WO2021155635A1 - 抗cd3和cd123双特异性抗体及其用途 - Google Patents

抗cd3和cd123双特异性抗体及其用途 Download PDF

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WO2021155635A1
WO2021155635A1 PCT/CN2020/082151 CN2020082151W WO2021155635A1 WO 2021155635 A1 WO2021155635 A1 WO 2021155635A1 CN 2020082151 W CN2020082151 W CN 2020082151W WO 2021155635 A1 WO2021155635 A1 WO 2021155635A1
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seq
antibody
binding portion
heavy chain
antigen
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PCT/CN2020/082151
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French (fr)
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刘志刚
郝小勃
张雪萍
刘玉兰
郭晶晶
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北京智仁美博生物科技有限公司
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Priority to KR1020227030639A priority Critical patent/KR20220137723A/ko
Priority to JP2022547913A priority patent/JP7457822B2/ja
Priority to EP20918015.7A priority patent/EP4101867A4/en
Priority to US17/797,656 priority patent/US20230071422A1/en
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • 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
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • 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
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    • C07K2317/74—Inducing cell proliferation
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • This application generally relates to the field of antibody drugs. Specifically, the application provides bispecific antibodies containing an antigen binding portion against human CD3E and/or an antigen binding portion against human CD123 and their medical and biological uses.
  • Bispecific antibody is a type of artificial antibody that contains two different antigen binding sites. Bispecific antibodies are widely used in the field of biomedicine, especially tumor immunotherapy.
  • a bispecific antibody targeting CD3 has one arm that binds to the CD3E subunit in the TCR receptor complex on the surface of T cells, and the other arm targets tumor antigens. In this way, bispecific antibodies can redirect T cells to specifically kill tumor cells in a non-histocompatibility complex (MHC) dependent manner.
  • MHC non-histocompatibility complex
  • bispecific antibodies There are many platforms for bispecific antibodies and their structures are complex.
  • the antibody structure can be divided into two categories: Fc segment and non-Fc segment.
  • Bispecific antibodies without Fc segment are composed of the VH and VL regions of two antibodies or are composed of Fab fragments.
  • the main representatives of this type of bispecific antibodies are BiTE, DART, TandAbs, bi-nanobody, etc.
  • the advantage of this type of bispecific antibody is that there is no mismatch between light and heavy chains, but the disadvantage is that the half-life is short and clinical application is inconvenient.
  • Bispecific antibodies with an Fc segment retain the structure of a traditional monoclonal antibody and can mediate the biological functions of the Fc segment.
  • bispecific antibodies are KIH IgG, crossmab, DVD-Ig, Triomab, etc., which have long in vivo half-lives and can have ADCC and CDC activities (Hongyan Liu, Abhishek Saxena, Sachdev S. Sidhu, et al. Fc engineering for Development) Therapeutic Bispecifc Antibodies and Novel Scaffolds.Front.Immunol.2017; 8:38).
  • the present application provides a bispecific antibody, which comprises an antigen binding portion against human CD3E, and the antigen binding portion against human CD3E comprises:
  • HCDR1 (heavy chain CDR1) shown in SEQ ID NO:1
  • HCDR2 (heavy chain CDR2) shown in SEQ ID NO: 2,
  • HCDR3 (heavy chain CDR3) shown in SEQ ID NO: 3,
  • LCDR1 light chain CDR1
  • LCDR2 (light chain CDR2) as shown in SEQ ID NO: 5, and
  • LCDR3 (light chain CDR3) shown in SEQ ID NO: 6;
  • HCDR and LCDR are defined according to Kabat.
  • the present application provides a bispecific antibody, which comprises an antigen-binding portion against human CD123, and the antigen-binding portion against human CD123 comprises:
  • HCDR1 (heavy chain CDR1) shown in SEQ ID NO: 7,
  • HCDR2 (heavy chain CDR2) shown in SEQ ID NO: 8,
  • LCDR1 light chain CDR1
  • LCDR2 (light chain CDR2) as shown in SEQ ID NO: 5, and
  • LCDR3 (light chain CDR3) shown in SEQ ID NO: 6;
  • HCDR and LCDR are defined according to Kabat.
  • the present application provides a bispecific antibody, which comprises an antigen-binding portion directed against human CD3E and an antigen-binding portion directed against human CD123.
  • the antigen binding portion for human CD3E comprises:
  • HCDR and LCDR are defined according to Kabat.
  • the antigen binding portion for human CD123 comprises:
  • HCDR and LCDR are defined according to Kabat.
  • the antigen binding portion for human CD3E and the antigen binding portion for human CD123 comprise the same light chain variable region.
  • the bispecific antibody is an IgG1 antibody, which comprises two heavy chain constant regions with the same hinge region, and the amino acid sequence of the hinge region is shown in SEQ ID NO: 10.
  • the bispecific antibody is an IgG1 antibody comprising a first heavy chain constant region and a second heavy chain constant region, wherein the 354th and 366th sections of the first heavy chain constant region
  • the amino acids at positions C and W are respectively C and W, and the amino acids at positions 349, 366, 368, and 407 of the constant region of the second heavy chain are C, S, A, and V respectively; the amino acid positions of the antibody constant region are determined according to EU numbering.
  • the bispecific antibody is an IgG1 antibody comprising a first heavy chain constant region and a second heavy chain constant region, wherein the first heavy chain constant region and the second heavy chain
  • the amino acids at positions 234, 235, and 331 of the constant region are F, E, and S respectively; the amino acid positions of the antibody constant region are determined according to EU numbering.
  • the antigen binding portion for human CD3E comprises a heavy chain variable region as shown in SEQ ID NO: 11 and a light chain as shown in SEQ ID NO: 12 Variable region.
  • the antigen binding portion for human CD123 comprises the heavy chain variable region shown in SEQ ID NO: 13 and the light chain variable region shown in SEQ ID NO: 12 Variable area.
  • the antigen binding portion for human CD3E comprises a single chain antibody (scfv) or a Fab fragment.
  • the antigen binding portion against human CD123 comprises a single chain antibody (scfv) or a Fab fragment.
  • the antibody has a first arm and a second arm, wherein the first arm comprises an antigen binding portion for human CD3E, and the second arm comprises an antigen binding portion for human CD123 :
  • the first arm includes the heavy chain variable region amino acid sequence shown in SEQ ID NO: 11, the heavy chain constant region amino acid sequence shown in SEQ ID NO: 31, and the light chain shown in SEQ ID NO: 12 The amino acid sequence of the variable region and the amino acid sequence of the light chain constant region as shown in SEQ ID NO: 32;
  • the second arm includes the heavy chain variable region amino acid sequence shown in SEQ ID NO: 13, the heavy chain constant region amino acid sequence shown in SEQ ID NO: 30, and the light chain shown in SEQ ID NO: 12 The amino acid sequence of the variable region and the amino acid sequence of the light chain constant region as shown in SEQ ID NO: 32.
  • the present application provides a pharmaceutical composition comprising the bispecific antibody described in any one of the first to third aspects.
  • the pharmaceutical composition is used to prevent or treat CD123-positive tumors.
  • this application provides the use of the bispecific antibody according to any one of the first to third aspects or the pharmaceutical composition according to the fourth aspect in the preparation of a medicine for the prevention or treatment of CD123-positive tumors use.
  • the present application provides a method for preventing or treating CD123-positive tumors, including administering the bispecific antibody according to any one of the first to third aspects or the drug according to the fourth aspect to an individual in need combination.
  • Figure 1 shows the results of using flow cytometry to analyze the specific binding of recombinant anti-CD123 monoclonal antibody to CD123 on the cell surface.
  • Figure 2 shows the results of using flow cytometry to analyze the binding of the H7A3 humanized mutant H7A3-h2-m5+L27E5 to CD123 on the surface of MV-4-11 cells.
  • Figure 3 shows the results of analyzing the binding of the bispecific antibody CD3E ⁇ CD123 to the two antigens of CD3E and CD123 by ELISA.
  • Figure 4 shows the results of using flow cytometry to analyze the ability of the bispecific antibody CD3E ⁇ CD123 to bind to CD3E on the surface of Jurkat-Dual cells.
  • Figure 5 shows the results of using flow cytometry to analyze the ability of the bispecific antibody CD3E ⁇ CD123 to bind CD123 on the surface of MV-4-11 cells.
  • Figure 6 shows the results of the bispecific antibody CD3 ⁇ CD123 on Jurkat-Dual activation in the presence of CD123-positive tumor cells.
  • Figure A shows the bispecific antibody CD3 ⁇ CD123, anti-CD3E monoclonal antibody H3B8+L27E5, and anti-CD123 monoclonal antibody.
  • Figure B is the CD123 positive target Jurkat-Dual activation results of bispecific antibody CD3 ⁇ CD123, bispecific antibody control sample Xmab14045 and HIgG in the presence of cells, and bispecific antibody CD3 ⁇ CD123 and bispecific antibody control sample in the presence of CD123-negative target cells.
  • Figure 7 shows the results of the killing of CD123-positive tumor cells by purified T cells mediated by the bispecific antibody CD3E ⁇ CD123.
  • Figure 8 shows the results of using flow cytometry to detect the bispecific antibody CD3E ⁇ CD123 that specifically activates T cells and up-regulates the expression of CD69 in the presence of CD123-positive tumor cells.
  • Figure 9 shows the results of using flow cytometry to detect the bispecific antibody CD3E ⁇ CD123 promoting T cell proliferation in the presence of CD123-positive tumor cells.
  • Figure A shows the bispecific antibody CD3 ⁇ CD123 and anti-CD3E monoclonal antibody H3B8+
  • L27E5, anti-CD123 monoclonal antibody H7A3-h2-m5+L27E5, and anti-CD3E monoclonal antibody H3B8+L27E5 and anti-CD123 monoclonal antibody H7A3-h2-m5+L27E5 promote T cells in the presence of CD123-positive tumor cells
  • Figure B is the result of the bispecific antibody CD3 ⁇ CD123 and the bispecific antibody control sample Xmab14045 promoting T cell proliferation in the presence of CD123-positive tumor cells.
  • Figure 10 shows the change of tumor volume in hCD34+ humanized MV-4-11 cell tumor model mice treated with bispecific antibody CD3E ⁇ CD123.
  • SEQ ID NO: 1 is the amino acid sequence of HCDR1 of the heavy chain variable region H3B8 of the anti-human CD3E monoclonal antibody H3B8+L27E5.
  • SEQ ID NO: 2 is the amino acid sequence of HCDR2 of the heavy chain variable region H3B8 of the anti-human CD3E monoclonal antibody H3B8+L27E5.
  • SEQ ID NO: 3 is the amino acid sequence of HCDR3 of the heavy chain variable region H3B8 of the anti-human CD3E monoclonal antibody H3B8+L27E5.
  • SEQ ID NO: 4 is the amino acid sequence of LCDR1 of the light chain variable region L27E5.
  • SEQ ID NO: 5 is the amino acid sequence of LCDR2 of the light chain variable region L27E5.
  • SEQ ID NO: 6 is the amino acid sequence of LCDR3 of the light chain variable region L27E5.
  • SEQ ID NO: 7 is the amino acid sequence of HCDR1 of the heavy chain variable region H7A3-h2-m5 of the anti-human CD123 monoclonal antibody H7A3-h2-m5+L27E5.
  • SEQ ID NO: 8 is the amino acid sequence of HCDR2 of the heavy chain variable region H7A3-h2-m5 of the anti-human CD123 monoclonal antibody H7A3-h2-m5+L27E5.
  • SEQ ID NO: 9 is the amino acid sequence of HCDR3 of the heavy chain variable region H7A3-h2-m5 of the anti-human CD123 monoclonal antibody H7A3-h2-m5+L27E5.
  • SEQ ID NO: 10 is the amino acid sequence of the hinge region.
  • SEQ ID NO: 11 is the amino acid sequence of the humanized heavy chain mutant H3B8 of the rat monoclonal antibody WM03-C6.
  • SEQ ID NO: 12 is the amino acid sequence of the humanized light chain mutant L27E5 of the rat monoclonal antibody WM03-C6.
  • SEQ ID NO: 13 is the amino acid sequence of the humanized version H7A3-h2-m5.
  • SEQ ID NO: 14 is the amino acid sequence of the extracellular region of human (Homo sapiens) CD3E (hCD3E).
  • SEQ ID NO: 15 is the amino acid sequence of the extracellular region of Homo sapiens CD3D (hCD3D).
  • SEQ ID NO: 16 is the amino acid sequence of the CD3E extracellular domain (mfCD3E) of Macaca fascicularis.
  • SEQ ID NO: 17 is the amino acid sequence of the cynomolgus monkey (Macaca fascicularis) CD3D extracellular domain (mfCD3D).
  • SEQ ID NO: 18 is the amino acid sequence of the extracellular region of mouse (Mus musculus) CD3E (mCD3E).
  • SEQ ID NO: 19 is the amino acid sequence of the extracellular region of mouse (Mus musculus) CD3D (mCD3D).
  • SEQ ID NO: 20 is the amino acid sequence of the extracellular region of human (Homo sapiens) CD123 subtype 1 (hCD123-SP1).
  • SEQ ID NO: 21 is the amino acid sequence of the cynomolgus monkey (Macaca fascicularis) CD123 subtype 1 extracellular domain (mfCD123-SP1).
  • SEQ ID NO: 22 is the amino acid sequence of the extracellular region of mouse (Mus musculus) CD123 subtype 1 (mCD123-SP1).
  • SEQ ID NO: 23 is the amino acid sequence of the His tag.
  • SEQ ID NO: 24 is the amino acid sequence of the Fc segment (mFc) of the mouse (Mus musculus) IgG2a antibody.
  • SEQ ID NO: 25 is the amino acid sequence of the Fc mutant FcK of the heterodimer human IgG1 subtype.
  • SEQ ID NO: 26 is the amino acid sequence of the Fc mutant FcH of the heterodimer human IgG1 subtype.
  • SEQ ID NO: 27 is the amino acid sequence of the heavy chain constant region of a human (Homo sapiens) IgG1 subtype antibody.
  • SEQ ID NO: 28 is the amino acid sequence of human IgG1 subtype antibody heavy chain constant region mutant IgG1H.
  • SEQ ID NO: 29 is the amino acid sequence of human IgG1 subtype antibody heavy chain constant region mutant IgG1K.
  • SEQ ID NO: 30 is the amino acid sequence of human IgG1 subtype antibody heavy chain constant region mutant IgG1m3-H.
  • SEQ ID NO: 31 is the amino acid sequence of human IgG1 subtype antibody heavy chain constant region mutant IgG1m3-K.
  • SEQ ID NO: 32 is the amino acid sequence of the human (Homo sapiens) ⁇ subtype light chain constant region.
  • SEQ ID NO: 33 is the amino acid sequence of the human (Homo sapiens) ⁇ subtype light chain constant region.
