状腺癌、肾盂和输尿管的移行迁移细胞癌、移行迁移肾盂和输尿管癌、滋养层细胞瘤、输尿管和肾盂细胞癌、输尿管癌、子宫癌、子宫肉瘤、阴道癌、视通路和下丘脑神经胶质瘤、阴门癌、瓦尔登斯特伦巨球蛋白血症、韦尔姆斯氏瘤和位于以上所列出的器官系统中的除赘瘤以外任何其它过度增生性疾病。
本文中所描述和要求保护的偶联物、组合物和方法可用于治疗恶性或恶变前病状和预防进展成赘生性或恶性状态，包括但不限于上文所描述的那些病症。指示此类用途用于已知或疑似提前进展成赘瘤或癌症的病状，特别是在已发生由增生、化生或最特别的是发育不良组成的非赘生性细胞生长的情况下(关于此类异常生长病状的综述，参见Robbins和Angell,BASIC PATHOLOGY,第2版,W.B.Saunders Co.,Philadelphia,第68-79页(1976))。
发育不良往往是癌症的前兆，并且主要发现于上皮中。它是非赘生性细胞生长的最无序形式，涉及个别细胞一致性和细胞结构定向的丧失。在存在慢性刺激或炎症的情况下，特征性地发生发育不良。可以治疗的发育不良病症包括但不限于：无汗性外胚层发育不良、前面部发育不良、窒息性胸廓发育不良、心房-手指发育不良、支气管肺发育不良、大脑发育不良、 宫颈发育不良、软骨外胚层发育不良、锁骨颅骨发育不良、先天性外胚层发育不良、颅骨干发育不良、颅腕跗发育不良、颅骨干骺端发育不良、牙本质发育不良、骨干结构不良、外胚层发育不良、牙釉质发育不良、脑性眼球发育不全、偏侧骨骺发育不良、多发性骨骺发育不良、点状骨骺发育不良、上皮异常增生、面指生殖器发育不良、家族性颌骨纤维性发育不良、家族性白色皱褶性发育不良、纤维肌肉发育不良、骨纤维性结构不良、旺盛骨性发育不良、遗传性肾视网膜发育不良、有汗性外胚层发育不良、少汗性外胚层发育不良、淋巴细胞减少性胸腺发育不良、乳腺发育不良、下颌骨颜面发育不良、干骺端发育不良、蒙蒂尼氏发育不良、单骨纤维性骨发育不良、粘液上皮发育不良、多发性骨骺发育不良、眼耳脊椎发育不良、眼齿指发育不良、眼椎骨发育不全、牙原性发育不良、眼下颌骨发育不良、根尖周牙骨质结构不良、多骨纤维性结构不良、假性软骨发育不全性脊椎骨骺发育不良、视网膜发育不良、中隔-眼发育不良、脊椎骨骺发育不良和脑室径向发育不良。
可以治疗的其它赘生前病症包括但不限于良性异常增生性病症(例如良性肿瘤、纤维囊性病状、组织肥大、肠息肉或腺瘤和食道发育不良)、粘膜白斑病、角化病、博文氏病、农夫皮肤、日光性唇炎和日光性角化病。
在优选的实施方案中，使用本发明的方法抑制癌症，特别是上文所列出的癌症的生长、进展和/或转移。
其它过度增生性疾病、病症和/或病状包括但不限于恶性病和相关病症的进展和/或转移，诸如白血病(包括急性白血病(例如急性淋巴细胞性白血病、急性骨髓细胞性白血病(包括成骨髓细胞性、早幼粒细胞性、骨髓单核细胞性、单核细胞性和红白血病))和慢性白血病(例如慢性骨髓细胞性(粒细胞性)白血病和慢性淋巴细胞性白血病))、真性红细胞增多症、淋巴瘤(例如何杰金氏病和非何杰金氏病)、多发性骨髓瘤、瓦尔登斯特伦巨球蛋白血症、重链病和实体肿瘤，包括但不限于肉瘤和癌瘤，诸如纤维肉瘤、粘液肉瘤、脂肪肉瘤、软骨肉瘤、骨原性肉瘤、脊索瘤、血管肉瘤、内皮肉瘤、淋巴管肉瘤、淋巴管内皮肉瘤、滑膜瘤、间皮瘤、尤因氏瘤、平滑肌肉瘤、横纹肌肉瘤、结肠癌、胰腺癌、乳癌、卵巢癌、前列腺癌、鳞状细胞癌、基底细胞癌、腺癌、汗腺癌、皮脂腺癌、乳头状癌、乳头状腺癌、囊腺癌、 髓样癌、支气管癌、肾细胞癌、肝细胞瘤、胆管癌、绒毛膜癌、精原细胞瘤、胚胎癌、韦尔姆斯氏瘤、宫颈癌、睾丸癌、肺癌、小细胞肺癌、膀胱癌、上皮癌、神经胶质瘤、星形细胞瘤、成神经管细胞瘤、颅咽管瘤、室管膜瘤、松果体瘤、成血管细胞瘤、听神经瘤、少突神经胶质瘤、脑脊膜瘤、黑素瘤、成神经细胞瘤和成视网膜细胞瘤。
药盒/试剂盒
