WO2019127346A1 - 募集抗体并靶向肿瘤细胞的双功能分子 - Google Patents

募集抗体并靶向肿瘤细胞的双功能分子 Download PDF

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WO2019127346A1
WO2019127346A1 PCT/CN2017/119779 CN2017119779W WO2019127346A1 WO 2019127346 A1 WO2019127346 A1 WO 2019127346A1 CN 2017119779 W CN2017119779 W CN 2017119779W WO 2019127346 A1 WO2019127346 A1 WO 2019127346A1
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group
bifunctional molecule
molecule according
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吴志猛
周志昉
洪皓飞
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江南大学
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Priority to PCT/CN2017/119779 priority Critical patent/WO2019127346A1/zh
Priority to CN201780085083.0A priority patent/CN111971060B/zh
Priority to US16/650,078 priority patent/US20210198381A1/en
Publication of WO2019127346A1 publication Critical patent/WO2019127346A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
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    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • 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
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • 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
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to a bifunctional molecule for recruiting antibodies and targeting tumor cells, and belongs to the technical field of biopharmaceuticals.
  • VHH nanobodies
  • RGD arginine-glycine-aspartate sequence-containing polypeptides
  • the antibody recruitment strategy consists in: (1) utilizing highly abundant antibodies naturally present in the body, such as dinitrobenzene (DNP), galactose- ⁇ -1,3-galactose ( ⁇ Gal), rhamnose (Rha) and An antibody corresponding to an antigen such as phosphorylcholine (PC); (2) using a ZZ domain molecule that specifically binds to immunoglobulin (IgG) to capture antibodies in serum, mediating immune activity of multiple pathways of the immune system Kill the cells of interest.
  • DNP dinitrobenzene
  • ⁇ Gal galactose- ⁇ -1,3-galactose
  • Rha rhamnose
  • PC phosphorylcholine
  • IgG immunoglobulin
  • the present inventors have developed a series of pharmaceutical compounds containing different linkers for use in immunotherapy of tumors.
  • a first object of the present invention is to provide a series of nanobody-based antibody recruitment bifunctional molecules for tumor immunotherapy, the bifunctional molecule having the structural formula shown in Formula 1,
  • A is a structure capable of recruiting an antibody, and includes a hapten, a polypeptide, a Z/ZZ domain affinity peptide capable of binding to an antibody, and the like; and B is a substance capable of binding to a cell surface, including but not limited to an oligopeptide, Polypeptides, Nanobodies, fragments having antibody function;
  • Linker is a linking structure linking A and B, including but not limited to chemical bonds or molecular linkers.
  • A is an antibody hapten structure that binds naturally to the human body.
  • the naturally occurring antibodies in the human body include, but are not limited to, DNP and Rha groups.
  • B is RGD and derivatives thereof, including but not limited to RGD cyclic peptides; said RGD is arginine-glycine-aspartate.
  • B is NGR and a derivative thereof, and the NGR is asparagine-glycine-arginine.
  • B is LHRH and a derivative thereof; said LHRH is a progesterone-releasing hormone.
  • B comprises a Nanobody against a tumor cell epidermal growth factor receptor EGFR, a Nanobody against HER2, a Nanobody against a prostate specific membrane antigen (PSMA), or A fusion protein of the above Nanobody with Z(ZZ) domain.
  • a in the bifunctional molecule comprises two different haptens or a Z/ZZ domain affinity peptide.
  • the structural formula of the bifunctional molecule is: Among them, A1 and A2 are two different hapten structures capable of binding to naturally occurring antibodies in humans; B contains tumor-targeting Nanobodies; Linker is a linking structure linking A and B, including but not limited to chemical bonds or molecular connections body.
  • the naturally occurring antibodies in the human body include, but are not limited to, DNP and Rha groups.
  • B comprises a Nanobody against a tumor cell epidermal growth factor receptor EGFR, a Nanobody against HER2, a Nanobody against a prostate specific membrane antigen (PSMA), or A fusion protein of the above Nanobody with Z(ZZ) domain.
  • B is linked to the linker by a sortase A enzyme.
  • B with LPXTGn and a linker with Gn are connected by sortaseA
  • the structural formula of the linker is Or -LPXTGn-; said -LPXTGn- is -leucine-valine-X-threonine-n glycine; wherein n is an integer from 1 to 10; n1 is an integer from 1 to 4; n2 is an integer from 2 to 2 9; n3 is an integer 2-9; X is O or S or N.
  • Near the X-end is a hapten structure, the other end is B, and B is connected by SortaseA.
  • the haptens are the same or different near the X-terminus, and the other end is B, and B is linked by SortaseA.
  • the structural formula of the bifunctional molecule is:
  • A is an antibody-binding end which has a hapten structure capable of binding to an existing antibody in a patient;
  • B contains a Nanobody capable of binding to a cell;
  • L 1 and L 2 are a linker, and contain one or more bifunctional linkers a group CON;
  • MULTICON is a difunctional or polyfunctional linker group, when MCON ⁇ 1, MULTICON connects at least one A to B via a linker L 1 and/or L 2 ;
  • MCON is an integer from 0 to 10; n and n' are each independently an integer from 1 to 15, usually from 2 to 10, usually from 2 to 5, more usually from 2 to 3, or 2, 3, 4, 5 or 6; NL1 and NL2 are each an integer of 0 to 10, and n ⁇ NL1, and n' ⁇ NL2.
  • the MCON is 1, 2 or 3.
  • the MCON is one.
  • n is 1, 2 or 3 and n' is 1 or 2.
  • NL1 is 1 and NL2 is 1.
  • MCON is 0, NL1 is 1 and NL2 is 1.
  • MCON is 0, n is 1 and n' is 1.
  • NL1 is one.
  • NL2 is one.
  • the B comprises an immunoglobulin single variable domain, or a Nanobody, or a polypeptide that can bind to a cell surface target and derivatives thereof; the polypeptide and its derivatives The object can target a target on the cell surface.
  • the polypeptide of B can be targeted to bind to a cell surface target; the cell is a diseased cell, including but not limited to a cancer cell.
  • the structure of B is: Wherein X is any amino acid and a derivative thereof, the -LPXTGn- is -leucine-valine-X-threonine-n glycine; X is O or S or N; n is 1-100 The integer.
  • the bifunctional linker group CON comprises:
  • X 2 is O, S, NR 4 , S(O), S(O) 2 , -S(O) 2 O, -OS(O) 2 or O S(O) 2 O;
  • X 3 is O, S And NR 4 ; and
  • R 4 is H, a C 1 -C 3 alkyl or alkanol group, or a -C(O)(C 1 -C 3 ) group; or a pharmaceutically acceptable salt thereof.
  • the bifunctional linker group CON is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • CL is m is an integer from 0 to 12, typically 0, 1, 2, 3, 4, 5, 6; and IL is 0 or 1, usually 1.
  • the linker L 1 or L 2 comprises a polyethylene glycol (PEG) linker, a polypropylene glycol linker or a polyethylene glycol-co-polypropylene polymer, the length of which It is from 1 to 100 units.
  • PEG polyethylene glycol
  • the linker L 1 or L 2 is a polyproline linker or a collagen linker: wherein the polyvaline linker has the structural formula The collagen linker structure is Where n is from 1 to 100.
  • the linker L 1 or L 2 is Any one of wherein; R a is H, C 1 -C 3 alkyl or alkanol or forms a ring with R 3 , R 3 is a side chain derived from an amino acid; m is an integer from 1 to 100; And m" is an integer from 0 to 25; any of the above groups may be further linked by an amide group, a ketone group, an amine group or an amino acid.
  • the linker L 1 or L 2 is Wherein R a is H or C 1 -C 3 alkyl, usually CH 3 , most typically H; m is an integer from 1 to 12, usually 1, 2, 3, 4, 5 or 6; m" is an integer 1, 2, 3, 4, 5 or 6, usually 6; t is 1, 2, 3, 4, 5 or 6; and iL is 0 or 1, wherein the linker is optionally attached to the A group at one end And at the other end optionally attached to the B group.
  • the linker L 1 or L 2 is Wherein q is an integer from 0 to 12; and q' is from 1 to 12; iL is 0 or 1; and R L is an amino acid or an oligopeptide;
  • each X S is independently S, O or NR S ;
  • R S is H or C 1 -C 3 alkyl;
  • S C is CH 2 , CH 2 O or CH 2 CH 2 O;
  • i is 0 or 1;
  • m s is 0, 1, 2, 3, 4, 5 or 6;
  • Z and Z' are each independently a bond, including -(CH 2 ) i -S, -(CH 2 ) i -NR, Or optionally bonded to another linker, linker, A group or B group; each R is H, or an alkyl or alkanol group; each R2 is independently H or alkyl; each Y independently Is a bond, O, S or NR; each i is independently from 0 to 100;
  • D is Or key, or Or a polypropylene glycol or polypropylene-co-polyethylene glycol linking group having 1 to 100 diols; wherein Z, Z' and D are each not a bond; each i is independently 0 to 100; 1 to 100; m is an integer from 1 to 100; and n is an integer from 1 to 100; m' is from 1 to 100; m" is an integer from 0 to 25, usually from 1 to 10, usually from 1 to 8; more usually 1, 2, 3, 4, 5 or 6; n' is an integer from 1 to 100, or from 1 to 75, or from 1 to 60, or from 1 to 5, or from 1 to 50, or from 1 to 45, or from 1 to 40, Or 2 to 35, or 3 to 30, or 1 to 15, or 1 to 10, or 1 to 8, or 1 to 6, or 1, 2, 3, 4 or 5; X i is O, S or NR; R is H, or an alkyl or alkanol group; R a is H, C 1 -C 3 alkyl or al
  • the bifunctional or polyfunctional linker group MULTICON group comprises:
  • X 2 is O, S, NR 4 , S(O), S(O) 2 , -S(O) 2 O, -OS(O) 2 or O S(O) 2 O;
  • X 3 is O, S And NR 4 ; and
  • R 4 is H, a C 1 -C 3 alkyl or alkanol group, or a -C(O)(C 1 -C 3 ) group; or a pharmaceutically acceptable salt thereof.
  • L 1 and/or L 2 are a group; wherein R a is H or CH 3 ; m is an integer from 1 to 12; m" is an integer of 1, 2, 3, 4, 5 or 6; t is 0, 1, 2, 3, 4, 5 or 6 ; and iL is 0 or 1.
  • L 1 and/or L 2 are Wherein R a is H; m" is an integer of 1, 2, 3, 4, 5 or 6 and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.
