WO2022083723A1 - 抗cd73的抗体及其用途 - Google Patents

抗cd73的抗体及其用途 Download PDF

Info

Publication number
WO2022083723A1
WO2022083723A1 PCT/CN2021/125564 CN2021125564W WO2022083723A1 WO 2022083723 A1 WO2022083723 A1 WO 2022083723A1 CN 2021125564 W CN2021125564 W CN 2021125564W WO 2022083723 A1 WO2022083723 A1 WO 2022083723A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
antibody
amino acid
sequence
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2021/125564
Other languages
English (en)
French (fr)
Inventor
李百勇
夏瑜
王忠民
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Akeso Pharmaceuticals Inc
Original Assignee
Akeso Biopharma Inc
Akeso Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to CA3196299A priority Critical patent/CA3196299A1/en
Priority to EP21882137.9A priority patent/EP4234579A4/en
Priority to KR1020237017266A priority patent/KR20230125774A/ko
Priority to IL302182A priority patent/IL302182A/en
Priority to US18/250,010 priority patent/US20240018254A1/en
Priority to JP2023548995A priority patent/JP2023546743A/ja
Application filed by Akeso Biopharma Inc, Akeso Pharmaceuticals Inc filed Critical Akeso Biopharma Inc
Priority to AU2021363350A priority patent/AU2021363350A1/en
Priority to MX2023004713A priority patent/MX2023004713A/es
Publication of WO2022083723A1 publication Critical patent/WO2022083723A1/zh
Priority to ZA2023/04591A priority patent/ZA202304591B/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • 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
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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 [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the field of immunology.
  • it relates to anti-CD73 antibodies and uses thereof.
  • Ecto-5'-nucleotidase namely CD73 protein
  • CD73 protein is a multifunctional glycoprotein with a molecular weight of 70KD encoded by the NT5E gene.
  • phosphatidy linositol, GPI is anchored to the cell membrane (Zimmermann H. Biochem J. 1992;285:345-365).
  • CD73 is widely distributed on the surface of human tissue cells. Current studies have found that CD73 is highly expressed in a variety of solid tumors, such as cancer cells, dendritic cells, regulatory T cells (Treg), natural killer cells (NK cells), Myeloid-derived suppressor cells (MDSC), tumor-associated macrophages (TAM), etc. Hypoxia induces the upregulation of molecules such as hypoxia-inducible factor-1 (HIF-1), which in turn leads to the widespread expression of CD73 in the tumor microenvironment (Synnestvedt K, et al. J Clin Invest. 2002; 110:993-1002.). Analysis of clinical tumor samples showed that high CD73 expression is a potential biomarker that is closely associated with poor prognosis in various types of tumors, including breast, lung, ovarian, renal, gastric, head and neck cancers, etc.
  • HIF-1 hypoxia-inducible factor-1
  • CD73 has both hydrolase activity and non-hydrolase activity.
  • the enzymatic and non-enzymatic functions of CD73 exist in tumor-related processes, and promote each other to maintain tumor progression. More and more studies have found that CD73 is a key regulator of tumor cell proliferation, metastasis and invasion in vitro, tumor angiogenesis and tumor immune escape in vivo.
  • CD39 upstream of CD73 can catalyze the production of adenosine monophosphate (AMP) from ATP, which is converted to adenosine by CD73, and adenosine binds to the downstream adenosine receptor (A2AR), A2AR
  • AMP adenosine monophosphate
  • A2AR adenosine receptor
  • PKA protein kinase A
  • Csk kinase By activating protein kinase A (PKA) and Csk kinase, it inhibits a series of signaling pathways related to immune activation, such as LCK, MAPK, and PKC, and inhibits the immune killing effect of T cells, thereby exerting an immunosuppressive effect (Antonioli L, et al. Nat Rev Cancer. 2013;13:842-857.).
  • the transmembrane receptor PD-1 (programmed cell death-1) is a member of the CD28 gene family and is expressed on activated T cells, B cells and myeloid cells.
  • Cells are expressed, including T cells, B cells and endothelial cells and epithelial cells, PDL2 is only expressed in antigen-presenting cells such as dendritic cells and macrophages.
  • the PD-1/PDL1 signaling pathway plays an important role in regulating immune tolerance, microbial infection and tumor immune escape.
  • the expression of PD-1 is mainly in immune cells such as T cells, while the ligand PDL1 of PD-1 is mainly highly expressed in many human tumor tissues.
  • Blocking the PD-1/PDL1 signaling pathway can activate the suppressed T cells to attack cancer cells. Blocking PD-1/PDL1 signaling can promote the proliferation of tumor antigen-specific T cells and play a role in killing tumor cells, thereby inhibiting local tumor growth (Julie R et al., 2012, N Engl J Med. 366: 2455-2465 ).
  • tumors expressing high PDL1 are accompanied by cancers that are difficult to detect (Hamanishi et al., 2007, Proc. Natl. Acad. Sci. USA 104:3360-5).
  • An effective method to implement is to modulate the expression of PD-1 by injecting anti-PD-1 antibody in vivo. Due to the broad-spectrum anti-tumor prospects and amazing efficacy of PD-1 antibodies, the industry generally believes that antibodies targeting the PD-1 pathway will bring breakthroughs in the treatment of various tumors: for the treatment of non-small cell lung cancer, renal cell Cancer, ovarian cancer, melanoma (Homet M.B., Parisi G., et al., 2015, Semin Oncol. 42(3):466-473), leukemia and anemia (Held SA, Heine A, et al., 2013 , Curr Cancer Drug Targets. 13(7):768-74).
  • Cytotoxic T lymphocyte-associated antigen-4 also referred to as CTLA4
  • CD28 molecules are closely related in gene structure, chromosomal location, sequence homology and gene expression, both of which are costimulatory Receptor for molecule B7, mainly expressed on the surface of activated T cells.
  • CTLA4 and B7 can inhibit the activation of mouse and human T cells and play a negative regulatory role in T cell activation.
  • CTLA4 antibody or anti-CTLA4 monoclonal antibody
  • CTLA4 ligand can prevent CTLA4 from binding to its natural ligand, thereby blocking CTLA4's negative regulatory signaling to T cells and enhancing T cell responsiveness to various antigens, in this regard
  • the results of in vivo and in vitro studies were basically consistent.
  • CTLA4 monoclonal antibodies in clinical trials or approved for the treatment of prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, malignant melanoma, etc. (Grosso JF., Jure-Kunkel MN., 2013 , Cancer Immun. 13:5.).
  • CTLA4 and CTLA4 antibodies as important factors affecting the functional status of T cells, play a role by interfering with the immune microenvironment of the body.
  • CTLA4 antibody can specifically relieve the immune suppression of CTLA4, activate T cells, and induce IL-2 production.
  • CTLA4 antibody can have a specific therapeutic effect on diseases, and exert a high curative effect, supplementing the insufficiency of traditional drugs, thus opening up a new way of gene therapy.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Fab segment of an antibody binds to the antigenic epitope of virus-infected cells or tumor cells, and its Fc segment interacts with killer cells (NK cells). , macrophages, etc.) on the surface of Fc receptors (Fc Receptor, FcR) binding, mediating the killing cells to directly kill target cells.
  • NK cells killer cells
  • Fc Receptor, FcR Fc Receptor
  • CDC complement dependent cytotoxicity
  • complement-dependent cytotoxicity means that when the antibody specifically binds to the corresponding antigen on the surface of the cell membrane, a complex is formed to activate the complement system, and then MAC is formed on the surface of the target cell, resulting in the lysis of the target cell.
  • Complement can lead to the lysis of a variety of bacteria and other pathogenic organisms, and is an important defense mechanism of the body against pathogenic organisms.
  • Fc receptors are immunoglobulin family proteins that are expressed on the surface of specific immune cells and are used to recognize the Fc region of antibodies to mediate immune responses. After the Fab region of the antibody recognizes the antigen, the Fc region of the antibody binds to Fc receptors on immune cells (such as killer cells) to initiate immune cell response functions, such as phagocytosis and ADCC. According to the different types of antibodies recognized by Fc receptors and expressing cells, Fc ⁇ RIIIa was found to be closely related to the ADCC effect. Fc ⁇ RIIIa is the most dominant molecule mediating ADCC (Hogarth PM, Pietersz GA. 2012, NATURE REVIEWS DRUG DISCOVERY, 11(4):311-331).
  • the IgG family consists of four members, IgG1, IgG2, IgG3 and IgG4, which have amino acid differences in the fragment crystallizable (Fc) region of their heavy chain constant regions, resulting in their different affinities with Fc ⁇ Rs.
  • IgG1 is the most subtype in the human body and the most used subtype in monoclonal antibody drugs. IgG1 can bind to various Fc ⁇ Rs and can trigger ADCC and CDC effects. Antibodies in the presence of ADCC and/or CDC may lead to unwanted targeted tissue damage, with negative effects affecting drug pharmacology.
  • the inventors used a mammalian cell expression system to express recombinant human CD73 as an antigen to immunize mice, and obtained hybridoma cells by fusing mouse spleen cells with myeloma cells.
  • the inventors obtained the hybridoma cell line LT014 by screening a large number of samples (the deposit number is CCTCC NO: C2018137).
  • the hybridoma cell line LT014 can secrete and produce a specific monoclonal antibody (named 19F3) that specifically binds to human CD73, and the monoclonal antibody can be very effectively inhibited in a non-substrate competition manner
  • the enzymatic reaction of CD73 reduces the production of adenosine, promotes the activity of T cells, and exerts the effect of inhibiting tumor growth.
  • the inventors creatively prepared humanized antibodies against human CD73 (named 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM), 19F3H2L3 and 19F3H2L3 (hG1DM)), in addition, the anti-CD73 antibodies introduced amino acid mutations to eliminate ADCC and CDC effects, avoiding unwanted toxic effects mediated by antibodies.
  • the inventors also surprisingly found that the combination of the antibody of the present invention and the anti-PD-1/CTLA-4 bispecific antibody has the pharmacological effect of effectively inhibiting the proliferation of tumor cells, which is better than that of the anti-PD-1/CTLA-4 bispecific Sexual antibody monotherapy or anti-CD73 antibody monotherapy.
  • Another aspect of the present invention also relates to an antibody, wherein the anti-CD73 antibody comprises:
  • the anti-CD73 antibody comprises
  • HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% with the sequence , 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or have one or more sequences compared to said sequence (preferably 1, 2 or 3) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions), or consisting of,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 16, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% with the sequence , 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or have one or more sequences compared to said sequence (preferably 1, 2 or 3) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions), or consisting of,
  • HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 17, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% with the sequence , 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or have one or more sequences compared to said sequence (preferably 1, 2 or 3) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions), or consisting of,
  • LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% with said sequence , 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or have one or more sequences compared to said sequence (preferably 1, 2 or 3) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions), or consisting of,
  • LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 19, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% with said sequence , 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or have one or more sequences compared to said sequence (preferably 1, 2 or 3) the amino acid sequence of conservative amino acid mutations (preferably substitutions, insertions or deletions), or consisting of, and
  • LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% with said sequence , 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or have one or more sequences compared to said sequence
  • the amino acid sequence of preferably 1, 2 or 3 conservative amino acid mutations (preferably substitutions, insertions or deletions), or consisting of.
  • the heavy chain variable region of the antibody comprises or consists of the following sequences:
  • SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:10, and SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:10 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity
  • the sequence of SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 10 has one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions); and
  • the light chain variable region of the antibody comprises or consists of the following sequences:
  • SEQ ID NO: 4 SEQ ID NO: 8, SEQ ID NO: 12, or SEQ ID NO: 14, and SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, or SEQ ID NO: 14 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98% or 99% sequence identity, or compared to the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12 or SEQ ID NO: 14 having one or more Amino acid sequence of multiple (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions).
