WO2020252907A1 - 抗cd3e和bcma双特异性抗体及其用途 - Google Patents

抗cd3e和bcma双特异性抗体及其用途 Download PDF

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WO2020252907A1
WO2020252907A1 PCT/CN2019/102817 CN2019102817W WO2020252907A1 WO 2020252907 A1 WO2020252907 A1 WO 2020252907A1 CN 2019102817 W CN2019102817 W CN 2019102817W WO 2020252907 A1 WO2020252907 A1 WO 2020252907A1
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seq
binding portion
antibody
antigen binding
human
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刘志刚
万姝南
刘玉兰
郝小勃
胡俊杰
郭晶晶
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Beijing Wisdomab Biotechnology Co Ltd
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Beijing Wisdomab Biotechnology Co Ltd
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Publication of WO2020252907A1 publication Critical patent/WO2020252907A1/zh
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Priority to ZA2022/00784A priority patent/ZA202200784B/en
Priority to US19/225,370 priority patent/US20250289901A1/en
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    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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
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    • C07K16/2803—Immunoglobulins [IG], 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 [IG], 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
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    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
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    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/54—F(ab')2
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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
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
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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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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • This application generally relates to the field of antibody medicines. Specifically, this application provides bispecific antibodies containing an antigen binding portion for human CD3E and/or an antigen binding portion for human BCMA and their medical and biological uses.
  • Bispecific antibody is a type of artificial antibody, which contains two different antigen binding sites. Bispecific antibodies are widely used in the field of biomedicine, especially tumor immunotherapy. A bispecific antibody targeting CD3. One arm can bind to the CD3E subunit in the TCR receptor complex on the surface of T cells to provide the first signal to activate T cells, and the other arm targets tumor antigens. Bispecific antibodies can bring tumor cells and T cells closer together, and directly kill tumor cells while activating T cells.
  • 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 such 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 Fc segment retain the structure of traditional monoclonal antibodies 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,
  • 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 by Kabat.
  • the present application provides a bispecific antibody, which comprises an antigen-binding portion against human BCMA, and the antigen-binding portion against human BCMA comprises:
  • HCDR1 (heavy chain CDR1) shown in SEQ ID NO: 7,
  • HCDR2 (heavy chain CDR2) shown in SEQ ID NO: 8,
  • HCDR3 (heavy chain CDR3) as shown in SEQ ID NO: 9,
  • 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 by Kabat.
  • the present application provides a bispecific antibody, which comprises an antigen binding portion for human CD3E and an antigen binding portion for human BCMA.
  • the antigen binding portion for human CD3E comprises:
  • HCDR and LCDR are defined by Kabat.
  • the antigen binding portion for human BCMA comprises:
  • HCDR and LCDR are defined by Kabat.
  • the antigen binding portion for human CD3E and the antigen binding portion for human BCMA 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: 15 .
  • the bispecific antibody is an IgG1 antibody, which comprises a first heavy chain constant region and a second heavy chain constant region, wherein the 354th and The amino acids at position 366 are C and W, respectively, and the amino acids at positions 349, 366, 368, and 407 of the second heavy chain constant region 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, which comprises a first heavy chain constant region and a second heavy chain constant region, wherein the first and second heavy chain constant regions are
  • the amino acids at positions 234, 235 and 331 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 includes a heavy chain variable region as shown in SEQ ID NO: 12 and a light chain as shown in SEQ ID NO: 14 Variable region.
  • the antigen binding portion for human BCMA includes the heavy chain variable region shown in SEQ ID NO: 10 and the light chain variable region shown in SEQ ID NO: 14 Change area.
  • the antigen binding portion for human CD3E comprises a single chain antibody (scfv) or a Fab fragment.
  • the antigen binding portion against human BCMA 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 BCMA unit:
  • the first arm includes the heavy chain variable region amino acid sequence shown in SEQ ID NO: 12, the heavy chain constant region amino acid sequence shown in SEQ ID NO: 19, and the light chain shown in SEQ ID NO: 14
  • the second arm includes the heavy chain variable region amino acid sequence shown in SEQ ID NO: 10, the heavy chain constant region amino acid sequence shown in SEQ ID NO: 18, and the light chain shown in SEQ ID NO: 14 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: 20.
  • this application provides a pharmaceutical composition comprising the bispecific antibody described in any one of the first to third aspects.
  • 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 medicament for preventing or treating multiple myeloma .
  • the present application provides a method for preventing or treating multiple myeloma, comprising 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 the ELISA method to detect the simultaneous binding of the bispecific antibody CD3ExBCMA to CD3E and BCMA.
  • Figure 2 shows the results of using flow cytometry to analyze the binding of the bispecific antibody CD3ExBCMA to the surface of Jurkat human acute T lymphocyte leukemia cells.
  • Figure 3 shows the results of using flow cytometry to analyze the binding of the bispecific antibody CD3ExBCMA to BCMA on the surface of NCI-H929 human plasma cell leukemia cells.
  • Figure 4 shows that the bispecific antibody CD3ExBCMA mediates the specific activation of BCMA-positive tumor cells on jurkat-dual cells.
  • Figure 5 shows the results of the bispecific antibody CD3ExBCMA mediated the killing effect of PBMC on BCMA-positive tumor cells.
  • Figure 6 shows the results of the bispecific antibody CD3ExBCMA stimulated the expression of CD69 on the surface of T cells in the presence of BCMA positive tumor cells.
  • Figure 7 shows the results of changes in tumor volume of PBMC humanized NCI-H929 human myeloma model mice treated with the bispecific antibody CD3ExBCMA.
  • Figure 8 shows the changes in body weight of PBMC humanized NCI-H929 human myeloma model mice treated with the bispecific antibody CD3ExBCMA.
  • Figure 9 shows the changes in body weight of hCD34+ humanized RPMI-8226 human myeloma model mice treated with the bispecific antibody CD3ExBCMA.
  • Figure 10 shows the change of tumor volume in hCD34+ humanized RPMI-8226 human myeloma model mice treated with the bispecific antibody CD3ExBCMA.
  • SEQ ID NO: 1 is the amino acid sequence of HCDR1 of the heavy chain variable region H10B7 of the anti-human CD3E monoclonal antibody H10B7+L1G10.
  • SEQ ID NO: 2 is the amino acid sequence of HCDR2 of the heavy chain variable region H10B7 of the anti-human CD3E monoclonal antibody H10B7+L1G10.
  • SEQ ID NO: 3 is the amino acid sequence of HCDR3 of the heavy chain variable region H10B7 of the anti-human CD3E monoclonal antibody H10B7+L1G10.
  • SEQ ID NO: 4 is the amino acid sequence of LCDR1 of the light chain variable region L9B9.
  • SEQ ID NO: 5 is the amino acid sequence of LCDR2 of the light chain variable region L9B9.
  • SEQ ID NO: 6 is the amino acid sequence of LCDR3 of the light chain variable region L9B9.
  • SEQ ID NO: 7 is the amino acid sequence of HCDR1 of the heavy chain variable region mutant H13F1 of anti-human BCMA monoclonal antibody C4.
  • SEQ ID NO: 8 is the amino acid sequence of HCDR2 of the heavy chain variable region mutant H13F1 of the anti-human BCMA monoclonal antibody C4.
  • SEQ ID NO: 9 is the amino acid sequence of HCDR3 of the heavy chain variable region mutant H13F1 of anti-human BCMA monoclonal antibody C4.