  • L 1 and/or L 2 in the compound is a polyethylene glycol linkage having a length of from 1 to 12 diol units, or extends to the second via the CON group.
  • a polyethylene glycol linkage of a polyethylene glycol linkage the polyethylene glycol linkage has a length of from 1 to 12 diol units and the second polyethylene glycol linkage has a length of from 1 to 12 Glycol unit.
  • the CON group is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • A has a moiety consisting of:
  • DNP group is optionally attached to the amino acid moiety through an X group;
  • X is O, CH 2 , NR 1 , SO, SO 2 , -S(O) 2 O, -OS(O) 2 or -OS (O) 2 O.
  • A has a moiety of the formula:
  • Y' is H or NO 2 ;
  • X is O, CH 2 , NR 1 , SO, SO 2 , -S(O) 2 O, -OS(O) 2 or -OS(O) 2 O;
  • R 1 Is a H, C 1 -C 3 alkyl or -C(O)(C 1 -C 3 ) group;
  • Xs is OH or NHAc;
  • X R is O or S;
  • X b is a chemical bond O, CH 2 , NR 1 or S;
  • X' is CH 2 , O, NR 1 ' (R 1 ' is H or C 1 -C 3 alkyl) or S;
  • X" is O, CH 2 , NR 1 (R 1 is H, C The 1- C 3 alkyl or -C(O)(C 1 -C 3 ) group)
  • Z is a chemical bond, a monosaccharide, a disaccharide, an oligo
  • the recruitment structures used in the present invention are all conventional small molecules or are easily fermented to obtain ZZ domain, are easily available, and can effectively recruit related antibodies in human serum.
  • the targeting structures used in the present invention are all readily synthesized polypeptides, cyclic peptides or antibody proteins which are easily obtained by fermentation, and all of them can achieve high affinity and high specific binding with antigens related to tumor surface, and their binding ability No less than traditional monoclonal antibodies.
  • connection mode of the recruitment structure and the targeting structure is not complicated, and the product is also easy to be purified.
  • both the Sortase A and the click chemistry can be used to rapidly modify the molecule in a physiological environment.
  • the bifunctional molecules provided by the invention can specifically target tumor cell cells, and the semi-binding constant is usually less than 10 nM, and can effectively recruit anti-DNP, anti-Rha and other antibodies, and mediate CDC or ADCC to varying degrees. Achieve killing of tumor cells (20%-90%).
  • the present invention also links the recruitment structure and the targeting structure through a series of different chains (including different chain lengths, chain types), and evaluates different chains, different recruitment structures (types, quantities), and targeting structures (types, quantities).
  • the effect of the tumor killing effect of the final compound is optimized by the series of bifunctional molecules, which can provide valuable foundation and reference for the development of similar tumor drugs in the future.
  • FIG. 1 Mass spectrum of the target product; (A) GGG-triEGDNP, (B) GGG-hexEGDNP;
  • Figure 2 is a flow cytogram; wherein the ordinate MFI refers to the mean fluorescence intensity, the abscissa in the left panel indicates that the corresponding compound is incubated with the cells, and the negative control indicates that the cells are incubated with an equal volume of PBS; Indicates the type of cell being incubated.
  • Figure 3 shows the tumor killing effect; among them, (A) ADCC effect; (B) CDC effect; the ordinate corresponds to the cell lysis rate, the abscissa indicates that the corresponding compound is incubated with the cells, and the negative control indicates that the same volume of PBS is used. The cells were incubated.
  • Figure 4 is the target product mass spectrum (A) GGG-triEGRha, (B) GGG-hexEGRha;
  • Figure 5 is a flow cytogram; wherein the ordinate MFI refers to the mean fluorescence intensity, the abscissa in the left panel indicates that the corresponding compound is incubated with the cells, and the negative control indicates that the cells are incubated with an equal volume of PBS; Indicates the type of cell being incubated.
  • Figure 6 shows the tumor killing effect; (A) ADCC effect; (B) CDC effect; the ordinate corresponds to the cell lysis rate, the abscissa indicates that the corresponding compound is incubated with the cells, and the negative control indicates that the cells are treated with an equal volume of PBS. Incubate.
  • Figure 7 shows the flow cytometry, CDC, and ADCC results of MCF-7 cells; the ordinate on the ordinate indicates the fluorescence intensity, the lysis% corresponds to the cell lysis rate, and the abscissa indicates that the corresponding compound is incubated with the cells. Negative control indicates The cells were incubated with an equal volume of PBS.
  • Figure 8 is a mass spectrometry result of the compound NGR-DNP
  • Figure 9 shows the flow cytometry, CDC, and ADCC results of MCF-7 cells; the ordinate on the ordinate represents the fluorescence intensity, the lysis% corresponds to the cell lysis rate, and the abscissa indicates that the corresponding compound is incubated with the cells, Negative control indicates The cells were incubated with an equal volume of PBS.
  • Figure 10 is mass spectral data of RGD-DNP (panel A) and RGD-linker-DNP (panel B);
  • Figure 11 shows the results of cell experiments;
  • A is the flow cytometry, CDC, and ADCC results of HEK-293 cells; the ordinate on the ordinate represents the fluorescence intensity, the lysis% corresponds to the cell lysis rate, and the abscissa indicates the corresponding Compounds were incubated with cells and Negative control indicated incubation of cells with an equal volume of PBS.
  • B Flow cytometry experiments of HT-29, SKOV-3 and HEK-293 cells with RGD-linker-DNP; ordinate MFI indicates fluorescence intensity, and abscissa indicates the type of cells incubated, wherein negative control indicates SKOV3 cells were incubated with an equal volume of PBS.
  • Example 1 takes anti-EGFR nanobody (7D12) and GGG-triEGDNP as examples
  • the high resolution of the target product is shown in Figure 1.
  • the molecular weight detected by GGG-triEGDNP was 487.1792 (M+H + ), and the molecular weight detected by GGG-hexEGDNP was 619.2580 (M+H + ), which was consistent with the prediction, indicating that the synthesized product was correct.
  • the cells were centrifuged, the medium was discarded, and the medium was resuspended in a buffer (10-100 mM Tris-HCl or PBS, pH 7-8), and the wall was broken by ultrasonication. After breaking the wall, the wall was removed with a nickel column. Purification and desalting using a desalting column or ultrafiltration or dialysis. Purification of the desalted 7D12 by SDS-PAGE showed that there was only one band and the molecular weight was consistent with the prediction (16.7 KD).
  • DNP was modified in 7D12 in Tris-HCl buffer, and reacted at 4-37 °C for 10-120 min. After reaction, unreacted 7D12 was removed with Ni + magnetic beads. Excess small molecules are removed by dialysis or exclusion. The product is purified (can be filtered by dialysis, ultrafiltration and exclusion), sterile filtered and stored at -20 °C until use.
  • Cell culture commercialized cells A431 (high expression of EGFR), MCF7 (low expression of EGFR) were cultured in DMEM complete medium, 4T1 (high expression of mouse EGFR) was cultured in 1640 complete medium, and all cell culture conditions were cultured. 37 ° C, 5% CO 2 ;
  • ADCC experiment The experimental procedure is as follows: 1 Add 100 ⁇ L of A431 cell suspension (10000 cells/well) to the corresponding wells of a 96-well plate. After the cells are fully attached, add diluted 7D12 or 7D12-triEGDNP or 7D12-hexEGDNP. Immediately add a certain concentration of IgG; 2 after incubation for a period of time, add freshly extracted PBMC cells at a certain effective target ratio for a certain period of time; 3 centrifuge to remove supernatant LDH is a method for detection.
  • Fig. 3(A) The results are shown in Fig. 3(A). Compared with the cell viability of other controls, the killing rates were 31% and 37% after treatment with 7D12-triEGDNP or 7D12-hexEGDNP, respectively, while the cell loss of the control group was only about 5%, indicating Both products of the synthesis can induce the ADCC effect, and the killing power of the two is almost the same.
  • the experimental procedure is to add 100 ⁇ L of A431 cell suspension (5000 cells/well) to the corresponding wells of a 96-well plate. After the cells are fully attached, add diluted 7D12 or 7D12-triEGDNP or 7D12-hexEGDNP, and Immediately after adding a certain concentration of IgG2 for a period of time, a certain amount of complement was added, and the cell activity was detected by the 3cck8 method for a certain period of time, and converted into corresponding cytotoxicity.
  • the killing rates were 21% and 27%, respectively, after treatment with 7D12-triEGDNP or 7D12-hexEGDNP, compared with the cell viability of the other controls, while the cell death rate of the control group was lower than 5 %, indicating that both products synthesized can induce CDC effects.
  • the CDC effect induced by 7D12-hexEGDNP was slightly stronger than 7D12-triEGDNP.
  • the cells were centrifuged, the medium was discarded, and the medium was resuspended in a buffer (10-100 mM Tris-HCl or PBS, pH 7-8), and the wall was broken by ultrasonication. After breaking the wall, the wall was removed with a nickel column. Purification and desalting using a desalting column or ultrafiltration or dialysis.
  • the Rha modification of C7b in Tris-HCl buffer was carried out at 4-37 ° C for 10-120 min. After the reaction, the unreacted C7b was removed with Ni + magnetic beads, and the excess small molecules were removed. The product is purified by dialysis or exclusion, and the product is purified (can be filtered by dialysis, ultrafiltration and exclusion), then sterile filtered and stored at -20 ° C for use.
  • the flow results indicate that the synthesized C7b-triEGRha and C7b-hexEGRha can specifically recognize and bind to HER2 on the target cell surface.
  • the addition of C7b without hapten modification has no fluorescence intensity.
  • ADCC experiment The experimental procedure is to add 100 ⁇ L of SKBR3 cell suspension (10000 cells/well) to the corresponding wells of a 96-well plate. After the cells are completely attached, add diluted C7b or Rha-modified C7b, and immediately add a certain amount. After incubating the concentration of anti-Rha IgG2 for a period of time, the freshly extracted PBMC cells were added at a certain effective target ratio, and the LDH was centrifuged for a certain period of time to determine the cytotoxicity.
  • Fig. 6(A) indicate that the synthesized C7b-triEGRha and C7b-hexEGRha can induce ADCC effects, and their corresponding killing rates are 32% and 43%, respectively, and the ADCC effect induced by C7b-hexEGRha is stronger than C7b- TriEGRha.
  • C7b-triEGRha and C7b-hexEGRha can induce significant CDC effects relative to the cell viability of other control groups (killing rate is less than 5%), and their killing rates reached 36%, respectively. And 47%, and the CDC effect induced by C7b-hexEGRha is stronger than C7b-triEGRha.
