WO2022247933A1 - ANTI-SIRPα ANTIBODY AND USE THEREOF - Google Patents

ANTI-SIRPα ANTIBODY AND USE THEREOF Download PDF

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WO2022247933A1
WO2022247933A1 PCT/CN2022/095634 CN2022095634W WO2022247933A1 WO 2022247933 A1 WO2022247933 A1 WO 2022247933A1 CN 2022095634 W CN2022095634 W CN 2022095634W WO 2022247933 A1 WO2022247933 A1 WO 2022247933A1
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seq
antibody
antigen
binding fragment
amino acid
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PCT/CN2022/095634
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French (fr)
Chinese (zh)
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陈振埕
李闯
郑丹丹
陈俊有
汪志炜
张慧
黄贤明
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百奥泰生物制药股份有限公司
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Priority to CN202280035718.7A priority Critical patent/CN117355539A/en
Publication of WO2022247933A1 publication Critical patent/WO2022247933A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to an anti-SIRP ⁇ antibody and its application.
  • SIRP ⁇ There are five proteins in the SIRP family, namely SIRP ⁇ , SIRP ⁇ 1, SIRP ⁇ , SIRP ⁇ 2 and SIRP ⁇ , and only SIRP ⁇ and SIRP ⁇ can bind to the ligand CD47.
  • the intracellular domain of SIRP ⁇ lacks a signaling motif associated with a phosphatase that associates with DNAX activator protein 12 (DAP12), a dimeric adapter protein with a basic side in the transmembrane domain that binds SIRP ⁇
  • DAP12 DNAX activator protein 12
  • ITAM tyrosine activation motif
  • SIRP ⁇ does not bind to DAP12, is mainly expressed on the surface of T cells, interacts with CD47, and participates in the activation of T cells.
  • SIRP ⁇ also known as signal regulatory protein ⁇ (SIRP ⁇ ), CD172a or SHPS-1
  • SIRP ⁇ signal regulatory protein ⁇
  • CD172a CD172a
  • SHPS-1 signal regulatory protein ⁇
  • SIRP ⁇ is a single transmembrane protein.
  • the extracellular region of SIRP ⁇ contains an IgV domain and two IgC domains (IgC1, IgC2).
  • IgC1, IgC2 The region where SIRP ⁇ binds to the ligand CD47 is an IgV domain;
  • ITIM tyrosine Kinase inhibitory motif
  • Human SIRP ⁇ (hSIRP ⁇ ) gene has 8 variants: V1, V2, V3, V4, V5, V6, V8 and V9, of which V1, V2 and V8 account for more than 90%. There is a 13 amino acid difference in the extracellular IgV domain of hSIRP ⁇ between V1, V2 and V8 types.
  • CD47 on the surface of the target cell binds to the inhibitory receptor SIRP ⁇ , which promotes the phosphorylation of the intracellular domain of SIRP ⁇ , recruits and activates the protein tyrosine phosphatases SHP-1 and SHP-2, and finally leads to the inhibition of the function of myosin IIA, resulting in " Don't eat me” signal.
  • SIRP ⁇ inhibitory receptor
  • SHP-1 and SHP-2 protein tyrosine phosphatases
  • myosin IIA resulting in " Don't eat me” signal.
  • the present invention provides anti-SIRP ⁇ antibodies or antigen-binding fragments. These antibodies or antigen-binding fragments can specifically bind SIRP ⁇ , block SIRP ⁇ /CD47 signaling pathway, and contribute to the immune system's clearance function.
  • Some embodiments provide antibodies or antigen-binding fragments that specifically bind SIRP ⁇ , SIRP ⁇ , and SIRP ⁇ while blocking the SIRP ⁇ /CD47 signaling pathway.
  • the antibody or antigen-binding fragment binds human SIRP ⁇ , monkey SIRP ⁇ , and mouse SIRP ⁇ . In some embodiments, the antibody or antigen-binding fragment binds human SIRP ⁇ . In some embodiments, the human SIRP ⁇ includes V1 type SIRP ⁇ , V2 type SIRP ⁇ , and V8 type SIRP ⁇ .
  • Some embodiments provide antibodies or antigen-binding fragments that bind SIRP ⁇ without affecting T cell proliferation.
  • Some embodiments provide an antibody or antigen-binding fragment that specifically binds SIRP ⁇ and comprising one or more of the following amino acid sequences:
  • HCDR1 comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 1;
  • HCDR2 comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 2;
  • HCDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 3-8 or an amino acid having a single site substitution, deletion or insertion compared to any one of SEQ ID NOs: 3-8 sequence, or consisting of it;
  • LCDR1 comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 9;
  • LCDR2 comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 10;
  • LCDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 11-15 or an amino acid having a single site substitution, deletion or insertion compared to any one of SEQ ID NOs: 11-15 sequence, or consists of it.
  • antibodies or antigen-binding fragments that specifically bind SIRP ⁇ and comprising:
  • HCDR1 comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 1;
  • HCDR2 comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 2;
  • HCDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 3-8 or an amino acid having a single site substitution, deletion or insertion compared to any one of SEQ ID NOs: 3-8 sequence, or consists of it.
  • HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1 or consists of it
  • HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2 or consists of it
  • HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3 An amino acid sequence or consisting of it.
  • HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1
  • HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 2
  • HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 4 An amino acid sequence or consisting of it.
  • HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1 or consists of it
  • HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2 or consists of it
  • HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 5 An amino acid sequence or consisting of it.
  • HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1 or consists of it
  • HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2 or consists of it
  • HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 6 An amino acid sequence or consisting of it.
  • HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1 or consists of it
  • HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2 or consists of it
  • HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 7 An amino acid sequence or consisting of it.
  • HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1 or consists of it
  • HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2 or consists of it
  • HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 8 An amino acid sequence or consisting of it.
  • Some embodiments provide an antibody or antigen-binding fragment that specifically binds SIRP ⁇ and comprising:
  • LCDR1 comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 9;
  • LCDR2 which comprises or consists of the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 10;
  • LCDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 11-15 or an amino acid having a single site substitution, deletion or insertion compared to any one of SEQ ID NOs: 11-15 sequence, or consists of it.
  • LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9 or consists of it
  • LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10 or consists of it
  • LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11 An amino acid sequence or consisting of it.
  • LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9 or consists of it
  • LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10 or consists of it
  • LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 12 An amino acid sequence or consisting of it.
  • LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9 or consists of it
  • LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10 or consists of it
  • LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 13 An amino acid sequence or consisting of it.
  • LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9 or consists of it
  • LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10 or consists of it
  • LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 14 An amino acid sequence or consisting of it.
  • LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9 or consists of it
  • LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10 or consists of it
  • LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 15 An amino acid sequence or consisting of it.
  • Some embodiments provide an antibody or antigen-binding fragment that specifically binds SIRP ⁇ and comprising:
  • HCDR1 comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 1;
  • HCDR2 comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 2;
  • HCDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 3-8 or an amino acid having a single site substitution, deletion or insertion compared to any one of SEQ ID NOs: 3-8 sequence, or consisting of it;
  • LCDR1 comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 9;
  • LCDR2 which comprises or consists of the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 10;
  • LCDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 11-15 or an amino acid having a single site substitution, deletion or insertion compared to any one of SEQ ID NOs: 11-15 sequence, or consists of it.
  • the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 3, HCDR3 as shown in SEQ ID NO: LCDR1 shown in NO: 9, LCDR2 shown in SEQ ID NO: 10 and LCDR3 shown in SEQ ID NO: 11.
  • the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 4, HCDR3 as shown in SEQ ID NO: LCDR1 shown in NO: 9, LCDR2 shown in SEQ ID NO: 10 and LCDR3 shown in SEQ ID NO: 11.
  • the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 5, HCDR3 as shown in SEQ ID NO: LCDR1 shown in NO: 9, LCDR2 shown in SEQ ID NO: 10 and LCDR3 shown in SEQ ID NO: 11.
  • the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 6, HCDR3 as shown in SEQ ID NO: LCDR1 shown in NO: 9, LCDR2 shown in SEQ ID NO: 10 and LCDR3 shown in SEQ ID NO: 11.
  • the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 7, HCDR3 as shown in SEQ ID NO: LCDR1 shown in NO: 9, LCDR2 shown in SEQ ID NO: 10 and LCDR3 shown in SEQ ID NO: 11.
  • the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 8, HCDR3 as shown in SEQ ID NO: LCDR1 shown in NO: 9, LCDR2 shown in SEQ ID NO: 10 and LCDR3 shown in SEQ ID NO: 11.
  • the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 6, HCDR3 as shown in SEQ ID NO: LCDR1 shown in NO: 9, LCDR2 shown in SEQ ID NO: 10 and LCDR3 shown in SEQ ID NO: 12.
  • the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 6, HCDR3 as shown in SEQ ID NO: LCDR1 shown in NO: 9, LCDR2 shown in SEQ ID NO: 10 and LCDR3 shown in SEQ ID NO: 13.
  • the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 6, HCDR3 as shown in SEQ ID NO: LCDR1 shown in NO: 9, LCDR2 shown in SEQ ID NO: 10 and LCDR3 shown in SEQ ID NO: 14.
  • the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 6, HCDR3 as shown in SEQ ID NO: LCDR1 shown in NO: 9, LCDR2 shown in SEQ ID NO: 10 and LCDR3 shown in SEQ ID NO: 15.
  • the substitutions are conservative amino acid substitutions.
  • Some embodiments provide an antibody or antigen-binding fragment that specifically binds SIRP ⁇ , the heavy chain variable region of the antibody or antigen-binding fragment comprising any one of SEQ ID NOs: 16-21
  • the sequence shown a sequence having at least 80% identity compared to the sequence shown in any one of SEQ ID NO: 16-21, or having one sequence compared to the sequence shown in any one of SEQ ID NO: 16-21 or an amino acid sequence with multiple conservative amino acid substitutions, or consists of it.
  • Some embodiments provide an antibody or antigen-binding fragment that specifically binds SIRP ⁇ , the light chain variable region of the antibody or antigen-binding fragment comprising any one of SEQ ID NOs: 22-26
  • the sequence shown a sequence having at least 80% identity compared to the sequence shown in any one of SEQ ID NO: 22-26, or having one sequence compared to the sequence shown in any one of SEQ ID NO: 22-26 or an amino acid sequence with multiple conservative amino acid substitutions, or consists of it.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 16 and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 22 sequence shown.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 17, and the light chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO: 22 sequence shown.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 18, and the light chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO: 22 sequence shown.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 19, and the light chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO: 22 sequence shown.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 20
  • the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 22 sequence shown.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 21, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 22 sequence shown.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 19, and the light chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO: 23 sequence shown.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 19, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 24 sequence shown.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 19, and the light chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO: 25 sequence shown.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 19, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 26 sequence shown.
  • the antibody or antigen-binding fragment further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.
  • the light chain constant region is a kappa or lambda chain constant region.
  • the antibody or antigen-binding fragment is an isotype of IgG, IgM, IgA, IgE, or IgD.
  • the isotype is IgGl, IgG2, IgG3 or IgG4.
  • the antibody or antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.
  • the Fc is a variant Fc region.
  • the variant Fc region has one or more amino acid modifications, such as substitutions, deletions or insertions, relative to the parental Fc region.
  • the amino acid modification of the Fc region alters effector function activity relative to the activity of the parental Fc region.
  • the variant Fc region may have altered (increased or decreased) antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization, or cell binding.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-mediated cytotoxicity
  • phagocytosis opsonization, or cell binding.
  • amino acid modifications of the Fc region can alter the affinity of the variant Fc region for Fc ⁇ R (Fc ⁇ receptor) relative to the parent Fc region.
  • the Fc region is derived from IgGl or IgG4.
  • the Fc region mutation is N297A, L234A, or L235A (Eu numbering).
  • the Fc region mutation is E345R or S440Y (Eu numbering).
  • the antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is a scFv, Fab or F(ab) 2 . In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody.
  • the heavy chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in any one of SEQ ID NOs: 27-29, or any of SEQ ID NOs: 27-29 or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 27-29, or consisting of a sequence having at least 80% identity compared to the sequence shown in Item 1; and / or
  • the light chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 30, or a sequence with at least 80% identity compared with the sequence shown in SEQ ID NO: 30, or a sequence with SEQ ID NO: 30.
  • the amino acid sequence shown in ID NO: 30 has one or more conservative amino acid substitutions compared to the sequence, or consists of it.
  • the constant region of the antibody or antigen-binding fragment is derived from IgG1.
  • the heavy chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 27, and the light chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: the sequence shown in 30.
  • the constant region of the antibody or antigen-binding fragment is derived from IgG2.
  • the heavy chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 28, and the light chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: the sequence shown in 30.
  • the constant region of the antibody or antigen-binding fragment is derived from IgG4.
  • the heavy chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 29
  • the light chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: the sequence shown in 30.
  • the heavy chain of the antibody comprises an amino acid sequence as shown in any one of SEQ ID NO: 31-36, compared with the sequence shown in any one of SEQ ID NO: 31-36 has A sequence of at least 80% identity, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 31-36, or consisting of; and/or
  • the light chain of the antibody comprises an amino acid sequence as shown in any one of SEQ ID NO: 39-43, which has at least 80% identity compared with the sequence shown in any one of SEQ ID NO: 39-43 sequence, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 39-43, or consisting of.
  • the heavy chain of the antibody comprises an amino acid sequence as shown in any one of SEQ ID NO: 31-36; and/or
  • the light chain of the antibody comprises an amino acid sequence as shown in any one of SEQ ID NO: 39-43.
  • the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 31, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 39.
  • the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 32
  • the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 39.
  • the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 33
  • the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 39.
  • the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34
  • the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 39.
  • the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 35
  • the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 39.
  • the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 36
  • the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 39.
  • the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34
  • the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 40.
  • the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34
  • the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 41.
  • the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34
  • the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 42.
  • the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34
  • the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 43.
  • the antibody or antigen-binding fragment is a monoclonal antibody (including a full-length monoclonal antibody) or a multispecific antibody or antigen-binding fragment (eg, a bispecific antibody or antigen-binding fragment).
  • the antibody has two heavy chains of identical sequence and two light chains of identical sequence, and the Fc regions pair to form disulfide bonds.
  • the present invention also provides nucleic acid encoding the antibody or antigen-binding fragment.
  • the nucleic acid is an isolated nucleic acid.
  • the nucleic acid sequence is selected from the nucleic acid sequences listed in Table 6.
  • the present invention also provides a vector comprising the nucleic acid.
  • the vector comprising the nucleic acid is a nucleic acid fragment, plasmid, phage, or virus.
  • the vector is an isolated vector.
  • the present invention also provides a host cell comprising the nucleic acid or vector.
  • the host cell is an isolated host cell.
  • the host cells are CHO cells, HEK cells (such as HEK293F cells), BHK cells, Cos1 cells, Cos7 cells, CV1 cells or murine L cells.
  • the present invention also provides a method for producing the antibody or antigen-binding fragment, which comprises culturing a host cell comprising a nucleic acid encoding the antibody or antigen-binding fragment in a culture medium.
  • the method further comprises purifying the antibody or antigen-binding fragment. Purification can be carried out by conventional methods, such as centrifuging the cell suspension first, collecting the supernatant, and centrifuging again to further remove impurities. Methods such as Protein A affinity column and ion exchange column can be used to purify antibody protein.
  • the present invention also provides a pharmaceutical composition, which comprises the above-mentioned antibody or antigen-binding fragment, and pharmaceutically acceptable auxiliary materials.
  • the present invention also provides methods and uses for preventing or treating immune cell dysfunction diseases (such as phagocytic cell dysfunction diseases).
  • immune cell dysfunction diseases such as phagocytic cell dysfunction diseases.
  • a method for treating or improving an immune cell dysfunction disease such as a phagocytic cell dysfunction disease
  • the method comprising administering an effective dose of the anti-SIRP ⁇ antibody or antigen-binding fragment to a patient.
  • use of the antibody or antigen-binding fragment for treating or improving immune cell dysfunction diseases (such as phagocytic cell dysfunction diseases) is provided.
  • use of the antibody or antigen-binding fragment in the preparation of a medicament for treating or improving immune cell dysfunction (such as phagocyte dysfunction) is provided.
  • the phagocytic cell dysfunction diseases include, but are not limited to, cancer, chronic infection, and autoimmune diseases.
  • cancers include, but are not limited to, cancers with infiltrating myeloid cells, particularly with infiltrating MDSC and/or TAM cells.
  • the cancer is selected from lung cancer, mesothelioma cancer, ovarian cancer, liver cancer, bladder cancer, brain cancer, breast cancer, colon cancer, sarcoma, pancreatic cancer, head and neck cancer, kidney cancer, thymoma, glioma , melanoma and hematological cancers.
  • the cancer is selected from lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma), leukemia (lymphoblastic leukemia and myelogenous leukemia), and myeloma.
  • lymphoma Hodgkin's lymphoma and non-Hodgkin's lymphoma
  • leukemia leukemia and myelogenous leukemia
  • myeloma myeloma
  • the invention also provides diagnostic methods and uses.
  • a method of detecting SIRP ⁇ expression in a sample is provided, the sample is contacted with the anti-SIRP ⁇ antibody or antigen-binding fragment, such that the antibody or antigen-binding fragment binds SIRP ⁇ , and the binding is detected, i.e., in the sample SIRP ⁇ content.
  • the use of the anti-SIRP ⁇ antibody or antigen-binding fragment in the preparation of a kit for diagnosing or prognosing immune cell dysfunction diseases (such as cancer) is provided.
  • a diagnostic or prognostic kit comprising the anti-SIRP ⁇ antibody or antigen-binding fragment
  • the kit further includes a second antibody that specifically recognizes the anti-SIRP ⁇ antibody
  • the second antibody also includes a detectable label, such as a radioactive isotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme
  • the kit is used to detect the presence of SIRP ⁇ in a sample or levels thereof; optionally, the kit further includes antibodies or antigen-binding fragments directed against other antigens, and/or cytotoxic agents, and optionally, instructions for use.
  • the invention provides an anti-SIRP ⁇ antibody or antigen-binding fragment and application thereof.
  • the antibody or antigen-binding fragment of the invention can specifically bind to SIRP ⁇ , block the SIRP ⁇ /CD47 signaling pathway, and help the immune system to clear tumor cells.
  • the antibody or antigen-binding fragment of the present invention can be used to treat or improve cancer, and can also be used for cancer diagnosis and prognosis.
  • Figure 1 shows that scFv blocks the binding of CD47 to SIRP ⁇ .
  • Figure 2 shows the binding of anti-SIRP ⁇ antibody to PBMC.
  • Figure 3 shows the effect of anti-SIRP ⁇ antibody on T cell proliferation;
  • Figure 3A shows CD4 + CD25 + T cells, and
  • Figure 3B shows CD8 + CD25 + T cells;
  • AB56 indicates antibody Ab56, and
  • HEFLB indicates antibody 18D5 HEFLB.
  • an entity refers to one or more such entities, for example "an antibody” should be understood as one or more antibodies, therefore, the term “a” (or “an” ), “one or more” and “at least one” may be used interchangeably herein.
  • compositions, methods, etc. means that compositions, methods, etc. include the listed elements, such as components or steps, but not exclude others.
  • Consisting essentially of means that the compositions and methods exclude other elements that substantially affect the characteristics of the combination, but do not exclude elements that do not substantially affect the composition or method.
  • Consisting of means excluding elements not specifically listed.
  • polypeptide is intended to encompass the singular as well as the plural “polypeptides” and refers to a molecule formed of amino acid monomers linked linearly by amide bonds (also known as peptide bonds).
  • polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product.
  • the definition of “polypeptide” includes peptide, dipeptide, tripeptide, oligopeptide, "protein”, “amino acid chain” or any other term used to refer to a chain of two or more amino acids, and the term “polypeptide” may Used in place of, or interchangeably with, any of the above terms.
  • polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-natural Amino acid modifications that occur.
  • a polypeptide may be derived from natural biological sources or produced by recombinant techniques, but it need not be translated from a specified nucleic acid sequence, it may be produced by any means including chemical synthesis.
  • amino acid refers to an organic compound containing both amino and carboxyl groups, such as an ⁇ -amino acid, which can be encoded by a nucleic acid directly or in the form of a precursor.
  • a single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called “degeneracy of the genetic code”.
  • Amino acids include natural amino acids and unnatural amino acids.
  • Natural amino acids include alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine amino acid (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I ), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).
  • a “conservative amino acid substitution” refers to the replacement of one amino acid residue with another amino acid residue containing a side chain (R group) of similar chemical properties (eg, charge or hydrophobicity). In general, conservative amino acid substitutions are unlikely to substantially alter the functional properties of a protein.
  • classes of amino acids that contain chemically similar side chains include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, Arginine and histidine; 6) acidic side chains: aspartic acid and glutamic acid.
  • the number of amino acids in the "conservative amino acid substitution of VL, VH” is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15 conservative amino acid substitutions, or a range between any two of these values (inclusive), or any value therein.
  • the number of amino acids in the "heavy chain constant region, light chain constant region, heavy chain or light chain conservative amino acid substitution” is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15, about 18, about 19, about 22, about 24, about 25, about 29 , about 31, about 35, about 38, about 41, about 45 conservative amino acid substitutions, or a range between any two of these values (inclusive) or any value therein.
  • isolated used in the present invention with respect to cells, nucleic acids, polypeptides, antibodies, etc., for example, "isolated" DNA, RNA, polypeptides, antibodies refers to the isolated components of the cell's natural environment, such as DNA or RNA. One or more of the isolated molecules.
  • isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or cell culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • isolated nucleic acid is intended to include fragments of nucleic acid that do not occur in nature, and do not exist in nature.
  • isolated is also used herein to refer to cells or polypeptides that are separated from other cellular proteins or tissues.
  • Isolated polypeptide is intended to include purified and recombinant polypeptides.
  • Isolated polypeptides, antibodies, etc. will usually be prepared by at least one purification step.
  • the purity of the isolated nucleic acid, polypeptide, antibody, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or some of these values The range (inclusive) between any two values of , or any value therein.
  • polypeptides or polynucleotides refers to polypeptides or polynucleotides, meaning forms of polypeptides or polynucleotides that do not occur in nature, non-limiting examples can be produced by combination of polynucleotides or polynucleotides that do not normally exist or peptide.
  • Homology refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the alignable positions in each sequence. When a position in the sequences being compared is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
  • At least 80% identity is about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, About 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or these A range (inclusive) between any two values in Numeric or any value therein.
  • a polynucleotide or polynucleotide sequence has a certain percentage (eg 90%, 95%, 98% or 99%) "identity or sequence identity" with another sequence
  • identity or sequence identity refers to the percentage of bases (or amino acids) that are identical in the two sequences being compared when the sequences are aligned. This alignment and percent identity or sequence identity can be determined using visual inspection or software programs known in the art, such as those described by Ausubel et al.eds. (2007) in Current Protocols in Molecular Biology. It is preferred to use the default parameters for the alignment.
  • Biologically equivalent polynucleotides are polynucleotides that share the above indicated percentages of identity and encode a polypeptide having the same or similar biological activity.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), or when a polynucleotide In the case of RNA, thymine is replaced by uracil (U).
  • a "polynucleotide sequence” may be denoted by the letters of the polynucleotide molecule. This letter designation can be entered into a database in a computer with a central processing unit and used in bioinformatics applications such as for functional genomics and homology searches.
  • polynucleotide refers to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or their analog.
  • a polynucleotide can have any three-dimensional structure and can perform any function, known or unknown.
  • polynucleotides genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribose Somatic RNA, ribozyme, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • nucleotides can be made before or after assembly of the polynucleotide.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • Polynucleotides may be further modified after polymerization, for example by conjugation with labeling components.
  • the term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of a polynucleotide of the present disclosure includes the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.
  • encoding when applied to a polynucleotide refers to a polynucleotide which is said to "encode” a polypeptide which, in its native state or when manipulated by methods well known to those skilled in the art, is transcribed and/or Or translation may result in the polypeptide and/or fragments thereof.
  • Antibodies and antigen-binding fragments disclosed in the present invention include but are not limited to polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, single-chain antibodies, epitope-binding fragments (such as Fab, Fab' and F(ab') 2 ), single chain Fvs (scFv).
  • Antibody and antigen-binding fragment refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen.
  • Antibodies can be whole antibodies and any antigen-binding fragments thereof or single chains thereof.
  • the term “antibody” thus includes any protein or peptide whose molecule contains at least a portion of an immunoglobulin molecule that has the biological activity to bind an antigen.
  • Antibodies and antigen-binding fragments include, but are not limited to, complementarity determining regions (CDRs), heavy chain variable regions (VH), light chain variable regions (VL), heavy chain constant regions of heavy or light chains or ligand-binding portions thereof (CH), light chain constant region (CL), framework region (FR) or any portion thereof, or at least a portion of a binding protein.
  • the CDR regions include the CDR regions of the light chain (LCDR1-3) and the CDR regions of the heavy chain (HCDR1-3).
  • Antibodies and antigen-binding fragments can specifically recognize and bind to one or more (eg, two) antigen polypeptides or polypeptide complexes.
  • Antibodies or antigen-binding fragments that specifically recognize and bind multiple (eg, two) antigens may be referred to as multispecific (eg, bispecific) antibodies or antigen-binding fragments.
  • antibody fragment refers to a part of an antibody, and the composition of the antibody fragment of the present invention may be similar to F(ab') 2 , F(ab) 2 , Fab', Fab in monospecific antibody fragments , Fv, scFv, etc. Regardless of their structure, antibody fragments bind to the same antigen recognized by the intact antibody.
  • antibody fragment includes aptamers, Spiegelmers and diabodies.
  • antiigen-binding fragment also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.
  • Single-chain variable fragment refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin. In some aspects, these regions are linked to short linker peptides of 10 to about 25 amino acids. Linkers can be rich in glycine for flexibility, and serine or threonine for solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa. Although the protein has had its constant regions removed and a linker introduced, it retains the specificity of the original immunoglobulin. scFv molecules are generally known in the art and are described, for example, in US Patent No. 5,892,019.
  • antibody includes a wide variety of polypeptides that can be distinguished biochemically.
  • classes of heavy chains include gamma, mu, alpha, delta, or epsilon ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ), with subclasses (eg, ⁇ 1- ⁇ 4).
  • the nature of this chain determines the "class” of the antibody as IgG, IgM, IgA, IgG or IgE, respectively.
  • the immunoglobulin subclasses (isotypes), eg, IgGl, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and the functional specificities conferred are also known. All immunoglobulin classes are within the scope of the present disclosure. In some embodiments, the immunoglobulin molecule is of the IgG class.
  • Light chains can be classified as kappa ( ⁇ ) or lambda ( ⁇ ). Each heavy chain can be associated with a kappa or lambda light chain.
  • ⁇ kappa
  • lambda ⁇
  • Each heavy chain can be associated with a kappa or lambda light chain.
  • immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, their light and heavy chains are joined by covalent bonds, and the "tail" portions of the two heavy chains are linked by covalent disulfide bonds or non-covalent bonding.
  • the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain.
  • the variable region of the immunoglobulin kappa light chain is V ⁇ ; the variable region of the immunoglobulin lambda light chain is V ⁇ .
  • Both light and heavy chains are divided into regions of structural and functional homology.
  • the terms "constant” and “variable” are used according to function.
  • the light chain variable (VL) and heavy chain variable (VH) portions determine antigen recognition and specificity.
  • the constant regions of the light and heavy chains confer important biological properties such as secretion, transplacental movement, Fc receptor binding, complement fixation, etc. By convention, the numbering of constant regions increases as they become farther away from the antigen-binding site or amino terminus of the antibody.
  • the N-terminal portion is the variable region and the C-terminal portion is the constant region; the CH3 and CL domains actually comprise the carboxy-terminal ends of the heavy and light chains, respectively.
  • each antigen-binding domain In naturally occurring antibodies, the six “complementarity determining regions" or “CDRs” present in each antigen-binding domain are short, A non-contiguous sequence of amino acids that specifically binds to an antigen. The remaining other amino acids in the antigen-binding domain, referred to as the "framework" regions, show less inter-molecular variability.
  • the framework regions mostly adopt a ⁇ -sheet conformation, and the CDRs form loop structures attached to them, or in some cases form part of the ⁇ -sheet structure. Thus, the framework regions position the CDRs in the correct orientation by forming a scaffold through non-covalent interchain interactions.
  • the antigen-binding domain with the CDRs in specific positions forms a surface complementary to the epitope on the antigen that facilitates the non-covalent binding of the antibody to its antigenic epitope.
  • a surface complementary to the epitope on the antigen that facilitates the non-covalent binding of the antibody to its antigenic epitope.
  • those of ordinary skill in the art can identify the amino acids comprising CDR and framework regions by known methods (see Kabat, E., et al., U.S. Department of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).
  • CDR complementarity determining regions
  • CDRs as defined by Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. Nevertheless, it is within the scope of the invention to use either definition to refer to the CDRs of an antibody or variant thereof.
  • the exact residue numbers comprising a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can generally determine which specific residues are included in the CDRs based on the amino acid sequence of the variable region of the antibody.
  • Kabat et al. also defined a numbering system applicable to the variable region sequences of any antibody.
  • One of ordinary skill in the art can apply this "Kabat numbering" system to any variable region sequence independently of other experimental data other than the sequence itself.
  • “Kabat numbering” refers to the numbering system proposed by Kabat et al., U.S. Dept. of Health and Human Services in "Sequence of Proteins of Immunological Interest” (1983).
  • Antibodies can also use the EU or Chothia numbering system.
  • Antibodies disclosed herein may be derived from any animal, including birds and mammals.
  • the antibody is of human, murine, donkey, rabbit, goat, camel, llama, horse or chicken origin.
  • the variable regions may be of condricthoid origin (eg, from sharks).
  • a "heavy chain constant region” includes at least one of a CH1 domain, a hinge (eg, upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment.
  • the heavy chain constant regions of antibodies can be derived from different immunoglobulin molecules.
  • the heavy chain constant region of a polypeptide can include a CH1 domain derived from an IgG 1 molecule and a hinge region derived from an IgG 3 molecule.