各种实施方案可涉及含有适合治疗或诊断患者的患病组织的组分的药盒/试剂盒。示例性药盒/试剂盒可以含有如本文中所描述的一种或多种偶联物、白细胞和/或其他治疗剂。如果未配制含有供施用的多种组分的组合物以便经由消化道递送，诸如通过口服递送，则可以包括能够通过一些其它途径递送试剂盒组分的装置。用于诸如肠胃外递送的应用的一种类型装置是注射器，其用于将组合物注入受治疗者体内。还可以使用吸入装置。在某些实施方案中，治疗剂可以呈含有无菌液体制剂或冻干制剂的预填充式注射器或自动注射笔形式提供。
药盒/试剂盒组分可以包装在一起或分隔于两个或更多个容器中。在一些实施方案中，容器可以是含有适合于复原的组合物的无菌冻干制剂的小瓶。药盒/试剂盒还可以含有一种或多种适合于复原和/或稀释其它试剂的缓冲液。可以使用的其它容器包括但不限于袋、盘、盒、管等等。药盒/试剂盒组分可以无菌包装和维持于容器中。可以包括的另一个组件是给药盒/试剂盒使用者的说明书。
实施例
提供以下实施例用于说明本发明，而非限制本发明的权利要求。
实施例1.双特异性结合偶联物抗CD56-PLGA-抗CD20的制备
制备了具有偶联于不同纳米颗粒(PLGA、PLA和PCL)的抗CD56抗体和抗CD20抗体的双特异性结合偶联物抗CD56-PLGA-抗CD20。
双特异性结合偶联物抗CD56-PLGA-抗CD20的具体制备方法如下：
(1)PLGA纳米颗粒的制备：用丙酮将PLGA完全溶解至浓度为5mg/mL，按照丙酮和去离子水1:4的体积比，在1000rpm/min磁力搅拌的状态下将PLGA与丙酮的溶液加入去离子水中，形成均匀的乳浊液，然后继续搅拌至丙酮挥发；
(2)PLGA纳米颗粒的收集：以8000rpm/min离心10min收集制备的较大颗粒的纳米颗粒；再以15000rpm/min离心10min收集得到较小颗粒的纳米颗粒。弃去较大颗粒的纳米颗粒，较小颗粒的纳米颗粒用去离子水重悬，重复操作2次洗涤纳米颗粒。使用较小颗粒的纳米颗粒分别进行如下操作；
(3)PLGA纳米颗粒的活化：用5mg/mL的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺混合溶剂于室温下活化PLGA纳米颗粒1h；
(4)PLGA纳米颗粒连接抗体：活化后的纳米材料通过离心收集，然后用0.1M、pH＝8.0的杜氏磷酸盐缓冲溶液洗涤纳米材料1次。将需要连接的抗CD56单克隆抗体(单抗)和抗CD20单抗(两种单抗均购于同立海源生物)等量混合加入到连接反应液，然后用含有所述抗CD56单抗和抗CD20单抗的连接反应液重悬纳米材料，于室温下连接反应进行0.5h。反应结束后离心收集纳米材料，用杜氏磷酸盐缓冲溶液洗涤纳米材料2次，然后再重悬到杜氏磷酸盐缓冲溶液中放4℃保存备用。
双特异性结合偶联物抗CD56-PLA-抗CD20的具体制备方法如下：
(1)PLA纳米颗粒的制备：用甲醇将PLA完全溶解至浓度为15mg/mL，按照甲醇和去离子水1:4的体积比，在500rpm/min磁力搅拌的状态下将PLA与甲醇的溶液加入去离子水中，形成均匀的乳浊液，然后继续搅拌至甲醇挥发；
(2)PLA纳米颗粒的收集：以10000rpm/min离心10min收集制备的纳米颗粒，然后用去离子水重悬，重复操作2次洗涤纳米颗粒；
(3)PLA纳米颗粒的活化：用1mg/mL的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺混合溶剂于室温活化PLA纳米颗粒0.5h；
(4)PLA纳米颗粒连接抗体：活化后的纳米材料通过离心收集，然后用0.1M、pH＝8.0的杜氏磷酸盐缓冲溶液洗涤纳米材料1次，将需要连接的抗CD56单抗和抗CD20单抗等量混合加入到连接反应液。然后用含有所述抗CD56单抗和抗CD20单抗的连接反应液重悬纳米材料，于室温进行连接反应5h。反应结束后离心收集纳米材料，用杜氏磷酸盐缓冲溶液洗涤纳米 材料2次，然后再重悬到杜氏磷酸盐缓冲溶液中放4℃保存备用。
双特异性结合偶联物抗CD56-PCL-抗CD20的具体制备方法如下：
