WO2020253393A1 - 共价多特异性抗体 - Google Patents

共价多特异性抗体 Download PDF

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WO2020253393A1
WO2020253393A1 PCT/CN2020/087668 CN2020087668W WO2020253393A1 WO 2020253393 A1 WO2020253393 A1 WO 2020253393A1 CN 2020087668 W CN2020087668 W CN 2020087668W WO 2020253393 A1 WO2020253393 A1 WO 2020253393A1
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polypeptide
domain
amino acid
target
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PCT/CN2020/087668
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French (fr)
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周桢昊
张洁
王晓庆
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成都恩沐生物科技有限公司
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Priority to US17/596,789 priority Critical patent/US20230192898A1/en
Priority to EP20826093.5A priority patent/EP3988575A4/en
Priority to JP2021576262A priority patent/JP2022537823A/ja
Publication of WO2020253393A1 publication Critical patent/WO2020253393A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • 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
    • 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
    • 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
    • C07K2317/50—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to a novel covalent multispecific antibody with higher stability and its therapeutic use.
  • Monoclonal antibodies have a wide range of diagnostic and therapeutic potentials in clinical practical applications against cancer and other diseases. Whether in the form of naked antibodies or in the form of conjugates bound to cytotoxic agents (eg, radioisotopes, drugs, toxins, or prodrug converting enzymes), monoclonal antibodies play an important role in cancer immunotherapy. These methods are in the process of efficacy evaluation, with different degrees of development and different degrees of clinical success. Naked mAb can achieve a clinical response by inducing cytotoxicity after binding to cell surface proteins overexpressed on cancer cells. Studies have shown that these therapeutic effects can be achieved by controlling tumor growth through programmed cell death (apoptosis) or by inducing anti-tumor immune responses.
  • apoptosis programmed cell death
  • Therapeutic antibodies have been used clinically for more than 20 years.
  • anti-tumor antibody drugs in clinical use include: Rituxan (Rituxan (1997)), Herceptin (Herceptin (1998)), Mylotarg (2000), Campath (2001), Zevalin (2002), Beckxer (2003), Vastin (Avastin (2004)), Erbitux (2004), Vectibix (2006); Arzerra (2009); Benlysta (2011); Yervoy (2011); Adcetris (2011); Perjeta (2012); Kadcyla (2013), Opdivo (2014), Keytruda (2014), Tecentriq (2016).
  • These antibodies mainly target EGFR, Her2, CD20 or VEGF, and the recently discovered PD1 or PD-L1.
  • Multifunctional antibodies are constructed based on traditional antibodies through complex design and molecular engineering, and have multiple antigen binding capabilities.
  • the therapeutic effect produced by a single multifunctional antibody molecule is the same as the therapeutic effect produced by the combination of several traditional antibodies.
  • the advantages of multifunctional antibodies far exceed the simple stacking of several traditional antibodies.
  • the simultaneous conjugation of multiple selected targets can produce beneficial effects over traditional antibodies.
  • Blinatumomab (CD3 x CD19, Amgen), which targets CD3 and CD19, can effectively engage T cells through its CD3-recognition Fv in killing tumor cells expressing CD19, which is effective in ALL (acute lymphatic Leukemia) and other indications have shown remarkable efficacy beyond traditional antibodies.
  • Bonatumumab was approved by the US FDA for the treatment of ALL in 2014.
  • Bispecific antibodies are produced by chemical cross-linking, hybrid-hybridoma or transfectionoma, or the exchange of disulfide at the hinge of two different Fab's.
  • the first method produces products that are heterogeneous and difficult to define.
  • the second method requires a large amount of purification of bispecific antibodies obtained from multiple hybrid antibody by-products, and this purification step may affect cell cross-linking activity.
  • the disulfide exchange method is essentially only applicable to F(ab') 2 and is therefore limited by the susceptibility of monoclonal antibodies to enzymatic digestion and cleavage.
  • Fab's have almost no affinity for each other, a very high protein concentration is required to form disulfide bonds between Fab's.
  • the disulfide exchange method has been modified by using Ellman's reagent to use one of the Fab's to modify the other before oxidation, thus reducing the occurrence of homodimerization.
  • heterodimeric F(ab') 2 is rarely produced in yields higher than 50%.
  • unfavorable safety issues, low response speed and limited effectiveness are the current status of antibody drugs. These unfavorable factors may come from the non-target effects on normal tissues/cells due to the epitope of the antibody derived from its own antigen, the inhibitory microenvironment of immune effector cells, unexpected Fc-mediated effector functions, etc. Wait. Therefore, there is still a need in the art for high-purity multispecific (e.g., trispecific) antibodies with good therapeutic utility.
  • the present invention provides an engineered antibody, which comprises:
  • a first polypeptide comprising in the direction from N-terminus to C-terminus: a second light chain variable domain VL2 that binds to a second target and a first heavy chain variable that binds to the first target.
  • a second polypeptide comprising in the direction from N-terminus to C-terminus: a first light chain variable domain VL1 that binds to the first target and a second heavy chain variable domain that binds to the second target
  • the domain VH2 includes the hinge domain of cysteine and the CH2-CH3 domain of IgG, where VL1 and VH2 are connected by a linker;
  • a third polypeptide which includes, from the N-terminus to the C-terminus, a hinge domain containing cysteine and a CH2-CH3 domain of IgG,
  • VL1 and VH1 combine to form a domain capable of binding to a first target, and the first target is CD3;
  • VL2 and VH2 combine to form a domain capable of binding to a second target, and the second target is CD19;
  • VL2 and VH2 are covalently linked by disulfide bonds, and VL2 and VH2 each independently include one or more substitutions introducing charged amino acids, which are electrostatically charged for the formation of homodimers. Academically disadvantaged
  • the hinge domain of the second polypeptide chain and the hinge domain of the third polypeptide chain are covalently connected by a disulfide bond.
  • the amino acid sequence of VL1 is SEQ ID NO.:1
  • the amino acid sequence of VH1 is SEQ ID NO.:2
  • the amino acid sequence of VL2 is SEQ ID NO.:3
  • the amino acid sequence of VH2 is SEQ ID NO.: 4.
  • the amino acid sequence of the first polypeptide is SEQ ID NO.: 5
  • the amino acid sequence of the second polypeptide is SEQ ID NO.: 6
  • the amino acid sequence of the third polypeptide is SEQ ID NO.: 7 .
  • the present invention also provides an engineered antibody, which comprises:
  • a first polypeptide comprising in the direction from N-terminus to C-terminus: a second light chain variable domain VL2 that binds to a second target and a first heavy chain variable that binds to the first target.
  • a second polypeptide comprising in the direction from N-terminus to C-terminus: a first light chain variable domain VL1 that binds to the first target and a second heavy chain variable domain that binds to the second target
  • the domain VH2 includes the hinge domain of cysteine and the CH2-CH3 domain of IgG, where VL1 and VH2 are connected by a linker;
  • a third polypeptide in the direction from the N-terminus to the C-terminus, including: the third heavy chain variable domain VH3 that binds to the third target, the CH1 domain of IgG, and the cysteine-containing The hinge domain and the CH2-CH3 domain of IgG, where VH3 and CH1 are connected by a linker; and
  • a fourth polypeptide which includes in the direction from the N-terminus to the C-terminus: a third light chain variable domain VL3 that binds to the third target and a light chain constant domain containing cysteine CL, where VL3 and CL are connected by a linker;
  • VL1 and VH1 combine to form a domain capable of binding to the first target
  • VL2 and VH2 combine to form a domain capable of binding to the second target
  • VL3 and VH3 combine to form a domain capable of binding to the third target
  • VL2 and VH2 are covalently connected by a disulfide bond, and VL2 and VH2 each independently comprise one or more substitutions introducing charged amino acids, and the substitution of the charged amino acids is essential for the formation of homodimers. Is electrostatically unfavorable;
  • CH1 and CL are covalently connected through a disulfide bond
  • the hinge domain of the second polypeptide chain and the hinge domain of the third polypeptide chain are covalently connected by a disulfide bond.
  • the first target is CD3, the second target is CD19, and the third target is CD20.
  • the amino acid sequence of VL1 is SEQ ID NO.: 1
  • the amino acid sequence of VH1 is SEQ ID NO.: 2
  • the amino acid sequence of VL2 is SEQ ID NO.: 3
  • the amino acid sequence of VH2 is SEQ ID NO.: 4
  • VL3 The amino acid sequence is SEQ ID NO.: 8, and the amino acid sequence of VH3 is SEQ ID NO.: 9.
  • the amino acid sequence of the first polypeptide is SEQ ID NO.: 10
  • the amino acid sequence of the second polypeptide is SEQ ID NO.: 11
  • the amino acid sequence of the third polypeptide is SEQ ID NO.: 12
  • the amino acid sequence of the fourth polypeptide is SEQ ID NO.: 13.
  • the first target is CD20
  • the second target is CD19
  • the third target is CD3.