  • the heavy chain constant region may comprise a hinge region derived in part from an IgG 1 molecule and in part from an IgG 3 molecule.
  • part of the heavy chain may comprise a chimeric hinge region derived partly from an IgG 1 molecule and partly from an IgG4 molecule.
  • a “light chain constant region” includes a portion of the amino acid sequence from an antibody light chain.
  • the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain.
  • a “light chain-heavy chain pair” refers to a collection of light and heavy chains that can form dimers through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain. The four chains are linked by disulfide bonds in a "Y" configuration, with the light chain starting at the mouth of the "Y” and continuing through the variable region surrounding the heavy chain.
  • a "VH domain” includes the amino-terminal variable domain of an immunoglobulin heavy chain, and a "CH1 domain” includes the first (mostly amino-terminal) constant region of an immunoglobulin heavy chain.
  • N297 in the two CH2 domains of the complete natural IgG molecule is connected to a branched carbohydrate chain.
  • the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.
  • a "hinge region” includes part of the heavy chain region connecting the CH1 domain and the CH2 domain.
  • the hinge region comprises approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently.
  • the hinge region can be subdivided into three distinct domains: upper, middle and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).
  • Disulfide bond refers to a covalent bond formed between two sulfur atoms.
  • a thiol group of cysteine can form a disulfide bond or bridge with a second thiol group.
  • the CH1 and CL regions are linked by disulfide bonds.
  • a “chimeric antibody” refers to any antibody whose variable regions are obtained or derived from a first species and whose constant regions (which may be complete, partial or modified) are derived from a second species.
  • the variable regions are of non-human origin (eg, mouse or primate) and the constant regions are of human origin.
  • Specific binding or “specific for” generally refers to the complementary binding of an antibody or antigen-binding fragment to a specific antigen through its antigen-binding domain and epitope to form a relatively stable complex.
  • Specificity can be expressed in terms of the relative affinity with which an antibody or antigen-binding fragment binds to a particular antigen or epitope. For example, antibody “A” may be said to have a higher specificity for that antigen than antibody “B” if it has a greater relative affinity for the same antigen than antibody "B”.
  • Specific binding can be described by an equilibrium dissociation constant ( KD ), with a smaller KD implying a tighter binding.
  • An antibody that "specifically binds" antigen a includes an equilibrium dissociation constant K D of antigen a of less than or equal to about 100 nM, less than or equal to about 10 nM, less than or equal to about 5 nM, less than or equal to about 1 nM.
  • Treatment means therapeutic treatment and prophylactic or preventive measures, the purpose of which is to prevent, slow down, ameliorate or stop an undesirable physiological change or disorder, such as the progression of a disease, including but not limited to the following whether detectable or undetectable Relief of symptoms, reduction of disease extent, stabilization of disease state (i.e. not worsening), delay or slowing of disease progression, amelioration, remission, alleviation or disappearance of disease state (whether partial or total), prolongation and Expected survival without treatment, etc.
  • Patients in need of treatment include those who already have a condition or disorder, are susceptible to having a condition or disorder, or are in need of prevention of the condition or disorder, and can or are expected to benefit from the administration of an antibody or pharmaceutical composition disclosed herein for detection , patients who benefit from the diagnostic process and/or treatment.
  • Patient refers to any mammal in need of diagnosis, prognosis, or treatment, including humans, dogs, cats, rabbits, mice, horses, cattle, and the like.
  • EC s0 half maximum effect concentration (concentration for 50% of maximal effect, EC 50 ) refers to the concentration that can cause 50% of the maximum effect.
  • IC50 means 50% inhibitory concentration, ie the concentration of drug or inhibitor required to inhibit a given biological process by half.
  • the "parental Fc region" in the present invention can be a naturally occurring Fc region, and the gene encoding the Fc region can be from human, mouse, rabbit, camel, monkey, preferably human and mouse; for example, the parental Fc region is SEQ ID NO: 31 , SEQ ID NO:32, SEQ ID NO:33 or the Fc region of SEQ ID NO:34.
  • the present invention provides antibodies or antigen-binding fragments with high affinity for SIRP ⁇ protein.
  • the antibody or antigen-binding fragment of the present invention exhibits effective binding activity, biological activity, and can be used for therapeutic and diagnostic purposes.
  • these antibodies or antigen-binding fragments can effectively block the SIRP ⁇ /CD47 signaling pathway, activate phagocytosis of phagocytes, and be used to treat various types of cancer or infection and other related diseases.
  • the antibody or antigen-binding fragment is a scFv.
  • the heavy chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 16
  • the light chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 22.
  • the heavy chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 17, and the light chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 22.
  • the heavy chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 18, and the light chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 22.
  • the heavy chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 19, and the light chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 22.
  • the heavy chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 20
  • the light chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 22.
  • the heavy chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 21, and the light chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 22.
  • the heavy chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 19, and the light chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 23.
  • the heavy chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 19, and the light chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 24.
  • the heavy chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 19, and the light chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 25.
  • the heavy chain variable region of the scFv fragment comprises the sequence set forth in SEQ ID NO:19
  • the light chain variable region of the scFv fragment comprises the sequence set forth in SEQ ID NO:26.
  • the linker connecting the variable region of the heavy chain and the variable region of the light chain in the scFv fragment is (G 4 S) n . In some embodiments, n is 1, 2, 3, 4 or 5.
  • the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 31, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 39.
  • the heavy chain of the antibody or antigen-binding fragment comprises a sequence other than Fc in the amino acid sequence of SEQ ID NO: 31, and the light chain of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: 39 sequence shown.
  • the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 32, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 39.
  • the heavy chain of the antibody or antigen-binding fragment comprises a sequence other than Fc in the amino acid sequence of SEQ ID NO: 32, and the light chain of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: 39 sequence shown.
  • the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 33
  • the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 39.
  • the heavy chain of the antibody or antigen-binding fragment comprises a sequence other than Fc in the amino acid sequence of SEQ ID NO: 33
  • the light chain of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: 39 sequence shown.
  • the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34
  • the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 39.
  • the heavy chain of the antibody or antigen-binding fragment comprises a sequence other than Fc in the amino acid sequence of SEQ ID NO: 34
  • the light chain of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: 39 sequence shown.
  • the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 35
  • the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 39.
  • the heavy chain of the antibody or antigen-binding fragment comprises a sequence other than Fc in the amino acid sequence of SEQ ID NO: 35
  • the light chain of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: 39 sequence shown.
  • the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 36
  • the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 39.
  • the heavy chain of the antibody or antigen-binding fragment comprises a sequence other than Fc in the amino acid sequence of SEQ ID NO: 36
  • the light chain of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: 39 sequence shown.
  • the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34
  • the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 40
  • the heavy chain of the antibody or antigen-binding fragment comprises a sequence other than Fc in the amino acid sequence of SEQ ID NO: 34
  • the light chain of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: 40 sequence shown.
  • the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34
  • the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 41
  • the heavy chain of the antibody or antigen-binding fragment comprises a sequence other than Fc in the amino acid sequence of SEQ ID NO: 34
  • the light chain of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: 41 sequence shown.
  • the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34
  • the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 42
  • the heavy chain of the antibody or antigen-binding fragment comprises a sequence other than Fc in the amino acid sequence of SEQ ID NO: 34
  • the light chain of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: 42 sequence shown.
  • the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34
  • the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 43
  • the heavy chain of the antibody or antigen-binding fragment comprises a sequence other than Fc in the amino acid sequence of SEQ ID NO: 34
  • the light chain of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: 43 sequence shown.
  • an antibody of the invention contains two heavy chains (or heavy chain fragments) with the same sequence and two light chains (or light chain fragments) with the same sequence.
  • the sequences of the antibodies or antigen-binding fragments disclosed in the present invention can be replaced, and the amino acid sequence after replacement is different from the naturally occurring amino acid sequence of the antibody.
  • the substituted amino acid sequence may be similar to the original sequence, such as having a certain proportion of identity with the original sequence, for example, it may be about 80%, about 85%, or about 90% identical to the original sequence. , about 95%, about 98%, about 99%, or a range between any two of these values (inclusive), or any value therein.
  • an antibody or antigen-binding fragment comprises an amino acid sequence with one or more modification groups.
  • an antibody or antigen-binding fragment disclosed herein may contain a flexible linker sequence, or may be modified to add functional groups (eg, PEG, drug, toxin, or tag).
  • the antibodies, antigen-binding fragments disclosed herein include derivatives that are modified, that is, modified by covalent linkage of any type of molecule to the antibody or antigen-binding fragment, wherein the covalent linkage does not prevent the antibody or antigen-binding fragment from binding to the epitope combined.
  • Examples including, but not limited to, antibodies or antigen-binding fragments may be glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytically cleaved, linked to Cell ligands or other proteins, etc.
  • the antibodies, antigen-binding fragments and modified derivatives thereof disclosed in the present invention include their salts with acids and/or bases.
  • an antibody or antigen-binding fragment can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.
  • Antibodies or antigen-binding fragments can be detectably labeled by coupling them to chemiluminescent compounds. The presence of the chemiluminescently labeled antibody or antigen-binding fragment is then determined by detecting the luminescence that occurs during the course of the chemical reaction.
  • chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.
  • the invention also discloses polynucleotides or nucleic acid molecules encoding the antibodies, antigen-binding fragments, and derivatives thereof of the invention.
  • the polynucleotide disclosed in the present invention can encode heavy chain variable region, light chain variable region, Fc region, part of heavy chain variable region, part of light chain variable region, heavy chain or light chain, etc. Methods of making antibodies are well known in the art and described herein.
  • antibodies are produced that do not elicit an adverse immune response in the animal (eg, human) to be treated.
  • the antibodies, antigen-binding fragments, or derivatives disclosed herein are modified to reduce their immunogenicity using art-recognized techniques.
  • antibodies can be humanized, primatized, deimmunized or chimeric antibodies can be prepared. These types of antibodies are derived from non-human antibodies, usually murine or primate antibodies, which retain or substantially retain the antigen-binding properties of the parent antibody but are less immunogenic in humans.
  • framework residues in the human framework regions will be replaced by corresponding residues from the CDR donor antibody, such as residues that improve antigen binding.
  • framework substitutions can be identified by methods known in the art, such as by modeling the interaction of CDRs and framework residues to identify framework residues important for antigen binding and by sequence alignment to identify abnormal framework residues at specific positions. (Refer to US Patent 5,585,089; Riechmann et al., Nature 332:323 (1988); the entire contents of which are incorporated herein by reference).
  • Antibodies can be humanized using various techniques known in the art, such as CDR grafting (EP 239,400; WO 91/09967; US Patents 5,225,539, 5,530,101 and 5,585,089), repair or surface rearrangement (EP 592,106; EP 519,596; Padlan, et al., Molecular Immunology 28 (4/5): 489-498 (1991); Studnicka et al., Protein Engineering 7 (6): 805-814 (1994); Roguska, et al., Proc. Natl . Sci. USA 91:969-973 (1994)), and chain rearrangements (US Patent 5,565,332), the entire contents of which are incorporated herein by reference.
  • CDR grafting EP 239,400; WO 91/09967; US Patents 5,225,539, 5,530,101 and 5,585,089)
  • repair or surface rearrangement EP 592,106; EP 519,596; Padlan, et al.,
  • Deimmunization can also be used to reduce the immunogenicity of antibodies.
  • the term "deimmunization” includes altering antibodies to modify T cell epitopes (see eg WO/9852976 A1 and WO/0034317 A2).
  • the heavy and light chain variable region sequences from a starting antibody are analyzed and a human T cell epitope "map" from each variable region is generated, showing the epitopes relative to the complementarity determining regions (CDRs) and the positions of other key residues within the sequence.
  • CDRs complementarity determining regions
  • Individual T-cell epitopes from T-cell epitope maps are analyzed to identify alternative amino acid substitutions with lower risk of altering antibody activity.
  • a series of alternative heavy chain variable region sequences and light chain variable region sequences comprising combinations of amino acid substitutions are designed and these sequences are subsequently incorporated into a series of binding polypeptides.
  • Genes for the complete heavy and light chains containing the modified variable and human constant regions are then cloned into expression vectors, and the plasmids are subsequently transformed into cell lines to produce complete antibodies.
  • Antibodies are then compared using appropriate biochemical and biological assays to identify the best antibody.
  • the binding specificity of the antibodies or antigen-binding fragments disclosed in the present invention can be detected by in vitro experiments, such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • in vitro experiments such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • scFv can refer to the technology of producing single chain unit (US Patent 4,694,778; Bird, Science 242:423-442 (1988), Huston et al., Proc.Natl.Acad.Sci.USA 55:5879-5883 (1988) and Ward et al., Nature 334:544-554 (1989) and Nie et al., Antibody Therapeutics 3(1):78-62 (2020)).
  • Single-chain fusion peptides are generated by amino acid bridging of the heavy and light chain fragments of the Fv region to form single-chain units.
  • the technique of assembling functional Fv fragments in E. coli can also be used (Skerra et al., Science 242:1038-1041 (1988)).
  • scFv single-chain Fv
  • antibodies include, for example, U.S. Patents 4,946,778 and 5,258,498, and Huston et al., Methods in Enzymology 203:46-88 (1991), Shu et al., Proc. Natl. Sci. USA 90: 1995-1999 (1993) and Skerra et al., Science 240: 1038-1040 (1988).
  • chimeric, humanized or fully human antibodies may be used.
  • Chimeric antibodies are molecules in which different parts of the antibody are derived from different animal species, such as antibodies that have the variable regions of a murine monoclonal antibody and the constant regions of a human immunoglobulin.
  • Methods for producing chimeric antibodies are known in the art, see Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191 -202 (1989); Neuberger et al., Nature 372:604-608 (1984); Takeda et al., Nature 314:452-454 (1985); and U.S. Patents 5,807,715, 4,816,567 and 4,816,397, the entire contents of which are incorporated by reference Incorporated into this article.
  • Antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries derived from immunoglobulin sequences. See also U.S. Patents 4,444,887 and 4,716,111, and PCT Publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741, each The entire content of the patent is incorporated herein by reference.
  • DNA encoding the desired monoclonal antibody can be isolated and sequenced using conventional methods (e.g., using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). sequencing. Isolated and subcloned hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce other immunoglobulins middle.
  • prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce other immunoglobulins middle.
  • Isolated DNA (which may be synthetic as described herein) can also be used to prepare the constant and variable region sequences of antibodies as described in US Pat. No. 5,658,570, the entire contents of which are incorporated herein by reference. This method extracts RNA from selected cells and converts it into cDNA, which is then amplified by PCR using Ig-specific primers. Suitable probes for this purpose are also mentioned in US Patent No. 5,658,570.
  • one or more CDRs of an antibody of the invention can be inserted into a framework region, eg, into a human framework region, to construct a humanized non-fully human antibody.
  • the framework regions may be naturally occurring or consensus framework regions, preferably human framework regions (see Chothia et al., J. Mol. Biol. 278:457-479 (1998) for a list of human framework regions).
  • Some polynucleotides may encode an antibody that specifically binds at least one epitope of an antigen of interest produced by a combination of framework regions and CDRs.
  • One or more amino acid substitutions may be made within the framework regions, and the amino acid substitutions may be selected to improve binding of the antibody to its antigen.
  • substitution or deletion of cysteine residues in one or more variable regions that participate in interchain disulfide bond formation can be performed in this way, thereby producing antibody molecules lacking one or more interchain disulfide bonds.
  • Other modifications to polynucleotides within the skill of the art are also encompassed in the present invention.
  • Antibodies can be prepared using conventional recombinant DNA techniques. Antibody-producing vectors, cell lines, and the like can be selected, constructed, and cultured using techniques known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, such as Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, D.L. hacker, F.M. Wurm, in Reference Module in Life Sciences, 2017, which in its entirety includes The supplementary content is incorporated by reference in its entirety.
  • the DNA encoding the antibody can be designed and synthesized according to the amino acid sequence of the antibody described herein in a conventional manner, placed into an expression vector, and then transfected into a host cell, and cultured in a medium to produce the transfected host cell.
  • Monoclonal antibodies Monoclonal antibodies.
  • an antibody expression vector includes at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing flanking the inserted sequence.
  • High-efficiency transcription can be obtained through the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and early promoters of cytomegalovirus, and other cellular promoters such as muscle Kinetin promoter.
  • Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or Plncx, pcDNA3.1(+/-), pcDNA/Zeo(+/-), pcDNA3.1/Hygro(+/-), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2 etc.
  • Commonly used mammalian cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells and CHO cells, etc.
  • the inserted gene fragment needs to contain selection markers, common selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other selection genes, so as to facilitate transfection Screening of successful cell isolation.
  • selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other selection genes, so as to facilitate transfection Screening of successful cell isolation.
  • the constructed plasmid is transfected into host cells without the above-mentioned genes, and cultured in a selective medium, the successfully transfected cells grow in large numbers and produce the desired target protein.
  • mutations can be introduced into the nucleotide sequence encoding the antibodies of the present invention using standard techniques known to those skilled in the art, including but not limited to site-directed mutagenesis and PCR-mediated mutations resulting in amino acid substitutions.
  • Variants include derivatives
  • mutations can be introduced randomly along all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity.
  • the invention also provides treatment methods and uses.
  • methods for treating or improving various types of cancers, chronic infections, and autoimmune diseases and other related diseases are provided, the methods comprising administering an effective dose of an anti-SIRP ⁇ antibody or an antigen-binding antibody to a patient in need thereof. fragment.
  • the application of anti-SIRP ⁇ antibody or antigen-binding fragment in treating or improving related diseases such as cancer, chronic infection and autoimmune disease is provided.
  • the use of the anti-SIRP ⁇ antibody or antigen-binding fragment in the preparation of medicines for treating or improving related diseases such as cancer, chronic infection and autoimmune diseases is provided.
  • the specific dosage and treatment regimen for any particular patient will depend on various factors, including the specific antibody or derivative used, the patient's age and weight, general health, sex, and diet, as well as the time of administration, frequency of excretion, drug combination, and the severity of the particular disease being treated. These factors are in the judgment of the medical caregiver, who is within the purview of those of ordinary skill in the art.
  • the dosage will also depend on the individual patient to be treated, the route of administration, the type of formulation, the nature of the compound employed, the severity of the disease and the effect desired.
  • the dosage used can be determined by principles of pharmacology and pharmacokinetics well known in the art.
  • the antibody of the present invention is administered to a patient at a dose of 0.01 mg/kg to 100 mg/kg of the patient's body weight each time. In some embodiments, the administration is every 1 week, 2 weeks, 3 weeks, or monthly.
  • compositions include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural, and oral administration.
  • the pharmaceutical compositions may be administered by any convenient route, such as by infusion or bolus injection, absorbed through the epithelium or mucous membranes (eg, oral mucosa, rectal and intestinal mucosa, etc.), and may be co-administered with other biologically active agents.
  • compositions containing an antibody or antigen-binding fragment of the invention may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (e.g., by powder, ointment , drops or transdermal patch), orally or by oral or nasal spray.
  • parenteral refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • the mode of administration can be systemic administration or local administration.
  • Antibodies or antigen-binding fragments of the invention may be administered topically to the area in need of treatment; by, but not limited to, topical application during surgery, for example in conjunction with post-operative wound dressing, by injection, by catheter, by suppository or by means of implants, which are porous, non-porous or gel-like materials comprising membranes (such as silicone rubber membranes) or fibers.
  • implants which are porous, non-porous or gel-like materials comprising membranes (such as silicone rubber membranes) or fibers.
  • care when administering proteins of the invention, including antibodies, care must be taken to use materials that do not absorb the protein.
  • compositions of the invention comprise a nucleic acid or polynucleotide encoding an antibody or antigen-binding fragment, which can be administered in vivo to facilitate its encoded expression by constructing it as part of a suitable nucleic acid expression vector.
  • a suitable nucleic acid expression vector Expression of the protein, followed by administration of the above-mentioned part of the vector to make it an intracellular part, for example by using a retroviral vector (see US Pat. No.
  • nucleic acid can be introduced into the cell by homologous recombination and integrated into the host cell DNA for expression.
  • the dose and frequency of administration of antibodies of the invention can be reduced by enhancing the uptake and tissue penetration of antibodies or antigen-binding fragments through modifications such as lipidation.
  • Various known delivery systems can be used to administer the antibodies, antigen-binding fragments or derivatives of the invention, or polynucleotides encoding the same, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds, subject Body-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of nucleic acids as part of retroviruses or other vectors, etc.
  • an anti-SIRP ⁇ antibody or antigen-binding fragment of the invention may be used in combination with other therapeutic or prophylactic regimens for the treatment of cancer, including administration of one or more antibodies or antigen-binding fragments of the invention, in combination with one or more other therapeutic Agents or methods used together or in combination.
  • other treatment regimens include, but are not limited to, radiation therapy, chemotherapy, hormone therapy, and the like.
  • the antibodies can be administered simultaneously or separately from the other therapeutic agents.
  • the antibody or antigen-binding fragment of the invention can be administered before or after another other therapeutic agent is administered.
  • anticancer agents that can be administered with the antibodies or antigen-binding fragments of the invention include, but are not limited to: 5-fluorouracil, acivicin, aldesleukin, hexamethylmelamine, aminoglutethimide, amsacrine Pyridine, Anastrozole, Antramycin, Asparaginase, Azacitidine, Azathepa, Azomycin, Batimastat, Bicalutamide, Bleomycin Sulfate, Buquina Sodium , bropirimine, busulfan, carboplatin, carmustine, carrubicin hydrochloride, kazellexine, sildifenagore, chlorambucil, siromycin, cisplatin, cladri Bine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride,
  • the therapeutic antibody that can be administered with the antibody or antigen-binding fragment of the invention is an immune checkpoint blocker or activator of immune cells (B or T lymphocytes), and the therapeutic antibody includes, but is not limited to: Anti-PD-L1 antibody, anti-PD1 antibody, anti-CTLA-4 antibody, anti-CD137 antibody, anti-CD2 antibody, anti-CD28 antibody, anti-CD40 antibody, anti-HVEM antibody, anti-BTLA antibody, anti-CD160 antibody, anti-TIGIT antibody, anti-TIM -1/3 antibody, anti-LAG-3 antibody, anti-2B4 antibody, anti-OX40 antibody, anti-CD40 antibody, anti-CD40-L antibody, anti-ICOS antibody, anti-ICOS-L antibody.
  • B or T lymphocytes immune checkpoint blocker or activator of immune cells
  • compositions comprise effective doses of anti-SIRP ⁇ antibodies or antigen-binding fragments and pharmaceutically acceptable auxiliary materials.
  • the pharmaceutical composition comprises 0.1%-90% of an anti-SIRP ⁇ antibody or antigen-binding fragment.
  • the pharmaceutical composition further comprises an anti-cancer agent (eg, an immune checkpoint inhibitor or activator).
  • the term "pharmaceutically acceptable” refers to a substance approved by a governmental regulatory agency or listed in a recognized pharmacopoeia for use in animals, especially in humans.
  • pharmaceutically acceptable excipients generally refer to any type of non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials or formulation aids, etc.
  • adjuvant refers to a diluent, adjuvant, excipient or carrier with which the active ingredient can be administered to a patient.
  • Such pharmaceutical excipients can be sterile liquids, such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, Propylene, ethylene glycol, water, ethanol, etc.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates.
  • Antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and tonicity adjusting agents such as sodium chloride or dextrose are also contemplated.
  • These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • compositions will contain a clinically effective dose of the antibody or antigen-binding fragment, preferably in purified form, together with an appropriate amount of carrier to provide a form suitable for administration to the patient.
  • the formulation should be suitable for the mode of administration.
  • the preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • the composition is formulated into a pharmaceutical composition suitable for intravenous injection to human body according to conventional procedures.
  • Compositions for intravenous administration are generally solutions in sterile isotonic aqueous buffer.
  • the composition may also contain a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site.
  • the active ingredients are presented alone or in combination in unit dosage form, eg, as a dry lyophilized powder or water-free concentrate, in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent.
  • the composition may be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water or saline for injection can be used so that the active ingredient can be mixed before administration.
  • the compounds of the present invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include salts derived from anions such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts derived from such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2 - Salts of cations such as ethylaminoethanol, histidine, procaine and the like.
  • the amino acid sequence (P78324) of human V1 type SIRP ⁇ was obtained from the Uniprot protein database, and the amino acid sequence of the extracellular region of human V1 type SIRP ⁇ was from the 1st to the 373rd amino acid residue.
  • the amino acid sequence (D86043) of V2-type SIRP ⁇ was obtained from the GeneBank database, and the amino acid sequence of the extracellular region of human V2-type SIRP ⁇ is the amino acid residues 1 to 369.
  • the amino acid sequence of human IgG1-Fc (P01857) was obtained from the Uniprot protein database, and its amino acid sequence is from the 104th to the 330th amino acid residues.
  • the nucleotide sequence was connected to SIRP ⁇ and Fc by EcoRI and Hind III double enzyme digestion respectively, and inserted into the pCDNA3.1 vector (purchased from Invitrogen Company), respectively to obtain recombinant plasmids: pCDNA-SIRP ⁇ V1-Fc, pCDNA-SIRP ⁇ V2 -Fc and pCDNA-SIRP ⁇ V8-Fc.
  • HEK293F cells purchased from ATCC
  • PEI polyetherimide
  • hSIRP ⁇ V1-Fc, hSIRP ⁇ V2-Fc and hSIRP ⁇ V ⁇ -Fc were obtained by purification Protein samples, used in the following examples.
  • VH gene and VL gene in unimmunized human peripheral blood lymphocytes were amplified by polymerase chain reaction (polymerase chain reaction, PCR), and then by overlapping polymerase chain reaction (Overlapping polymerase chain reaction) , overlap PCR) randomly combined heavy chain VH and light chain VL into a single chain Fv (single chain Fv, scFv); scFv was inserted into a phagemid vector through restriction sites to construct a phage library (stock capacity greater than 10 11 ), displayed on Phage surfaces for antibody screening (see, for details, SHEETS et al. (1998) Cell Biology. 95:6157-6162).
  • the screening conditions used include: coupling the antigenic protein hSIRP ⁇ V1-Fc dissolved in PBS (phosphate-buffered saline, pH 8.2) to an immunotube (purchased from Nunc Company), and placing it overnight at 4°C; Add 100 ⁇ l of blocking solution (5% BSA (bovine serum albumin)) and block at room temperature for 2 hours; after 2 hours, add 100 ⁇ l of phage (5 ⁇ 10 13 PFU) for 1 hour at room temperature; use PBST (containing 0.05% Tween- 20 PBS buffer) to wash 20 times to wash off non-binding phage; use 100mM TEA (triethylamine, Sigma) eluent to dissociate the phage specifically bound to the antigenic protein hSIRP ⁇ V1-Fc, and then infect E.
  • PBS phosphate-buffered saline, pH 8.2
  • BSA bovine serum albumin
  • the antigen concentration in the first round was 50 ⁇ g/ml
  • the antigen concentration in the second round was 10 ⁇ g/ml
  • the antigen concentration was 10 ⁇ g/ml.
  • the enriched phage specifically binding to the antigen protein hSIRP ⁇ V1-Fc was obtained.
  • the ELISA detection method is as follows: the antigen SIRP ⁇ -His (Baipu Saisi, product number SIA-H5225) is coated on the microtiter plate at a concentration of 2 ⁇ g/ml one day in advance, 100 ⁇ l/well, coated overnight at 4 ° C, and coated with 5 ⁇ g the next day.
  • % skimmed milk powder was blocked at 37°C for 2 hours, and then used for detection; take 100 ⁇ l of the above supernatant to the ELISA plate, and incubate at room temperature for 1 hour; wash off unbound antibodies with PBST, and add Myc-Tag(9B11) Mouse mAb (HRP Conjugate) (Cell Signaling Technology, Inc; catalog number is 2040S), put it at room temperature for 1 hour; wash off unbound antibody with PBST, add TMB (tetramethylbenzidine) chromogenic solution (Shanghai Shenggong, Cat. No. E66100), after 15 minutes at room temperature, 0.1M sulfuric acid was added to terminate the reaction, and the absorbance was read at a wavelength of 450nm by a microplate reader. The positive judgment standard was that the OD value was higher than 0.5.
  • the same ELISA detection method as above take 50 ⁇ l of the supernatant of the above-identified positive clones, mix it with 50 ng/ml CD47-Fc-biotin (Beijing Biosciences Biotechnology Co., Ltd., product number is CD7-H82F6), and then add SIRP ⁇ -His-coated ELISA plate, incubate at room temperature for 1 hour; wash unbound ligand with PBST, add Streptavidin-HRP (Jackson ImmunoResearch, product number 016-050-084), and place at room temperature for 1 hour; wash with PBST Wash away unbound antibodies, add TMB chromogenic solution, place at room temperature for 15 minutes, add 0.1M sulfuric acid to stop the reaction, and read the absorbance value.
  • CD47-Fc-biotin Beijing Biosciences Biotechnology Co., Ltd., product number is CD7-H82F6
  • SIRP ⁇ -His-coated ELISA plate incubate at
  • scFvs that are positive for binding and can block the binding of CD47 and SIRP ⁇ (as shown in Figure 1) were detected and screened, such as: scFv (P22-1), scFv (P22-19), scFv (P22-34), scFv (P22-63), scFv(P22-15), scFv(P22-32), scFv(Ab56), scFv(P39-1), scFv(P39-3) and scFv(P39-95), and analyzed by sequencing clone sequence.
  • the VH and VL sequences in the scFv are connected by the sequences shown in Table 1 through a linker (G 4 S) 3 .
  • the reference antibody KWAR23 is derived from WO2015138600A2, and the reference antibody 18D5HEFLB is derived from WO2017178653A2.
  • the amino acid sequences and nucleic acid sequences related to the antibody examples are shown in Tables 2-6; the CDR region is marked with a single underline in Table 4, and the Fc region is marked with a single underline in Table 5.