(1)PCL纳米颗粒的制备：用异丙醇将PCL完全溶解至浓度为30mg/mL，按照异丙醇和去离子水1:4的体积比，在1500rpm/min磁力搅拌的状态下将PCL与异丙醇的溶液加入去离子水中，形成均匀的乳浊液，然后继续搅拌至异丙醇挥发；
(2)PCL纳米颗粒的收集：以15000rpm/min离心收集制备的纳米颗粒，然后用去离子水重悬，重复操作2次洗涤纳米颗粒；
(3)PCL纳米颗粒的活化：用10mg/mL的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺混合溶剂室温活化PLGA纳米颗粒2h；
(4)PCL纳米颗粒连接抗体：活化后的纳米材料通过离心收集，然后用0.1M、pH＝8.0的杜氏磷酸盐缓冲溶液洗涤纳米材料1次。将需要连接的抗CD56单抗和抗CD20单抗等量混合加入到连接反应液，然后用含有所述抗CD56单抗和抗CD20单抗的连接反应液重悬纳米材料，室温连接反应3h。反应结束后离心收集纳米材料，用杜氏磷酸盐缓冲溶液洗涤纳米材料2次，然后再重悬到杜氏磷酸盐缓冲溶液中放4℃保存备用。
实施例2.其他双特异性结合偶联物的制备
按照类似于实施例1中所述的方法，制备了其他双特异性结合偶联物—抗CD16-PLGA-抗MUC1、抗CD56-PLGA-抗MUC1、抗CD16-PLGA-抗CD19、抗CD56-PLGA-抗CD19、抗CD16-PLGA-抗CD20和抗CD56-PLGA-抗CD20。其中所用的抗体皆为市售的单克隆抗体。
此外，按照类似于实施例1中所述的方法，还制备了抗CD56-PLA-抗CD19、抗CD56-PCL-抗CD19、抗CD16-PLA-抗CD20、抗CD16-PCL-抗CD20、抗CD16-PLA-抗MUC1和抗CD16-PCL-抗MUC1。
实施例3.双特异性结合偶联物对癌细胞的杀伤效力
对实施例1和实施例2中制备的双特异性结合偶联物杀伤肿瘤细胞的能力进行了评估。
具体而言，于96孔板中，以5000个靶细胞/孔培养12h后，弃原培养基。用无细胞因子的X-vivo 15培养基(购于lonza公司)调整NK细胞(用 健康献血者的外周血单核细胞诱导扩增获得)密度，使100μl体积NK细胞数量为靶细胞4倍(效靶比为4:1)。加100μl NK细胞悬液到癌细胞培养板中，并加入10μl制备的双特异性结合偶联物(双特异性结合偶联物含量为0.2mg，含单抗成分共0.2μg)，于培养箱中孵育8h。然后加入CCK-8试剂，按照试剂说明进行孵育。用酶标仪检测450nm处吸光值。对数据进行统计分析，按照以下公式计算DC-CIK细胞对癌细胞的杀伤率。
杀伤率＝[1-(实验组-效应对照组)/(靶对照组-空白对照组)]×100％
其中，空白对照表示加入培养基；靶对照组表示加入靶细胞+培养基；效应对照组表示加入效应细胞+培养基；实验组表示效应细胞+靶细胞+培养基+双特异性结合偶联物。
结果显示于表1-2和图2-7中。
表1.双特异性结合偶联物对肿瘤细胞的杀伤能力(效靶比为4:1)
Figure PCTCN2016101984-appb-000001
注：HKC—人胚肾上皮细胞；A549—人非小细胞肺癌细胞；HepG2—人肝癌细胞；HT29—人结肠癌细胞；AGS—人胃腺癌细胞；Hela—人宫颈癌细胞；5637—人膀胱癌细胞；OS-RC-2—人肾癌细胞。
表2.双特异性结合偶联物对肿瘤细胞的杀伤能力(效靶比为4:1)
Figure PCTCN2016101984-appb-000002
注：Raji—黑人Burkitt淋巴瘤细胞；RAMOS—人B淋巴细胞瘤细胞。
抗CD56-PLA-抗CD19和抗CD56-PCL-抗CD19对于黑人Burkitt淋巴瘤细胞和人B淋巴细胞瘤细胞表现出83％以上的杀伤率(具体数据未显示)。
抗CD16-PLA-抗CD20和抗CD16-PCL-抗CD20对于黑人Burkitt淋巴瘤细胞和人B淋巴细胞瘤细胞表现出83％以上的杀伤率(具体数据未显示)。