  • the amino acid sequence of VL1 is SEQ ID NO.: 8
  • the amino acid sequence of VH1 is SEQ ID NO.: 9
  • the amino acid sequence of VL2 is SEQ ID NO.: 3
  • the amino acid sequence of VH2 is SEQ ID NO.: 4, VL3
  • the amino acid sequence is SEQ ID NO.: 1, and the VH3 amino acid sequence is SEQ ID NO.: 2.
  • the amino acid sequence of the first polypeptide is SEQ ID NO.: 14
  • the amino acid sequence of the second polypeptide is SEQ ID NO.: 15
  • the amino acid sequence of the third polypeptide is SEQ ID NO.: 16
  • the amino acid sequence of the fourth polypeptide is SEQ ID NO.: 17.
  • Fig. 1 is a schematic diagram of the structure of a bispecific antibody according to an embodiment of the present invention.
  • Figure 2 shows the killing effect of antibody A of the present invention on Raji cells.
  • Figure 3 shows the tumor suppressive effect of administration of antibody A of the present invention.
  • Figure 4 shows the effect of administration of antibody A of the present invention on the body weight of mice.
  • Figure 5 is a schematic diagram of the structure of a trispecific antibody according to an embodiment of the present invention.
  • Figure 6 shows the killing effect of antibody #1 and antibody #2 of the present invention on Raji cells.
  • Figure 7 shows the killing effect of antibody #1 and antibody #2 of the present invention on K562 cells.
  • Figure 8 shows the effects of antibody #1 and antibody #2 of the present invention on the body weight of mice under different concentration conditions.
  • Figure 9 shows the tumor inhibitory effects of antibody #1 and antibody #2 of the present invention under different concentration conditions.
  • the present invention provides bispecific engineered antibodies and trispecific engineered antibodies, which have disulfide bonds between VH and VL, mutations in selected amino acids based on their electrostatic properties, and Knob-in-hole structure in the Fc fragment.
  • the present invention provides a bispecific engineered antibody, which comprises:
  • a first polypeptide comprising in the direction from N-terminus to C-terminus: a second light chain variable domain VL2 that binds to a second target and a first heavy chain variable that binds to the first target.
  • a second polypeptide comprising in the direction from N-terminus to C-terminus: a first light chain variable domain VL1 that binds to the first target and a second heavy chain variable domain that binds to the second target
  • the domain VH2 includes the hinge domain of cysteine and the CH2-CH3 domain of IgG, where VL1 and VH2 are connected by a linker;
  • a third polypeptide which includes, from the N-terminus to the C-terminus, a hinge domain containing cysteine and a CH2-CH3 domain of IgG,
  • VL1 and VH1 combine to form a domain capable of binding to a first target, and the first target is CD3;
  • VL2 and VH2 combine to form a domain capable of binding to a second target, and the second target is CD19;
  • VL2 and VH2 are covalently linked by disulfide bonds, and VL2 and VH2 each independently include one or more substitutions introducing charged amino acids, which are electrostatically charged for the formation of homodimers. Academically disadvantaged
  • the hinge domain of the second polypeptide chain and the hinge domain of the third polypeptide chain are covalently connected by a disulfide bond.
  • the first polypeptide has the following structure in the direction from N-terminus to C-terminus: VL2-linker-VH1.
  • the second polypeptide has the following structure in the direction from N-terminus to C-terminus: VL1-linker-VH2-hinge region-CH2-CH3.
  • the third polypeptide has the following structure in the direction from N-terminus to C-terminus: hinge region-CH2-CH3.
  • the linker in the first polypeptide, the linker in the second polypeptide, and the linker in the third polypeptide have the following sequence: RTVAA, or GGGGS, or GGSGGS, or GGSGGSGGS.
  • the hinge region in the second polypeptide and the hinge region in the third polypeptide comprise hinges derived from IgG1, IgG2, IgG3, IgG4 or IgA.
  • VL2 includes the following amino acid substitutions: Cys is substituted for Gln100, Lys is substituted for Gln38, and VH2 includes the following amino acid substitutions: Asp is substituted for Gln39, and Cys is substituted for Gly44.
  • One of the CH3 domain of the second polypeptide and the CH3 domain of the third polypeptide contains Trp instead of Thr366, and the other CH3 domain contains Ser, Ala and Val instead of Thr366, Leu368, Tyr407, respectively.
  • the bispecific antibody of the present invention can recognize two antigens at the same time. It has a disulfide bond between VH and VL and mutations in selected amino acids based on their electrostatic properties.
  • the CH2CH3 domain of the second polypeptide chain and the CH2CH3 domain of the third polypeptide chain form a tenon-and-mortise structure.
  • the present invention provides a trispecific engineered antibody, which comprises:
  • a first polypeptide comprising in the direction from N-terminus to C-terminus: a second light chain variable domain VL2 that binds to a second target and a first heavy chain variable that binds to the first target.
  • a second polypeptide comprising in the direction from N-terminus to C-terminus: a first light chain variable domain VL1 that binds to the first target and a second heavy chain variable domain that binds to the second target
  • the domain VH2 includes the hinge domain of cysteine and the CH2-CH3 domain of IgG, where VL1 and VH2 are connected by a linker;
  • a third polypeptide in the direction from the N-terminus to the C-terminus, including: the third heavy chain variable domain VH3 that binds to the third target, the CH1 domain of IgG, and the cysteine-containing The hinge domain and the CH2-CH3 domain of IgG, where VH3 and CH1 are connected by a linker; and
  • a fourth polypeptide which includes in the direction from the N-terminus to the C-terminus: a third light chain variable domain VL3 that binds to the third target and a light chain constant domain containing cysteine CL, where VL3 and CL are connected by a linker;
  • VL1 and VH1 combine to form a domain capable of binding to the first target
  • VL2 and VH2 combine to form a domain capable of binding to the second target
  • VL3 and VH3 combine to form a domain capable of binding to the third target
  • VL2 and VH2 are covalently connected by a disulfide bond, and VL2 and VH2 independently comprise one or more substitutions introducing charged amino acids, and the substitution of the charged amino acids is essential for the formation of homodimers. Electrostatically unfavorable;
  • CH1 and CL are covalently connected through a disulfide bond
  • the hinge domain of the second polypeptide chain and the hinge domain of the third polypeptide chain are covalently connected by a disulfide bond.
  • the first polypeptide has the following structure in the direction from N-terminus to C-terminus: VL2-linker-VH1.
  • the second polypeptide has the following structure in the direction from N-terminus to C-terminus: VL1-linker-VH2-hinge region-CH2-CH3.
  • the third polypeptide has the following structure in the direction from N-terminus to C-terminus: VH3-linker-CH1-hinge region-CH2-CH3.
  • the fourth polypeptide has the following structure in the direction from N-terminus to C-terminus: VL3-linker-CL.
  • the linker in the first polypeptide, the linker in the second polypeptide, the linker in the third polypeptide, and the linker in the fourth polypeptide have the following sequence: RTVAA, or GGGGS, or GGSGGS, or GGSGGSGGS.
  • the hinge region in the second polypeptide and the hinge region in the third polypeptide comprise hinges derived from IgG1, IgG2, IgG3, IgG4 or IgA.
  • VL2 includes the following amino acid substitutions: Cys is substituted for Gln100, Lys is substituted for Gln38, and VH2 includes the following amino acid substitutions: Asp is substituted for Gln39, and Cys is substituted for Gly44.
  • One of the CH3 domain of the second polypeptide and the CH3 domain of the third polypeptide contains Trp instead of Thr366, and the other CH3 domain contains Ser, Ala and Val instead of Thr366, Leu368, Tyr407, respectively.
  • the trispecific antibody of the present invention can recognize three antigens at the same time. It has a disulfide bond between VH and VL and mutations in selected amino acids based on their electrostatic properties.
  • the first The CH3 domain of the second polypeptide chain and the CH3 domain of the third polypeptide chain form a tenon-and-mortise structure.
  • VL2 and VH2 are covalently linked through a disulfide bond.
  • the FR of VL2 and the FR of VH2 are covalently linked by disulfide bonds.
  • a cysteine mutation is introduced at the VL-VH interface to form a disulfide bond between VL and VH, so that VL and VH are covalently linked, thereby improving the stability of the antibody.
  • Position 100 of VL2 and position 44 of VH2 were substituted with cysteine.
  • the substituted cysteine forms a disulfide bond connecting the VL2 and VH2 of the bispecific antibody and the trispecific antibody.
  • the bispecific antibody and trispecific antibody of the present invention also have one or more amino acid substitutions with different charge properties on VL2 and VH2.
  • VL2 is replaced by a negatively charged amino acid
  • VH2 is replaced by a positively charged amino acid
  • VL2 is replaced by a positively charged amino acid
  • VH2 is replaced by a negatively charged amino acid.
  • the negatively charged amino acid is aspartic acid or glutamic acid
  • the positively charged amino acid is lysine or arginine.
  • Gln38 on VL2 is replaced by Lys, and Gln39 on VH2 is replaced by Asp, thereby forming a charged amino acid substitution.