  • the heavy chain nucleic acid sequence and light chain nucleic acid sequence corresponding to the above antibody were artificially synthesized, and then the heavy chain nucleic acid sequence and the light chain nucleic acid sequence were respectively inserted into the pCHO1.0 plasmid (purchased from Invitrogen) by EcoRI and HindIII double enzyme digestion ligation, A recombinant plasmid for expressing the whole antibody was obtained.
  • the above recombinant plasmids were transferred into the CHO-S cell line, and the supernatant was collected after 11 days of culture, and immobilized metal affinity chromatography was used on a Protein A column (GE Healthcare). (IMAC) for purification, and the purity of the purified antibody protein is >90%.
  • the purified antibodies were detected by gel electrophoresis, antibody P22-1, P22-19, P22-34, P22-63, antibody P22-15, antibody P22-32, antibody Ab56, antibody P39-1, antibody P39-3 and Antibody P39-95 is a single substance, and its molecular weight is consistent with the theoretical value.
  • the purified antibodies were sequenced, antibody P22-1, P22-19, P22-34, P22-63, antibody P22-15, antibody P22-32, antibody Ab56, antibody P39-1, antibody P39-3 and antibody P39- 95 sequencing results were identical to the expected sequence.
  • the purified antibody is used for affinity detection and biological activity identification, etc.
  • Embodiment 4 ELISA detects the combination of antibody and antigen
  • Example 5 FLOW flow cytometry detects the binding of antibodies to cells expressing SIRP ⁇
  • CHO-SIRP ⁇ cells (0.5 ⁇ 10 6 cells) were incubated with antibodies (initial concentration 100 nM, 2.5-fold serial dilution) in PBS buffer (containing 0.1% BSA) on ice for 40 minutes. Then the cells were washed twice, and incubated with the secondary antibody Goat anti-Human IgG Fc Secondary Antibody, PE (eBioscience, catalog number: 12-4998-82) in PBS on ice for 25 minutes. Cells were washed twice and analyzed by flow cytometry on a cytoflex system (Beckman).
  • antibodies P22-1, P22-19, P22-34 and P22-63 can all bind SIRP ⁇ -His.
  • Example 8 ELISA detection antibody Ab56 combined with different antigen SIRP
  • the detection results show that the antibody Ab56 can bind to hSIRP ⁇ V1-Fc, hSIRP ⁇ V2-Fc and hSIRP ⁇ V ⁇ -Fc, and the EC 50 of the binding are 0.0898nM, 0.0783nM and 0.2166nM, respectively.
  • the test results show that the antibody Ab56 is compatible with hSIRP ⁇ (Bypsy, product number SIA-H5225), hSIRP ⁇ 1 (Bypsy, product number SIA-H5257), hSIRP ⁇ (Bypsy , Cat. No. SIG-H5253) can be combined, and their binding EC 50 are 0.0384nM, 0.0236nM and 0.026nM, respectively.
  • the test results showed that the antibody Ab56 was compatible with monkey SIRP ⁇ (Baipusi, product number: SIA-C52H7), mouse SIRP ⁇ (Shenzhou, product number 50956-M08H) and rat SIRP ⁇ ( Sino Biological, 80270-R08H) binding EC 50 are 0.0179nM, 0.5755nM and 5.784nM, respectively.
  • PBMC Human peripheral blood mononuclear cells
  • E-AB-F1230E APC Anti-Human CD14 Antibody[M5E2] (elabscience, Cat. No. E-AB-F1209E), APC Anti-Human CD19 Antibody [CB19] (elabscience, Cat. No. E-AB-F1004E) and APC Anti-Human CD56 Antibody [MEM188] (elabscience, Cat. No. E-AB-F1006E).
  • antibody Ab56 was able to bind to T cells (CD3 + cells) and monocytes (CD14 + cells), but not to B cells (CD19 + cells) and NK cells (CD56 + cells).
  • CD3 + cells Human peripheral blood mononuclear cells were extracted from the purified blood of healthy volunteers, and CD3 + cells were separated and purified by magnetic bead sorting; CD3 + cells were centrifuged at 1500 rpm for 8 min, and a final concentration of 0.25 ⁇ M CFSE (hydroxyl Fluorescein diacetate succinimide lipid, fluorescent dye), labeling at 37°C for 15min, and adding RPMI-1640 complete medium (Gibco) containing 10% FBS to terminate, and standing at room temperature for 5min; centrifuge and wash with RPMI-1640 Adjust the cell density to 1 million/ml with complete medium, and add T cell stimulation activated magnetic beads, 100 ⁇ l/well; dilute the antibody to 40 ⁇ g/mL, 100ul/well; incubate at 37°C, 5% CO 2 for 72 hours; Cells were washed twice with FACS buffer (PBS containing 2% BSA and 0.1% NaN 3 (PH7.4)), using APC Anti-Human CD8
  • E-AB-F1110E PE Anti-Human CD4 Antibody [RPA-T4] (elabscience, catalog number is E-AB-F1109D) and PerCP/Cyanine5.5 Anti-Human CD25 Antibody [BC96] elabscience, catalog number is E-AB-F1194J) for labeling, 1% paraformaldehyde fixed; flow cytometry for detection and analysis.
  • RPA-T4 PE Anti-Human CD4 Antibody
  • BC96 PerCP/Cyanine5.5 Anti-Human CD25 Antibody
  • antibody Ab56 had no effect on the proliferation of CD4 + T cells and CD8 + T cells, which also indicated that T cell proliferation was not affected by the binding of antibody Ab56 to SIRP ⁇ .

Abstract

An anti-SIRPα antibody or an antigen-binding fragment thereof, and the use thereof. The antibody or the antigen-binding fragment thereof can specifically bind to SIRPα and block an SIRPα/CD47 signaling pathway, helps the immune system to remove tumor cells, and is also used in the diagnosis or prognosis of tumors or cancers.

Description

[根据细则91更正 15.08.2022]　抗SIRPα抗体及其应用[Corrected under Rule 91 15.08.2022] Anti-SIRPα antibodies and their applications 技术领域technical field
[根据细则91更正 15.08.2022]　
本发明属于生物医药领域，尤其涉及抗SIRPα抗体及其应用。[Corrected 15.08.2022 under Rule 91]
The invention belongs to the field of biomedicine, and in particular relates to an anti-SIRPα antibody and its application.
背景技术Background technique
SIRP家族一共有5种蛋白，分别是SIRPα、SIRPβ1、SIRPγ、SIRPβ2和SIRPδ，仅SIRPα和SIRPγ能够与配体CD47结合。SIRPβ胞内区缺乏与磷酸酶缔合的信号传导基序，该蛋白与DNAX激活蛋白12(DAP12)缔合，DAP12是二聚体衔接蛋白，其可结合SIRPβ的跨膜区中具有碱性侧链的氨基酸，通过酪氨酸的激活基序(ITAM)传递激活信号。SIRPγ不与DAP12结合，主要表达于T细胞表面，与CD47相互作用，参与T细胞的活化。There are five proteins in the SIRP family, namely SIRPα, SIRPβ1, SIRPγ, SIRPβ2 and SIRPδ, and only SIRPα and SIRPγ can bind to the ligand CD47. The intracellular domain of SIRPβ lacks a signaling motif associated with a phosphatase that associates with DNAX activator protein 12 (DAP12), a dimeric adapter protein with a basic side in the transmembrane domain that binds SIRPβ The chain of amino acids transmits the activation signal through the tyrosine activation motif (ITAM). SIRPγ does not bind to DAP12, is mainly expressed on the surface of T cells, interacts with CD47, and participates in the activation of T cells.
SIRPα也被称为信号调控蛋白α(SIRPα)、CD172a或SHPS-1，其主要表达于髓系细胞表面，如单核细胞、巨噬细胞、粒细胞、树突细胞。SIRPα属于单次跨膜蛋白，SIRPα胞外区包含一个IgV结构域和两个IgC结构域(IgC1、IgC2)，SIRPα与配体CD47结合的区域是IgV结构域；SIRPα胞内区含有酪氨酸激酶抑制基序(ITIM)，促进酪氨酸磷酸酶SHP-1和SHP-2结合，参与髓系细胞的抑制。SIRPα, also known as signal regulatory protein α (SIRPα), CD172a or SHPS-1, is mainly expressed on the surface of myeloid cells, such as monocytes, macrophages, granulocytes, and dendritic cells. SIRPα is a single transmembrane protein. The extracellular region of SIRPα contains an IgV domain and two IgC domains (IgC1, IgC2). The region where SIRPα binds to the ligand CD47 is an IgV domain; the intracellular region of SIRPα contains tyrosine Kinase inhibitory motif (ITIM), which promotes the binding of tyrosine phosphatases SHP-1 and SHP-2, is involved in the inhibition of myeloid cells.
人SIRPα(即hSIRPα)基因有8种变体：V1型、V2型、V3型、V4型、V5型、V6型、V8型和V9型，其中V1、V2和V8型占90％以上。V1、V2和V8型的hSIRPα的细胞外IgV结构域存在13个氨基酸的差异。Human SIRPα (hSIRPα) gene has 8 variants: V1, V2, V3, V4, V5, V6, V8 and V9, of which V1, V2 and V8 account for more than 90%. There is a 13 amino acid difference in the extracellular IgV domain of hSIRPα between V1, V2 and V8 types.
靶细胞表面CD47与抑制性受体SIRPα结合，促使SIRPα胞内区被磷酸化，招募并激活蛋白酪氨酸磷酸酶SHP-1和SHP-2，最终导致肌球蛋白IIA功能被抑制，产生“别吃我”信号。通过阻断SIRPα/CD47信号通路，抗SIRPα抗体或其抗原结合片段可以增强巨噬细胞的吞噬作用，有助于免疫系统清除肿瘤细胞。CD47 on the surface of the target cell binds to the inhibitory receptor SIRPα, which promotes the phosphorylation of the intracellular domain of SIRPα, recruits and activates the protein tyrosine phosphatases SHP-1 and SHP-2, and finally leads to the inhibition of the function of myosin IIA, resulting in " Don't eat me" signal. By blocking the SIRPα/CD47 signaling pathway, anti-SIRPα antibodies or their antigen-binding fragments can enhance the phagocytosis of macrophages and help the immune system to clear tumor cells.
发明内容Contents of the invention
本发明提供了抗SIRPα抗体或抗原结合片段，这些抗体或抗原结合片段可以特异性结合SIRPα，阻断SIRPα/CD47信号通路，有助于免疫系统的清除功能。The present invention provides anti-SIRPα antibodies or antigen-binding fragments. These antibodies or antigen-binding fragments can specifically bind SIRPα, block SIRPα/CD47 signaling pathway, and contribute to the immune system's clearance function.
一些实施方案提供了抗体或抗原结合片段，所述抗体或抗原结合片段特异性结合 SIRPα、SIRPβ和SIRPγ，同时阻断SIRPα/CD47信号通路。Some embodiments provide antibodies or antigen-binding fragments that specifically bind SIRPα, SIRPβ, and SIRPγ while blocking the SIRPα/CD47 signaling pathway.
在一些实施方案中，所述抗体或抗原结合片段结合人源SIRPα、猴源SIRPα和小鼠SIRPα。在一些实施方案中，所述抗体或抗原结合片段结合人源SIRPα。在一些实施方案中，人源SIRPα包括V1型SIRPα、V2型SIRPα和V8型SIRPα。In some embodiments, the antibody or antigen-binding fragment binds human SIRPα, monkey SIRPα, and mouse SIRPα. In some embodiments, the antibody or antigen-binding fragment binds human SIRPα. In some embodiments, the human SIRPα includes V1 type SIRPα, V2 type SIRPα, and V8 type SIRPα.
一些实施方案中提供了抗体或抗原结合片段，所述抗体或抗原结合片段结合SIRPγ，同时不影响T细胞增殖。Some embodiments provide antibodies or antigen-binding fragments that bind SIRPγ without affecting T cell proliferation.
一些实施方案提供了抗体或抗原结合片段，所述抗体或抗原结合片段特异性结合SIRPα，并且包含以下中的一个或多个氨基酸序列：Some embodiments provide an antibody or antigen-binding fragment that specifically binds SIRPα and comprising one or more of the following amino acid sequences:
(a)HCDR1，其包含如SEQ ID NO：1所示的氨基酸序列或与SEQ ID NO：1相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；(a) HCDR1 comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 1;
(b)HCDR2，其包含如SEQ ID NO：2所示的氨基酸序列或与SEQ ID NO：2相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；(b) HCDR2 comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 2;
(c)HCDR3，其包含如SEQ ID NO：3-8中任一项所示的氨基酸序列或与SEQ ID NO：3-8中任一项相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；(c) HCDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 3-8 or an amino acid having a single site substitution, deletion or insertion compared to any one of SEQ ID NOs: 3-8 sequence, or consisting of it;
(d)LCDR1，其包含如SEQ ID NO：9所示的氨基酸序列或与SEQ ID NO：9相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；(d) LCDR1 comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 9;
(e)LCDR2，其包含如SEQ ID NO：10所示的氨基酸序列或与SEQ ID NO：10相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；(e) LCDR2 comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 10;
(f)LCDR3，其包含如SEQ ID NO：11-15中任一项所示的氨基酸序列或与SEQ ID NO：11-15中任一项相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成。(f) LCDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 11-15 or an amino acid having a single site substitution, deletion or insertion compared to any one of SEQ ID NOs: 11-15 sequence, or consists of it.
一些实施方案中提供了抗体或抗原结合片段，所述抗体或抗原结合片段特异性结合SIRPα，并且包含：Provided in some embodiments are antibodies or antigen-binding fragments that specifically bind SIRPα and comprising:
(a)HCDR1，其包含如SEQ ID NO：1所示的氨基酸序列或与SEQ ID NO：1相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；(a) HCDR1 comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 1;
(b)HCDR2，其包含如SEQ ID NO：2所示的氨基酸序列或与SEQ ID NO：2相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；和(b) HCDR2 comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 2; and
(c)HCDR3，其包含如SEQ ID NO：3-8中任一项所示的氨基酸序列或与SEQ ID NO：3-8中任一项相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成。(c) HCDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 3-8 or an amino acid having a single site substitution, deletion or insertion compared to any one of SEQ ID NOs: 3-8 sequence, or consists of it.
在一些实施方案中，HCDR1包含SEQ ID NO：1所示的氨基酸序列或由其组成，HCDR2包含SEQ ID NO：2所示的氨基酸序列或由其组成，HCDR3包含SEQ ID NO：3所示的氨基酸序列或由其组成。In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1 or consists of it, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2 or consists of it, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3 An amino acid sequence or consisting of it.
在一些实施方案中，HCDR1包含SEQ ID NO：1所示的氨基酸序列或由其组成，HCDR2包含SEQ ID NO：2所示的氨基酸序列或由其组成，HCDR3包含SEQ ID NO：4 所示的氨基酸序列或由其组成。In some embodiments, HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 4 An amino acid sequence or consisting of it.
在一些实施方案中，HCDR1包含SEQ ID NO：1所示的氨基酸序列或由其组成，HCDR2包含SEQ ID NO：2所示的氨基酸序列或由其组成，HCDR3包含SEQ ID NO：5所示的氨基酸序列或由其组成。In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1 or consists of it, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2 or consists of it, HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 5 An amino acid sequence or consisting of it.
在一些实施方案中，HCDR1包含SEQ ID NO：1所示的氨基酸序列或由其组成，HCDR2包含SEQ ID NO：2所示的氨基酸序列或由其组成，HCDR3包含SEQ ID NO：6所示的氨基酸序列或由其组成。In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1 or consists of it, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2 or consists of it, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 6 An amino acid sequence or consisting of it.
在一些实施方案中，HCDR1包含SEQ ID NO：1所示的氨基酸序列或由其组成，HCDR2包含SEQ ID NO：2所示的氨基酸序列或由其组成，HCDR3包含SEQ ID NO：7所示的氨基酸序列或由其组成。In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1 or consists of it, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2 or consists of it, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 7 An amino acid sequence or consisting of it.
在一些实施方案中，HCDR1包含SEQ ID NO：1所示的氨基酸序列或由其组成，HCDR2包含SEQ ID NO：2所示的氨基酸序列或由其组成，HCDR3包含SEQ ID NO：8所示的氨基酸序列或由其组成。In some embodiments, HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1 or consists of it, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2 or consists of it, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 8 An amino acid sequence or consisting of it.
一些实施方案提供了抗体或抗原结合片段，所述抗体或抗原结合片段特异性结合SIRPα，并且包含：Some embodiments provide an antibody or antigen-binding fragment that specifically binds SIRPα and comprising:
(d)LCDR1，其包含如SEQ ID NO：9所示的氨基酸序列或与SEQ ID NO：9相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；(d) LCDR1 comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 9;
(e)LCDR2，其包含如SEQ ID NO：10所示的氨基酸序列或与SEQ ID NO：10相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；和(e) LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 10; and
(f)LCDR3，其包含如SEQ ID NO：11-15中任一项所示的氨基酸序列或与SEQ ID NO：11-15中任一项相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成。(f) LCDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 11-15 or an amino acid having a single site substitution, deletion or insertion compared to any one of SEQ ID NOs: 11-15 sequence, or consists of it.
在一些实施方案中，LCDR1包含SEQ ID NO：9所示的氨基酸序列或由其组成，LCDR2包含SEQ ID NO：10所示的氨基酸序列或由其组成，LCDR3包含SEQ ID NO：11所示的氨基酸序列或由其组成。In some embodiments, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9 or consists of it, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10 or consists of it, LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11 An amino acid sequence or consisting of it.
在一些实施方案中，LCDR1包含SEQ ID NO：9所示的氨基酸序列或由其组成，LCDR2包含SEQ ID NO：10所示的氨基酸序列或由其组成，LCDR3包含SEQ ID NO：12所示的氨基酸序列或由其组成。In some embodiments, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9 or consists of it, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10 or consists of it, LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 12 An amino acid sequence or consisting of it.
在一些实施方案中，LCDR1包含SEQ ID NO：9所示的氨基酸序列或由其组成，LCDR2包含SEQ ID NO：10所示的氨基酸序列或由其组成，LCDR3包含SEQ ID NO：13所示的氨基酸序列或由其组成。In some embodiments, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9 or consists of it, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10 or consists of it, LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 13 An amino acid sequence or consisting of it.
在一些实施方案中，LCDR1包含SEQ ID NO：9所示的氨基酸序列或由其组成，LCDR2包含SEQ ID NO：10所示的氨基酸序列或由其组成，LCDR3包含SEQ ID NO：14所示的氨基酸序列或由其组成。In some embodiments, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9 or consists of it, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10 or consists of it, LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 14 An amino acid sequence or consisting of it.
在一些实施方案中，LCDR1包含SEQ ID NO：9所示的氨基酸序列或由其组成， LCDR2包含SEQ ID NO：10所示的氨基酸序列或由其组成，LCDR3包含SEQ ID NO：15所示的氨基酸序列或由其组成。In some embodiments, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9 or consists of it, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10 or consists of it, LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 15 An amino acid sequence or consisting of it.
一些实施方案提供了抗体或抗原结合片段，所述抗体或抗原结合片段特异性结合SIRPα，并且包含：Some embodiments provide an antibody or antigen-binding fragment that specifically binds SIRPα and comprising:
(a)HCDR1，其包含如SEQ ID NO：1所示的氨基酸序列或与SEQ ID NO：1相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；(a) HCDR1 comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 1;
(b)HCDR2，其包含如SEQ ID NO：2所示的氨基酸序列或与SEQ ID NO：2相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；(b) HCDR2 comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 2;
(c)HCDR3，其包含如SEQ ID NO：3-8中任一项所示的氨基酸序列或与SEQ ID NO：3-8中任一项相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；(c) HCDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 3-8 or an amino acid having a single site substitution, deletion or insertion compared to any one of SEQ ID NOs: 3-8 sequence, or consisting of it;
(d)LCDR1，其包含如SEQ ID NO：9所示的氨基酸序列或与SEQ ID NO：9相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；(d) LCDR1 comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 9;
(e)LCDR2，其包含如SEQ ID NO：10所示的氨基酸序列或与SEQ ID NO：10相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；和(e) LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 10; and
(f)LCDR3，其包含如SEQ ID NO：11-15中任一项所示的氨基酸序列或与SEQ ID NO：11-15中任一项相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成。(f) LCDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 11-15 or an amino acid having a single site substitution, deletion or insertion compared to any one of SEQ ID NOs: 11-15 sequence, or consists of it.
在一些实施方案中，所述抗体或抗原结合片段包含如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：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：11所示的LCDR3。In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 3, HCDR3 as shown in SEQ ID NO: LCDR1 shown in NO: 9, LCDR2 shown in SEQ ID NO: 10 and LCDR3 shown in SEQ ID NO: 11.
在一些实施方案中，所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：4所示的HCDR3、如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：11所示的LCDR3。In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 4, HCDR3 as shown in SEQ ID NO: LCDR1 shown in NO: 9, LCDR2 shown in SEQ ID NO: 10 and LCDR3 shown in SEQ ID NO: 11.
在一些实施方案中，所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：5所示的HCDR3、如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：11所示的LCDR3。In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 5, HCDR3 as shown in SEQ ID NO: LCDR1 shown in NO: 9, LCDR2 shown in SEQ ID NO: 10 and LCDR3 shown in SEQ ID NO: 11.
在一些实施方案中，所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：6所示的HCDR3、如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：11所示的LCDR3。In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 6, HCDR3 as shown in SEQ ID NO: LCDR1 shown in NO: 9, LCDR2 shown in SEQ ID NO: 10 and LCDR3 shown in SEQ ID NO: 11.
在一些实施方案中，所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：7所示的HCDR3、如SEQ ID  NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：11所示的LCDR3。In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 7, HCDR3 as shown in SEQ ID NO: LCDR1 shown in NO: 9, LCDR2 shown in SEQ ID NO: 10 and LCDR3 shown in SEQ ID NO: 11.
在一些实施方案中，所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：8所示的HCDR3、如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：11所示的LCDR3。In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 8, HCDR3 as shown in SEQ ID NO: LCDR1 shown in NO: 9, LCDR2 shown in SEQ ID NO: 10 and LCDR3 shown in SEQ ID NO: 11.
在一些实施方案中，所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：6所示的HCDR3、如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：12所示的LCDR3。In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 6, HCDR3 as shown in SEQ ID NO: LCDR1 shown in NO: 9, LCDR2 shown in SEQ ID NO: 10 and LCDR3 shown in SEQ ID NO: 12.
在一些实施方案中，所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：6所示的HCDR3、如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：13所示的LCDR3。In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 6, HCDR3 as shown in SEQ ID NO: LCDR1 shown in NO: 9, LCDR2 shown in SEQ ID NO: 10 and LCDR3 shown in SEQ ID NO: 13.
在一些实施方案中，所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：6所示的HCDR3、如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：14所示的LCDR3。In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 6, HCDR3 as shown in SEQ ID NO: LCDR1 shown in NO: 9, LCDR2 shown in SEQ ID NO: 10 and LCDR3 shown in SEQ ID NO: 14.
在一些实施方案中，所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：6所示的HCDR3、如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：15所示的LCDR3。In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 6, HCDR3 as shown in SEQ ID NO: LCDR1 shown in NO: 9, LCDR2 shown in SEQ ID NO: 10 and LCDR3 shown in SEQ ID NO: 15.
在一些实施方案中，所述取代为保守氨基酸取代。In some embodiments, the substitutions are conservative amino acid substitutions.
一些实施方案提供了抗体或抗体原结合片段，所述抗体或抗原结合片段特异性结合SIRPα，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：16-21中任一项所示的序列，与SEQ ID NO：16-21中任一项所示序列相比具有至少80％同一性的序列，或与SEQ ID NO：16-21中任一项所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列，或由其组成。Some embodiments provide an antibody or antigen-binding fragment that specifically binds SIRPα, the heavy chain variable region of the antibody or antigen-binding fragment comprising any one of SEQ ID NOs: 16-21 The sequence shown, a sequence having at least 80% identity compared to the sequence shown in any one of SEQ ID NO: 16-21, or having one sequence compared to the sequence shown in any one of SEQ ID NO: 16-21 or an amino acid sequence with multiple conservative amino acid substitutions, or consists of it.
一些实施方案提供了抗体或抗体原结合片段，所述抗体或抗原结合片段特异性结合SIRPα，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：22-26中任一项所示的序列，与SEQ ID NO：22-26中任一项所示序列相比具有至少80％同一性的序列，或与SEQ ID NO：22-26中任一项所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列，或由其组成。Some embodiments provide an antibody or antigen-binding fragment that specifically binds SIRPα, the light chain variable region of the antibody or antigen-binding fragment comprising any one of SEQ ID NOs: 22-26 The sequence shown, a sequence having at least 80% identity compared to the sequence shown in any one of SEQ ID NO: 22-26, or having one sequence compared to the sequence shown in any one of SEQ ID NO: 22-26 or an amino acid sequence with multiple conservative amino acid substitutions, or consists of it.
在一些实施方案中，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：16所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：22所示的序列。In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 16, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 22 sequence shown.
在一些实施方案中，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：17所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：22所示的序列。In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 17, and the light chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO: 22 sequence shown.
在一些实施方案中，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：18所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：22所示的序列。In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 18, and the light chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO: 22 sequence shown.
在一些实施方案中，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：19所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：22所示的序列。In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 19, and the light chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO: 22 sequence shown.
在一些实施方案中，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：20所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：22所示的序列。In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 20, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 22 sequence shown.
在一些实施方案中，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：21所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：22所示的序列。In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 21, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 22 sequence shown.
在一些实施方案中，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：19所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：23所示的序列。In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 19, and the light chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO: 23 sequence shown.
在一些实施方案中，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：19所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：24所示的序列。In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 19, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 24 sequence shown.
在一些实施方案中，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：19所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：25所示的序列。In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 19, and the light chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO: 25 sequence shown.
在一些实施方案中，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：19所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：26所示的序列。In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 19, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 26 sequence shown.
在一些实施方案中，抗体或抗原结合片段还包含重链恒定区、轻链恒定区、Fc区或其组合。在一些实施方案中，轻链恒定区是κ或λ链恒定区。在一些实施方案中，抗体或抗原结合片段是IgG、IgM、IgA、IgE或IgD其中一种同种型。在一些实施方案中，同种型是IgG1、IgG2、IgG3或IgG4。在一些实施方案中，抗体或抗原结合片段是鼠源抗体、嵌合抗体、人源化抗体或全人源抗体。In some embodiments, the antibody or antigen-binding fragment further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In some embodiments, the light chain constant region is a kappa or lambda chain constant region. In some embodiments, the antibody or antigen-binding fragment is an isotype of IgG, IgM, IgA, IgE, or IgD. In some embodiments, the isotype is IgGl, IgG2, IgG3 or IgG4. In some embodiments, the antibody or antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.
在一些实施方案中，Fc是变体Fc区。在一些实施方案中，相对于亲本Fc区，变体Fc区具有一个或多个氨基酸修饰，如取代、缺失或插入。在一些实施方案中，相对于亲本Fc区活性，Fc区的氨基酸修饰改变了效应功能活性。在一些实施方案中，变体Fc区可以具有改变(增加的或降低的)的抗体依赖性细胞毒性(ADCC)、补体介导的细胞毒性(CDC)、吞噬作用、调理作用或细胞结合。在一些实施方案中，相对于亲本Fc区，Fc区氨基酸修饰可以改变变体Fc区对FcγR(Fcγ受体)的亲和力。在一些实施方案中，所述Fc区来源于IgG1或IgG4。在一些实施方案中，Fc区突变是N297A、L234A或L235A(Eu编号)。在一些实施方案中，Fc区突变是E345R或S440Y(Eu编号)。In some embodiments, the Fc is a variant Fc region. In some embodiments, the variant Fc region has one or more amino acid modifications, such as substitutions, deletions or insertions, relative to the parental Fc region. In some embodiments, the amino acid modification of the Fc region alters effector function activity relative to the activity of the parental Fc region. In some embodiments, the variant Fc region may have altered (increased or decreased) antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization, or cell binding. In some embodiments, amino acid modifications of the Fc region can alter the affinity of the variant Fc region for FcγR (Fcγ receptor) relative to the parent Fc region. In some embodiments, the Fc region is derived from IgGl or IgG4. In some embodiments, the Fc region mutation is N297A, L234A, or L235A (Eu numbering). In some embodiments, the Fc region mutation is E345R or S440Y (Eu numbering).
在一些实施方案中，所述抗体或抗原结合片段为分离的抗体或抗原结合片段。在一些实施方案中，所述抗体或抗原结合片段为scFv、Fab或F(ab) 2。在一些实施方案中，所述抗体或抗原结合片段为单克隆抗体。 In some embodiments, the antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is a scFv, Fab or F(ab) 2 . In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody.
在一些实施方案中，所述抗体或抗原结合片段的重链恒定区包含氨基酸序列如SEQ ID NO：27-29中任一项所示的序列，或与SEQ ID NO：27-29中任一项所示序列相比具有至少80％同一性的序列，或与SEQ ID NO：27-29中任一项所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列，或由其组成；和/或In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in any one of SEQ ID NOs: 27-29, or any of SEQ ID NOs: 27-29 or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 27-29, or consisting of a sequence having at least 80% identity compared to the sequence shown in Item 1; and / or
所述抗体或抗原结合片段的轻链恒定区包含氨基酸序列如SEQ ID NO：30所示的序列，或与SEQ ID NO：30所示序列相比具有至少80％同一性的序列，或与SEQ ID NO：30所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列，或由其组成。The light chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 30, or a sequence with at least 80% identity compared with the sequence shown in SEQ ID NO: 30, or a sequence with SEQ ID NO: 30. The amino acid sequence shown in ID NO: 30 has one or more conservative amino acid substitutions compared to the sequence, or consists of it.
在一些实施方案中，所述抗体或抗原结合片段的恒定区来源于IgG1。在一些实施方案中，所述抗体或抗原结合片段的重链恒定区包含氨基酸序列如SEQ ID NO：27所示的序列，所述抗体或抗原结合片段的轻链恒定区包含氨基酸序列如SEQ ID NO：30所示的序列。In some embodiments, the constant region of the antibody or antigen-binding fragment is derived from IgG1. In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 27, and the light chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: the sequence shown in 30.