  • SEQ ID NO: 34 is the amino acid sequence of the heavy chain variable region of monoclonal antibody WM03-C6.
  • SEQ ID NO: 35 is the amino acid sequence of the light chain variable region of monoclonal antibody WM03-C6.
  • SEQ ID NO: 36 is the amino acid sequence of the anti-human CD123 single-chain antibody S8F3.
  • SEQ ID NO: 37 is the amino acid sequence of the heavy chain variable region S8F3VH of the anti-human CD123 single-chain antibody S8F3.
  • SEQ ID NO: 38 is the heavy chain amino acid sequence of the anti-CD123 antibody CSL362.
  • SEQ ID NO: 39 is the amino acid sequence of the light chain of the anti-CD123 antibody CSL362.
  • SEQ ID NO: 40 is the amino acid sequence of the CD123-binding heavy chain of Xmab14045 of the IgG1m3 subtype containing the Hole mutation.
  • SEQ ID NO: 41 is the amino acid sequence of the CD3E-binding scFv structure of Xmab14045 of IgG1m3 subtype with Knob mutation.
  • SEQ ID NO: 42 is the amino acid sequence of the light chain of Xmab14045.
  • amino acid position numbering refers to the EU numbering definition of human IgG1 antibody, which is well known and easily found by those skilled in the art.
  • a mutation refers to a mutation generated relative to the natural antibody sequence.
  • Fc fragment refers to a part of the constant region of an antibody heavy chain, including the hinge region, the CH2 fragment and the CH3 fragment of the constant region.
  • the Fc fragment is the amino acid sequence of positions 216-447 in the constant region of the antibody.
  • Fab fragment antigen binding fragment
  • Fab part or similar terms as used herein refer to the antibody fragments that can bind to the antigen produced by the treatment of intact antibodies with papain, including the intact light chain (VL-CL ), heavy chain variable region and CH1 fragment (VH-CH1).
  • single chain antibody scfv, single chain fragment variable
  • VH heavy chain variable region
  • VL light chain variable region
  • a flexible linker is usually designed between the variable region of the heavy chain and the variable region of the light chain so that the variable region of the heavy chain and the variable region of the light chain can be folded into the correct conformation capable of binding the antigen.
  • antigen-binding portion refers to the part of the antibody structure that determines the antigen-binding ability.
  • the main part of the antibody structure that determines the antigen-binding ability is the CDR, so the CDR is also the core component of the antigen-binding part.
  • examples of the "antigen binding portion” include, but are not limited to, single-chain antibodies (scfv) or Fab fragments.
  • bispecific antibody refers to an antibody that has the ability to bind two different antigens, which can consist of two Fc fragments and two antigen binding parts respectively fused to them.
  • the "bispecific antibody” herein refers to a bispecific antibody based on a human IgG1 antibody, and in addition to the altered structure described herein, it has the basic characteristics and functions of a human IgG1 antibody.
  • bispecific antibodies herein can also be based on other immunoglobulin subtypes, such as human IgG2 antibodies.
  • CDR complementarity determining region
  • CDR complementarity determining region
  • VH or VL the complementarity determining region
  • Chothia definition see, for example, Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991); A1-Lazikani et al., J. Mol. Biol. 273: 927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86: 9268-9272 (1989)).
  • the sequence of the CDR regions in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition.
  • Kabat is used to define CDR sequences.
  • variable region sequence of a given antibody the CDR region sequence in the variable region sequence can be analyzed in a variety of ways, for example, it can be determined using the online software Abysis (http://www.abysis.org/).
  • binding refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an epitope.
  • the CD3 molecule is an important differentiation antigen on the T cell membrane and a characteristic mark of mature T cells.
  • CD3 is composed of four chains of ⁇ , ⁇ , ⁇ and ⁇ or five chains of ⁇ , ⁇ , ⁇ , ⁇ and ⁇ ( ⁇ and ⁇ are homologous isomers), with 3 types of CD3 ⁇ , CD3 ⁇ and CD3 ⁇ (or CD3 ⁇ )
  • the dimer is composed and expressed on the T cell membrane.
  • the three chains of CD3 ⁇ , ⁇ and ⁇ contain highly conserved acidic amino acid residues ( ⁇ is glutamic acid, ⁇ and ⁇ are aspartic acid), which can interact with the basicity of T cell receptor (TCR) ⁇ and ⁇ chains.
  • TCR-CD3 complex structure Amino acid residues are connected by non-covalent bonds through salt bridges to form a stable TCR-CD3 complex structure.
  • the complex can transmit T cell activation signals and stabilize the TCR structure.
  • ITAM immunoglobulin-like tyrosine activation motif
  • the intracellular region of each chain of CD3 contains ITAM (immunoreceptor tyrosine activation motif) structure, which is the basis of CD3 molecule-mediated intracellular signal transduction.
  • CD3 has the function of transmitting the activation signal generated by the TCR recognition antigen and is the first signal to induce T cell activation.
  • CD123 also known as human interleukin-3 (IL-3) receptor alpha chain
  • IL-3 receptor alpha chain is a member of the cytokine receptor superfamily, with a molecular weight of about 40KDa, and is a type I transmembrane glycoprotein.
  • Interleukin-3 receptor is a heterodimer formed by ⁇ chain (CD123) and ⁇ chain (CD131). After IL-3 binds to CD123, CD131 provides signal transduction, which regulates the functions of hematopoietic cells and immune cells and stimulates endothelial cell proliferation (Testa et al, Biomark Res. 2:4 (2014)).
  • CD123 is mainly expressed in bone marrow progenitor cells, plasmacytoid dendritic cells, monocytes, basophils, and a small amount of B cell subgroups (Munoz L et al, Haematologica. 86(12): 1261-9). About 80% of AML patients' primordial cells overexpress CD123. Studies have shown that the overexpression of CD123 antigen corresponds to the poor prognosis and lower remission rate of AML (Testa U et al, Blood. 2002; 100(8)). Although most AML patients respond well to the initial treatment, there are still some patients (60-80%) who need consolidation therapy to achieve complete remission.
  • the present application provides a bispecific antibody, which comprises an antigen binding portion against human CD3E, and the antigen binding portion against human CD3E comprises:
  • HCDR and LCDR are defined according to Kabat.
  • the present application provides a bispecific antibody, which comprises an antigen-binding portion against human CD123, and the antigen-binding portion against human CD123 comprises:
  • HCDR and LCDR are defined according to Kabat.
  • the present application provides a bispecific antibody, which comprises an antigen-binding portion directed against human CD3E and an antigen-binding portion directed against human CD123.
  • the antigen binding portion for human CD3E comprises:
  • HCDR and LCDR are defined according to Kabat.
  • the antigen binding portion for human CD123 comprises:
  • HCDR and LCDR are defined according to Kabat.
  • the antigen binding portion for human CD3E and the antigen binding portion for human CD123 comprise the same light chain variable region.
  • the antigen binding portion for human CD3E and the antigen binding portion for human CD123 comprise the same light chain. This embodiment is conducive to the correct assembly of the light chain and the heavy chain, It is also a preferred embodiment.
  • the bispecific antibody is an IgG1 antibody, comprising two heavy chain constant regions with the same hinge region, and the amino acid sequence of the hinge region is shown in SEQ ID NO: 10, which Replace the sequence 216-230 of the constant region of natural human IgG1 antibody, and the amino acid position of the constant region of the antibody is determined according to EU numbering.
  • the bispecific antibody is an IgG1 antibody, which comprises a first heavy chain constant region and a second heavy chain constant region, wherein the positions at positions 354 and 366 of the first heavy chain constant region
  • the amino acids are C and W, and the amino acids at positions 349, 366, 368, and 407 of the constant region of the second heavy chain are C, S, A, and V, respectively; the amino acid positions of the antibody constant region are determined according to EU numbering.
  • the structure of the bispecific antibody can be optimized from the following two perspectives: one is the heteromerization of the heavy chain, and the other is the correct assembly of the light and heavy chains.
  • the two Fc fragments contain mutations capable of ensuring heavy chain heteromerization.
  • KIH technology knock-in-hole, KIH
  • KIH technology refers to the transformation of the amino acid sequence of the CH3 region to form a structure that is conducive to the pairing of heterogeneous half-antibodies, which can form a bispecific antibody while maintaining the structure of a normal antibody as much as possible.
  • the KIH technology utilized includes making the amino acids at positions 354 and 366 of one Fc fragment be C and W, respectively, and the amino acids at positions 349, 366, 368, and 407 of the other Fc fragment are C and C, respectively. , S, A and V.
  • KIH technology for example, see "An efficient route to human bispecific IgG", A. Margaret Merchant et al., Nature Biotechnology, Volume 16, 1998, and the full text of this document is incorporated herein by reference.
  • the bispecific antibody is an IgG1 antibody comprising a first heavy chain constant region and a second heavy chain constant region, wherein the first heavy chain constant region and the second heavy chain constant region
  • the amino acids at positions 234, 235, and 331 of the antibody are F, E, and S respectively; the amino acid positions of the antibody constant region are determined according to EU numbering.
  • the amino acids at positions 234, 235, and 331 of the CH2 fragments of the two heavy chain constant regions are F, E, and S, respectively, which can reduce antibody-dependent cells mediated by the Fc segment of an antibody.
  • Toxic effects ADCC
  • the binding affinity of human IgG for its high affinity Fc receptor is determined by multiple amino acids in the CH2 domain and is modulated by the hinge region
  • Stephen M. Canfield et al., J .Exp.Med.Volume 173,1991 the full text of this document is incorporated into this article by reference.
  • the antigen-binding portion for human CD3E includes the HCDR1 shown in SEQ ID NO: 11 (including the HCDR1 shown in SEQ ID NO: 1 and the HCDR2 shown in SEQ ID NO: 2 And the heavy chain variable region shown in SEQ ID NO: 3) and the light chain variable region shown in SEQ ID NO: 12 (including the LCDR1 shown in SEQ ID NO: 4). LCDR2 shown in ID NO: 5 and LCDR3 shown in SEQ ID NO: 6).
  • the antigen binding portion for human CD123 comprises SEQ ID NO: 13 (including HCDR 1 as shown in SEQ ID NO: 7, HCDR 2 as shown in SEQ ID NO: 8 and The heavy chain variable region shown in the HCDR3 shown in SEQ ID NO: 9) and the light chain variable region shown in SEQ ID NO: 12 (including the LCDR1 shown in SEQ ID NO: 4) and the light chain variable region shown in SEQ ID NO: 4 LCDR2 shown in NO: 5 and LCDR3 shown in SEQ ID NO: 6).
  • the antigen binding portion for human CD3E comprises a single chain antibody (scfv) or a Fab fragment.
  • the antigen binding portion against human CD123 comprises a single chain antibody (scfv) or a Fab fragment.
  • the bispecific antibody has two different antigen binding parts against two different antigens, and the antigen binding part can contain two forms of single-chain antibody (scfv) or Fab fragments, the bispecific antibody is directed against two different antigens.
  • the configuration of the antigen binding part of the sex antibody can have four combinations: Fab+Fab, Fab+scfv, scfv+Fab, and scfv+scfv.
  • the antigen binding portion for human CD3E comprises a Fab fragment
  • the antigen binding portion for human CD123 comprises a Fab fragment
  • the antigen binding portion for human CD3E comprises a Fab fragment
  • the antigen binding portion for human CD123 comprises a single chain antibody (scfv).
  • the antigen binding portion against human CD3E comprises a single chain antibody (scfv), and the antigen binding portion against human CD123 comprises a Fab fragment.
  • the antigen binding portion against human CD3E comprises a single chain antibody (scfv), and the antigen binding portion against human CD123 comprises a single chain antibody (scfv).
  • the bispecific antibody is also described as having two “arms", with the middle as the boundary, the bispecific antibody can be divided into two arms.
  • the arm of the bispecific antibody can be composed of an Fc fragment and an antigen binding portion (Fab fragment or single chain antibody).
  • Fab fragment or single chain antibody For the arm composed of Fc fragment and Fab fragment, its structure is similar to that of a normal antibody, containing complete heavy and light chains. Therefore, the structure of such an arm can be expressed as Fc+Fab or heavy chain (Fc+ Heavy chain variable region and CH1 fragment in Fab) + light chain (light chain part in Fab).
  • both arms contain antigen binding portions in the form of Fab fragments, the structure of the bispecific antibody thus formed is close to that of a natural antibody, which is a preferred embodiment.
  • the antibody has a first arm and a second arm, wherein the first arm contains an antigen binding portion for human CD3E, and the second arm contains an antigen binding portion for human CD123:
  • the first arm includes the heavy chain variable region amino acid sequence shown in SEQ ID NO: 11, the heavy chain constant region amino acid sequence shown in SEQ ID NO: 31, and the light chain shown in SEQ ID NO: 12 The amino acid sequence of the variable region and the amino acid sequence of the light chain constant region as shown in SEQ ID NO: 32;
  • the second arm includes the heavy chain variable region amino acid sequence shown in SEQ ID NO: 13, the heavy chain constant region amino acid sequence shown in SEQ ID NO: 30, and the light chain shown in SEQ ID NO: 12 The amino acid sequence of the variable region and the amino acid sequence of the light chain constant region as shown in SEQ ID NO: 32.
  • the heavy chain constant region of the bispecific antibody is human IgG1 subtype or various mutants of selected human IgG1 subtype, for example: IgG1H, IgG1K, IgG1m3-H Or IgG1m3-K.
  • the light chain constant region of the bispecific antibody is of human kappa subtype or human lambda subtype, preferably human kappa subtype.
  • the present application provides a pharmaceutical composition comprising the bispecific antibody described in any one of the first to third aspects.
  • the pharmaceutical composition further includes a pharmaceutically acceptable carrier, excipient, diluent, and the like.
  • the pharmaceutical composition is used to prevent or treat CD123-positive tumors, such as acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell tumors (BPDCN).
  • CD123-positive tumors such as acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell tumors (BPDCN).
  • AML acute myeloid leukemia
  • BPDCN blastic plasmacytoid dendritic cell tumors
  • the pharmaceutical composition may also include lubricants, such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifiers; suspending agents; preservatives, such as benzoic acid, sorbic acid, and calcium propionate ; Sweeteners and/or flavoring agents, etc.
  • lubricants such as talc, magnesium stearate, and mineral oil
  • wetting agents such as talc, magnesium stearate, and mineral oil
  • emulsifiers such as talc, magnesium stearate, and mineral oil
  • suspending agents such as benzoic acid, sorbic acid, and calcium propionate
  • preservatives such as benzoic acid, sorbic acid, and calcium propionate
  • Sweeteners and/or flavoring agents etc.
  • the pharmaceutical composition in this application can be formulated in the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories, or capsules.
  • any physiologically acceptable mode of administration may be used to deliver the pharmaceutical composition of the present application.