抗CD16-PLA-抗MUC1和抗CD16-PCL-抗MUC1对于人非小细胞肺癌细胞表现出高于95％的杀伤率；对于人膀胱癌细胞和人乳腺癌细胞表现出高于92％的杀伤率；对于人宫颈癌细胞和人胃腺癌细胞均表现出接近85％的杀伤率；对于人结肠癌细胞、人肾癌细胞和人肝癌细胞均表现出80％以上的杀伤率。
实施例4体外抗肿瘤实验
按照前述实施例的方法，制备通过PLGA连接的抗CD3和/或抗CD16和抗MUC1双特异性或三特异性结合偶联物(抗CD3-PLGA-抗MUC1、抗CD16-PLGA-抗MUC1或抗CD3加抗CD16-PLGA-抗MUC1，下文分别简称为CD3-MUC1组、CD16-MUC1组和CD3/CD16-MUC1组)，其中所用的抗体均为市售抗体。
对所述双特异性或三特异性结合偶联物辅助NK杀伤A549细胞的能力进行了评估。具体而言，采集健康人外周血50ml，分离单核细胞(PBMC)，然后加入NK细胞因子进行诱导和扩增培养。细胞培养至第21天时检测NK细胞比例。检测结果如下：其中NK细胞比例为86.9％，CD3+T细胞比例为10.2％(参见图8)。于96孔板中，将作为靶细胞的A549细胞以5000个细胞/孔培养12h，然后弃原培养基。利用无细胞因子的X-vivo 15培养基调整NK细胞密度，使100μl体积NK细胞数量为靶细胞4倍(效靶比为4:1)。加100μl NK细胞悬液到癌细胞培养板中，并加入10μl所制备的双特异性结合偶联物(双特异性结合偶联物制剂含量为0.2mg，含单抗成分共0.2μg)。于培养箱中孵育8h，然后加入CCK-8试剂，按照试剂说明进行孵育，用酶标仪检测450nm处吸光值。对数据进行统计分析，按照以下公式计算NK细胞对癌细胞的杀伤率。
杀伤率＝[1-(实验组-效应对照组)/(靶对照组-空白对照组)]×100％
实验结果表明(参见图9)：NK、CD3单抗+NK、CD16单抗+NK、MUC1单 抗+NK呈现出一定疗效，统计结果显示与对照组相比肿瘤抑制率分别为26.1％、33.02％、26.22％、37.51％，各组之间无显著差异。CD3-MUC1、CD16-MUC1、CD3/CD16-MUC1组辅助NK细胞对肿瘤抑制率分别为71.02％、81.84％、90.91％，均呈现出良好的疗效，其中CD3/CD16-MUC1>CD16-MUC1>CD3-MUC1,也刚好与NK细胞中细胞成分相关，NK普通表达CD16，且NK细胞的比例远大于CD3+T细胞比例，所以用CD16做为NK的识别位点比较合适。因为目前体外诱导的NK细胞，NK比例为86.9％左右，其中还有部分其他效应细胞，包括CD3+T细胞比例为10.2％，所以同时连接CD16和CD3，其抗瘤效果更优。
实施例5.体内抗肿瘤实验
对实施例4中所述的双特异性或三特异性结合偶联物于体内抗肿瘤的效力进行了评估。具体而言，在健康Balb-c裸鼠(清洁级，雌性，四周龄，体重18-22g，购自北京维通利华实验动物技术有限公司)腋下接种人肺腺癌细胞A549，接种3-4周后取瘤体直径约0.5×0.5cm的裸鼠90只随机分成8组用于实验(分组如下所示)。
Figure PCTCN2016101984-appb-000003
于第1、2、3天分别采用尾静脉注射方式给药。各实验组每次注射20μl抗体或结合偶联物制剂，制剂中含有每种单抗12.5μg，即单抗组每只 动物注射0.25μg抗体，双特异性结合偶联物组每只动物注射的抗体总量为0.50μg，三特异性结合偶联物组每只动物注射的抗体总量为0.75μg。对照组注射同等体积的生理盐水。
从第一天起开始测量记录瘤体体积。计算双特异性/三特异性结合偶联物对肿瘤的治疗效果。
图10显示统计肿瘤体积变化情况。