在一些实施方案中，所述抗体或抗原结合片段的恒定区来源于IgG2。在一些实施方案中，所述抗体或抗原结合片段的重链恒定区包含氨基酸序列如SEQ ID NO：28所示的序列，所述抗体或抗原结合片段的轻链恒定区包含氨基酸序列如SEQ ID NO：30所示的序列。In some embodiments, the constant region of the antibody or antigen-binding fragment is derived from IgG2. In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 28, and the light chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: the sequence shown in 30.
在一些实施方案中，所述抗体或抗原结合片段的恒定区来源于IgG4。在一些实施方案中，所述抗体或抗原结合片段的重链恒定区包含氨基酸序列如SEQ ID NO：29所示的序列，所述抗体或抗原结合片段的轻链恒定区包含氨基酸序列如SEQ ID NO：30所示的序列。In some embodiments, the constant region of the antibody or antigen-binding fragment is derived from IgG4. In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 29, and the light chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: the sequence shown in 30.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：31-36中任一项所示的序列，与SEQ ID NO：31-36中任一项所示序列相比具有至少80％同一性的序列，或与SEQ ID NO：31-36中任一项所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列，或由其组成；和/或In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in any one of SEQ ID NO: 31-36, compared with the sequence shown in any one of SEQ ID NO: 31-36 has A sequence of at least 80% identity, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 31-36, or consisting of; and/or
所述抗体的轻链包含氨基酸序列如SEQ ID NO：39-43中任一项所示的序列，与SEQ ID NO：39-43中任一项所示序列相比具有至少80％同一性的序列，或与SEQ ID NO：39-43中任一项所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列，或由其组成。The light chain of the antibody comprises an amino acid sequence as shown in any one of SEQ ID NO: 39-43, which has at least 80% identity compared with the sequence shown in any one of SEQ ID NO: 39-43 sequence, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 39-43, or consisting of.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：31-36中任一项所示的序列；和/或In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in any one of SEQ ID NO: 31-36; and/or
所述抗体的轻链包含氨基酸序列如SEQ ID NO：39-43中任一项所示的序列。The light chain of the antibody comprises an amino acid sequence as shown in any one of SEQ ID NO: 39-43.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：31所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：39所示的序列。In some embodiments, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 31, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 39.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：32所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：39所示的序列。In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 32, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 39.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：33所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：39所示的序列。In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 33, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 39.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：34所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：39所示的序列。In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 39.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：35所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：39所示的序列。In some embodiments, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 35, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 39.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：36所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：39所示的序列。In some embodiments, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 36, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 39.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：34所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：40所示的序列。In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 40.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：34所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：41所示的序列。In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 41.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：34所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：42所示的序列。In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 42.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：34所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：43所示的序列。In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 43.
在一些实施方案中，所述抗体或抗原结合片段为单克隆抗体(包括全长单克隆抗体)或多特异性抗体或抗原结合片段(例如双特异性抗体或抗原结合片段)。In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody (including a full-length monoclonal antibody) or a multispecific antibody or antigen-binding fragment (eg, a bispecific antibody or antigen-binding fragment).
在一些实施方案中，所述抗体具有两条序列相同的重链和两条序列相同的轻链，Fc区配对形成二硫键。In some embodiments, the antibody has two heavy chains of identical sequence and two light chains of identical sequence, and the Fc regions pair to form disulfide bonds.
本发明还提供了编码所述的抗体或抗原结合片段的核酸。在一些实施方案中，所述核酸为分离的核酸。在一些实施方案中，所述核酸序列选自表6所列的核酸序列。The present invention also provides nucleic acid encoding the antibody or antigen-binding fragment. In some embodiments, the nucleic acid is an isolated nucleic acid. In some embodiments, the nucleic acid sequence is selected from the nucleic acid sequences listed in Table 6.
本发明还提供了包含所述的核酸的载体。在一些实施方案中，包含所述核酸的载体为核酸片段、质粒、噬菌体或病毒。在一些实施方案中，所述载体为分离的载体。The present invention also provides a vector comprising the nucleic acid. In some embodiments, the vector comprising the nucleic acid is a nucleic acid fragment, plasmid, phage, or virus. In some embodiments, the vector is an isolated vector.
本发明还提供了包含所述核酸或载体的宿主细胞。在一些实施方案中，所述宿主细胞为分离的宿主细胞。在一些实施方案中，所述宿主细胞为CHO细胞、HEK细胞(如HEK293F细胞)、BHK细胞、Cos1细胞、Cos7细胞、CV1细胞或鼠L细胞。The present invention also provides a host cell comprising the nucleic acid or vector. In some embodiments, the host cell is an isolated host cell. In some embodiments, the host cells are CHO cells, HEK cells (such as HEK293F cells), BHK cells, Cos1 cells, Cos7 cells, CV1 cells or murine L cells.
本发明还提供了制备所述抗体或抗原结合片段的方法，其包括在培养基里培养包含编码所述抗体或抗原结合片段的核酸的宿主细胞。在一些实施方案中，所述方法还包括纯化所述抗体或抗原结合片段。纯化可以采用常规方法进行，例如先离心细胞悬液并收集上清液，再次离心进一步去除杂质。Protein A亲和柱和离子交换柱等方法可以用于纯化抗体蛋白。The present invention also provides a method for producing the antibody or antigen-binding fragment, which comprises culturing a host cell comprising a nucleic acid encoding the antibody or antigen-binding fragment in a culture medium. In some embodiments, the method further comprises purifying the antibody or antigen-binding fragment. Purification can be carried out by conventional methods, such as centrifuging the cell suspension first, collecting the supernatant, and centrifuging again to further remove impurities. Methods such as Protein A affinity column and ion exchange column can be used to purify antibody protein.
本发明还提供了药物组合物，所述药物组合物包含上述的抗体或抗原结合片段，以及药学上可接受的辅料。The present invention also provides a pharmaceutical composition, which comprises the above-mentioned antibody or antigen-binding fragment, and pharmaceutically acceptable auxiliary materials.
[根据细则91更正 15.08.2022]　
本发明还提供了预防或治疗免疫细胞功能障碍疾病(如吞噬细胞功能障碍疾病)的方法和用途。在一些实施方案中，提供了用于治疗或改善免疫细胞功能障碍疾病(如吞噬细胞功能障碍疾病)的方法，所述方法包括向患者施用有效剂量的所述抗SIRPα抗体或抗原结合片段。在一些实施方案中，提供了所述抗体或抗原结合片段在用于治疗或改善免疫细胞功能障碍疾病(如吞噬细胞功能障碍疾病)中的应用。在一些实施方案中，提供了所述抗体或抗原结合片段在制备用于治疗或改善免疫细胞功能障碍疾病(如吞噬细胞功能障碍疾病)的药物中的应用。[Corrected 15.08.2022 under Rule 91]
The present invention also provides methods and uses for preventing or treating immune cell dysfunction diseases (such as phagocytic cell dysfunction diseases). In some embodiments, there is provided a method for treating or improving an immune cell dysfunction disease (such as a phagocytic cell dysfunction disease), the method comprising administering an effective dose of the anti-SIRPα antibody or antigen-binding fragment to a patient. In some embodiments, use of the antibody or antigen-binding fragment for treating or improving immune cell dysfunction diseases (such as phagocytic cell dysfunction diseases) is provided. In some embodiments, use of the antibody or antigen-binding fragment in the preparation of a medicament for treating or improving immune cell dysfunction (such as phagocyte dysfunction) is provided.
在一些实施方案中，所述吞噬细胞功能障碍疾病包括但不限于：癌症、慢性感染、和自生免疫疾病。在一些实施方案中，癌症包括但不限于具有浸润性骨髓细胞(特别是具有浸润性MDSC和/或TAM细胞)的癌症。在一些实施方案中，癌症选自肺癌、间皮瘤癌、卵巢癌、肝癌、膀胱癌、脑癌、乳腺癌、结肠癌、肉瘤、胰腺癌、头颈癌、肾癌、胸腺瘤、胶质瘤、黑色素瘤和血液系统癌症。在一些实施方案中，癌症选自淋巴瘤(霍奇金淋巴瘤和非霍奇金淋巴瘤)、白血病(淋巴细胞白血病和髓细胞性白血病)和骨髓瘤。In some embodiments, the phagocytic cell dysfunction diseases include, but are not limited to, cancer, chronic infection, and autoimmune diseases. In some embodiments, cancers include, but are not limited to, cancers with infiltrating myeloid cells, particularly with infiltrating MDSC and/or TAM cells. In some embodiments, the cancer is selected from lung cancer, mesothelioma cancer, ovarian cancer, liver cancer, bladder cancer, brain cancer, breast cancer, colon cancer, sarcoma, pancreatic cancer, head and neck cancer, kidney cancer, thymoma, glioma , melanoma and hematological cancers. In some embodiments, the cancer is selected from lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma), leukemia (lymphoblastic leukemia and myelogenous leukemia), and myeloma.
[根据细则91更正 15.08.2022]　
本发明还提供了诊断方法和用途。在一些实施方案中，提供了检测样品中SIRPα表达的方法，使样品与所述抗SIRPα抗体或抗原结合片段进行接触，使得所述抗体或抗原结合片段结合SIRPα，并检测其结合，即样品中SIRPα的含量。在一些实施方案中，提供了所述抗SIRPα抗体或抗原结合片段在制备用于诊断或预后免疫细胞功能障碍疾病(如癌症)试剂盒中的应用。在一些实施方案中，提供了一种包含所述抗SIRPα抗体或抗原结合片段的诊断或预后试剂盒；任选的，所述试剂盒还包括第二抗体，其特异性识别所述抗SIRPα抗体；任选的，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、化学发光物质、有色物质或酶；任选的，所述试剂盒用于检测SIRPα在样品中的存在或其水平；任选的，所述试剂盒还包括针对其它抗原的抗体或抗原结合片段，和/或细胞毒性剂，和任选的，使用说明书。[Corrected 15.08.2022 under Rule 91]
The invention also provides diagnostic methods and uses. In some embodiments, a method of detecting SIRPα expression in a sample is provided, the sample is contacted with the anti-SIRPα antibody or antigen-binding fragment, such that the antibody or antigen-binding fragment binds SIRPα, and the binding is detected, i.e., in the sample SIRPα content. In some embodiments, the use of the anti-SIRPα antibody or antigen-binding fragment in the preparation of a kit for diagnosing or prognosing immune cell dysfunction diseases (such as cancer) is provided. In some embodiments, a diagnostic or prognostic kit comprising the anti-SIRPα antibody or antigen-binding fragment is provided; optionally, the kit further includes a second antibody that specifically recognizes the anti-SIRPα antibody Optionally, the second antibody also includes a detectable label, such as a radioactive isotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme; Optionally, the kit is used to detect the presence of SIRPα in a sample or levels thereof; optionally, the kit further includes antibodies or antigen-binding fragments directed against other antigens, and/or cytotoxic agents, and optionally, instructions for use.
[根据细则91更正 15.08.2022]　
本发明提供了抗SIRPα抗体或抗原结合片段及其应用，本发明抗体或抗原结合片段可以特异性结合SIRPα，阻断SIRPα/CD47信号通路，有助于免疫系统清除肿瘤细 胞。本发明抗体或抗原结合片段可以用于治疗或改善癌症，也可以用于癌症诊断和预后。[Corrected 15.08.2022 under Rule 91]
The invention provides an anti-SIRPα antibody or antigen-binding fragment and application thereof. The antibody or antigen-binding fragment of the invention can specifically bind to SIRPα, block the SIRPα/CD47 signaling pathway, and help the immune system to clear tumor cells. The antibody or antigen-binding fragment of the present invention can be used to treat or improve cancer, and can also be used for cancer diagnosis and prognosis.
附图说明Description of drawings
图1示scFv阻断CD47与SIRPα的结合。Figure 1 shows that scFv blocks the binding of CD47 to SIRPα.
[根据细则91更正 15.08.2022]　
图2示抗SIRPα抗体与PBMC结合的情况。[Corrected 15.08.2022 under Rule 91]
Figure 2 shows the binding of anti-SIRPα antibody to PBMC.
[根据细则91更正 15.08.2022]　
图3示抗SIRPα抗体对T细胞增殖的影响；图3A中显示的是CD4 +CD25 +T细胞，图3B中显示的是CD8 +CD25 +T细胞；AB56表示抗体Ab56，HEFLB表示抗体18D5 HEFLB。[Corrected 15.08.2022 under Rule 91]
Figure 3 shows the effect of anti-SIRPα antibody on T cell proliferation; Figure 3A shows CD4 + CD25 + T cells, and Figure 3B shows CD8 + CD25 + T cells; AB56 indicates antibody Ab56, and HEFLB indicates antibody 18D5 HEFLB.
术语the term
除非另作说明，否则下列的每一个术语应当具有下文所述的含义。Unless otherwise specified, each of the following terms shall have the meaning set forth below.
定义definition
应当注意的是，术语“一种”实体是指一种或多种该实体，例如“一种抗体”应当被理解为一种或多种抗体，因此，术语“一种”(或“一个”)、“一种或多种”和“至少一种”可以在本文中互换使用。It should be noted that the term "an" entity refers to one or more such entities, for example "an antibody" should be understood as one or more antibodies, therefore, the term "a" (or "an" ), "one or more" and "at least one" may be used interchangeably herein.
本文所用的术语″包含″或″包括″意味着组合物和方法等包括所列举的元素，例如组份或步骤，但不排除其它。″基本上由......组成″意味着组合物和方法排除对组合的特征有根本影响的其它元素，但不排除对组合物或方法无本质上影响的元素。″由......组成″意味着排除未特别列举的元素。As used herein, the term "comprises" or "comprises" means that compositions, methods, etc. include the listed elements, such as components or steps, but not exclude others. "Consisting essentially of" means that the compositions and methods exclude other elements that substantially affect the characteristics of the combination, but do not exclude elements that do not substantially affect the composition or method. "Consisting of" means excluding elements not specifically listed.
术语“多肽”旨在涵盖单数的“多肽”以及复数的“多肽”，并且是指由通过酰胺键(也称为肽键)线性连接的氨基酸单体形成的分子。术语“多肽”是指两个或更多个氨基酸的任何单条链或多条链，并且不涉及产物的特定长度。因此，“多肽”的定义中包括肽、二肽、三肽、寡肽、“蛋白质”、“氨基酸链”或用于指两个或多个氨基酸链的任何其他术语，并且术语“多肽”可以用来代替上述任何一个术语，或者与上述任何一个术语交替使用。术语“多肽”也意在指多肽表达后修饰的产物，包括但不限于糖基化、乙酰化、磷酸化、酰胺化、通过已知的保护/封闭基团衍生化、蛋白水解切割或非天然发生的氨基酸修饰。多肽可以源自天然生物来源或通过重组技术产生，但其不必从指定的核酸序列翻译所得，它可能以包括化学合成的任何方式产生。The term "polypeptide" is intended to encompass the singular as well as the plural "polypeptides" and refers to a molecule formed of amino acid monomers linked linearly by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, the definition of "polypeptide" includes peptide, dipeptide, tripeptide, oligopeptide, "protein", "amino acid chain" or any other term used to refer to a chain of two or more amino acids, and the term "polypeptide" may Used in place of, or interchangeably with, any of the above terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-natural Amino acid modifications that occur. A polypeptide may be derived from natural biological sources or produced by recombinant techniques, but it need not be translated from a specified nucleic acid sequence, it may be produced by any means including chemical synthesis.
“氨基酸”是指既含氨基又含羧基的有机化合物，比如α-氨基酸，其可直接或以前体的形式由核酸编码。单个氨基酸由三个核苷酸(所谓的密码子或碱基三联体)组成的 核酸编码。每一个氨基酸由至少一个密码子编码。相同氨基酸由不同密码子编码称为“遗传密码的简并性”。氨基酸包括天然氨基酸和非天然氨基酸。天然氨基酸包括丙氨酸(三字母代码：ala，一字母代码：A)、精氨酸(arg，R)、天冬酰胺(asn，N)、天冬氨酸(asp，D)、半胱氨酸(cys，C)、谷氨酰胺(gln，Q)、谷氨酸(glu，E)、甘氨酸(gly，G)、组氨酸(his，H)、异亮氨酸(ile，I)、亮氨酸(leu，L)、赖氨酸(lys，K)、甲硫氨酸(met，M)、苯丙氨酸(phe，F)、脯氨酸(pro，P)、丝氨酸(ser，S)、苏氨酸(thr，T)、色氨酸(trp，W)、酪氨酸(tyr，Y)和缬氨酸(val，V)。"Amino acid" refers to an organic compound containing both amino and carboxyl groups, such as an α-amino acid, which can be encoded by a nucleic acid directly or in the form of a precursor. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called "degeneracy of the genetic code". Amino acids include natural amino acids and unnatural amino acids. Natural amino acids include alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine amino acid (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I ), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).
“保守氨基酸取代”是指一个氨基酸残基被另一个含有化学性质(例如电荷或疏水性)相似的侧链(R基团)的氨基酸残基所取代。一般而言，保守氨基酸取代不大会在实质上改变蛋白质的功能性质。含有化学性质相似侧链的氨基酸类别的实例包括：1)脂族侧链：甘氨酸、丙氨酸、缬氨酸、亮氨酸和异亮氨酸；2)脂族羟基侧链：丝氨酸和苏氨酸；3)含酰胺的侧链：天冬酰胺和谷氨酰胺；4)芳族侧链：苯丙氨酸、酪氨酸和色氨酸；5)碱性侧链：赖氨酸、精氨酸和组氨酸；6)酸性侧链：天冬氨酸和谷氨酸。A "conservative amino acid substitution" refers to the replacement of one amino acid residue with another amino acid residue containing a side chain (R group) of similar chemical properties (eg, charge or hydrophobicity). In general, conservative amino acid substitutions are unlikely to substantially alter the functional properties of a protein. Examples of classes of amino acids that contain chemically similar side chains include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, Arginine and histidine; 6) acidic side chains: aspartic acid and glutamic acid.
“VL、VH的保守氨基酸取代”的氨基酸数目为约1个、约2个、约3个、约4个、约5个、约6个、约8个、约9个、约10个、约11个、约13个、约14个、约15个保守氨基酸取代，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。“重链恒定区、轻链恒定区、重链或轻链的保守氨基酸取代”的氨基酸数目为约1个、约2个、约3个、约4个、约5个、约6个、约8个、约9个、约10个、约11个、约13个、约14个、约15个、约18个、约19个、约22个、约24个、约25个、约29个、约31个、约35个、约38个、约41个、约45个保守氨基酸取代，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。The number of amino acids in the "conservative amino acid substitution of VL, VH" is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15 conservative amino acid substitutions, or a range between any two of these values (inclusive), or any value therein. The number of amino acids in the "heavy chain constant region, light chain constant region, heavy chain or light chain conservative amino acid substitution" is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15, about 18, about 19, about 22, about 24, about 25, about 29 , about 31, about 35, about 38, about 41, about 45 conservative amino acid substitutions, or a range between any two of these values (inclusive) or any value therein.
本发明中关于细胞、核酸、多肽、抗体等所使用的术语“分离的”，例如“分离的”DNA、RNA、多肽、抗体是指分别于细胞天然环境中的其它组分如DNA或RNA中的一种或多种所分离的分子。本发明使用的术语“分离的”还指当通过重组DNA技术产生时基本上不含细胞材料、病毒材料或细胞培养基的核酸或肽，或化学合成时的化学前体或其他化学品。此外，“分离的核酸”意在包括不以天然状态存在的核酸片段，并且不会以天然状态存在。术语“分离的”在本发明中也用于指从其他细胞蛋白质或组织分离的细胞或多肽。分离的多肽意在包括纯化的和重组的多肽。分离的多肽、抗体等通常通过至少一个纯化步骤制备。在一些实施方案中，分离的核酸、多肽、抗体等的纯度至少为约50％、约60％、约70％、约80％、约90％、约95％、约99％，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。The term "isolated" used in the present invention with respect to cells, nucleic acids, polypeptides, antibodies, etc., for example, "isolated" DNA, RNA, polypeptides, antibodies refers to the isolated components of the cell's natural environment, such as DNA or RNA. One or more of the isolated molecules. The term "isolated" as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or cell culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" is intended to include fragments of nucleic acid that do not occur in nature, and do not exist in nature. The term "isolated" is also used herein to refer to cells or polypeptides that are separated from other cellular proteins or tissues. Isolated polypeptide is intended to include purified and recombinant polypeptides. Isolated polypeptides, antibodies, etc. will usually be prepared by at least one purification step. In some embodiments, the purity of the isolated nucleic acid, polypeptide, antibody, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or some of these values The range (inclusive) between any two values of , or any value therein.
术语“重组”涉及多肽或多聚核苷酸，意指天然不存在的多肽或多聚核苷酸的形式， 不受限制的实施例可以通过组合产生通常并不存在的多聚核苷酸或多肽。The term "recombinant" refers to polypeptides or polynucleotides, meaning forms of polypeptides or polynucleotides that do not occur in nature, non-limiting examples can be produced by combination of polynucleotides or polynucleotides that do not normally exist or peptide.
“同源性”或“同一性”或“相似性”是指两个肽之间或两个核酸分子之间的序列相似性。可以通过比较每个序列中可以比对的位置来确定同源性。当被比较的序列中的位置被相同的碱基或氨基酸占据时，则分子在该位置是同源的。序列之间的同源程度是由序列共有的匹配或同源位置的数目组成的一个函数。"Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the alignable positions in each sequence. When a position in the sequences being compared is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
“至少80％同一性”为约80％同一性、约81％同一性、约82％同一性、约83％同一性、约85％同一性、约86％同一性、约87％同一性、约88％同一性、约90％同一性、约91％同一性、约92％同一性、约94％同一性、约95％同一性、约98％同一性、约99％同一性，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。"At least 80% identity" is about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, About 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or these A range (inclusive) between any two values in Numeric or any value therein.
多聚核苷酸或多聚核苷酸序列(或多肽或抗体序列)与另一序列有具有一定百分比(例如90％、95％、98％或者99％)的“同一性或序列同一性”是指当序列比对时，所比较的两个序列中该百分比的碱基(或氨基酸)相同。可以使用目测或本领域已知的软件程序来确定该比对和同一性百分比或序列同一性，比如Ausubel et al.eds.(2007)在Current Protocols in Molecular Biology中所述的软件程序。优选使用默认参数进行比对。其中一种比对程序是使用默认参数的BLAST，例如BLASTN和BLASTP，两者使用下列默认参数：Geneticcode＝standard；filter＝none；strand＝both；cutoff＝60；expect＝10；Matrix＝BLOSUM62；Descriptions＝50sequences；sortby＝HIGHSCORE；Databases＝non-redundant；GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR。生物学上等同的多聚核苷酸是具有上述指定百分比的同一性并编码具有相同或相似生物学活性的多肽的多聚核苷酸。A polynucleotide or polynucleotide sequence (or polypeptide or antibody sequence) has a certain percentage (eg 90%, 95%, 98% or 99%) "identity or sequence identity" with another sequence Refers to the percentage of bases (or amino acids) that are identical in the two sequences being compared when the sequences are aligned. This alignment and percent identity or sequence identity can be determined using visual inspection or software programs known in the art, such as those described by Ausubel et al.eds. (2007) in Current Protocols in Molecular Biology. It is preferred to use the default parameters for the alignment. One such alignment program is BLAST with default parameters, such as BLASTN and BLASTP, both of which use the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions =50sequences; sortby=HIGHSCORE; Databases=non-redundant; GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are polynucleotides that share the above indicated percentages of identity and encode a polypeptide having the same or similar biological activity.
多聚核苷酸是由四个核苷酸碱基的特定序列组成：腺嘌呤(A)、胞嘧啶(C)、鸟嘌呤(G)、胸腺嘧啶(T)，或当多聚核苷酸是RNA时胸腺嘧啶换为尿嘧啶(U)。“多聚核苷酸序列”可以以多聚核苷酸分子的字母表示。该字母表示可以被输入到具有中央处理单元的计算机中的数据库中，并用于生物信息学应用，例如用于功能基因组学和同源性搜索。A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), or when a polynucleotide In the case of RNA, thymine is replaced by uracil (U). A "polynucleotide sequence" may be denoted by the letters of the polynucleotide molecule. This letter designation can be entered into a database in a computer with a central processing unit and used in bioinformatics applications such as for functional genomics and homology searches.
术语“多聚核苷酸”、“核酸”和“寡核苷酸”可互换使用，是指任何长度的核苷酸的聚合形式，无论是脱氧核糖核苷酸还是核糖核苷酸或其类似物。多聚核苷酸可以具有任何三维结构并且可以执行已知或未知的任何功能。以下是不受限制的多聚核苷酸的实施例：基因或基因片段(例如探针、引物、EST或SAGE标签)、外显子、内含子、信使RNA(mRNA)、转运RNA、核糖体RNA、核糖酶、cDNA、dsRNA、siRNA、miRNA、重组多聚核苷酸、分支的多聚核苷酸、质粒、载体、任何序列的分离的DNA、任何序列的分离的RNA、核酸探针和引物。多聚核苷酸可以包含修饰的核苷酸，例如甲基化的核苷酸和核苷酸类似物。如果存在该修饰，则对核苷酸的结构修饰可以在组 装多聚核苷酸之前或之后进行。核苷酸的序列可以被非核苷酸组分中断。聚合后可以进一步修饰多聚核苷酸，例如通过与标记组分缀合。这个术语也指双链和单链分子。除另有说明或要求外，本公开的任何多聚核苷酸的实施例包括双链形式和已知或预测构成双链形式的两种可互补单链形式中的每一种。The terms "polynucleotide", "nucleic acid" and "oligonucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or their analog. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribose Somatic RNA, ribozyme, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. Structural modifications to the nucleotides, if present, can be made before or after assembly of the polynucleotide. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, for example by conjugation with labeling components. The term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of a polynucleotide of the present disclosure includes the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.
术语“编码”应用于多聚核苷酸时，是指被称为“编码”多肽的多聚核苷酸，在其天然状态或当通过本领域技术人员公知的方法操作时，经转录和/或翻译可以产生该多肽和/或其片段。The term "encoding" when applied to a polynucleotide refers to a polynucleotide which is said to "encode" a polypeptide which, in its native state or when manipulated by methods well known to those skilled in the art, is transcribed and/or Or translation may result in the polypeptide and/or fragments thereof.
本发明公开的抗体、抗原结合片段包括但不限于多克隆、单克隆、多特异性、全人源、人源化、灵长类化、嵌合抗体、单链抗体、表位结合片段(例如Fab、Fab′和F(ab′) 2)、单链Fvs(scFv)。 Antibodies and antigen-binding fragments disclosed in the present invention include but are not limited to polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, single-chain antibodies, epitope-binding fragments (such as Fab, Fab' and F(ab') 2 ), single chain Fvs (scFv).
“抗体”、“抗原结合片段”是指特异性识别和结合抗原的多肽或多肽复合物。抗体可以是完整的抗体及其任何抗原结合片段或其单链。因此术语“抗体”包括分子中含有具有与抗原结合的生物学活性的免疫球蛋白分子的至少一部分的任何蛋白质或肽。抗体和抗原结合片段包括但不局限重链或轻链或其配体结合部分的互补决定区(CDR)、重链可变区(VH)、轻链可变区(VL)、重链恒定区(CH)、轻链恒定区(CL)、框架区(FR)或其任何部分，或结合蛋白的至少一部分。CDR区包括轻链的CDR区(LCDR1-3)和重链的CDR区(HCDR1-3)。抗体及抗原结合片段可以特异性识别和结合一个或多个(如两个)抗原的多肽或多肽复合物。特异性识别和结合多个(如两个)抗原的抗体或抗原结合片段可以被称为多特异性(如双特异性)抗体或抗原结合片段。"Antibody" and "antigen-binding fragment" refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. Antibodies can be whole antibodies and any antigen-binding fragments thereof or single chains thereof. The term "antibody" thus includes any protein or peptide whose molecule contains at least a portion of an immunoglobulin molecule that has the biological activity to bind an antigen. Antibodies and antigen-binding fragments include, but are not limited to, complementarity determining regions (CDRs), heavy chain variable regions (VH), light chain variable regions (VL), heavy chain constant regions of heavy or light chains or ligand-binding portions thereof (CH), light chain constant region (CL), framework region (FR) or any portion thereof, or at least a portion of a binding protein. The CDR regions include the CDR regions of the light chain (LCDR1-3) and the CDR regions of the heavy chain (HCDR1-3). Antibodies and antigen-binding fragments can specifically recognize and bind to one or more (eg, two) antigen polypeptides or polypeptide complexes. Antibodies or antigen-binding fragments that specifically recognize and bind multiple (eg, two) antigens may be referred to as multispecific (eg, bispecific) antibodies or antigen-binding fragments.
术语“抗体片段”或“抗原结合片段”指抗体的一部分，本发明抗体片段的组成形式可类似于单特异性抗体片段中的F(ab’) 2、F(ab) 2、Fab′、Fab、Fv、scFv等。不管其结构如何，抗体片段与被完整抗体识别的同一抗原结合。术语“抗体片段”包括适体、镜像异构体和双价抗体。术语“抗原结合片段”还包括通过与特定抗原结合形成复合物起抗体作用的任何合成或基因工程蛋白质。 The term "antibody fragment" or "antigen-binding fragment" refers to a part of an antibody, and the composition of the antibody fragment of the present invention may be similar to F(ab') 2 , F(ab) 2 , Fab', Fab in monospecific antibody fragments , Fv, scFv, etc. Regardless of their structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, Spiegelmers and diabodies. The term "antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.