  • Rhamnose is coupled to the LHRH polypeptide and targeted to kill tumor cells by recruiting antibodies present in the body to mediate immune effects.
  • LHRH-Rhamnose conjugate (LHRH-Rhamnose, LHRH-Rha) is:
  • D-lysine (D-Lys(N 3 )) with azide group (-N 3 ) was carried out at the position of 6-glycine without affecting its activity. Substituted to become a [6-D-Lys(N 3 )]-LHRH polypeptide molecule (the amino acid sequence is: pyroglutamate-histidine-tryptophan-serine-tyrosine-D-lysine (side) Chain azide group -N 3 )-leucine-arginine-valine-glycine-amino group, Glp-His-Trp-Ser-Tyr-D-Lys(N 3 )-Leu-Arg-Pro -Gly-NH 2 ), wherein Glp is pyroglutamic acid.
  • the crude peptide was obtained by precipitation with ice diethyl ether, and then purified by HPLC to obtain the peptide [D-Lys6]-LHRH, which was confirmed by mass spectrometry, and [M+Na + ] was 1301.6, which was the correct product.
  • the synthesized compound is The synthetic route is as follows:
  • Embodiment of cell experiment cell culture: commercial cells MCF-7 (LHRH-R high expression) and UCI-107 (LHRH non-expression) were cultured in DMEM complete medium, all cell culture conditions were 37 ° C, 5% CO 2 .
  • Flow cytometry The experimental procedure was as follows: trypsinize the cells, resuspend the cells by flow, dilute the cells to 1 ⁇ 10 5 , and take 400 ⁇ L of cells (ie 40,000 cells) to the sterile EP tube 2 for final concentration.
  • LHRH, LHRH-Rha, LHRH-TEG-Rha and LHRH-HEG-Rha compounds for the blank group, add the corresponding volume of PBS, and immediately add anti-antibody at a concentration of 20 ⁇ g/mL in all groups.
  • Anti-Rha antibody incubate for 1 h on ice, then wash the cells, add fluorescent secondary antibody, incubate for 0.5 h on ice; 3 thoroughly wash the cells with flow buffer, resuspend, and test with instrument .
  • Flow cytometry, ADCC, CDC results are shown in Figure 7.
  • the results show that LHRH-Rha, LHRH-TEG-Rha and LHRH-HEG-Rha conjugated compounds can recruit anti-Rha antibodies and target high expression of LHRH.
  • MCF-7 tumor cells, and mediate the immune response CDC and ADCC further kill MCF-7 tumor cells, and these three compounds are mediated by the same immune killing effect (CDC-mediated killing rate is about 26%, ADCC The induced killing rate is about 33%.
  • the length of the connecting arm has little effect on the activity.
  • the NGR polypeptide is a polypeptide comprising an asparaginic acid-arginine-glycine sequence.
  • the linear NGR polypeptides are CYGGRGNG, CNGRCVSGCAGRC, GNGRGGVRSSSRTPSDKYC and the like.
  • the NGR polypeptide is a targeting peptide that binds to the CD13 receptor on tumor neovascular endothelial cells and is capable of effectively targeting tumor cells. Therefore, the present invention contemplates the synthesis of bifunctional molecules based on NGR-based antibodies, coupling the structure of DNP to NGR, and mediated immune response targets by recruiting antibodies against DNP and targeting tumor angiogenic endothelial cells with high expression of CD13. To kill tumor cells.
  • Embodiment of Cell Experiment Cell culture: Commercialized cells HUVEC (high expression of CD13) and cultured in DMEM complete medium, all cell culture conditions were 37 ° C, 5% CO 2 .
  • the flow cytometry experiment was performed by trypsinizing the cells, resuspending the cells by flow, diluting the cells to 1 ⁇ 10 5 , and taking 400 ⁇ L of cells (ie, 40,000 cells) to the sterile EP tube 2 to a final concentration of 250 nM.
  • NGR oligopeptide NGR peptide, amino acid sequence: GGGCNGRC
  • NGR-DNP NGR-DNP
  • PBS PBS
  • anti-DNP anti-DNP
  • -DNP antibody incubate for 1 h on ice, then wash the cells and incubate with fluorescent secondary antibody for 0.5 h on ice; 3 wash the cells thoroughly with flow buffer, resuspend and test with the instrument.
  • NGR peptide amino acid sequence: GGGCNGRC
  • CDC immune response CDC and ADCC (cell lysis rate about 60%) further kill tumor cells.
  • Integrins are highly expressed on the surface of various tumor cells, but are not expressed or expressed in normal cells, and integrin plays an important role in adhesion, migration, invasion and tumor angiogenesis of tumor cells. Therefore, integrins are An important target for cancer treatment. Since the integrin receptor-specific ligand arginine-glycine-aspartate (Arg-Gly-Asp, RGD) is capable of specifically binding to integrins, RGD and its derivatives have been used to develop tumor-targeted therapeutic strategies.
  • Arg-Gly-Asp arginine-glycine-aspartate
  • the synthesized compound was confirmed to be correct by mass spectrometry, and the mass spectrum is shown in FIG.
  • the actual molecular weight of RGD-DNP is 769.78, and 770.38 (M+H + ) and 792.26 (M+Na + ) appearing in the mass spectrum are consistent with the prediction; the molecular weight of RGD-linker-DNP is 882.3, the quality appearing in the mass spectrum.
  • Both 453.57 (M+Na + H + ) and 883.44 (M+H + ) and 905.44 (M+Na + ) were consistent with the predictions, indicating that the product was synthesized correctly.
  • the activity detection protocols for the compounds RGD-DNP and RGD-linker-DNP are as follows: including flow cytometry, CDC experiments, and ADCC experiments.
  • the specific implementation scheme is as follows:
  • Cell assays Cell culture: Commercialized cells HEK-293 (integrin ⁇ v ⁇ 3 high expression) SKOV-3 (integrin ⁇ v ⁇ 3 and ⁇ v ⁇ 5 are expressed) and HT-29 (integrin ⁇ v ⁇ 5 high expression) cultured in DMEM completely Medium, all cell culture conditions were 37 ° C, 5% CO 2 .
  • Flow cytometry The experimental procedure was as follows: trypsinize the cells, resuspend the cells by flow, dilute the cells to 1 ⁇ 10 5 , and take 400 ⁇ L of cells (ie 40,000 cells) to the sterile EP tube 2 for final concentration.
  • RGD-DNP and RGD-linker-DNP compounds for the blank group, add the corresponding volume of PBS, and immediately add anti-DNP (anti-) at a concentration of 20 ⁇ g/mL in all groups.
  • DNP anti-DNP
  • DNP antibody incubated on ice for 1 h, then wash the cells, add fluorescent secondary antibody, incubate for 0.5 h on ice; 3 thoroughly wash the cells with flow buffer, resuspend, and test with the instrument.
  • Example of ADCC experiment 1 Add 100 ⁇ L of HEK-293, SKOV-3 and HT-29 cell suspension (10000/well) to the corresponding wells of a 96-well plate. After the cells are fully attached, add RGD at a final concentration of 250 nM. Analogs, RGD-DNP and RGD-linker-DNP compounds, and immediately add a certain concentration of anti-DNP antibody 2 after incubation for a period of time, add freshly extracted PBMC cells according to a certain target ratio, and process for 3 minutes to centrifuge. Clear, cytotoxicity was measured by LDH method.
  • the longer RRG-linker-DNP compound with the long arm is more effective in killing the virus (the short link arm corresponds to CDC and ADCC killing rate of only 21% and 18%).
  • the CDC and ADCC kill rates for the long link arms reached 42% and 38%, consistent with the results of flow cytometry experiments. This result indicates that the recruitment of DNP antibodies requires that the DNP moiety be exposed outside of the RGD-integrin complex. If the linker is too short, it will affect the binding of the anti-DNP antibody to the RGD-integrin complex. Thus, the results of the present invention optimize the length of a connecting arm.