本实验前22天测量的结果表明：NK、CD3单抗+NK、CD16单抗+NK、MUC1单抗+NK呈现出一定疗效，目前统计结果显示与对照组相比肿瘤抑制率分别为39.24％、36.19％、33.49％、37.01％，各组之间无显著差异。CD3-MUC1、CD16-MUC1、CD3/CD16-MUC1辅助NK细胞对肿瘤抑制率分别为81.22％、88.24％、95.15％，均呈现出良好的疗效，其中CD3/CD16-MUC1>CD16-MUC1>CD3-MUC1,也刚好与NK细胞中细胞成分相关，NK普通表达CD16，其比例远大于CD3+T细胞比例，所以用CD16做为NK的识别位点比较合适。因为目前体外诱导的NK细胞，NK比例为86.9％左右，其中还有部分其他效应细胞，包括CD3+T细胞比例为10.2％，所以同时连接CD16和CD3，其抗瘤效果更优。
***
根据本公开内容，不需要过度实验就可以制备和使用本文中所公开和要求保护的所有偶联物、组合物和方法。尽管已经就优选实施方案描述了组合物和方法，但本领域技术人员应显而易见，可在不背离本发明的理念、精神和范围的情况下对本文中所描述的偶联物、组合物和方法以及方法中的步骤或步骤顺序施加变化。更具体地说，可以用化学上和生理学上相关的某些试剂来替代本文中所描述的试剂，同时可实现相同或类似的结果。认为对本领域技术人员显而易见的所有此类相似的取代和修改都在如由所附权利要求所限定的本发明精神、范围和理念之内。

Claims (18)

  1. 一种多特异性结合偶联物，其包含：
    a)至少一种结合细胞毒性效应细胞上受体、共受体、抗原和/或细胞标志物的第一结合部分，所述细胞毒性效应细胞优选是细胞毒性细胞，所述细胞毒性细胞优选是白细胞，所述白细胞优选选自巨噬细胞、嗜中性粒细胞、嗜酸性粒细胞、NK细胞、B细胞和T细胞；和
    b)至少一种结合靶细胞上受体、共受体、抗原和/或细胞标志物的第二结合部分，所述靶细胞优选为患病细胞或病原体；
    其中所述第一结合部分可选自小分子、细胞靶向分子、配体、蛋白质、肽、类肽、DNA适体、肽核酸、维生素、底物或底物类似物、抗体或其片段，优选为配体、受体、适体、抗体或其片段；所述第二结合部分可选自小分子、细胞靶向分子、配体、蛋白质、肽、类肽、DNA适体、肽核酸、维生素、底物或底物类似物、抗体或其片段，优选为配体、受体、适体、抗体或其片段；
    其中所述第一结合部分和所述第二结合部分偶联于纳米微粒上，所述纳米微粒优选为生物可降解纳米材料，更优选为聚乳酸-羟基乙酸、聚乳酸、聚己内酯、聚丁二醇丁二酸酯、聚苯胺、聚碳酸酯、乙丙交酯共聚物或乙交酯己内酯共聚物中的任意一种或至少两种的混合物。
  2. 根据权利要求1所述的偶联物，其包含：
    a)至少一种结合白细胞上受体、共受体、抗原和/或细胞标志物的第一结合部分，所述受体、共受体、抗原和/或细胞标志物优选选自ADAM17、CD2、CD3、CD4、CD5、CD6、CD8、CD11a、CD11b、CD14、CD16、CD16b、CD25、CD28、CD30、CD32a、CD38、CD40、CD40L、CD44、CD45、CD56、CD57、CD64、CD69、CD74、CD89、CD90、CD137、CD177、CEACAM6、CEACAM8、HLA-DRα链、SLC44A2以及LSECtin、CTLA4、PD1、PD-L1、LAG3、B7-H3、B7-H4、KIR和TIM3；和
    b)至少一种结合患病细胞或病原体上受体、共受体、抗原和/或细胞标志物的第二结合部分。