“单链可变片段”或“scFv”是指免疫球蛋白的重链(VH)和轻链(VL)的可变区的融合蛋白。在一些方面，这些区域与10个至约25个氨基酸的短接头肽连接。接头可以富含甘氨酸以增加柔韧性，以及富含丝氨酸或苏氨酸以增加溶解性，并且可以连接VH的N端和VL的C端，反之亦然。尽管该蛋白质被除去了恒定区和引入了接头，但其保留了原始免疫球蛋白的特异性。scFv分子通常是本领域中已知的，例如在美国专利5,892,019中有相关描述。"Single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin. In some aspects, these regions are linked to short linker peptides of 10 to about 25 amino acids. Linkers can be rich in glycine for flexibility, and serine or threonine for solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa. Although the protein has had its constant regions removed and a linker introduced, it retains the specificity of the original immunoglobulin. scFv molecules are generally known in the art and are described, for example, in US Patent No. 5,892,019.
术语“抗体”包括可以在生物化学上区分的各种广泛种类的多肽。本领域技术人员 将会理解，重链的类别包括gamma、mu、alpha、delta或epsilon(γ、μ、α、δ、ε)，其中还有一些亚类(例如γ1-γ4)。该链的性质决定了抗体的“种类”分别为IgG、IgM、IgA、IgG或IgE。免疫球蛋白亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgG5等已被充分表征并且赋予的功能特异性也已知。所有的免疫球蛋白种类都在本发明公开的保护范围内。在一些实施方案中，免疫球蛋白分子为IgG种类。The term "antibody" includes a wide variety of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that classes of heavy chains include gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with subclasses (eg, γ1-γ4). The nature of this chain determines the "class" of the antibody as IgG, IgM, IgA, IgG or IgE, respectively. The immunoglobulin subclasses (isotypes), eg, IgGl, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and the functional specificities conferred are also known. All immunoglobulin classes are within the scope of the present disclosure. In some embodiments, the immunoglobulin molecule is of the IgG class.
轻链可以分为kappa(κ)或lambda(λ)。每个重链可以与κ或λ轻链结合。一般来说，当由杂交瘤，B细胞或基因工程宿主细胞生产免疫球蛋白时，其轻链和重链通过共价键结合，两条重链的“尾巴”部分通过共价二硫键或非共价键结合。在重链中，氨基酸序列从Y构型的叉状末端的N末端延伸至每条链底部的C末端。免疫球蛋白κ轻链可变区为Vκ；免疫球蛋白λ轻链可变区为V λ。 Light chains can be classified as kappa (κ) or lambda (λ). Each heavy chain can be associated with a kappa or lambda light chain. Generally, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, their light and heavy chains are joined by covalent bonds, and the "tail" portions of the two heavy chains are linked by covalent disulfide bonds or non-covalent bonding. In the heavy chains, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain. The variable region of the immunoglobulin kappa light chain is Vκ; the variable region of the immunoglobulin lambda light chain is Vλ .
轻链和重链都分成结构和功能同源性的区域。术语“恒定的”和“可变的”根据功能被使用。轻链可变区(VL)和重链可变区(VH)部分决定了抗原识别和特异性。轻链和重链的恒定区赋予重要的生物学性质，如分泌、经胎盘移动、Fc受体结合、补体结合等。按照惯例，恒定区的编号随着它们变得更远离抗体的抗原结合位点或氨基末端而增加。N端部分是可变区，C端部分是恒定区；CH3和CL结构域实际上分别包含重链和轻链的羧基端。Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used according to function. The light chain variable (VL) and heavy chain variable (VH) portions determine antigen recognition and specificity. The constant regions of the light and heavy chains confer important biological properties such as secretion, transplacental movement, Fc receptor binding, complement fixation, etc. By convention, the numbering of constant regions increases as they become farther away from the antigen-binding site or amino terminus of the antibody. The N-terminal portion is the variable region and the C-terminal portion is the constant region; the CH3 and CL domains actually comprise the carboxy-terminal ends of the heavy and light chains, respectively.
在天然存在的抗体中，假设抗体在含水环境中呈现其三维构型时，存在于每个抗原结合域中的六个“互补决定区”或“CDR”是形成抗原结合结构域的短的、非连续的与抗原特异性结合的氨基酸序列。抗原结合结构域中被称为“构架”区域的剩余其它氨基酸显示出较小的分子间可变性。构架区大部分采用β-折叠构象，CDR形成与之连接的环状结构，或在某些情况下形成β折叠结构的一部分。因此，框架区通过形成支架从而通过链间非共价相互作用使CDR定位在正确的方位上。具有特定位置的CDR的抗原结合域形成了与抗原上的表位互补的表面，该互补表面促进抗体和其抗原表位的非共价结合。对于给定的重链或轻链可变区，本领域普通技术人员都可以通过已知方法鉴定出包含CDR和框架区的氨基酸(参见Kabat，E.，et al.，U.S.Department of Health and Human Services，Sequences of Proteins of Immunological Interest，(1983)和Chothia and Lesk，J.Mol.Biol.，196：901-917(1987))。In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen-binding domain are short, A non-contiguous sequence of amino acids that specifically binds to an antigen. The remaining other amino acids in the antigen-binding domain, referred to as the "framework" regions, show less inter-molecular variability. The framework regions mostly adopt a β-sheet conformation, and the CDRs form loop structures attached to them, or in some cases form part of the β-sheet structure. Thus, the framework regions position the CDRs in the correct orientation by forming a scaffold through non-covalent interchain interactions. The antigen-binding domain with the CDRs in specific positions forms a surface complementary to the epitope on the antigen that facilitates the non-covalent binding of the antibody to its antigenic epitope. For a given heavy chain or light chain variable region, those of ordinary skill in the art can identify the amino acids comprising CDR and framework regions by known methods (see Kabat, E., et al., U.S. Department of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).
在本领域中使用和/或接受的术语有两个或多个定义的情况下，除非明确地对立指出，否则本文使用的术语的定义包括所有这些含义。一个具体的例子是使用“互补决定区”(“CDR”)一词来描述在重链和轻链多肽的可变区内发现的非连续的抗原结合位点。这一特定区域在Kabat et al.，U.S.Dept.of Health and Human Services，Sequences of Proteins of Immunological Interest(1983)和Chothia等在J.Mol.Biol.196：901-917(1987)有相关描述，其通过引用全部并入本文。Where there are two or more definitions for a term used and/or accepted in the art, the definition of the term used herein includes all such meanings unless clearly indicated to the contrary. A specific example is the use of the term "complementarity determining regions" ("CDR") to describe the non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. This particular region is described in Kabat et al., U.S. Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al. in J. Mol. Biol. 196: 901-917 (1987), It is hereby incorporated by reference in its entirety.
根据Kabat和Chothia定义的CDR包括相互比较时的氨基酸残基的重叠或子集。尽管如此，应用任一定义来指代抗体或其变体的CDR都在本发明范围内。包含特定CDR的确切残基编号将根据CDR的序列和大小而变化。本领域技术人员通常可以根据抗体的可变区氨基酸序列确定出CDR包含哪些特定的残基。CDRs as defined by Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. Nevertheless, it is within the scope of the invention to use either definition to refer to the CDRs of an antibody or variant thereof. The exact residue numbers comprising a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can generally determine which specific residues are included in the CDRs based on the amino acid sequence of the variable region of the antibody.
Kabat等人还定义了适用于任何抗体的可变区序列的编号系统。本领域普通技术人员可以不依赖于序列本身以外的其他实验数据将该“Kabat编号”系统应用到任何可变区序列。“Kabat编号”是指由Kabat et al.，U.S.Dept.of Health and Human Services在“Sequence of Proteins of Immunological Interest”(1983)提出的编号系统。抗体还可以用EU或Chothia编号系统。Kabat et al. also defined a numbering system applicable to the variable region sequences of any antibody. One of ordinary skill in the art can apply this "Kabat numbering" system to any variable region sequence independently of other experimental data other than the sequence itself. "Kabat numbering" refers to the numbering system proposed by Kabat et al., U.S. Dept. of Health and Human Services in "Sequence of Proteins of Immunological Interest" (1983). Antibodies can also use the EU or Chothia numbering system.
本发明公开的抗体可以来源于任何动物，包括鸟类和哺乳动物。较佳地，抗体是人源、鼠源、驴源、兔源、山羊源、骆驼源、美洲驼源、马源或鸡源抗体。在另一实施方案中，可变区可以是软骨鱼纲(condricthoid)来源(例如来自鲨鱼)。Antibodies disclosed herein may be derived from any animal, including birds and mammals. Preferably, the antibody is of human, murine, donkey, rabbit, goat, camel, llama, horse or chicken origin. In another embodiment, the variable regions may be of condricthoid origin (eg, from sharks).
“重链恒定区”包括CH1结构域、铰链(例如上、中和/或下铰链区)结构域、CH2结构域、CH3结构域，或变体或片段中的至少一种。抗体的重链恒定区可以来源于不同的免疫球蛋白分子。例如，多肽的重链恒定区可以包括源自IgG 1分子的CH1结构域和源自IgG 3分子的铰链区。在另一实施方案中，重链恒定区可以包括部分源自IgG 1分子和部分源自IgG 3分子的铰链区。在另一实施方案中，部分重链可以包括部分源自IgG 1分子和部分源自IgG 4分子的嵌合铰链区。 A "heavy chain constant region" includes at least one of a CH1 domain, a hinge (eg, upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment. The heavy chain constant regions of antibodies can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide can include a CH1 domain derived from an IgG 1 molecule and a hinge region derived from an IgG 3 molecule. In another embodiment, the heavy chain constant region may comprise a hinge region derived in part from an IgG 1 molecule and in part from an IgG 3 molecule. In another embodiment, part of the heavy chain may comprise a chimeric hinge region derived partly from an IgG 1 molecule and partly from an IgG4 molecule.
“轻链恒定区”包括来自抗体轻链的一部分氨基酸序列。较佳地，轻链恒定区包含恒定κ结构域或恒定λ结构域中的至少一个。“轻链-重链对”是指可通过轻链的CL结构域和重链的CH1结构域之间的二硫键形成二聚体的轻链和重链的集合。这四条链通过二硫键以“Y”构型连接，其中轻链从“Y”口开始并延续通过可变区包围重链。A "light chain constant region" includes a portion of the amino acid sequence from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain. A "light chain-heavy chain pair" refers to a collection of light and heavy chains that can form dimers through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain. The four chains are linked by disulfide bonds in a "Y" configuration, with the light chain starting at the mouth of the "Y" and continuing through the variable region surrounding the heavy chain.
“VH结构域”包括免疫球蛋白重链的氨基末端可变结构域，“CH1结构域”包括免疫球蛋白重链的第一个(大部分氨基末端)恒定区。完整的天然IgG分子中两个CH2结构域中N297各连接一个分支碳水化合物链。CH3结构域从CH2结构域开始延伸到IgG分子的C-末端，大约包含108个残基。“铰链区”包括连接CH1结构域和CH2结构域的部分重链区域。所述铰链区包含约25个残基并且是有韧性的，从而使得两个N端抗原结合区能够独立移动。铰链区可以被细分为三个不同的结构域：上、中和下铰链结构域(Rouxetal.，J.Immunol 161：4083(1998))。A "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and a "CH1 domain" includes the first (mostly amino-terminal) constant region of an immunoglobulin heavy chain. N297 in the two CH2 domains of the complete natural IgG molecule is connected to a branched carbohydrate chain. The CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues. A "hinge region" includes part of the heavy chain region connecting the CH1 domain and the CH2 domain. The hinge region comprises approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: upper, middle and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).
“二硫键”指两个硫原子之间形成的共价键。半胱氨酸的硫醇基团可以与第二个硫醇基团形成二硫键或桥接。在大多数天然存在的IgG分子中，CH1和CL区通过二硫键连接。"Disulfide bond" refers to a covalent bond formed between two sulfur atoms. A thiol group of cysteine can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by disulfide bonds.
“嵌合抗体”指其可变区从第一个物种中获得或衍生，而其恒定区(可以是完整的、部分的或修饰过的)来源于第二个物种的任何抗体。某些实施方案中，可变区来自非人源(例如小鼠或灵长类动物)，而恒定区来自人源。A "chimeric antibody" refers to any antibody whose variable regions are obtained or derived from a first species and whose constant regions (which may be complete, partial or modified) are derived from a second species. In certain embodiments, the variable regions are of non-human origin (eg, mouse or primate) and the constant regions are of human origin.
“特异性结合”或“对......具有特异性”通常是指抗体或抗原结合片段与特定抗原通过其抗原结合结构域与表位互补性结合形成相对稳定的复合物。“特异性”可以用抗体或抗原结合片段与特定抗原或表位结合的相对亲和力表达。例如，如果抗体“A”比抗体“B”与同一抗原的相对亲和力大，可以认为抗体“A”比抗体“B”对该抗原具有更高的特异性。特异性结合可以用平衡解离常数(K D)来描述，较小的K D意味着较紧密的结合。确定两个分子是否特异性结合的方法是本领域内众所周知的，并包括例如平衡透析、表面等离子共振、生物膜层光学干涉测量法等。“特异性结合”抗原a的抗体包括与抗原a平衡解离常数K D小于或等于约100nM、小于或等于约10nM、小于或等于约5nM、小于或等于约1nM。 "Specific binding" or "specific for" generally refers to the complementary binding of an antibody or antigen-binding fragment to a specific antigen through its antigen-binding domain and epitope to form a relatively stable complex. "Specificity" can be expressed in terms of the relative affinity with which an antibody or antigen-binding fragment binds to a particular antigen or epitope. For example, antibody "A" may be said to have a higher specificity for that antigen than antibody "B" if it has a greater relative affinity for the same antigen than antibody "B". Specific binding can be described by an equilibrium dissociation constant ( KD ), with a smaller KD implying a tighter binding. Methods of determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, optical interferometry of biofilm layers, and the like. An antibody that "specifically binds" antigen a includes an equilibrium dissociation constant K D of antigen a of less than or equal to about 100 nM, less than or equal to about 10 nM, less than or equal to about 5 nM, less than or equal to about 1 nM.
“治疗”是指治疗性治疗和预防性或防治性措施，其目的是预防、减缓、改善或停止不良的生理改变或紊乱，例如疾病的进程，包括但不限于以下无论是可检测还是不可检测的结果，症状的缓解、疾病程度的减小、疾病状态的稳定(即不恶化)、疾病进展的延迟或减缓、疾病状态的改善、缓和、减轻或消失(无论是部分还是全部)、延长与不接受治疗时预期的生存期限等。需要治疗的患者包括已经患有病症或紊乱的患者，容易患有病症或紊乱的患者，或者需要预防该病症或紊乱的患者，可以或预期从施用本发明公开的抗体或药物组合物用于检测、诊断过程和/或治疗中受益的患者。"Treatment" means therapeutic treatment and prophylactic or preventive measures, the purpose of which is to prevent, slow down, ameliorate or stop an undesirable physiological change or disorder, such as the progression of a disease, including but not limited to the following whether detectable or undetectable Relief of symptoms, reduction of disease extent, stabilization of disease state (i.e. not worsening), delay or slowing of disease progression, amelioration, remission, alleviation or disappearance of disease state (whether partial or total), prolongation and Expected survival without treatment, etc. Patients in need of treatment include those who already have a condition or disorder, are susceptible to having a condition or disorder, or are in need of prevention of the condition or disorder, and can or are expected to benefit from the administration of an antibody or pharmaceutical composition disclosed herein for detection , patients who benefit from the diagnostic process and/or treatment.
“患者”指需要诊断、预后或治疗的任何哺乳动物，包括人类、狗、猫、兔子、鼠、马、牛等。"Patient" refers to any mammal in need of diagnosis, prognosis, or treatment, including humans, dogs, cats, rabbits, mice, horses, cattle, and the like.
“约”指相关技术领域技术人员容易知道的相应数值的常规误差范围。在一些实施方式中，本文中提到“约”指所描述的数值以及其±10％、±5％或±1％的范围。"About" refers to the usual error range for the corresponding value readily known to those skilled in the relevant art. In some embodiments, reference to "about" herein refers to the described numerical value and the range of ±10%, ±5% or ±1%.
“EC s0”即半最大效应浓度(concentration for 50％of maximal effect，EC 50)是指能引起50％最大效应的浓度。 "EC s0 ", half maximum effect concentration (concentration for 50% of maximal effect, EC 50 ) refers to the concentration that can cause 50% of the maximum effect.
“IC 50”表示50％抑制浓度，即对指定的生物过程抑制一半时所需的药物或者抑制剂的浓度。 " IC50 " means 50% inhibitory concentration, ie the concentration of drug or inhibitor required to inhibit a given biological process by half.
本发明中“亲本Fc区”可以为天然存在的Fc区，编码Fc区的基因可来自人、鼠、兔、骆驼、猴子，优选为人和小鼠；例如，亲本Fc区为SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：33或SEQ ID NO：34中Fc区。The "parental Fc region" in the present invention can be a naturally occurring Fc region, and the gene encoding the Fc region can be from human, mouse, rabbit, camel, monkey, preferably human and mouse; for example, the parental Fc region is SEQ ID NO: 31 , SEQ ID NO:32, SEQ ID NO:33 or the Fc region of SEQ ID NO:34.
本文提及出版物的相关描述均通过引用全部并入本文。The relevant descriptions of publications mentioned herein are incorporated by reference in their entirety.
抗SIRPα抗体anti-SIRPα antibody
本发明提供了对SIRPα蛋白具有高亲和力的抗体或抗原结合片段。本发明的抗体或抗原结合片段表现出有效的结合活性、生物学活性，并可用于治疗和诊断用途。比如，这些抗体或抗原结合片段可以有效阻断SIRPα/CD47信号通路，激活吞噬细胞的吞噬作用，用于治疗各种类型的癌症或感染等相关疾病。The present invention provides antibodies or antigen-binding fragments with high affinity for SIRPα protein. The antibody or antigen-binding fragment of the present invention exhibits effective binding activity, biological activity, and can be used for therapeutic and diagnostic purposes. For example, these antibodies or antigen-binding fragments can effectively block the SIRPα/CD47 signaling pathway, activate phagocytosis of phagocytes, and be used to treat various types of cancer or infection and other related diseases.
在一些实施方案中，所述抗体或抗原结合片段为scFv。In some embodiments, the antibody or antigen-binding fragment is a scFv.
在一些实施方案中，scFv片段的重链可变区包含SEQ ID NO：16所示的序列，scFv片段的轻链可变区包含SEQ ID NO：22所示的序列。In some embodiments, the heavy chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 16, and the light chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 22.
在一些实施方案中，scFv片段的重链可变区包含SEQ ID NO：17所示的序列，scFv片段的轻链可变区包含SEQ ID NO：22所示的序列。In some embodiments, the heavy chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 17, and the light chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 22.
在一些实施方案中，scFv片段的重链可变区包含SEQ ID NO：18所示的序列，scFv片段的轻链可变区包含SEQ ID NO：22所示的序列。In some embodiments, the heavy chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 18, and the light chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 22.
在一些实施方案中，scFv片段的重链可变区包含SEQ ID NO：19所示的序列，scFv片段的轻链可变区包含SEQ ID NO：22所示的序列。In some embodiments, the heavy chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 19, and the light chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 22.
在一些实施方案中，scFv片段的重链可变区包含SEQ ID NO：20所示的序列，scFv片段的轻链可变区包含SEQ ID NO：22所示的序列。In some embodiments, the heavy chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 20, and the light chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 22.
在一些实施方案中，scFv片段的重链可变区包含SEQ ID NO：21所示的序列，scFv片段的轻链可变区包含SEQ ID NO：22所示的序列。In some embodiments, the heavy chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 21, and the light chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 22.
在一些实施方案中，scFv片段的重链可变区包含SEQ ID NO：19所示的序列，scFv片段的轻链可变区包含SEQ ID NO：23所示的序列。In some embodiments, the heavy chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 19, and the light chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 23.
在一些实施方案中，scFv片段的重链可变区包含SEQ ID NO：19所示的序列，scFv片段的轻链可变区包含SEQ ID NO：24所示的序列。In some embodiments, the heavy chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 19, and the light chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 24.
在一些实施方案中，scFv片段的重链可变区包含SEQ ID NO：19所示的序列，scFv片段的轻链可变区包含SEQ ID NO：25所示的序列。In some embodiments, the heavy chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 19, and the light chain variable region of the scFv fragment comprises the sequence shown in SEQ ID NO: 25.
在一些实施方案中，scFv片段的重链可变区包含SEQ ID NO：19所示的序列，scFv片段的轻链可变区包含SEQ ID NO：26所示的序列。在一些实施方案中，scFv片段中连接重链可变区和轻链可变区的连接子为(G 4S) n。在一些实施方案中，n为1、2、3、4或5。 In some embodiments, the heavy chain variable region of the scFv fragment comprises the sequence set forth in SEQ ID NO:19, and the light chain variable region of the scFv fragment comprises the sequence set forth in SEQ ID NO:26. In some embodiments, the linker connecting the variable region of the heavy chain and the variable region of the light chain in the scFv fragment is (G 4 S) n . In some embodiments, n is 1, 2, 3, 4 or 5.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：31所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：39所示的序列。在一些实施方案中，所述抗体或抗原结合片段的重链包含氨基酸序列SEQ ID NO：31中除Fc以外的序列，所述抗体或抗原结合片段的轻链包含氨基酸序列如SEQ ID NO：39所示的序列。In some embodiments, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 31, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 39. In some embodiments, the heavy chain of the antibody or antigen-binding fragment comprises a sequence other than Fc in the amino acid sequence of SEQ ID NO: 31, and the light chain of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: 39 sequence shown.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：32所示的序 列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：39所示的序列。在一些实施方案中，所述抗体或抗原结合片段的重链包含氨基酸序列SEQ ID NO：32中除Fc以外的序列，所述抗体或抗原结合片段的轻链包含氨基酸序列如SEQ ID NO：39所示的序列。In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 32, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 39. In some embodiments, the heavy chain of the antibody or antigen-binding fragment comprises a sequence other than Fc in the amino acid sequence of SEQ ID NO: 32, and the light chain of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: 39 sequence shown.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：33所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：39所示的序列。在一些实施方案中，所述抗体或抗原结合片段的重链包含氨基酸序列SEQ ID NO：33中除Fc以外的序列，所述抗体或抗原结合片段的轻链包含氨基酸序列如SEQ ID NO：39所示的序列。In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 33, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 39. In some embodiments, the heavy chain of the antibody or antigen-binding fragment comprises a sequence other than Fc in the amino acid sequence of SEQ ID NO: 33, and the light chain of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: 39 sequence shown.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：34所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：39所示的序列。在一些实施方案中，所述抗体或抗原结合片段的重链包含氨基酸序列SEQ ID NO：34中除Fc以外的序列，所述抗体或抗原结合片段的轻链包含氨基酸序列如SEQ ID NO：39所示的序列。In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 39. In some embodiments, the heavy chain of the antibody or antigen-binding fragment comprises a sequence other than Fc in the amino acid sequence of SEQ ID NO: 34, and the light chain of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: 39 sequence shown.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：35所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：39所示的序列。在一些实施方案中，所述抗体或抗原结合片段的重链包含氨基酸序列SEQ ID NO：35中除Fc以外的序列，所述抗体或抗原结合片段的轻链包含氨基酸序列如SEQ ID NO：39所示的序列。In some embodiments, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 35, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 39. In some embodiments, the heavy chain of the antibody or antigen-binding fragment comprises a sequence other than Fc in the amino acid sequence of SEQ ID NO: 35, and the light chain of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: 39 sequence shown.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：36所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：39所示的序列。在一些实施方案中，所述抗体或抗原结合片段的重链包含氨基酸序列SEQ ID NO：36中除Fc以外的序列，所述抗体或抗原结合片段的轻链包含氨基酸序列如SEQ ID NO：39所示的序列。In some embodiments, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 36, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 39. In some embodiments, the heavy chain of the antibody or antigen-binding fragment comprises a sequence other than Fc in the amino acid sequence of SEQ ID NO: 36, and the light chain of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: 39 sequence shown.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：34所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：40所示的序列。在一些实施方案中，所述抗体或抗原结合片段的重链包含氨基酸序列SEQ ID NO：34中除Fc以外的序列，所述抗体或抗原结合片段的轻链包含氨基酸序列如SEQ ID NO：40所示的序列。In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 40. In some embodiments, the heavy chain of the antibody or antigen-binding fragment comprises a sequence other than Fc in the amino acid sequence of SEQ ID NO: 34, and the light chain of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: 40 sequence shown.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：34所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：41所示的序列。在一些实施方案中，所述抗体或抗原结合片段的重链包含氨基酸序列SEQ ID NO：34中除Fc以外的序列，所述抗体或抗原结合片段的轻链包含氨基酸序列如SEQ ID NO：41所示的序列。In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 41. In some embodiments, the heavy chain of the antibody or antigen-binding fragment comprises a sequence other than Fc in the amino acid sequence of SEQ ID NO: 34, and the light chain of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: 41 sequence shown.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：34所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：42所示的序列。在一些实施方案中，所述抗体或抗原结合片段的重链包含氨基酸序列SEQ ID NO：34中除Fc以外的序列，所述抗体或抗原结合片段的轻链包含氨基酸序列如SEQ ID NO：42所示的序列。In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 42. In some embodiments, the heavy chain of the antibody or antigen-binding fragment comprises a sequence other than Fc in the amino acid sequence of SEQ ID NO: 34, and the light chain of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: 42 sequence shown.
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO：34所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：43所示的序列。在一些实施方案中，所述抗体或抗原结合片段的重链包含氨基酸序列SEQ ID NO：34中除Fc以外的序列，所述抗体或抗原结合片段的轻链包含氨基酸序列如SEQ ID NO：43所示的序列。In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 43. In some embodiments, the heavy chain of the antibody or antigen-binding fragment comprises a sequence other than Fc in the amino acid sequence of SEQ ID NO: 34, and the light chain of the antibody or antigen-binding fragment comprises an amino acid sequence such as SEQ ID NO: 43 sequence shown.
在一些实施方案中，本发明抗体含有两条序列相同的重链(或重链片段)和两条序列相同的轻链(或轻链片段)。In some embodiments, an antibody of the invention contains two heavy chains (or heavy chain fragments) with the same sequence and two light chains (or light chain fragments) with the same sequence.
本领域普通技术人员还应当理解，本发明所公开抗体或抗原结合片段序列是可以被替换的，替换后其氨基酸序列不同于该抗体的天然存在的氨基酸序列。例如，替换后的氨基酸序列可以是与起始序列相似的，比如与起始序列具有一定比例的同一性，比如它可以与起始序列的同一性是约80％、约85％、约90％、约95％、约98％、约99％，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。Those of ordinary skill in the art should also understand that the sequences of the antibodies or antigen-binding fragments disclosed in the present invention can be replaced, and the amino acid sequence after replacement is different from the naturally occurring amino acid sequence of the antibody. For example, the substituted amino acid sequence may be similar to the original sequence, such as having a certain proportion of identity with the original sequence, for example, it may be about 80%, about 85%, or about 90% identical to the original sequence. , about 95%, about 98%, about 99%, or a range between any two of these values (inclusive), or any value therein.
在一些实施方案中，抗体或抗原结合片段包含氨基酸序列具有一个或多个修饰基团。例如，本发明公开的抗体或抗原结合片段可以包含有韧性的接头序列，或者可以被修饰以添加功能性基团(例如PEG、药物、毒素或标签)。In some embodiments, an antibody or antigen-binding fragment comprises an amino acid sequence with one or more modification groups. For example, an antibody or antigen-binding fragment disclosed herein may contain a flexible linker sequence, or may be modified to add functional groups (eg, PEG, drug, toxin, or tag).
本发明公开的抗体、抗原结合片段包括被修饰的衍生物，即通过任何类型的分子与抗体或抗原结合片段的共价连接进行修饰，其中共价连接不会阻止抗体或抗原结合片段与表位结合。包括但不限制以下实例，抗体或抗原结合片段可以被糖基化、乙酰化、聚乙二醇化、磷酸化、酰胺化、通过已知的保护/封闭基团衍生化、蛋白水解切割、连接至细胞配体或其他蛋白质等。众多化学修饰中的任一种修饰可以通过现有技术进行，包括但不限于特异性化学裂解、乙酰化、甲酰化、衣霉素的代谢合成等。本发明公开的抗体、抗原结合片段及其被修饰的衍生物包括其与酸和/或碱形成的盐。The antibodies, antigen-binding fragments disclosed herein include derivatives that are modified, that is, modified by covalent linkage of any type of molecule to the antibody or antigen-binding fragment, wherein the covalent linkage does not prevent the antibody or antigen-binding fragment from binding to the epitope combined. Examples including, but not limited to, antibodies or antigen-binding fragments may be glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytically cleaved, linked to Cell ligands or other proteins, etc. Any of the numerous chemical modifications can be performed by existing techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. The antibodies, antigen-binding fragments and modified derivatives thereof disclosed in the present invention include their salts with acids and/or bases.
在一些实施方案中，抗体或抗原结合片段可以与治疗剂、药物前体、肽、蛋白质、酶、病毒、脂类、生物反应调节剂、药剂或PEG缀合。In some embodiments, an antibody or antigen-binding fragment can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.
抗体或抗原结合片段可通过将其偶联至化学发光化合物来被可检测地标记。然后通过检测在化学反应过程中出现的发光从而确定化学发光标记的抗体或抗原结合片段的存在。化学发光标记化合物的实例包括鲁米诺、异鲁米诺、芳香吖啶酯、咪唑、吖啶盐和草酸酯。Antibodies or antigen-binding fragments can be detectably labeled by coupling them to chemiluminescent compounds. The presence of the chemiluminescently labeled antibody or antigen-binding fragment is then determined by detecting the luminescence that occurs during the course of the chemical reaction. Examples of chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.
编码抗体的多聚核苷酸和抗体的制备方法Antibody-encoding polynucleotides and antibody production methods
本发明还公开了编码本发明所述抗体、抗原结合片段、及其衍生物的多聚核苷酸或核酸分子。本发明公开的多聚核苷酸可以编码重链可变区、轻链可变区、Fc区、部分重链可变区、部分轻链可变区、重链或轻链等。制备抗体的方法是本领域公知的并且在本发明中有所描述。The invention also discloses polynucleotides or nucleic acid molecules encoding the antibodies, antigen-binding fragments, and derivatives thereof of the invention. The polynucleotide disclosed in the present invention can encode heavy chain variable region, light chain variable region, Fc region, part of heavy chain variable region, part of light chain variable region, heavy chain or light chain, etc. Methods of making antibodies are well known in the art and described herein.