  • SEQ ID NO: 10 is the amino acid sequence of the heavy chain variable region mutant H13F1 of the anti-human BCMA monoclonal antibody C4.
  • SEQ ID NO: 11 is the amino acid sequence of the heavy chain variable region C4VH of the anti-human BCMA monoclonal antibody C4.
  • SEQ ID NO: 12 is the amino acid sequence of the heavy chain variable region H10B7 of the anti-human CD3E monoclonal antibody H10B7+L1G10.
  • SEQ ID NO: 13 is the amino acid sequence of the light chain variable region L1G10 of the anti-human CD3E monoclonal antibody H10B7+L1G10.
  • SEQ ID NO: 14 is the amino acid sequence of the light chain variable region L9B9.
  • SEQ ID NO: 15 is the amino acid sequence of the hinge region.
  • SEQ ID NO: 16 is the amino acid sequence of the light chain variable region C4VK of the anti-human BCMA monoclonal antibody C4.
  • SEQ ID NO: 17 is the amino acid sequence of human IgG1 subtype antibody heavy chain constant region mutant IgG1K.
  • SEQ ID NO: 18 is the amino acid sequence of human IgG1 subtype antibody heavy chain constant region mutant IgG1m3-H.
  • SEQ ID NO: 19 is the amino acid sequence of human IgG1 subtype antibody heavy chain constant region mutant IgG1m3-K.
  • SEQ ID NO: 20 is the amino acid sequence of the constant region of the human kappa subtype light chain.
  • SEQ ID NO: 21 is the amino acid sequence of the human lambda subtype light chain constant region.
  • SEQ ID NO: 22 is the amino acid sequence of the extracellular region of human CD3E.
  • SEQ ID NO: 23 is the amino acid sequence of the extracellular region of human CD3D.
  • SEQ ID NO: 24 is the amino acid sequence of the extracellular region of monkey CD3E.
  • SEQ ID NO: 25 is the amino acid sequence of the extracellular region of monkey CD3D.
  • SEQ ID NO: 26 is the amino acid sequence of the extracellular region of mouse CD3E.
  • SEQ ID NO: 27 is the amino acid sequence of the extracellular region of mouse CD3D.
  • SEQ ID NO: 28 is the amino acid sequence of the extracellular domain of human BCMA.
  • SEQ ID NO: 29 is the amino acid sequence of the extracellular region of monkey BCMA.
  • SEQ ID NO: 30 is the amino acid sequence of the extracellular region of mouse BCMA.
  • SEQ ID NO: 31 is the amino acid sequence of the His tag.
  • SEQ ID NO: 32 is the amino acid sequence of the Fc segment (mFc) of the mouse antibody IgG2a.
  • SEQ ID NO: 33 is the amino acid sequence of the Fc mutant FcK of the heterodimer human IgG1 subtype.
  • SEQ ID NO: 34 is the amino acid sequence of the Fc mutant FcH of the heterodimer human IgG1 subtype.
  • SEQ ID NO: 35 is the amino acid sequence of the heavy chain constant region of a human IgG1 subtype antibody.
  • SEQ ID NO: 36 is the amino acid sequence of human IgG1 subtype antibody heavy chain constant region mutant IgG1H.
  • 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 refers to antibody fragments that are capable of binding to the antigen produced by treating a complete antibody with papain, including complete light chain (VL-CL) ), heavy chain variable region and CH1 fragment (VH-CH1).
  • 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.
  • 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 may 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. USA86: 9268-9272 (1989)).
  • the sequence of the CDR regions in the VH and VL sequences can be determined according to the Kabat definition or 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 various 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 homoisomers), 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 binding motif
  • 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.
  • BCMA B cell maturation antigen
  • TNF receptor superfamily As a non-glycosylated type III transmembrane protein receptor, BCMA consists of 184 amino acid residues. The intracellular region contains 80 amino acid residues, and the extracellular region has only one carbohydrate recognition domain. As a specific antigen on the surface of plasma cells, BCMA is involved in the maturation and differentiation of B cells, and as an essential substance in the long-term survival of plasma cells.
  • BCMA proliferation-inducing ligand
  • BAFF B cell activating factor
  • BCMA BCMA-derived neurotrophic factor
  • the expression of BCMA is selective. It is not expressed in naive B cells, memory B cells, CD34+ hematopoietic cells and other normal tissues. It is selectively induced and expressed in the differentiation process of plasma cells, mainly expressed in plasmacytoid dendritic cells and Bone marrow plasma cells. Multiple myeloma is a B-cell malignant tumor caused by malignant cell proliferation and canceration. It is mainly manifested as the uncontrolled expansion of plasma cells in the bone marrow, producing a large number of monoclonal immunoglobulins, leading to a series of symptoms such as bone destruction, elevated blood calcium, anemia, kidney damage, and immune decline.
  • BCMA B Cell Maturation Antigen
  • 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 by Kabat.
  • the present application provides a bispecific antibody, which comprises an antigen-binding portion against human BCMA, and the antigen-binding portion against human BCMA comprises:
  • HCDR and LCDR are defined by Kabat.
  • the present application provides a bispecific antibody, which comprises an antigen binding portion for human CD3E and an antigen binding portion for human BCMA.
  • the antigen binding portion for human CD3E comprises:
  • HCDR and LCDR are defined by Kabat.
  • the antigen binding portion for human BCMA comprises:
  • HCDR and LCDR are defined by Kabat.
  • the antigen binding portion for human CD3E and the antigen binding portion for human BCMA comprise the same light chain variable region.
  • the antigen binding portion for human CD3E and the antigen binding portion for human BCMA 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: 15, It replaces 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 positions 354 and 366 of the first heavy chain constant region
  • the amino acids of 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 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 used 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 another 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, which is incorporated in this document in its entirety by reference.
  • the bispecific antibody is an IgG1 antibody, which comprises a first heavy chain constant region and a second heavy chain constant region, wherein the 234th of the first and second heavy chain constant regions
  • the amino acids at positions, 235 and 331 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 SEQ ID NO: 12 (including HCDR1 as SEQ ID NO: 1 and HCDR2 as 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: 14 (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 BCMA comprises SEQ ID NO: 10 (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 SEQ ID NO: 9 (HCDR3) and the light chain variable region shown in SEQ ID NO: 14 (including the LCDR1 shown in SEQ ID NO: 4) LCDR2 shown in NO: 5 and LCDR3 shown in SEQ ID NO: 6).
  • SEQ ID NO: 10 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 SEQ ID NO: 9 (HCDR3) and the light chain variable region shown in SEQ ID NO: 14 (including the LCDR1 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 for human BCMA comprises a single chain antibody (scfv) or a Fab fragment.
  • the bispecific antibody has two different antigen binding parts for two different antigens, and the antigen binding part can contain two forms of single-chain antibody (scfv) or Fab fragments, when directed against two given antigens, the bispecific
  • 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 BCMA comprises a Fab fragment
  • the antigen binding portion for human CD3E comprises a Fab fragment
  • the antigen binding portion for human BCMA 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 BCMA comprises a Fab fragment.
  • the antigen binding portion for human CD3E comprises a single chain antibody (scfv)
  • the antigen binding portion for human BCMA 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 as 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 BCMA:
  • the first arm includes the heavy chain variable region amino acid sequence shown in SEQ ID NO: 12, the heavy chain constant region amino acid sequence shown in SEQ ID NO: 19, and the light chain shown in SEQ ID NO: 14
  • the second arm includes the heavy chain variable region amino acid sequence shown in SEQ ID NO: 10, the heavy chain constant region amino acid sequence shown in SEQ ID NO: 18, and the light chain shown in SEQ ID NO: 14 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: 20.