  • the hinge domain of the second polypeptide chain and the hinge domain of the third polypeptide chain are covalently connected by a disulfide bond.
  • the CH2CH3 domain of the second polypeptide chain and the CH2CH3 domain of the third polypeptide chain form a tenon-and-mortise structure.
  • the tenon-and-mortise structure is also called the "protuberance-into-cavity" strategy, which is used to design the interface between the hetero-oligomeric second and third polypeptides.
  • the preferred interface comprises at least a part of the CH3 domain of an antibody constant domain.
  • “Bumps” are constructed by replacing small amino acid side chains in the interface of the second polypeptide with larger side chains (eg, tyrosine or tryptophan).
  • a complementary "lumen” that is the same or similar to the size of the protrusion is optionally established at the interface of the third polypeptide by substituting a smaller amino acid (for example, alanine or threonine) for a larger amino acid side chain on.
  • the CH3 domain of the second polypeptide contains Trp instead of Thr366, and the CH3 domain of the third polypeptide contains Ser, Ala and Val instead of Thr366, Leu368, and Tyr407, respectively, to form a tenon joint. structure.
  • the CH3 domain of the second polypeptide contains Ser, Ala and Val replace Thr366, Leu368, and Tyr407, respectively, and the CH3 domain of the third polypeptide contains Trp instead of Thr366, thereby forming a tenon ⁇ structure.
  • All forms of antibodies are based on the heavy and light chains of IgG antibodies, which can be prepared using methods known in the art.
  • the method usually includes the following steps: constructing an expression cassette for the heavy chain gene and the light chain gene, and combining the two genes Co-transfected into a suitable cell system to produce recombinant antibodies and prepare stable and high-yield cell clones, and the cells are fermented to produce cGMP final antibody products.
  • the present invention is further illustrated by the following embodiments, but is not limited thereto.
  • the example section below illustrates the preparation of the engineered antibody of the present invention.
  • This example illustrates the construction of CD3X and CD19 bispecific antibodies.
  • the amino acid sequences of VH and VL of CD3 and CD19 are listed in SEQ ID NO.1 to SEQ ID NO.4 (CD3 VL: SEQ ID NO.:1, CD3 VH: SEQ ID NO.: 2, CD19 VL: SEQ ID NO.: 3, CD19 VH: SEQ ID NO.: 4).
  • the target gene was first constructed in the pUC57 vector, and then subcloned into the pTGE5 vector.
  • DNA was prepared by Maxiprep for transfection.
  • the CHO3E7 cells were cultured and passaged at a concentration of 0.3 ⁇ 10 6 cells/ml. Transfection is performed when the cell density reaches 1.8-2.5 ⁇ 10 6 cells/ml. First, add 300 ⁇ l of DNA heavy chain and light chain to 50 ml of Freestyle CHO medium and mix gently. Then add 3 mg of PEI transfection reagent and mix gently for more than 3 minutes.
  • the mixture was allowed to stand at 37°C for 7 minutes, and then added to 450 ml of cell suspension to obtain a total volume of 500 ml. After 24 hours, 25 ml of TN1 (mother liquor concentration of 200 g/L) was added to the mixture. Then, take 1ml of the suspension on day 1, day 3 and day 5 for testing. Take 50 ⁇ l sample for cell count, and centrifuge the remaining sample at 3000 rpm for 5 minutes, and then leave the supernatant at -20°C. On day 6, the culture was harvested and centrifuged at 5500 rpm for 30 minutes. The supernatant was separated, filtered through a 0.22 ⁇ m filter, and the protein was further purified.
  • TN1 mother liquor concentration of 200 g/L
  • Chromatography column 5ml Monofinity A resin (GenScript, lot number L00433) chromatography column; equilibration buffer A: 20mM PB, 150mM NaCl, pH7.2; washing buffer B: 50mM citric acid, pH3.5; neutralization buffer C: 1M Tris-HCl, pH 9.0; flow rate: 2 ml/min; gradient: 100% gradient elution. After separation, 0.155 ml of Neutralization Buffer C was added to each 1 ml fraction. The collected protein solution was dialyzed in PBS (pH 7.2) at 4°C for 16 hours.
  • PBS pH 7.2
  • the CD3 X CD19 bispecific antibody (antibody A, structure diagram is shown in Figure 1) including the following three polypeptide chains is constructed:
  • the first polypeptide (SEQ ID NO.5): CD19 VL-Linker-CD3 VH
  • the second polypeptide (SEQ ID NO.6): CD3 VL-linker-CD19 VH-hinge region-CH2CH3
  • the third polypeptide C (SEQ ID NO.7): Hinge region-CH2CH3
  • the obtained antibody A is purified by size exclusion (SEC), and its purity is above 95%.
  • the binding affinity of antibody A to human CD3 ⁇ antigen and human CD19 antigen was tested, and ka, kd and KD values were calculated.
  • the capture method was used for affinity evaluation.
  • human CD19 as a ligand, it is captured on a chip coupled with an antihistamine antibody.
  • 5 candidate drugs of different concentrations were injected as analytes for affinity analysis.
  • the CD3 ⁇ antigen and CD19 antigen were solidified on the chip, and the affinity test of antibody A was carried out using the BIAcore method. The results are as follows:
  • lymphocytes as effector cells, the antibody-mediated killing effect on target cells (Raji cells) was analyzed.
  • the operating procedures are described below.
  • PBMC peripheral blood mononuclear cells
  • the Stemcell separation kit was used to further separate CD4+T cells and CD8+T cells from PBMC.
  • PBMC, CD4+ T cells and CD8+ T cells were resuspended in the cell culture medium and the cell density and cell survival rate were measured.
  • the cell culture medium was used to adjust the cell density to 6 ⁇ 10 6 viable cells/mL, and then 50 ⁇ L/well of the cell suspension was added to the flat-bottom 96-well plate.
  • the ratio of effector cells to target cells (E/T) is 20:1, which is used in experiments.
  • Cell culture medium RPMI 1640 suspended with 10% HI-FBS and 1% penicillin-streptomycin.
  • the passage density of Raji cells is 2 ⁇ 10 5 cells/mL, and they will be used for experiments after 4 days of passage growth. Transfer an appropriate amount of cell suspension to a 50ml centrifuge tube and centrifuge at 200g at room temperature for 5 minutes. The cells were resuspended in cell culture medium and the cell density and cell viability were measured. The cell density was adjusted to 3 ⁇ 10 5 viable cells/mL with the cell culture medium, and then 50 ⁇ L/well of the cell suspension was added to the flat bottom 96-well plate in which Raji cells already existed.
  • Antibody preparation Dilute antibody A and blinatumomab, MGD011 and RG6026 as controls to different concentrations in the cell culture medium. Add 50 ⁇ L of cell culture medium or diluted solution to the indicated wells to obtain a final concentration of 0 pM, 1 pM or 100 pM.
  • the flat-bottom 96-well plate with antibody, target cells and effector cells was placed in a 37°C, 5% CO2 incubator, and samples were taken at 4 hours, 20 hours and 40 hours for detection. The samples were centrifuged at 350g for 5 minutes, the cells were resuspended and stained with PI. Add 10 ⁇ L of counting beads to each well, and then analyze the sample by flow cytometry. The analysis results showed that the EC50 value of antibody A was 1.086 pM, the EC50 value of blinatumomab was 5.476 pM, the EC50 value of MGD011 was 1.721 pM, and the EC50 value of RG6026 was 0.6701 pM. The killing effect of antibody A on target cells is shown in Figure 2.
  • TRTV/CRTV (%) is the relative tumor growth rate, that is, at a certain point in time, the ratio between the tumor volume of the treatment group and the tumor volume of the control group receiving PBS.
  • TRTV and CRTV are the tumor volume (TV) of the treatment group and the control group at a certain time point, respectively.
  • This example illustrates the construction of CD3 X CD19 X CD20 trispecific antibodies.
  • the VH and VL sequences of CD3, CD19 and CD20 are in SEQ ID NO. 1 to SEQ ID NO. 4 and SEQ ID NO.: 8 and SEQ ID NO.: 9 Listed in (CD3 VL: SEQ ID NO.:1, CD3 VH: SEQ ID NO.: 2, CD19 VL: SEQ ID NO.: 3, CD19 VH: SEQ ID NO.: 4, CD20 VL: SEQ ID NO .:8,CD20 VH:SEQ ID NO.:9).
  • Polypeptide A (SEQ ID NO.10): CD19 VL-Linker-CD3 VH
  • Polypeptide B (SEQ ID NO.11): CD3 VL-linker-CD19 VH-hinge area-CH2CH3
  • Polypeptide C (SEQ ID NO.12): CD20 VH—CH1—hinge area—CH2CH3
  • Polypeptide D (SEQ ID NO.13): CD20 VL-CL
  • CD3 X CD19 X CD20 trispecific antibody including the following four polypeptide chains:
  • Polypeptide E (SEQ ID NO.14): CD19 VL-Linker-CD20 VH
  • Polypeptide F (SEQ ID NO.15): CD20 VL-linker-CD19 VH-hinge area-CH2CH3
  • Polypeptide G (SEQ ID NO.16): CD3 VH—CH1—hinge area—CH2CH3
  • Polypeptide H (SEQ ID NO.17): CD3 VL-CL
  • the obtained antibody #1 and antibody #2 were purified by size exclusion (SEC), and their purity was above 90%.