  • These modes of administration include, but are not limited to: oral administration, parenteral administration, nasal administration, rectal administration Medicine, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, etc.
  • a pharmaceutical composition for therapeutic use can be formulated in the form of a lyophilized preparation or an aqueous solution by mixing reagents with the required purity with pharmaceutically acceptable carriers, excipients, etc., as appropriate. storage.
  • this application provides the use of the bispecific antibody according to any one of the first to third aspects or the pharmaceutical composition according to the fourth aspect in the preparation of a medicine for the prevention or treatment of CD123-positive tumors use.
  • the CD123-positive tumor is selected from acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell tumor (BPDCN).
  • AML acute myeloid leukemia
  • BPDCN blastic plasmacytoid dendritic cell tumor
  • the present application provides a method for preventing or treating CD123-positive tumors, including administering the bispecific antibody according to any one of the first to third aspects or the drug according to the fourth aspect to an individual in need combination.
  • the CD123-positive tumor is selected from acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell tumor (BPDCN).
  • AML acute myeloid leukemia
  • BPDCN blastic plasmacytoid dendritic cell tumor
  • CD3E ⁇ CD123 bispecific antibody requires a variety of different recombinant proteins, including the extracellular domain of human CD3E (hCD3E, SEQ ID NO: 14) and the extracellular domain of human CD3D (hCD3D, SEQ ID NO).
  • monkey CD3E extracellular domain mfCD3E, SEQ ID NO: 16
  • monkey CD3D extracellular domain mfCD3D, SEQ ID NO: 17
  • mouse CD3E extracellular domain mCD3E, SEQ ID NO: 18
  • Mouse CD3D extracellular region mCD3D, SEQ ID NO: 19
  • human CD123 subtype 1 extracellular region hCD123-SP1, SEQ ID NO: 20
  • monkey CD123 subtype 1 extracellular region mfCD123-SP1, SEQ ID NO: 21
  • mouse CD123 subtype 1 extracellular region mCD123-SP1, SEQ ID NO: 22.
  • recombinant proteins have a large number of post-translational modifications (such as glycosylation or disulfide bonds, etc.), so the use of mammalian cell expression systems will be more conducive to maintaining the structure and function of the recombinant protein.
  • non-antibody recombinant proteins are added with a His tag (SEQ ID NO: 23) at the C-terminus, or the Fc segment of mouse antibody IgG2a (mFc, SEQ ID NO: 24), or based on KIH (Knob- Into-Hole technology forms a heterodimer Fc mutant of human IgG1 subtype (FcK, SEQ ID NO: 25 or FcH, SEQ ID NO: 26).
  • the heavy chain constant region of the antibody can be human IgG1 subtype (SEQ ID NO: 27) or various mutants of selected human IgG1 subtype, such as: IgG1H (SEQ ID NO: 28), IgG1K (SEQ ID NO: 29), IgG1m3-H (SEQ ID NO: 30) or IgG1m3-K (SEQ ID NO: 31), the light chain constant region is human ⁇ subtype (SEQ ID NO: 32) or human ⁇ subtype Type (SEQ ID NO: 33).
  • the genes including His tag, mFc or Fc encoding genes of the above-mentioned various recombinant proteins are designed and synthesized.
  • the recombinant protein expressed by His tag fusion uses a metal chelating affinity chromatography column (such as GE's HisTrap FF, etc.) to purify the recombinant protein in the culture supernatant in one step.
  • the recombinant protein and recombinant antibody expressed by mFc fusion are purified in one step using a ProteinA/G affinity chromatography column (such as Mabselect SURE from GE). Then use a desalting column (such as Hitrap desaulting from GE) to replace the recombinant protein storage buffer with PBS (pH 7.0) or other suitable buffers. If necessary, the antibody sample can be filtered and sterilized, and then aliquoted and stored at -20°C.
  • the light chain variable region of a specific anti-CD3E monoclonal antibody was selected to be matched with the mouse heavy chain variable region that has been affinity matured in the CD123 antigen in vivo, using conventional molecular biology methods Construct a single-chain antibody (scFv) library for screening specific antibodies against CD123.
  • scFv single-chain antibody
  • mice After 6-8 weeks of age BALB/c mice were cross-immunized with hCD123-SP1-His and mfCD123-SP1-His recombinant proteins, spleen cells were collected.
  • Use mouse lymphocyte separation solution (Daktronics Biotechnology Co., Ltd., CAT#DKW33-R0100) to separate mouse spleen lymphocytes, and use the total cell RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., CAT#DP430), extract the total RNA from the separated lymphocytes.
  • the first strand cDNA synthesis kit (Thermo Scientific, CAT#K1621) was used to synthesize the heavy chain variable region of the antibody.
  • PCR amplification obtains rat monoclonal antibody WM03-C6 that specifically recognizes CD3E of human and cynomolgus monkeys (see the sequence of monoclonal antibody 20E5-F10 in patent WO_2016_116626_A1.
  • the amino acid sequence of the heavy chain variable region is as follows: As shown in SEQ ID NO: 34, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 35)
  • the mouse heavy chain variable region obtained by immunization with the light chain variable region and CD123 recombinant antigen, and then using overlap extension PCR technology
  • scFv single-chain antibody
  • clone the prepared mouse single-chain antibody gene into the vector pADSCFV-S for the experimental technology flow, please refer to Example 1 of Chinese Patent Application No. 201510097117.0
  • the storage capacity of this antibody library reaches 1.2 ⁇ 10E8, and the correct rate is 65%.
  • Example 2 Using the recombinant hCD123-SP1-his prepared in Example 1 as the antigen, using a solid-phase screening strategy (refer to the experimental protocol for phage display: General Experimental Guide / (United States) Clarkson (Clackson, T.), (United States) Lowman (Lowman) , HB) edited; Ma Lan et al. Chemical Industry Press, 2008.5) Screening of the phage library displaying mouse single-chain antibodies constructed in Example 2 was carried out through binding, elution, neutralization, infection, and amplification. After three rounds of screening, a single-chain antibody S8F3 (SEQ ID NO: 36) that specifically binds to human CD123 was finally obtained.
  • S8F3 SEQ ID NO: 36
  • nucleotide sequences of the heavy chain variable region S8F3VH (SEQ ID NO: 37) and light chain variable region WM03-C6VK (SEQ ID NO: 35) encoding S8F3 were cloned into the fusion.
  • Eukaryotic expression vectors with nucleotide sequences encoding human heavy chain constant regions and light chain constant regions such as pcDNA3.1 from Invitrogen, combined to express the full antibody S8F3VH+C6VK.
  • the anti-CD123 antibody CSL362 (heavy chain amino acid sequence shown in SEQ ID NO: 38 and light chain amino acid sequence shown in SEQ ID NO: 39) was prepared with reference to US patent US_2014_0178364_A1 as a positive control antibody for subsequent research.
  • Biacore X100 was used to determine the affinity of anti-CD123 antibodies by surface plasmon resonance technology.
  • Amino coupling kit (BR-1000-50), human antibody capture kit (BR-1008-39), CM5 chip (BR100012) and pH7.4 10 ⁇ HBS-EP (BR100669) and other related reagents and consumables are available Purchased from GE healthcare.
  • KG-1a human acute myeloid leukemia cells, purchased from Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences
  • KG-1a human acute myeloid leukemia cells, purchased from Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences
  • PBS buffer containing 1% BSA after centrifugation /mL
  • 100 ⁇ L/well was plated in a 96-well V bottom plate, and the supernatant was removed after centrifugation.
  • test sample S8F3VH+C6VK the control sample CSL362 and HIgG irrelevant antibody (GenScript A01006, human IgG control (full molecule), Purifie) with PBS to the final concentration of 5 ⁇ g/mL and 0.5 ⁇ g/mL, and add Incubate the cells in the wells at 4°C for 1 hour. Then wash with 200 ⁇ L PBS for 3 times, add goat anti-human IgG-FITC (Zhongshan Jinqiao, ZF-0308) secondary antibody (100 ⁇ L/well), and incubate for 30 minutes at 4°C in the dark.
  • HIgG irrelevant antibody GeneScript A01006, human IgG control (full molecule), Purifie
  • the rat monoclonal antibody WM03-C6 was humanized to reduce its immunogenicity.
  • the humanization program adopts the classic frame transplantation strategy (J Immunol.169, 1119-1125, 2002).
  • the heavy chain variable region and light chain variable region of WM03-C6 were compared with the human antibody germline gene sequence in the IMGT database, and the appropriate germline gene sequence was selected to provide antibody framework regions 1 to 3 (FR1+ FR2+FR3), select the appropriate J region gene sequence to provide framework region 4 (FR4).
  • This template can be selected based on a variety of factors, such as: the relative total length of the antibody, the size of the CDR, the amino acid residues located at the junction between the antibody framework region (FR) and the hypervariable region (CDR), and the overall sequence homology Sex and so on.
  • the selected template can be a mixture of multiple sequences or can be a shared template, in order to maintain the proper conformation of the parental complementarity determining region (CDR) as much as possible.
  • the humanized antibody light and heavy chains were respectively designed for mutations.
  • the humanized light chain mutant L27E5 SEQ ID NO: 12
  • the humanized heavy chain mutant H3B8 SEQ ID NO: 11
  • Example 3.2 couple the anti-human IgG (Fc) antibody to the surface of the CM5 chip, dilute the anti-CD3E antibody (WM03-C6 and H3B8+L27E5) to 0.5-1 ⁇ g/mL, and inject 10 ⁇ L/min to ensure about 350-400RU
  • the antibody is captured by the anti-human Fc antibody.
  • set hCD3E-his to a series of concentration gradients for example, 6.17nM, 18.5nM, 55.6nM, 167nM, 500nM
  • the binding time is 120s, and the dissociation The time is 600-1800s, and the surface of the chip is regenerated by injecting 3M MgCl 2 at 10 ⁇ L/min for 30 s.
  • the association rate (K on ) and the dissociation rate (K off ) were calculated by fitting the association and dissociation sensorgrams with a 1:1 association model.
  • the dissociation equilibrium constant (KD) is calculated with the ratio K off /K on. The fitting results are shown in Table 2.
  • the S8F3 heavy chain variable region was subjected to in vitro affinity maturation.
  • a library of CDR3 mutations based on S8F3VH was constructed by introducing mutations into the CDR3 of the variable region of the S8F3 heavy chain.
  • the designed mutation scheme is shown in Table 3. The construction capacity is 1.7 ⁇ 10E8, and the correct rate is 86%.
  • the humanization of the heavy chain H7A3 of the anti-human CD123 murine monoclonal antibody adopts the classic framework transplantation strategy.
  • the humanized version of H7A3VH-h2 was obtained by CDR grafting the heavy chain variable region of H7A3.
  • some key amino acids in the framework region of the humanized antibody such as I69, R71, T73 , A75 was back-mutated, and finally the humanized version H7A3-h2-m5 (SEQ ID NO: 13) was obtained.
  • the antibody variable region gene is designed and synthesized, cloned into a eukaryotic expression vector and cooperated with the common light chain L27E5 to express the human IgG1 version of the full antibody.
  • MV-4-11 human acute monocytic leukemia cells, purchased from Nanjing Kebai Biotechnology Co., Ltd.
  • PBS buffer containing 1% BSA after centrifugation /mL
  • 100 ⁇ L/well was plated in a 96-well V bottom plate, and the supernatant was removed after centrifugation.
  • test sample H7A3-h2-m5+L27E5, the control sample CSL362, and HIgG irrelevant antibody were all prepared with PBS to a final concentration of 100 nM starting with a 3-fold gradient Dilute to 9 concentrations, add them to wells containing cells, and incubate at 4°C for 1 hour. Then wash with 200 ⁇ L PBS for 3 times, add goat anti-human IgG-FITC (ZF-0308), 100 ⁇ L/well, and incubate for 30 minutes at 4°C in the dark.
  • H7A3-h2-m5+L27E5 can bind CD123-positive cells MV-4-11 well, with a KD value of 3.1 nM.
  • nucleotide sequences encoding the heavy chain variable region H3B8 of the CD3E monoclonal antibody and the heavy chain variable region H7A3-h2-m5 of the CD123 monoclonal antibody were cloned into a suitable eukaryotic expression vector to construct a common light chain-based Heterodimer.
  • the nucleotide sequence encoding the heavy chain variable region of the CD3E antibody is cloned into the eukaryotic expression vector fused with the nucleotide sequence encoding the Knob mutation IgG1 constant region IgG1m3-K, which will encode the heavy chain variable region of the CD123 antibody
  • the nucleotide sequence was cloned into a eukaryotic expression vector containing the IgG1m3-H nucleotide sequence of the IgG1 constant region encoding the Hole mutation, and the nucleotide sequence encoding the variable region VK of the common light chain L27E5 was cloned into a human fusion with encoding
  • the eukaryotic expression vector of the nucleotide sequence of the light chain constant region CK was cloned into a human fusion with encoding The eukaryotic expression vector of the nucleotide sequence of the light chain constant region CK.
  • the constructed three eukaryotic expression vectors expressing H3B8-IgG1m3-K, expressing H7A3-h2-m5-IgG1m3-H and expressing L27E5-CK were co-transfected into HEK293F cells using liposomes, and under serum-free suspension culture conditions Cultivate for 3-5 days, and then harvest the culture supernatant by centrifugation and other methods.
  • the bispecific antibody in the culture supernatant is purified with a ProteinA/G affinity chromatography column (such as GE's Mabselect SURE, etc.), and then a desalting column (such as GE's Hitrap desaulting, etc.) is used to replace the recombinant protein storage buffer with PBS (pH7.0) or other suitable buffer.
  • the desalted protein solution was purified by size exclusion chromatography (SEC) using Superdex200 (GE) to obtain the target protein. If necessary, the antibody sample can be filtered and sterilized, and then aliquoted and stored at -20°C for later use.
  • the conventional ELISA method was used to detect the simultaneous binding of CD3 ⁇ CD123 bispecific antibody (CD3 ⁇ CD123 BsAb) to both CD3E and CD123 antigens.
  • CD3 ⁇ CD123 BsAb CD3 ⁇ CD123 bispecific antibody
  • the CD123-SP1-mFc antigen was coated on a 96-well ELISA plate (3 ⁇ g/mL, 100 ⁇ L/well), and coated overnight at 4°C.
  • bispecific antibody control sample Xmab14045 Dilute the sample with PBS, bispecific antibody control sample Xmab14045, anti-CD3E monoclonal antibody H3B8+L27E5, HIgG irrelevant antibody (GenScript A01006, human IgG control (full molecule, Purifie) and CD3 ⁇ CD123 bispecific antibody (CD3) ⁇ CD123BsAb) are all set at 400nM as the starting concentration, the first 3 points are diluted 2 times, and the last 5 points are diluted 3 times, a total of 8 concentrations, add to the wells containing cells, incubate at 4°C for 1 hour.