  • 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 human ⁇ subtype or human ⁇ subtype, preferably human ⁇ subtype.
  • this 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 multiple myeloma.
  • 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 the present application can be formulated into the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories, or capsules.
  • any physiologically acceptable mode of administration can 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 medicament for preventing or treating multiple myeloma .
  • the present application provides a method for preventing or treating multiple myeloma, comprising 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.
  • CD3ExBCMA bispecific antibodies require a variety of different recombinant proteins, including human CD3E extracellular domain (hCD3E, SEQ ID NO: 22), human CD3D extracellular domain (hCD3D, SEQ ID NO: 23) , Monkey CD3E extracellular region (mfCD3E, SEQ ID NO: 24), monkey CD3D extracellular region (mfCD3D, SEQ ID NO: 25), mouse CD3E extracellular region (mCD3E, SEQ ID NO: 26), mouse CD3D Extracellular region (mCD3D, SEQ ID NO: 27) and human BCMA extracellular region (hBCMA, SEQ ID NO: 28), monkey BCMA extracellular region (mfBCMA, SEQ ID NO: 29), mouse BCMA extracellular region ( mBCMA, SEQ ID NO: 30).
  • human CD3E extracellular domain hCD3E, SEQ ID NO: 22
  • human CD3D extracellular domain hCD3D, SEQ ID NO: 23
  • 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 have a His tag (SEQ ID NO: 31) added to the C-terminus, or the Fc segment of mouse antibody IgG2a (mFc, SEQ ID NO: 32), or based on KIH (Knob- Into-Hole technology forms a heterodimer Fc mutant of human IgG1 subtype (FcK, SEQ ID NO: 33 or FcH, SEQ ID NO: 34).
  • the heavy chain constant region of the antibody can be human IgG1 subtype (SEQ ID NO: 35) or various mutants of selected human IgG1 subtype, such as: IgG1H (SEQ ID NO: 36), IgG1K (SEQ ID NO: 17), IgG1m3-H (SEQ ID NO: 18) or IgG1m3-K (SEQ ID NO: 19), the light chain constant region is human ⁇ subtype (SEQ ID NO: 20) or human ⁇ subtype Type (SEQ ID NO: 21)
  • 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 is purified by a metal chelating affinity chromatography column (such as HisTrap FF of GE Company, etc.) in the culture supernatant.
  • the recombinant protein and recombinant antibody expressed by mFc fusion are purified in one step using ProteinA/G affinity chromatography columns (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.
  • H10B7+L1G10 is a monoclonal antibody that binds to human CD3E screened by human antibody library technology.
  • the amino acid sequence of the heavy chain variable region H10B7 of H10B7+L1G10 is SEQ ID NO: 12, and the amino acid sequence of the light chain variable region L1G10 is SEQ ID NO: 13 (see the amino acid sequence shown in SEQ ID NO: 19 and SEQ ID NO: 20 in Chinese Patent Application No. 201910372193.6).
  • C4 is a monoclonal antibody targeting the tumor antigen BCMA.
  • the amino acid sequence of the heavy chain variable region C4VH is SEQ ID NO: 11, and the light chain variable region C4VK amino acid sequence is SEQ ID NO: 16 (see the monoclonal antibody CA8- in US9273141B2 J7M0 sequence).
  • the light chain replacement strategy is adopted (for the experimental technology flow, please refer to Example 4 in Chinese Patent Application No. 201510097117.0) to use the heavy chain variable region of the anti-CD3E monoclonal antibody H10B7+L1G10
  • CD3E/CD3D was used as the screening antigen, and the original light chain library was screened and enriched for 2 rounds.
  • BCMA was used as the antigen.
  • the CD3D-enriched light chain library was subjected to two rounds of screening and enrichment, and finally the obtained light chain was identified, and a common light chain variable region L9B9 (SEQ ID NO:) that can maintain the activities of anti-CD3E antibodies and anti-BCMA antibodies 14).
  • the heavy chain variable region H10B7, C4 heavy chain variable region C4VH and light chain variable region L9B9 were cloned into the eukaryotic fusion of human IgG1 heavy chain constant region and kappa light chain constant region, respectively Expression vector, combined to express full antibodies H10B7+L9B9 and C4VH+L9B9.
  • Biacore X100 was used to determine the affinity of anti-BCMA antibodies (C4 and C4VH+L9B9) 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.
  • 1M ethanolamine was injected to block unreacted groups.
  • set a series of concentration gradients for hBCMA-his for example, 0.617nM, 1.85nM, 5.56nM, 16.7nM, 50nM
  • the binding time is 120s.
  • the separation time is 1800s, and the surface of the chip is regenerated by injecting 3M MgCl 2 solution for 30s at 10 ⁇ L/min.
  • the constructed C4VH-CDR3 mutant library was screened and enriched for a total of 3 rounds by solid phase screening using hBCMA-His antigen.
  • a heavy chain variable region mutant H13F1 (SEQ ID NO: 10) with increased affinity was obtained.
  • C4 heavy chain variable region mutant H13F1 and light chain variable region L9B9 were cloned into nucleosides encoding human heavy chain constant region and light chain constant region, respectively Eukaryotic expression vector of acid sequence, combined to express whole antibody.
  • affinity analysis of C4 mutants was performed using Biacore X100, and the results are shown in Table 3.
  • nucleotide sequences encoding the heavy chain variable region H10B7 of the anti-CD3E monoclonal antibody and the heavy chain variable region H13F1 of the anti-BCMA monoclonal antibody were cloned into a suitable eukaryotic expression vector to construct a heterologous based on the common light chain. Dimer.
  • the nucleotide sequence encoding the heavy chain variable region of the anti-CD3E antibody is cloned into the eukaryotic expression vector fused with the nucleotide sequence encoding the Knob mutation IgG1 constant region IgG1m3-K, and the heavy chain encoding the anti-BCMA antibody can be
  • the nucleotide sequence of the variable region is cloned into a eukaryotic expression vector containing the nucleotide sequence of the IgG1 constant region IgG1m3-H encoding the Hole mutation, and the nucleotide sequence of the variable region VK of the common light chain L9B9 is cloned to the fusion
  • the constructed three eukaryotic expression vectors expressing H10B7-IgG1m3-K, expressing H13F1-IgG1m3-H and expressing L9B9VK-CK were co-transfected into HEK293F cells using liposomes and cultured under serum-free suspension culture conditions for 3-5 Day, 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 Mabselect SURE from GE), and then a desalting column (such as Hitrap desaulting from GE) is used to replace the recombinant protein storage buffer with PBS (pH7.0) or other suitable buffers.
  • 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 anti-BCMA monoclonal antibody H13F1+L9B9, anti-CD3E monoclonal antibody H10B7+L9B9 and CD3ExBCMA bispecific antibodies were used for affinity determination by surface plasmon resonance technology using Biacore X100.
  • anti-CD3E monoclonal antibody When determining the affinity of the anti-CD3E monoclonal antibody and the CD3ExBCMA bispecific antibody to the CD3E antigen, couple the anti-human Fab antibody (human Fab capture kit, GE, 28-9583-25) to the surface of the CM5 chip, and dilute the antibody protein to 0.5-1 ⁇ g/mL, 10 ⁇ L/min injection, anti-CD3E monoclonal antibody ensures that about 70RU of antibody is captured by anti-human Fab antibody, and CD3xBCMA bispecific antibody ensures that about 150RU of antibody is captured by anti-human Fab antibody.