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Abstract

本发明公开了募集抗体并靶向肿瘤细胞的双功能分子，属于生物制药技术领域。本发明的双功能分子包含至少一个能够结合到体内已有的抗体的募集结构和一个选择性结合肿瘤细胞的靶向性部分。根据本发明的化合物选择性结合至癌细胞表面，并通过募集结构招募已有抗体，介导患者中对癌细胞的体液应答和细胞应答反应。根据本发明的化合物的这一生理作用，癌细胞可以被免疫调节，为肿瘤免疫治疗提供了新的策略。

Description

募集抗体并靶向肿瘤细胞的双功能分子 技术领域
本发明涉及募集抗体并靶向肿瘤细胞的双功能分子，属于生物制药技术领域。
背景技术
近年来，癌症的发病率和死亡率都迅速增加，2015年我国新增肿瘤患者400多万，死亡人数280多万。为了解决传统疗法靶向性低、副作用大等问题，增强抗癌药物的靶向性并降低其副作用被认为是有效的改进策略。
目前市面上应用最广的为基于单克隆抗体的靶向药物，例如用于治疗霍杰金淋巴瘤的利妥昔单抗。然而，自身巨大的分子量(150KD)和昂贵的生产的成本所带来的缺陷则限制了单克隆抗体的肿瘤治疗效果和在临床上的应用。虽然之后分子量更小的抗体片段如单链抗体(ScFv)、抗原结合片段(Fab)得以开发，但它们相对完整单克隆抗体亲和力和稳定性都有所欠缺。
因此，开发和利用亲和性和稳定性都不亚于的小分子靶向性抗体/化合物是解决这一问题的关键。在此类小分子中，纳米抗体(VHH)、含精氨酸-甘氨酸-天冬氨酸序列的多肽(RGD)等都已经被广泛证明具有良好的靶向性。
另一方面，为了降低传统药物对正常细胞的毒性，解决肿瘤细胞的耐药性等问题，调动自身免疫系统对靶细胞进行杀伤的免疫疗法广受关注。其中，抗体募集策略在于：(1)利用体内自然存在的高丰度的抗体，如二硝基苯(DNP)，galactose-α-1,3-galactose(αGal)，鼠李糖(Rha)和磷酰胆碱(PC)等抗原对应的抗体；(2)利用ZZ domain此类可以特异性结合免疫球蛋白(IgG)的分子捕捉血清内游离的抗体，介导免疫系统多重途径的免疫活性从而杀伤目的细胞。
此外，通过何种方式，选用何种linker对靶向部分和募集部分进行连接仍不具备完善的理论研究体系和值得借鉴的成熟经验。
发明内容
基于上述肿瘤靶向的分子和抗体募集分子策略，本发明开发了一系列含不同linker的药用化合物，用于肿瘤的免疫治疗。
本发明的第一个目的是提供一系列用于肿瘤免疫治疗的基于nanobody的抗体募集双功能分子，所述双功能分子的结构式如式1所示，
式1：
Figure PCTCN2017119779-appb-000001
其中，A为能够招募抗体的结构，包括能够与抗体结合 的半抗原、多肽、Z/ZZ domain亲和肽及其类似物；B为能够与细胞表面结合的物质，包括但不限于寡肽、多肽、纳米抗体、具有抗体功能的片段；Linker为连接A和B的连接结构，包括但不限于化学键或分子连接体。
在本发明的一种实施方式中，A为能结合人体内天然存在的抗体半抗原结构。
在本发明的一种实施方式中，所述人体内天然存在的抗体包括但不限于DNP和Rha基团。
在本发明的一种实施方式中，B为RGD及其衍生物，包括但不限于RGD环肽；所述RGD为精氨酸-甘氨酸-天冬氨酸。
在本发明的一种实施方式中，B为NGR及其衍生物，所述NGR为天冬酰胺-甘氨酸-精氨酸。
在本发明的一种实施方式中，B为LHRH及其衍生物；所述LHRH是促黄体酮激素释放激素。
在本发明的一种实施方式中，B包括抗肿瘤细胞表皮生长因子受体EGFR的纳米抗体、抗HER2的纳米抗体、抗前列腺特异性膜抗原(prostate specific membrane antigen,PSMA)的纳米抗体，或上述纳米抗体与Z(ZZ)domain的融合蛋白。
在本发明的一种实施方式中，所述双功能分子中A包括两种不同的半抗原或Z/ZZ domain亲和肽。
在本发明的一种实施方式中，所述双功能分子的结构式为：
Figure PCTCN2017119779-appb-000002
其中，A1和A2为两种不同的能够结合人体内天然存在的抗体的半抗原结构；B含有靶向肿瘤的纳米抗体；Linker为连接A和B的连接结构，包括但不限于化学键或分子连接体。
在本发明的一种实施方式中，所述人体内天然存在的抗体包括但不限于DNP和Rha基团。
在本发明的一种实施方式中，B包括抗肿瘤细胞表皮生长因子受体EGFR的纳米抗体、抗HER2的纳米抗体、抗前列腺特异性膜抗原(prostate specific membrane antigen,PSMA)的纳米抗体，或上述纳米抗体与Z(ZZ)domain的融合蛋白。
在本发明的一种实施方式中，B与linker通过sortaseA酶进行连接。
在本发明的一种实施方式中，带有LPXTGn的B和带有Gn的linker通过sortaseA连接
在本发明的一种实施方式中，linker的结构式为
Figure PCTCN2017119779-appb-000003
Figure PCTCN2017119779-appb-000004
或-LPXTGn-；所述-LPXTGn-为-亮氨酸-脯氨酸-X-苏氨酸-n个甘氨酸；其中n为整数1～10；n1为整数1～4；n2为整数2-9；n3为整数2-9；X为O或S或N。
在本发明的一种实施方式中，
Figure PCTCN2017119779-appb-000005
靠近X端为半抗原结构，另一端为B，B通过SortaseA连接。
在本发明的一种实施方式中，
Figure PCTCN2017119779-appb-000006
靠近X端为相同或不同的半抗原，另一端为B，B通过SortaseA连接。
-LPXTGn-适用于SortaseA将纳米抗体和Z(ZZ)domain直接进行连接，所述纳米抗体连接在Z/ZZ domain的N端或C端。
在本发明的一种实施方式中，所述双功能分子的结构式为：
Figure PCTCN2017119779-appb-000007
其中，A是抗体结合末端，其具有能结合到病人体内已有抗体的半抗原结构；B含有能够结合细胞的纳米抗体；L 1和L 2为连接基，含有一个或多个双官能连接体基团CON；MULTICON为双官能或多官能连接体基团，当MCON≥1时，MULTICON通过连接基L 1和/或L 2将至少一个A与B连接；
MCON为整数0～10；n和n’各自独立地为整数1～15，通常为2～10，通常为2～5，更通常为2～3，或2、3、4、5或6；NL1和NL2各自为整数0～10，且n≥NL1，且n’≥NL2。
在本发明的一种实施方式中，MCON为1、2或3。
在本发明的一种实施方式中，MCON为1。
在本发明的一种实施方式中，n为1、2或3，且n’为1或2。
在本发明的一种实施方式中，NL1为1且NL2为1。
在本发明的一种实施方式中，MCON为0，NL1为1且NL2为1。
在本发明的一种实施方式中，MCON为0，n为1且n’为1。
在本发明的一种实施方式中，NL1为1。
在本发明的一种实施方式中，NL2为1。
在本发明的一种实施方式中，所述B包含一种免疫球蛋白单可变结构域，或纳米抗体，或可以靶向结合细胞表面靶标的多肽及其衍生物；所述多肽及其衍生物可以靶向细胞表面的靶标。
在本发明的一种实施方式中，所述B的多肽可以靶向结合细胞表面靶标；所述细胞是有病细胞，包括但不限于癌细胞。
在本发明的一种实施方式中，B的结构为：
Figure PCTCN2017119779-appb-000008
其中，X为任一氨基酸及其衍生物，所述-LPXTGn-为-亮氨酸-脯氨酸-X-苏氨酸-n个甘氨酸；X为O或S或N；n为1-100的整数。
在本发明的一种实施方式中，所述双官能连接体基团CON包括：
Figure PCTCN2017119779-appb-000009
其中X 2为O、S、NR 4、S(O)、S(O) 2、-S(O) 2O、-OS(O) 2或O S(O) 2O；X 3为O、S、NR 4；且R 4为H、C 1-C 3烷基或烷醇基，或-C(O)(C 1-C 3)基团；或其可药用盐。
在本发明的一种实施方式中，所述双官能连接体基团CON为
Figure PCTCN2017119779-appb-000010
其中CL为
Figure PCTCN2017119779-appb-000011
m为整数0～12，通常为0、1、2、3、4、5、6；且IL为0或1，通常为1。
在本发明的一种实施方式中，所述连接基L 1或L 2包括聚乙二醇(PEG)连接基、聚丙二醇连接基或聚乙二醇-共-聚丙烯聚合物，它们的长度为1至100单位。
在本发明的一种实施方式中，所述连接基L 1或L 2为聚脯氨酸连接基或胶原连接基：其 中，聚脯氨酸连接基结构式为
Figure PCTCN2017119779-appb-000012
胶原连接基结构式为
Figure PCTCN2017119779-appb-000013
其中n为1至100。
在本发明的一种实施方式中，所述连接基L 1或L 2为
Figure PCTCN2017119779-appb-000014
中的任一种；其中，R a是H、C 1-C 3烷基或烷醇或与R 3形成一环，R 3为衍生自氨基酸的侧链；m是从1到100的整数；且m”是从0到25的整数；上述任一基团均可进一步通过酰胺基、酮基、胺基或氨基酸连接。
在本发明的一种实施方式中，所述连接基L 1或L 2为
Figure PCTCN2017119779-appb-000015
其中，R a是H或C 1-C 3烷基，通常为CH 3，最通常为H；m为整数1至12，通常为1、2、3、4、5或6；m”为整数1、2、3、4、5或6，通常为6；t为1、2、3、4、5或6；且iL为0或1，其中所述连接基任选在一端连接至A基团，且在另一端任选连接至B基团。
在本发明的一种实施方式中，所述连接基L 1或L 2为
Figure PCTCN2017119779-appb-000016
Figure PCTCN2017119779-appb-000017
其中q为整数0～12；且q’为1～12；iL为0或1；且R L为氨基酸或寡肽；
或根据以下化学结构的连接基琥珀酰亚胺：
Figure PCTCN2017119779-appb-000018
其中各X S独立地为S、O或N-R S；R S为H或C 1-C 3烷基；S C为CH 2、CH 2O或CH 2CH 2O；i为0或1；且m s为0、1、2、3、4、5或6；
或根据以下化学式的连接基：
Figure PCTCN2017119779-appb-000019
其中Z和Z’各自独立地为键，包括-(CH 2) i-S，-(CH 2) i-N-R，
Figure PCTCN2017119779-appb-000020
Figure PCTCN2017119779-appb-000021
或任选键合至另一连接基、连接体、A基团或B基团；每个R为H，或烷基或烷醇基；每个R2独立地为H或烷基；每个Y独立地为键、O、S或N-R；每个i独立地为0至100；
D为
Figure PCTCN2017119779-appb-000022
或键，或
Figure PCTCN2017119779-appb-000023
或具有1至100个二醇但愿的聚丙二醇或聚丙烯-共-聚乙二醇连接基；其中Z，Z’和D各自不同时为键；每个i独立地为0～100；j为1至100；m为整数1～100；且n为整数1～100；m’为1～100；m”为整数0～25，通常为1～10，通常为1～8；更通常为1、2、3、4、5或6；n’为整数1～100，或1～75，或1～60，或1～5，或1～50，或1～45，或1～40，或2～35，或3～30，或1～15，或1～10，或1～8，或1～6，或1、2、3、4或5；X i为O，S或N-R；R为H，或烷基或烷醇基；R a是H、C 1-C 3烷基或烷醇或与R 3形成一环，R 3是衍生自氨基酸的侧链；或他们药学上可接受的盐、溶剂化物或多晶型物。
在本发明的一种实施方式中，所述双官能或多官能连接体基团MULTICON基团包括：
Figure PCTCN2017119779-appb-000024
Figure PCTCN2017119779-appb-000025
其中Y 4为C-H或N；且各X”独立地衍生自亲电或亲核基团，包括但不限于(CH 2) n ”O、(CH 2) n ”N RCON、(CH 2) n ”S、(CH 2) n ”、(CH 2) n ”C＝O或CON基团；该取代基RCON为H或C 1-C 3烷基，尤其是H或CH 3，且n”为0、1、2或3；r为整数1至12；且所述CON基团，如果存在的话，结构为
Figure PCTCN2017119779-appb-000026
其中X 2为O、S、NR 4、S(O)、S(O) 2、-S(O) 2O、-OS(O) 2或O S(O) 2O；X 3为O、S、NR 4；且R 4为H、C 1-C 3烷基或烷醇基，或-C(O)(C 1-C 3)基团；或其可药用盐。
在本发明的一种实施方式中，L 1和/或L 2为