  • the capture method was used for affinity evaluation.
  • human CD19 as a ligand, it is captured on a chip coupled with an antihistamine antibody.
  • 5 candidate drugs of different concentrations were injected as analytes for affinity analysis.
  • the CD3 ⁇ antigen and CD19 antigen were solidified on the chip, and the affinity test of antibody #1 was carried out using the BIAcore method. The results are as follows:
  • lymphocytes as effector cells, the antibody-mediated killing effect on target cells (Raji cells) was analyzed.
  • the operating procedures are described below.
  • PBMC peripheral blood mononuclear cells
  • the Stemcell separation kit was used to further separate CD4+T cells and CD8+T cells from PBMC.
  • PBMC, CD4+ T cells and CD8+ T cells were resuspended in the cell culture medium and the cell density and cell survival rate were measured.
  • the cell culture medium was used to adjust the cell density to 6 ⁇ 10 6 viable cells/mL, and then 50 ⁇ L/well of the cell suspension was added to the flat-bottom 96-well plate.
  • the ratio of effector cells to target cells (E/T) is 20:1, which is used in experiments.
  • Cell culture medium RPMI 1640 suspended with 10% HI-FBS and 1% penicillin-streptomycin.
  • the passage density of Raji cells is 2 ⁇ 10 5 cells/mL, and they will be used for experiments after 4 days of passage growth. Transfer an appropriate amount of cell suspension to a 50ml centrifuge tube and centrifuge at 200g at room temperature for 5 minutes. The cells were resuspended in cell culture medium and the cell density and cell viability were measured. The cell density was adjusted to 3 ⁇ 10 5 viable cells/mL with the cell culture medium, and then 50 ⁇ L/well of the cell suspension was added to the flat bottom 96-well plate in which Raji cells already existed.
  • Antibody preparation Dilute antibody #1, antibody #2, and the bispecific antibody CD3 X CD19 as a control to different concentrations in the cell culture medium. Add 50 ⁇ L of cell culture medium or diluted solution to the indicated wells to obtain a final concentration of 0 pM, 1 pM or 100 pM.
  • the flat-bottom 96-well plate with antibody, target cells and effector cells was placed in a 37°C, 5% CO2 incubator, and samples were taken at 4 hours, 20 hours and 40 hours for detection. The samples were centrifuged at 350g for 5 minutes, the cells were resuspended and stained with PI. Add 10 ⁇ L of counting beads to each well, and then analyze the sample by flow cytometry. The analysis results show that the EC50 value of antibody #2 is 971.8 pM, the EC50 value of antibody #1 is 1.423 pM, and the killing effects of antibody #1 and antibody #2 on target cells are shown in FIG. 6.
  • CD20 was stably transferred to the CD19-CD20-double negative cell K562 as the target cell, and lymphocytes were used as the effector cell to analyze the antibody mediated against the target cell (K562 cell). Killing effect.
  • K562-CD20 cells were resuspended in 1640+2% FBS medium, centrifuged at 1000 rpm for 5 minutes, counted, and planted 10000 cells/100ul per well in a 96-well cell culture plate. Take PBMC, centrifuge at 1000 rpm for 5 minutes, resuspend in 1640+2% FBS and count, add 100000 cells/100ul to each well of the above cell plate.
  • the antibody was diluted with PBS, 10 times gradient dilution 8 points, each well was added 10ul to the cell culture plate, each concentration of two replicate wells, 37 °C, 5% CO2 incubator for 4 hours. Take out the substrate in the test kit, add 12ml buffer to each bottle and resuspend.
  • TRTV/CRTV (%) is the relative tumor growth rate, that is, at a certain point in time, the ratio between the tumor volume of the treatment group and the tumor volume of the control group receiving PBS.
  • TRTV and CRTV are the tumor volume (TV) of the treatment group and the control group at a certain time point, respectively.

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Abstract

本发明提供了具有较高的稳定性的新型双特异性抗体和三特异性抗体及其治疗用途。