  • anti-human Fab antibody human Fab capture kit, GE, 28-9583-25
  • the conventional ELISA method was used to detect the ability of the bispecific antibody CD3ExBCMA (CD3ExBCMA BsAb) to simultaneously bind CD3E and BCMA.
  • a 96-well ELISA plate (3 ⁇ g/mL, 100 ⁇ L/well) was coated with CD3E-FcK/CD3D-FcH antigen, and coated overnight in a refrigerator at 4°C. After blocking with blocking solution PBS-0.1% Tween20-3% milk at 37°C for 1 hour, add anti-BCMA monoclonal antibody H13F1+L9B9, anti-CD3E monoclonal antibody H10B7+L9B9 and CD3ExBCMA bispecific antibody (10 ⁇ g/mL, 100 ⁇ L/well), each with two duplicate wells, incubate at 37°C for 1 hour.
  • NCI-H929 human plasma cell leukemia cells (high expression of BCMA, purchased from Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences) in logarithmic growth phase were collected. After centrifugation, resuspend in 1640 medium to 4 ⁇ 10 5 cells/mL, and spread 50 ⁇ L/well in the cell plate. Collect Jurkat-dual cells (purchased from Invivogen) in the logarithmic phase, centrifuge and resuspend to 1 ⁇ 10 6 cells/mL with 1640 medium, and add 100 ⁇ L/well to the cell plate to obtain a final E:T of 5:1 proportion.
  • the bispecific antibody CD3ExBCMA 50 ⁇ L/well
  • an initial concentration of 10 ⁇ g/mL, 3-fold dilution, 10 concentration points or an initial concentration of 5 ⁇ g/mL, 3-fold dilution, 10 concentration points
  • Anti-CD3E monoclonal antibody H10B7+L9B9 50 ⁇ L/well
  • a combination of anti-CD3E monoclonal antibody H10B7+L9B9 and anti-BCMA monoclonal antibody H13F1+L9B9 the initial concentration of monoclonal antibodies is 5 ⁇ g/mL, 3-fold gradient Dilution, 10 concentration points.
  • PBMC peripheral blood mononuclear cells
  • the blood of normal volunteers (50 mL each) was collected.
  • the blood collected was provided by the inventor and his colleagues as volunteers. All volunteers had signed an informed consent form.
  • the inclusion criteria for volunteers are:
  • Ficoll density gradient centrifugation was used to separate PBMC from the whole blood of volunteers and cultured in 1640 medium.
  • NCI-H929 human plasma cell leukemia cells high BCMA expression
  • RPMI-8226 human multiple myeloma cells moderate BCMA expression
  • HL60 human acute promyelocytic leukemia cells BCMA negative
  • the cells in the logarithmic growth phase were collected by centrifugation and resuspended in 1640 medium to 4 ⁇ 10 5 cells/mL, and 50 ⁇ L/well was plated in the cell plate.
  • Non-Radioactive Cytotoxicity Assay detects and analyzes the killing rate of T cells to tumor cells mediated by bispecific antibodies.
  • the results showed that in the presence of the bispecific antibody CD3ExBCMA, the effector cells had a significant killing effect on the highly expressed NCI-H929 cells and the moderately expressed RPMI-8226 cells, but had no killing effect on the negative HL60 ( Figure 5). It shows that the bispecific antibody CD3ExBCMA can effectively mediate the killing of T cells to cells with different expression levels of BCMA, but cannot mediate the killing of BCMA-negative cells.
  • NCI-H929 human plasma cell leukemia cells high BCMA expression
  • RPMI-8226 human multiple myeloma cells moderate BCMA expression
  • HL60 human acute promyelocytic leukemia cells BCMA negative
  • the blood of normal volunteers (50 mL each) was collected.
  • the blood collected was provided by the inventor and his colleagues as volunteers. All volunteers had signed an informed consent form.
  • the inclusion criteria for volunteers are:
  • PBMC peripheral blood mononuclear cells
  • the test is divided into bispecific antibody CD3ExBCMA 0.1mg/kg group (group 2), bispecific antibody CD3ExBCMA 0.02mg/kg group (group 3) and vehicle control
  • group (group 1) consists of 3 groups, each with 6 animals, and they are given by tail vein injection, once.
  • the dose of the second group was changed to 0.5 mg/kg
  • the dose of the third group was changed to 0.1 mg/kg
  • the dose was administered three more times at the frequency of twice a week.
  • TGI relative tumor inhibition rate
  • the efficacy is evaluated, and the safety is evaluated based on the change of animal weight and death.
  • the bispecific antibody CD3ExBCMA has a significant inhibitory effect on tumor growth at a dose of 0.1/0.5mg/kg, with a relative tumor inhibition rate TGI (%) of 95%, p ⁇ 0.001 (Figure 7).
  • mice 20 20-24 week old female hCD34+ humanized mice (purchased from Pengli Biotech), mix 100 ⁇ L 1x10 7 RPMI8226 cells and 100 ⁇ L Matrigel and inoculate the mice on the right side of the back by subcutaneous injection. Use 3 -4% isoflurane anesthetize the mice.
  • the tumor grew to an average of about 50-80 mm 3
  • 16 tumor-bearing mice were randomly divided into 2 groups according to the ratio of hCD34+ in the peripheral blood and the tumor volume, with 8 mice in each group. The day of group administration is defined as day 0.
  • the test is divided into two groups, the bispecific antibody CD3ExBCMA 0.01mg/kg administration group and the negative control group (IgG1m3, 0.01mg/kg), each group has 8 animals, which are given by tail vein injection, and given at the frequency of once a week. Medicine 4 times. According to the relative tumor inhibition rate (TGI), the efficacy is evaluated, and the safety is evaluated based on the change of animal weight and death.