  • bispecific antibody control sample Xmab14045 Dilute the sample with PBS, bispecific antibody control sample Xmab14045, anti-CD123 monoclonal antibody H7A3-h2-m5+L27E5, CD3 ⁇ CD123 bispecific antibody (CD3 ⁇ CD123 BsAb) and HIgG irrelevant antibody (GenScript A01006, human
  • the IgG control (whole molecule, Purifie) started with a final concentration of 100nM, diluted by 3 times, a total of 9 concentration points, added to a 96-well plate containing cells, incubated at 4°C for 1 hour.
  • Collect MV-4-11 cells (CD123 + cells) in the logarithmic growth phase, resuspend them to 2 ⁇ 10 6 cells/mL with 1640 medium after centrifugation, spread 50 ⁇ L/well on the cell plate, and remove the supernatant after centrifugation .
  • Collect Jurkat-Dual cells purchased from Invivogen in the logarithmic phase, centrifuge and resuspend them to 2 ⁇ 10 6 cells/mL with 1640 medium, and add 50 ⁇ L/well to the cell plate to obtain a final E:T of 1:1 Proportion.
  • CD3 ⁇ CD123 bispecific antibody (CD3 ⁇ CD123 BsAb, 50 ⁇ L/well) with an initial concentration of 6 nM, 4-fold gradient dilution, and 8 concentration points.
  • the system control is equipped with a bispecific antibody control sample Xmab14045, anti-CD3E monoclonal antibody H3B8+L27E5, anti-CD123 monoclonal antibody H7A3-h2-m5+L27E5, H3B8+L27E5 and a combination group of H7A3-h2-m5+L27E5 (H3B8 +L27E5&H7A3-h2-m5+L27E5) and CD123-negative target cell (BAF3 cells) control group and HIgG irrelevant antibody (GenScript A01006, human IgG control (full molecule), Purifie), using the same concentration as CD3 ⁇ CD123 bispecific The concentration of sex antibodies.
  • Example 12 CD3 ⁇ CD123 bispecific antibody mediates the expression of purified T cell surface activation molecules and kills CD123-positive tumor cells
  • PBMC peripheral blood mononuclear cells
  • the selection criteria for volunteers are:
  • Ficoll density gradient centrifugation was used to separate PBMCs from volunteers' whole blood cells and cultured in 1640 medium.
  • MV-4-11 cells in the logarithmic phase were collected, centrifuged and resuspended in 1640 medium to 1 ⁇ 10 6 cells/mL, and 50 ⁇ L/well was plated in the cell plate. Then add the CD3 ⁇ CD123 bispecific antibody (CD3 ⁇ CD123 BsAb, 50 ⁇ L/well) with an initial concentration of 1 nM, 4-fold gradient dilution, and 8 concentration points.
  • CD3 ⁇ CD123 bispecific antibody CD3 ⁇ CD123 BsAb, 50 ⁇ L/well
  • T cell negative sorting kit instructions (BD IMaq human T lymphocyte enrichment set-DM, BD, 557874) to obtain purified T cells (5 ⁇ 10 6 cells/mL, 50 ⁇ L/well) from PBMC, and the final effect
  • the target ratio is 5:1.
  • MV-4-11 cells MV-4-11 cells
  • T single effector cell control
  • a single medium blank control MV-4-11 cells
  • the volume was filled up to 150 ⁇ L with medium.
  • Non-Radioactive Cytotoxicity Assay promega, G1780
  • the results show that the CD3 ⁇ CD123 bispecific antibody can specifically mediate the killing of CD123-positive tumor cells by T cells, but has no killing effect on CD123-negative tumor cells (BAF3 cells) ( Figure 7).
  • CD3 ⁇ CD123 bispecific antibody could specifically up-regulate the expression of CD69 on the surface of T cells in the presence of positive tumor cells, while CD123-negative tumor cells (BAF3 cells) could not up-regulate the expression of CD69 on the surface of T cells ( Figure 8).
  • PBMC Peripheral Blood Mononuclear Cells
  • Purified T cells were sorted from PBMC according to the instructions of the T cell negative sorting kit (BD IMaq human T lymphocyte enrichment set-DM, BD, 557874).
  • BD IMaq human T lymphocyte enrichment set-DM BD, 557874.
  • CFSE staining instructions to stain the purified T cells (CFSE, eBioscience, 65-0850-84), resuspend in 1640 medium to 2 ⁇ 10 6 cells/mL, and add 50 ⁇ L/well to the cell culture plate.
  • Collect MV-4-11 (CD123 + cells) in the logarithmic growth phase resuspend them to 5 ⁇ 10 5 cells/mL with 1640 medium after centrifugation, and spread 50 ⁇ L/well on the cell plate.
  • the final effect-to-target ratio is 4 :1.
  • CD3 ⁇ CD123 bispecific antibody (CD3 ⁇ CD123 BsAb, 50 ⁇ L/well) and Xmab14045 with the starting concentration of 0.25nM, 4-fold dilution, and 8 concentration points; in addition, a control of 0.25nM concentration point is set up, respectively It is a single anti-CD3E monoclonal antibody H3B8+L27E5, a single anti-CD123 monoclonal antibody H7A3-h2-m5+L27E5 and a combination of H3B8+L27E5 and H7A3-h2-m5+L27E5 (H3B8+L27E5&H7A3-h2-m5+L27E5 ); In addition, a control of CD3 ⁇ CD123 bispecific antibody at a concentration of 0.25 nM in the presence of CD123-negative tumor cells (BAF3 cells) is also set.
  • BAF3 cells CD123-negative tumor cells
  • the test is divided into 4 groups of CD3 ⁇ CD123 bispecific antibody 0.01mg/kg, 0.1mg/kg, 0.5mg/kg and the negative control group (IgG1m3, 0.5mg/kg), each group has 8 animals, according to the 0th day, Three days, seven days, 14 days, and 21 days, the drug was administered by tail vein injection for a total of 5 times.
  • the efficacy is evaluated based on the relative tumor inhibition rate (TGI), and the safety is evaluated based on the changes in animal weight and death.
  • TGI tumor inhibition rate
  • the CD3 ⁇ CD123 bispecific antibody 0.1mg/kg dose group (G3 group) showed a slow increase in tumor volume after administration, and the relative tumor inhibition rate TGI (%) was 52.08%, compared with the negative control group (G1 group) in the same period.
  • Significant difference (p ⁇ 0.05) is the 0.01 mg/kg dose group of CD3 ⁇ CD123 bispecific antibody.
  • the CD3 ⁇ CD123 bispecific antibody has a significant dose-effect relationship with tumor growth. The tumor growth of each group of animals at each time point is shown in Figure 10.

Abstract

提供了包含针对人CD3E的抗原结合部和/或针对人CD123的抗原结合部的双特异性抗体，还提供了该双特异性抗体的医学和生物学用途。

Description

抗CD3和CD123双特异性抗体及其用途
相关申请的交叉引用
本申请要求于2020年2月5日递交的中国专利申请第202010080449.9号的优先权，其全部内容通过引用整体并入本文。
技术领域
本申请大体涉及抗体药物领域，具体而言，本申请提供了包含针对人CD3E的抗原结合部和/或针对人CD123的抗原结合部的双特异性抗体及其医学和生物学用途。
背景技术
双特异性抗体(bispecific antibody，BsAb)是一类人工抗体，其包含两个不同的抗原结合位点。双特异性抗体在生物医药领域，尤其是肿瘤免疫治疗方面应用广泛。靶向CD3的双特异性抗体，一条臂能够结合T细胞表面TCR受体复合物中的CD3E亚基，另一条臂靶向肿瘤抗原。通过此种方式，双特异性抗体能够以非组织相容性复合物(MHC)依赖方式，重新导向T细胞特异性杀伤肿瘤细胞。
双特异性抗体的平台众多，结构复杂。以抗体结构区分可以分为有Fc段和无Fc段两大类。无Fc段的双特异性抗体由两个抗体的VH区及VL区组成或者由Fab片段组成，此类双特异性抗体的主要代表有BiTE、DART、TandAbs、bi-nanobody等。此类双特异性抗体的优势在于无轻重链错配，缺点在于半衰期短，临床应用不方便。有Fc段的双特异性抗体保留了传统单克隆抗体的结构，并可以介导Fc段的生物学功能。此类双特异性抗体的代表有KIH IgG、crossmab、DVD-Ig、Triomab等，体内半衰期长且可以具有ADCC、CDC活性(Hongyan Liu,Abhishek Saxena,Sachdev S.Sidhu,et al.Fc engineering for Developing Therapeutic Bispecifc Antibodies and Novel Scaffolds.Front.Immunol.2017；8:38)。
因此，鉴于双特异性抗体具有广泛的适用性，本领域需要开发新的双特异性抗体。
发明内容
第一方面，本申请提供了双特异性抗体，其包含针对人CD3E的抗原结合部，所述针对人CD3E的抗原结合部包含：
如SEQ ID NO:1所示的HCDR1(重链CDR1)，
如SEQ ID NO:2所示的HCDR2(重链CDR2)，
如SEQ ID NO:3所示的HCDR3(重链CDR3)，