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO:2
  • amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:4 Show;
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 6, and the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 8;
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 10
  • amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 12; or
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 10
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 14.
  • the heavy chain constant region of the antibody is the Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region is the Ig kappa chain C region, ACCESSION: P01834.
  • the heavy chain constant region of the antibody is a leucine to alanine point introduced at position 234 based on the Ig gamma-1 chain C region, ACCESSION:P01857 Mutation (L234A), a point mutation (L235A) from leucine to alanine was introduced at position 235; the light chain constant region is the C region of the Ig kappa chain, ACCESSION: P01834, the amino acid sequence is as shown in SEQ ID NO: 22 Show.
  • variable regions of the light and heavy chains determine antigen binding; the variable regions of each chain contain three hypervariable regions, called complementarity determining regions (CDRs) (the CDRs of the heavy chain (H) include HCDR1, HCDR2, HCDR3 , the CDRs of the light chain (L) include LCDR1, LCDR2, LCDR3; it is named by Kabat et al., see Bethesda M.d., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 1991; 1-3:91-3242).
  • CDRs complementarity determining regions
  • the CDRs can also be defined by the IMGT numbering system, see Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc.
  • IMGT/3Dstructure-DB and IMGT/DomainGapAlign a database and a tool for immunoglobulins or antibodies, T cell receptors , MHC, IgSF and MhcSF. Nucleic acids research 2009;38(suppl_1):D301-D307.
  • amino acid sequences of the CDR regions of the monoclonal antibody sequences are analyzed according to the IMGT definitions by technical means well known to those skilled in the art, for example, by the VBASE2 database.
  • the antibodies 19F3, 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM), and 19F3H2L3 (hG1DM) involved in the present invention have the same CDRs:
  • amino acid sequences of the three CDR regions in the variable region of its heavy chain are as follows:
  • HCDR1 GYSFTGYT (SEQ ID NO: 15),
  • HCDR2 INPYNAGT (SEQ ID NO: 16),
  • HCDR3 ARSEYRYGGDYFDY (SEQ ID NO: 17);
  • amino acid sequences of the three CDR regions of the light chain variable region are as follows:
  • LCDR1 QSLLNSSNQKNY (SEQ ID NO: 18),
  • LCDR2 FAS (SEQ ID NO: 19),
  • LCDR3 QQHYDTPYT (SEQ ID NO: 20).
  • the antibody is a monoclonal antibody.
  • the antibody is a humanized antibody, a chimeric antibody, a multispecific antibody (eg, a bispecific antibody).
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') 2 , Fd, Fv, dAb, Fab/c, complementarity determining region fragments, single chain antibodies (eg, scFv ), humanized antibodies, chimeric antibodies or bispecific antibodies.
  • Another aspect of the invention pertains to isolated polypeptides selected from the group consisting of:
  • polypeptide comprising the sequences shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, wherein the polypeptide, as part of an anti-CD73 antibody, specifically binds to CD73, the antibody Also included are the sequences shown in SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20.
  • An isolated polypeptide comprising or having at least 80%, 81%, 82%, 83%, 84% with the sequence shown in SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 10 %, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in sequence or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence
  • the amino acid sequence of the polypeptide, wherein the polypeptide is used as a part of an anti-CD73 antibody that specifically binds to CD73, and the antibody also corresponds to the sequence shown in SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 12 or with Said sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
  • An isolated polypeptide comprising or having at least 80%, 81%, 82%, 83%, 84% with the sequence shown in SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 12 %, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in sequence or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence
  • the amino acid sequence of the polypeptide, as part of an anti-CD73 antibody specifically binds to CD73, and the antibody also corresponds to the sequence shown in SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 10 or with Said sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%
  • An isolated polypeptide comprising or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% with the sequence shown in SEQ ID NO: 10 %, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or compared to said sequence
  • the antibody also corresponds to the sequence shown in SEQ ID NO: 14 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or with Said
  • polypeptide comprising or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% with the sequence shown in SEQ ID NO: 14 %, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or compared to said sequence
  • Another aspect of the present invention pertains to an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of any one of the present invention, or the isolated polypeptide.
  • Yet another aspect of the present invention relates to a vector comprising the isolated nucleic acid molecule of the present invention.
  • Yet another aspect of the present invention relates to a host cell comprising an isolated nucleic acid molecule of the present invention, or a vector of the present invention.
  • Yet another aspect of the present invention relates to a conjugate comprising an antibody and a coupling moiety, wherein the antibody is the antibody or antigen-binding fragment thereof of any one of the present invention, and the coupling moiety is a purification tag (eg His tag), a detectable label; preferably, the coupling moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, polyethylene glycol or an enzyme.
  • the antibody is the antibody or antigen-binding fragment thereof of any one of the present invention
  • the coupling moiety is a purification tag (eg His tag), a detectable label; preferably, the coupling moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, polyethylene glycol or an enzyme.
  • Yet another aspect of the present invention relates to a fusion protein or a multispecific antibody (preferably a bispecific antibody) comprising the antibody or antigen-binding fragment thereof of any one of the present invention.
  • kits comprising the antibody or antigen-binding fragment thereof of any one of the present invention, the conjugate, fusion protein or multispecific antibody of the present invention; preferably, the reagent
  • the cassette also includes a second antibody that specifically recognizes the antibody; optionally, the second antibody further includes a detectable label, such as a radioisotope, fluorescent substance, chemiluminescent substance, colored substance, or enzyme.
  • a further aspect of the present invention relates to the use of the antibody or antigen-binding fragment thereof, conjugate, fusion protein or multispecific antibody of the present invention in the preparation of a kit for use in The presence or level of CD73 in the sample is detected.
  • compositions comprising the antibody or antigen-binding fragment thereof of any one of the present invention, the conjugate, fusion protein or multispecific antibody of the present invention; optionally, the
  • the pharmaceutical composition also includes a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.
  • a further aspect of the present invention relates to the use of the antibody or antigen-binding fragment thereof, conjugate, fusion protein or multispecific antibody of the present invention in the preparation of the treatment and/or prevention of tumors (such as solid tumors, preferably Non-small cell lung cancer, prostate cancer (including metastatic castration-resistant prostate cancer (mCRPC)), triple-negative breast cancer, ovarian cancer, colorectal cancer (including nodal microsatellite stabilization (MSS) and mismatch repair deficiency/micro Satellite instability (dMMR/MSI-high), gastric cancer (including nodal microsatellite stable (MSS) and mismatch repair deficiency/microsatellite instability (dMMR/MSI-high)), melanoma, head and neck cancer, Use in a medicament for renal cell carcinoma or pancreatic duct adenocarcinoma, or in the preparation of a medicament for diagnosing tumors.
  • tumors such as solid tumors, preferably Non-small cell lung cancer, prostate cancer (
  • Yet another aspect of the present invention relates to the hybridoma cell line LT014, which is deposited in the China Center for Type Culture Collection (CCTCC) with the deposit number CCTCC NO: C2018137.
  • CTCC China Center for Type Culture Collection
  • kits comprising (1) the antibody or antigen-binding fragment thereof of the present invention, the conjugate of the present invention or the fusion protein or multispecific antibody of the present invention, and (2) Anti-PD-1/CTLA-4 bispecific antibody, and optionally, instructions for use.
  • Yet another aspect of the present invention relates to a method for treating and/or preventing tumors, comprising administering to a patient a therapeutically effective amount of Drug A and a therapeutically effective amount of Drug B, wherein Drug A comprises the antibody or antigen-binding fragment thereof of the present invention,
  • Drug A comprises the antibody or antigen-binding fragment thereof of the present invention
  • the conjugate of the present invention or the fusion protein or multispecific antibody of the present invention, the drug B comprises an anti-PD-1/CTLA-4 bispecific antibody, preferably, the drug A and the drug B are administered simultaneously or sequentially , wherein the sequential administration is that drug A is administered first or drug B is administered first.
  • the heavy chain amino acid sequence of the anti-PD-1/CTLA-4 bispecific antibody is shown in SEQ ID NO:35, and the light chain amino acid sequence is shown in SEQ ID NO:36.
  • EC 50 refers to the concentration for 50% of maximal effect, which refers to the concentration that elicits 50% of the maximal effect.
  • antibody refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having one "light” (L) chain and one "heavy” (H) chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region ( VH ) and a heavy chain constant region ( CH ).
  • the heavy chain constant region consists of 3 domains ( CH1 , CH2 and CH3 ).
  • Each light chain consists of a light chain variable region ( VL ) and a light chain constant region ( CL ).
  • the light chain constant region consists of one domain, CL .
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus.
  • the assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda Md (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al.
  • IMGT/3Dstructure-DB and IMGT/DomainGapAlign a database and a tool for immunoglobulins or antibodies, T Definition of cell receptors, MHC, IgSF and MhcSF.”Nucleic acids research 2009;38(suppl_1):D301-D307.
  • antibody is not limited by any particular method of producing an antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies.
  • Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3, or IgG4 subtype
  • IgAl IgA2, IgD, IgE, or IgM antibodies.
  • the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, that is, excluding natural mutations that may arise spontaneously, A population of identical antibody molecules.
  • Monoclonal antibodies are highly specific for a single epitope on an antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which generally comprise at least two or more different antibodies that generally recognize different epitopes on an antigen.
  • Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. ( G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. nature, 1975; 256(5517): 495), but can also be obtained by recombinant DNA technology (eg, see US Patent 4,816,567).
  • humanized antibody refers to the replacement of all or part of the CDR regions of a human immunoglobulin (acceptor antibody) with the CDR regions of a non-human antibody (donor antibody)
  • the antibody or antibody fragment of which the donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity.
  • some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of corresponding non-human antibodies, or by amino acid residues of other antibodies, to further improve or optimize the performance of the antibody.
  • isolated refers to artificially obtained from the natural state. If an "isolated” substance or component occurs in nature, it may be due to a change in its natural environment, or separation of the substance from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolated of.
  • isolated or isolated
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1 derived artificial chromosomes (PACs) ; Phage such as ⁇ phage or M13 phage and animal viruses.
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PACs P1 derived artificial chromosomes
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses eg, adeno-associated viruses
  • herpesviruses eg, herpes simplex virus
  • poxviruses baculoviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolar virus eg SV40
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and
  • the term "host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc., Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, GS cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus, etc.