  • TGI tumor inhibition rate

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Abstract

双特异性抗体，其包含针对人CD3E的抗原结合部和/或人BCMA的抗原结合部。此外，提供所述双特异性抗体的医学和生物学用途。

Description

抗CD3E和BCMA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2019年6月19日递交的中国专利申请第201910532734.7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BCMA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，提供激活T细胞的第一信号，另一条臂靶向肿瘤抗原。双特异性抗体可以将肿瘤细胞和T细胞拉近，激活T细胞的同时直接杀伤肿瘤细胞。
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BCMA的抗原结合部，所述针对人BCMA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BCMA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BCMA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BCMA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，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:15所示。
在第三方面的一些实施方案中，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；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，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:12所示的重链可变区和如SEQ ID NO:14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BCMA的抗原结合部包含SEQ ID NO:10所示的重链可变区和如SEQ ID NO:14所示的轻链可变区。
在上述任一方面的一些实施方案中，其中所述针对人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，其中所述针对人BCMA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，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BCMA的抗原结合部：
所述第一臂包含如SEQ ID NO:12所示的重链可变区氨基酸序列、如SEQ ID NO:19所示的重链恒定区氨基酸序列、如SEQ ID NO:14所示的轻链可变区氨基酸序列和如SEQ ID NO:20所示的轻链恒定区氨基酸序列；
所述第二臂包含如SEQ ID NO:10所示的重链可变区氨基酸序列、如SEQ ID NO:18所示的重链恒定区氨基酸序列、如SEQ ID NO:14所示的轻链可变区氨基酸序列和如SEQ ID NO:20所示的轻链恒定区氨基酸序列。
第四方面，本申请提供药物组合物，其包含第一至第三方面中任一方面所述的双特异性抗体。
第五方面，本申请提供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多发性骨髓瘤的药物中的用途。
第六方面，本申请提供了预防或治疗多发性骨髓瘤的方法，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ELISA方法检测双特异性抗体CD3ExBCMA同时结合CD3E和BCMA两种抗原的结果。
图2显示了利用流式细胞术分析双特异性抗体CD3ExBCMA与Jurkat人急性T淋巴细胞白血病细胞表面CD3E结合的结果。
图3显示了利用流式细胞术分析双特异性抗体CD3ExBCMA与NCI-H929人浆细胞白血病细胞表面BCMA结合的结果。
图4显示了双特异性抗体CD3ExBCMA介导BCMA阳性肿瘤细胞对jurkat-dual细胞的特异性激活。
图5显示了双特异性抗体CD3ExBCMA介导PBMC对BCMA阳性肿瘤细胞的杀伤作用结果。
图6显示了双特异性抗体CD3ExBCMA在BCMA阳性肿瘤细胞存在时刺激T细胞表面CD69表达的结果。
图7显示了双特异性抗体CD3ExBCMA治疗的PBMC人源化NCI-H929人源骨髓瘤模型小鼠肿瘤体积的变化结果。
图8显示了双特异性抗体CD3ExBCMA治疗的PBMC人源化NCI-H929人源骨髓瘤模型小鼠体重的变化。
图9显示了双特异性抗体CD3ExBCMA治疗的hCD34+人源化RPMI-8226人源骨髓瘤模型小鼠体重的变化。
图10显示了双特异性抗体CD3ExBCMA治疗的hCD34+人源化RPMI-8226人源骨髓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H10B7+L1G10的重链可变区H10B7的HCDR1的氨基酸序列。
SEQ ID NO:2为抗人CD3E单克隆抗体H10B7+L1G10的重链可变区H10B7的HCDR2的氨基酸序列。
SEQ ID NO:3为抗人CD3E单克隆抗体H10B7+L1G10的重链可变区H10B7的HCDR3的氨基酸序列。
SEQ ID NO:4为轻链可变区L9B9的LCDR1的氨基酸序列。
SEQ ID NO:5为轻链可变区L9B9的LCDR2的氨基酸序列。
SEQ ID NO:6为轻链可变区L9B9的LCDR3的氨基酸序列。
SEQ ID NO:7为抗人BCMA单克隆抗体C4的重链可变区突变体 H13F1的HCDR1的氨基酸序列。
SEQ ID NO:8为抗人BCMA单克隆抗体C4的重链可变区突变体H13F1的HCDR2的氨基酸序列。
SEQ ID NO:9为抗人BCMA单克隆抗体C4的重链可变区突变体H13F1的HCDR3的氨基酸序列。
SEQ ID NO:10为抗人BCMA单克隆抗体C4的重链可变区突变体H13F1的氨基酸序列。
SEQ ID NO:11为抗人BCMA单克隆抗体C4的重链可变区C4VH的氨基酸序列。
SEQ ID NO:12为抗人CD3E单克隆抗体H10B7+L1G10的重链可变区H10B7的氨基酸序列。
SEQ ID NO:13为抗人CD3E单克隆抗体H10B7+L1G10的轻链可变区L1G10的氨基酸序列。
SEQ ID NO:14为轻链可变区L9B9的氨基酸序列。
SEQ ID NO:15为铰链区的氨基酸序列。
SEQ ID NO:16为抗人BCMA单克隆抗体C4的轻链可变区C4VK的氨基酸序列。
SEQ ID NO:17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:18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:19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:20为人κ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:21为人λ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:22为人CD3E胞外区的氨基酸序列。
SEQ ID NO:23为人CD3D胞外区的氨基酸序列。
SEQ ID NO:24为猴CD3E胞外区的氨基酸序列。
SEQ ID NO:25为猴CD3D胞外区的氨基酸序列。
SEQ ID NO:26为小鼠CD3E胞外区的氨基酸序列。
SEQ ID NO:27为小鼠CD3D胞外区的氨基酸序列。
SEQ ID NO:28为人BCMA胞外区的氨基酸序列。
SEQ ID NO:29为猴BCMA胞外区的氨基酸序列。
SEQ ID NO:30为小鼠BCMA胞外区的氨基酸序列。
SEQ ID NO:31为His标签的氨基酸序列。
SEQ ID NO:32为小鼠抗体IgG2a的Fc段(mFc)的氨基酸序列。
SEQ ID NO:33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:34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:35为人IgG1亚型抗体重链恒定区的氨基酸序列。
SEQ ID NO:36为人IgG1亚型抗体重链恒定区突变体IgG1H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ζ四种链或γ、δ、ε、ζ和η(ζ和η为同源异构体)五种链组成，以CD3γε、CD3δε和CD3ζζ(或CD3ζη)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。