Description

共价多特异性抗体
本申请要求于2019年6月20日提交的、申请号为201910535703.7、发明名称为“共价多特异性抗体”的中国专利申请的优先权，其全部内容通过引用并入本文。
技术领域
本发明涉及具有较高的稳定性的新型共价多特异性抗体及其治疗用途。
背景技术
单克隆抗体(mAb)在针对癌症和其他疾病的临床实际应用方面具有广泛的诊断和治疗潜力。无论是以裸抗体的形式还是以与细胞毒性剂(例如，放射性同位素，药物，毒素或前药转化酶)结合的偶联物的形式，单克隆抗体均在癌症免疫疗法中发挥重要作用。这些方法均处于疗效评估过程中，具有不同程度的发展并获得不同程度的临床成功。裸mAb可通过在与癌细胞上过表达的细胞表面蛋白结合之后诱导细胞毒性作用而实现临床反应。已有研究表明，通过经由程序性细胞死亡(凋亡)来控制肿瘤生长或通过诱导抗肿瘤免疫反应来实现这些治疗作用。
由于抗体的特异性靶向并介导效应子功能这一独特性质，自1975年Cesar Milstein和Georges J.F.Kohler的单克隆抗体技术的发明以来，已研发出用作针对疾病的靶向免疫疗法的抗体药物。目前已有超过60个被批准的基于抗体的生物药物，其全球年销售额超过50亿美元。当代抗体药物的成功应用已形成了药物产业并使得公共健康得到了很大改善。除了针对新型靶点的抗体药物的研发之外，最优的联合疗法和创新性的双特异性抗体的研发也有着广阔的前景。
治疗性抗体已在临床应用超过20年。目前，临床使用的抗肿瘤抗体药物包括：美罗华(Rituxan(1997)),赫赛汀(Herceptin(1998)),Mylotarg(2000),Campath(2001),Zevalin(2002),Bexxer(2003),阿瓦斯汀(Avastin(2004)),爱 必妥(Erbitux(2004)),Vectibix(2006)；Arzerra(2009)；Benlysta(2011)；Yervoy(2011)；Adcetris(2011)；Perjeta(2012)；Kadcyla(2013),Opdivo(2014),Keytruda(2014),Tecentriq(2016)。这些抗体主要靶向EGFR,Her2，CD20或VEGF，以及最近发现的PD1或PD-L1。
多功能抗体是基于传统抗体通过复杂的设计和分子工程而构建的，其具有多抗原结合能力。从实际应用方面来说，单个多功能抗体分子所产生的治疗效果与若干个传统抗体的组合所产生的治疗效果相同。然而，多功能抗体的优势远超过了若干个传统抗体的简单叠加。通过新的且独一无二的机理，所选择的多个靶点的同时接合可产生优于传统抗体的有益作用。例如，靶向CD3和CD19的博纳吐单抗(Blinatumomab，CD3 x CD19，Amgen)在杀伤表达CD19的肿瘤细胞方面可通过其CD3-识别Fv而有效接合T细胞，其在ALL(急性淋巴性白血病)等适应症上展现出了超越传统抗体的显著疗效。博纳吐单抗已于2014年被美国FDA批准上市用于治疗ALL。
双特异性抗体通过化学交联、杂交-杂交瘤或转染瘤，或在两个不同的Fab’的铰链处进行二硫化物的交换而产生。第一种方法生成异源性且难以界定的产品。第二种方法需要对由多种杂交抗体副产物得到的双特异性抗体进行大量纯化，该纯化步骤可能会影响细胞交联活性。二硫化物交换方法实质上仅仅应用于F(ab’) 2并且因此受到单克隆抗体易受到酶消化裂解的影响的限制。而且，因为Fab’彼此几乎没有亲和性，所以，形成Fab’间二硫键需要非常高的蛋白质浓度。二硫化物交换方法已通过使用Ellman试剂进行了改良，以在氧化前采用Fab’中的一个修饰另一个，这样，降低了同源二聚化的发生。然而，即便进行了这样的改良，在高于50％的产率中，异二聚F(ab’) 2也鲜有产生。然而，不利的安全问题，低反应速度和有限的有效性是目前抗体药物的现状。这些不利因素可能来自于由于抗体的表位来自自身抗原而产生的对正常组织/细胞的非靶效应，免疫效应子细胞的抑制性微环境，未预料到的Fc介导的效应子功能，等等。因此，本领域仍然需要具有良好治疗效用的高纯度多特异性(例如，三特异性)抗体。
发明内容
一方面，本发明提供一种工程化抗体，其包含：
(i)第一多肽，其在N-末端至C-末端的方向上包括：结合第二靶点的第二轻链可变结构域VL2和结合第一靶点的第一重链可变结构域VH1，其中，VL2和VH1通过连接体连接；
(ii)第二多肽，其在N-末端至C-末端的方向上包括：结合第一靶点的第一轻链可变结构域VL1和结合第二靶点的第二重链可变结构域VH2，包含半胱氨酸的铰链结构域以及IgG的CH2-CH3结构域，其中，VL1和VH2通过连接体连接；
(iii)第三多肽，其在N-末端至C-末端的方向上包括：包含半胱氨酸的铰链结构域以及IgG的CH2-CH3结构域，
其中：
VL1和VH1结合形成能够结合第一靶点的结构域，所述第一靶点是CD3；
VL2和VH2结合形成能够结合第二靶点的结构域，所述第二靶点是CD19；
VL2和VH2通过二硫键共价连接，并且，VL2和VH2分别独立地包含引入带电荷的氨基酸的一个或多个取代，所述带电荷的氨基酸的取代对于同源二聚体的形成是静电学上不利的；
所述第二多肽链的铰链结构域和所述第三多肽链的铰链结构域通过二硫键共价连接。
在上文所述的抗体中，VL1的氨基酸序列为SEQ ID NO.:1，VH1的氨基酸序列为SEQ ID NO.:2，VL2的氨基酸序列为SEQ ID NO.:3，VH2的氨基酸序列为SEQ ID NO.:4。所述第一多肽的氨基酸序列为SEQ ID NO.:5，所述第二多肽的氨基酸序列为SEQ ID NO.:6，所述第三多肽的氨基酸序列为SEQ ID NO.:7。
另一方面，本发明还提供一种工程化抗体，其包含：
(i)第一多肽，其在N-末端至C-末端的方向上包括：结合第二靶点的第二轻链可变结构域VL2和结合第一靶点的第一重链可变结构域VH1，其中，VL2和VH1通过连接体连接；
(ii)第二多肽，其在N-末端至C-末端的方向上包括：结合第一靶点的第一轻链可变结构域VL1和结合第二靶点的第二重链可变结构域VH2，包含半胱氨酸的铰链结构域以及IgG的CH2-CH3结构域，其中，VL1和VH2通过连接 体连接；
(iii)第三多肽，其在N-末端至C-末端的方向上包括：结合第三靶点的第三重链可变结构域VH3，IgG的CH1结构域，包含半胱氨酸的铰链结构域以及IgG的CH2-CH3结构域，其中，VH3和CH1通过连接体连接；以及
(iv)第四多肽，其在N-末端至C末端的方向上包括：结合所述第三靶点的第三轻链可变结构域VL3和包含半胱氨酸的轻链恒定结构域CL，其中VL3和CL通过连接体连接；
其中，VL1和VH1结合形成能够结合第一靶点的结构域；
其中，VL2和VH2结合形成能够结合第二靶点的结构域；
其中，VL3和VH3结合形成能够结合第三靶点的结构域；
其中，VL2和VH2通过二硫键共价连接，并且，VL2和VH2分别独立地包含引入带电荷的氨基酸的一个或多个取代，所述带电荷的氨基酸的取代对于同源二聚体的形成是静电学上不利的；
其中，CH1和CL通过二硫键共价连接；
其中，所述第二多肽链的铰链结构域和所述第三多肽链的铰链结构域通过二硫键共价连接。
在上文所述的抗体的一种实施方式中，所述第一靶点为CD3，所述第二靶点为CD19，所述第三靶点为CD20。VL1的氨基酸序列为SEQ ID NO.:1，VH1的氨基酸序列为SEQ ID NO.:2，VL2的氨基酸序列为SEQ ID NO.:3，VH2的氨基酸序列为SEQ ID NO.:4，VL3的氨基酸序列为SEQ ID NO.:8，VH3的氨基酸序列为SEQ ID NO.:9。所述第一多肽的氨基酸序列为SEQ ID NO.:10，所述第二多肽的氨基酸序列为SEQ ID NO.:11，所述第三多肽的氨基酸序列为SEQ ID NO.:12，所述第四多肽的氨基酸序列为SEQ ID NO.:13。
在上文所述的抗体的一种实施方式中，所述第一靶点为CD20，所述第二靶点为CD19，所述第三靶点为CD3。VL1的氨基酸序列为SEQ ID NO.:8，VH1的氨基酸序列为SEQ ID NO.:9，VL2的氨基酸序列为SEQ ID NO.:3，VH2的氨基酸序列为SEQ ID NO.:4，VL3的氨基酸序列为SEQ ID NO.:1，VH3氨基酸序列为SEQ ID NO.:2。所述第一多肽的氨基酸序列为SEQ ID NO.:14，所述第二多肽的氨基酸序列为SEQ ID NO.:15，所述第三多肽的氨基酸序列为SEQ ID NO.:16，所述第四多肽的氨基酸序列为SEQ ID NO.:17。
附图说明
本发明的新特征在所附的权利要求中具体列出。通过参考下文的具体实施方式部分以及附图将会更好地理解本发明的特征和优势，下文的具体实施方式部分列出了示例性的实施方式，其中应用了本发明的原理，本发明的附图如下：
图1是根据本发明的一种实施方式的双特异性抗体的结构示意图。
图2显示了本发明的抗体A对Raji细胞的杀伤效果。
图3显示了给药本发明的抗体A对肿瘤的抑制作用。
图4显示了给药本发明的抗体A对小鼠体重的影响。
图5是根据本发明的一种实施方式的三特异性抗体的结构示意图。
图6显示了本发明的抗体#1和抗体#2对Raji细胞的杀伤效果。
图7显示了本发明的抗体#1和抗体#2对K562细胞的杀伤效果。