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, GS cells, BHK cells, HEK 293 cells or human cells.
  • an antibody that specifically binds an antigen refers to an antibody that is less than about 10-5 M, such as less than about 10-6 M, 10-7 M, Binds the antigen with an affinity (K D ) of 10-8 M, 10-9 M, or 10-10 M or less.
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen.
  • antibodies exhibit a dissociation equilibrium constant (K D ) of less than about 10-5 M, eg, less than about 10-6 M, 10-7 M, 10-8 M, 10-9 M, or 10-10 M or less Binds antigen (eg, PD-1 protein).
  • KD can be determined using methods known to those skilled in the art, eg, using a Fortebio Molecular Interactometer.
  • amino acids are generally represented by one-letter and three-letter abbreviations well known in the art.
  • alanine can be represented by A or Ala.
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well known in the art (see e.g. Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ions Strength enhancer.
  • pH adjusting agents include but are not limited to phosphate buffers; surfactants include but are not limited to cationic, anionic or nonionic surfactants such as Tween-80; ionic strength enhancers include but are not limited to sodium chloride.
  • the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect.
  • a disease-prophylactically effective amount refers to an amount sufficient to prevent, arrest, or delay the onset of a disease (eg, a tumor);
  • a therapeutically-effective amount refers to an amount sufficient to cure or at least partially prevent the development of a disease in a patient already suffering from the disease. The amount of disease and its complications.
  • the monoclonal antibody of the present invention can specifically bind to CD73 well, and can very effectively inhibit the enzymatic reaction of CD73 in a non-substrate competition manner, reduce the production of adenosine, and promote the activity of T cells and the tumor suppressing effect.
  • the combination of the antibody of the present invention and the anti-PD-1/CTLA-4 bispecific antibody has the pharmacological effect of effectively inhibiting the proliferation of tumor cells, which is better than that of the anti-PD-1/CTLA-4 bispecific antibody single drug or anti-PD-1/CTLA-4 bispecific antibody.
  • CD73 antibody monotherapy is better than that of the anti-PD-1/CTLA-4 bispecific antibody single drug or anti-PD-1/CTLA-4 bispecific antibody.
  • Anti-CD73 antibodies effectively inhibit CD73 enzymatic activity.
  • the BALB/c mice used were purchased from the Guangdong Provincial Medical Laboratory Animal Center.
  • the used positive control antibody MEDI9447 (Oleclumab) is produced from Zhongshan Kangfang Bio-Pharmaceutical Co., Ltd., and its sequence is the same as the International Nonproprietary Names for Pharmaceutical Substances (INN) published by Medlmmune Limited on the WHO website. The antibody sequence recorded in the same (World Health Organization (2016). "International Nonproprietary Names for Pharmaceutical Substances (INN). Proposed INN: List 116" (PDF). WHO Drug Information. 30 (4), P661-662.).
  • the heavy chain constant region of the wild IgG1 control antibody used as a positive control antibody adopts the Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region adopts the Ig kappa chain C region, ACCESSION: P01834.
  • the C1q used was purchased from fizgerald, article number: A16050201;
  • the Fc ⁇ RIIIa-bio used was purchased from Sino Biological, article number: LC09JA0407;
  • CD73 5'-nuclease specific inhibitor APCP (alpha, beta-methylene adenosine-5'-diphosphate, 5'- ⁇ , ⁇ -methylene-diphosphate Adenosine) from sigma, catalog number: M3763-10MG.
  • the isotype control antibody used is the sequence of human anti-Hen Egg Lysozyme IgG (anti-HEL antibody, or human IgG, hIgG for short, or isotype control) Variable region sequence from Fab F10.6.6 sequence in Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies published by Acierno et al. (Acierno et al. J Mol Biol. 2007; 374(1): 130-146.);
  • the antigen used for preparing the anti-CD73 antibody was human NT5E-His (NT5E is Genbank ID: NP_002517.1, position: 1-552, produced by Zhongshan Kangfang Biomedical Co., Ltd.).
  • the spleen cells of the immunized mice were fused with mouse myeloma cells to prepare hybridoma cells.
  • NT5E is GenbankID: NP_002517.1, position: 1-552
  • -Biotin N5E-Biotin, prepared by Zhongshan Kangfang Biopharmaceutical Co., Ltd.
  • the hybridoma cells were screened by indirect ELISA method, and obtained Hybridoma cells secreting antibodies that specifically bind to CD73.
  • a stable hybridoma cell line was obtained by limiting dilution method.
  • the above hybridoma cell line was named as hybridoma cell line LT014, and the monoclonal antibody secreted by it was named as 19F3.
  • Hybridoma cell line LT014 also known as CD73-19F3
  • CTCC China Center for Type Culture Collection
  • the deposit number is CCTCC NO: C2018137
  • the deposit address is China. Wuhan. Wuhan University , Zip Code: 430072.
  • the LT014 cell lines prepared above were separately cultured with CD medium (Chemical Defined Medium) (CD medium, containing 1% penicillin streptomycin, cultured in 5% CO 2 , 37° C. cell incubator). After 7 days, the cell culture supernatant was collected, subjected to high-speed centrifugation, vacuum filtration through a microporous membrane, and purified using a HiTrap protein A HP column to obtain antibody 19F3.
  • CD medium Chemical Defined Medium
  • mRNA was extracted from the LT014 cell line cultured in Example 1 according to the method of the cultured cell bacterial total RNA extraction kit (Tiangen, Cat. No. DP430).
  • the PCR amplification product was directly cloned by TA, and the specific operation was carried out by referring to the instructions of the pEASY-T1 Cloning Kit (Transgen CT101).
  • the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the length is 363 bp.
  • the sequence of heavy chain CDR1 is shown in SEQ ID NO: 15
  • the sequence of heavy chain CDR2 is shown in SEQ ID NO: 16
  • the sequence of heavy chain CDR3 is shown in SEQ ID NO: 17.
  • the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO: 3, and the length is 339 bp.
  • the sequence of light chain CDR1 is shown in SEQ ID NO: 18, the sequence of light chain CDR2 is shown in SEQ ID NO: 19, and the sequence of light chain CDR3 is shown in SEQ ID NO: 20.
  • the designed variable region sequences of the above-mentioned humanized antibodies are as follows:
  • the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO: 5, and the length is 363 bp.
  • the encoded amino acid sequence is shown in SEQ ID NO: 6, and the length is 121aa, wherein the sequence of the heavy chain CDR1 is shown in SEQ ID NO: 15, the sequence of the heavy chain CDR2 is shown in SEQ ID NO: 16, and the heavy chain CDR3 The sequence is shown in SEQ ID NO: 17.
  • the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO: 7, and the length is 339 bp.
  • the encoded amino acid sequence is shown in SEQ ID NO: 8, and the length is 113aa, wherein the sequence of light chain CDR1 is shown in SEQ ID NO: 18, the sequence of light chain CDR2 is shown in SEQ ID NO: 19, and the light chain CDR3 The sequence is shown in SEQ ID NO: 20.
  • the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO: 9, and the length is 363 bp.
  • the encoded amino acid sequence is shown in SEQ ID NO: 10, and the length is 121aa, wherein the sequence of heavy chain CDR1 is shown in SEQ ID NO: 15, the sequence of heavy chain CDR2 is shown in SEQ ID NO: 16, and the heavy chain CDR3 The sequence is shown in SEQ ID NO: 17.
  • the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO: 11, and the length is 339 bp.
  • the encoded amino acid sequence is shown in SEQ ID NO: 12, and the length is 113aa, wherein the sequence of light chain CDR1 is shown in SEQ ID NO: 18, the sequence of light chain CDR2 is shown in SEQ ID NO: 19, and the light chain CDR3 The sequence is shown in SEQ ID NO: 20.
  • the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO: 9, and the length is 363 bp.
  • the encoded amino acid sequence is shown in SEQ ID NO: 10, and the length is 121aa, wherein the sequence of heavy chain CDR1 is shown in SEQ ID NO: 15, the sequence of heavy chain CDR2 is shown in SEQ ID NO: 16, and the heavy chain CDR3 The sequence is shown in SEQ ID NO: 17.
  • the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO: 13, and the length is 339 bp.
  • the encoded amino acid sequence is shown in SEQ ID NO: 14, and the length is 113aa, wherein the sequence of light chain CDR1 is shown in SEQ ID NO: 18, the sequence of light chain CDR2 is shown in SEQ ID NO: 19, and the light chain CDR3 The sequence is shown in SEQ ID NO: 20.
  • the light chain constant region of the antibodies of 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM) and 19F3H2L3 (hG1DM) is the Ig kappa chain C region, ACCESSION: P01834.
  • the heavy chain constant region is based on the Ig gamma-1 chain C region, ACCESSION: P01857, and a leucine-to-alanine point mutation (L234A) was introduced at position 234, and leucine was introduced at position 235.
  • Amino acid to alanine point mutation (L235A) (SEQ ID NO: 21), resulting in humanized antibodies designated 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM) and 19F3H2L3 (hG1DM).
  • the 19F3H1L1 (hG1DM) heavy chain cDNA and light chain cDNA, 19F3H2L2 (hG1DM) heavy chain cDNA and light chain cDNA, and 19F3H2L3 (hG1DM) heavy chain cDNA and light chain cDNA were cloned into pUC57simple (GenScript). Company provided) vectors, respectively obtained pUC57simple-19F3H1 (hG1DM), pUC57simple-19F3L1; pUC57simple-19F3H2 (hG1DM), pUC57simple-19F3L2 and pUC57simple-19F3L3.
  • the recombinant plasmids containing the corresponding light and heavy chains were designed to combine the genes (pcDNA3.1-19F3H1(hG1DM)/pcDNA3.1-19F3L1, pcDNA3.1-19F3H2(hG1DM)/pcDNA3.1-19F3L2 and pcDNA3.1-19F3H2 (hG1DM)/pcDNA3.1-19F3L3 were co-transfected into 293F cells and the culture medium was collected for purification. After the sequencing was verified correctly, an endotoxin-free expression plasmid was prepared and the plasmid was transiently transfected into HEK293 cells for antibody expression. After 7 days of culture The cell culture medium was collected, and the humanized antibody was obtained by affinity purification with a Protein A column.
  • Example 4 Dynamic affinity determination of anti-CD73 antibodies to C1q and FcyRIIIa.
  • the sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 50 ⁇ g/mL of antibody was immobilized on the FAB2G sensor, the immobilization height was about 2.0 nm, the sensor was equilibrated in the buffer for 60 s, and then immobilized on the sensor.
  • the antibody above binds to the antigen C1q, the antigen concentration is 0.63-10 nM (two-fold gradient dilution), the time is 60s, and the antigen-antibody is dissociated in the buffer for 60s.
  • the sensor was regenerated using 10 mM glycine, pH 1.7, for 5 s, and repeated 4 times.
  • the vibration rate of the sample plate is 1000 rpm
  • the detection temperature is 30 degrees
  • the detection frequency is 0.6 Hz.
  • Data were analyzed with a 1:1 model fit to yield affinity constants.
  • the data acquisition software was Fortebio Data Acq ⁇ isition 7.0
  • the data analysis software was Fortebio Data Analysis 7.0.
  • the sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4.
  • 0.5 ⁇ g/mL Fc ⁇ RIIIa (from Sino Biological) was immobilized on the SA sensor for 120 s, and the sensor was equilibrated in the buffer for 60 s.
  • the CD16a immobilized on the sensor binds to each antibody, and the antibody concentration is 31.3-500 nM (two-fold gradient dilution) for 60 s, and the antibody antigen is dissociated in the buffer for 60 s.
  • the sensor was regenerated using 10 mM NaOH.
  • the detection temperature is 30°C and the frequency is 0.6Hz. Data were analyzed with a 1:1 model fit to yield affinity constants.
  • Example 5 Anti-CD73 antibody combined with anti-CTLA-4/PD-1 bispecific antibody effectively blocks the growth of tumor cells
  • Mouse colon cancer cells MC38-hPDL1/hCD73 (provided by Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were recovered, and the recovery generation was Pn+3.
  • MC38-hPDL1/hCD73 cells in logarithmic growth phase were collected, the culture medium was removed, and the cells were washed twice with PBS and then inoculated into C57BL/6-hPD1/hPDL1/hCD73 mice (provided by Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) (before tumor loading,
  • the survival rates of MC38-hPDL1/hCD73 cells after tumor-bearing were: 99.1% and 96.4%, respectively), inoculation volume: 2*106/100 ⁇ L/cell, inoculation location: right forelimb of mice.
  • mice On the 5th day after inoculation, when the average tumor volume reached 86.02 mm3 , 32 mice were randomly divided into 4 groups of 8 mice according to the tumor volume. The day of grouping was defined as Day 0, and the administration started on Day 0 according to Table 3.
  • the experimental steps are as follows: take log-phase MDA-MB-231 cells (derived from ATCC, HTB-26) in good condition, resuspend and count the cells with serum-free RPMI-1640 medium; Inoculate to 96-well plate, 3*10 4 cells/100 ⁇ l/well; dilute the antibody with serum-free RPMI-1640 medium, the initial concentration is 200 ⁇ g/ml, and carry out gradient dilution by 2.5 times; add the antibody to the 96-well plate, each Well 50 ⁇ l, incubate at 37°C for 1 hour.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Endocrinology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

