WO2022247933A1 - ANTICORPS ANTI-SIRPα ET SON UTILISATION - Google Patents

ANTICORPS ANTI-SIRPα ET SON UTILISATION 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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English (en)
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/zh
Publication of WO2022247933A1 publication Critical patent/WO2022247933A1/fr

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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

L'invention concerne un anticorps anti-SIRPα ou un fragment de liaison à l'antigène de celui-ci, et son utilisation. L'anticorps ou le fragment de liaison à l'antigène de celui-ci peut se lier de manière spécifique à SIRPα et bloquer une voie de signalisation SIRPα/CD47, aide le système immunitaire à éliminer les cellules tumorales, et est également utilisé dans le diagnostic ou le pronostic de tumeurs ou de cancers.
PCT/CN2022/095634 2021-05-28 2022-05-27 ANTICORPS ANTI-SIRPα ET SON UTILISATION WO2022247933A1 (fr)

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