Figure PCTCN2017119779-appb-000027
基团；其中R a为H或CH 3；m为整数1～12；m”为整数1、2、3、4、5或6；t为0、1、2、3、4、5或6；且iL为0或1。
在本发明的一种实施方式中，L 1和/或L 2为
Figure PCTCN2017119779-appb-000028
其中R a为H；m”为整数1、2、3、4、5或6且m为1、2、3、4、5、6、7、8、9、10、11或12。
在本发明的一种实施方式中，所述化合物中L 1和/或L 2为长度为1～12个二醇单元的聚乙二醇连接基，或通过所述CON基团延伸至第二聚乙二醇连接基的聚乙二醇连接基；所述聚乙二醇连接基的长度为1～12个二醇单元且所述第二聚乙二醇连接基为长度为1～12个二醇单元。
在本发明的一种实施方式中，所述CON基团为
Figure PCTCN2017119779-appb-000029
在本发明的一种实施方式中，A具有以下结构的部分组成的基团：
Figure PCTCN2017119779-appb-000030
其中DNP基团任选地通过X基团连接到所述氨基酸部分；X是O、CH 2、NR 1、SO、SO 2、-S(O) 2O、-OS(O) 2或-OS(O) 2O。
在本发明的一种实施方式中，A具有以下化学式的部分：
Figure PCTCN2017119779-appb-000031
其中，Y’是H或NO 2；X是O、CH 2、NR 1、SO、SO 2、-S(O) 2O、-OS(O) 2或-OS(O) 2O；R 1是H、C 1-C 3烷基或-C(O)(C 1-C 3)基团；Xs是OH或NHAc；X R是O或者S；X b是一化学键O、CH 2、NR 1或S；X’是CH 2、O、N-R 1’(R 1’是H或C 1-C 3烷基)或S；X”是O、CH 2、NR 1(R 1是H、C 1-C 3烷基或-C(O)(C 1-C 3)基团)Z是一化学键、单糖、二糖、寡糖、糖蛋白或糖脂。
有益效果：(1)本发明所采用的募集结构均为常规的小分子或易于发酵获得ZZ domain, 易于获得，并可以有效募集人体血清内相关的抗体。
(2)本发明所采用的靶向结构均为易于合成的多肽、环肽或者易于发酵获得的抗体蛋白，它们均可以和肿瘤表面相关的抗原实现高亲和、高特异地结合，其结合能力不亚于传统的单克隆抗体。
(3)本发明将募集结构和靶向结构的连接方式并不复杂，产物也易于纯化，如利用Sortase A和点击化学均可以在生理环境下实现对分子的快速修饰。
(4)本发明提供的双功能分子均可以特异性靶向肿瘤细胞细胞，且半结合常数通常低于10nM，并可以有效募集抗DNP、抗Rha等抗体，通过介导CDC或ADCC不同程度地实现对肿瘤细胞的杀伤(20％-90％)。本发明还通过一系列不同的链(包括不同链长、链类型)对募集结构和靶向结构进行连接，评估不同链、不同募集结构(种类、数量)和靶向结构(种类、数量)对终化合物肿瘤杀伤作用的影响，优选出该系列双功能分子，可以为以后相类似肿瘤药物的开发提供宝贵的基础和借鉴。
附图说明
图1目标产物的质谱；(A)GGG-triEGDNP,(B)GGG-hexEGDNP；
图2为流式细胞图；其中纵坐标MFI指平均荧光强度，左图中横坐标表示用对应的化合物与细胞进行孵育，Negative control表示用等体积的PBS对细胞进行孵育；右图中横坐标表示所孵育的细胞种类。
图3为肿瘤杀伤效应；其中，(A)ADCC效果；(B)CDC效果；纵坐标对应为细胞裂解率，横坐标表示用对应的化合物与细胞进行孵育，Negative control表示用等体积的PBS对细胞进行孵育。
图4为目标产物质谱(A)GGG-triEGRha,(B)GGG-hexEGRha；
图5为流式细胞图；其中纵坐标MFI指平均荧光强度，左图中横坐标表示用对应的化合物与细胞进行孵育，Negative control表示用等体积的PBS对细胞进行孵育；右图中横坐标表示所孵育的细胞种类。
图6为肿瘤杀伤效应；(A)ADCC效果；(B)CDC效果；纵坐标对应为细胞裂解率，横坐标表示用对应的化合物与细胞进行孵育，Negative control表示用等体积的PBS对细胞进行孵育。
图7为MCF-7细胞的流式细胞仪、CDC、ADCC实验结果；纵坐标MFI表示荧光强度，lysis％对应为细胞裂解率，横坐标均表示用对应的化合物与细胞进行孵育，Negative control表示用等体积的PBS对细胞进行孵育。
图8为化合物NGR-DNP的质谱结果；
图9为MCF-7细胞的流式细胞仪、CDC、ADCC实验结果；纵坐标MFI表示荧光强度，lysis％对应为细胞裂解率，横坐标均表示用对应的化合物与细胞进行孵育，Negative control表示用等体积的PBS对细胞进行孵育。
图10为RGD-DNP(图A)和RGD-linker-DNP(图B)的质谱数据；
图11为细胞实验结果；其中，(A)为HEK-293细胞的流式细胞仪、CDC、ADCC实验结果；纵坐标MFI表示荧光强度，lysis％对应为细胞裂解率，横坐标均表示用对应的化合物与细胞进行孵育，Negative control表示用等体积的PBS对细胞进行孵育。(B)为HT-29、SKOV-3和HEK-293细胞与RGD-linker-DNP的流式细胞结合实验；纵坐标MFI表示荧光强度，横坐标表示所孵育的细胞种类，其中negative control表示用等体积的PBS对SKOV3细胞进行孵育。
具体实施方式
以下实施例用来说明本发明，但不用来限制本发明的范围。
实施例1以anti-EGFR nanobody(7D12)和GGG-triEGDNP为例
1、A-linker 1的合成(化合物结构为：
Figure PCTCN2017119779-appb-000032
以A为DNP，n1＝2或5，n2＝2，X＝O为例)，具体的合成路线以及每步相应的条件和产率如下：
Figure PCTCN2017119779-appb-000033
Figure PCTCN2017119779-appb-000034
目标产物的高分辨如图1所示。其中GGG-triEGDNP检测到的分子量为487.1792(M+H +)，GGG-hexEGDNP检测到的分子量为619.2580(M+H +)，均和预测一致，表明所合成的产物正确。
2、anti-EGFR nanobody(7D12)的发酵生产：
合成7D12的氨基酸序列(参考NCBI Sequence ID:4KRL_B)：
maqvkleesgggsvqtggslrltcaasgrtsrsygmgwfrqapgkerefvsgiswrgdstgyadsvkgrftisrdnakntvdlqmnslkpedtaiyycaaaagsawygtlyeydywgqgtqvtvssaaaeqkliseedlngaalpetgghhhhhh
发酵具体实施方式为：将编码7D12的基因克隆至pET28a商业载体上并转化至商业宿主E.coli BL21(DE3)。挑取单菌落进行接种，接种培养基为LB，并添加0.1％的卡那霉素，37℃过夜培养；按1-10％的量进行转接，发酵培养基为TB培养基，待OD＝0.6-1.2时进行诱导，IPTG终浓度为0.1-1.0mM,温度转为16-30℃，诱导24h。发酵结束后进行离心，弃置培养基，用缓冲液(10-100mM Tris-Hcl或者PBS，pH 7-8)进行重悬，并用超声进行破壁，破壁后取破壁上清用镍柱进行纯化，并用脱盐柱或者超滤或者透析进行脱盐。纯化脱盐后的7D12通过SDS-PAGE进行验证，结果显示只有一条带，且分子量和预测相符(16.7KD)。
3、7D12-triEGDNP和7D12-hexEGDNP的合成：
在增强型SortaseA(SrtA)的作用下，在Tris-HCl缓冲液当中对7D12进行DNP的修饰，在4-37℃，反应10-120min，反应后未反应的7D12用Ni +磁珠进行去除，多余的小分子则通过透析或者排阻法除去，产物经纯化(可以通过透析、超滤和排阻过滤)后无菌过滤后保存在-20℃备用。
4、细胞实验
(1)细胞的培养：商业化细胞A431(EGFR高表达)，MCF7(EGFR低表达)培养在DMEM完全培养基，4T1(鼠源EGFR高表达)培养在1640完全培养基，所有细胞培养条件均为37℃，5％CO 2；
(2)流式细胞实验：实验流程为①用胰酶消化细胞，流式液重悬计数，稀释细胞到1*10 5个，取400μL细胞(即40000个细胞)加到无菌EP管②加入终浓度为250nM的7D12或DNP修饰的7D12,对于空白组(Negative)，加入对应体积的PBS,并在所有组中立即加入终浓度为20μg/mL的抗DNP抗体，冰上孵育1h③用流式缓冲液彻底温和洗涤细胞，重悬，用仪器进行检测。
流式结果如图2所示，相对于空白对照，加入未经半抗原修饰的7D12在荧光强度上并没有明显升高，证明未经修饰的7D12不具备募集抗体的能力；此外，经过7D12-triEGDNP 或7D12-hexEGDNP处理后，A431荧光明显增加，而MCF7则无明显增加，这说明了了所构建的7D12-triEGDNP和7D12-hexEGDNP可以特异性结合细胞表面的EGFR，值得注意的是，由于4T1组经7D12-triEGDNP或7D12-hexEGDNP处理后，荧光强度也无明显增加，则证明了所合成的7D12-triEGDNP所识别的为人源EGFR，而非鼠源。
(3)ADCC实验：实验流程为：①在96孔板相应孔加入100μL A431细胞悬浮液(10000个/孔)，待细胞完全贴壁后，加入稀释的7D12或7D12-triEGDNP或7D12-hexEGDNP,并立即加入一定浓度的IgG；②孵育一段时间后，按一定的效靶比加入新鲜提取的PBMC细胞，处理一定时间；③离心去上清LDH是方法检测。
结果如图3(A)，相对于其它对照的细胞活性，以7D12-triEGDNP或7D12-hexEGDNP处理后，杀伤率分别为31％和37％，而对照组的细胞损失仅有5％左右，表明合成的两种产物均可以诱导ADCC效应，而且两者的杀伤力度相差无几。
(4)CDC实验：实验流程为①在96孔板相应孔加入100μL A431细胞悬浮液(5000个/孔)，待细胞完全贴壁后，加入稀释的7D12或7D12-triEGDNP或7D12-hexEGDNP,并立即加入一定浓度的IgG②孵育一段时间后，加入一定量的补体，处理一定时间③cck8法检测细胞活性，并转化成相应的细胞毒性。
结果如图3(B)所示，相对于其它对照的细胞活性，以7D12-triEGDNP或7D12-hexEGDNP处理后，杀伤率分别为21％和27％，而对照组的细胞死亡率则低于5％，表明合成的两种产物均可以诱导CDC效应。并且7D12-hexEGDNP所诱导的CDC效果略强于7D12-triEGDNP。
实施例2
以anti-HER2 nanobody(C7b)和GGG-triEGRha、GGG-hexEGRha为例。
1、A-linker 1的合成(以A为Rha，n1＝2或5,n2＝2,X＝O为例条)，具体的合成路线以及每步相应的条件和产率如下：
Figure PCTCN2017119779-appb-000035
产物的高分辨如图4所示。其中GGG-triEGRha检测到的分子量为467.2354(M+H +)，GGG-hexEGRha检测到的分子量为599.3145(M+H +)，均和预测一致，表明所合成的产物正确。
2、anti-HER2 nanobody(C7b)的发酵生产：
合成C7b的氨基酸序列(参考NCBI Sequence ID:AFN61318.1)：
mqvqlvqsggglvqaggslrlscaasgrtfssyamawfrqapgkerefvaaiswsganiyvadsvkgrftisrdnakdtvylqmnslkpedtavyycavklgfapveerqydywgqgtqvtvsslpetgg
发酵具体实施方式为：将编码C7b的基因克隆至pET22b商业载体上并转化至商业宿主E.coli BL21(DE3)。挑取单菌落进行接种，接种培养基为LB，并添加0.1％的氨苄抗生素，37℃过夜培养；按1-10％的量进行转接，发酵培养基为TB培养基，待OD＝0.6-1.2时进行诱导，IPTG终浓度为0.1-1.0mM,温度转为16-30℃，诱导24h。发酵结束后进行离心，弃置培养基，用缓冲液(10-100mM Tris-Hcl或者PBS，pH 7-8)进行重悬，并用超声进行破壁，破壁后取破壁上清用镍柱进行纯化，并用脱盐柱或者超滤或者透析进行脱盐。
3、C7b-triEGRha和C7b-hexEGRha的合成
在增强型SrtA的作用下，在Tris-HCl缓冲液当中对C7b进行Rha修饰，在为4-37℃，反应10-120min，反应后未反应的C7b用Ni+磁珠进行去除，多余的小分子则通过透析或者排阻法除去，产物经纯化(可以通过透析、超滤和排阻过滤)后无菌过滤后保存在-20℃备用。
4、细胞实验
(1)细胞的培养：商业化细胞SKBR3(HER2高表达)MCF7(HER2低表达)培养在DMEM完全培养基，所有细胞培养条件均为37℃，5％CO2.