抗CD73抗体及其应用，所述抗体的重链可变区包含氨基酸序列如SEQ ID NOs：15-17所示的HCDR1-HCDR3；并且所述抗体的轻链可变区包含氨基酸序列如SEQ ID NOs：18-20所示的LCDR1-LCDR3。

Description

抗CD73的抗体及其用途 技术领域
本发明涉及免疫学领域。具体地，涉及抗CD73抗体及其用途。
背景技术
胞外-5’-核苷酸酶(Ecto-5’-nucleotidase)，即CD73蛋白，是NT5E基因编码的一种蛋白分子量为70KD的多功能糖蛋白，其通过糖基磷脂酰肌醇(glyocsyl phosphatidy linositol，GPI)锚定于细胞膜上(Zimmermann H.Biochem J.1992；285：345-365)。
CD73广泛分布在人体组织细胞表面，目前研究已经发现CD73高表达于多种实体肿瘤，如肿瘤微环境的癌细胞、树突细胞、调节型T细胞(Treg)、自然杀伤细胞(NK细胞)、髓系起源的抑制细胞(MDSC)、肿瘤相关巨噬细胞(TAM)等。低氧诱导低氧诱导因子-1(HIF-1)等分子的上调，进而导致CD73在肿瘤微环境广泛表达(Synnestvedt K，et al.J Clin Invest.2002；110：993-1002.)。对临床肿瘤样本的分析显示，CD73高表达是一种潜在的生物标志物，与多种类型肿瘤的不良预后密切相关，包括乳腺癌、肺癌、卵巢癌、肾癌、胃癌、头颈癌等。
CD73既有水解酶活性，又有非水解酶作用。CD73的酶与非酶功能同时存在于肿瘤的相关过程中，并且互相促进，维持肿瘤的演进。越来越多的研究发现，CD73是体外肿瘤细胞增殖、转移和侵袭，体内肿瘤血管生成和肿瘤免疫逃逸机制的关键调控分子，其中很重要的一个免疫抑制机制是由CD73-腺苷(Adenosine)代谢信号通路介导的，CD73上游的CD39可以催化ATP产生腺苷单磷酸(AMP)，所产生的AMP被CD73转化为腺苷，而腺苷会结合下游的腺苷受体(A2AR)，A2AR通过激活蛋白激酶A(PKA)和Csk激酶，抑制LCK、MAPK、PKC等一系列与免疫激活相关的信号通路，抑制T细胞的免疫杀伤作用，从而发挥免疫抑制作用(Antonioli L，et al.Nat Rev Cancer.2013；13：842-857.)。
跨膜受体PD-1(程序性细胞死亡-1)是CD28基因家族成员之一，在 活化的T细胞，B细胞以及骨髓系细胞都有表达。PD-1的配体PDL1(Programmed cell death 1 ligand 1，亦简称为PDL-1)和PDL2(Programmed cell death 1 ligand 2，亦简称为PDL-2)均属于B7超家族，其中PDL1在多种细胞都有表达，包括T细胞，B细胞以及内皮细胞和上皮细胞，PDL2则仅表达于抗原呈递细胞如树突状细胞和巨噬细胞。
PD-1/PDL1信号通路在调节免疫耐受、微生物感染及肿瘤免疫逃逸中发挥重要作用。PD-1的表达主要在T细胞等免疫细胞，而PD-1的配体PDL1主要在许多人类肿瘤组织呈高表达。阻断PD-1/PDL1信号通路可使被抑制的T细胞激活，进而攻击癌细胞。阻断PD-1/PDL1信号可以促进肿瘤抗原特异性T细胞的增殖，发挥杀伤肿瘤细胞的作用，进而抑制局部肿瘤生长(Julie R et al.，2012，N Engl J Med.366：2455-2465)。另外，高表达PDL1的肿瘤伴随着很难被检测到的癌症(Hamanishi et al.，2007，Proc.Natl.Acad.Sci.USA 104：3360-5)。一种实施有效的方法是通过体内注射抗PD-1抗体对PD-1的表达进行调控。由于PD-1抗体的广谱抗肿瘤前景和惊人的药效，业界普遍认为针对PD-1通路的抗体将带来治疗多种肿瘤的突破性进展：用于治疗非小细胞性肺癌，肾细胞癌，卵巢癌，黑色素瘤(Homet M.B.，Parisi G.，et al.，2015，Semin Oncol.42(3)：466-473)，白血病以及贫血病(Held SA，Heine A，et al.，2013，Curr Cancer Drug Targets.13(7)：768-74)。
细胞毒性T淋巴细胞相关抗原-4(cytotoxic T lymphocyte sociated antigen 4，亦简称为CTLA4)与CD28分子在基因结构、染色体定位、序列的同源性及基因表达具有十分相近的关系，都是共刺激分子B7的受体，主要表达于被激活T细胞表面。CTLA4与B7结合后能抑制小鼠和人T细胞的激活，在T细胞活化中起负调节作用。
CTLA4抗体(或抗CTLA4单克隆抗体)或CTLA4配体可以阻止CTLA4与其天然配体结合，从而封闭CTLA4对T细胞负性调节信号的传导，增强T细胞对各种抗原的反应性，在这方面体内与体外研究结果基本一致。目前已有CTLA4单克隆抗体处于临床试验阶段或被批准用于治疗前列腺癌，膀胱癌，结肠直肠癌，胃肠道癌，肝癌，恶性黑色素瘤等(Grosso JF.，Jure-Kunkel MN.，2013，Cancer Immun.13：5.)。
CTLA4及CTLA4抗体作为T细胞功能状况的重要影响因素，通过干预机体免疫微环境发挥作用。体外和体内试验中，CTLA4抗体可特异地解除CTLA4对机体免疫抑制，激活T细胞，诱导IL-2产生，在抗肿瘤及寄生虫等疾病的基因治疗中有广泛应用前景。CTLA4抗体可对疾病产生特异性治疗作用，并发挥较高疗效，补充传统用药的不足，由此开辟基因治疗的新途径。
ADCC(antibody-dependent cell-mediated cytotoxicity)即抗体依赖的细胞介导的细胞毒性作用，是指抗体的Fab段结合病毒感染的细胞或肿瘤细胞的抗原表位，其Fc段与杀伤细胞(NK细胞、巨噬细胞等)表面的Fc受体(Fc Receptor，FcR)结合，介导杀伤细胞直接杀伤靶细胞。
CDC(complement dependent cytotoxicity)即补体依赖的细胞毒性，是指当抗体与细胞膜表面相应抗原特异性结合后，形成复合物而激活补体系统，进而在靶细胞表面形成MAC，导致靶细胞溶解。补体能导致多种细菌和其他病原生物细胞的溶解，是机体抵抗病原生物感染的重要防御机制。
Fc受体是表达在特定免疫细胞表面，用于识别抗体Fc区域介导免疫应答的免疫球蛋白家族蛋白。抗体Fab区域识别抗原后，其抗体Fc区域与免疫细胞(如杀伤细胞)上的Fc受体结合，启动免疫细胞的应答功能，如吞噬作用及ADCC。根据Fc受体识别抗体类型及表达细胞的不同，发现FcγRIIIa与ADCC效应密切相关。FcγRIIIa是介导ADCC的最主要分子(Hogarth PM，Pietersz GA.2012，NATURE REVIEWS DRUG DISCOVERY，11(4)：311-331)。
IgG家族包含四个成员，IgG1、IgG2、IgG3和IgG4，它们重链恒定区的可结晶片段(fragment crystallizable，Fc)区域存在氨基酸的差异，导致它们与FcγRs的亲和力各不相同。IgG1是人体内最多的亚型，也是单抗药物中用的最多的亚型，IgG1能够结合各种FcγRs，能够引发ADCC以及CDC效应。抗体存在ADCC和/或CDC可能会导致不必要的靶向组织损伤，具有影响药物药理学负面效应。
发明内容
本发明人经过深入的研究和创造性的劳动，利用哺乳动物细胞表达系统表达出重组的人CD73作为抗原免疫小鼠，经小鼠脾脏细胞与骨髓瘤细胞融合获得杂交瘤细胞。发明人通过对大量样本的筛选，得到了杂交瘤细胞株LT014(保藏编号为CCTCC NO：C2018137)。
本发明人惊奇地发现，杂交瘤细胞株LT014能够分泌产生与人CD73特异性结合的特异性单克隆抗体(命名为19F3)，并且该单克隆抗体能够十分有效地以非底物竞争的方式抑制CD73的酶活反应，降低腺苷的产生，促进T细胞活性，发挥抑制肿瘤生长的效果。
进一步地，本发明人创造性地制得了抗人CD73的人源化抗体(命名为19F3H1L1(hG1DM)、19F3H2L2(hG1DM)、19F3H2L3及19F3H2L3(hG1DM))，此外，抗CD73抗体引入了氨基酸突变以消除ADCC和CDC效应，避免了由抗体介导的不必要的毒性作用。
本发明人还惊奇地发现，本发明的抗体与抗PD-1/CTLA-4双特异性抗体联用具有有效抑制肿瘤细胞增殖的药理学效果，优于抗PD-1/CTLA-4双特异性抗体单药或者抗CD73抗体单药。
本发明的另一个方面还涉及一种抗体，其中，所述抗CD73抗体包含：
SEQ ID NO：2、SEQ ID NO：6或SEQ ID NO：10所示的重链可变区的HCDR1，HCDR2和HCDR3；和SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：12或SEQ ID NO：14所示的轻链可变区的LCDR1，LCDR2和LCDR3；
优选地，根据IMGT编号系统，所述抗CD73抗体包含
HCDR1，其包含SEQ ID NO：15所示的氨基酸序列，与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成，
HCDR2，其包含SEQ ID NO：16所示的氨基酸序列，与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、 90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成，
HCDR3，其包含SEQ ID NO：17所示的氨基酸序列，与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成，
LCDR1，其包含SEQ ID NO：18所示的氨基酸序列，与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成，
LCDR2，其包含SEQ ID NO：19所示的氨基酸序列，与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成，和
LCDR3，其包含SEQ ID NO：20所示的氨基酸序列，与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成。
在本发明的一些实施方案中，
所述抗体的重链可变区包含下述序列，或由下述序列组成：
SEQ ID NO：2、SEQ ID NO：6或SEQ ID NO：10，与SEQ ID NO：2、SEQ ID NO：6或SEQ ID NO：10具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NO：2、SEQ ID NO：6或SEQ ID NO：10所示的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；并且
所述抗体的轻链可变区包含下述序列，或由下述序列组成：
SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：12或SEQ ID NO：14，与SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：12或SEQ ID NO：14具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：12或SEQ ID NO：14所示的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列。
在本发明的一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO：2所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：4所示；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：6所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：8所示；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：10所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：12所示；或
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：10所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：14所示。
在本发明的一些实施方案中，所述抗体的重链恒定区为Ig gamma-1链C区，ACCESSION：P01857；轻链恒定区为Ig kappa链C区，ACCESSION：P01834。
在本发明的一些实施方案中，所述抗体的重链恒定区为在Ig gamma-1链C区，ACCESSION：P01857的基础上在第234号位点引进了 亮氨酸到丙氨酸的点突变(L234A)，第235号位点引进了亮氨酸到丙氨酸的点突变(L235A)；轻链恒定区为Ig kappa链C区，ACCESSION：P01834，氨基酸序列如SEQ ID NO：22所示。
轻链和重链的可变区决定抗原的结合；每条链的可变区均含有三个高变区，称互补决定区(CDR)(重链(H)的CDR包含HCDR1、HCDR2、HCDR3，轻链(L)的CDR包含LCDR1、LCDR2、LCDR3；其由Kabat等人命名，见Bethesda M.d.，Sequences of Proteins of Immunological Interest，Fifth Edition，NIH Publication 1991；1-3：91-3242)。
优选地，CDR也可以由IMGT编号系统定义，请参见Ehrenmann，Francois，Quentin Kaas，and Marie-Paule Lefranc.IMGT/3Dstructure-DB and IMGT/DomainGapAlign：a database and a tool for immunoglobulins or antibodies，T cell receptors，MHC，IgSF and MhcSF.Nucleic acids research 2009；38(suppl_1)：D301-D307。
通过本领域技术人员所熟知的技术手段，例如通过VBASE2数据库根据IMGT定义分析单克隆抗体序列的CDR区的氨基酸序列。
本发明涉及的抗体19F3、19F3H1L1(hG1DM)、19F3H2L2(hG1DM)、19F3H2L3(hG1DM)具有相同的CDR：
其重链可变区的3个CDR区的氨基酸序列如下：
HCDR1：GYSFTGYT(SEQ ID NO：15)，
HCDR2：INPYNAGT(SEQ ID NO：16)，
HCDR3：ARSEYRYGGDYFDY(SEQ ID NO：17)；
其轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1：QSLLNSSNQKNY(SEQ ID NO：18)，
LCDR2：FAS(SEQ ID NO：19)，
LCDR3：QQHYDTPYT(SEQ ID NO：20)。
在本发明的一些实施方案中，所述抗体为单克隆抗体。
在本发明的一些实施方案中，所述抗体为人源化抗体、嵌合抗体、多特异性抗体(例如双特异性抗体)。
在本发明的一些实施方式中，所述抗原结合片段选自Fab、Fab′、F(ab′) 2、Fd、Fv、dAb、Fab/c、互补决定区片段、单链抗体(例如，scFv)、 人源化抗体、嵌合抗体或双特异性抗体。
本发明的另一个方面涉及分离的多肽，其选自以下各项组成的组：
(1)分离的多肽，其包含SEQ ID NO：15，SEQ ID NO：16和SEQ ID NO：17所示的序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还包含SEQ ID NO：18；SEQ ID NO：19；SEQ ID NO：20所示的序列。
(2)分离的多肽，其包含SEQ ID NO：18；SEQ ID NO：19；SEQ ID NO：20所示的序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还包含SEQ ID NO：15，SEQ ID NO：16和SEQ ID NO：17所示的序列。
(3)分离的多肽，其包含SEQ ID NO：2、SEQ ID NO：6或SEQ ID NO：10所示的序列或与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还分别对应包含SEQ ID NO：4、SEQ ID NO：8或SEQ ID NO：12所示的序列或与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列。
(4)分离的多肽，其包含SEQ ID NO：4、SEQ ID NO：8或SEQ ID NO：12所示的序列或与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还分别对应包含SEQ ID NO：2、SEQ ID NO：6或SEQ ID NO：10所示的序列或与所述序列具有至少80％， 81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列。
(5)分离的多肽，其包含SEQ ID NO：10所示的序列或与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还分别对应包含SEQ ID NO：14所示的序列或与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列。
(6)分离的多肽，其包含SEQ ID NO：14所示的序列或与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还分别对应包含SEQ ID NO：10所示的序列或与所述序列具有至少80％，81％，82％，83％，84％，85％，86％、87％、88％、89％、90％，优选至少91％，92％，93％，94％，95％，96％，97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列。
本发明的另一方面涉及一种分离的核酸分子，其编码本发明任一项所述的抗体或其抗原结合片段，或所述分离的多肽。
本发明的再一方面涉及一种载体，其包含本发明的分离的核酸分子。
本发明的再一方面涉及一种宿主细胞，其包含本发明的分离的核酸分子，或者本发明的载体。
本发明的再一方面涉及一种偶联物，其包括抗体以及偶联部分，其中，所述抗体为本发明任一项所述的抗体或其抗原结合片段，所述偶联部分为纯化标签(如His标签)，可检测的标记；优选地，所述偶联部分为放射性同位素、荧光物质、化学发光物质、有色物质、聚乙二醇或酶。
本发明的再一个方面，涉及融合蛋白或多特异性抗体(优选双特异性抗体)，其包含本发明任一项所述的抗体或其抗原结合片段。
本发明的再一方面涉及一种试剂盒，其包括本发明任一项所述的抗体或其抗原结合片段、本发明的偶联物、融合蛋白或多特异性抗体；优选地，所述试剂盒还包括第二抗体，其特异性识别所述抗体；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、化学发光物质、有色物质或酶。
本发明的再一方面涉及本发明任一项所述的抗体或其抗原结合片段、本发明的偶联物、融合蛋白或多特异性抗体在制备试剂盒中的用途，所述试剂盒用于检测CD73在样品中的存在或其水平。
本发明的再一方面涉及一种药物组合物，其包含本发明任一项所述的抗体或其抗原结合片段、本发明的偶联物、融合蛋白或多特异性抗体；可选地，所述药物组合物还包括药学上可接受的载体和/或赋形剂。优选地，所述药物组合物为适于通过皮下注射、皮内注射、静脉内注射、肌内注射或病灶内注射施用的形式。
本发明的再一方面涉及本发明任一项所述的抗体或其抗原结合片段、本发明的偶联物、融合蛋白或多特异性抗体在制备治疗和/或预防肿瘤(例如实体瘤，优选非小细胞肺癌、前列腺癌(包括转移性去势抵抗前列腺癌(mCRPC))、三阴性性乳腺癌、卵巢癌、结直肠癌(包括结微卫星稳定(MSS)和错配修复功能缺陷/微卫星不稳定(dMMR/MSI-high)型)、胃癌(包括结微卫星稳定(MSS)和错配修复功能缺陷/微卫星不稳定(dMMR/MSI-high)型)、黑色素癌、头颈癌、 肾细胞癌或胰管腺癌的药物中的用途，或者在制备用于诊断肿瘤的药物中的用途。
本发明的再一方面涉及杂交瘤细胞株LT014，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2018137。
本发明的再一方面涉及试剂盒，其包含(1)本发明所述的抗体或其抗原结合片段、本发明所述的偶联物或本发明所述的融合蛋白或多特异性抗体，和(2)抗PD-1/CTLA-4双特异性抗体，和任选地，使用说明书。
本发明的再一方面涉及治疗和/或预防肿瘤的方法，包括对患者施用治疗有效量的药物A和治疗有效量的药物B，其中药物A包含本发明所述的抗体或其抗原结合片段、本发明所述的偶联物或本发明所述的融合蛋白或多特异性抗体，药物B包含抗PD-1/CTLA-4双特异性抗体，优选地，药物A和药物B同时或顺序施用，其中所述顺序施用为先施用药物A或先施用药物B。
在本发明的一些实施方案中，所述抗PD-1/CTLA-4双特异性抗体的重链氨基酸序列如SEQ ID NO：35所示，轻链氨基酸序列如SEQ ID NO：36所示。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语EC 50是指半最大效应浓度(concentration for 50％of maximal effect)，是指能引起50％最大效应的浓度。
如本文中所使用的，术语“抗体”是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(V H)和重链恒定区(C H)组成。重链恒定区由3个结构域(C H1、C H2和C H3)组成。各轻 链由轻链可变区(V L)和轻链恒定区(C L)组成。轻链恒定区由一个结构域C L组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。V H和V L区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各V H和V L由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(V H和V L)分别形成抗原结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health，Bethesda M.d.(1987and 1991))，或Chothia&Lesk J.Mol.Biol.1987；196：901-917；Chothia等人Nature 1989；342：878-883或者IMGT编号系统定义，见Ehrenmann，Francois，Quentin Kaas，and Marie-Paule Lefranc.″IMGT/3Dstructure-DB and IMGT/DomainGapAlign：a database and a tool for immunoglobulins or antibodies，T cell receptors，MHC，IgSF and MhcSF.″Nucleic acids research 2009；38(suppl_1)：D301-D307.的定义。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，特别地，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(
Figure PCTCN2021125564-appb-000001
G，Milstein C.Continuous cultures of fused cells secreting antibody of predefined specificity[J].nature，1975；256(5517)：495)，但也可采用重组DNA技术获得(如参见U.S.Patent 4,816,567)。
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR 区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones et al.，Nature 1986；321：522-525；Reichmann et al.，Nature 1988；332：323-329；Presta，Curr.Op.Struct.Biol.，1992；2：593-596；和Clark M.Antibody humanization：a case of the‘Emperor’s new clothes’？[J].Immunol.Today，2000；21(8)：397-402。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草杆菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，GS细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的亲和力(K D)结合该抗原。
如本文中所使用的，术语“K D”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的解离平衡常数(K D)结合抗原(例如，PD-1蛋白)。可以使用本领域技术人员知悉的方法测定K D，例如使用Fortebio分子相互作用仪测定。
如本文中所使用的，术语“单克隆抗体”和“单抗”具有相同的含义且可互换使用；术语“多克隆抗体”和“多抗”具有相同的含义且可互换使用；术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington′s Pharmaceutical Sciences.Edited by Gennaro AR，19th ed.Pennsylvania：Mack Publishing Company，1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如肿瘤)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。
发明的有益效果：
本发明的单克隆抗体能够很好地特异性与CD73结合，并且能够十分有效地、以非底物竞争的方式抑制CD73的酶活反应，降低腺苷的产生，促进T细胞活性及肿瘤抑制效果。同时，本发明的抗体与抗PD-1/CTLA-4双特异性抗体联用具有有效抑制肿瘤细胞增殖的药理学效果，优于抗PD-1/CTLA-4双特异性抗体单药或者抗CD73抗体单药。
附图说明
图1. 19F3H2L3(hG1DM)与C1q结合的动态亲和力数据拟合曲线。
图2.MEDI9447与C1q结合的动态亲和力数据拟合曲线。
图3.IgG1野生型抗体与C1q结合的动态亲和力数据拟合曲线。
图4. 19F3H2L3(hG1DM)与FcγRIIIa结合的动态亲和力数据拟合曲线。
图5.MEDI9447与FcγRIIIa结合的动态亲和力数据拟合曲线。
图6.IgG1野生型抗体与FcγRIIIa结合的动态亲和力数据拟合曲线。
图7.分组后第23天时各组小鼠肿瘤重量。**P＜0.01。
图8.各组动物实验期间体重变化率，与Day 0天相比。
图9.抗CD73抗体有效抑制CD73酶学活性。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或按照产品说明书进行。所用试剂或仪器未注明生产厂商者，为可以通过市场购买获得的常规产品。
在本发明的下述实施例中，使用的BALB/c小鼠购自广东省医学实验动物中心。
在本发明的下述实施例中，所用的阳性对照抗体MEDI9447(Oleclumab)，生产自中山康方生物医药有限公司，其序列与Medlmmune Limited在WHO网站公开登载的International Nonproprietary Names for Pharmaceutical Substances(INN)中记载的抗体序列相同(World Health Organization(2016).″International Nonproprietary Names for Pharmaceutical Substances(INN).Proposed INN：List 116″(PDF).WHO Drug Information.30(4)，P661-662.)。
在本发明的下述实施例中，所用的阳性对照抗体野生IgG1对照抗体的重链恒定区采用Ig gamma-1 chain C region，ACCESSION：P01857；轻链恒定区采用Ig kappa chain C region，ACCESSION：P01834。
在本发明的下述实施例中，所用的C1q购自fizgerald，货号：A16050201；
在本发明的下述实施例中，所用的FcγRIIIa-bio购自Sino Biological，货号：LC09JA0407；
本发明的下述实施例中，所用的CD73(5′-核酸酶)特异性抑制剂APCP(alpha，beta-methylene adenosine-5′-diphosphate，5’-α，β-亚甲基-二磷酸腺苷)来源于sigma，货号：M3763-10MG。