  3. 根据权利要求2所述的偶联物，其中所述患病细胞为肿瘤细胞，所述患病细胞上的受体、共受体、抗原和/或细胞标志物选自：碳酸酐酶IX、甲胎蛋白、α-辅肌动蛋白-4、A3、A33抗体特异性抗原、AFP、ART-4、B7、Ba 733、BAGE、BrE3-抗原、CA125、CAMEL、CAP-1、CASP-8/m、、CCCL19、CCCL21、CD1、CD1a、CD2、CD3、CD4、CD5、CD8、CD11A、CD14、CD15、CD16、CD18、CD19、CD20、CD21、CD22、CD23、CD25、CD29、CD30、CD32b、CD33、CD37、CD38、CD40、CD40L、CD45、CD46、CD52、CD54、CD55、CD59、CD64、CD66a-e、CD67、CD70、CD70L、CD74、CD79a、CD79b、CD80、CD83、CD95、CD126、CD132、CD133、CD138、CD147、CD154、CDC27、CDK-4/m、CDKN2A、CXCR4、CXCR7、CXCL12、HIF-1α、结肠特异性抗原-p(CSAp)、CEA(CEACAM5)、CEACAM6、c-met、DAM、EGFR、EGFRvIII、EGP-1、EGP-2、ELF2-M、Ep-CAM、Flt-1、Flt-3、叶酸受体、G250抗原、GAGE、gp100、GROB、HLA-DR、HM1.24、人绒毛膜促性腺激素(HCG)和其亚单位、HER2/neu、HMGB-1、低氧可诱导因子(HIF-1)、HSP70-2M、HST-2、Ia、IGF-1R、IFN-γ、IFN-α、IFN-β、IL-2、IL-4R、IL-6R、IL-13R、IL-15R、IL-17R、IL-18R、IL-6、IL-8、IL-12、IL-15、IL-17、IL-18、IL-23、IL-25、胰岛素样生长因子-1(IGF-1)、KC4-抗原、KS-1-抗原、KS1-4、Le-Y、LDR/FUT、巨噬细胞迁移抑制因子(MIF)、MAGE、MAGE-3、MART-1、MART-2、NY-ESO-1、TRAG-3、mCRP、MCP-1、MIP-1A、MIP-1B、MIF、MUC1、MUC2、MUC3、MUC4、MUC5ac、MUC13、MUC16、MUM-1/2、MUM-3、NCA66、NCA95、NCA90、胰腺癌粘蛋白、胎盘生长因子、p53、PLAGL2、前列腺酸性磷酸酶、PSA、PRAME、PSMA、PlGF、ILGF、ILGF-1R、IL-6、IL-25、RS5、RANTES、T101、SAGE、S100、存活蛋白、存活蛋白-2B、TAC、TAG-72、腱生蛋白、TRAIL受体、TNF-α、Tn抗原、汤姆森-弗里德里希抗原、肿瘤坏死抗原、TROP-2、VEGFR、ED-B纤连蛋白、WT-1、17-1A-抗原、补体因子C3、C3a、C3b、C5a、C5、血管生成标记物、bcl-2、bcl-6、Kras、cMET、致癌基因产物。
  4. 根据权利要求2所述的偶联物，其中所述患病细胞为肿瘤细胞，所述患病细胞上的受体、共受体、抗原和/或细胞标志物选自胆囊收缩素B受体、促性腺激素释放激素受体、促生长素抑制素受体2、avb3整联蛋白、胃泌素释放肽受体、神经激肽1受体、黑皮质素1受体、神经降压肽受 体、神经肽Y受体和C型凝集素样分子1的细胞表面蛋白。
  5. 根据权利要求1-4中任一项所述的偶联物，其中所述第一结合部分结合CD16或CD56。
  6. 根据权利要求2所述的偶联物，其中所述病原体选自：HIV病毒、结核分枝杆菌(mycobacterium tuberculosis)、无乳链球菌(Streptococcus agalactiae)、抗甲氧西林性金黄色葡萄球菌(meticillin-resistant Staphylococcus aureus)、嗜肺军团菌(Legionella pneumophilia)、酿脓链球菌(Streptococcus pyogenes)、大肠杆菌(Escherichia coli)、淋病奈瑟氏菌(Neisseria gonorrhoeae)、脑膜炎奈瑟氏菌(Neisseria meningitidis)、肺炎球菌(Pneumococcus)、新型隐球菌(Cryptococcus neoformans)、荚膜组织胞浆菌(Histoplasma capsulatum)、B型流感嗜血杆菌(Hemophilis