图8显示了本发明的抗体#1和抗体#2在不同浓度条件下对小鼠体重的影响。
图9显示了本发明的抗体#1和抗体#2在不同浓度条件下的肿瘤抑制作用。
具体实施方式
总体上，本发明提供双特异性工程化抗体和三特异性工程化抗体，其具有位于VH和VL之间的二硫键，基于它们的静电性质而在所选择的氨基酸上出现的突变以及位于Fc片段中的榫卯(knob-in-hole)结构。
一方面，本发明提供一种双特异性工程化抗体，其包含：
(i)第一多肽，其在N-末端至C-末端的方向上包括：结合第二靶点的第二轻链可变结构域VL2和结合第一靶点的第一重链可变结构域VH1，其中，VL2和VH1通过连接体连接；
(ii)第二多肽，其在N-末端至C-末端的方向上包括：结合第一靶点的第一轻链可变结构域VL1和结合第二靶点的第二重链可变结构域VH2，包含半胱氨酸的铰链结构域以及IgG的CH2-CH3结构域，其中，VL1和VH2通过连接 体连接；
(iii)第三多肽，其在N-末端至C-末端的方向上包括：包含半胱氨酸的铰链结构域以及IgG的CH2-CH3结构域，
其中：
VL1和VH1结合形成能够结合第一靶点的结构域，所述第一靶点是CD3；
VL2和VH2结合形成能够结合第二靶点的结构域，所述第二靶点是CD19；
VL2和VH2通过二硫键共价连接，并且，VL2和VH2分别独立地包含引入带电荷的氨基酸的一个或多个取代，所述带电荷的氨基酸的取代对于同源二聚体的形成是静电学上不利的；
所述第二多肽链的铰链结构域和所述第三多肽链的铰链结构域通过二硫键共价连接。
在一些实施方式中，所述第一多肽在N-末端至C-末端的方向上具有如下结构：VL2-连接体-VH1。
在一些实施方式中，所述第二多肽在N-末端至C-末端的方向上具有如下结构：VL1-连接体-VH2-铰链区-CH2-CH3。
在一些实施方式中，所述第三多肽在N-末端至C-末端的方向上具有如下结构：铰链区-CH2-CH3。
所述第一多肽中的连接体、所述第二多肽中的连接体和所述第三多肽中的连接体具有如下序列：RTVAA，或GGGGS，或GGSGGS，或GGSGGSGGS。
所述第二多肽中的铰链区和所述第三多肽中的铰链区包含来自IgG1，IgG2，IgG3，IgG4或IgA的铰链。
在本发明的一种示例性的实施方式中，VL2包括下列氨基酸取代：Cys取代Gln100，Lys取代Gln38，VH2包括下列氨基酸取代：Asp取代Gln39，Cys取代Gly44。所述第二多肽的CH3结构域和所述第三多肽的CH3结构域中的一个包含Trp取代Thr366，另一个CH3结构域包含Ser，Ala和Val分别取代Thr366，Leu368，Tyr407。
本发明的双特异性抗体能够同时识别两个个抗原，其具有位于VH和VL之间的二硫键以及基于它们的静电性质而在所选择的氨基酸上出现的突变，在一些实施方式中，第二多肽链的CH2CH3结构域和第三多肽链的CH2CH3结构域形成榫卯结构。
另一方面，本发明提供三特异性工程化抗体，其包含：
(i)第一多肽，其在N-末端至C-末端的方向上包括：结合第二靶点的第二轻链可变结构域VL2和结合第一靶点的第一重链可变结构域VH1，其中，VL2和VH1通过连接体连接；
(ii)第二多肽，其在N-末端至C-末端的方向上包括：结合第一靶点的第一轻链可变结构域VL1和结合第二靶点的第二重链可变结构域VH2，包含半胱氨酸的铰链结构域以及IgG的CH2-CH3结构域，其中，VL1和VH2通过连接体连接；
(iii)第三多肽，其在N-末端至C-末端的方向上包括：结合第三靶点的第三重链可变结构域VH3，IgG的CH1结构域，包含半胱氨酸的铰链结构域以及IgG的CH2-CH3结构域，其中，VH3和CH1通过连接体连接；以及
(iv)第四多肽，其在N-末端至C末端的方向上包括：结合所述第三靶点的第三轻链可变结构域VL3和包含半胱氨酸的轻链恒定结构域CL，其中，VL3和CL通过连接体连接；
其中，VL1和VH1结合形成能够结合第一靶点的结构域；
其中，VL2和VH2结合形成能够结合第二靶点的结构域；
其中，VL3和VH3结合形成能够结合第三靶点的结构域；
其中，VL2和VH2通过二硫键共价连接，并且，VL2和VH2独立地包含引入带电荷的氨基酸的一个或多个取代，所述带电荷的氨基酸的取代对于同源二聚体的形成是静电学上不利的；
其中，CH1和CL通过二硫键共价连接；
其中，所述第二多肽链的铰链结构域和所述第三多肽链的铰链结构域通过二硫键共价连接。
在一些实施方式中，所述第一多肽在N-末端至C-末端的方向上具有如下结构：VL2-连接体-VH1。
在一些实施方式中，所述第二多肽在N-末端至C-末端的方向上具有如下结构：VL1-连接体-VH2-铰链区-CH2-CH3。
在一些实施方式中，所述第三多肽在N-末端至C-末端的方向上具有如下结构：VH3-连接体-CH1-铰链区-CH2-CH3。
在一些实施方式中，所述第四多肽在N-末端至C-末端的方向上具有如下 结构：VL3-连接体-CL。
所述第一多肽中的连接体、所述第二多肽中的连接体、所述第三多肽中的连接体和所述第四多肽中的连接体具有如下序列：RTVAA，或GGGGS，或GGSGGS，或GGSGGSGGS。
所述第二多肽中的铰链区和所述第三多肽中的铰链区包含来自IgG1，IgG2，IgG3，IgG4或IgA的铰链。
在本发明的一种示例性的实施方式中，VL2包括下列氨基酸取代：Cys取代Gln100，Lys取代Gln38，VH2包括下列氨基酸取代：Asp取代Gln39，Cys取代Gly44。所述第二多肽的CH3结构域和所述第三多肽的CH3结构域中的一个包含Trp取代Thr366，另一个CH3结构域包含Ser，Ala和Val分别取代Thr366，Leu368，Tyr407。
本发明的三特异性抗体能够同时识别三个抗原，其具有位于VH和VL之间的二硫键以及基于它们的静电性质而在所选择的氨基酸上出现的突变，在一些实施方式中，第二多肽链的CH3结构域和第三多肽链的CH3结构域形成榫卯结构。
二硫键
在本发明的一些实施方式中，VL2和VH2通过二硫键共价连接。在一些实施方式中，VL2的FR和VH2的FR通过二硫键共价连接。在VL-VH界面上引入半胱氨酸突变，从而在VL和VH之间形成二硫键，这样使VL和VH共价连接，从而改善了抗体的稳定性。
VL2的位置100和VH2的位置44被半胱氨酸取代。取代的半胱氨酸形成连接双特异性抗体和三特异性抗体的VL2和VH2的二硫键。
采用带电荷的氨基酸的取代
除了在VL2和VH2之间引入二硫键之外，本发明的双特异性抗体和三特异性抗体的VL2和VH2上还具有不同电荷性质的一个或多个氨基酸的取代。
早期研究显示，二硫键的引入大大提高了抗体的稳定性，但是仍然会有部 分比例的轻重链之间会以非共价的方式结合，从而会影响产物的纯度。在本发明的三特异性抗体中，考虑到了区域静电作用力的影响，在VH2和VL2上分别引入具有不同电荷性质的一个或多个氨基酸的取代，这样使不期望的非共价结合方式最小化，进一步改善产物的稳定性和纯度。
在本发明的一些实施方式中，VL2被带负电荷的氨基酸取代，VH2被带正电荷的氨基酸取代。在本发明的一些实施方式中，VL2被带正电荷的氨基酸取代，VH2被带负电荷的氨基酸取代。带负电荷的氨基酸是天冬氨酸或谷氨酸，带正电荷的氨基酸是赖氨酸或精氨酸。
在本发明的一种示例性的实施方式中，VL2上的Gln38被Lys取代，VH2上的Gln39被Asp取代，从而形成带电荷的氨基酸的取代。
榫卯结构(knobs-in-hole)
在本发明的双特异性抗体和三特异性抗体中，所述第二多肽链的铰链结构域和所述第三多肽链的铰链结构域通过二硫键共价连接。在一些实施方式中，第二多肽链的CH2CH3结构域和第三多肽链的CH2CH3结构域形成榫卯结构。
榫卯结构也称为“凸起-进入-内腔(protuberance-into-cavity)”策略，其用于设计异源寡聚的第二和第三多肽之间的界面。
总体而言，优选的界面包含抗体恒定结构域的CH3结构域的至少一部分。“凸起”通过采用较大的侧链(例如，酪氨酸或色氨酸)取代所述第二多肽的界面中的小氨基酸侧链来构建。与凸起的尺寸相同或类似的互补性“内腔”通过用较小的氨基酸(例如，丙氨酸或苏氨酸)取代较大的氨基酸侧链任选地建立在第三多肽的界面上。在定位和尺寸合适的凸起或内腔存在于第二或第三多肽的界面上时，仅需要在邻近的界面上分别设计对应的内腔或凸起。参见美国专利US8,216,805，其公开的内容通过引用并入本文。
在一种示例性的实施方式中，第二多肽的CH3结构域包含Trp取代Thr366，第三多肽的CH3结构域中包含Ser，Ala和Val分别取代Thr366，Leu368，Tyr407，从而形成榫卯结构。
在另一种示例性的实施方式中，第二多肽的CH3结构域包含Ser，Ala和Val 分别取代Thr366，Leu368，Tyr407，第三多肽的CH3结构域中包含Trp取代Thr366，从而形成榫卯结构。
抗体的制备
抗体的所有形式均基于IgG抗体的重链和轻链，其可使用本领域已知的方法制备，所述方法通常包括如下步骤：构建重链基因和轻链基因的表达盒，将两个基因共转染至合适的细胞系统以生成重组抗体并制备稳定的且高产率的细胞克隆，细胞发酵生成cGMP最终抗体产物。
实施例
本发明进一步通过下文的实施例来举例说明，但不限于此。下文的实施例部分举例说明了本发明的工程化抗体的制备。
实施例1.双特异性抗体的构建