(2)流式细胞实验：实验流程为①用胰酶消化细胞，流式液重悬计数，稀释细胞到1*10^5个，取400μL细胞(即40000个细胞)加到无菌EP管②加入终浓度为250nM的C7b或Rha修饰C7b,对于空白组(Negative)，加入对应体积的PBS,并在所有组中立即加入中浓度为20μg/mL的抗鼠李糖(anti-Rha)抗体，冰上孵育1h③用流式缓冲液彻底温和洗涤细胞，重悬，用仪器进行检测。
流式结果(图5)表明所合成的C7b-triEGRha和C7b-hexEGRha可以专一性识别并结合靶细胞表面的HER2，相对于空白对照，加入未经半抗原修饰的C7b在荧光强度上并没有明显升高，证明未经修饰的C7b不具备募集抗体的能力；而经过C7b-triEGRha和C7b-hexEGRha处理后，SKBR3荧光强度增加最多，MCF7则无明显增加，这说明了所构建的C7b-triEGRha和C7b-hexEGRha可以特异性结合细胞表面的HER2。
(3)ADCC实验：实验流程为①在96孔板相应孔加入100μL SKBR3细胞悬浮液(10000个/孔)，待细胞完全贴壁后，加入稀释的C7b或者Rha修饰的C7b,并立即加入一定浓度的anti-Rha IgG②孵育一段时间后，按一定的效靶比加入新鲜提取的PBMC细胞，处理一定时间③离心去上清LDH是方法检测细胞毒性。
结果(图6(A))表明合成的C7b-triEGRha和C7b-hexEGRha均可以诱导ADCC效应，它们对应的杀伤率分别为32％和43％，且C7b-hexEGRha所诱导的ADCC效果强于C7b-triEGRha。
(4)CDC实验：实验流程简单概括为：①在96孔板相应孔加入100μL A431细胞悬浮液(5000个/孔)，待细胞完全贴壁后，加入稀释的C7b或者Rha修饰的C7b,并立即加入终浓度为20％的商业化正常人血清(已检测内有anti-Rha抗体)，处理一定时间③cck8法检测细胞活性，行转化成相应的细胞毒性。
结果如图6(B)所示，相对于其它对照组的细胞活性(杀伤率低于5％)，C7b-triEGRha和C7b-hexEGRha可以诱明显的CDC效应，它们的杀伤率分别达到了36％和47％，且C7b-hexEGRha所诱导的CDC效果强于C7b-triEGRha。
实施例3
将鼠李糖(Rhamnose,Rha)偶联到LHRH多肽上，通过招募体内已有的抗体介导免疫效应来靶向杀死肿瘤细胞。
合成LHRH-鼠李糖偶联物(LHRH-Rhamnose，LHRH-Rha)的实施方式为：
基于LHRH构效关系的研究，在不影响其活性的基础上，在6位甘氨酸的位置进行带叠 氮基团(-N 3)的D型赖氨酸(D-Lys(N 3))的取代，成为[6-D-Lys(N 3)]-LHRH多肽分子(其氨基酸序列为：焦谷氨酸-组氨酸-色氨酸-丝氨酸-酪氨酸-D型赖氨酸(侧链带叠氮基团-N 3)-亮氨酸-精氨酸-脯氨酸-甘氨酸-氨基，Glp-His-Trp-Ser-Tyr-D-Lys(N 3)-Leu-Arg-Pro-Gly-NH 2)，其中，Glp为焦谷氨酸。
选取Rink-amide MBHA树脂作为固相载体，基于Fmoc合成策略，按照Glp-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-Gly-NH2的序列(其中，Glp为焦谷氨酸，无Fmoc保护)，利用TBTU作为缩合剂，从C端Gly开始到N端Glp逐步缩合。序列合成后，加入切割试剂(TFA:Tis:H 2O＝95:2.5:2.5)将多肽从树脂上切割下来。利用冰乙醚沉淀得到粗肽，再通过HPLC进行纯化，获得多肽[D-Lys6]-LHRH，质谱验证,[M+Na +]为1301.6，为正确产物。
其中，带叠氮的D型赖氨酸的合成的实施方式为(质谱数据[M+Na +]417.1，为正确化合物)：
Figure PCTCN2017119779-appb-000036
合成鼠李糖Rha衍生物的实施方式为：Rha分子进行寡聚聚乙二醇(选用n＝0，2，5)连接臂进行组装，该连接臂的另一端为炔基通过点击反应偶联到上述LHRH多肽衍生物中，合成的化合物为
Figure PCTCN2017119779-appb-000037
合成路线如下：
Figure PCTCN2017119779-appb-000038
硫酸铜、维生素C钠催化的条件下，Rha衍生物与LHRH衍生物通过点击反应获得LHRH-Rha偶联产物，质谱验证偶联成功。连接臂linker为寡聚聚乙二醇(选用n＝0，2，5)，分别表示为LHRH-Rha(质谱分子量[M+Na +]为1560.7),LHRH-TEG-Rha(质谱分子量[M+Na +]为1648.8),LHRH-HEG-Rha(质谱分子量[M+Na +]为1780.9)。
细胞实验的实施方案：细胞的培养：商业化细胞MCF-7(LHRH-R高表达)和UCI-107(LHRH不表达)培养在DMEM完全培养基，所有细胞培养条件均为37℃，5％CO 2。流式细胞实验：实验流程为①用胰酶消化细胞，流式液重悬计数，稀释细胞到1×10 5个，取400μL细胞(即40000个细胞)加到无菌EP管②加入终浓度为250nM的LHRH、LHRH-Rha、LHRH-TEG-Rha和LHRH-HEG-Rha化合物，对于空白组(Negative)，加入对应体积的PBS,并在所有组中立即加入中浓度为20μg/mL的抗鼠李糖(anti-Rha)抗体，冰上孵育1h，随后洗涤细胞后加入带荧光的二抗，冰上孵育0.5h；③用流式缓冲液彻底温和洗涤细胞，重悬，用仪器进行检测。
ADCC实验的实施方案：①在96孔板相应孔加入100μL MCF-7或UCI-107细胞悬浮液(10000个/孔)，待细胞完全贴壁后，加入终浓度为250nM的LHRH、LHRH-Rha、LHRH-TEG-Rha和LHRH-HEG-Rha化合物，并立即加入一定浓度的anti-Rha抗体②孵育一段时间后，按一定的效靶比加入新鲜提取的PBMC细胞，处理一定时间③离心去上清，用LDH方法检测细胞毒性。
CDC实验的实施方案：①在96孔板相应孔加入100μL MCF-7或UCI-107细胞悬浮液(5000个/孔)，待细胞完全贴壁后，加入终浓度为250nM的LHRH、LHRH-Rha、LHRH-TEG-Rha和LHRH-HEG-Rha化合物，并同时加入一定浓度的anti-Rha抗体，在37度孵育2小时候，加入终浓度为10％的补体溶液，37度孵育1个小时。孵育结束后，直接用cck8法检测细胞活性。
流式细胞仪、ADCC、CDC实验结果如7所示，实验结果表明，LHRH-Rha,LHRH-TEG-Rha和LHRH-HEG-Rha偶联化合物都能招募抗Rha抗体并靶向LHRH高表达的MCF-7肿瘤细胞，并介导免疫反应CDC和ADCC进一步杀伤MCF-7肿瘤细胞，且这三个化合物所介导的免疫杀伤效果相当(CDC介导的杀伤率均在26％左右，ADCC介导的杀伤率均在33％左右)，在本实施例中，连接臂的长短对活性的影响较小。
实施例4
NGR多肽即含天门冬酰胺酸-精氨酸-甘氨酸序列的多肽。直链状的NGR多肽有CYGGRGNG，CNGRCVSGCAGRC，GNGRGGVRSSSRTPSDKYC等。NGR多肽是一种能与肿瘤新生血管内皮细胞上的CD13受体结合的靶向肽，能够有效地靶向肿瘤细胞。因此， 本发明设计合成了基于NGR的抗体募集双功能分子，将DNP的结构偶联到NGR上，通过招募抗DNP的抗体并靶向CD13高表达的肿瘤新生血管内皮细胞，介导免疫反应靶向杀伤肿瘤细胞。
合成NGR-DNP偶联物的实施方式如下：
Figure PCTCN2017119779-appb-000039
化合物3(DNP-TEG-COOH)的合成：在0℃条件下将化合物2(DNP-TEG-COOEt)0.35g溶于1:1的四氢呋喃/水溶液(15ml/15ml)中，加入碳酸钾0.4g反应过夜。反应结束后抽干四氢呋喃，以20ml二氯甲烷DCM萃取3次，产物呈盐状态溶于水中，以薄层色谱监测是否萃净水中杂质。以1mol/ml的盐酸中和水相，至PH试纸显红色。抽干后用二氯甲烷溶解过滤。若仍有少量杂质可过硅胶柱。产物0.26g，产率80％。
化合物4的合成：先合成肽链GGGCNGRC(G、C、N、R分别代表氨基酸甘氨酸、半胱氨酸、天门冬酰胺、精氨酸)，以Fmoc-Cys(Acm)-Wang resin(0.1mmol)树脂为固相载体将树脂在固相合成管中溶胀过夜，加入20％哌啶/DMF溶液(5ml)在摇床上摇15min脱去Fmoc，然后将Fmoc-Arg-OH(0.25mmol)，HBTU(0.25mmol),Dipea(0.5mmol)溶于5mlDMF溶液，加入树脂中，摇床上摇2h，以茚三酮溶液监测是否连上。再次脱去Fmoc保护基，重复上述步骤直到肽链合成。
然后再将0.25mmol的化合物3，HBTU(0.25mmol),Dipea(0.5mmol)溶于5ml DMF溶液，加入树脂反应2h，监测反应结束。化合物5的合成：将化合物4在95％的三氟乙酸TFA溶液(TFA:Tis:H2O＝95:2.5:2.5)中，反应2h切去树脂，用大量冰乙醚沉淀离心，除去乙醚，冻干。HPLC监测。化合物6的合成：将化合物5溶于乙酸中，加入碘I 2氧化，反应2-3h。HPLC监测反应进行，反应结束后用水猝灭，三氯甲烷CHCl3洗涤多次除去大多数碘I 2。水相抽干后以半制备柱纯化。图8为化合物NGR-DNP的质谱结果，可见，有预测分子量973的峰出现，表明产物合成成功