influenzae B)、苍白密螺旋体(Treponema pallidum)、莱姆病螺旋体(Lyme disease spirochetes)、铜绿假单胞菌(Pseudomonas aeruginosa)、麻风分枝杆菌(Mycobacterium leprae)、流产布鲁氏菌(Brucella abortus)、狂犬病病毒、流感病毒、巨细胞病毒、I型单纯疱疹病毒、II型单纯疱疹病毒、人血清细小样病毒、呼吸道合胞病毒、水痘-带状疱疹病毒、乙型肝炎病毒、丙型肝炎病毒、麻疹病毒、腺病毒、人T细胞白血病病毒、埃-巴二氏病毒、鼠白血病病毒、腮腺炎病毒、水泡性口膜炎病毒、辛德毕斯病毒、淋巴细胞性脉络丛脑膜炎病毒、疣病毒、蓝舌病病毒、仙台病毒、猫白血病毒、呼肠孤病毒、脊髓灰质炎病毒、猿猴病毒40、小鼠乳腺瘤病毒、登革热病毒、风疹病毒、西尼罗河病毒、恶性疟原虫(Plasmodium falciparum)、间日疟原虫(Plasmodium vivax)、刚地弓形虫(Toxoplasma gondii)、让氏锥虫(Trypanosoma rangeli)、克氏锥虫(Trypanosoma cruzi)、罗德西亚锥虫(Trypanosoma rhodesiensei)、布氏锥虫(Trypanosoma brucei)、曼森氏裂体吸虫(Schistosoma mansoni)、日本裂体吸虫(Schistosoma japonicum)、牛巴贝虫(Babesia bovis)、艾美耳球虫(Elmeria tenella)、旋盘尾丝虫(Onchocerca volvulus)、热带利什曼虫(Leishmania tropica)、旋毛虫(Trichinella spiralis)、小泰勒虫(Theileria parva)、泡状带绦虫(Taenia hydatigena)、羊绦虫(Taenia ovis)、牛肉绦虫(Taenia  saginata)、细粒棘球绦虫(Echinococcus granulosus)、科氏中殖孔绦虫(Mesocestoides corti)、关节炎支原体(Mycoplasma arthritidis)、鼻支原体(M.hyorhinis)、口腔支原体(M.orale)、精氨酸支原体(M.arginini)、拉氏无胆甾原体(Acholeplasma laidlawii)、唾液支原体(M.salivarium)和肺炎支原体(M.pneumoniae)。
  7. 根据权利要求1-5中任一项所述的偶联物，其中所述第二结合部分结合选自AFP、CD19、CD20、CD38、MUC1、EGFR和HER2/neu的抗原。
  8. 根据权利要求1-5中任一项所述的偶联物，其中所述第二结合部分为抗体或其片段，所述抗体选自hR1(抗IGF-1R)、hPAM4(抗粘蛋白)、KC4(抗粘蛋白)、hA20(抗CD20)、hA19(抗CD19)、hIMMU31(抗AFP)、hLL1(抗CD74)、hLL2(抗CD22)、RFB4(抗CD22)、hMu-9(抗CSAp)、hL243(抗HLA-DR)、hMN-14(抗CEACAM5)、hMN-15(抗CEACAM6)、hRS7(抗TROP-2)、hMN-3(抗CEACAM6)、CC49(抗TAG-72)、J591(抗PSMA)、D2/B(抗PSMA)、G250(抗碳酸酐酶IX)、英夫利昔单抗(抗TNF-α)、聚乙二醇化赛妥珠单抗(certolizumab pegol)(抗TNF-α)、阿达木单抗(抗TNF-α)、阿仑单抗(抗CD52)、贝伐单抗(抗VEGF)、西妥昔单抗(抗EGFR)、吉姆单抗(抗CD33)、替伊莫单抗(抗CD20)、帕尼单抗(panitumumab)(抗EGFR)、利妥昔单抗(抗CD20)、托西莫单抗(抗CD20)、GA101(抗CD20)、曲妥单抗(抗HER2/neu)、托珠单抗(tocilizumab)(抗IL-6受体)、巴利昔单抗(抗CD25)、达克珠单抗(抗CD25)、依法利珠单抗(efalizumab)(抗CD11a)、莫罗单抗(muromonab)-CD3(抗CD3受体)、那他珠单抗(抗α4整合素)、BWA-3(抗组蛋白H2A/H4)、LG2-1(抗组蛋白H3)、MRA12(抗组蛋白H1)、PR1-1(抗组蛋白H2B)、LG11-2(抗组蛋白H2B)、LG2-2(抗组蛋白H2B)、P4/D10(抗gp120)和奥玛珠单抗(omalizumab)(抗IgE)。