在本发明的下述实施例中，所用的同型对照抗体为人抗鸡蛋溶酶体抗体(human anti-Hen Egg Lysozyme IgG，即anti-HEL抗体，或者human IgG，简称hIgG，或者同型对照)的序列来自于Acierno等人发表的Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies中Fab F10.6.6序列的可变区序列(Acierno等人.J Mol Biol.2007；374(1)：130-146.)；
实施例1：抗CD73抗体19F3的制备
1、杂交瘤细胞株LT014的制备
制备抗CD73抗体所用的抗原为人NT5E-His(NT5E为GenbankID：NP_002517.1，位置：1-552，由中山康方生物医药有限公司制备)。取免疫后的小鼠的脾细胞与小鼠骨髓瘤细胞融合，制成杂交瘤细胞。 以人NT5E(NT5E为GenbankID：NP_002517.1，位置：1-552)-Biotin(NT5E-Biotin，由中山康方生物医药有限公司制备)作为抗原，对杂交瘤细胞进行间接ELISA法筛选，获得能够分泌与CD73特异性结合的抗体的杂交瘤细胞。对筛选得到的杂交瘤细胞，经过有限稀释法得到稳定的杂交瘤细胞株。将以上杂交瘤细胞株命名为杂交瘤细胞株LT014，其分泌的单克隆抗体命名为19F3。
杂交瘤细胞株LT014(又称CD73-19F3)，其于2018年6月21日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2018137，保藏地址为中国.武汉.武汉大学，邮编：430072。
2、抗CD73抗体19F3的制备
用CD培养基(Chemical Defined Medium)对上面制得的LT014细胞株分别进行培养(CD培养基，内含1％青链霉素，于5％CO 2，37℃细胞培养箱中进行培养)。7天后收集细胞培养上清，通过高速离心、微孔滤膜抽真空过滤，并使用HiTrap protein A HP柱进行纯化，制得抗体19F3。
实施例2：抗CD73的抗体19F3的序列分析
按照培养细胞细菌总RNA提取试剂盒(Tiangen，货号DP430)的方法，从实施例1中培养的LT014细胞株中提取mRNA。
按照Invitrogen
Figure PCTCN2021125564-appb-000002
III First-Strand Synthesis System for RT-PCR试剂盒说明书合成cDNA，并进行PCR扩增。
PCR扩增产物直接进行TA克隆，具体操作参考pEASY-T1 Cloning Kit(Transgen CT101)试剂盒说明书进行。
将TA克隆的产物直接进行测序，抗CD73的抗体19F3的序列分析测序结果如下：
重链可变区的核酸序列如SEQ ID NO：1所示，长度为363bp。
其编码的氨基酸序列如SEQ ID NO：2所示，长度为121aa；
其中重链CDR1的序列如SEQ ID NO：15所示，重链CDR2的序列如SEQ ID NO：16所示，重链CDR3的序列如SEQ ID NO：17所示。
轻链可变区的核酸序列如SEQ ID NO：3所示，长度为339bp。
其编码的氨基酸序列如SEQ ID NO：4所示，长度为113aa；
其中轻链CDR1的序列如SEQ ID NO：18所示，轻链CDR2的序列如SEQ ID NO：19所示，轻链CDR3的序列如SEQ ID NO：20所示。
实施例3：抗人CD73的人源化抗体的轻链和重链设计和制备
根据人CD73蛋白的三维晶体结构(Hage T，Reinemer P，Sebald W.Crystals of a 1∶1 complex between human interleukin-4 and the extracellular domain of its receptor alpha chain.Eur J Biochem.1998；258(2)：831-6.)以及实施例2获得的抗体19F3的序列，通过计算机模拟抗体模型，根据模型设计突变，得到19F3H1L1(hG1DM)、19F3H2L2(hG1DM)、19F3H2L3(hG1DM)的可变区序列；
上述人源化抗体的设计的可变区序列如下：
(1)人源化单克隆抗体19F3H1L1(hG1DM)的重链可变区和轻链可变区序列
重链可变区的核酸序列如SEQ ID NO：5所示，长度为363bp。
其编码的氨基酸序列如SEQ ID NO：6所示，长度为121aa，其中重链CDR1的序列如SEQ ID NO：15所示，重链CDR2的序列如SEQ ID NO：16所示，重链CDR3的序列如SEQ ID NO：17所示。
轻链可变区的核酸序列如SEQ ID NO：7所示，长度为339bp。
其编码的氨基酸序列如SEQ ID NO：8所示，长度为113aa，其中轻链CDR1的序列如SEQ ID NO：18所示，轻链CDR2的序列如SEQ ID NO：19所示，轻链CDR3的序列如SEQ ID NO：20所示。
(2)人源化单克隆抗体19F3H2L2(hG1DM)的重链可变区和轻链可变区序列
重链可变区的核酸序列如SEQ ID NO：9所示，长度为363bp。
其编码的氨基酸序列如SEQ ID NO：10所示，长度为121aa，其中重链CDR1的序列如SEQ ID NO：15所示，重链CDR2的序列如SEQ ID NO：16所示，重链CDR3的序列如SEQ ID NO：17所示。
轻链可变区的核酸序列如SEQ ID NO：11所示，长度为339bp。
其编码的氨基酸序列如SEQ ID NO：12所示，长度为113aa，其中轻链CDR1的序列如SEQ ID NO：18所示，轻链CDR2的序列如SEQ ID NO：19 所示，轻链CDR3的序列如SEQ ID NO：20所示。
(3)人源化单克隆抗体19F3H2L3(hG1DM)的重链可变区和轻链可变区序列
重链可变区的核酸序列如SEQ ID NO：9所示，长度为363bp。
其编码的氨基酸序列如SEQ ID NO：10所示，长度为121aa，其中重链CDR1的序列如SEQ ID NO：15所示，重链CDR2的序列如SEQ ID NO：16所示，重链CDR3的序列如SEQ ID NO：17所示。
轻链可变区的核酸序列如SEQ ID NO：13所示，长度为339bp。
其编码的氨基酸序列如SEQ ID NO：14所示，长度为113aa，其中轻链CDR1的序列如SEQ ID NO：18所示，轻链CDR2的序列如SEQ ID NO：19所示，轻链CDR3的序列如SEQ ID NO：20所示。
3.人源化19F3H1L1(hG1DM)、19F3H2L2(hG1DM)和19F3H2L3(hG1DM)的制备
19F3H1L1(hG1DM)、19F3H2L2(hG1DM)和19F3H2L3(hG1DM)的抗体的轻链恒定区为Ig kappa链C区，ACCESSION：P01834。
重链恒定区在Ig gamma-1 chain C region，ACCESSION：P01857的基础上，在第234号位点引进了亮氨酸到丙氨酸的点突变(L234A)，第235号位点引进了亮氨酸到丙氨酸的点突变(L235A)(SEQ ID NO：21)，获得了命名为19F3H1L1(hG1DM)、19F3H2L2(hG1DM)和19F3H2L3(hG1DM)的人源化抗体。
将19F3H1L1(hG1DM)重链cDNA和轻链的cDNA、19F3H2L2(hG1DM)重链cDNA和轻链的cDNA、以及19F3H2L3(hG1DM)的重链cDNA和轻链的cDNA，分别克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-19F3H1(hG1DM)、pUC57simple-19F3L1；pUC57simple-19F3H2(hG1DM)、pUC57simple-19F3L2和pUC57simple-19F3L3。参照《分子克隆实验指南(第二版)》介绍的标准技术，EcoRI&HindIII酶切合成的重、轻链全长基因，通过限制酶(EcoRI&HindIII)的酶切亚克隆到表达载体pcDNA3.1中获得表达质粒pcDNA3.1-19F3H1(hG1DM)，pcDNA3.1-19F3L1，pcDNA3.1-19F3H2(hG1DM)，pcDNA3.1-19F3L2，和pcDNA3.1-19F3L3， 并进一步对重组表达质粒的重/轻链基因进行测序分析。随后将含有相应的轻、重链重组质粒设计基因组合(pcDNA3.1-19F3H1(hG1DM)/pcDNA3.1-19F3L1，pcDNA3.1-19F3H2(hG1DM)/pcDNA3.1-19F3L2和pcDNA3.1-19F3H2(hG1DM)/pcDNA3.1-19F3L3分别共转染293F细胞后收集培养液进行纯化。测序验证正确后，制备去内毒素级别的表达质粒并将质粒瞬时转染HEK293细胞进行抗体表达，培养7天后收集细胞培养液，采用Protein A柱进行亲和纯化获得人源化抗体。
实施例4：抗CD73抗体与C1q和FcγRIIIa的动态亲和力测定。
(1)抗CD73抗体与C1q的动态亲和力测定
样品稀释缓冲液为PBS，0.02％Tween-20，0.1％BSA，pH7.4。50μg/mL的抗体固定在FAB2G传感器上，固定高度约为2.0nm，传感器在缓冲液中平衡60s，固定在传感器上的抗体与抗原C1q结合，抗原浓度为0.63-10nM(两倍梯度稀释)，时间60s，抗原抗体在缓冲液中解离，时间60s。传感器使用10mM甘氨酸，pH1.7再生，时间5s，重复4次。样品板震动速率为1000rpm，检测温度为30度，检测频率为0.6Hz。数据以1∶1模型拟合分析，得到亲和力常数。数据采集软件为Fortebio Data Acqμisition 7.0，数据分析软件为Fortebio Data Analysis 7.0。
结果如表1和图1-3所示，19F3H2L3(hG1DM)和MEDI9447与C1q均无结合活性。
Figure PCTCN2021125564-appb-000003
KD＝kdis/kon
(2)抗CD73抗体与FcγRIIIa的动态亲和力测定
样品稀释缓冲液为PBS，0.02％Tween-20，0.1％BSA，pH7.4。0.5μg/mL 的FcγRIIIa(来源于Sino Biological)固定在SA传感器上，时间120s，传感器在缓冲液中平衡60s，固定在传感器上的CD16a与各抗体结合，抗体浓度为31.3-500nM(两倍梯度稀释)，时间60s，抗体抗原在缓冲液中解离，时间60s。传感器使用10mM NaOH再生。检测温度为30℃，频率为0.6Hz。数据以1∶1模型拟合分析，得到亲和力常数。
结果如表2和图4至6所示，19F3H2L3(hG1DM)与FcγRIIIa无结合，而MEDI9447与FcγRIIIa具有结合活性。
Figure PCTCN2021125564-appb-000004
KD＝kdis/kon
实施例5：抗CD73抗体联合抗CTLA-4/PD-1双特异性抗体有效的阻断肿瘤细胞的生长
本实验考察了抗CD73抗体即19F3H2L3(hG1DM)联合抗PD-1/CTLA-4双特异性抗体BiAb004(hG1TM)(其重链氨基酸序列如SEQ ID NO：35所示，轻链氨基酸序列如SEQ ID NO：36所示，轻链及重链CDR氨基酸序列如SEQ ID NO：23至SEQ ID NO：34所示)抑制肿瘤生长的药理学活性。
结果如图7所示，抗CD73抗体联合抗PD-1/CTLA-4双特异性抗体较抗CD73抗体单药、抗PD-1/CTLA-4双特异性抗体单药显示出更为优异的抑制肿瘤作用，并且对小鼠体重无显著影响(图8)。
表3.实验设计
Figure PCTCN2021125564-appb-000005
Figure PCTCN2021125564-appb-000006
将小鼠结肠癌细胞MC38-hPDL1/hCD73(江苏集萃药康生物科技有限公司提供)复苏，复苏代次为Pn+3代。收集对数生长期的MC38-hPDL1/hCD73细胞，去除培养液并用PBS洗两次后接种C57BL/6-hPD1/hPDL1/hCD73小鼠(江苏集萃药康生物科技有限公司提供)(荷瘤前、荷瘤后MC38-hPDL1/hCD73细胞存活率分别为：99.1％及96.4％)，接种量：2*106/100μL/只，接种位置：小鼠右前肢。接种后第5天，平均肿瘤体积达到86.02mm 3时，32只小鼠根据肿瘤体积随机分成4组，每组8只。分组当天定义为Day 0天，并按照表3于Day 0天开始给药。
细胞接种后，每周常规监测肿瘤对动物正常行为的影响。给药日期：Day 0、Day 3、Day 7、Day 10、Day 14、Day 17、Day 22天。Day 0、Day3、Day 6、Day 10、Day 13、Day 17、Day 20、Day 23天观测肿瘤大小并称量小鼠体重。在Day23实验结束后，挖取各组动物的肿瘤组织称重。小鼠体重变化率、肿瘤重量等实验结果以平均值±标准误差(Mean±SEM)表示。采用独立样本T检验比较不同治疗组与对照组相比有无显著性差异，P＜0.05被认为具有显著性差异。数据使用Graphpad或Excel进行做图。
实施例6：抗CD73抗体对抑制细胞内源表达CD73酶活性的检测
实验步骤如下：取状态良好的对数期MDA-MB-231细胞(来源于ATCC，HTB-26)，用无血清的RPMI-1640培养液将细胞重悬、计数；将MDA-MB-231细胞接种至96孔板，3*10 4个细胞/100μl/孔；用无血清RPMI-1640培养液稀释抗体，起始浓度200μg/ml，按2.5倍进行梯度稀释；将抗体加入96孔板，每孔50μl，37℃孵育1小时。1小时后，每孔加入50μl RPMI-1640稀释的600μM AMP；3小时后取25μl细胞培养液上清，转移至新96孔板，每孔加入25μl 100μM的ATP；和50μl CTG(
Figure PCTCN2021125564-appb-000007
One Solution Assay，promega，Cat：G8461)显色液，进行显色，多标记微孔板检测仪(PerkinElmer，货号：2140-0020)读取数 据。同型对照抗体及CD73特异性抑制剂APCP分别作为阴性对照及阳性对照。
实验结果：结果如图9所示，19F3、19F3H2L3(hG1DM)、19F3H2L3(hG1DM)和19F3H2L3(hG1DM)均可呈剂量依赖性抑制MDA-MB-231内源表达的CD73酶催化AMP为腺苷A的活性，从而剂量依赖性降低产生的平均萤光强度RLU。
以上实验结果说明加入的AMP在无CD73抗体处理情况下，可被MDA-MB-231内源表达于细胞表面的CD73酶催化而转化为腺苷，从而解除对荧光素酶活性的抑制。而加入抗体后，由于CD73被抗体结合，而降低了其酶催化活性，使AMP不能转化为腺苷。提示抗CD73抗体以非底物竞争的方式有效抑制CD73的酶活反应，降低腺苷的产生。