B细胞成熟抗原(B cell maturation antigen，BCMA)是TNF受体超家族的第17号成员，作为一种非糖基化的III型跨膜蛋白受体，BCMA由184个氨基酸残基组成，其胞内区含80个氨基酸残基，胞外区只有一个糖类识别结构域。BCMA作为一种浆细胞表面特异抗原，参与B细胞成熟分化，并作为必需物质参与浆细胞长期存活。BCMA、TACI和BAFFR分别和诱导增殖配体(APRIL)、B细胞激活因子(BAFF)两种配体结合，通过NFκB通路参与激活p38、Elk、c-Jun等信号转导分子，影响B细胞成熟、生长和存活。但BCMA对B细胞存活并不是关键的，有资料显示BCMA敲除后小鼠浆细胞的短期免疫球蛋白的产生、早期体液免疫反应及B淋巴细胞的发育等并不受影响(Christine M.Coquery,Loren D.Erickson.Regulatory Roles of the Tumor Necrosis Factor  Receptor BCMA.Crit Rev Immunol.2012；32(4):287–305)。BCMA的表达具有选择性，在幼稚B细胞、记忆B细胞、CD34+造血细胞及其他正常组织中均未表达，在浆细胞的分化过程中选择性诱导表达，主要表达于浆细胞样树突细胞和骨髓浆细胞上。多发性骨髓瘤是一种细胞恶性增殖、癌变引起的B细胞恶性肿瘤。其主要表现为浆细胞在骨髓中不受控制的扩增，产生大量的单克隆免疫球蛋白，导致骨破坏、血钙升高、贫血、肾损害、免疫下降等一系列症状。BCMA在骨髓瘤细胞上的表达量显著高于浆细胞及浆母细胞，从单克隆丙种球蛋白病到冒烟型骨髓瘤到多发性骨髓瘤，BCMA在整个浆细胞恶性疾病病程进程中都高度、广泛表达(Shih-Feng Cho,Kenneth C.Anderson,Yu-Tzu Tai.Targeting B Cell Maturation Antigen(BCMA)in Multiple Myeloma:Potential Uses of BCMA-Based immunotherapy.Front.Immunol.2018；9:1821)。
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BCMA的抗原结合部，所述针对人BCMA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BCMA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BCMA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BCMA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BCMA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，其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:15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，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，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，其中第一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:12(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:14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BCMA的抗原结合部包含SEQ ID NO:10(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:14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BCMA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BCMA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BCMA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BCMA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BCMA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，其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BCMA的抗原结合部：
所述第一臂包含如SEQ ID NO:12所示的重链可变区氨基酸序列、如SEQ ID NO:19所示的重链恒定区氨基酸序列、如SEQ ID NO:14所示的轻链可变区氨基酸序列和如SEQ ID NO:20所示的轻链恒定区氨基酸序列；
所述第二臂包含如SEQ ID NO:10所示的重链可变区氨基酸序列、如SEQ ID NO:18所示的重链恒定区氨基酸序列、如SEQ ID NO:14所示的轻链可变区氨基酸序列和如SEQ ID NO:20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，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，药物组合物用于预防或治疗多发性骨髓 瘤。
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多发性骨髓瘤的药物中的用途。
第六方面，本申请提供了预防或治疗多发性骨髓瘤的方法，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。
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CD3ExBCMA双特异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：22)、人CD3D胞外区(hCD3D，SEQ ID NO：23)、猴CD3E胞外区(mfCD3E， SEQ ID NO：24)、猴CD3D胞外区(mfCD3D，SEQ ID NO：25)、小鼠CD3E胞外区(mCD3E，SEQ ID NO：26)、小鼠CD3D胞外区(mCD3D，SEQ ID NO：27)以及人BCMA胞外区(hBCMA，SEQ ID NO：28)、猴BCMA胞外区(mfBCMA，SEQ ID NO：29)、小鼠BCMA胞外区(mBCMA，SEQ ID NO：30)。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系统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：31)，或者小鼠抗体IgG2a的Fc段(mFc，SEQ ID NO：32)，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：33或者FcH，SEQ ID NO：34)。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：35)或者选定的人IgG1亚型的各种突变体，如：IgG1H(SEQ ID NO：36)、IgG1K(SEQ ID NO：17)、IgG1m3-H(SEQ ID NO：18)或者IgG1m3-K(SEQ ID NO：19)，轻链恒定区是人κ亚型(SEQ ID NO：20)或人λ亚型(SEQ ID NO：21)
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 共同轻链的筛选及鉴定
2.1共同轻链的筛选
H10B7+L1G10为利用人抗体库技术筛选获得的结合人CD3E的单克隆抗体，H10B7+L1G10的重链可变区H10B7氨基酸序列为SEQ ID NO：12，轻链可变区L1G10氨基酸序列为SEQ ID NO：13(参见中国专利申请第201910372193.6号中SEQ ID NO：19和SEQ ID NO：20所示的氨基酸序列)。
C4为靶向肿瘤抗原BCMA的单克隆抗体，重链可变区C4VH氨基酸序列为SEQ ID NO：11，轻链可变区C4VK氨基酸序列为SEQ ID NO：16(参见US9273141B2中单克隆抗体CA8-J7M0的序列)。
单克隆抗体C4和H10B7+L1G10各自的抗体功能和性质已经过实验确认。
基于已建立的双载体噬菌体呈现系统，采用轻链置换的策略(实验技术流程可参见中国专利申请第201510097117.0号中的实施例4)，以抗CD3E单克隆抗体H10B7+L1G10的重链可变区H10B7为基础，以CD3E/CD3D作为筛选抗原，对原始轻链库进行2轮的筛选和富集；然后以抗BCMA单克隆抗体C4的重链为基础，用BCMA作为抗原，对上述经CD3E/CD3D富集后的轻链库进行2轮筛选和富集，最后对获得的轻链进行鉴定，得到能够同时保持抗CD3E抗体和抗BCMA抗体活性的共同轻链可变区L9B9(SEQ ID NO：14)。
利用常规的分子生物学手段，将重链可变区H10B7、C4的重链可变区C4VH及轻链可变区L9B9分别克隆至融合有人IgG1重链恒定区和κ轻链恒定区的真核表达载体，组合表达全抗体H10B7+L9B9和C4VH+L9B9。
2.2共同轻链BCMA抗体C4VH+L9B9结合人BCMA亲和力测定
利用Biacore X100通过表面等离子共振技术测定抗BCMA抗体(C4和C4VH+L9B9)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乙酸钠溶液稀释至25μg/mL，之后以流速10μL/min注射以实现大约多至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BCMA抗体至0.5-1μg/mL，10μL/min注射，保证400RU左右的抗体被抗人Fc的抗体捕获。然后将hBCMA-his设置一系列的浓度梯度(例如0.617nM、1.85nM、5.56nM、16.7nM、50nM)，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1800s，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版本，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(Kon)和解离速率(Koff)。以比率Koff/Kon计算解离平衡常数(KD)。拟合结果如表1所示。
表1.抗BCMA抗体结合人BCMA亲和力常数
  Kon Koff KD