如SEQ ID NO:4所示的LCDR1(轻链CDR1)，
如SEQ ID NO:5所示的LCDR2(轻链CDR2)，和
如SEQ ID NO:6所示的LCDR3(轻链CDR3)；
其中，HCDR和LCDR根据Kabat定义。
第二方面，本申请提供了双特异性抗体，其包含针对人CD123的抗原结合部，所述针对人CD123的抗原结合部包含：
如SEQ ID NO:7所示的HCDR1(重链CDR1)，
如SEQ ID NO:8所示的HCDR2(重链CDR2)，
如SEQ ID NO:9所示的HCDR3(重链CDR3)，
如SEQ ID NO:4所示的LCDR1(轻链CDR1)，
如SEQ ID NO:5所示的LCDR2(轻链CDR2)，和
如SEQ ID NO:6所示的LCDR3(轻链CDR3)；
其中，HCDR和LCDR根据Kabat定义。
第三方面，本申请提供了双特异性抗体，其包含针对人CD3E的抗原结合部和针对人CD123的抗原结合部。
在第三方面的一些实施方案中，所述针对人CD3E的抗原结合部包含：
如SEQ ID NO:1所示的HCDR1，
如SEQ ID NO:2所示的HCDR2，
如SEQ ID NO:3所示的HCDR3，
如SEQ ID NO:4所示的LCDR1，
如SEQ ID NO:5所示的LCDR2，和
如SEQ ID NO:6所示的LCDR3；
其中，HCDR和LCDR根据Kabat定义。
在第三方面的一些实施方案中，所述针对人CD123的抗原结合部包含：
如SEQ ID NO:7所示的HCDR1，
如SEQ ID NO:8所示的HCDR2，
如SEQ ID NO:9所示的HCDR3，
如SEQ ID NO:4所示的LCDR1，
如SEQ ID NO:5所示的LCDR2，和
如SEQ ID NO:6所示的LCDR3；
其中，HCDR和LCDR根据Kabat定义。
在第三方面的一些实施方案中，所述针对人CD3E的抗原结合部和所述针对人CD123的抗原结合部包含相同的轻链可变区。
在第三方面的一些实施方案中，所述双特异性抗体是IgG1抗体，其包含两种具有相同铰链区的重链恒定区，所述铰链区的氨基酸序列如SEQ ID NO:10所示。
在第三方面的一些实施方案中，所述双特异性抗体是IgG1抗体，其包含第一重链恒定区和第二重链恒定区，其中所述第一重链恒定区的第354和366位的氨基酸分别为C和W，所述第二重链恒定区的第349、366、368和407位的氨基酸分别为C、S、A和V；抗体恒定区氨基酸位置按照EU numbering确定。
在第三方面的一些实施方案中，所述双特异性抗体是IgG1抗体，其包含第一重链恒定区和第二重链恒定区，其中所述第一重链恒定区和第二重链恒定区的第234、235和331位的氨基酸分别为F、E和S；抗体恒定区氨基酸位置按照EU numbering确定。
在第一方面和第三方面的一些实施方案中，所述针对人CD3E的抗原结合部包含如SEQ ID NO:11所示的重链可变区和如SEQ ID NO:12所示的轻链可变区。
在第二方面和第三方面的一些实施方案中，所述针对人CD123的抗原结合部包含SEQ ID NO:13所示的重链可变区和如SEQ ID NO:12所示的轻链可变区。
在上述任一方面的一些实施方案中，所述针对人CD3E的抗原结合部包含单链抗体(scfv)或Fab片段。
在上述任一方面的一些实施方案中，所述针对人CD123的抗原结合部包含单链抗体(scfv)或Fab片段。
在第三方面的一些实施方案中，所述抗体具有第一臂和第二臂，其中所述第一臂包含针对人CD3E的抗原结合部，所述第二臂包含针对人CD123的抗原结合部：
所述第一臂包含如SEQ ID NO:11所示的重链可变区氨基酸序列、如SEQ ID NO:31所 示的重链恒定区氨基酸序列、如SEQ ID NO:12所示的轻链可变区氨基酸序列和如SEQ ID NO:32所示的轻链恒定区氨基酸序列；
所述第二臂包含如SEQ ID NO:13所示的重链可变区氨基酸序列、如SEQ ID NO:30所示的重链恒定区氨基酸序列、如SEQ ID NO:12所示的轻链可变区氨基酸序列和如SEQ ID NO:32所示的轻链恒定区氨基酸序列。
第四方面，本申请提供了药物组合物，其包含第一至第三方面中任一方面所述的双特异性抗体。
在第四方面的一些实施方案中，所述药物组合物用于预防或治疗CD123阳性肿瘤。
第五方面，本申请提供了第一至第三方面中任一方面所述的双特异性抗体、或者第四方面所述的药物组合物在制备用于预防或治疗CD123阳性肿瘤的药物中的用途。
第六方面，本申请提供了预防或治疗CD123阳性肿瘤的方法，包括向有需要的个体给予第一至第三方面中任一方面所述的双特异性抗体、或者第四方面所述的药物组合物。
附图说明
图1显示了采用流式细胞仪分析重组抗CD123单克隆抗体特异性与细胞表面的CD123结合的结果。
图2显示了采用流式细胞仪分析H7A3人源化突变体H7A3-h2-m5+L27E5结合MV-4-11细胞表面的CD123的结果。
图3显示了利用ELISA分析双特异性抗体CD3E×CD123与CD3E和CD123两种抗原结合的结果。
图4显示了采用流式细胞仪分析双特异性抗体CD3E×CD123结合Jurkat-Dual细胞表面的CD3E的能力的结果。
图5显示了采用流式细胞仪分析双特异性抗体CD3E×CD123结合MV-4-11细胞表面的CD123的能力的结果。
图6显示了双特异性抗体CD3×CD123在CD123阳性肿瘤细胞存在时对Jurkat-Dual激活的结果，其中图A为双特异性抗体CD3×CD123、抗CD3E单克隆抗体H3B8+L27E5、抗CD123单克隆抗体H7A3-h2-m5+L27E5、以及抗CD3E单克隆抗体H3B8+L27E5和抗CD123单克隆抗体H7A3-h2-m5+L27E5的联合组对Jurkat-Dual激活的结果；图B为在CD123阳性靶细胞存在时双特异性抗体CD3×CD123、双特异性抗体对照样品Xmab14045以及HIgG对Jurkat-Dual激活的结果，以及在CD123阴性靶细胞存在时双特异性抗体CD3×CD123和双特异性抗体对照样品Xmab14045对Jurkat-Dual激活的结果。
图7显示了双特异性抗体CD3E×CD123介导的纯化T细胞对CD123阳性肿瘤细胞的杀伤的结果。
图8显示了采用流式细胞仪检测双特异性抗体CD3E×CD123在CD123阳性肿瘤细胞存在时特异性活化T细胞，上调CD69的表达的结果。
图9显示了采用流式细胞仪检测双特异性抗体CD3E×CD123在CD123阳性肿瘤细胞存在时促进T细胞增殖的结果，其中图A为双特异性抗体CD3×CD123、抗CD3E单克隆抗体H3B8+L27E5、抗CD123单克隆抗体H7A3-h2-m5+L27E5、以及抗CD3E单克隆抗体H3B8+L27E5和抗CD123单克隆抗体H7A3-h2-m5+L27E5的联合组在CD123阳性肿瘤细胞存在时促进T细胞增殖的结果；图B为双特异性抗体CD3×CD123和双特异性抗体对照样品Xmab14045在CD123阳性肿瘤细胞存在时促进T细胞增殖的结果。
图10显示了双特异性抗体CD3E×CD123治疗的hCD34 +人源化MV-4-11细胞肿瘤模型小鼠肿瘤体积的变化。
序列说明
SEQ ID NO:1为抗人CD3E单克隆抗体H3B8+L27E5的重链可变区H3B8的HCDR1的氨基酸序列。
SEQ ID NO:2为抗人CD3E单克隆抗体H3B8+L27E5的重链可变区H3B8的HCDR2的氨基酸序列。
SEQ ID NO:3为抗人CD3E单克隆抗体H3B8+L27E5的重链可变区H3B8的HCDR3的氨基酸序列。
SEQ ID NO:4为轻链可变区L27E5的LCDR1的氨基酸序列。
SEQ ID NO:5为轻链可变区L27E5的LCDR2的氨基酸序列。
SEQ ID NO:6为轻链可变区L27E5的LCDR3的氨基酸序列。
SEQ ID NO:7为抗人CD123单克隆抗体H7A3-h2-m5+L27E5的重链可变区H7A3-h2-m5的HCDR1的氨基酸序列。
SEQ ID NO:8为抗人CD123单克隆抗体H7A3-h2-m5+L27E5的重链可变区H7A3-h2-m5的HCDR2的氨基酸序列。
SEQ ID NO:9为抗人CD123单克隆抗体H7A3-h2-m5+L27E5的重链可变区H7A3-h2-m5的HCDR3的氨基酸序列。
SEQ ID NO:10为铰链区的氨基酸序列。
SEQ ID NO:11为大鼠单克隆抗体WM03-C6的人源化重链突变体H3B8的氨基酸序列。
SEQ ID NO:12为大鼠单克隆抗体WM03-C6的人源化的轻链突变体L27E5的氨基酸序列。
SEQ ID NO:13为人源化版本H7A3-h2-m5的氨基酸序列。
SEQ ID NO:14为人(Homo sapiens)CD3E胞外区(hCD3E)的氨基酸序列。
SEQ ID NO:15为人(Homo sapiens)CD3D胞外区(hCD3D)的氨基酸序列。
SEQ ID NO:16为食蟹猴(Macaca fascicularis)CD3E胞外区(mfCD3E)的氨基酸序列。
SEQ ID NO:17为食蟹猴(Macaca fascicularis)CD3D胞外区(mfCD3D)的氨基酸序列。
SEQ ID NO:18为小鼠(Mus musculus)CD3E胞外区(mCD3E)的氨基酸序列。
SEQ ID NO:19为小鼠(Mus musculus)CD3D胞外区(mCD3D)的氨基酸序列。
SEQ ID NO:20为人(Homo sapiens)CD123亚型1胞外区(hCD123-SP1)的氨基酸序列。
SEQ ID NO:21为食蟹猴(Macaca fascicularis)CD123亚型1胞外区(mfCD123-SP1)的氨基酸序列。
SEQ ID NO:22为小鼠(Mus musculus)CD123亚型1胞外区(mCD123-SP1)的氨基酸序列。
SEQ ID NO:23为His标签的氨基酸序列。
SEQ ID NO:24为小鼠(Mus musculus)IgG2a抗体的Fc段(mFc)的氨基酸序列。
SEQ ID NO:25为异源二聚体的人IgG1亚型的Fc突变体FcK的氨基酸序列。
SEQ ID NO:26为异源二聚体的人IgG1亚型的Fc突变体FcH的氨基酸序列。
SEQ ID NO:27为人(Homo sapiens)IgG1亚型抗体重链恒定区的氨基酸序列。
SEQ ID NO:28为人IgG1亚型抗体重链恒定区突变体IgG1H的氨基酸序列。
SEQ ID NO:29为人IgG1亚型抗体重链恒定区突变体IgG1K的氨基酸序列。
SEQ ID NO:30为人IgG1亚型抗体重链恒定区突变体IgG1m3-H的氨基酸序列。
SEQ ID NO:31为人IgG1亚型抗体重链恒定区突变体IgG1m3-K的氨基酸序列。
SEQ ID NO:32为人(Homo sapiens)κ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:33为人(Homo sapiens)λ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:34为单克隆抗体WM03-C6的重链可变区氨基酸序列。
SEQ ID NO:35为单克隆抗体WM03-C6的轻链可变区的氨基酸序列。
SEQ ID NO:36为抗人CD123的单链抗体S8F3的氨基酸序列。
SEQ ID NO:37为抗人CD123的单链抗体S8F3的重链可变区S8F3VH的氨基酸序列。
SEQ ID NO:38为抗CD123抗体CSL362的重链氨基酸序列。
SEQ ID NO:39为抗CD123抗体CSL362的轻链氨基酸序列。
SEQ ID NO:40为包含有Hole突变的IgG1m3亚型的Xmab14045的结合CD123的重链的氨基酸序列。
SEQ ID NO:41为包含有Knob突变的IgG1m3亚型的Xmab14045的结合CD3E的scFv结构的氨基酸序列。
SEQ ID NO:42为Xmab14045的轻链的氨基酸序列。
发明详述
定义
提供以下定义和方法用以更好地界定本申请以及在本申请实践中指导本领域普通技术人员。除非另外指明，本申请中所用的术语具有本领域技术人员通常所理解的含义。本文所引用的所有专利文献、学术论文及其他公开出版物，其中的全部内容整体并入本文作为参考。
在本文描述抗体结构时，涉及氨基酸位置编号的描述参照人IgG1抗体的EU numbering定义，这是本领域技术人员公知且容易查询到的。此外，在本文结合EU numbering位置描述突变时，是指相对于天然抗体序列产生的突变。
本文所用术语“Fc片段”、“Fc结构域”、“Fc部分”或类似的术语是指抗体重链恒定区的一部分，包括铰链区(hinge)、恒定区的CH2片段和CH3片段。参照人IgG1抗体的EU numbering定义，Fc片段是抗体恒定区中第216-447位的氨基酸序列。
本文所用术语“Fab(fragment antigen binding)片段”、“Fab部分”或类似的术语是指完整的抗体用木瓜蛋白酶处理后产生的能够与抗原结合的抗体片段，包括完整的轻链(VL-CL)、重链可变区和CH1片段(VH-CH1)。
本文所用术语“单链抗体(scfv,single chain fragment variable)”是指一般利用基因工程技术构建的单链结构的抗体，包含重链可变区(VH)和轻链可变区(VL)的一条多肽链。在重链可变区和轻链可变区之间通常会设计一段柔性的连接肽(linker)以便重链可变区和轻链可变区可以折叠成为能够结合抗原的正确构象。
本文所用术语“抗原结合部”是指抗体结构中决定抗原结合能力的部分。本领域技术人员能够理解，抗体结构中决定抗原结合能力的主要部分是CDR，因此CDR也是抗原结合部的核心组成部分。在双特异性抗体构建中，“抗原结合部”的例子包括但不限于单链抗体(scfv)或Fab片段。
本文所用术语“双特异性抗体”是指具有结合两种不同抗原能力的抗体，其可以由两个Fc片段以及分别与其融合的两个抗原结合部组成。
在一些实施方案中，本文中的“双特异性抗体”是指基于人IgG1抗体的双特异性抗体，并且除了本文说明的改变结构之外，其具备人IgG1抗体的基本特征和功能。本领域技术人员公知本文中的“双特异性抗体”也可以基于其他免疫球蛋白亚型，例如人IgG2抗体。
本领域技术人员公知，互补决定区(CDR，通常有CDR1、CDR2及CDR3)是可变区中对抗体的亲和力和特异性影响最大的区域。VH或VL的CDR序列有两种常见的定义方式，即kabat定义和Chothia定义(参阅例如Kabat,“Sequences of Proteins of Immunological Interest”，National Institutes of Health,Bethesda，Md.(1991)；A1-Lazikani  et al.，J.Mol.Biol.273:927-948(1997)；以及Martin et al.，Proc.Natl.Acad.Sci.USA86:9268-9272(1989))。对于给定抗体的可变区序列，可以根据Kabat定义或者Chothia定义来确定VH和VL序列中CDR区序列。在本申请的实施方案中，利用Kabat定义CDR序列。
对于给定抗体的可变区序列，可以通过多种方式分析可变区序列中CDR区序列，例如可以利用在线软件Abysis确定(http://www.abysis.org/)。
本文所用术语“特异性结合”，是指两个分子之间的非随机结合反应，例如抗体至抗原表位的结合。