本实施例举例说明CD3X CD19双特异性抗体的构建方式。在构建本实施例的双特异性抗体的过程中，CD3和CD19的VH和VL的氨基酸序列在SEQ ID NO.1至SEQ ID NO.4中列出(CD3 VL:SEQ ID NO.:1,CD3 VH:SEQ ID NO.:2,CD19 VL:SEQ ID NO.:3,CD19 VH:SEQ ID NO.:4)。
构建方法：使用OptimumGene对密码子的序列进行优化。首先在pUC57载体中构建目标基因，随后再亚克隆至pTGE5载体中。通过Maxiprep制备DNA用于转染。培养CHO3E7细胞并将其以0.3x10 6个细胞/ml的浓度进行传代。当细胞密度达到1.8-2.5x10 6个细胞/ml时进行转染。首先，分别将300μl DNA重链和轻链添加至50ml Freestyle CHO培养基中并轻摇混合。随后加入3mg PEI转染试剂并轻摇混合3分钟以上。将混合物在37℃下静置7分钟，随后将其加至450ml细胞悬浮液中，得到总体积500ml。24小时后，将25ml TN1(母液浓度200g/L)加至混合物中。转然后第1天、第3天和第5天分别取1ml悬浮液进行检测。取50μl样品进行细胞计数，剩余样品在3000rpm条件下离心处理5分钟，随 后将上清液留于-20℃。第6天，收获培养物并且在5500rpm条件下离心处理30分钟。分离上清液、通过0.22μm过滤器过滤并进一步纯化蛋白质。层析柱：5ml Monofinity A树脂(GenScript,批号L00433)层析柱；平衡缓冲液A：20mM PB,150mM NaCl,pH7.2；洗涤缓冲液B：50mM柠檬酸，pH3.5；中和缓冲液C：1M Tris-HCl，pH9.0；流速：2ml/min；梯度：100％梯度洗脱。分离之后，将0.155ml中和缓冲液C加至各个1ml组分中。收集的蛋白质溶液在4℃下PBS(pH7.2)中进行透析持续16小时。
根据上述方法构建得到包括如下三条多肽链的CD3 X CD19双特异性抗体(抗体A，结构示意图如图1所示)：
第一多肽(SEQ ID NO.5)：CD19 VL–连接体–CD3 VH
第二多肽(SEQ ID NO.6)：CD3 VL–连接体–CD19 VH–铰链区-CH2CH3
第三多肽C(SEQ ID NO.7)：铰链区–CH2CH3
得到的抗体A通过尺寸排阻(SEC)进行纯化，其纯度在95％以上。
实施例2.亲和力测试
使用BIAcore方法，对抗体A与人CD3ε抗原和人CD19抗原的结合亲和力进行测试，计算ka，kd和KD值。本实施例使用捕获法进行亲和力评价。通过将人CD19作为配体，使其捕获于偶联有抗组胺抗体的芯片上。随后将5个不同浓度的候选药物作为分析物进样，进行亲和力分析。将CD3ε抗原和CD19抗原固化在芯片上，采用BIAcore方法对抗体A进行亲合力测试，结果如下：
抗原 ka(1/Ms) kd(1/s) KD(M)
CD3 4.876E+5 0.01530 3.137E-8
CD19 3.653E+5 6.686E-4 1.830E-9
实施例3.针对Raji细胞的细胞杀伤分析
使用淋巴细胞作为效应细胞，分析对靶细胞(Raji细胞)的抗体介导的杀伤作用。操作规程如下描述。
准备效应细胞：通过密度梯度离心从血液中新鲜分离外周血单核细胞(PBMC)。使用Stemcell分离试剂盒从PBMC中进一步分别分离CD4+T细胞和CD8+T细胞。将PBMC，CD4+T细胞和CD8+T细胞分别重悬于细胞培养基中并且检测细胞密度和细胞存活率。细胞培养基用于将细胞密度调节至6X10 6个活细胞/mL，随后将50μL/孔的细胞悬浮液加至平底96孔板中。效应细胞与靶细胞的比例(E/T)为20:1，用于实验。细胞培养基：悬浮有10％HI-FBS和1％青霉素-链霉素的RPMI 1640。
准备靶细胞：Raji细胞的传代密度为2x10 5个细胞/mL，并在传代生长4天后开始用于实验。将适当量的细胞悬浮液转移至50ml离心管中并在室温、200g条件下离心处理5分钟。将细胞重悬于细胞培养基中并检测细胞密度和细胞存活率。用细胞培养基调节细胞密度为3x10 5个活细胞/mL，随后将50μL/孔的细胞悬浮液加至其中已存在Raji细胞的平底96孔板中。
抗体的准备：在细胞培养基中将抗体A以及作为对照的博纳吐单抗(blinatumomab)，MGD011及RG6026稀释至不同浓度。将50μL细胞培养基或稀释的溶液加至指示的孔中以得到最终浓度0pM，1pM或100pM。
将带有抗体、靶细胞和效应细胞的平底96孔板放置于37℃、5％CO2的孵育器中，在4小时、20小时和40小时取样进行检测。在350g条件下对样品进行离心处理持续5分钟，将细胞重悬并用PI染色。向每个孔中加入10μL计数珠，随后通过流式细胞仪对样品进行分析。分析结果显示，抗体A的EC50值为1.086pM，博纳吐单抗(blinatumomab)EC50值为5.476pM，MGD011EC50值为1.721pM，RG6026的EC50值为0.6701pM。抗体A对靶细胞的杀伤作用如图2所示。
实施例3.体内药效评价
在Jeko-1/NCG Mixeno模型中测试抗体A的体内抗肿瘤作用。在最开始(第0天)，将悬浮于100μL 1:1PBS/凝胶的5x10 6个Jeko-1细胞接种于动物右侧背 部皮下。接种后3天(第3天)，将1x10 7/0.1ml PBMC注射至动物腹腔。当肿瘤的平均体积达到100mm 3时，给药抗体样品。对四种抗体(博纳吐单抗@0.5mg/kg,抗体A@0.3mg/kg,MGD011@0.3mg/kg,RG6026@0.7mg/kg)以及一个对照组(pH 6.0PBS)进行测试，6个动物/组。所有样品通过尾静脉进行静脉注射给药。所有抗体和载剂每周给药两次，连续给药3周。基于相对肿瘤抑制(TGIRTV)进行疗效评价，根据动物体重变化和死亡情况进行安全性评价。
相对肿瘤生长抑制率TGIRTV(％)：TGIRTV＝1-TRTV/CRTV(％)。TRTV/CRTV(％)是相对肿瘤生长速率，即，在某一时间点，接受治疗组的肿瘤体积与接受PBS的对照组的肿瘤体积之间的比值。TRTV和CRTV分别是某一时间点治疗组和对照组的肿瘤体积(TV)。
接种后40天结束实验。如图3所示，所有治疗(抗体)组显示出对肿瘤生长的显著抑制。并且，如图4所示，所有治疗(抗体)组均未显示出显著的体重降低，这也说明，治疗用抗体没有显著的体内毒性作用。
实施例4.三特异性抗体的构建
本实施例举例说明CD3 X CD19 X CD20三特异性抗体的构建方式。在构建本实施例的三特异性抗体的过程中，CD3，CD19和CD20的VH和VL序列在SEQ ID NO.1至SEQ ID NO.4以及SEQ ID NO.:8和SEQ ID NO.:9中列出(CD3 VL:SEQ ID NO.:1,CD3 VH:SEQ ID NO.:2,CD19 VL:SEQ ID NO.:3,CD19 VH:SEQ ID NO.:4,CD20 VL:SEQ ID NO.:8,CD20 VH:SEQ ID NO.:9)。
根据实施例1记载的构建方法构建得到包括如下四条多肽链的CD3 X CD19 X CD20三特异性抗体(抗体#1)：
多肽A(SEQ ID NO.10)：CD19 VL–连接体–CD3 VH
多肽B(SEQ ID NO.11)：CD3 VL–连接体–CD19 VH–铰链区-CH2CH3
多肽C(SEQ ID NO.12)：CD20 VH–CH1–铰链区–CH2CH3
多肽D(SEQ ID NO.13)：CD20 VL–CL
采用上述相同的方法，仅对CD3，CD19和CD20的位置进行交换，构建得到包括如下四条多肽链的CD3 X CD19 X CD20三特异性抗体(抗体#2)：
多肽E(SEQ ID NO.14)：CD19 VL–连接体–CD20 VH
多肽F(SEQ ID NO.15)：CD20 VL–连接体–CD19 VH–铰链区-CH2CH3
多肽G(SEQ ID NO.16)：CD3 VH–CH1–铰链区–CH2CH3
多肽H(SEQ ID NO.17)：CD3 VL–CL
得到的抗体#1和抗体#2通过尺寸排阻(SEC)进行纯化，其纯度均在90％以上。
实施例5.亲和力测试
使用BIAcore方法，对抗体#1与人CD3ε抗原和人CD19抗原的结合亲和力进行测试，计算ka，kd和KD值。本实施例使用捕获法进行亲和力评价。通过将人CD19作为配体，使其捕获于偶联有抗组胺抗体的芯片上。随后将5个不同浓度的候选药物作为分析物进样，进行亲和力分析。将CD3ε抗原和CD19抗原固化在芯片上，采用BIAcore方法对抗体#1进行亲合力测试，结果如下：
抗原 ka(1/Ms) kd(1/s) KD(M)
CD3 3.342E+5 0.008349 2.498E-8
CD19 2.068E+5 2.727E-4 1.319E-9
实施例6.针对Raji细胞的细胞杀伤分析
使用淋巴细胞作为效应细胞，分析对靶细胞(Raji细胞)的抗体介导的杀伤作用。操作规程如下描述。
准备效应细胞：通过密度梯度离心从血液中新鲜分离外周血单核细胞(PBMC)。使用Stemcell分离试剂盒从PBMC中进一步分别分离CD4+T细胞和 CD8+T细胞。将PBMC，CD4+T细胞和CD8+T细胞分别重悬于细胞培养基中并且检测细胞密度和细胞存活率。细胞培养基用于将细胞密度调节至6X10 6个活细胞/mL，随后将50μL/孔的细胞悬浮液加至平底96孔板中。效应细胞与靶细胞的比例(E/T)为20:1，用于实验。细胞培养基：悬浮有10％HI-FBS和1％青霉素-链霉素的RPMI 1640。