C4 4.333E+5 7.948E-5 1.834E-10
C4VH+L9B9 3.238E+5 9.554E-4 2.950E-9
实施例3 BCMA抗体亲和力成熟
3.1 C4重链突变库的筛选
利用常规分子生物学手段，通过在重链可变区C4VH的CDR3区引入突变，构建基于重链可变区C4VH的CDR3突变库，设计的突变方案如表2所示，构建库容1.2×10E8，正确率为86.7％。
表2.基于C4重链可变区C4VH的CDR3突变库的突变方案
Figure PCTCN2019102817-appb-000001
Figure PCTCN2019102817-appb-000002
基于双载体噬菌体展示系统(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hBCMA-His抗原对构建的C4VH-CDR3突变库共进行3轮筛选富集，最终得到亲和力提高的重链可变区突变体H13F1(SEQ ID NO:10)。
3.2 C4重链突变体亲和力测定
利用常规的分子生物学手段，将编码C4重链可变区突变体H13F1和轻链可变区L9B9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的真核表达载体，组合表达全抗体。参照实施例2.2，利用Biacore X100对C4突变体(IgG1亚型)进行亲和力分析，结果见表3。
表3.抗BCMA抗体结合人BCMA亲和力常数
  Kon Koff KD
C4VH+L9B9 5.307E+5 8.156E-4 1.537E-9
H13F1+L9B9 7.85E+5 4.284E-4 5.429E-10
实施例4 双特异性抗体的制备
分别将编码抗CD3E单克隆抗体的重链可变区H10B7和抗BCMA单克隆抗体的重链可变区H13F1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。即将编码抗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，将编码抗BCMA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的核苷酸序列的真核表达载体，将编码共同轻链L9B9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的的核苷酸序列真核表达载体。
将构建的表达H10B7-IgG1m3-K、表达H13F1-IgG1m3-H和表达L9B9VK-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5 双特异性抗体的亲和力分析
参照实施例2.2，利用Biacore X100通过表面等离子共振技术对抗BCMA单克隆抗体H13F1+L9B9、抗CD3E单克隆抗体H10B7+L9B9和CD3ExBCMA双特异性抗体进行亲和力测定。
测定抗BCMA单克隆抗体和CD3ExBCMA双特异性抗体对BCMA抗原的亲和力时，将抗人IgG(Fc)抗体偶联至CM5芯片表面，稀释抗体蛋白至0.5-1μg/mL，10μL/min注射，BCMA单克隆抗体保证400RU左右的抗体被抗人Fc的抗体捕获，CD3ExBCMA双特异性抗体保证800RU左右的抗体被抗人Fc的抗体捕获。然后将BCMA-his设置一系列的浓度梯度(例如0.617nM、1.85nM、5.56nM、16.7nM、50nM)，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1800s，以10μL/min注射3M的MgCl 2溶液30s对芯片表面进行再生。亲和力拟合结果见表4和表5。
表4.抗BCMA单克隆抗体H13F1+L9B9和CD3xBCMA双特异性抗体结合人BCMA的亲和力常数
  Kon Koff KD
H13F1+L9B9 1.174E+6 4.161E-4 3.544E-10
CD3ExBCMA 1.063E+6 3.236E-4 3.045E-10
表5.抗BCMA单克隆抗体H13F1+L9B9和CD3xBCMA双特异性抗体结合猴BCMA的亲和力常数
  Kon Koff KD
H13F1+L9B9 6.63E+5 3.289E-3 4.961E-9
CD3ExBCMA 6.551E+5 3.26E-3 4.976E-9
测定抗CD3E单克隆抗体和CD3ExBCMA双特异性抗体对CD3E抗原的亲和力时，将抗人Fab抗体(人Fab捕获试剂盒，GE，28-9583-25)偶联至CM5芯片表面，稀释抗体蛋白至0.5-1μg/mL，10μL/min注射，抗CD3E单克隆抗体保证70RU左右的抗体被抗人Fab的抗体捕获，CD3xBCMA双特异性抗体保证150RU左右的抗体被抗人Fab的抗体捕获。将人CD3E异源二聚体CD3E-FcK/CD3D-FcH设置一系列的浓度梯度(例如12.5nM、25nM、50nM、100nM、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600s，以10μL/min注射10mM、pH 2.1的甘氨酸-HCl 60s对芯片表面进行再生。亲和力拟合结果如表6所示。
表6.抗CD3E单克隆抗体H10B7+L9B9和CD3ExBCMA双特异性抗体结合人CD3E亲和力常数
  Kon Koff KD
H10B7+L9B9 1.750E+5 3.075E-3 1.757E-8
CD3ExBCMA 1.179E+5 2.838E-3 2.408E-8
实施例6 双特异性抗体同时识别CD3E和BCMA两种抗原的能力鉴 定
利用常规ELISA方法检测双特异性抗体CD3ExBCMA(CD3ExBCMA BsAb)对CD3E和BCMA两种抗原同时结合的能力。
用CD3E-FcK/CD3D-FcH抗原包被96孔ELISA板(3μg/mL、100μL/孔)，4℃冰箱包被过夜。利用封闭液PBS-0.1％Tween20-3％牛奶在37℃封闭1小时后，分别加入抗BCMA单克隆抗体H13F1+L9B9、抗CD3E单克隆抗体H10B7+L9B9和CD3ExBCMA双特异性抗体(10μg/mL、100μL/孔)，各两个复孔，37℃孵育1小时。用PBS-0.1％Tween20洗涤ELISA板，然后加入BCMA-His抗原(1μg/mL、100μL/孔)，37℃孵育1小时。用PBS-0.1％Tween20洗涤ELISA板，然后加入HRP小鼠抗his IgG(康为世纪，cw0285M)，37℃孵育1小时。用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，利用酶标仪测定492nm/630nm双波长处的光密度值。ELISA分析结果如图1所示，CD3ExBCMA双特异性抗体能够同时识别CD3E和BCMA两种抗原。
实施例7 双特异性抗体识别细胞表面CD3E和BCMA的能力鉴定
取生长对数期的Jurkat人急性T淋巴细胞白血病细胞(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。取抗CD3E单克隆抗体H10B7+L9B9和双特异性抗体CD3ExBCMA进行梯度稀释，抗CD3E单克隆抗体3μg/mL，3倍梯度稀释，8个浓度点；双特异性抗体6μg/mL，3倍梯度稀释，8个浓度点。取100μL抗CD3E单克隆抗体或100μL双特异性抗体，加入含细胞的孔中，4℃孵育1小时。然后用200μL PBS溶液洗3遍，孵育羊抗人IgG-FITC(中杉金桥，ZF-0308)100μL每孔，4℃避光30分钟。然后用200μL PBS溶液洗3遍，100μL PBS溶液重悬后用流式细胞仪(ACEA，Novocyte)检测FITC通道。结果显示CD3ExBCMA双特异性抗体能很好地结合CD3阳性细胞Jurkat(图2)。双特异性抗体CD3ExBCMA的KD值为16.79nM，抗CD3E单抗的KD值为1.43nM。
取生长对数期的NCI-H929人浆细胞白血病细胞(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。取抗BCMA单克隆抗体H13F1+L9B9和双特异性抗体CD3ExBCMA梯度稀释，抗BCMA单克隆抗体10μg/mL，3倍梯度，8个浓度点；双特异性抗体20μg/mL，3倍梯度，8个浓度点。取100μL抗BCMA单克隆抗体或100μL双特异性抗体，加入含细胞的孔中，4℃孵育1小时。然后用200μL PBS溶液洗3遍，孵育羊抗人IgG-FITC(中杉金桥，ZF-0308)100μL每孔，4℃避光30分钟。后用200μL PBS溶液洗3遍，100μL PBS溶液重悬后用流式细胞仪(ACEA，Novocyte)检测FITC通道。结果显示双特异性抗体CD3ExBCMA能很好的结合BCMA阳性细胞NCI-H929(图3)。双特异性抗体CD3ExBCMA的KD值为8.27nM，抗BCMA单克隆抗体的KD值为8.47nM。
实施例8 双特异性抗体介导BCMA阳性肿瘤细胞对Jurkat-Dual细胞的特异性激活
收集生长对数期的NCI-H929人浆细胞白血病细胞(BCMA高表达，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1640培养基重悬至4×10 5个/mL，50μL/孔铺于细胞板中。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1×10 6个/mL，100μL/孔加入细胞板中以获得5：1的最终E:T比例。然后加入起始浓度为10μg/mL，3倍梯度稀释，10个浓度点的双特异性抗体CD3ExBCMA(50μL/孔)，或者起始浓度为5μg/mL，3倍梯度稀释，10个浓度点的抗CD3E单克隆抗体H10B7+L9B9(50μL/孔)，或者抗CD3E单克隆抗体H10B7+L9B9和抗BCMA单克隆抗体H13F1+L9B9的联合，单克隆抗体均为起始浓度5μg/mL，3倍梯度稀释，10个浓度点。孵育20小时后，取上清，参照QUANTI-Luc TM说明书(QUANTI-Luc TM，Invivogen，rep-qlc2)检测和分析CD3ExBCMA双特异性抗体、抗CD3E单克隆抗体、抗CD3E单克隆抗体和抗BCMA单克隆抗体联合，这3种不同条件下介导的BCMA阳性肿瘤细胞对 Jurkat-Dual细胞的特异性激活。结果显示，只有CD3ExBCMA双特异性抗体可以介导BCMA阳性肿瘤细胞激活Jurkat-Dual细胞，单独的抗CD3E单克隆抗体、或者抗CD3E单克隆抗体和抗BCMA单克隆抗体联合的时候，都不能介导BCMA阳性肿瘤细胞激活Jurkat-Dual细胞(图4)。