CD3分子是T细胞膜上的重要分化抗原，也是成熟T细胞的特征性标志。CD3由γ、δ、ε和δ四种链或γ、δ、ε、δ和ε(δ和ε为同源异构体)五种链组成，以CD3γε、CD3δε和CD3δδ(或CD3δε)3种二聚体组成并表达于T细胞膜上。CD3γ、δ和ε三条链中含有高度保守的酸性氨基酸残基(γ为谷氨酸，δ和ε为天冬氨酸)，可以与T细胞受体(TCR)α和β链上的碱性氨基酸残基通过盐桥以非共价键相连形成稳定的TCR-CD3复合物结构。该复合物可以传导T细胞激活信号并稳定TCR结构。CD3各链的胞内区中均含有ITAM(免疫受体酪氨酸活化基序)结构，该结构是CD3分子介导细胞内信号转导的基础。当TCR特异性识别并结合抗原(MHC分子提呈的抗原肽)后，T细胞内的酪氨酸蛋白激酶磷酸化ITAM上的酪氨酸残基，募集含有SH2结构域的酪氨酸蛋白激酶(ZAP-70)，将信号转导到T细胞胞浆内，启动细胞内活化机制，因此CD3有传导TCR识别抗原产生的激活信号的功能，是诱导T细胞活化的第一信号。
CD123又称为人白细胞介素-3(IL-3)受体α链，属于细胞因子受体超家族成员，分子量约40KDa，为Ⅰ型跨膜糖蛋白。白细胞介素-3受体是由α链(CD123)和β链(CD131)形成的异二聚体。IL-3与CD123结合后，由CD131提供信号转导，进而调节造血细胞和免疫细胞的功能并刺激内皮细胞增殖(Testa et al,Biomark Res.2:4(2014))。
CD123主要表达在骨髓祖细胞、浆细胞样树突状细胞、单核细胞、嗜碱性粒细胞和少量的B细胞亚群(Munoz L et al,Haematologica.86(12):1261-9)。大约80％的AML患者原幼细胞过表达CD123，研究显示CD123抗原的过表达对应AML的不良预后和较低的缓解率(Testa U et al,Blood.2002；100(8))。虽然大多数AML患者对初始治疗响应良好，但仍有部分患者(60-80％)需要巩固治疗才能达到完全缓解。
第一方面，本申请提供了双特异性抗体，其包含针对人CD3E的抗原结合部，所述针对人CD3E的抗原结合部包含：
如SEQ ID NO:1所示的HCDR1，
如SEQ ID NO:2所示的HCDR2，
如SEQ ID NO:3所示的HCDR3，
如SEQ ID NO:4所示的LCDR1，
如SEQ ID NO:5所示的LCDR2，和
如SEQ ID NO:6所示的LCDR3；
其中，HCDR和LCDR根据Kabat定义。
第二方面，本申请提供了双特异性抗体，其包含针对人CD123的抗原结合部，所述针对人CD123的抗原结合部包含：
如SEQ ID NO:7所示的HCDR1，
如SEQ ID NO:8所示的HCDR2，
如SEQ ID NO:9所示的HCDR3，
如SEQ ID NO:4所示的LCDR1，
如SEQ ID NO:5所示的LCDR2，和
如SEQ ID NO:6所示的LCDR3；
其中，HCDR和LCDR根据Kabat定义。
第三方面，本申请提供了双特异性抗体，其包含针对人CD3E的抗原结合部和针对人CD123的抗原结合部。
在第三方面的一些实施方案中，所述针对人CD3E的抗原结合部包含：
如SEQ ID NO:1所示的HCDR1，
如SEQ ID NO:2所示的HCDR2，
如SEQ ID NO:3所示的HCDR3，
如SEQ ID NO:4所示的LCDR1，
如SEQ ID NO:5所示的LCDR2，和
如SEQ ID NO:6所示的LCDR3；
其中，HCDR和LCDR根据Kabat定义。
在第三方面的一些实施方案中，所述针对人CD123的抗原结合部包含：
如SEQ ID NO:7所示的HCDR1，
如SEQ ID NO:8所示的HCDR2，
如SEQ ID NO:9所示的HCDR3，
如SEQ ID NO:4所示的LCDR1，
如SEQ ID NO:5所示的LCDR2，和
如SEQ ID NO:6所示的LCDR3；
其中，HCDR和LCDR根据Kabat定义。
在第三方面的一些实施方案中，所述针对人CD3E的抗原结合部和所述针对人CD123的抗原结合部包含相同的轻链可变区。
在第三方面的一些具体实施方案中，所述针对人CD3E的抗原结合部和所述针对人CD123的抗原结合部包含相同的轻链，该实施方案有利于轻链和重链的正确装配，也是优选的一种实施方案。
在第三方面的一些实施方案中，所述双特异性抗体是IgG1抗体，包含两种具有相同铰链区的重链恒定区，所述铰链区的氨基酸序列如SEQ ID NO:10所示，其替换天然人IgG1抗体恒定区的第216-230位序列，抗体恒定区氨基酸位置按照EU numbering确定。
在第三方面的一些实施方案中，所述双特异性抗体是IgG1抗体，其包含第一重链恒定区和第二重链恒定区，其中第一重链恒定区的第354和366位的氨基酸分别为C和W，第二重链恒定区的第349、366、368和407位的氨基酸分别为C、S、A和V；抗体恒定区氨基酸位置按照EU numbering确定。
当构建保留抗体Fc结构域的双特异性抗体时，可以从以下两个角度优化双特异性抗体的结构：一是重链异聚化，二是轻链和重链的正确装配。在一些实施方案中，两种Fc片段包含能够确保重链异聚化的突变。KIH技术(knob-in-hole，KIH)是解决重链异聚化的一种策略。通常，KIH技术是指通过改造CH3区的氨基酸序列，形成有利于异种半抗体相互配对的结构，可以在构成双特异性抗体的同时又尽可能地保持正常抗体的结构。在一些实施方案中，所利用的KIH技术包括，使一个Fc片段的第354和366位的氨基酸分别为C和W，另一个Fc片段的第349、366、368和407位的氨基酸分别为C、S、A和V。关于KIH技术的指导，例如可参见“An efficient route to human bispecific IgG”,A.Margaret Merchant et al.,Nature Biotechnology,Volume 16,1998，通过引用的方式将该文献全文并入本文。
在第三方面的一些实施方案中，所述双特异性抗体是IgG1抗体，其包含第一重链恒定区和第二重链恒定区，其中第一重链恒定区和第二重链恒定区的第234、235和331位的氨基酸分别为F、E和S；抗体恒定区氨基酸位置按照EU numbering确定。
在第三方面的一些实施方案中，两种重链恒定区的CH2片段的第234、235和331位的氨 基酸分别为F、E和S，其能够降低抗体Fc段介导的抗体依赖性细胞毒作用(ADCC)，从而可能减少双特异性抗体在体内导致的副作用。关于上述突变的指导，例如可参见“The binding affinity of human IgG for its high affinity Fc receptor is determined by multiple amino acids in the CH2 domain and is modulated by the hinge region”,Stephen M.Canfield et al.,J.Exp.Med.Volume 173,1991，通过引用的方式将该文献全文并入本文。
在第一和第三方面的一些实施方案中，针对人CD3E的抗原结合部包含如SEQ ID NO:11(包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:2所示的HCDR2和如SEQ ID NO:3所示的HCDR3)所示的重链可变区和如SEQ ID NO:12所示的轻链可变区(包含如SEQ ID NO:4所示的LCDR1、如SEQ ID NO:5所示的LCDR2和如SEQ ID NO:6所示的LCDR3)。
在第二和第三方面的一些实施方案中，针对人CD123的抗原结合部包含SEQ ID NO:13(包含如SEQ ID NO:7所示的HCDR1、如SEQ ID NO:8所示的HCDR2和如SEQ ID NO:9所示的HCDR3)所示的重链可变区和如SEQ ID NO:12所示的轻链可变区(包含如SEQ ID NO:4所示的LCDR1、如SEQ ID NO:5所示的LCDR2和如SEQ ID NO:6所示的LCDR3)。
在上述任一方面的一些实施方案中，所述针对人CD3E的抗原结合部包含单链抗体(scfv)或Fab片段。
在上述任一方面的一些实施方案中，所述针对人CD123的抗原结合部包含单链抗体(scfv)或Fab片段。
由于双特异性抗体具有针对两种不同抗原的两个不同抗原结合部，而抗原结合部可以包含单链抗体(scfv)或Fab片段两种形式，那么针对给定的两种抗原时，双特异性抗体的抗原结合部配置可以具有四种组合方式：Fab+Fab、Fab+scfv、scfv+Fab和scfv+scfv。
在上述任一方面的一些具体实施方案中，所述针对人CD3E的抗原结合部包含Fab片段，所述针对人CD123的抗原结合部包含Fab片段。
在上述任一方面的一些具体实施方案中，所述针对人CD3E的抗原结合部包含Fab片段，所述针对人CD123的抗原结合部包含单链抗体(scfv)。
在上述任一方面的一些具体实施方案中，所述针对人CD3E的抗原结合部包含单链抗体(scfv)，所述针对人CD123的抗原结合部包含Fab片段。
在上述任一方面的一些具体实施方案中，所述针对人CD3E的抗原结合部包含单链抗体(scfv)，所述针对人CD123的抗原结合部包含单链抗体(scfv)。
在本文中，还将双特异性抗体描述为具有两个“臂”，以中间为界，可以将双特异性抗体分为两个臂。双特异性抗体的臂可以由Fc片段和抗原结合部(Fab片段或单链抗体)组成。对于由Fc片段和Fab片段组成的臂，其结构类似于通常的抗体，含有完整的重链和轻链，因此这样的臂的结构可以表示为Fc+Fab，也可以表示为重链(Fc+Fab中的重链可变区和CH1片段)+轻链(Fab中的轻链部分)。当两个臂都含有Fab片段形式的抗原结合部时，由此形成的双特异性抗体的结构接近于天然抗体，是优选的一种实施方案。
在第三方面的一些实施方案中，所述抗体具有第一臂和第二臂，其中第一臂包含针对人CD3E的抗原结合部，第二臂包含针对人CD123的抗原结合部：
所述第一臂包含如SEQ ID NO:11所示的重链可变区氨基酸序列、如SEQ ID NO:31所示的重链恒定区氨基酸序列、如SEQ ID NO:12所示的轻链可变区氨基酸序列和如SEQ ID NO:32所示的轻链恒定区氨基酸序列；
所述第二臂包含如SEQ ID NO:13所示的重链可变区氨基酸序列、如SEQ ID NO:30所示的重链恒定区氨基酸序列、如SEQ ID NO:12所示的轻链可变区氨基酸序列和如SEQ ID NO:32所示的轻链恒定区氨基酸序列。
在上述任一方面的一些实施方案中，所述双特异性抗体的重链恒定区是人IgG1亚型或者 选定的人IgG1亚型的各种突变体，例如：IgG1H、IgG1K、IgG1m3-H或者IgG1m3-K。
在上述任一方面的一些实施方案中，所述双特异性抗体的轻链恒定区是人κ亚型或人λ亚型，优选为人κ亚型。
第四方面，本申请提供了药物组合物，其包含第一至第三方面中任一方面所述的双特异性抗体。
在一些实施方案中，所述药物组合物还包含药学可接受的载体、赋形剂、稀释剂等。
在一些实施方案中，所述药物组合物用于预防或治疗CD123阳性肿瘤，例如急性髓系白血病(AML)和母细胞性浆细胞样树突状细胞肿瘤(BPDCN)。
在一些实施方案中，药物组合物还可包含润滑剂，如滑石粉、硬脂酸镁和矿物油；润湿剂；乳化剂；悬浮剂；防腐剂，如苯甲酸、山梨酸和丙酸钙；增甜剂和/或调味剂等。
在一些实施方案中，可将本申请中的药物组合物配制为片剂、丸剂、粉剂、锭剂、酏剂、悬液、乳剂、溶液、糖浆、栓剂或胶囊等形式。
在一些实施方案中，可以利用任何生理上可接受的给药方式递送本申请的药物组合物，这些给药方式包括但不限于：口服给药、肠胃外给药、经鼻给药、直肠给药、腹膜内给药、血管内注射、皮下给药、经皮给药、吸入给药等。
在一些实施方案中，可以通过混合具有所需纯度的试剂与视情况的药学上可接受的载体、赋形剂等，以冻干制剂或水溶液的形式配制用于治疗用途的药物组合物用于存储。
第五方面，本申请提供了第一至第三方面中任一方面所述的双特异性抗体、或者第四方面所述的药物组合物在制备用于预防或治疗CD123阳性肿瘤的药物中的用途。
在第五方面的一些实施方案中，所述CD123阳性肿瘤选自急性髓系白血病(AML)和母细胞性浆细胞样树突状细胞肿瘤(BPDCN)。
第六方面，本申请提供了预防或治疗CD123阳性肿瘤的方法，包括向有需要的个体给予第一至第三方面中任一方面所述的双特异性抗体、或者第四方面所述的药物组合物。
在第六方面的一些实施方案中，所述CD123阳性肿瘤选自急性髓系白血病(AML)和母细胞性浆细胞样树突状细胞肿瘤(BPDCN)。
应当理解，以上详细描述仅为了使本领域技术人员更清楚地了解本申请的内容，而并非意图在任何方面加以限制。本领域技术人员能够对所述实施方案进行各种改动和变化。
以下实施例仅用于说明而非限制本申请范围的目的。
实施例
实施例1重组蛋白的制备
制备和鉴定CD3E×CD123双特异性抗体的过程中需要用到多种不同的重组蛋白，包括人CD3E胞外区(hCD3E，SEQ ID NO：14)、人CD3D胞外区(hCD3D，SEQ ID NO：15)、猴CD3E胞外区(mfCD3E，SEQ ID NO：16)、猴CD3D胞外区(mfCD3D，SEQ ID NO：17)、小鼠CD3E胞外区(mCD3E，SEQ ID NO：18)、小鼠CD3D胞外区(mCD3D，SEQ ID NO：19)以及人CD123亚型1胞外区(hCD123-SP1，SEQ ID NO：20)、猴CD123亚型1胞外区(mfCD123-SP1，SEQ ID NO：21)、小鼠CD123亚型1胞外区(mCD123-SP1，SEQ ID NO：22)。这些重组蛋白都有大量的翻译后修饰(如糖基化或二硫键等)，因而利用哺乳动物细胞表达***将更有利于保持重组蛋白的结构和功能。此外，为了方便纯化，非抗体类的重组蛋白在C端添加His标签(SEQ ID NO：23)，或者小鼠抗体IgG2a的Fc段(mFc，SEQ ID NO：24)，或者基于KIH(Knob-Into-Hole)技术形成异源二聚体的人IgG1亚型的Fc突变体(FcK， SEQ ID NO：25或者FcH，SEQ ID NO：26)。在制备重组抗体时，抗体重链恒定区可以是人IgG1亚型(SEQ ID NO：27)或者选定的人IgG1亚型的各种突变体，如：IgG1H(SEQ ID NO：28)、IgG1K(SEQ ID NO：29)、IgG1m3-H(SEQ ID NO：30)或者IgG1m3-K(SEQ ID NO：31)，轻链恒定区是人κ亚型(SEQ ID NO：32)或人λ亚型(SEQ ID NO：33)。
根据Uniprot数据库的各种目的重组蛋白的氨基酸序列，设计并合成上述各种重组蛋白的基因(包含His标签、mFc或者Fc编码基因)。利用常规的分子生物学技术将合成的各种重组蛋白基因克隆至合适的真核表达载体(如invitrogen公司的pcDNA3.1等)，然后利用脂质体(如invitrogen公司的293fectin等)或其它转染试剂(如PEI等)将制备的重组蛋白表达质粒转染入HEK293细胞(如invitrogen公司的HEK293F)，在无血清悬浮培养条件下培养3-5天。然后通过离心等方式收获培养上清。
His标签融合表达的重组蛋白利用金属螯合亲和层析柱(如GE公司的HisTrap FF等)对培养上清中的重组蛋白进行一步纯化。而mFc融合表达的重组蛋白和重组抗体用ProteinA/G亲和层析柱(如GE公司的Mabselect SURE等)进行一步纯化。然后利用脱盐柱(如GE公司的Hitrap desaulting等)将重组蛋白保存缓冲液置换为PBS(pH7.0)或者其它合适的缓冲液。必要时，可以对抗体样品进行过滤除菌，然后分装保存于-20℃。
实施例2基于固定轻链的CD123小鼠免疫库构建
为构建基于共同轻链的CD3E×CD123双特异性抗体，选取特定的抗CD3E单克隆抗体轻链可变区配合经过CD123抗原体内亲和力成熟的小鼠重链可变区，利用常规分子生物学手段构建单链抗体(scFv)库，用于筛选针对CD123的特异性抗体。