在某些实施方案中，制备的抗体不会在待治疗的动物(例如人类)中引起有害的免疫应答。在一些实施方案中，本发明公开的抗体、抗原结合片段、或衍生物使用本领域公认的技术修饰以降低其免疫原性。例如，抗体可以被人源化、灵长类化、去免 疫化或者可以制备嵌合抗体。这些类型的抗体来源于非人抗体，通常是鼠类或灵长类抗体，其保留或基本保留亲本抗体的抗原结合特性但在人体中免疫原性较低。其可以通过多种方法来实现，包括(a)将整个非人源的可变区移植到人源的恒定区以产生嵌合抗体；(b)将一个或多个非人类互补决定区(CDR)的至少一部分移植到人源的框架和恒定区中，保留或不保留关键的框架残基；或(c)移植整个非人源的可变区，但通过用类人源的部分置换表面残基从而“隐藏”它们。通常人框架区中的框架残基将被来自CDR供体抗体的相应残基取代，比如能够改善抗原结合的残基。这些框架替换可以通过本领域公知的方法鉴定，例如通过模拟CDR和框架残基的相互作用以鉴定对抗原结合起重要作用的框架残基和通过序列对比以鉴定特定位置上异常的框架残基。(参考美国专利5,585,089；Riechmann et al.，Nature 332：323(1988)；其全部内容通过引用并入本文)。可以使用本领域公知的多种技术使抗体人源化，例如CDR移植(EP 239,400；WO 91/09967；美国专利5,225,539，5,530,101和5,585,089)，修复或者表面重排(EP592,106；EP519,596；Padlan，et al.，Molecular Immunology 28(4/5)：489-498(1991)；Studnicka et al.，Protein Engineering 7(6)：805-814(1994)；Roguska，et al.，Proc.Natl.Sci.USA 91：969-973(1994))，以及链的重排(美国专利5,565,332)，其全部内容通过引用并入本文。In certain embodiments, antibodies are produced that do not elicit an adverse immune response in the animal (eg, human) to be treated. In some embodiments, the antibodies, antigen-binding fragments, or derivatives disclosed herein are modified to reduce their immunogenicity using art-recognized techniques. For example, antibodies can be humanized, primatized, deimmunized or chimeric antibodies can be prepared. These types of antibodies are derived from non-human antibodies, usually murine or primate antibodies, which retain or substantially retain the antigen-binding properties of the parent antibody but are less immunogenic in humans. This can be achieved in a variety of ways, including (a) grafting entire non-human variable domains to human constant regions to generate chimeric antibodies; (b) grafting one or more non-human complementarity determining regions (CDRs) ) at least a portion of the human framework and constant regions, with or without key framework residues; or (c) grafting of the entire non-human variable region, but by replacing the surface residues with human-like parts base to "hide" them. Typically framework residues in the human framework regions will be replaced by corresponding residues from the CDR donor antibody, such as residues that improve antigen binding. These framework substitutions can be identified by methods known in the art, such as by modeling the interaction of CDRs and framework residues to identify framework residues important for antigen binding and by sequence alignment to identify abnormal framework residues at specific positions. (Refer to US Patent 5,585,089; Riechmann et al., Nature 332:323 (1988); the entire contents of which are incorporated herein by reference). Antibodies can be humanized using various techniques known in the art, such as CDR grafting (EP 239,400; WO 91/09967; US Patents 5,225,539, 5,530,101 and 5,585,089), repair or surface rearrangement (EP 592,106; EP 519,596; Padlan, et al., Molecular Immunology 28 (4/5): 489-498 (1991); Studnicka et al., Protein Engineering 7 (6): 805-814 (1994); Roguska, et al., Proc. Natl . Sci. USA 91:969-973 (1994)), and chain rearrangements (US Patent 5,565,332), the entire contents of which are incorporated herein by reference.
去免疫化也可用于降低抗体的免疫原性。在本发明中，术语“去免疫化”包括改变抗体以修饰T细胞表位(参见例如WO/9852976 A1和WO/0034317 A2)。例如，分析来自起始抗体的重链可变区序列和轻链可变区序列，并产生来自每个可变区的人T细胞表位“图谱”，显示表位相对于互补决定区(CDRs)和序列内其它关键残基的位置。分析来自T细胞表位图的单个T细胞表位，以鉴定具有较低改变抗体活性风险的可选择的氨基酸取代。设计包含氨基酸取代组合的一系列可选的重链可变区序列和轻链可变区序列，随后将这些序列掺入到一系列结合多肽中。然后将包含修饰过的可变区和人类恒定区的完整重链和轻链的基因克隆到表达载体中，随后将质粒转入细胞系以产生完整的抗体。然后利用合适的生物化学和生物学实验中比较抗体，鉴定出最佳的抗体。Deimmunization can also be used to reduce the immunogenicity of antibodies. In the present invention, the term "deimmunization" includes altering antibodies to modify T cell epitopes (see eg WO/9852976 A1 and WO/0034317 A2). For example, the heavy and light chain variable region sequences from a starting antibody are analyzed and a human T cell epitope "map" from each variable region is generated, showing the epitopes relative to the complementarity determining regions (CDRs) and the positions of other key residues within the sequence. Individual T-cell epitopes from T-cell epitope maps are analyzed to identify alternative amino acid substitutions with lower risk of altering antibody activity. A series of alternative heavy chain variable region sequences and light chain variable region sequences comprising combinations of amino acid substitutions are designed and these sequences are subsequently incorporated into a series of binding polypeptides. Genes for the complete heavy and light chains containing the modified variable and human constant regions are then cloned into expression vectors, and the plasmids are subsequently transformed into cell lines to produce complete antibodies. Antibodies are then compared using appropriate biochemical and biological assays to identify the best antibody.
本发明公开的抗体或抗原结合片段的结合特异性可以通过体外实验，例如免疫共沉淀、放射免疫实验(RIA)或酶联免疫吸附实验(ELISA)来检测。The binding specificity of the antibodies or antigen-binding fragments disclosed in the present invention can be detected by in vitro experiments, such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
scFv的制备可参见生产单链单元的技术(美国专利4,694,778；Bird，Science 242：423-442(1988)、Huston et al.，Proc.Natl.Acad.Sci.USA 55：5879-5883(1988)和Ward et al.，Nature 334：544-554(1989)和Nie et al.，Antibody Therapeutics 3(1)：78-62(2020))。通过氨基酸桥接Fv区的重链和轻链片段形成单链单元，产生单链融合肽。也可以使用在大肠杆菌中组装功能性Fv片段的技术(Skerra et al.，Science 242：1038-1041(1988))。The preparation of scFv can refer to the technology of producing single chain unit (US Patent 4,694,778; Bird, Science 242:423-442 (1988), Huston et al., Proc.Natl.Acad.Sci.USA 55:5879-5883 (1988) and Ward et al., Nature 334:544-554 (1989) and Nie et al., Antibody Therapeutics 3(1):78-62 (2020)). Single-chain fusion peptides are generated by amino acid bridging of the heavy and light chain fragments of the Fv region to form single-chain units. The technique of assembling functional Fv fragments in E. coli can also be used (Skerra et al., Science 242:1038-1041 (1988)).
可用于生产单链Fv(scFv)和抗体的技术的实例包括如美国专利4,946,778和5,258,498，以及Huston et al.，Methods in Enzymology 203：46-88(1991)、Shu et al.，Proc.Natl.Sci.USA 90：1995-1999(1993)和Skerra et al.，Science 240：1038-1040(1988)中所述。对于包括在人体内使用抗体和体外检测实验的某些用途，可以使用嵌合抗体、人源化抗体或全人源抗体。嵌合抗体是抗体的不同部分源自不同动物物种的一类分子，例如具有鼠源单克隆抗体的可变区和人源免疫球蛋白恒定区的抗体。生产嵌合抗体的方法是本领域已知的，参见Morrison，Science 229：1202(1985)；Oi et al.，BioTechniques 4：214(1986)；Gillies et al.，J.Immunol.Methods 125：191-202(1989)；Neuberger et al.，Nature 372：604-608(1984)；Takeda et al.，Nature 314：452-454(1985)；和美国专利5,807,715、4,816,567和4,816,397，其全部内容通过引用并入本文。Examples of techniques that can be used to produce single-chain Fv (scFv) and antibodies include, for example, U.S. Patents 4,946,778 and 5,258,498, and Huston et al., Methods in Enzymology 203:46-88 (1991), Shu et al., Proc. Natl. Sci. USA 90: 1995-1999 (1993) and Skerra et al., Science 240: 1038-1040 (1988). For certain uses involving the use of antibodies in humans and in vitro detection assays, chimeric, humanized or fully human antibodies may be used. Chimeric antibodies are molecules in which different parts of the antibody are derived from different animal species, such as antibodies that have the variable regions of a murine monoclonal antibody and the constant regions of a human immunoglobulin. Methods for producing chimeric antibodies are known in the art, see Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191 -202 (1989); Neuberger et al., Nature 372:604-608 (1984); Takeda et al., Nature 314:452-454 (1985); and U.S. Patents 5,807,715, 4,816,567 and 4,816,397, the entire contents of which are incorporated by reference Incorporated into this article.
此外，在Newman，Biotechnology 10：1455-1460(1992)中公开了另一种生产重组抗体的高效方法，特别地，该技术能产生含有猴可变区和人恒定区序列的灵长类抗体，该参考文献的全部内容通过引用并入本文。此外，该技术也在美国专利5,658,570、5,693,780和5,756,096中有所提及，每个专利的全部内容通过引用并入本文。In addition, another highly efficient method for producing recombinant antibodies is disclosed in Newman, Biotechnology 10: 1455-1460 (1992). In particular, this technique can produce primate antibodies containing monkey variable region and human constant region sequences, The entire content of this reference is incorporated herein by reference. Additionally, this technology is also mentioned in US Patent Nos. 5,658,570, 5,693,780, and 5,756,096, each of which is incorporated herein by reference in its entirety.
抗体可以通过本领域已知的多种方法制备，包括使用来自免疫球蛋白序列的抗体文库进行的噬菌体展示方法。也可参考美国专利4,444,887和4,716,111，以及PCT公布文本WO 98/46645、WO 98/50433、WO 98/24893、WO 98/16654、WO 96/34096、WO 96/33735和WO 91/10741，每个专利的全部内容通过引用并入本文。Antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries derived from immunoglobulin sequences. See also U.S. Patents 4,444,887 and 4,716,111, and PCT Publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741, each The entire content of the patent is incorporated herein by reference.
在另一实施方案中，使用常规方法(例如使用能够特异性结合编码鼠抗体重链和轻链的基因的寡核苷酸探针)，可以分离编码所需单克隆抗体的DNA并对其进行测序。分离的和亚克隆的杂交瘤细胞可以作为此类DNA的来源。一旦分离出来，DNA可以被置于表达载体中然后被转染到原核或真核宿主细胞如大肠杆菌细胞、猿猴COS细胞、中国仓鼠卵巢(CHO)细胞或不产生其他免疫球蛋白的骨髓瘤细胞中。分离的DNA(如本文所述可以是合成的)也可用于制备抗体的恒定区和可变区的序列，如美国专利5,658,570中所述，其全部内容通过引用并入本文。该方法从所选细胞中提取RNA并转化成cDNA，然后使用Ig特异性引物通过PCR技术进行扩增。适于此目的的合适的探针在美国专利5,658,570中也有所提及。In another embodiment, DNA encoding the desired monoclonal antibody can be isolated and sequenced using conventional methods (e.g., using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). sequencing. Isolated and subcloned hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce other immunoglobulins middle. Isolated DNA (which may be synthetic as described herein) can also be used to prepare the constant and variable region sequences of antibodies as described in US Pat. No. 5,658,570, the entire contents of which are incorporated herein by reference. This method extracts RNA from selected cells and converts it into cDNA, which is then amplified by PCR using Ig-specific primers. Suitable probes for this purpose are also mentioned in US Patent No. 5,658,570.
此外，使用常规重组DNA技术，可将本发明的抗体的一个或多个CDR插入框架区，例如插入到人类框架区以构建人源化非全人源抗体。框架区可以是天然存在的或共有的框架区，优选人类框架区(参见Chothia et al.，J.Mol.Biol.278：457-479(1998)，其列出一系列人类框架区)。一些多核苷酸可以编码框架区和CDR组合产生的与目标抗原的至少一个表位特异性结合的抗体。在框架区内可以进行一个或多个氨基酸取代，可以选择能够改善抗体与其抗原结合的氨基酸取代。另外，可用此法进行参与链间二 硫键形成的一个或多个可变区中半胱氨酸残基的取代或缺失，从而产生缺少一个或多个链间二硫键的抗体分子。本领域技术范围内的对多核苷酸进行的其他改变也涵盖于本发明中。In addition, using conventional recombinant DNA techniques, one or more CDRs of an antibody of the invention can be inserted into a framework region, eg, into a human framework region, to construct a humanized non-fully human antibody. The framework regions may be naturally occurring or consensus framework regions, preferably human framework regions (see Chothia et al., J. Mol. Biol. 278:457-479 (1998) for a list of human framework regions). Some polynucleotides may encode an antibody that specifically binds at least one epitope of an antigen of interest produced by a combination of framework regions and CDRs. One or more amino acid substitutions may be made within the framework regions, and the amino acid substitutions may be selected to improve binding of the antibody to its antigen. In addition, substitution or deletion of cysteine residues in one or more variable regions that participate in interchain disulfide bond formation can be performed in this way, thereby producing antibody molecules lacking one or more interchain disulfide bonds. Other modifications to polynucleotides within the skill of the art are also encompassed in the present invention.
抗体可以通过使用常规重组DNA技术制备。使用本领域技术人员公知的技术可以选择、构建和培养生产抗体的载体及细胞系等。这些技术在各种实验室手册和主要出版物中均有描述，例如Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells，D.L.Hacker，F.M.Wurm，in Reference Module in Life Sciences，2017，其全部内容包括补充内容通过引用并入全文。Antibodies can be prepared using conventional recombinant DNA techniques. Antibody-producing vectors, cell lines, and the like can be selected, constructed, and cultured using techniques known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, such as Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, D.L. Hacker, F.M. Wurm, in Reference Module in Life Sciences, 2017, which in its entirety includes The supplementary content is incorporated by reference in its entirety.
在一些实施方案中，可以按常规方法根据本文所述抗体氨基酸序列设计合成编码抗体的DNA，将其置入表达载体中，然后转染宿主细胞，在培养基中培养被转染的宿主细胞产生单克隆抗体。在一些实施方案中，表达抗体载体包括至少一个启动子元件，抗体编码序列，转录终止信号和polyA尾。其他元件包括增强子，Kozak序列及插入序列两侧RNA剪接的供体和受体位点。可以通过SV40的前期和后期启动子，来自逆转录病毒的长末端重复序列如RSV、HTLV1、HIVI及巨细胞病毒的早期启动子来获得高效的转录，也可应用其它一些细胞的启动子如肌动蛋白启动子。合适的表达载体可包括pIRES1neo，pRetro-Off，pRetro-On，PLXSN，或者Plncx，pcDNA3.1(+/-)，pcDNA/Zeo(+/-)，pcDNA3.1/Hygro(+/-)，PSVL，PMSG，pRSVcat，pSV2dhfr，pBC12MI和pCS2等。常使用的哺乳动物细胞包括293细胞，Cos1细胞，Cos7细胞，CV1细胞，鼠L细胞和CHO细胞等。In some embodiments, the DNA encoding the antibody can be designed and synthesized according to the amino acid sequence of the antibody described herein in a conventional manner, placed into an expression vector, and then transfected into a host cell, and cultured in a medium to produce the transfected host cell. Monoclonal antibodies. In some embodiments, an antibody expression vector includes at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing flanking the inserted sequence. High-efficiency transcription can be obtained through the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and early promoters of cytomegalovirus, and other cellular promoters such as muscle Kinetin promoter. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or Plncx, pcDNA3.1(+/-), pcDNA/Zeo(+/-), pcDNA3.1/Hygro(+/-), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2 etc. Commonly used mammalian cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells and CHO cells, etc.
在一些实施方案中，插入基因片段需含有筛选标记，常见的筛选标记包括二氢叶酸还原酶，谷氨酰胺合成酶，新霉素抗性，潮霉素抗性等筛选基因，以便于转染成功的细胞的筛选分离。将构建好的质粒转染到无上述基因的宿主细胞，经过选择性培养基培养，转染成功的细胞大量生长，产生想要获得的目的蛋白。In some embodiments, the inserted gene fragment needs to contain selection markers, common selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other selection genes, so as to facilitate transfection Screening of successful cell isolation. The constructed plasmid is transfected into host cells without the above-mentioned genes, and cultured in a selective medium, the successfully transfected cells grow in large numbers and produce the desired target protein.
此外，可以使用本领域技术人员已知的标准技术在编码本发明所述抗体的核苷酸序列中引入突变，包括但不限于导致氨基酸取代的定点突变和PCR介导的突变。变体(包括衍生物)编码相对于原重链可变区和轻链可变区来说少于50个氨基酸的取代、少于40个氨基酸的替换、少于30个氨基酸的取代、少于25个氨基酸的取代、少于20个氨基酸的取代、少于15个氨基酸的取代、少于10个氨基酸的取代、少于5个氨基酸的取代、少于4个氨基酸的取代、少于3个氨基酸的取代或少于2个氨基酸的取代。或者可以沿着全部或部分编码序列时随机引入突变，例如通过饱和突变，以及可以筛选所得突变体的生物活性以鉴定保留活性的突变体。In addition, mutations can be introduced into the nucleotide sequence encoding the antibodies of the present invention using standard techniques known to those skilled in the art, including but not limited to site-directed mutagenesis and PCR-mediated mutations resulting in amino acid substitutions. Variants (including derivatives) encode substitutions of less than 50 amino acids, substitutions of less than 40 amino acids, substitutions of less than 30 amino acids, substitutions of less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions Amino acid substitutions or substitutions of less than 2 amino acids. Alternatively mutations can be introduced randomly along all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity.
治疗方法treatment method
本发明还提供了治疗方法和用途。在一些实施方案中，提供了用于治疗或改善各种类型的癌症、慢性感染和自身免疫疾病等相关疾病的方法，所述方法包括向有需要的患者施用有效剂量的抗SIRPα抗体或抗原结合片段。在一些实施方案中，提供了抗SIRPα抗体或抗原结合片段在用于治疗或改善癌症、慢性感染和自身免疫疾病等相关疾病中的应用。在一些实施方案中，提供了所述抗SIRPα抗体或抗原结合片段在制备用于治疗或改善癌症、慢性感染和自身免疫疾病等相关疾病的药物中的应用。The invention also provides treatment methods and uses. In some embodiments, methods for treating or improving various types of cancers, chronic infections, and autoimmune diseases and other related diseases are provided, the methods comprising administering an effective dose of an anti-SIRPα antibody or an antigen-binding antibody to a patient in need thereof. fragment. In some embodiments, the application of anti-SIRPα antibody or antigen-binding fragment in treating or improving related diseases such as cancer, chronic infection and autoimmune disease is provided. In some embodiments, the use of the anti-SIRPα antibody or antigen-binding fragment in the preparation of medicines for treating or improving related diseases such as cancer, chronic infection and autoimmune diseases is provided.
对于任何特定患者的具体剂量和治疗方案将取决于各种因素，包括所使用的特定抗体或衍生物、患者的年龄和体重、一般健康状况、性别和饮食，以及给药时间、排泄频率、药物组合，以及所治疗的特定疾病的严重程度。由包括在本领域普通技术人员范围内的医疗护理人员对这些因素进行判断。所述剂量还将取决于待治疗的个体患者、给药途径、制剂类型、所用化合物的特性、疾病的严重程度以及所需的效果。所用剂量可以通过本领域熟知的药理学和药代动力学原理确定。在一些实施方案中，本发明抗体施用于患者的剂量为每次0.01mg/kg至100mg/kg患者体重。在一些实施方案中，每1星期、2星期、3星期、或每月给药一次。The specific dosage and treatment regimen for any particular patient will depend on various factors, including the specific antibody or derivative used, the patient's age and weight, general health, sex, and diet, as well as the time of administration, frequency of excretion, drug combination, and the severity of the particular disease being treated. These factors are in the judgment of the medical caregiver, who is within the purview of those of ordinary skill in the art. The dosage will also depend on the individual patient to be treated, the route of administration, the type of formulation, the nature of the compound employed, the severity of the disease and the effect desired. The dosage used can be determined by principles of pharmacology and pharmacokinetics well known in the art. In some embodiments, the antibody of the present invention is administered to a patient at a dose of 0.01 mg/kg to 100 mg/kg of the patient's body weight each time. In some embodiments, the administration is every 1 week, 2 weeks, 3 weeks, or monthly.
抗体、抗原结合片段或衍生物的施用方法包括但不限于真皮内、肌肉、腹腔、静脉、皮下、鼻腔、硬脊膜外和口服施用。药物组合物可以通过任何方便的途径施用，例如通过输注或推注，通过上皮或皮肤粘膜(例如口腔粘膜、直肠和肠粘膜等)吸收，并且可以与其他生物活性剂共同施用。因此，含有本发明抗体或抗原结合片段的药物组合物可以口服给药、直肠给药、肠胃外给药、脑池内给药、阴道内给药、腹腔内给药、外敷(如通过粉末，软膏，滴剂或透皮贴剂)、口腔给药或通过口服或鼻腔喷雾给药。Methods of administration of antibodies, antigen-binding fragments or derivatives include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural, and oral administration. The pharmaceutical compositions may be administered by any convenient route, such as by infusion or bolus injection, absorbed through the epithelium or mucous membranes (eg, oral mucosa, rectal and intestinal mucosa, etc.), and may be co-administered with other biologically active agents. Accordingly, pharmaceutical compositions containing an antibody or antigen-binding fragment of the invention may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (e.g., by powder, ointment , drops or transdermal patch), orally or by oral or nasal spray.
本发明使用的术语“肠胃外”是指包括静脉内、肌肉内、腹腔内、胸骨内、皮下和关节内注射和输注的施用方式。施用方式可以是全身施用或局部施用。The term "parenteral" as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. The mode of administration can be systemic administration or local administration.
本发明抗体或抗原结合片段可以局部施用于需要治疗的区域；可以通过但不限于以下方式：手术期间局部施用，例如与手术后伤口敷料联合的局部应用，通过注射，通过导管，借助栓剂或借助植入物来实现，所述植入物是多孔的、无孔的或凝胶状的材料，包括膜(例如硅橡胶膜)或纤维。在一些实施方式中，当施用本发明的蛋白质(包括抗体)时，必须注意使用不吸收蛋白质的材料。Antibodies or antigen-binding fragments of the invention may be administered topically to the area in need of treatment; by, but not limited to, topical application during surgery, for example in conjunction with post-operative wound dressing, by injection, by catheter, by suppository or by means of implants, which are porous, non-porous or gel-like materials comprising membranes (such as silicone rubber membranes) or fibers. In some embodiments, when administering proteins of the invention, including antibodies, care must be taken to use materials that do not absorb the protein.
在一些实施方案中，本发明组合物包含编码抗体或抗原结合片段的核酸或多聚核苷酸，可以通过将其构建为合适的核酸表达载体的一部分来体内施用所述核酸以促进其编码的蛋白质的表达，然后通过下述方式施用上述部分载体使其变为胞内部分，例 如通过使用逆转录病毒载体(参见美国专利4,980,286)，或通过直接注射，或通过使用微粒轰击(例如基因枪；Biolistic，Dupont)，或用脂质或细胞表面受体或转染试剂包被，或者通过与已知进入细胞核的同源异型盒类肽连接施用(参见例如Joliot et al.，1991，Proc.Natl.Acad.Sci.USA 88：1864-1868)等等。可选地，核酸可以通过同源重组在引入细胞内并整合至宿主细胞DNA中用于表达。In some embodiments, the compositions of the invention comprise a nucleic acid or polynucleotide encoding an antibody or antigen-binding fragment, which can be administered in vivo to facilitate its encoded expression by constructing it as part of a suitable nucleic acid expression vector. Expression of the protein, followed by administration of the above-mentioned part of the vector to make it an intracellular part, for example by using a retroviral vector (see US Pat. No. 4,980,286), or by direct injection, or by using particle bombardment (such as a gene gun; Biolistic, Dupont), or coated with lipid or cell surface receptors or transfection reagents, or administered via linkage to homeobox peptides known to enter the nucleus (see for example Joliot et al., 1991, Proc. Natl .Acad.Sci.USA 88:1864-1868) and so on. Alternatively, the nucleic acid can be introduced into the cell by homologous recombination and integrated into the host cell DNA for expression.
可以通过例如脂质化等修饰来增强抗体或抗原结合片段的摄取和组织穿透能力，从而减少本发明抗体的施用的剂量和频率。各种已知输送系统可用于施用本发明抗体、抗原结合片段或衍生物或编码其的多核苷酸，例如包封于脂质体、微粒、微胶囊、能够表达所述化合物的重组细胞、受体介导的内吞作用(参见例如Wu and Wu，1987，J.Biol.Chem.262：4429-4432)、作为逆转录病毒或其它载体的一部分的核酸的构建等。The dose and frequency of administration of antibodies of the invention can be reduced by enhancing the uptake and tissue penetration of antibodies or antigen-binding fragments through modifications such as lipidation. Various known delivery systems can be used to administer the antibodies, antigen-binding fragments or derivatives of the invention, or polynucleotides encoding the same, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds, subject Body-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of nucleic acids as part of retroviruses or other vectors, etc.
联合疗法combination therapy
在一些实施方案中，本发明抗SIRPα抗体或抗原结合片段可以结合其它治疗或预防方案用于治疗癌症，包括施用一种或多种本发明抗体或抗原结合片段，以及一种或多种其它治疗剂或方法一起使用或组合使用。在一些实施方案中，其他治疗方案包括但不限于放射疗法、化学疗法、激素疗法等。对于组合治疗，抗体可以与其它治疗剂可同时或分开施用。当分开施用时，可以在施用另一种其它治疗剂之前或之后施用本发明抗体或抗原结合片段。In some embodiments, an anti-SIRPα antibody or antigen-binding fragment of the invention may be used in combination with other therapeutic or prophylactic regimens for the treatment of cancer, including administration of one or more antibodies or antigen-binding fragments of the invention, in combination with one or more other therapeutic Agents or methods used together or in combination. In some embodiments, other treatment regimens include, but are not limited to, radiation therapy, chemotherapy, hormone therapy, and the like. For combination therapy, the antibodies can be administered simultaneously or separately from the other therapeutic agents. When administered separately, the antibody or antigen-binding fragment of the invention can be administered before or after another other therapeutic agent is administered.
在一些实施方案中，可以与本发明抗体或抗原结合片段一起施用的抗癌剂包括但不限于：5-氟尿嘧啶、阿西维辛、阿地白介素、六甲蜜胺、氨鲁米特、安吖啶、阿那曲唑、安曲霉素、天冬酰胺酶、阿扎胞苷、阿扎替派、阿佐霉素、巴马司他、比卡鲁胺、硫酸博来霉素、布喹那钠、溴匹立明、白消安、卡铂、卡莫司汀、盐酸卡柔比星、卡折来新、西地芬戈、苯丁酸氮芥、西罗霉素、顺铂、克拉屈滨、甲横酸克立那托(crisnatol mesylate)、环磷酰胺、阿糖胞苷、达卡巴嗪、更生霉素、盐酸柔红霉素、地西他滨、右奥马铂、地扎胍宁、甲磺酸地扎胍宁(dezaguanine mesylate)、地吖醌、多西他赛、多柔比星、盐酸阿霉素、屈洛昔芬、柠檬酸屈洛昔芬、丙酸屈他雄酮、达佐霉素、依达曲沙、盐酸依氟鸟氨酸、恩洛铂、恩普氨酯、依匹哌啶、盐酸表柔比星、厄布洛唑、盐酸伊索比星、雌莫司汀、雌莫司汀磷酸钠、依他硝唑、依托泊苷、磷酸依托泊苷、法扎拉滨、芬维A胺、氟尿苷、磷酸氟达拉滨、氟尿嘧啶、氟西他滨、磷喹酮、福司曲星钠、吉西他滨、盐酸吉西他滨、羟基脲、盐酸伊达比星、异环磷酰胺、伊莫福新、白介素II(包括重组白介素II)、干扰素α-2a、干扰素α-2b、干扰素α-m、干扰素α-n3、干扰素β-Ia、干扰素γ-I b、异丙铂、盐酸伊立替康、醋酸兰瑞肽、来曲唑、亮丙瑞林乙酸盐、盐酸利阿唑、洛美曲索钠、洛莫司汀、盐酸洛索蒽醌、马索罗酚、盐酸氮芥、醋酸甲地孕酮、乙酸甲烯雌醇、美法仑、美诺立尔、巯基嘌呤、甲氨蝶呤、甲氨蝶呤 钠、氯苯氨啶、美妥替哌、丝裂霉素、米托司培、米托坦、盐酸米托蒽醌、麦考酚酸、诺考达唑、奥马铂、紫杉醇、培门冬酶、紫菜霉素(porfromycin)、泼尼莫司汀、盐酸丙卡巴肼、嘌呤霉素、罗谷亚胺、盐酸沙芬戈、司莫司汀、辛曲秦、司泊索非钠、司帕霉素、螺莫司汀、螺铂、链黑霉素、链脲菌素、磺氯苯脲、太利苏霉素、替加氟、盐酸替洛蒽醌、替莫泊芬、替尼泊苷、替罗昔隆、睾内酯、硫咪嘌呤、硫鸟嘌呤、噻替派、噻唑呋林、替拉扎明、拓扑替康、三甲曲沙、葡萄糖醛酸三甲曲沙、曲普瑞林、乌拉莫司汀、乌瑞替派、伐普肽、维替泊芬(verteporfn)、硫酸长春碱、硫酸长春新碱、长春地辛、硫酸长春地辛、硫酸长春匹定、硫酸长春甘酯、硫酸长春罗辛、酒石酸长春瑞滨、硫酸长春罗定、硫酸长春利定、伏氯唑、折尼铂、净司他丁和盐酸佐柔比星等。In some embodiments, anticancer agents that can be administered with the antibodies or antigen-binding fragments of the invention include, but are not limited to: 5-fluorouracil, acivicin, aldesleukin, hexamethylmelamine, aminoglutethimide, amsacrine Pyridine, Anastrozole, Antramycin, Asparaginase, Azacitidine, Azathepa, Azomycin, Batimastat, Bicalutamide, Bleomycin Sulfate, Buquina Sodium , bropirimine, busulfan, carboplatin, carmustine, carrubicin hydrochloride, kazellexine, sildifenagore, chlorambucil, siromycin, cisplatin, cladri Bine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine, dexomaplatin, dezaguanine, Dezaguanine mesylate, deacrine, docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, drostanolone propionate, Dazomycin, edatrexate, eflornithine hydrochloride, enloplatin, enprabamate, epilipridine, epirubicin hydrochloride, ebrozole, esorubicin hydrochloride, estramox Stine, estramustine sodium phosphate, etanidazole, etoposide, etoposide phosphate, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, flucitabine , fosquinone, fostrixin sodium, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, imofosin, interleukin II (including recombinant interleukin II), interferon α-2a, interference Interferon α-2b, interferon α-m, interferon α-n3, interferon β-Ia, interferon γ-I b, isoproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide Relin Acetate, Liarazole Hydrochloride, Lometresol Sodium, Lomustine, Loxoanthraquinone Hydrochloride, Masoprofen, Nitrogen Mustard Hydrochloride, Megestrol Acetate, Methylestradiol Acetate, Phalan, Menolil, Mercaptopurine, Methotrexate, Methotrexate Sodium, Chlorpheniramine, Metutepa, Mitomycin, Mitospex, Mitotane, Mitoxrene Hydrochloride Quinone, mycophenolic acid, nocodazole, omaplatin, paclitaxel, pegaspargase, porfromycin, prednimustine, procarbazine hydrochloride, puromycin, roglutamic acid, hydrochloric acid Safinge, semustine, octrazine, sporsofi sodium, spamycin, spiromustine, spiroplatinum, streptomycin, streptozotocin, sulfonylurea, terisu Mycin, tegafur, tiloxantrone hydrochloride, temoporfin, teniposide, tiroxiron, testolactone, thiomethoprine, thioguanine, thiotepa, thiazofurin, tila Zamine, topotecan, trimetrexate, trimetrexate glucuronate, triptorelin, uramustine, uretipa, vapretide, verteporfn, vinblastine sulfate, sulfuric acid Vincristine, vindesine, vindesine sulfate, vinblastine sulfate, vinblastine sulfate, vinca rosin sulfate, vinorelbine tartrate, vinblastine sulfate, vinblastine sulfate, vorozole, zeniplatin , net astatin and Zorubicin hydrochloride, etc.