  9. 根据权利要求1-5中任一项所述的偶联物，其中所述第二抗体选自hA19、hR1、hPAM4、hA20(维妥珠单抗(veltuzumab))、hIMMU31、hLL1(米拉珠单抗(milatuzumab))、hLL2(依帕珠单抗)、hMu-9、hL243、hMN-14、hMN-15、hRS7和hMN-3。
  10. 根据权利要求1-9中任一项所述的偶联物，其中所述纳米微粒为聚乳酸-羟基乙酸、聚乳酸和/或聚己内酯。
  11. 一种药物组合物，其包含权利要求1-10中任一项所述的偶联物。
  12. 根据权利要求11所述的药物组合物，其还包含白细胞，所述白细胞优选包含NK细胞。
  13. 根据权利要求11或12所述的药物组合物，其还包含其他治疗剂，所述治疗剂优选选自抗体、抗体片段、药物、毒素、酶、细胞毒性剂、抗血管生成剂、促细胞凋亡剂、抗生素、激素、免疫调节剂、细胞因子、趋化因子、反义寡核苷酸、小干扰RNA(siRNA)、硼化合物和放射性同位素。
  14. 一种治疗有需要的受治疗者的疾病或病症的方法，其包括向所述受治疗者施用前述任一项权利要求所述的多特异性结合偶联物或药物组合物。
  15. 根据权利要求14的方法，其中其中所述疾病或病症是癌症，优选为选自以下的癌症：非何杰金氏淋巴瘤、B细胞淋巴瘤、B细胞白血病、T细胞淋巴瘤、T细胞白血病、急性淋巴细胞性白血病、慢性淋巴细胞性白血病、伯基特淋巴瘤、何杰金氏淋巴瘤、毛细胞白血病、急性骨髓性白血病、慢性骨髓性白血病、多发性骨髓瘤、神经胶质瘤、华氏巨球蛋白血症、癌瘤、黑素瘤、肉瘤、神经胶质瘤、皮肤癌、口腔癌、胃肠道癌、结肠癌、胃癌、肺癌、肺癌、乳腺癌、卵巢癌、前列腺癌、子宫癌、子宫内膜癌、子宫颈癌、膀胱癌、胰腺癌、骨癌、肝癌、胆囊癌、肾癌、睾丸癌、上皮癌、结直肠癌、胃癌、脑癌、胶质母细胞瘤、胰腺癌、髓样白血病、宫颈癌、甲状腺髓样癌、星形细胞瘤、前列腺癌、膀胱癌、神经内分泌癌、胃肠胰肿瘤、外分泌胰腺癌和尤因肉瘤。
  16. 根据权利要求14或15所述的方法，其还包括向所述受治疗者施用其他治疗剂，所述治疗剂优选选自抗体、抗体片段、药物、毒素、酶、细胞毒性剂、抗血管生成剂、促细胞凋亡剂、抗生素、激素、免疫调节剂、细胞因子、趋化因子、反义寡核苷酸、小干扰RNA(siRNA)、硼化合物和放射性同位素。
  17. 根据权利要求1-13任一项所述的偶联物或药物组合物在制备用于治疗有需要的受治疗者的疾病或病症的药物中的用途。
  18. 根据权利要求17的用途，其中其中所述疾病或病症是癌症，优选为选自以下的癌症：非何杰金氏淋巴瘤、B细胞淋巴瘤、B细胞白血病、T 细胞淋巴瘤、T细胞白血病、急性淋巴细胞性白血病、慢性淋巴细胞性白血病、伯基特淋巴瘤、何杰金氏淋巴瘤、毛细胞白血病、急性骨髓性白血病、慢性骨髓性白血病、多发性骨髓瘤、神经胶质瘤、华氏巨球蛋白血症、癌瘤、黑素瘤、肉瘤、神经胶质瘤、皮肤癌、口腔癌、胃肠道癌、结肠癌、胃癌、肺癌、肺癌、乳腺癌、卵巢癌、前列腺癌、子宫癌、子宫内膜癌、子宫颈癌、膀胱癌、胰腺癌、骨癌、肝癌、胆囊癌、肾癌、睾丸癌、上皮癌、结直肠癌、胃癌、脑癌、胶质母细胞瘤、胰腺癌、髓样白血病、宫颈癌、甲状腺髓样癌、星形细胞瘤、前列腺癌、膀胱癌、神经内分泌癌、胃肠胰肿瘤、外分泌胰腺癌和尤因肉瘤。
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