利用hCD123-SP1-His和mfCD123-SP1-His重组蛋白交叉免疫6-8周龄的BALB/c小鼠后，收集脾细胞。使用小鼠淋巴细胞分离液(达科为生物技术股份有限公司，CAT#DKW33-R0100)对小鼠脾脏淋巴细胞进行分离，利用细胞总RNA提取试剂盒(天根生化科技(北京)有限公司，CAT#DP430)，将分离的淋巴细胞进行总RNA的提取。以提取的总RNA为模板，利用第一链cDNA合成试剂盒(Thermo scientific，CAT#K1621)合成抗体的重链可变区。利用常规分子生物学手段，PCR扩增得到特异性识别人和食蟹猴CD3E的大鼠单克隆抗体WM03-C6(参见专利WO_2016_116626_A1中单克隆抗体20E5-F10的序列，重链可变区氨基酸序列如SEQ ID NO：34所示，轻链可变区氨基酸序列如SEQ ID NO：35所示)轻链可变区和CD123重组抗原免疫得到的小鼠重链可变区，然后利用重叠延伸PCR技术，构建单链抗体(scFv)，将制备的小鼠单链抗体基因克隆至载体pADSCFV-S(实验技术流程可参见中国专利申请第201510097117.0号的实施例1)，构建scFv库。此抗体库的库容达到1.2×10E8，正确率为65％。
实施例3固定轻链的CD123小鼠免疫库的筛选
3.1抗人CD123小鼠单链抗体的筛选
以实施例1制备的重组hCD123-SP1-his为抗原，利用固相筛选策略(实验方案参考噬菌体展示：通用实验指南/(美)克拉克森(Clackson,T.),(美)洛曼(Lowman,H.B.)编；马岚等译。化学工业出版社，2008.5)筛选实施例2构建的展示小鼠单链抗体的噬菌体库，通过结合、洗脱、中和、感染、扩增的方式共进行三轮筛选，最终获得1株特异性结合人CD123的单链抗体S8F3(SEQ ID NO：36)。
利用常规的分子生物学手段，将编码S8F3的重链可变区S8F3VH(SEQ ID NO：37)和轻链可变区WM03-C6VK(SEQ ID NO：35)的核苷酸序列分别克隆至融合有编码人重链恒定区和轻链恒定区的核苷酸序列真核表达载体(如invitrogen公司的pcDNA3.1等)，组合表达全抗体S8F3VH+C6VK。同时，参照美国专利US_2014_0178364_A1制备抗CD123抗体 CSL362(重链氨基酸序列如SEQ ID NO：38所示，轻链氨基酸序列如SEQ ID NO：39所示)作为阳性对照抗体用于后续研究。
3.2重组抗CD123单克隆抗体的亲和力分析
利用Biacore X100通过表面等离子共振技术测定抗CD123抗体的亲和力。氨基偶联试剂盒(BR-1000-50)、人抗体捕获试剂盒(BR-1008-39)、CM5芯片(BR100012)和pH7.4的10×HBS-EP(BR100669)等相关试剂和耗材均购自GE healthcare。依照试剂盒中的说明书，用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride，EDC)和N-羟基琥珀酰亚胺(N-Hydroxysuccinimide，NHS)对羧基化CM5芯片表面进行活化，将抗人IgG(Fc)抗体(捕获抗体)用10mM pH5.0乙酸钠稀释至25μg/mL，之后以流速10μL/min注射以实现大约10000个响应单位(RU)的偶联量。注射捕获抗体之后，注射1M乙醇胺以封闭未反应的基团。对于动力学测量，稀释抗CD123抗体(S8F3VH+C6VK和CSL362)至0.5-1μg/mL，10μL/min注射，保证100RU左右的抗体被抗人Fc的抗体捕获。然后将hCD123-SP1-his设置一系列的浓度梯度(例如2.47nM、7.4nM、22.2nM、66.7nM、200nM)，于25℃下30μL/min从低浓度到高浓度进行注射，结合时间为120s，解离时间为600-2400s，以10μL/min注射3M的MgCl 2溶液30s对芯片表面进行再生。使用Biacore X100评估软件2.0.1版，通过1:1结合模型拟合结合和解离传感图来计算结合速率(K on)和解离速率(K off)。以比率K off/K on计算解离平衡常数(KD)。拟合结果如表1所示。
表1.重组抗CD123单克隆抗体结合hCD123-SP1-his亲和力常数
  K on K off KD
S8F3VH+C6VK 4.546E+5 3.651E-3 8.030E-9
CSL362 1.324E+5 7.931E-5 5.991E-10
3.3重组抗CD123单克隆抗体结合细胞表面CD123抗原的鉴定
取生长对数期的KG-1a(人急性髓系白血病细胞，购自中国医学科学院基础医学研究所细胞资源中心)，离心后用含1％BSA的PBS缓冲液重悬至2×10 6个/mL，100μL/孔铺于96孔V底板中，离心后去掉上清。用PBS将待测样品S8F3VH+C6VK、对照样品CSL362以及HIgG无关抗体(金斯瑞A01006，人IgG对照(全分子)，Purifie)分别配制为5μg/mL和0.5μg/mL的终浓度，加入含细胞的孔中，4℃孵育1小时。然后用200μL PBS清洗3遍，加入羊抗人IgG-FITC(中杉金桥，ZF-0308)二抗(100μL/孔)，4℃避光孵育30分钟。然后用200μL PBS清洗3遍，100μL PBS重悬后用流式细胞仪(ACEA，Novocyte)检测FITC通道。结果显示S8F3VH+C6VK能特异性结合CD123阳性细胞KG-1a(图1)。
实施例4 WM03-C6单克隆抗体的人源化和活性鉴定
4.1 WM03-C6人源化设计
对大鼠单克隆抗体WM03-C6进行人源化研究以降低其免疫原性。人源化方案采取经典的框架移植策略(J immunol.169,1119-1125,2002)。将WM03-C6的重链可变区和轻链可变区分别与IMGT数据库中的人抗体胚系基因序列相比较，选择合适的胚系基因序列以提供抗体的框架区1至3(FR1+FR2+FR3)，选择合适的J区基因序列以提供框架区4(FR4)。这个模板可以根据多种因素选出，如：抗体的相对总长度、CDR的大小、位于抗体框架区(FR)和超变区(CDR)之间连接处的氨基酸残基、序列整体的同源性等。所选的模板可以是多个序列的混合物或者可以是共有模板，目的是尽可能维持亲本互补决定区(CDR)的合适构象。同时，为避免抗体超变区(CDR)中脱氨基位点NG可能带来的蛋白异质性，对人源化后的抗体轻重链分别进行了突变设计。最终得到了人源化的轻链突变体L27E5(SEQ ID NO：12)和人源化 重链突变体H3B8(SEQ ID NO：11)。
4.2 WM03-C6人源化单克隆抗体的亲和力分析
参照实施例3.2，将抗人IgG(Fc)抗体偶联至CM5芯片表面，稀释抗CD3E抗体(WM03-C6和H3B8+L27E5)至0.5-1μg/mL，10μL/min注射，保证350-400RU左右的抗体被抗人Fc的抗体捕获。然后将hCD3E-his设置一系列的浓度梯度(例如6.17nM、18.5nM、55.6nM、167nM、500nM)，在25℃下30μL/min从低浓度到高浓度进行注射，结合时间为120s，解离时间为600-1800s，以10μL/min注射3M的MgCl 2 30s对芯片表面进行再生。使用Biacore X100评估软件2.0.1版，通过1:1结合模型拟合结合和解离传感图来计算结合速率(K on)和解离速率(K off)。以比率K off/K on计算解离平衡常数(KD)。拟合结果如表2所示。
表2.WM03-C6人源化单克隆抗体结合hCD3E-his亲和力常数
  K on K off KD
WM03-C6 2.716E+4 2.879E-4 1.060E-8
H3B8+L27E5 2.084E+4 9.928E-4 4.763E-8
实施例5 S8F3的体外亲和力成熟
为提高双特异性抗体针对靶抗原的特异性，增强双特异性抗体在给药过程中的组织分布和杀伤效率，对S8F3重链可变区进行了体外亲和力成熟。利用常规分子生物学手段，通过在S8F3重链可变区的CDR3中引入突变，构建了基于S8F3VH的CDR3突变库。设计的突变方案如表3所示，构建库容为1.7×10E8，正确率86％。
表3.S8F3VH-CDR3突变库设计方案
初始氨基酸 设计氨基酸 简并密码子
L LFVI NTC
R RTKSN AVW
Y YF TWC
G GSDNIV RDT
N NDTA RMC
Y YF TWC
G GVAD GNT
D DNYSTA DMC
A ATNDIV RHT
M MVTAKE RHG
D DNYSTA DMC
D DYFV KWT
基于双载体噬菌体展示***(实验技术流程参见中国专利申请第201510097117.0号中的实施例5)，通过固相筛选的方法，利用hCD123-SP1抗原对构建的S8F3VH-CDR3突变库共进行了3轮筛选富集，最终得到亲和力提高的重链可变区突变体H7A3。将得到的编码H7A3可变区的核苷酸序列克隆至融合有编码人重链恒定区的核苷酸序列的真核表达载体，配合L27E5轻链表达载体组合表达全抗体。
参照实施例3.2，用Biacore X100对S8F3重链突变体H7A3进行亲和力测定，结果如表4所示。
表4.S8F3重链突变体结合hCD123-SP1-his亲和力常数
  K on K off KD
S8F3VH+L27E5 4.384E+4 1.455E-4 3.319E-9
H7A3+L27E5 2.698E+5 6.638E-5 2.46E-10
实施例6 H7A3的人源化及其活性鉴定
抗人CD123鼠单克隆抗体重链H7A3的人源化采用经典的框架移植策略。参照实施例4，对H7A3的重链可变区进行CDR移植得到人源化版本H7A3VH-h2，同时为保证抗体的构象和亲和力，对人源化抗体框架区一些关键氨基酸如I69、R71、T73、A75进行了回复突变，最终得到人源化版本H7A3-h2-m5(SEQ ID NO：13)。根据人源化抗体的氨基酸序列设计并合成抗体可变区基因，克隆至真核表达载体并配合共同轻链L27E5表达人IgG1版本全抗体。
参考实施例3.2，用Biacore X100对H7A3人源化版本H7A3-h2-m5进行亲和力测定，结果如表5所示。
表5.H7A3人源化版本结合hCD123-SP1-his亲和力常数
  K on K off KD
H7A3+L27E5 3.054E+5 6.889E-5 2.256E-10
H7A3-h2-m5+L27E5 3.144E+5 7.671E-5 2.440E-10
取生长对数期的MV-4-11(人急性单核细胞白血病细胞，购自南京科佰生物科技有限公司)，离心后用含1％BSA的PBS缓冲液重悬至2×10 6个/mL，100μL/孔铺于96孔V底板中，离心后去掉上清。用PBS将待测样品H7A3-h2-m5+L27E5、对照样品CSL362以及HIgG无关抗体(金斯瑞A01006，人IgG对照(全分子)，Purifie)均配制成终浓度为100nM起始，3倍梯度稀释，共9个浓度，加入含细胞的孔中，4℃孵育1小时。然后用200μL PBS清洗3遍，加入羊抗人IgG-FITC(中杉金桥，ZF-0308)，100μL/孔，4℃避光孵育30分钟。然后用200μL PBS清洗3遍，100μL PBS重悬后用流式细胞仪(ACEA，Novocyte)检测FITC通道。结果如图2所示，H7A3-h2-m5+L27E5能很好的结合CD123阳性细胞MV-4-11，KD值为3.1nM。
实施例7双特异性抗体的制备
分别将编码CD3E单克隆抗体的重链可变区H3B8和CD123单克隆抗体的重链可变区H7A3-h2-m5的核苷酸序列克隆至合适的真核表达载体，构建基于共同轻链的异源二聚体。即将编码CD3E抗体的重链可变区的核苷酸序列克隆至融合有编码Knob突变的IgG1恒定区IgG1m3-K的核苷酸序列的真核表达载体，将编码CD123抗体的重链可变区的核苷酸序列克隆至含有编码Hole突变的IgG1恒定区IgG1m3-H核苷酸序列的真核表达载体，将编码共同轻链L27E5的可变区VK的核苷酸序列克隆至融合有编码人轻链恒定区CK的核苷酸序列的真核表达载体。同时为了比较候选分子与临床在研的CD3E×CD123双特异性抗体的生物学活性，参照专利WO2017210443，构建了基于相同Fc结构的Xmab14045(SEQ ID NO：40、SEQ ID NO：41、SEQ ID NO：42)。
将构建的表达H3B8-IgG1m3-K、表达H7A3-h2-m5-IgG1m3-H和表达L27E5-CK的3个真核表达载体利用脂质体共转染入HEK293F细胞，在无血清悬浮培养条件下培养3-5天，然后通过离心等方式收获培养上清。培养上清中双特异性抗体用ProteinA/G亲和层析柱(如GE公司的Mabselect SURE等)进行纯化，然后利用脱盐柱(如GE公司的Hitrap desaulting等)将重组蛋白保存缓冲液置换为PBS(pH7.0)或者其它合适的缓冲液。将脱盐后蛋白溶液通过尺寸排阻层析(SEC)使用Superdex200(GE)纯化得到目的蛋白。必要时，可以对抗体样品进行过滤除菌，然后分装保存于-20℃备用。
实施例8双特异性抗体的亲和力分析
参照实施例3.2和4.2，利用Biacore X100通过表面等离子共振技术对抗CD3E单克隆抗体H3B8+L27E5、抗CD123单克隆抗体H7A3-h2-m5+L27E5、CD3×CD123双特异性抗体(CD3×CD123 BsAb)及Xmab14045对照抗体进行亲和力测定，亲和力拟合结果见表6和表7。
表6.CD3×CD123双特异性抗体结合hCD123-SP1-his亲和力常数
  K on K off KD
H7A3-h2-m5+L27E5 2.337E+5 5.137E-5 2.198E-10
CD3×CD123 BsAb 2.254E+5 4.015E-5 1.781E-10
Xmab14045 9.671E+4 8.188E-5 8.467E-10
表7.CD3×CD123双特异性抗体结合CD3E亲和力常数
Figure PCTCN2020082151-appb-000001
实施例9双特异性抗体同时识别CD3E和CD123两种抗原的能力鉴定
利用常规ELISA方法检测CD3×CD123双特异性抗体(CD3×CD123 BsAb)对CD3E和CD123两种抗原的同时结合。将CD123-SP1-mFc抗原包被96孔ELISA板(3μg/mL，100μL/孔)，4℃包被过夜。利用封闭液PBS-0.1％Tween20-3％牛奶在37℃下封闭1小时后，分别加入抗CD123单克隆抗体H7A3-h2-m5+L27E5、抗CD3E单克隆抗体H3B8+L27E5和CD3×CD123双特异性抗体(10μg/mL，100μL/孔)，各两个复孔，37℃孵育1小时。PBS-0.1％Tween20洗涤ELISA板，然后加入CD3E-his抗原(1μg/mL，100μL/孔)，37℃孵育1小时。PBS-0.1％Tween20洗涤ELISA板，然后加入HRP小鼠抗his IgG(北京康为世纪生物科技有限公司，cw0285M)，37℃孵育1小时。PBS-0.1％Tween20洗涤ELISA板，加入OPD底物显色液，5-10分钟后用1M的H 2SO 4终止显色，酶标仪492nm/630nm双波长测定光密度值。ELISA分析结果如图3所示，CD3×CD123双特异性抗体能够同时识别CD3E和CD123两种抗原。
实施例10双特异性抗体识别细胞表面CD3E和CD123的能力鉴定
10.1双特异性抗体识别细胞表面CD3E能力鉴定
取生长对数期的Jurkat-Dual细胞(购自Invivogen，jktd-isnf)，离心后用含1％BSA的PBS缓冲液重悬至2×10 6个/mL，100μL/孔铺于96孔V底板中，离心后去掉上清。用PBS稀释样品，双特异性抗体对照样品Xmab14045、抗CD3E单克隆抗体H3B8+L27E5、HIgG无关抗体(金斯瑞A01006，人IgG对照(全分子，Purifie)和CD3×CD123双特异性抗体(CD3×CD123BsAb)均以400nM为起始浓度，前3个点2倍稀释，后5个浓度点3倍稀释，共计8个浓度，加入含细胞的孔中，4℃孵育1小时。然后用200μL PBS清洗3遍，加入羊抗人IgG-FITC(中杉金桥，ZF-0308)100μL/孔，4℃避光孵育30分钟。然后用200μL PBS清洗3遍，100μL PBS重悬后用流式细胞仪(ACEA，Novocyte)检测FITC通道。结果显示CD3×CD123双特异性抗体可以结合Jurkat-Dual细胞表面的CD3E，结合强度弱于对照样品Xmab14045，与实施例9中亲和力测定结果一致(图4)。