实施例9 双特异性抗体介导T细胞对BCMA阳性肿瘤细胞的杀伤
9.1人外周血单核细胞(PBMC)的分离
采集正常志愿者的血液(各50mL)，其中所采集的血液由发明人及其同事作为志愿者提供，所有志愿者均已签署知情同意书。志愿者的纳入标准为：
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中分离得到PBMC，并培养于1640培养基中。
9.2双特异性抗体介导PBMC杀伤BCMA阳性肿瘤细胞的检测
NCI-H929人浆细胞白血病细胞(BCMA高表达)、RPMI-8226人多发性骨髓瘤细胞(BCMA中度表达)、HL60人急性早幼粒白血病细胞(BCMA阴性)均购自中国医学科学院基础医学研究所细胞资源中心。收集生长对数期的细胞离心后用1640培养基重悬至4×10 5个/mL，50μL/孔铺于细胞板中。然后加入起始浓度为1μg/mL，4倍梯度稀释，10个浓度点的双特异性抗体CD3ExBCMA，50μL每孔，最后加入100μL/孔的PBMC(效应物)以获得5:1的最终E:T比例。同时设置单独靶细胞对照(NCI-H929细胞、RPMI-8226细胞或者HL60细胞)、单独效应细胞对照(PBMC)、单独培养基空白对照，并用培养基将体积补齐至200μL。孵育20小时后，取上清，参照
Figure PCTCN2019102817-appb-000003
非放射性细胞毒性检测试剂说明书(
Figure PCTCN2019102817-appb-000004
Non-Radioactive Cytotoxicity  Assay，promega，G1780)检测和分析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，在双特异性抗体CD3ExBCMA存在时效应细胞对高表达的NCI-H929细胞和中度表达的RPMI-8226细胞都有明显的杀伤，对阴性的HL60没有表现出杀伤作用(图5)，说明双特异性抗体CD3ExBCMA可以有效介导T细胞对BCMA阳性表达量不同细胞的杀伤，不能介导对BCMA阴性细胞的杀伤。
实施例10 双特异性抗体特异刺激T细胞表面活化分子表达
收集生长对数期的NCI-H929人浆细胞白血病细胞(BCMA高表达)、RPMI-8226人多发性骨髓瘤细胞(BCMA中度表达)、HL60人急性早幼粒白血病细胞(BCMA阴性)。离心后用1640培养基重悬至4×10 5个/mL，50μL/孔铺于细胞板中。然后加入起始浓度为1μg/mL，4倍梯度稀释，10个浓度点的双特异性抗体CD3ExBCMA，50μL/孔，最后加入100μL/孔的PBMC(效应物)以获得5:1的最终E:T比例。孵育20小时后，350g离心5分钟并用PBS洗1遍，用抗人CD3(ebioscience,17-0037-42)和抗人CD69(ebioscience,11-0069-42)流式抗体4℃避光孵育30分钟，然后用200μL PBS溶液洗2遍，100μL PBS溶液重悬后上流式(ACEA，Novocyte)检测，比较用双特异性抗体CD3ExBCMA处理后CD3阳性细胞群中活化标志物CD69的表达差异。结果显示，双特异性抗体CD3ExBCMA在高表达BCMA的NCI-H929或中度表达BCMA的RPMI-8226存在的条件下对T细胞有特异性活化，双特异性抗体CD3ExBCMA在阴性的HL60存在条件下不能活化T细胞(图6)，说明双特异性抗体CD3ExBCMA在BCMA阳性表达量不同的细胞存在条件下可以有效活化T细胞，在BCMA阴性细胞存在的条件下无法活化T细胞。
实施例11 双特异性抗体在PBMC免疫重建模型小鼠体内的抑瘤活性
采集正常志愿者的血液(各50mL)，其中所采集的血液由发明人及其同事作为志愿者提供，所有志愿者均已签署知情同意书。志愿者 的纳入标准为：
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。
使用Ficoll密度梯度离心法从健康人外周血中分离人外周血单个核细胞(PBMC)。选取25只7-8周龄雌性NPG小鼠(北京维通达生物技术有限公司)，在NPG小鼠右侧皮下接种5×10 6个NCI-H929细胞，接种当天定义为第0天。在肿瘤细胞接种2h后，每只小鼠腹腔接种5×10 6个人PBMC。待肿瘤平均体积95mm 3时，根据肿瘤大小随机分组，试验分为双特异性抗体CD3ExBCMA 0.1mg/kg组(第2组)、双特异抗体CD3ExBCMA 0.02mg/kg组(第3组)及溶媒对照组(第1组)共3组，每组6只，尾静脉注射给药，给药一次。第一次给药4天后，第2组剂量更改为0.5mg/kg，第3组剂量更改为0.1mg/kg，按每周两次给药频率再给药三次。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。
结果显示双特异性抗体CD3ExBCMA在0.1/0.5mg/kg剂量下，具有显著抑制肿瘤生长的作用，相对肿瘤抑制率TGI(％)为95％，p<0.001(图7)。同时双特异性抗体CD3ExBCMA在0.02/0.1mg/kg和0.1/0.5mg/kg剂量下，各治疗组均无动物死亡，没有表现明显的药物毒性，治疗期间耐受良好(图8)。
实施例12 双特异性抗体在hCD34+人源化模型小鼠体内的抑瘤活性
选取20只20-24周雌性hCD34+人源化小鼠(购自澎立生物)，将100μL 1ⅹ10 7个RPMI8226细胞和100μL 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，16只荷瘤小鼠根据外周血中hCD34+的比例和肿瘤体积被随机分成2组，每组8只。分组给药当天定义为第0天。试验分为双特异性抗体CD3ExBCMA 0.01mg/kg给药组及阴性对照组(IgG1m3，0.01mg/kg)2组，每组8只，尾静脉注射 给药，按每周一次给药频率共给药4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34天)，和阴性对照组(IgG1m3i.v.0.01mg/kg剂量组，G1组)相比，给药组(G2组)动物体重无显著性差异(P>0.05)，各组动物在各个时间点体重变化趋势见图9。双特异性抗体CD3ExBCMA在0.01mg/kg剂量下，具有显著抑制肿瘤生长的作用，相对肿瘤抑制率TGI(％)为61.17％。肿瘤生长情况见图10。
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 (12)

  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BCMA的抗原结合部，所述针对人BCMA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BCMA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BCMA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BCMA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:15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:12所示的重链可变区和如SEQ ID NO:14所示的轻链可变区；和/或
    所述针对人BCMA的抗原结合部包含SEQ ID NO:10所示的重链可变区和如SEQ ID NO:14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BCMA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BCMA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BCMA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BCMA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BCMA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BCMA的抗原结合部：
    所述第一臂包含如SEQ ID NO:12所示的重链可变区氨基酸序列、如SEQ ID NO:19所示的重链恒定区氨基酸序列、如SEQ ID NO:14所示的轻链可变区氨基酸序列和如SEQ ID NO:20所示的轻链恒定区氨基酸序列；
    所述第二臂包含如SEQ ID NO:10所示的重链可变区氨基酸序列、如SEQ ID NO:18所示的重链恒定区氨基酸序列、如SEQ ID NO:14所示的轻链可变区氨基酸序列和如SEQ ID NO:20所示的轻链恒定区氨基酸序列。
  9. 药物组合物，其包含权利要求1-8中任一项所述的双特异性抗体。
  10. 如权利要求9所述的药物组合物，其用于预防或治疗多发性骨髓瘤。
  11. 权利要求1-8中任一项所述的双特异性抗体或者权利要求9或10所述的药物组合物在制备用于预防或治疗多发性骨髓瘤的药物中的用途。
  12. 预防或治疗多发性骨髓瘤的方法，其包括向有需要的个体给予权利要求1-8中任一项所述的双特异性抗体，或权利要求9或10所述的药物组合物。
PCT/CN2019/102817 2019-06-19 2019-08-27 抗cd3e和bcma双特异性抗体及其用途 Ceased WO2020252907A1 (zh)

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