Claims (18)

  1. 抗CD73(例如人CD73)抗体或其抗原结合片段，其中，根据IMGT编号系统，所述抗CD73抗体包含：
    SEQ ID NO：2、SEQ ID NO：6或SEQ ID NO：10所示的重链可变区的HCDR1，HCDR2和HCDR3；和SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：12或SEQ ID NO：14所示的轻链可变区的LCDR1，LCDR2和LCDR3；
    优选地，根据IMGT编号系统，所述抗CD73抗体包含
    HCDR1，其包含SEQ ID NO：15所示的氨基酸序列，与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成，
    HCDR2，其包含SEQ ID NO：16所示的氨基酸序列，与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成，
    HCDR3，其包含SEQ ID NO：17所示的氨基酸序列，与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成，
    LCDR1，其包含SEQ ID NO：18所示的氨基酸序列，与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、 90％，优选至少91％、92％、93％、94％、95％、96％、97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成，
    LCDR2，其包含SEQ ID NO：19所示的氨基酸序列，与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％，98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成，和
    LCDR3，其包含SEQ ID NO：20所示的氨基酸序列，与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，或由其组成。
  2. 权利要求1所述的抗CD73抗体或其抗原结合片段，所述抗体的重链可变区包含下述序列，或由下述序列组成：
    SEQ ID NO：2、SEQ ID NO：6或SEQ ID NO：10，与SEQ ID NO：2、SEQ ID NO：6或SEQ ID NO：10具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与SEQ ID NO：2、SEQ ID NO：6或SEQ ID NO：10所示的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列；并且
    所述抗体的轻链可变区包含下述序列，或由下述序列组成：
    SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：12或SEQ ID NO：14，与SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：12或SEQ ID NO：14具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％ 序列同一性的序列，或与SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：12或SEQ ID NO：14所示的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列。
  3. 权利要求1或2所述的抗CD73抗体或其抗原结合片段，其中
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO：2所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：4所示；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO：6所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：8所示；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO：10所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：12所示；或
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO：10所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：14所示。
  4. 权利要求1或2所述的抗CD73抗体或其抗原结合片段，其中所述抗体的重链恒定区为Ig gamma-1链C区，ACCESSION：P01857；轻链恒定区为Ig kappa链C区，ACCESSION：P01834。
  5. 权利要求1或2所述的抗CD73抗体或其抗原结合片段，其中所述抗体为单克隆抗体、人源化抗体、嵌合抗体、多特异性抗体(例如双特异性抗体)。
  6. 权利要求1或2所述的抗CD73抗体或其抗原结合片段，所述抗原结合片段选自Fab、Fab′、F(ab′) 2、Fd、Fv、dAb、Fab/c、互补决定区片段、单链抗体(例如，scFv)、人源化抗体、嵌合抗体或双特异性抗体。
  7. 分离的多肽，其选自以下各项组成的组：
    (1)分离的多肽，其包含SEQ ID NO：15，SEQ ID NO：16和SEQ ID NO：17所示的序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还包含SEQ ID NO：18；SEQ ID NO：19；SEQ ID NO：20所示的序列。
    (2)分离的多肽，其包含SEQ ID NO：18；SEQ ID NO：19；SEQ ID NO：20所示的序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合 CD73，所述抗体还包含SEQ ID NO：15，SEQ ID NO：16和SEQ ID NO：17所示的序列。
    (3)分离的多肽，其包含SEQ ID NO：2、SEQ ID NO：6或SEQ ID NO：10所示的序列或与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还分别对应包含SEQ ID NO：4、SEQ ID NO：8或SEQ ID NO：12所示的序列或与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列。
    (4)分离的多肽，其包含SEQ ID NO：4、SEQ ID NO：8或SEQ ID NO：12所示的序列或与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还分别对应包含SEQ ID NO：2、SEQ ID NO：6或SEQ ID NO：10所示的序列或与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列。
    (5)分离的多肽，其包含SEQ ID NO：10所示的序列或与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、 5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还分别对应包含SEQ ID NO：14所示的序列或与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列。
    (6)分离的多肽，其包含SEQ ID NO：14所示的序列或与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列，其中所述多肽作为抗CD73抗体的一部分，特异性结合CD73，所述抗体还分别对应包含SEQ ID NO：10所示的序列或与所述序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％，优选至少91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，或与所述序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9或10个)保守氨基酸突变(优选置换，插入或缺失)的氨基酸序列。
  8. 核酸分子，其编码权利要求1-6任一项所述的抗体或其抗原结合片段，或权利要求7所述分离的多肽。
  9. 载体，其包含权利要求8所述的核酸分子。
  10. 宿主细胞，其包含权利要求8所述的核酸分子，或权利要求9所述的载体。
  11. 偶联物，其包含权利要求1-6任一项所述的抗体或其抗原结合片段以及偶联部分，其中，所述偶联部分为纯化标签(如His标签)，可检测的标记；优选地，所述偶联部分为放射性同位素、荧光物质、化学发光物质、有色物质、聚乙二醇或酶。
  12. 融合蛋白或多特异性抗体(优选双特异性抗体)，其包含权利 要求1-6任一项所述的抗体或其抗原结合片段。
  13. 试剂盒，其包括权利要求1-6任一项所述的抗体或其抗原结合片段、权利要求11所述的偶联物或权利要求12所述的融合蛋白或多特异性抗体；优选地，所述试剂盒还包括第二抗体，其特异性识别所述抗体；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、化学发光物质、有色物质或酶；优选地，所述试剂盒用于检测CD73在样品中的存在或其水平。
  14. 药物组合物，其包含权利要求1-6任一项所述的抗体或其抗原结合片段、权利要求11所述的偶联物或权利要求12所述的融合蛋白或多特异性抗体；可选地，所述药物组合物还包括药学上可接受的载体和/或赋形剂。优选地，所述药物组合物为适于通过皮下注射、皮内注射、静脉内注射、肌内注射或病灶内注射施用的形式。
  15. 权利要求1-6任一项所述的抗体或其抗原结合片段、权利要求11所述的偶联物或权利要求12所述的融合蛋白或多特异性抗体在制备治疗和/或预防肿瘤(例如实体瘤，优选非小细胞肺癌、前列腺癌(包括转移性去势抵抗前列腺癌(mCRPC))、三阴性性乳腺癌、卵巢癌、结直肠癌(包括结微卫星稳定(MSS)和错配修复功能缺陷/微卫星不稳定(dMMR/MSI-high)型)、胃癌(包括结微卫星稳定(MSS)和错配修复功能缺陷/微卫星不稳定(dMMR/MSI-high)型)、黑色素癌、头颈癌、肾细胞癌或胰管腺癌的药物中的用途，或者在制备用于诊断肿瘤的药物中的用途。
  16. 杂交瘤细胞株，其选自：
    LT014，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2018137。
  17. 试剂盒，其包含(1)权利要求1-6任一项所述的抗体或其抗原结合片段、权利要求11所述的偶联物或权利要求12所述的融合蛋白或多特异性抗体，和(2)抗PD-1/CTLA-4双特异性抗体，和任选地，使用说明书，优选地，所述抗PD-1/CTLA-4双特异性抗体的重链氨基酸序列如SEQ ID NO：35所示，轻链氨基酸序列如SEQ ID NO：36所示。
  18. 治疗和/或预防肿瘤的方法，包括对患者施用治疗有效量的药物 A和治疗有效量的药物B，其中药物A包含权利要求1-6任一项所述的抗体或其抗原结合片段、权利要求11所述的偶联物或权利要求12所述的融合蛋白或多特异性抗体，药物B包含抗PD-1/CTLA-4双特异性抗体，优选地，药物A和药物B同时或顺序施用，其中所述顺序施用为先施用药物A或先施用药物B，优选地，所述抗PD-1/CTLA-4双特异性抗体的重链氨基酸序列如SEQ ID NO：35所示，轻链氨基酸序列如SEQ ID NO：36所示。
PCT/CN2021/125564 2020-10-23 2021-10-22 抗cd73的抗体及其用途 Ceased WO2022083723A1 (zh)

Priority Applications (9)

Application Number Priority Date Filing Date Title
EP21882137.9A EP4234579A4 (en) 2020-10-23 2021-10-22 ANTI-CD73 ANTIBODY AND ITS USE
KR1020237017266A KR20230125774A (ko) 2020-10-23 2021-10-22 항-cd73 항체 및 이의 용도
IL302182A IL302182A (en) 2020-10-23 2021-10-22 Anti-CD73 antibody and its use
US18/250,010 US20240018254A1 (en) 2020-10-23 2021-10-22 Anti-cd73 antibody and use thereof
JP2023548995A JP2023546743A (ja) 2020-10-23 2021-10-22 抗ｃｄ７３抗体及びその使用
CA3196299A CA3196299A1 (en) 2020-10-23 2021-10-22 Anti-cd73 antibody and use thereof
AU2021363350A AU2021363350A1 (en) 2020-10-23 2021-10-22 Anti-cd73 antibody and use thereof
MX2023004713A MX2023004713A (es) 2020-10-23 2021-10-22 Anticuerpo anti-cd73 y uso del mismo.
ZA2023/04591A ZA202304591B (en) 2020-10-23 2023-04-20 Anti-cd73 antibody and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011152518 2020-10-23
CN202011152518.9 2020-10-23

Publications (1)

Publication Number Publication Date
WO2022083723A1 true WO2022083723A1 (zh) 2022-04-28

Family

ID=81291626

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/125564 Ceased WO2022083723A1 (zh) 2020-10-23 2021-10-22 抗cd73的抗体及其用途

Country Status (11)

Country Link
US (1) US20240018254A1 (zh)
EP (1) EP4234579A4 (zh)
JP (1) JP2023546743A (zh)
KR (1) KR20230125774A (zh)
CN (2) CN120590533A (zh)
AU (1) AU2021363350A1 (zh)
CA (1) CA3196299A1 (zh)
IL (1) IL302182A (zh)
MX (1) MX2023004713A (zh)
WO (1) WO2022083723A1 (zh)
ZA (1) ZA202304591B (zh)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024040194A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
WO2024249954A1 (en) 2023-05-31 2024-12-05 Capstan Therapeutics, Inc. Lipid nanoparticle formulations and compositions
WO2025076127A1 (en) 2023-10-05 2025-04-10 Capstan Therapeutics, Inc. Constrained ionizable cationic lipids and lipid nanoparticles
WO2025076113A1 (en) 2023-10-05 2025-04-10 Capstan Therapeutics, Inc. Ionizable cationic lipids with conserved spacing and lipid nanoparticles
WO2025179294A2 (en) 2024-02-22 2025-08-28 Capstan Therapeutics, Inc. Immune engineering amplification
WO2025217454A2 (en) 2024-04-11 2025-10-16 Capstan Therapeutics, Inc. Ionizable cationic lipids and lipid nanoparticles
WO2025217452A1 (en) 2024-04-11 2025-10-16 Capstan Therapeutics, Inc. Constrained ionizable cationic lipids and lipid nanoparticles

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR112022002028A2 (pt) * 2019-08-02 2022-04-12 Cttq Akeso Shanghai Biomed Tech Co Ltd Anticorpo anti-pd-1 e uso farmacêutico do mesmo