准备靶细胞：Raji细胞的传代密度为2x10 5个细胞/mL，并在传代生长4天后开始用于实验。将适当量的细胞悬浮液转移至50ml离心管中并在室温、200g条件下离心处理5分钟。将细胞重悬于细胞培养基中并检测细胞密度和细胞存活率。用细胞培养基调节细胞密度为3x10 5个活细胞/mL，随后将50μL/孔的细胞悬浮液加至其中已存在Raji细胞的平底96孔板中。
抗体的准备：在细胞培养基中将抗体#1、抗体#2以及作为对照的双特异性抗体CD3 X CD19稀释至不同浓度。将50μL细胞培养基或稀释的溶液加至指示的孔中以得到最终浓度0pM，1pM或100pM。
将带有抗体、靶细胞和效应细胞的平底96孔板放置于37℃、5％CO2的孵育器中，在4小时、20小时和40小时取样进行检测。在350g条件下对样品进行离心处理持续5分钟，将细胞重悬并用PI染色。向每个孔中加入10μL计数珠，随后通过流式细胞仪对样品进行分析。分析结果显示，抗体#2的EC50值为971.8pM，抗体#1的EC50值为1.423pM，并且抗体#1和抗体#2对靶细胞的杀伤作用如图6所示。
实施例7.针对K562细胞的细胞杀伤分析
为了观察CD3XCD19XCD20不依赖CD19的细胞杀伤作用，在CD19-CD20-双阴细胞K562上稳转入CD20作为靶细胞，使用淋巴细胞作为效应细胞，来分析针对靶细胞(K562细胞)的抗体介导的杀伤作用。
将K562-CD20细胞离心后用1640+2％FBS培养基重悬，1000rpm离心5分钟，计数，在96孔细胞培养板中每孔种10000cells/100ul。取PBMC，1000rpm离心5分钟，用1640+2％FBS重悬计数，在上述细胞板中每孔再加入100000cells/100ul。用PBS分别稀释抗体，10倍梯度稀释8个点，每孔分别加入10ul到细胞培养板中，每个浓度两复孔，37℃，5％CO2培养箱中培养4h。取出检测试剂盒中的 底物，每瓶加入12ml buffer重悬。将培养板1500rpm离心5分钟，每孔取上清50uL至新的培养板中，每孔在分别加入50ul新配置的底物，室温避光孵育10min，酶标仪检测OD490。分析结果显示，抗体#1的EC50值为1.222pM，并且抗体#1和对照抗体对靶细胞的杀伤作用如图7所示。
实施例8.体内药效评价
在Jeko-1/NCG Mixeno模型中测试抗体#1和抗体#2的体内抗肿瘤作用。在最开始(第0天)，将悬浮于100μL 1:1PBS/凝胶的5x10 6个Jeko-1细胞接种于动物右侧背部皮下。接种后3天(第3天)，将1x10 7/0.1ml PBMC注射至动物腹腔。当肿瘤的平均体积达到100mm 3时，给药抗体#1和抗体#2。对四种抗体(CD3 X CD19@0.5mg/kg,抗体#1@0.5mg/kg,抗体#1@3mg/kg,抗体#2@0.5mg/kg)以及一个对照组(pH 6.0 PBS)进行测试，6个动物/组。所有样品通过尾静脉进行静脉注射给药。所有抗体和载剂每周给药两次，连续给药3周。基于相对肿瘤抑制(TGIRTV)进行疗效评价，根据动物体重变化和死亡情况进行安全性评价。
相对肿瘤生长抑制率TGIRTV(％)：TGIRTV＝1-TRTV/CRTV(％)。TRTV/CRTV(％)是相对肿瘤生长速率，即，在某一时间点，接受治疗组的肿瘤体积与接受PBS的对照组的肿瘤体积之间的比值。TRTV和CRTV分别是某一时间点治疗组和对照组的肿瘤体积(TV)。
接种后28天结束实验。如图9所示，所有治疗(抗体)组显示出对肿瘤生长的显著抑制。并且，如图8所示，所有治疗(抗体)组均未显示出显著的体重降低，这也说明，治疗用抗体没有显著的体内毒性作用。
虽然本文描述并显示了本发明的示例性的实施方式，但是对于本领域技术人员而言显而易见的是，这些实施方式仅仅是举例说明。本领域技术人员可对这些实施方式作出多种改变、修改和替换，而不背离本发明。通过所附的权利要求来限定本发明的范围，并且这些权利要求范围内的方法和结构及其等同物也被所附的权利要求涵盖。

Claims (10)

  1. 一种工程化抗体，其包含：
    (i)第一多肽，其在N-末端至C-末端的方向上包括：结合第二靶点的第二轻链可变结构域VL2和结合第一靶点的第一重链可变结构域VH1，其中，VL2和VH1通过连接体连接；
    (ii)第二多肽，其在N-末端至C-末端的方向上包括：结合第一靶点的第一轻链可变结构域VL1和结合第二靶点的第二重链可变结构域VH2，包含半胱氨酸的铰链结构域以及IgG的CH2-CH3结构域，其中，VL1和VH2通过连接体连接；
    (iii)第三多肽，其在N-末端至C-末端的方向上包括：包含半胱氨酸的铰链结构域以及IgG的CH2-CH3结构域，
    其中：
    VL1和VH1结合形成能够结合第一靶点的结构域，所述第一靶点是CD3；
    VL2和VH2结合形成能够结合第二靶点的结构域，所述第二靶点是CD19；
    VL2和VH2通过二硫键共价连接，并且，VL2和VH2分别独立地包含引入带电荷的氨基酸的一个或多个取代，所述带电荷的氨基酸的取代对于同源二聚体的形成是静电学上不利的；
    所述第二多肽链的铰链结构域和所述第三多肽链的铰链结构域通过二硫键共价连接。
  2. 如权利要求1所述的抗体，其中，VL1的氨基酸序列为SEQ ID NO.:1，VH1的氨基酸序列为SEQ ID NO.:2，VL2的氨基酸序列为SEQ ID NO.:3，VH2的氨基酸序列为SEQ ID NO.:4。
  3. 如权利要求1所述的抗体，其中，所述第一多肽的氨基酸序列为SEQ ID NO.:5，所述第二多肽的氨基酸序列为SEQ ID NO.:6，所述第三多肽的氨基酸序列为SEQ ID NO.:7。
  4. 一种工程化抗体，其包含：
    (i)第一多肽，其在N-末端至C-末端的方向上包括：结合第二靶点的第二轻链可变结构域VL2和结合第一靶点的第一重链可变结构域VH1，其中，VL2和VH1通过连接体连接；
    (ii)第二多肽，其在N-末端至C-末端的方向上包括：结合第一靶点的第一轻链可变结构域VL1和结合第二靶点的第二重链可变结构域VH2，包含半胱氨酸的铰链结构域以及IgG的CH2-CH3结构域，其中，VL1和VH2通过连接体连接；
    (iii)第三多肽，其在N-末端至C-末端的方向上包括：结合第三靶点的第三重链可变结构域VH3，IgG的CH1结构域，包含半胱氨酸的铰链结构域以及IgG的CH2-CH3结构域，其中，VH3和CH1通过连接体连接；以及
    (iv)第四多肽，其在N-末端至C末端的方向上包括：结合所述第三靶点的第三轻链可变结构域VL3和包含半胱氨酸的轻链恒定结构域CL，其中VL3和CL通过连接体连接；
    其中，VL1和VH1结合形成能够结合第一靶点的结构域；
    其中，VL2和VH2结合形成能够结合第二靶点的结构域；
    其中，VL3和VH3结合形成能够结合第三靶点的结构域；
    其中，VL2和VH2通过二硫键共价连接，并且，VL2和VH2分别独立地包含引入带电荷的氨基酸的一个或多个取代，所述带电荷的氨基酸的取代对于同源二聚体的形成是静电学上不利的；
    其中，CH1和CL通过二硫键共价连接；
    其中，所述第二多肽链的铰链结构域和所述第三多肽链的铰链结构域通过二硫键共价连接。
  5. 如权利要求4所述的抗体，其中，所述第一靶点为CD3，所述第二靶点为CD19，所述第三靶点为CD20。
  6. 如权利要求4所述的抗体，其中，所述第一靶点为CD20，所述第二靶点为CD19，所述第三靶点为CD3。
  7. 如权利要求5所述的抗体，其中，VL1的氨基酸序列为SEQ ID NO.:1， VH1的氨基酸序列为SEQ ID NO.:2，VL2的氨基酸序列为SEQ ID NO.:3，VH2的氨基酸序列为SEQ ID NO.:4，VL3的氨基酸序列为SEQ ID NO.:8，VH3的氨基酸序列为SEQ ID NO.:9。
  8. 如权利要求6所述的抗体，其中，VL1的氨基酸序列为SEQ ID NO.:8，VH1的氨基酸序列为SEQ ID NO.:9，VL2的氨基酸序列为SEQ ID NO.:3，VH2的氨基酸序列为SEQ ID NO.:4，VL3的氨基酸序列为SEQ ID NO.:1，VH3氨基酸序列为SEQ ID NO.:2。
  9. 如权利要求5所述的抗体，其中，所述第一多肽的氨基酸序列为SEQ ID NO.:10，所述第二多肽的氨基酸序列为SEQ ID NO.:11，所述第三多肽的氨基酸序列为SEQ ID NO.:12，所述第四多肽的氨基酸序列为SEQ ID NO.:13。
  10. 如权利要求6所述的抗体，其中，所述第一多肽的氨基酸序列为SEQ ID NO.:14，所述第二多肽的氨基酸序列为SEQ ID NO.:15，所述第三多肽的氨基酸序列为SEQ ID NO.:16，所述第四多肽的氨基酸序列为SEQ ID NO.:17。
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