细胞实验的实施方案：细胞的培养：商业化细胞HUVEC(CD13高表达)和培养在DMEM完全培养基，所有细胞培养条件均为37℃，5％CO2。流式细胞实验流程为①用胰酶消化细 胞，流式液重悬计数，稀释细胞到1×10 5个，取400μL细胞(即40000个细胞)加到无菌EP管②加入终浓度为250nM的NGR寡肽(NGR peptide，其氨基酸序列：GGGCNGRC)或NGR-DNP，对于空白组(Negative)，加入对应体积的PBS,并在所有组中立即加入中浓度为20μg/mL的抗DNP(anti-DNP)抗体，冰上孵育1h，随后洗涤细胞后加入带荧光的二抗冰上孵育0.5h；③用流式缓冲液彻底温和洗涤细胞，重悬，用仪器进行检测。
ADCC实验的实施方案：①在96孔板相应孔加入100μL HUVEC细胞悬浮液(10000个/孔)，待细胞完全贴壁后，加入终浓度为250nM的NGR寡肽(NGR peptide，其氨基酸序列：GGGCNGRC)或NGR-DNP化合物，并立即加入一定浓度的anti-DNP抗体②孵育一段时间后，按一定的效靶比加入新鲜提取的PBMC细胞，处理一定时间③离心去上清，用LDH方法检测细胞毒性。
CDC实验的实施方案：①在96孔板相应孔加入100μL HUVEC细胞悬浮液(5000个/孔)，待细胞完全贴壁后，加入终浓度为250nM的NGR寡肽(NGR peptide，其氨基酸序列：GGGCNGRC)或NGR-DNP化合物，并同时加入一定浓度的anti-DNP抗体，在37度孵育2小时候，加入终浓度为10％的补体溶液，37度孵育1个小时。孵育结束后，直接用cck8法检测细胞活性。
NGR peptide(氨基酸序列：GGGCNGRC)和化合物NGR-DNP的流式细胞仪、ADCC、CDC实验结果如图9所示，实验结果表明，NGR-DNP化合物能招募抗体并靶向肿瘤细胞，并介导免疫反应CDC和ADCC(细胞裂解率约60％)进一步杀伤肿瘤细胞。
实施例5
整合素在多种肿瘤细胞表面高表达，而在正常细胞中低表达或不表达，且整合素在肿瘤细胞的粘附、迁移、浸润及肿瘤血管新生中起着重要作用，因此，整合素是肿瘤治疗的一个重要的靶点。由于整合素受体特异配体精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp，RGD)能够特异性结合整合素，RGD及其衍生物被用于开发肿瘤靶向治疗策略。在本发明中，我们设计合成了一系列基于RGD结构的抗体募集双功能分子，将DNP偶联到RGD或其衍生的多肽或环肽上，通过招募抗DNP的抗体，介导免疫杀伤作用并靶向杀死肿瘤细胞。
合成RGD-DNP偶联物的实施方案如下所示：
Figure PCTCN2017119779-appb-000040
合成RGD-linker-DNP的实施方案如下所示：
Figure PCTCN2017119779-appb-000041
通过质谱检测确认合成的化合物正确，质谱如图10所示。RGD-DNP的实际分子量为769.78，质谱中中出现的770.38(M+H +)和792.26(M+Na +)均与预测相符；另外RGD-linker-DNP的分子量为882.3，质谱中出现的质何比453.57(M+Na +H +)以及883.44(M+H +)以及905.44(M+Na +)均和预测相符，表明产物合成正确。
化合物RGD-DNP和RGD-linker-DNP的活性检测实施方案如下：包括流式细胞实验、CDC实验和ADCC实验。具体实施方案如下：
细胞实验的实施方案：细胞的培养：商业化细胞HEK-293(整合素αvβ3高表达)SKOV-3(整合素αvβ3和αvβ5都表达)和HT-29(整合素αvβ5高表达)培养在DMEM完全培养基，所有细胞培养条件均为37℃，5％CO2。流式细胞实验：实验流程为①用胰酶消化细胞，流 式液重悬计数，稀释细胞到1×10 5个，取400μL细胞(即40000个细胞)加到无菌EP管②加入终浓度为250nM的RGD类似物、RGD-DNP和RGD-linker-DNP化合物，对于空白组(Negative)，加入对应体积的PBS,并在所有组中立即加入中浓度为20μg/mL的抗DNP(anti-DNP)抗体，冰上孵育1h，随后洗涤细胞后加入带荧光的二抗，冰上孵育0.5h；③用流式缓冲液彻底温和洗涤细胞，重悬，用仪器进行检测。
ADCC实验的实施方案：①在96孔板相应孔加入100μL HEK-293、SKOV-3和HT-29细胞悬浮液(10000个/孔)，待细胞完全贴壁后，加入终浓度为250nM的RGD类似物、RGD-DNP和RGD-linker-DNP化合物，并立即加入一定浓度的anti-DNP抗体②孵育一段时间后，按一定的效靶比加入新鲜提取的PBMC细胞，处理一定时间③离心去上清，用LDH方法检测细胞毒性。
CDC实验的实施方案：①在96孔板相应孔加入100μL HEK-293、SKOV-3和HT-29细胞悬浮液(5000个/孔)，待细胞完全贴壁后，加入终浓度为250nM的RGD类似物、RGD-DNP和RGD-linker-DNP化合物，并同时加入一定浓度的anti-DNP抗体，在37度孵育2小时候，加入终浓度为10％的补体溶液，37度孵育1个小时。孵育结束后，直接用cck8法检测细胞活性。
流式细胞仪、ADCC、CDC实验结果如图11所示，实验结果表明，RGD-DNP和RGD-linker-DNP偶联化合物都能结合到HEK-293、SKOV-3和HT-29细胞，但是与细胞的结合能力略有差别，可能是因为每种细胞表达的整合素不一样。RGD-DNP和RGD-linker-DNP偶联化合物都能招募抗DNP抗体并靶向整合素高表达的HEK-293细胞，并介导免疫反应CDC和ADCC进一步杀伤这几款细胞，且这2个化合物所介导的免疫杀伤效果有一定区别，连接臂较长的RGD-linker-DNP化合物所介导的免疫杀伤效果更强(短连接臂对应的CDC和ADCC杀伤率仅为21％和18％，长连接臂对应的CDC和ADCC杀伤率则达到了42％和38％)，与流式细胞实验的结果相一致。这个结果表明，招募DNP的抗体需要将DNP这部分结构裸露在RGD-整合素的复合物外面，如果连接臂太短，将会影响抗DNP的抗体与RGD-整合素复合物的结合。因此，本发明结果优化了一个连接臂的长度。
Figure PCTCN2017119779-appb-000042
Figure PCTCN2017119779-appb-000043
Figure PCTCN2017119779-appb-000044
Figure PCTCN2017119779-appb-000045
Figure PCTCN2017119779-appb-000046

Claims (33)

  1. 一种双功能分子，其特征在于，结构式为：
    Figure PCTCN2017119779-appb-100001
    其中，A为能够招募抗体的结构，包括能够与抗体结合的半抗原、多肽或Z/ZZ domain亲和肽及其类似物；B为能够与细胞表面结合的物质，包括寡肽、多肽、环肽、纳米抗体或具有抗体功能的片段；A和B通过linker连接；linker包括连接基L 1和L 2；L 1或L 2含有一个或多个双官能连接体基团CON；MULTICON为双官能或多官能连接体基团，当MCON≥1时，MULTICON通过连接基L 1和/或L 2将至少一个A与B连接；
    MCON为整数0～10；n和n’各自独立地为整数1～15，通常为2～10，通常为2～5，更通常为2～3，或2、3、4、5或6；NL1和NL2各自为整数0～10，n≥NL1，且n’≥NL2。
  2. 根据权利要求1所述的双功能分子，其特征在于，A包括DNP或Rha基团。
  3. 根据权利要求1所述的双功能分子，其特征在于，B为RGD及其衍生物，所述RGD为精氨酸-甘氨酸-天冬氨酸。
  4. 根据权利要求3所述的双功能分子，其特征在于，B为至少一组RGD形成的环肽。
  5. 根据权利要求1所述的双功能分子，其特征在于，B为NGR及其衍生物，所述NGR为天冬酰胺-甘氨酸-精氨酸。
  6. 根据权利要求1所述的双功能分子，其特征在于，B为LHRH及其衍生物；所述LHRH是促黄体酮激素释放激素。
  7. 根据权利要求1所述的双功能分子，其特征在于，B包括抗肿瘤细胞表皮生长因子受体EGFR的纳米抗体、抗HER2的纳米抗体、抗前列腺特异性膜抗原的纳米抗体，或上述任一纳米抗体与Z(ZZ)domain的融合蛋白。
  8. 根据权利要求1所述的双功能分子，其特征在于，B可以靶向细胞表面的靶标。
  9. 根据权利要求8所述的双功能分子，其特征在于，所述细胞是发生病变的细胞。
  10. 根据权利要求9所述的双功能分子，其特征在于，所述发生病变的细胞为癌细胞。
  11. 根据权利要求8所述的双功能分子，其特征在于，B具有如下的结构：
    Figure PCTCN2017119779-appb-100002
    其中，X为任一氨基酸及其衍生物；n为1-100的整数；L、P、T、G分别为亮氨酸、脯氨酸、苏氨酸、甘氨酸。
  12. 根据权利要求11所述的双功能分子，其特征在于，B与linker通过sortase A酶进行连接。
  13. 根据权利要求1或12所述的双功能分子，其特征在于，linker的结构式为
    Figure PCTCN2017119779-appb-100003
    -LPXTGn-；所述-LPXTGn-为-亮氨酸-脯氨酸-X-苏氨酸-n个甘氨酸；其中n为整数1～10；n1为整数1～4；n2为整数2-9；n3为整数2-9；X为O或S或N。
  14. 根据权利要求13所述的双功能分子，其特征在于，