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
CN107001472A (zh) * 2014-11-10 2017-08-01 免疫医疗有限公司 对cd73具有特异性的结合分子及其用途
CN111434688A (zh) * 2019-01-11 2020-07-21 上海开拓者生物医药有限公司 Cd73抗体及其制备方法和应用
CN113527489A (zh) * 2020-04-22 2021-10-22 中山康方生物医药有限公司 抗cd73的抗体及其用途

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016131950A1 (en) * 2015-02-20 2016-08-25 Innate Pharma Cd73 blockade
EP3204417B1 (en) * 2014-10-10 2020-07-22 Innate Pharma Cd73 blockade
GB2538120A (en) * 2014-11-11 2016-11-09 Medimmune Ltd Therapeutic combinations comprising anti-CD73 antibodies and uses thereof
AU2015349878A1 (en) * 2014-11-21 2017-05-25 Bristol-Myers Squibb Company Antibodies against CD73 and uses thereof
CN104558137A (zh) * 2015-01-15 2015-04-29 浙江欧诗漫生物股份有限公司 一种珍珠蛋白的制备方法及该方法制备得到的水溶性珍珠蛋白和酸溶性珍珠蛋白
US11130817B2 (en) * 2015-10-12 2021-09-28 Innate Pharma CD73 blocking agents
CN114805575B (zh) * 2015-12-09 2024-07-23 科尔沃斯制药股份有限公司 人源化抗cd73抗体
EP3423494A1 (en) * 2016-03-04 2019-01-09 Bristol-Myers Squibb Company Combination therapy with anti-cd73 antibodies
CN106967172B (zh) * 2016-08-23 2019-01-08 康方药业有限公司 抗ctla4-抗pd-1 双功能抗体、其药物组合物及其用途
RU2754058C2 (ru) * 2016-12-13 2021-08-26 Астеллас Фарма Инк. Антитело к cd73 человека
CA3045376C (en) * 2017-01-24 2022-08-30 I-Mab Anti-cd73 antibodies and uses thereof
US11530273B2 (en) * 2017-05-23 2022-12-20 Helmholtz Zentrum München—Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Anti-CD73 monoclonal antibody, encoding nucleic acids and method for producing
MY204117A (en) * 2017-06-22 2024-08-08 Novartis Ag Antibody molecules to cd73 and uses thereof
EP3762030A4 (en) * 2018-03-09 2022-01-05 Phanes Therapeutics, Inc. ANTI-CD73 ANTIBODIES AND USES THEREOF
CA3176321A1 (en) * 2020-04-22 2021-10-28 Peng Zhang Anti-cd73-anti-pd-1 bispecific antibody and use thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
CN107001472A (zh) * 2014-11-10 2017-08-01 免疫医疗有限公司 对cd73具有特异性的结合分子及其用途
CN111434688A (zh) * 2019-01-11 2020-07-21 上海开拓者生物医药有限公司 Cd73抗体及其制备方法和应用
CN113527489A (zh) * 2020-04-22 2021-10-22 中山康方生物医药有限公司 抗cd73的抗体及其用途

Non-Patent Citations (27)

* Cited by examiner, † Cited by third party
Title
"Molecular Cloning: A Laboratory Manual"
"Remington's Pharmaceutical Sciences", 1995, MACK PUBLISHING COMPANY
ACIERNO ET AL.: "Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies", J MOL BIOL., vol. 374, no. 1, 2007, pages 130 - 146, XP022310713, DOI: 10.1016/j.jmb.2007.09.005
AKESO ( AKESOBIO AUSTRALIA PTY LTD ): "A Study of AK119 (Anti-CD73 Antibody), a Treatment for COVID-19, in Healthy Subjects", CLINICALTRIALS.GOV, NCBI, 18 August 2020 (2020-08-18), pages 1 - 6, XP055923817, Retrieved from the Internet <URL:https://clinicaltrials.gov/ct2/show/NCT04516564> [retrieved on 20220523] *
ANTONIOLI L ET AL., NAT REV CANCER, vol. 13, 2013, pages 842 - 857
B. ALLARD, S. POMMEY, M. J. SMYTH, J. STAGG: "Targeting CD73 Enhances the Antitumor Activity of Anti-PD-1 and Anti-CTLA-4 mAbs", CLINICAL CANCER RESEARCH, ASSOCIATION FOR CANCER RESEARCH, vol. 19, no. 20, 15 October 2013 (2013-10-15), pages 5626 - 5635, XP055216360, ISSN: 10780432, DOI: 10.1158/1078-0432.CCR-13-0545 *
CHOTHIA ET AL., NATURE, vol. 342, 1989, pages 878 - 883
CHOTHIALESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
CLARK M: "Antibody humanization: a case of the 'Emperor's new clothes'? [J", IMMUNOL. TODAY, vol. 21, no. 8, 2000, pages 397 - 402, XP004215168, DOI: 10.1016/S0167-5699(00)01680-7
DATABASE REGISTRY 8 December 2020 (2020-12-08), ANONYMOUS : "Immunoglobulin G1, anti-(human 5'-nucleotidase) (human-Mus musculus monoclonal AK119 .gamma.1-chain), disulfide with human-Mus musculus monoclonal AK119 .kappa.-chain, dimer (CA INDEX NAME)", XP055859690, retrieved from STN Database accession no. 2550560-20-6 *
EHRENMANN, FRANCOISQUENTIN KAASMARIE-PAULE LEFRANC: "IMGT/3Dstructure-DB and IMGT/DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF", NUCLEIC ACIDS RESEARCH, vol. 38, no. l, 2009, pages D301 - D307
HAGE TREINEMER PSEBALD W.: "Crystals of a 1:1 complex between human interleukin-4 and the extracellular domain of its receptor alpha chain", EUR JBIOCHEM., vol. 258, no. 2, 1998, pages 831 - 6, XP003018346, DOI: 10.1046/j.1432-1327.1998.2580831.x
HAMANISHI ET AL., PROC. NATL. ACAD. SCI. USA, vol. 104, 2007, pages 3360 - 5
HELD SAHEINE A ET AL., CURR CANCER DRUG TARGETS., vol. 13, no. 7, 2013, pages 768 - 74
HOGARTH PMPIETERSZ GA, NATURE REVIEWS DRUG DISCOVERY, vol. 11, no. 4, 2012, pages 311 - 331
HORNET M. B.PARISI G. ET AL., SEMIN ONCOL., vol. 42, no. 3, 2015, pages 466 - 473
JONES ET AL., NATURE, vol. 321, 1986, pages 522 - 525
JULIE R ET AL., A ENGL J MED., vol. 366, 2012, pages 2455 - 2465
KABAT: "Sequences of Proteins of Immunological Interest", vol. 1-3, 1991, NIH PUBLICATION, pages: 91 - 3242
KOHLER GMILSTEIN C: "Continuous cultures of fused cells secreting antibody of predefined specificity [J", NATURE, vol. 256, no. 5517, 1975, pages 495, XP037052082, DOI: 10.1038/256495a0
PRESTA, CURR. OP. STRUCT. BIOL., vol. 2, 1992, pages 593 - 596
REICHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 329
See also references of EP4234579A4
SYNNESTVEDT K ET AL., J CLIN INVEST., vol. 110, 2002, pages 993 - 1002
WORLD HEALTH ORGANIZATION, INTERNATIONAL NONPROPRIETARY NAMES FOR PHARMACEUTICAL SUBSTANCES (INN). PROPOSED INN: LIST 116, vol. 30, no. 4, 2016, pages 661 - 662
ZHANG SHU-GUANG, PEI-YAO ZHU; WEN-KE LIU; SI-YUAN DONG; LIN ZHANG: "Advances in research of targeting CD73 in tumor combined therapy", CHINA JOURNAL OF MODERN MEDICINE, ZHONGNAN DAXUE, CN, vol. 29, no. 16, 1 August 2019 (2019-08-01), CN , pages 38 - 43, XP055923819, ISSN: 1005-8982, DOI: 10.3969/j.issn.1005-8982.2019.16.007 *
ZIMMERMANN H., BIOCHEM J., vol. 285, 1992, pages 345 - 365

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024040194A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
WO2024040195A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
WO2024249954A1 (en) 2023-05-31 2024-12-05 Capstan Therapeutics, Inc. Lipid nanoparticle formulations and compositions
WO2025076127A1 (en) 2023-10-05 2025-04-10 Capstan Therapeutics, Inc. Constrained ionizable cationic lipids and lipid nanoparticles
WO2025076113A1 (en) 2023-10-05 2025-04-10 Capstan Therapeutics, Inc. Ionizable cationic lipids with conserved spacing and lipid nanoparticles
WO2025179294A2 (en) 2024-02-22 2025-08-28 Capstan Therapeutics, Inc. Immune engineering amplification
WO2025217454A2 (en) 2024-04-11 2025-10-16 Capstan Therapeutics, Inc. Ionizable cationic lipids and lipid nanoparticles
WO2025217452A1 (en) 2024-04-11 2025-10-16 Capstan Therapeutics, Inc. Constrained ionizable cationic lipids and lipid nanoparticles

Also Published As

Publication number Publication date
EP4234579A1 (en) 2023-08-30
CN114478768B (zh) 2025-05-30
EP4234579A4 (en) 2025-03-12
US20240018254A1 (en) 2024-01-18
ZA202304591B (en) 2025-08-27
KR20230125774A (ko) 2023-08-29
JP2023546743A (ja) 2023-11-07
IL302182A (en) 2023-06-01
CA3196299A1 (en) 2022-04-28
CN120590533A (zh) 2025-09-05
AU2021363350A1 (en) 2023-06-22
MX2023004713A (es) 2023-05-09
CN114478768A (zh) 2022-05-13

Similar Documents

Publication Publication Date Title
CN113527489B (zh) 抗cd73的抗体及其用途
CN114478768B (zh) 抗cd73的抗体及其用途
CN114181310B (zh) 抗tigit抗体、其药物组合物及用途
WO2018036473A1 (zh) 抗ctla4-抗pd-1双功能抗体、其药物组合物及其用途
WO2018036472A1 (zh) 一种抗pd1单克隆抗体、其药物组合物及其用途
WO2017071625A1 (zh) 一种抗pd-1单克隆抗体、其药物组合物及其用途
CN114075289A (zh) 抗cd73的抗体及其用途
JP2022547850A (ja) 抗ｔｉｇｉｔ免疫阻害剤及び応用
WO2022188832A1 (zh) 含有抗pd-1-抗vegfa双特异性抗体的药物组合及其用途
WO2023001303A1 (zh) 药物组合物及用途
WO2021213475A1 (zh) 抗cd73-抗pd-1双特异性抗体及其用途
WO2022089392A1 (zh) 抗tigit抗体、其药物组合物及用途
KR20230166096A (ko) Cldn18.2 항원 결합 단백질 및 이의 적용
CN114106182A (zh) 抗tigit的抗体及其用途
EP4321537A1 (en) Pharmaceutical composition and use thereof
WO2024051223A1 (zh) 药物组合及用途
IL319148A (en) Therapeutic combination and its use
CN115626956A (zh) Ctla-4结合分子及其用途
HK40093416A (zh) 抗cd73的抗体及其用途
EA051023B1 (ru) Фармацевтическая композиция и набор, содержащие антитело против cd73, и их применение в способе лечения опухоли
WO2023236968A1 (zh) Cd39/cd73双特异性抗原结合蛋白及其用途
AU2024244603A1 (en) Bispecific antibody, pharmaceutical composition, and use
HK40096182A (zh) 药物组合物及其用途
EA048409B1 (ru) Антитело против cd73 и его применение
HK40068895A (zh) 一种抗tigit免疫抑制剂及应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21882137

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 202317028528

Country of ref document: IN

ENP Entry into the national phase

Ref document number: 3196299

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2023548995

Country of ref document: JP

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112023007549

Country of ref document: BR

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021882137

Country of ref document: EP

Effective date: 20230523

ENP Entry into the national phase

Ref document number: 112023007549

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20230420

ENP Entry into the national phase

Ref document number: 2021363350

Country of ref document: AU

Date of ref document: 20211022

Kind code of ref document: A