在一些实施方案中，可以与本发明抗体或抗原结合片段一起施用的治疗性抗体为免疫细胞(B或T淋巴细胞)的免疫检查点阻断剂或活化剂，治疗性抗体包括但不限于：抗PD-L1抗体、抗PD1抗体、抗CTLA-4抗体、抗CD137抗体、抗CD2抗体、抗CD28抗体、抗CD40抗体、抗HVEM抗体、抗BTLA抗体、抗CD160抗体、抗TIGIT抗体、抗TIM-1/3抗体、抗LAG-3抗体、抗2B4抗体、抗OX40抗体、抗CD40抗体、抗CD40-L抗、抗ICOS抗体、抗ICOS-L抗体。In some embodiments, the therapeutic antibody that can be administered with the antibody or antigen-binding fragment of the invention is an immune checkpoint blocker or activator of immune cells (B or T lymphocytes), and the therapeutic antibody includes, but is not limited to: Anti-PD-L1 antibody, anti-PD1 antibody, anti-CTLA-4 antibody, anti-CD137 antibody, anti-CD2 antibody, anti-CD28 antibody, anti-CD40 antibody, anti-HVEM antibody, anti-BTLA antibody, anti-CD160 antibody, anti-TIGIT antibody, anti-TIM -1/3 antibody, anti-LAG-3 antibody, anti-2B4 antibody, anti-OX40 antibody, anti-CD40 antibody, anti-CD40-L antibody, anti-ICOS antibody, anti-ICOS-L antibody.
药物组合物pharmaceutical composition
本发明还提供了药物组合物。这样的组合物包含有效剂量的抗SIRPα抗体或抗原结合片段以及药学上可接受的辅料。在一些实施方案中，药物组合物包含0.1％-90％的抗SIRPα抗体或抗原结合片段。在一些实施方案中，药物组合物还包含抗癌剂(例如免疫检查点抑制剂或活化剂)。The present invention also provides pharmaceutical compositions. Such compositions comprise effective doses of anti-SIRPα antibodies or antigen-binding fragments and pharmaceutically acceptable auxiliary materials. In some embodiments, the pharmaceutical composition comprises 0.1%-90% of an anti-SIRPα antibody or antigen-binding fragment. In some embodiments, the pharmaceutical composition further comprises an anti-cancer agent (eg, an immune checkpoint inhibitor or activator).
在一些实施方案中，术语“药学上可接受的”是指由政府的监管机构批准的或公认药典中列出的用于动物，特别是用于人类的物质。此外，“药学上可接受的辅料”通常指是任何类型的无毒固体、半固体或液体填充剂、稀释剂、包封材料或制剂助剂等。In some embodiments, the term "pharmaceutically acceptable" refers to a substance approved by a governmental regulatory agency or listed in a recognized pharmacopoeia for use in animals, especially in humans. In addition, "pharmaceutically acceptable excipients" generally refer to any type of non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials or formulation aids, etc.
术语“辅料”是指可以与活性成分一起施用于患者的稀释剂、佐剂、赋形剂或载体。这此类药物辅料可以是无菌液体，如水和油，包括石油、动植物或合成来源的油，如花生油、大豆油、矿物油、芝麻油等。当药物组合物静脉内给药时，水是优选的载体。盐水溶液和葡萄糖水溶液和甘油溶液也可用作液体载体，特别是用于注射溶液。合适的药物赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、大米、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、甘油、丙烯、乙二醇、水、乙醇等。如有需要，组合物还可以含有少量的润湿剂或乳化剂，或pH缓冲剂如乙酸盐、柠檬酸盐或磷酸盐。抗菌剂如苯甲醇或对羟基苯甲酸甲酯、抗氧化剂如抗坏 血酸或亚硫酸氢钠、螯合剂如乙二胺四乙酸，以及调节张力的试剂如氯化钠或右旋葡萄糖也是可以预见的。这些组合物可以采取溶液、悬液、乳剂、片剂、丸剂、胶囊、散剂、缓释制剂等形式。该组合物可以用传统的粘合剂和载体如甘油三酯配制成栓剂。口服制剂可以包括标准载体，例如药物等级的甘露糖醇、乳糖、淀粉、硬脂酸镁、糖精钠、纤维素、碳酸镁等。合适的药物载体的实例在E.W.Martin的Remington′s Pharmaceutical Sciences中有描述，在此通过引用并入本发明。此类组合物将含有临床有效剂量的抗体或抗原结合片段，优选以纯化后的形式，连同合适数量的载体，以提供适合于患者的给药形式。该制剂应该适用于给药模式。制剂可以封装在安瓿瓶、一次性注射器或由玻璃或塑料制成的多剂量小瓶中。The term "adjuvant" refers to a diluent, adjuvant, excipient or carrier with which the active ingredient can be administered to a patient. Such pharmaceutical excipients can be sterile liquids, such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, Propylene, ethylene glycol, water, ethanol, etc. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and tonicity adjusting agents such as sodium chloride or dextrose are also contemplated. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin, which is hereby incorporated by reference. Such compositions will contain a clinically effective dose of the antibody or antigen-binding fragment, preferably in purified form, together with an appropriate amount of carrier to provide a form suitable for administration to the patient. The formulation should be suitable for the mode of administration. The preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
在一些实施方案中，根据常规步骤将组合物配制成适合静脉内注射于人体的药物组合物。用于静脉内给药的组合物通常是在无菌等渗水性缓冲液中的溶液。组合物还可包含增溶剂和局部麻醉剂如利多卡因，从而缓解注射部位的疼痛。一般而言，有效成分以单位剂量形式单独供给或混在一起供给，如以干燥的冻干粉末或无水浓缩物的形式装在可指示活性剂份量的密封容器(如安瓿瓶或小袋)中。在通过输注施用组合物的情况下，可以用含有无菌药用级水或盐水的输液瓶来分装组合物。在通过注射施用组合物的情况下，可以使用注射用的无菌水或盐水的安瓿瓶，使得可以在施用之前混合有效成分。In some embodiments, the composition is formulated into a pharmaceutical composition suitable for intravenous injection to human body according to conventional procedures. Compositions for intravenous administration are generally solutions in sterile isotonic aqueous buffer. The composition may also contain a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the active ingredients are presented alone or in combination in unit dosage form, eg, as a dry lyophilized powder or water-free concentrate, in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the composition is administered by infusion, the composition may be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of administering the composition by injection, an ampoule of sterile water or saline for injection can be used so that the active ingredient can be mixed before administration.
本发明的化合物可以配制成中性的或盐的形式。药学上可接受的盐包括与衍生自如盐酸、磷酸、乙酸、草酸、酒石酸等的阴离子形成的盐，以及与衍生自如钠、钾、铵、钙、氢氧化铁、异丙胺、三乙胺、2-乙氨基乙醇、组氨酸、普鲁卡因等的阳离子形成的盐。The compounds of the present invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts derived from anions such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts derived from such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2 - Salts of cations such as ethylaminoethanol, histidine, procaine and the like.
具体实施方式Detailed ways
以下通过具体的实施例进一步说明本发明的技术方案，具体实施例不代表对本发明保护范围的限制。其他人根据本发明理念所做出的一些非本质的修改或调整仍属于本发明的保护范围。The technical solutions of the present invention are further described below through specific examples, which do not represent limitations to the protection scope of the present invention. Some non-essential modifications or adjustments made by others according to the concept of the present invention still belong to the protection scope of the present invention.
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.
实施例1 SIRPα抗原的制备Example 1 Preparation of SIRPα antigen
从Uniprot蛋白数据库获取人V1型SIRPα的氨基酸序列(P78324)，人V1型SIRPα的胞外区的氨基酸序列是从第1位到第373位的氨基酸残基。从GeneBank数据库获取V2型SIRPα的氨基酸序列(D86043)，人V2型SIRPα的胞外区的氨基酸序列是第1位到第369位的氨基酸残基。从EMBL-Bank数据库获取V8型SIRPα的氨基酸序 列(NA18570_V8)，人V8型SIRPα的胞外区的氨基酸序列是从第1位到第369位的氨基酸残基。从Uniprot蛋白数据库获取人IgG1-Fc的氨基酸序列(P01857)，其氨基酸序列是从第104位到第330位的氨基酸残基。The amino acid sequence (P78324) of human V1 type SIRPα was obtained from the Uniprot protein database, and the amino acid sequence of the extracellular region of human V1 type SIRPα was from the 1st to the 373rd amino acid residue. The amino acid sequence (D86043) of V2-type SIRPα was obtained from the GeneBank database, and the amino acid sequence of the extracellular region of human V2-type SIRPα is the amino acid residues 1 to 369. Obtain the amino acid sequence (NA18570_V8) of V8 type SIRPα from EMBL-Bank database, the amino acid sequence of the extracellular region of human V8 type SIRPα is from the 1st to the 369th amino acid residue. The amino acid sequence of human IgG1-Fc (P01857) was obtained from the Uniprot protein database, and its amino acid sequence is from the 104th to the 330th amino acid residues.
然后人工合成(通用公司)V1型SIRPα胞外区(1-373)、V2型SIRPα胞外区(1-369)、V8型SIRPα(1-369)和Fc区(104-330)对应的核苷酸序列，再分别通过EcoRI和Hind III双酶切连接SIRPα和Fc，并插入到pCDNA3.1载体中(购自Invitrogen公司)，分别得到重组质粒：pCDNA-SIRPα V1-Fc、pCDNA-SIRPα V2-Fc和pCDNA-SIRPα V8-Fc。Then artificially synthesize (general company) V1 type SIRPα extracellular region (1-373), V2 type SIRPα extracellular region (1-369), V8 type SIRPα (1-369) and Fc region (104-330) corresponding core The nucleotide sequence was connected to SIRPα and Fc by EcoRI and Hind III double enzyme digestion respectively, and inserted into the pCDNA3.1 vector (purchased from Invitrogen Company), respectively to obtain recombinant plasmids: pCDNA-SIRPα V1-Fc, pCDNA-SIRPα V2 -Fc and pCDNA-SIRPα V8-Fc.
再将上述重组质粒通过PEI(聚醚酰亚胺)瞬转HEK293F细胞(购自ATCC)，培养7天后收集上清液，最后通过纯化得到hSIRPα V1-Fc、hSIRPα V2-Fc和hSIRPa Vα-Fc蛋白样品，用于下面实施例。Then the above recombinant plasmids were transiently transfected into HEK293F cells (purchased from ATCC) through PEI (polyetherimide), cultured for 7 days, and the supernatant was collected, and finally hSIRPα V1-Fc, hSIRPα V2-Fc and hSIRPα Vα-Fc were obtained by purification Protein samples, used in the following examples.
实施例2抗SIRPα抗体的筛选Example 2 Screening of anti-SIRPα antibodies
2.1噬菌体展示筛选2.1 Phage display screening
通过聚合酶链式反应(polymerase chain reaction，PCR)扩增未免疫的人外周血淋巴细胞(peripheral blood lymphocytes，PBLs)中VH基因和VL基因，再通过重叠聚合酶链式反应(Overlapping polymerase chain reaction，overlap PCR)将重链VH与轻链VL随机组合成单链抗体(single chain Fv，scFv)；scFv通过酶切位点插入至phagemid载体中构建成噬菌体库(库容大于10 11)，展示在噬菌体表面，用于抗体筛选(详见，SHEETS等人(1998)Cell Biology.95：6157-6162)。所使用的筛选条件包括：将溶解在PBS(磷酸缓冲盐溶液，pH8.2)中的抗原蛋白hSIRPα V1-Fc偶联在免疫管(购于Nunc公司)上，4℃放置过夜；第二天加入100μl封闭液(5％BSA(牛血清白蛋白))，室温封闭2小时；2小时后，加入100μl噬菌体(5×10 13PFU)，室温下作用1小时；用PBST(含0.05％Tween-20的PBS缓冲液)洗涤20次，以洗掉非结合的噬菌体；用100mM TEA(三乙胺，Sigma)洗脱液将与抗原蛋白hSIRPα V1-Fc特异性结合的噬菌体解离下来，之后感染处于对数期生长的大肠杆菌TG1细胞。37℃培养1小时，扩大培养噬菌体用于下一轮的筛选；上述筛选过程重复3轮：第1轮的抗原浓度为50μg/ml，第2轮的抗原浓度为10μg/ml，且第3轮的抗原浓度为10μg/ml。得到富集的与抗原蛋白hSIRPα V1-Fc特异性结合的噬菌体。 The VH gene and VL gene in unimmunized human peripheral blood lymphocytes (PBLs) were amplified by polymerase chain reaction (polymerase chain reaction, PCR), and then by overlapping polymerase chain reaction (Overlapping polymerase chain reaction) , overlap PCR) randomly combined heavy chain VH and light chain VL into a single chain Fv (single chain Fv, scFv); scFv was inserted into a phagemid vector through restriction sites to construct a phage library (stock capacity greater than 10 11 ), displayed on Phage surfaces for antibody screening (see, for details, SHEETS et al. (1998) Cell Biology. 95:6157-6162). The screening conditions used include: coupling the antigenic protein hSIRPα V1-Fc dissolved in PBS (phosphate-buffered saline, pH 8.2) to an immunotube (purchased from Nunc Company), and placing it overnight at 4°C; Add 100 μl of blocking solution (5% BSA (bovine serum albumin)) and block at room temperature for 2 hours; after 2 hours, add 100 μl of phage (5×10 13 PFU) for 1 hour at room temperature; use PBST (containing 0.05% Tween- 20 PBS buffer) to wash 20 times to wash off non-binding phage; use 100mM TEA (triethylamine, Sigma) eluent to dissociate the phage specifically bound to the antigenic protein hSIRPα V1-Fc, and then infect E. coli TG1 cells growing in logarithmic phase. Incubate at 37°C for 1 hour, and expand the cultured phages for the next round of screening; the above screening process was repeated for 3 rounds: the antigen concentration in the first round was 50 μg/ml, the antigen concentration in the second round was 10 μg/ml, and in the third round The antigen concentration was 10 μg/ml. The enriched phage specifically binding to the antigen protein hSIRPα V1-Fc was obtained.
2.2 ELISA鉴定阳性克隆2.2 Positive clones identified by ELISA
从含有上述3轮筛选后得到的噬菌体的细胞培养皿中，随机挑选单菌落并接种于含有氨苄青霉素的MagicMedia大肠杆菌表达培养基(Invitrogen)中。37℃培养菌落生长 至对数期后，培养温度调整为28℃培养过夜，第二天，在4000rpm、4℃条件离心30min，取上清用于ELISA检测。From the cell culture dish containing the phage obtained after the above three rounds of selection, a single colony was randomly selected and inoculated in MagicMedia E. coli expression medium (Invitrogen) containing ampicillin. After the colonies were grown at 37°C to the logarithmic phase, the culture temperature was adjusted to 28°C for overnight culture. On the second day, centrifuged at 4000rpm and 4°C for 30min, the supernatant was taken for ELISA detection.
ELISA检测方法为：抗原SIRPα-His(百普赛斯，货号SIA-H5225)提前一天以2μg/ml的浓度，100μl/孔包被于酶标板，4℃包被过夜，第二天用5％脱脂奶粉在37℃中封闭2小时，之后用于检测；取上述上清100μl到ELISA板，在室温下孵育1小时；用PBST洗去未结合的抗体，加入Myc-Tag(9B11)Mouse mAb(HRP Conjugate)(Cell Signaling Technology，Inc；目录号为2040S)，室温下放置1小时；用PBST洗去未结合的抗体，加入TMB(四甲基联苯胺)显色液(上海生工，货号为E66100)，室温下放置15min后加入0.1M硫酸终止反应，酶标仪在450nm波长读取吸收值。阳性判断标准为OD值高于0.5。The ELISA detection method is as follows: the antigen SIRPα-His (Baipu Saisi, product number SIA-H5225) is coated on the microtiter plate at a concentration of 2 μg/ml one day in advance, 100 μl/well, coated overnight at 4 ° C, and coated with 5 μg the next day. % skimmed milk powder was blocked at 37°C for 2 hours, and then used for detection; take 100 μl of the above supernatant to the ELISA plate, and incubate at room temperature for 1 hour; wash off unbound antibodies with PBST, and add Myc-Tag(9B11) Mouse mAb (HRP Conjugate) (Cell Signaling Technology, Inc; catalog number is 2040S), put it at room temperature for 1 hour; wash off unbound antibody with PBST, add TMB (tetramethylbenzidine) chromogenic solution (Shanghai Shenggong, Cat. No. E66100), after 15 minutes at room temperature, 0.1M sulfuric acid was added to terminate the reaction, and the absorbance was read at a wavelength of 450nm by a microplate reader. The positive judgment standard was that the OD value was higher than 0.5.
2.3筛选阻断CD47与SIRPα结合的scFv2.3 Screening scFv that blocks the binding of CD47 and SIRPα
同上所述ELISA检测方法，取上述鉴定为阳性的克隆上清50μl，与50ng/ml的CD47-Fc-biotin(北京百普赛斯生物科技股份有限公司，货号为CD7-H82F6)混合之后加入SIRPα-His包被的ELISA板中，室温下孵育1小时；用PBST洗去未结合的配体，加入Streptavidin-HRP(Jackson ImmunoResearch，货号为016-050-084)，室温下放置1小时；用PBST洗去未结合的抗体，加入TMB显色液，室温下放置15min后加入0.1M硫酸终止反应，读取吸收值。The same ELISA detection method as above, take 50 μl of the supernatant of the above-identified positive clones, mix it with 50 ng/ml CD47-Fc-biotin (Beijing Biosciences Biotechnology Co., Ltd., product number is CD7-H82F6), and then add SIRPα -His-coated ELISA plate, incubate at room temperature for 1 hour; wash unbound ligand with PBST, add Streptavidin-HRP (Jackson ImmunoResearch, product number 016-050-084), and place at room temperature for 1 hour; wash with PBST Wash away unbound antibodies, add TMB chromogenic solution, place at room temperature for 15 minutes, add 0.1M sulfuric acid to stop the reaction, and read the absorbance value.
检测筛选到结合呈阳性且能够阻断CD47与SIRPα结合(如图1所示)的多种scFv，如：scFv(P22-1)、scFv(P22-19)、scFv(P22-34)、scFv(P22-63)、scFv(P22-15)、scFv(P22-32)、scFv(Ab56)、scFv(P39-1)、scFv(P39-3)和scFv(P39-95)，并通过测序分析克隆序列。其中，scFv中VH和VL序列由表1所示序列并通过连接子(G 4S) 3连接。 A variety of scFvs that are positive for binding and can block the binding of CD47 and SIRPα (as shown in Figure 1) were detected and screened, such as: scFv (P22-1), scFv (P22-19), scFv (P22-34), scFv (P22-63), scFv(P22-15), scFv(P22-32), scFv(Ab56), scFv(P39-1), scFv(P39-3) and scFv(P39-95), and analyzed by sequencing clone sequence. Wherein, the VH and VL sequences in the scFv are connected by the sequences shown in Table 1 through a linker (G 4 S) 3 .
表1 scFv的组成Table 1 Composition of scFv
scFv编号scFv number VH序列号VH serial number VL序列号VL serial number
scFv(P22-1)scFv(P22-1) 1616 22twenty two
scFv(P22-19)scFv(P22-19) 1717 22twenty two
scFv(P22-34)scFv(P22-34) 1818 22twenty two
scFv(P22-63)scFv(P22-63) 1919 22twenty two
scFv(P22-15)scFv(P22-15) 2020 22twenty two
scFv(P22-32)scFv(P22-32) 21twenty one 22twenty two
scFv(Ab56)scFv(Ab56) 1919 23twenty three
scFv(P39-1)scFv(P39-1) 1919 24twenty four
scFv(P39-3)scFv(P39-3) 1919 2525
scFv(P39-95)scFv(P39-95) 1919 2626
实施例3抗体的制备The preparation of embodiment 3 antibody
依据上述筛选的scFv(P22-1)、scFv(P22-19)、scFv(P22-34)、scFv(P22-63)、、scFv(P22-15)、scFv(P22-32)、scFv(Ab56)、scFv(P39-1)、scFv(P39-3)和scFv(P39-95)，构建IgG1型单抗：抗体P22-1、抗体P22-19、抗体P22-34、抗体P22-63、抗体P22-15、抗体P22-32、抗体Ab56、抗体P39-1、抗体P39-3和抗体P39-95。参照抗体KWAR23来源于WO2015138600A2，参照抗体18D5HEFLB来源于WO2017178653A2。抗体实例相关的氨基酸序列和核酸序列见表2-6；表4中单下划线标出CDR区，表5中单下划线标出Fc区。scFv(P22-1), scFv(P22-19), scFv(P22-34), scFv(P22-63), scFv(P22-15), scFv(P22-32), scFv(Ab56) screened according to the above ), scFv(P39-1), scFv(P39-3) and scFv(P39-95), construct IgG1 monoclonal antibody: antibody P22-1, antibody P22-19, antibody P22-34, antibody P22-63, antibody P22-15, antibody P22-32, antibody Ab56, antibody P39-1, antibody P39-3, and antibody P39-95. The reference antibody KWAR23 is derived from WO2015138600A2, and the reference antibody 18D5HEFLB is derived from WO2017178653A2. The amino acid sequences and nucleic acid sequences related to the antibody examples are shown in Tables 2-6; the CDR region is marked with a single underline in Table 4, and the Fc region is marked with a single underline in Table 5.
人工合成得到上述抗体对应的重链核酸序列和轻链核酸序列，再通过EcoRI和HindIII双酶切连接分别把重链核酸序列和轻链核酸序列插入到pCHO1.0质粒中(购自Invitrogen)，得到用于表达全抗的重组质粒。采用Freedom CHO-S试剂盒(购自Invitrogen)，将上述重组质粒转入CHO-S细胞系中，培养11天后收集上清液，采用Protein A柱(GE Healthcare)的固定化金属亲和层析(IMAC)进行纯化，纯化后的抗体蛋白的纯度＞90％。The heavy chain nucleic acid sequence and light chain nucleic acid sequence corresponding to the above antibody were artificially synthesized, and then the heavy chain nucleic acid sequence and the light chain nucleic acid sequence were respectively inserted into the pCHO1.0 plasmid (purchased from Invitrogen) by EcoRI and HindIII double enzyme digestion ligation, A recombinant plasmid for expressing the whole antibody was obtained. Using the Freedom CHO-S kit (purchased from Invitrogen), the above recombinant plasmids were transferred into the CHO-S cell line, and the supernatant was collected after 11 days of culture, and immobilized metal affinity chromatography was used on a Protein A column (GE Healthcare). (IMAC) for purification, and the purity of the purified antibody protein is >90%.
对纯化后抗体进行凝胶电泳检测，抗体P22-1、P22-19、P22-34、P22-63、抗体P22-15、抗体P22-32、抗体Ab56、抗体P39-1、抗体P39-3和抗体P39-95为单一物质，分子量与理论值是一致的。对纯化后抗体进行测序，抗体P22-1、P22-19、P22-34、P22-63、抗体P22-15、抗体P22-32、抗体Ab56、抗体P39-1、抗体P39-3和抗体P39-95测序结果与预计的序列相同。纯化后抗体用于亲和力检测和生物活性鉴定等。The purified antibodies were detected by gel electrophoresis, antibody P22-1, P22-19, P22-34, P22-63, antibody P22-15, antibody P22-32, antibody Ab56, antibody P39-1, antibody P39-3 and Antibody P39-95 is a single substance, and its molecular weight is consistent with the theoretical value. The purified antibodies were sequenced, antibody P22-1, P22-19, P22-34, P22-63, antibody P22-15, antibody P22-32, antibody Ab56, antibody P39-1, antibody P39-3 and antibody P39- 95 sequencing results were identical to the expected sequence. The purified antibody is used for affinity detection and biological activity identification, etc.
表2抗SIRPα抗体的组成Table 2 Composition of anti-SIRPα antibodies
抗体编号Antibody number 重链SEQ ID NO(序列号)Heavy chain SEQ ID NO (serial number) 轻链SEQ ID NO(序列号)Light chain SEQ ID NO (serial number)
P22-1P22-1 3131 3939
P22-19P22-19 3232 3939
P22-34P22-34 3333 3939
P22-63P22-63 3434 3939
P22-15P22-15 3535 3939
P22-32P22-32 3636 3939
Ab56Ab56 3434 4040
P39-1P39-1 3434 4141
P39-3P39-3 3434 4242
P39-95P39-95 3434 4343
KWAR23KWAR23 3737 4444
18D5 HEFLB18D5 HEFLB 3838 4545
表3抗SIRPα抗体的CDR区Table 3 CDR regions of anti-SIRPα antibodies
Figure PCTCN2022095634-appb-000001
Figure PCTCN2022095634-appb-000001
表4抗SIRPα抗体的可变区和恒定区Table 4 Variable and constant regions of anti-SIRPα antibodies
Figure PCTCN2022095634-appb-000002
Figure PCTCN2022095634-appb-000002
Figure PCTCN2022095634-appb-000003
Figure PCTCN2022095634-appb-000003
Figure PCTCN2022095634-appb-000004
Figure PCTCN2022095634-appb-000004
表5抗SIRPα抗体的重链和轻链Table 5 Heavy chain and light chain of anti-SIRPα antibody
Figure PCTCN2022095634-appb-000005
Figure PCTCN2022095634-appb-000005
Figure PCTCN2022095634-appb-000006
Figure PCTCN2022095634-appb-000006
Figure PCTCN2022095634-appb-000007
Figure PCTCN2022095634-appb-000007
Figure PCTCN2022095634-appb-000008
Figure PCTCN2022095634-appb-000008
表6抗体相关核酸序列Table 6 Antibody related nucleic acid sequences
Figure PCTCN2022095634-appb-000009
Figure PCTCN2022095634-appb-000009
Figure PCTCN2022095634-appb-000010
Figure PCTCN2022095634-appb-000010
实施例4 ELISA检测抗体与抗原的结合Embodiment 4 ELISA detects the combination of antibody and antigen
[根据细则91更正 15.08.2022]　
采用实施例2中ELISA检测方法，检测结果显示抗体P22-1、P22-19、P22-34、和P22-63与SIRPα-his结合的EC 50分别为0.2428nM、0.1253nM、0.1287nM和0.1383nM。[Corrected 15.08.2022 under Rule 91]
Using the ELISA detection method in Example 2, the detection results show that the EC 50 of antibodies P22-1, P22-19, P22-34, and P22-63 binding to SIRPα-his are 0.2428nM, 0.1253nM, 0.1287nM and 0.1383nM, respectively .