    Figure PCTCN2017119779-appb-100004
    靠近X端为半抗原结构，另一端与B通过SortaseA连接。
  15. 根据权利要求13所述的双功能分子，其特征在于，
    Figure PCTCN2017119779-appb-100005
    靠近X端为相同或不同的半抗原，另一端与B通过SortaseA连接。
  16. 根据权利要求13所述的双功能分子，其特征在于，-LPXTGn-通过SortaseA将纳米抗体和Z/ZZ domain直接连接，所述纳米抗体连接在Z/ZZ domain的N端或C端。
  17. 根据权利要求1所述的双功能分子，其特征在于，所述双功能分子中A包括两种不同的能够招募抗体的结构，其结构式为：
    Figure PCTCN2017119779-appb-100006
  18. 根据权利要求1所述的双功能分子，其特征在于，所述双官能连接体基团CON包括：
    Figure PCTCN2017119779-appb-100007
    其中X 2为O、S、NR 4、S(O)、S(O) 2、-S(O) 2O、-OS(O) 2或O S(O) 2O；X 3为O、S、NR 4；且R 4为H、C 1-C 3烷基或烷醇基，或-C(O)(C 1-C 3)基团；或其可药用盐。
  19. 根据权利要求1所述的双功能分子，其特征在于，所述双官能连接体基团CON为
    Figure PCTCN2017119779-appb-100008
    其中CL为
    Figure PCTCN2017119779-appb-100009
    m为整数0～12，通常为0、1、2、3、4、5、6；且IL为0或1，通常为1。
  20. 根据权利要求1所述的双功能分子，其特征在于，所述连接基L 1或L 2包括聚乙二醇连接基、聚丙二醇连接基或聚乙二醇共聚丙烯聚合物，其长度为1至100单位。
  21. 根据权利要求1所述的双功能分子，其特征在于，所述连接基L 1或L 2为聚脯氨酸连接基或胶原连接基：其中，聚脯氨酸连接基结构式为
    Figure PCTCN2017119779-appb-100010
    胶原连接基结构式为
    Figure PCTCN2017119779-appb-100011
    其中n为1至100。
  22. 根据权利要求1所述的双功能分子，其特征在于，所述连接基L 1或L 2为
    Figure PCTCN2017119779-appb-100012
    中的任一种；其中，R a是H、C 1-C 3烷基或烷醇或与R 3形成一环，R 3为衍生自氨基酸的侧链；m是从1到100的整数；且m”是从0到25的整数；上述任一基团均可进一步通过酰胺基、酮基、胺基或氨基酸连接。
  23. 根据权利要求1所述的双功能分子，其特征在于，所述连接基L 1或L 2为
    Figure PCTCN2017119779-appb-100013
    其中，R a是H或C 1-C 3烷基，通常为CH 3，最通常为H；m为整数1至12，通常为1、2、3、4、5或6；m”为整数1、2、3、4、5或6，通常为6；t为1、2、3、4、5或6；且iL为0或1，其中所述连接基任选在一端连接至A基团，且在另一端任选连接至B基团。
  24. 根据权利要求1所述的双功能分子，其特征在于，所述连接基L 1或L 2为
    Figure PCTCN2017119779-appb-100014
    其中q为整数0～12；且q’为1～12；iL为0或1；且R L为氨基酸或寡肽；
    或根据以下化学结构的连接基琥珀酰亚胺：
    Figure PCTCN2017119779-appb-100015
    其中各X S独立地为S、O或N-R S；R S为H或C 1-C 3烷基；S C为CH 2、CH 2O或CH 2CH 2O；i为0或1；且m s为0、1、2、3、4、5或6；
    或根据以下化学式的连接基：
    Figure PCTCN2017119779-appb-100016
    其中Z和Z’各自独立地为键，包括-(CH 2) i-S，-(CH 2) i-N-R，
    Figure PCTCN2017119779-appb-100017
    -(CH 2) m′-；
    Figure PCTCN2017119779-appb-100018
    或任选键合至另一连接基、连接体、基团A或基团B；每个R为H，或烷基或烷醇基；每个R2独立地为H或烷基；每个Y独立地为键、O、S或N-R；每个i独立地为0至100；D为
    Figure PCTCN2017119779-appb-100019
    -(CH 2) m′-；
    Figure PCTCN2017119779-appb-100020
    或键，或
    Figure PCTCN2017119779-appb-100021
    Figure PCTCN2017119779-appb-100022
    或具有1至100个二醇但愿的聚丙二醇或聚丙烯共聚乙二醇连接基；其中Z，Z’和D各自不同时为键；每个i独立地为0～100；j为1至100；m为整数1～100；且n为整数1～100；m’为1～100；m”为整数0～25，通常为1～10，通常为1～8；更通常为1、2、3、4、5或6；n’为整数1～100，或1～75，或1～60，或1～5，或1～50，或1～45，或1～40，或2～35，或3～30，或1～15，或1～10，或1～8，或1～6，或1、2、3、4或5；X i为O，S或N-R；R为H，或烷基或烷醇基；R a是H、C 1-C 3烷基或烷醇或与R 3形成一环，R 3是衍生自氨基酸的侧链；或他们药学上可接受的盐、溶剂化物或多晶型物。
  25. 根据权利要求1所述的双功能分子，其特征在于，所述双官能或多官能连接体基团MULTICON基团包括：
    Figure PCTCN2017119779-appb-100023
    Figure PCTCN2017119779-appb-100024
    Figure PCTCN2017119779-appb-100025
    其中Y 4为C-H或N；且各X”独立地衍生自亲电或亲核基团，包括(CH 2) n”O、(CH 2) n”N RCON、(CH 2) n”S、(CH 2) n”、(CH 2) n”C＝O或CON基团；该取代基RCON为H或C 1-C 3烷基，尤其是H或CH 3，且n”为0、1、2或3；r为整数1至12；且所述CON基团，如果存在的话，结构为
    Figure PCTCN2017119779-appb-100026
    Figure PCTCN2017119779-appb-100027
    其中X 2为O、S、NR 4、S(O)、S(O) 2、-S(O) 2O、-OS(O) 2或O S(O) 2O；X 3为O、S、NR 4；且R 4为H、C 1-C 3烷基或烷醇基，或-C(O)(C 1-C 3)基团；或其可药用盐。
  26. 根据权利要求1所述的双功能分子，其特征在于，L 1和/或L 2为
    Figure PCTCN2017119779-appb-100028
    Figure PCTCN2017119779-appb-100029
    其中R a为H；m”为整数1、2、3、4、5或6且m为1、2、3、4、5、6、7、8、9、10、11或12。
  27. 根据权利要求1所述的双功能分子，其特征在于，所述化合物中L 1和/或L 2为长度为1～12个二醇单元的聚乙二醇连接基，或通过所述CON基团延伸至第二聚乙二醇连接基的聚乙二醇连接基；所述聚乙二醇连接基的长度为1～12个二醇单元且所述第二聚乙二醇连接基为长度为1～12个二醇单元。
  28. 根据权利要求1所述的双功能分子，其特征在于，A具有以下结构的部分组成的基团：
    Figure PCTCN2017119779-appb-100030
    其中DNP基团任选地通过X基团连接到所述氨基酸部分；X是O、CH 2、NR 1、SO、SO 2、-S(O) 2O、-OS(O) 2或-OS(O) 2O。
  29. 根据权利要求1所述的双功能分子，其特征在于，A具有以下化学式的部分：
    Figure PCTCN2017119779-appb-100031
    其中，Y’是H或NO 2；X是O、CH 2、NR 1、SO、SO 2、-S(O) 2O、-OS(O) 2或-OS(O) 2O；R 1是H、C 1-C 3烷基或-C(O)(C 1-C 3)基团；Xs是OH或NHAc；X R是O或者S；X b是一化学键O、CH 2、NR 1或S；X’是CH 2、O、N-R 1’(R 1’是H或C 1-C 3烷基)或S；X”是O、CH 2、NR 1(R 1是H、C 1-C 3烷基或-C(O)(C 1-C 3)基团)Z是一化学键、单糖、二糖、寡糖、糖蛋白或糖脂。
  30. 含有权利要求1～12，14～30任一所述双功能分子的药物组合物。
  31. 根据权利要求31所述的药物组合物，其特征在于，含有权利要求1～30任一所述的双功能分子和药学上可接受的载体。
  32. 权利要求1～12，14～30任一所述的双功能分子在制备用于抑制癌细胞的药物方面的 应用。
  33. 含有权利要求1～12，14～30任一所述的双功能分子的食品或保健品。
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