10.2双特异性抗体识别细胞表面CD123能力鉴定
取生长对数期的MV-4-11细胞，离心后用含1％BSA的PBS缓冲液重悬至2×10 6个/mL，100μL/孔铺于96孔V底板中，离心后去掉上清。用PBS稀释样品，双特异性抗体对照样品Xmab14045、抗CD123单克隆抗体H7A3-h2-m5+L27E5、CD3×CD123双特异性抗体(CD3×CD123 BsAb)和HIgG无关抗体(金斯瑞A01006，人IgG对照(全分子，Purifie)均以100nM终浓度起始，3倍梯度稀释，共9个浓度点，加入含细胞的96孔板中，4℃孵育1小时。然后用200μL PBS清洗3遍，加入羊抗人IgG-FITC(中杉金桥，ZF-0308)100μL/孔，4℃避光孵育30分钟。然后用200μL PBS清洗3遍，100μL PBS重悬后用流式细胞仪(ACEA，Novocyte)检测FITC通道。结果显示，CD3×CD123双特异性抗体能很好的结合CD123阳性细胞MV-4-11表面的CD123，KD值为7.08nM,与Xmab14045(KD＝6.07nM)相当(图5)。
实施例11双特异性抗体介导Jurkat-Dual细胞特异性激活
收集处于生长对数期的MV-4-11细胞(CD123 +细胞)，离心后用1640培养基重悬至2×10 6个/mL，50μL/孔铺于细胞板中，离心后去掉上清。收集生长对数期的Jurkat-Dual细胞(购自Invivogen)，离心后用1640培养基重悬至2×10 6个/mL，50μL/孔加入细胞板中以获得1：1的最终E:T比例。然后加入起始浓度为6nM，4倍梯度稀释，8个浓度点的CD3×CD123双特异性抗体(CD3×CD123 BsAb，50μL/孔)。***对照设置有双特异性抗体对照样品Xmab14045、抗CD3E单克隆抗体H3B8+L27E5、抗CD123单克隆抗体H7A3-h2-m5+L27E5、H3B8+L27E5和H7A3-h2-m5+L27E5的联合组(H3B8+L27E5&H7A3-h2-m5+L27E5)以及CD123阴性靶细胞(BAF3细胞)对照组以及HIgG无关抗体(金斯瑞A01006，人IgG对照(全分子)，Purifie)，使用浓度均同CD3×CD123双特异性抗体的浓度。孵育20小时后，取上清，参照QUANTI-Luc TM说明书(QUANTI-Luc，Invivogen，rep-qlc2)检测和分析不同条件下介导的肿瘤细胞对Jurkat-Dual细胞的特异性激活(图6A-B)。
实施例12 CD3×CD123双特异性抗体介导纯化的T细胞表面活化分子的表达和对CD123阳性肿瘤细胞的杀伤
12.1人外周血单核细胞(PBMC)的分离
采集正常志愿者的血液(各50mL),所有志愿者均已签订知情同意书。
志愿者入选标准为：
1.年龄大于18周岁；
2.无HIV、HBV感染；
3.血常规检测正常；
4.非孕妇或哺乳期妇女；
利用Ficoll密度梯度离心法从志愿者全血细胞中分离得到PBMC，并培养于1640培养基中。
12.2双特异性抗体介导纯化的T细胞杀伤CD123阳性肿瘤细胞的检测
收集生长对数期的MV-4-11细胞(CD123 +细胞)，离心后用1640培养基重悬至1×10 6个/mL，50μL/孔铺于细胞板中。然后加入起始浓度为1nM，4倍梯度稀释，8个浓度点的CD3×CD123双特异性抗体(CD3×CD123 BsAb，50μL/孔)。参照T细胞阴性分选试剂盒说明书(BD IMaq human T lymphocyte enrichment set-DM，BD，557874)从PBMC中分选获得纯化的T细胞(5×10 6个/mL，50μL/孔)，最终效靶比为5：1。同时设置单独靶细胞对照(MV-4-11细胞)、单独效应细胞对照(T)、单独培养基空白对照，并用培养基将体积补齐至150μL。孵育20小时后，取上清，参照
Figure PCTCN2020082151-appb-000002
非放射性细胞毒性检测试剂说明书(
Figure PCTCN2020082151-appb-000003
Non-Radioactive Cytotoxicity Assay，promega，G1780)检测和分析CD3×CD123双特异性抗体介导的T细胞对 肿瘤细胞的杀伤率。结果显示CD3×CD123双特异性抗体可以特异的介导T细胞对CD123阳性肿瘤细胞的杀伤，对CD123阴性肿瘤细胞(BAF3细胞)没有表现出杀伤效果(图7)。
12.3双特异性抗体特异性激活T细胞后表面活化分子表达的鉴定
在12.2实验中，取出上清后的细胞，PBS清洗2遍，加入抗人CD3-APC(ebioscience,17-0037-42)和抗人CD69-PE(ebioscience,11-0069-42)流式抗体4℃避光孵育30分钟，然后用PBS清洗2遍，100μL PBS重悬后上流式(ACEA，Novocyte)检测，比较CD3×CD123双特异性抗体(CD3×CD123 BsAb)处理后CD3阳性细胞群(MV-4-11细胞)中活化标志物CD69的表达差异。结果显示：CD3×CD123双特异性抗体在阳性肿瘤细胞存在下，可以特异的上调T细胞表面CD69的表达，CD123阴性肿瘤细胞(BAF3细胞)不能上调T细胞表面CD69的表达(图8)。
实施例13双特异性抗体介导的T细胞体外增殖实验
13.1人外周血单核细胞(PBMC)的分离
参照实施例12.1进行分选获得PBMC。
13.2双特异性抗体介导的T细胞体外增殖实验
参照T细胞阴性分选试剂盒说明书(BD IMaq human T lymphocyte enrichment set-DM,BD,557874)从PBMC中分选获得纯化的T细胞。参考CFSE染色说明书对纯化的T细胞进行染色(CFSE，eBioscience，65-0850-84)，用1640培养基重悬至2×10 6个/mL，50μL/孔加入细胞培养板。收集生长对数期的MV-4-11(CD123 +细胞)，离心后用1640培养基重悬至5×10 5个/mL，50μL/孔铺于细胞板中，最终的效靶比是4：1。然后加入起始浓度为0.25nM，4倍梯度稀释，8个浓度点的CD3×CD123双特异性抗体(CD3×CD123 BsAb，50μL/孔)和Xmab14045；另外设置了0.25nM浓度点的对照，分别为单独的抗CD3E单克隆抗体H3B8+L27E5、单独的抗CD123单克隆抗体H7A3-h2-m5+L27E5以及H3B8+L27E5和H7A3-h2-m5+L27E5的联合(H3B8+L27E5&H7A3-h2-m5+L27E5)；此外还设置了0.25nM浓度点的CD3×CD123双特异性抗体在CD123阴性肿瘤细胞(BAF3细胞)存在下作用的对照。孵育5天后，用PBS清洗2遍，用抗人CD3-APC(ebioscience,17-0037-42)流式抗体4℃避光孵育30分钟，然后用PBS清洗2遍，100μL PBS重悬后上流式(ACEA，Novocyte)检测T细胞增殖的情况(图9A-B)。
实施例14双特异性抗体在CD34 +小鼠模型中的活性鉴定
选取42只(32只分组，10只备用)20-24周雌性hCD34 +人源化小鼠(购自澎立生物医药技术(上海)有限公司)，将100μL的1×10 7个MV-4-11细胞和100μL的Matrigel混匀后通过皮下注射接种于小鼠背部右侧，接种前用3-4％异氟烷将小鼠麻醉。当肿瘤生长到平均约50-80mm 3左右时，32只荷瘤小鼠根据外周血中hCD45 +的比例和肿瘤体积被随机分成4组，每组8只。分组给药当天定义为第0天。试验分为CD3×CD123双特异性抗体0.01mg/kg、0.1mg/kg、0.5mg/kg组及阴性对照组(IgG1m3，0.5mg/kg)4组，每组8只，按照第0天、3天、7天、14天和第21天尾静脉注射给药，共给药5次。根据相对肿瘤抑制率(TGI)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。
实验过程中动物精神状态普遍良好，体内实验结束时(第24天)，和阴性对照组(IgG1m3i.v.0.5mg/kg剂量组，G1组)相比，给药组动物与对照组相比体重无显著性差异(P>0.05)。测试CD3×CD123双特异性抗体在0.5mg/kg剂量下(G2组)，具有显著抑制肿瘤生长的作用，相对肿瘤抑制率TGI(％)为97.35％；且有6只动物肿瘤几乎完全消退，与同期阴性对照组(G1组)相比具有极显著差异(p<0.001)。CD3×CD123双特异性抗体0.1mg/kg剂量组(G3组)动物给药后瘤体积增长缓慢，相对肿瘤抑制率TGI(％)为52.08％，与同期阴性对照组(G1组)相比具有显著 差异(p<0.05)，G4为CD3×CD123双特异性抗体0.01mg/kg剂量组。CD3×CD123双特异性抗体与肿瘤生长有着明显的量效关系，各组动物在各个时间点肿瘤生长情况如图10所示。其中，当与G1组相比，采用单因素方差分析/Dunnett t检验统计时，*表示P<0.05，**表示P<0.01，***表示P<0.001；采用重复测量/Bonferroni校正统计时，###表示P<0.001。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

Claims (13)

  1. 双特异性抗体，其包含针对人CD3E的抗原结合部，所述针对人CD3E的抗原结合部包含：
    如SEQ ID NO:1所示的HCDR1，
    如SEQ ID NO:2所示的HCDR2，
    如SEQ ID NO:3所示的HCDR3，
    如SEQ ID NO:4所示的LCDR1，
    如SEQ ID NO:5所示的LCDR2，和
    如SEQ ID NO:6所示的LCDR3；
    其中，HCDR和LCDR根据Kabat定义。
  2. 双特异性抗体，其包含针对人CD123的抗原结合部，所述针对人CD123的抗原结合部包含：
    如SEQ ID NO:7所示的HCDR1，
    如SEQ ID NO:8所示的HCDR2，
    如SEQ ID NO:9所示的HCDR3，
    如SEQ ID NO:4所示的LCDR1，
    如SEQ ID NO:5所示的LCDR2，和
    如SEQ ID NO:6所示的LCDR3；
    其中，HCDR和LCDR根据Kabat定义。
  3. 双特异性抗体，其包含针对人CD3E的抗原结合部和针对人CD123的抗原结合部；
    优选地，所述针对人CD3E的抗原结合部包含：
    如SEQ ID NO:1所示的HCDR1，
    如SEQ ID NO:2所示的HCDR2，
    如SEQ ID NO:3所示的HCDR3，
    如SEQ ID NO:4所示的LCDR1，
    如SEQ ID NO:5所示的LCDR2，和
    如SEQ ID NO:6所示的LCDR3；和/或
    所述针对人CD123的抗原结合部包含：
    如SEQ ID NO:7所示的HCDR1，
    如SEQ ID NO:8所示的HCDR2，
    如SEQ ID NO:9所示的HCDR3，
    如SEQ ID NO:4所示的LCDR1，
    如SEQ ID NO:5所示的LCDR2，和
    如SEQ ID NO:6所示的LCDR3；
    其中，HCDR和LCDR根据Kabat定义。
  4. 如权利要求3所述的双特异性抗体，其中所述针对人CD3E的抗原结合部和所述针对人CD123的抗原结合部包含相同的轻链可变区，优选地，包含相同的轻链；和/或
    所述双特异性抗体是IgG1抗体，其包含两种具有相同铰链区的重链恒定区，所述铰链区的氨基酸序列如SEQ ID NO:10所示。
  5. 如权利要求3或4所述的双特异性抗体，其中所述双特异性抗体是IgG1抗体，其包含第一重链恒定区和第二重链恒定区，其中
    所述第一重链恒定区的第354和366位的氨基酸分别为C和W，所述第二重链恒定区的第349、366、368和407位的氨基酸分别为C、S、A和V；和/或
    所述第一重链恒定区和第二重链恒定区的第234、235和331位的氨基酸分别为F、E和S；
    抗体恒定区氨基酸位置按照EU numbering确定。
  6. 如权利要求1、2和3-5中任一项所述的双特异性抗体，其中所述针对人CD3E的抗原结合部包含如SEQ ID NO:11所示的重链可变区和如SEQ ID NO:12所示的轻链可变区；和/或
    所述针对人CD123的抗原结合部包含SEQ ID NO:13所示的重链可变区和如SEQ ID NO:12所示的轻链可变区。
  7. 如权利要求1-6中任一项所述的双特异性抗体，其中所述针对人CD3E的抗原结合部和/或所述针对人CD123的抗原结合部包含单链抗体(scfv)或Fab片段；
    优选地，所述针对人CD3E的抗原结合部包含Fab片段，所述针对人CD123的抗原结合部包含Fab片段；或
    所述针对人CD3E的抗原结合部包含Fab片段，所述针对人CD123的抗原结合部包含单链抗体(scfv)；或
    所述针对人CD3E的抗原结合部包含单链抗体(scfv)，所述针对人CD123的抗原结合部包含Fab片段；或
    所述针对人CD3E的抗原结合部包含单链抗体(scfv)，所述针对人CD123的抗原结合部包含单链抗体(scfv)。
  8. 如权利要求3所述的双特异性抗体，其中所述抗体具有第一臂和第二臂，其中所述第一臂包含针对人CD3E的抗原结合部，所述第二臂包含针对人CD123的抗原结合部：
    所述第一臂包含如SEQ ID NO:11所示的重链可变区氨基酸序列、如SEQ ID NO:31所示的重链恒定区氨基酸序列、如SEQ ID NO:12所示的轻链可变区氨基酸序列和如SEQ ID NO:32所示的轻链恒定区氨基酸序列；
    所述第二臂包含如SEQ ID NO:13所示的重链可变区氨基酸序列、如SEQ ID NO:30所示的重链恒定区氨基酸序列、如SEQ ID NO:12所示的轻链可变区氨基酸序列和如SEQ ID NO:32所示的轻链恒定区氨基酸序列。
  9. 双特异性抗体，其包含针对人CD3E的抗原结合部和针对人CD123的抗原结合部；
    其中所述双特异性抗体是IgG1抗体，其包含两种具有相同铰链区的重链恒定区，所述铰链区的氨基酸序列如SEQ ID NO:10所示。
  10. 药物组合物，其包含权利要求1-9中任一项所述的双特异性抗体。
  11. 如权利要求10所述的药物组合物，其用于预防或治疗CD123阳性肿瘤；优选地，所述CD123阳性肿瘤选自急性髓系白血病(AML)和母细胞性浆细胞样树突状细胞肿瘤(BPDCN)。
  12. 权利要求1-9中任一项所述的双特异性抗体、或者权利要求10或11所述的药物组合物在制备用于预防或治疗CD123阳性肿瘤的药物中的用途；优选地，所述CD123阳性肿瘤选自急性髓系白血病(AML)和母细胞性浆细胞样树突状细胞肿瘤(BPDCN)。
  13. 预防或治疗CD123阳性肿瘤的方法，其包括向有需要的个体给予权利要求1-9中任一项所述的双特异性抗体、或者权利要求10或11所述的药物组合物；优选地，所述CD123阳性肿瘤选自急性髓系白血病(AML)和母细胞性浆细胞样树突状细胞肿瘤(BPDCN)。
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