[根据细则91更正 15.08.2022]　
实施例5 FLOW(流式)检测抗体与表达SIRPα细胞的结合 [Corrected 15.08.2022 under Rule 91]
Example 5 FLOW (flow cytometry) detects the binding of antibodies to cells expressing SIRPα
[根据细则91更正 15.08.2022]　
1)将含有人V1型SIRPα胞外端cDNA的pCMV载体(Invitrogen公司)转染CHO细胞，产生过表达人SIRPα-V1胞外端的CHO细胞，即CHO-SIRPα细胞。将CHO-SIRPα细胞(0.5×10 6个细胞)与抗体(初始浓度为100nM，2.5倍梯度稀释)在PBS缓冲液(含0.1％BSA)中置于冰上孵育40分钟。然后细胞洗涤两次，并与二抗Goat anti-Human IgG Fc Secondary Antibody，PE(eBioscience，货号：12-4998-82)在PBS中置于冰上孵育25分钟。细胞洗涤两次，在cytoflex系统(Beckman)上进行流式细胞术分析。[Corrected 15.08.2022 under Rule 91]
1) Transfect CHO cells with pCMV vector (Invitrogen) containing human V1 type SIRPα extracellular end cDNA to generate CHO cells overexpressing human SIRPα-V1 extracellular end, namely CHO-SIRPα cells. CHO-SIRPα cells (0.5×10 6 cells) were incubated with antibodies (initial concentration 100 nM, 2.5-fold serial dilution) in PBS buffer (containing 0.1% BSA) on ice for 40 minutes. Then the cells were washed twice, and incubated with the secondary antibody Goat anti-Human IgG Fc Secondary Antibody, PE (eBioscience, catalog number: 12-4998-82) in PBS on ice for 25 minutes. Cells were washed twice and analyzed by flow cytometry on a cytoflex system (Beckman).
[根据细则91更正 15.08.2022]　
结果显示，抗体P22-1、P22-19、P22-34和P22-63与CHO-SIRPα细胞结合的EC 50分别为17.98nM、2.832nM、2.532nM和2.64nM。[Corrected 15.08.2022 under Rule 91]
The results showed that the EC 50 of antibodies P22-1, P22-19, P22-34 and P22-63 binding to CHO-SIRPα cells were 17.98nM, 2.832nM, 2.532nM and 2.64nM, respectively.
[根据细则91更正 15.08.2022]　
2)采用上述相同方法，结果显示抗体Ab56和KWAR23与CHO-SIRPα细胞结合的EC 50分别为0.9686nM和0.765nM。[Corrected 15.08.2022 under Rule 91]
2) Using the same method as above, the results showed that the EC 50 of antibodies Ab56 and KWAR23 binding to CHO-SIRPα cells were 0.9686nM and 0.765nM, respectively.
实施例6 FLOW检测抗体阻断CD47与SIRPα的结合Example 6 FLOW detection antibody blocks the binding of CD47 and SIRPα
[根据细则91更正 15.08.2022]　
1)抗体(初始浓度为100nM，2.5倍梯度稀释)在PBS中与0.5μg/ml CD47-biotin混合之后，加入CHO-SIRPα细胞(0.5×10 6个)置于冰上孵育30分钟。然后细胞洗涤两次，并与二抗SA-PE Conjugate(eBioscience TM，Streptavidin PE Conjugate，货号：12-4317-87)在PBS中置于冰上孵育25分钟。细胞洗涤两次，在cytoflex系统(Beckman)上进行流式细胞术分析。[Corrected 15.08.2022 under Rule 91]
1) After the antibody (initial concentration is 100nM, 2.5-fold serial dilution) was mixed with 0.5 μg/ml CD47-biotin in PBS, CHO-SIRPα cells (0.5×10 6 ) were added and incubated on ice for 30 minutes. Cells were then washed twice and incubated with secondary antibody SA-PE Conjugate (eBioscience ™ , Streptavidin PE Conjugate, Cat. No.: 12-4317-87) in PBS on ice for 25 minutes. Cells were washed twice and analyzed by flow cytometry on a cytoflex system (Beckman).
[根据细则91更正 15.08.2022]　
结果显示，抗体P22-1、P22-19、P22-34和P22-63阻断CD47与CHO-SIRPα细胞结合的IC 50分别为44.61nM、12.26nM、10.86nM和10.31nM。[Corrected 15.08.2022 under Rule 91]
The results showed that the IC 50 of antibodies P22-1, P22-19, P22-34 and P22-63 blocking the binding of CD47 to CHO-SIRPα cells were 44.61nM, 12.26nM, 10.86nM and 10.31nM, respectively.
[根据细则91更正 15.08.2022]　
2)采用上述相同方法，结果显示抗体Ab56和KWAR23阻断CD47与CHO-SIRPα细胞结合的IC 50分别为3.847nM和4.757nM。[Corrected 15.08.2022 under Rule 91]
2) Using the same method as above, the results showed that the IC 50 of antibodies Ab56 and KWAR23 blocking the binding of CD47 to CHO-SIRPα cells were 3.847nM and 4.757nM, respectively.
实施例7抗SIRPα抗体的亲和力检测Example 7 Affinity Detection of Anti-SIRPα Antibody
1)采用生物光干涉实验测量抗SIRPα抗体的亲和力。ForteBio亲和力测定按照现有的常规方法(Estep，P等MAbs，2013，5(2)：270-8)。实验过程主要如下：先用protein A探头结合抗体(抗体浓度为20μg/ml)，PBS缓冲液里平衡后与不同浓度的SIRPα-His(43.75、87.5、175、350nM)结合得出结合常数K a，再转移至PBS缓冲液里解离得出解离常数K d，计算出结合解离平衡常数K D。 1) The affinity of anti-SIRPα antibody was measured by bio-light interference experiment. ForteBio affinity was determined according to existing conventional methods (Estep, P et al. MAbs, 2013, 5(2): 270-8). The main process of the experiment is as follows: first use the protein A probe to bind the antibody (the antibody concentration is 20 μg/ml), equilibrate in PBS buffer, and combine with different concentrations of SIRPα-His (43.75, 87.5, 175, 350nM) to obtain the binding constant K a , and then transferred to PBS buffer to dissociate to obtain the dissociation constant K d , and calculate the binding-dissociation equilibrium constant K D .
如表7所示，抗体P22-1、P22-19、P22-34和P22-63均能结合SIRPα-His。As shown in Table 7, antibodies P22-1, P22-19, P22-34 and P22-63 can all bind SIRPα-His.
表7抗体与SIRPα-His结合的亲和力常数Table 7 Affinity constants of antibodies binding to SIRPα-His
抗体Antibody K a(1/Ms) Ka (1/Ms) K d(1/s) K d (1/s) K D(M) K D (M)
P22-1P22-1 1.02E+061.02E+06 3.64E-023.64E-02 3.56E-083.56E-08
P22-19P22-19 1.25E+061.25E+06 5.68E-025.68E-02 4.55E-084.55E-08
P22-34P22-34 2.10E+062.10E+06 8.33E-028.33E-02 3.97E-083.97E-08
P22-63P22-63 1.43E+061.43E+06 4.65E-024.65E-02 3.25E-083.25E-08
2)采用上述相同方法检测抗体Ab56和KWAR23的亲和力常数，如表8所示，抗体Ab56结合SIRPα-His的亲和力优于参照抗体KWAR23。2) The affinity constants of antibodies Ab56 and KWAR23 were detected by the same method as above. As shown in Table 8, the binding affinity of antibody Ab56 to SIRPα-His is better than that of reference antibody KWAR23.
表8抗体Ab56与SIRPα-His结合的亲和力常数Table 8 Affinity constants of antibody Ab56 binding to SIRPα-His
抗体Antibody K a(1/Ms) Ka (1/Ms) K d(1/s) K d (1/s) K D(M) K D (M)
Ab56Ab56 4.67E+054.67E+05 1.29E-041.29E-04 2.77E-102.77E-10
KWAR23KWAR23 1.18E+061.18E+06 3.82E-033.82E-03 3.23E-093.23E-09
实施例8 ELISA检测抗体Ab56与不同抗原SIRP的结合Example 8 ELISA detection antibody Ab56 combined with different antigen SIRP
1)采用上述实施例中ELISA检测方法，检测结果显示抗体Ab56与hSIRPαV1-Fc、hSIRPα V2-Fc和hSIRPa Vα-Fc均能结合，其结合的EC 50分别为0.0898nM、0.0783nM和0.2166nM。 1) Using the ELISA detection method in the above example, the detection results show that the antibody Ab56 can bind to hSIRPαV1-Fc, hSIRPαV2-Fc and hSIRPαVα-Fc, and the EC 50 of the binding are 0.0898nM, 0.0783nM and 0.2166nM, respectively.
2)采用上述实施例中ELISA检测方法，检测结果显示抗体Ab56与hSIRPα(百普赛斯，货号SIA-H5225)、hSIRPβ1(百普赛斯，货号为SIA-H5257)、hSIRPγ(百普赛斯，货号为SIG-H5253)都能结合，其结合的EC 50分别为0.0384nM、0.0236nM和0.026nM。 2) Using the ELISA detection method in the above example, the test results show that the antibody Ab56 is compatible with hSIRPα (Bypsy, product number SIA-H5225), hSIRPβ1 (Bypsy, product number SIA-H5257), hSIRPγ (Bypsy , Cat. No. SIG-H5253) can be combined, and their binding EC 50 are 0.0384nM, 0.0236nM and 0.026nM, respectively.
3)采用上述实施例中ELISA检测方法，检测结果显示抗体Ab56与猴SIRPα(百普赛斯，货号为SIA-C52H7)、小鼠SIRPα(义翘神州，货号50956-M08H)和大鼠SIRPα(义翘神州，80270-R08H)结合的EC 50分别为0.0179nM、0.5755nM和5.784nM。 3) Using the ELISA detection method in the above example, the test results showed that the antibody Ab56 was compatible with monkey SIRPα (Baipusi, product number: SIA-C52H7), mouse SIRPα (Shenzhou, product number 50956-M08H) and rat SIRPα ( Sino Biological, 80270-R08H) binding EC 50 are 0.0179nM, 0.5755nM and 5.784nM, respectively.
实施例9抗体对免疫细胞的影响Example 9 Effects of Antibodies on Immune Cells
1)从健康志愿者的纯化血液中提取人外周血单个核细胞(PBMC)，将PBMC在4℃下采用100μg/ml IgG封闭30分钟，洗涤后用10μg/ml的生物素化的抗体染色30分钟，洗涤后用荧光标记的SA-PE抗体(eBioscience TM Streptavidin PE Conjugate，货号：12-4317-87)和标记免疫细胞的抗体在4℃再染色30分钟。洗涤后，在Cytoflex(Beckman Coulter)流式细胞仪上分析细胞。标记免疫细胞的抗体包括APC Anti-Human CD3[UCHT1](elabscience，货号E-AB-F1230E)，APC Anti-Human CD14 Antibody[M5E2](elabscience，货号E-AB-F1209E)，APC Anti-Human CD19 Antibody[CB19](elabscience，货号E-AB-F1004E)和APC Anti-Human CD56 Antibody[MEM188](elabscience，货号E-AB-F1006E)。 1) Human peripheral blood mononuclear cells (PBMC) were extracted from the purified blood of healthy volunteers, and the PBMC were blocked with 100 μg/ml IgG for 30 minutes at 4°C, washed and stained with 10 μg/ml biotinylated antibody for 30 minutes After washing, stain with fluorescently labeled SA-PE antibody (eBioscience TM Streptavidin PE Conjugate, Cat. No.: 12-4317-87) and antibody for labeling immune cells at 4°C for 30 minutes. After washing, cells were analyzed on a Cytoflex (Beckman Coulter) flow cytometer. Antibodies for labeling immune cells include APC Anti-Human CD3[UCHT1] (elabscience, Cat. No. E-AB-F1230E), APC Anti-Human CD14 Antibody[M5E2] (elabscience, Cat. No. E-AB-F1209E), APC Anti-Human CD19 Antibody [CB19] (elabscience, Cat. No. E-AB-F1004E) and APC Anti-Human CD56 Antibody [MEM188] (elabscience, Cat. No. E-AB-F1006E).
如图2所示，抗体Ab56能与T细胞(CD3 +细胞)和单核细胞(CD14 +细胞)结合，而与B细胞(CD19 +细胞)和NK细胞(CD56 +细胞)不结合。 As shown in Figure 2, antibody Ab56 was able to bind to T cells (CD3 + cells) and monocytes (CD14 + cells), but not to B cells (CD19 + cells) and NK cells (CD56 + cells).
2)从健康志愿者的纯化血液中提取人外周血单个核细胞，采用磁珠分选的方法从中分离纯化出CD3 +细胞；将CD3 +细胞以1500rpm离心8min，加入终浓度0.25μM CFSE(羟基荧光素二醋酸盐琥珀酰亚胺脂，荧光染料)，37℃标记15min，并加入含 10％FBS的RPMI-1640完全培养基(Gibco)终止，室温放置5min；离心洗涤之后用RPMI-1640完全培养基调整细胞密度到100万/ml，并加入T细胞刺激活化磁珠，100μl/孔；将抗体稀释至40μg/mL，100ul/孔；37℃、5％CO 2培养箱培养72小时；细胞用FACS缓冲液(含2％BSA和0.1％NaN 3的PBS(PH7.4))清洗两次，采用APC Anti-Human CD8a Antibody[OKT-8](elabscience，货号为E-AB-F1110E)，PE Anti-Human CD4 Antibody[RPA-T4](elabscience，货号为E-AB-F1109D)和PerCP/Cyanine5.5 Anti-Human CD25 Antibody[BC96]elabscience，货号为E-AB-F1194J)进行标记，1％多聚甲醛固定；流式细胞术进行检测分析。 2) Human peripheral blood mononuclear cells were extracted from the purified blood of healthy volunteers, and CD3 + cells were separated and purified by magnetic bead sorting; CD3 + cells were centrifuged at 1500 rpm for 8 min, and a final concentration of 0.25 μM CFSE (hydroxyl Fluorescein diacetate succinimide lipid, fluorescent dye), labeling at 37°C for 15min, and adding RPMI-1640 complete medium (Gibco) containing 10% FBS to terminate, and standing at room temperature for 5min; centrifuge and wash with RPMI-1640 Adjust the cell density to 1 million/ml with complete medium, and add T cell stimulation activated magnetic beads, 100 μl/well; dilute the antibody to 40 μg/mL, 100ul/well; incubate at 37°C, 5% CO 2 for 72 hours; Cells were washed twice with FACS buffer (PBS containing 2% BSA and 0.1% NaN 3 (PH7.4)), using APC Anti-Human CD8a Antibody [OKT-8] (elabscience, Cat. No. E-AB-F1110E) , PE Anti-Human CD4 Antibody [RPA-T4] (elabscience, catalog number is E-AB-F1109D) and PerCP/Cyanine5.5 Anti-Human CD25 Antibody [BC96] elabscience, catalog number is E-AB-F1194J) for labeling, 1% paraformaldehyde fixed; flow cytometry for detection and analysis.
如图3A和3B所示，抗体Ab56对CD4 +T细胞和CD8 +T细胞的增殖都没有影响，也表明T细胞增殖不受抗体Ab56与SIRPγ的结合的影响。 As shown in Figures 3A and 3B, antibody Ab56 had no effect on the proliferation of CD4 + T cells and CD8 + T cells, which also indicated that T cell proliferation was not affected by the binding of antibody Ab56 to SIRPγ.

Claims (13)

  1. 一种与SIRPα结合的抗体或抗原结合片段，所述抗体或抗原结合片段包含以下中的一个或多个氨基酸序列：An antibody or antigen-binding fragment that binds to SIRPα, said antibody or antigen-binding fragment comprising one or more of the following amino acid sequences:
    (a)HCDR1，其包含如SEQ ID NO：1所示的氨基酸序列或与SEQ ID NO：1相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；(a) HCDR1 comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 1;
    (b)HCDR2，其包含如SEQ ID NO：2所示的氨基酸序列或与SEQ ID NO：2相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；(b) HCDR2 comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 2;
    (c)HCDR3，其包含如SEQ ID NO：3-8中任一项所示的氨基酸序列或与SEQ ID NO：3-8中任一项相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；(c) HCDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 3-8 or an amino acid having a single site substitution, deletion or insertion compared to any one of SEQ ID NOs: 3-8 sequence, or consisting of it;
    (d)LCDR1，其包含如SEQ ID NO：9所示的氨基酸序列或与SEQ ID NO：9相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；(d) LCDR1 comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 9;
    (e)LCDR2，其包含如SEQ ID NO：10所示的氨基酸序列或与SEQ ID NO：10相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成；(e) LCDR2 comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having a single site substitution, deletion or insertion compared to SEQ ID NO: 10;
    (f)LCDR3，其包含如SEQ ID NO：11-15中任一项所示的氨基酸序列或与SEQ ID NO：11-15中任一项相比具有单一位点取代、缺失或插入的氨基酸序列，或由其组成。(f) LCDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 11-15 or an amino acid having a single site substitution, deletion or insertion compared to any one of SEQ ID NOs: 11-15 sequence, or consists of it.
  2. 一种与SIRPα结合的抗体或抗原结合片段，所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：3-8中任一项所示的HCDR3、如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：11-15中任一项所示的LCDR3中一个或多个。An antibody or antigen-binding fragment that binds to SIRPα, the antibody or antigen-binding fragment comprising HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR2 as shown in SEQ ID NO: 3-8 One of the HCDR3 shown in any one of the HCDR3, the LCDR1 shown in SEQ ID NO: 9, the LCDR2 shown in SEQ ID NO: 10, and the LCDR3 shown in any one of SEQ ID NOs: 11-15 or Multiple.
  3. 如权利要求1所述的抗体或抗原结合片段，所述抗体或抗原结合片段包含如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；或The antibody or antigen-binding fragment according to claim 1, said antibody or antigen-binding fragment comprising HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR2 as shown in SEQ ID NO: 3 HCDR3 indicated; or
    所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：4所示的HCDR3；或The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 4; or
    所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：5所示的HCDR3；或The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 5; or
    所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：6所示的HCDR3；或The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 6; or
    所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：7所示的HCDR3；或The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 7; or
    所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：8所示的HCDR3。The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 8.
  4. 如权利要求1-3任一项所述的抗体或抗原结合片段，所述抗体或抗原结合片段包含如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：11所示的LCDR3；或The antibody or antigen-binding fragment according to any one of claims 1-3, said antibody or antigen-binding fragment comprising LCDR1 as shown in SEQ ID NO: 9, LCDR2 as shown in SEQ ID NO: 10, and LCDR2 as shown in SEQ ID NO: 10 and ID NO: LCDR3 shown in 11; or
    所述抗体或抗原结合片段包含如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：12所示的LCDR3；或The antibody or antigen-binding fragment comprises LCDR1 as shown in SEQ ID NO: 9, LCDR2 as shown in SEQ ID NO: 10, and LCDR3 as shown in SEQ ID NO: 12; or
    所述抗体或抗原结合片段包含如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：13所示的LCDR3；或The antibody or antigen-binding fragment comprises LCDR1 as shown in SEQ ID NO: 9, LCDR2 as shown in SEQ ID NO: 10, and LCDR3 as shown in SEQ ID NO: 13; or
    所述抗体或抗原结合片段包含如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：14所示的LCDR3；或The antibody or antigen-binding fragment comprises LCDR1 as shown in SEQ ID NO: 9, LCDR2 as shown in SEQ ID NO: 10, and LCDR3 as shown in SEQ ID NO: 14; or
    所述抗体或抗原结合片段包含如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：15所示的LCDR3。The antibody or antigen-binding fragment comprises LCDR1 as shown in SEQ ID NO:9, LCDR2 as shown in SEQ ID NO:10 and LCDR3 as shown in SEQ ID NO:15.
  5. 一种与SIRPα结合的抗体或抗原结合片段，所述抗体或抗原结合片段包含如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：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：11所示的LCDR3；或An antibody or antigen-binding fragment that binds to SIRPα, the antibody or antigen-binding fragment comprising HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR2 as shown in SEQ ID NO: 3 HCDR3, LCDR1 as set forth in SEQ ID NO: 9, LCDR2 as set forth in SEQ ID NO: 10, and LCDR3 as set forth in SEQ ID NO: 11; or
    所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：4所示的HCDR3、如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：11所示的LCDR3；或The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 4, HCDR3 as shown in SEQ ID NO: 9 LCDR1, LCDR2 as set forth in SEQ ID NO: 10, and LCDR3 as set forth in SEQ ID NO: 11; or
    所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：5所示的HCDR3、如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：11所示的LCDR3；或The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 5, HCDR3 as shown in SEQ ID NO: 9 LCDR1, LCDR2 as set forth in SEQ ID NO: 10, and LCDR3 as set forth in SEQ ID NO: 11; or
    所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：6所示的HCDR3、如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：11所示的LCDR3；或The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 6, HCDR3 as shown in SEQ ID NO: 9 LCDR1, LCDR2 as set forth in SEQ ID NO: 10, and LCDR3 as set forth in SEQ ID NO: 11; or
    所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：6所示的HCDR3、如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：12所示的LCDR3； 或The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 6, HCDR3 as shown in SEQ ID NO: 9 LCDR1, LCDR2 as set forth in SEQ ID NO: 10, and LCDR3 as set forth in SEQ ID NO: 12; or
    所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：7所示的HCDR3、如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：11所示的LCDR3；或The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 7, HCDR3 as shown in SEQ ID NO: 9 LCDR1, LCDR2 as set forth in SEQ ID NO: 10, and LCDR3 as set forth in SEQ ID NO: 11; or
    所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：8所示的HCDR3、如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：11所示的LCDR3；或The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 8, HCDR3 as shown in SEQ ID NO: 9 LCDR1, LCDR2 as set forth in SEQ ID NO: 10, and LCDR3 as set forth in SEQ ID NO: 11; or
    所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：6所示的HCDR3、如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：13所示的LCDR3；或The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 6, HCDR3 as shown in SEQ ID NO: 9 LCDR1, LCDR2 as set forth in SEQ ID NO: 10, and LCDR3 as set forth in SEQ ID NO: 13; or
    所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：6所示的HCDR3、如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：14所示的LCDR3；或The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 6, HCDR3 as shown in SEQ ID NO: 9 LCDR1, LCDR2 as set forth in SEQ ID NO: 10, and LCDR3 as set forth in SEQ ID NO: 14; or
    所述抗体或抗原结合片段包含如SEQ ID NO：1所示的HCDR1、如SEQ ID NO：2所示的HCDR2、如SEQ ID NO：6所示的HCDR3、如SEQ ID NO：9所示的LCDR1、如SEQ ID NO：10所示的LCDR2和如SEQ ID NO：15所示的LCDR3。The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 6, HCDR3 as shown in SEQ ID NO: 9 LCDR1, LCDR2 as shown in SEQ ID NO: 10 and LCDR3 as shown in SEQ ID NO: 15.
  6. 如权利要求1-5任一项所述的抗体或抗原结合片段，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：16-21中任一项所示的序列，与SEQ ID NO：16-21中任一项所示序列相比具有至少80％同一性的序列，或与SEQ ID NO：16-21中任一项所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列，或由其组成；和/或The antibody or antigen-binding fragment according to any one of claims 1-5, wherein the heavy chain variable region of said antibody or antigen-binding fragment comprises the sequence shown in any one of SEQ ID NO: 16-21, and SEQ ID NO: A sequence having at least 80% identity compared to the sequence shown in any one of ID NOs: 16-21, or having one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 16-21 The amino acid sequence of, or consisting of; and/or
    所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：22-26中任一项所示的序列，与SEQ ID NO：22-26中任一项所示序列相比具有至少80％同一性的序列，或与SEQ ID NO：22-26中任一项所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列，或由其组成。The light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in any one of SEQ ID NOs: 22-26, and has at least 80% of the sequence shown in any one of SEQ ID NOs: 22-26. % identity, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 22-26, or consisting of.
  7. 一种与SIRPα结合的抗体或抗原结合片段，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：16所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：22所示的序列；或An antibody or antigen-binding fragment that binds to SIRPα, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 16, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 22; or
    所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：17所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：22所示的序列；或The heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 17, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 22; or
    所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：18所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：22所示的序列；或The heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 18, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 22; or
    所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：19所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：22所示的序列；或The heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 19, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 22; or
    所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：20所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：22所示的序列；或The heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 20, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 22; or
    所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：21所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：22所示的序列；或The heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 21, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 22; or
    所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：19所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：23所示的序列；或The heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 19, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 23; or
    所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：19所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：24所示的序列；或The heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 19, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 24; or
    所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：19所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：25所示的序列；或The heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 19, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 25; or
    所述抗体或抗原结合片段的重链可变区包含SEQ ID NO：19所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO：26所示的序列。The heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 19, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 26.
  8. 如权利要求1-7任一项所述的抗体或抗原结合片段，所述抗体或抗原结合片段还包括重链恒定区和/或轻链恒定区，所述重链恒定区包含氨基酸序列如SEQ ID NO：27-29中任一项所示的序列，或与SEQ ID NO：27-29中任一项所示序列相比具有至少80％同一性的序列成与SEQ ID NO：27-29中任一项所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列，或由其组成；和/或The antibody or antigen-binding fragment according to any one of claims 1-7, said antibody or antigen-binding fragment further comprising a heavy chain constant region and/or a light chain constant region, said heavy chain constant region comprising an amino acid sequence such as SEQ The sequence shown in any one of ID NOs: 27-29, or a sequence having at least 80% identity compared with the sequence shown in any one of SEQ ID NOs: 27-29 and SEQ ID NOs: 27-29 An amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in any one of, or consisting of; and/or
    所述轻链恒定区包含氨基酸序列如SEQ ID NO：30所示的序列，或与SEQ ID NO：30所示序列相比具有至少80％同一性的序列，或与SEQ ID NO：30所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列，或由其组成。The light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 30, or a sequence with at least 80% identity compared with the sequence shown in SEQ ID NO: 30, or a sequence shown in SEQ ID NO: 30 An amino acid sequence having, or consisting of, one or more conservative amino acid substitutions compared to the sequence.
  9. 一种与SIRPα结合的抗体，所述抗体的重链包含氨基酸序列如SEQ ID NO：31所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：39所示的序列；或An antibody that binds to SIRPα, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 31, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 39; or
    所述抗体的重链包含氨基酸序列如SEQ ID NO：32所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：39所示的序列；或The heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 32, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 39; or
    所述抗体的重链包含氨基酸序列如SEQ ID NO：33所示的序列，所述抗体的 轻链包含氨基酸序列如SEQ ID NO：39所示的序列；或The heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 33, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 39; or
    所述抗体的重链包含氨基酸序列如SEQ ID NO：34所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：39所示的序列；或The heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 39; or
    所述抗体的重链包含氨基酸序列如SEQ ID NO：35所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：39所示的序列；或The heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 35, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 39; or
    所述抗体的重链包含氨基酸序列如SEQ ID NO：36所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：39所示的序列；或The heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 36, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 39; or
    所述抗体的重链包含氨基酸序列如SEQ ID NO：34所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：40所示的序列；或The heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 40; or
    所述抗体的重链包含氨基酸序列如SEQ ID NO：34所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：41所示的序列；或The heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 41; or
    所述抗体的重链包含氨基酸序列如SEQ ID NO：34所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：42所示的序列；或The heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 34, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO: 42; or
    所述抗体的重链包含氨基酸序列如SEQ ID NO：34所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO：43所示的序列。The heavy chain of the antibody includes the amino acid sequence shown in SEQ ID NO: 34, and the light chain of the antibody includes the amino acid sequence shown in SEQ ID NO: 43.
  10. 一种生物材料，为a biological material for
    (1)核酸，其编码如权利要求1-7任一项所述的抗体或抗原结合片段，或权利要求8所述抗体；(1) a nucleic acid encoding the antibody or antigen-binding fragment of any one of claims 1-7, or the antibody of claim 8;
    (2)载体，其包含编码如权利要求1-7任一项所述的抗体或抗原结合片段或如权利要求8所述的抗体的核酸；或(2) a vector comprising a nucleic acid encoding the antibody or antigen-binding fragment of any one of claims 1-7 or the antibody of claim 8; or
    (3)宿主细胞，其包含编码如权利要求1-7任一项所述的抗体或抗原结合片段或如权利要求8所述的抗体的核酸。(3) A host cell comprising a nucleic acid encoding the antibody or antigen-binding fragment of any one of claims 1-7 or the antibody of claim 8.
  11. 一种药物组合物，其包含如权利要求1-8任一项所述的抗体或抗原结合片段，或如权利要求9所述抗体；或者，还包含药学上可接受的辅料。A pharmaceutical composition comprising the antibody or antigen-binding fragment according to any one of claims 1-8, or the antibody according to claim 9; or, further comprising pharmaceutically acceptable excipients.
  12. [根据细则91更正 15.08.2022]　
    诊断或预后试剂盒，其包含如权利要求1-8任一项所述的抗体或抗原结合片段或如权利要求9所述的抗体；任选的，所述试剂盒还包括第二抗体，其特异性识别所述抗SIRPα抗体；任选的，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、化学发光物质、有色物质或酶；任选的，所述试剂盒用于检测SIRPα在样品中的存在或其水平；任选的，所述试剂盒还包括针对其它抗原的抗体或抗原结合片段，和/或细胞毒性剂，和任选的，使用说明书。     [Corrected 15.08.2022 under Rule 91]
    A diagnostic or prognostic kit comprising the antibody or antigen-binding fragment as claimed in any one of claims 1-8 or the antibody as claimed in claim 9; optionally, the kit further comprises a second antibody comprising specifically recognize the anti-SIRPα antibody; optionally, the second antibody also includes a detectable label, such as a radioactive isotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme; optionally, the kit uses It is used to detect the presence or level of SIRPα in a sample; optionally, the kit further includes antibodies or antigen-binding fragments against other antigens, and/or cytotoxic agents, and optionally, instructions for use.
  13. 如权利要求1-8任一项所述的抗体或抗原结合片段、如权利要求9所述的抗体或如权利要求11所述的药物组合物在制备治疗或预防疾病的药物的用 途，其中所述疾病为免疫细胞功能障碍疾病，例如所述疾病为吞噬细胞功能障碍疾病；或者，所述疾病为癌症、感染或自身免疫病。Use of the antibody or antigen-binding fragment as claimed in any one of claims 1-8, the antibody as claimed in claim 9, or the pharmaceutical composition as claimed in claim 11 in the preparation of medicines for treating or preventing diseases, wherein the The disease is a disease of immune cell dysfunction, for example, the disease is a disease of phagocyte dysfunction; or, the disease is cancer, infection or autoimmune disease.
PCT/CN2022/095634 2021-05-28 2022-05-27 ANTI-SIRPα ANTIBODY AND USE THEREOF WO2022247933A1 (en)

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