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

抗cd73的抗体及其用途 Download PDF

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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
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seq
antibody
amino acid
sequence
acid sequence
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French (fr)
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李百勇
夏瑜
王忠民
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Akeso Pharmaceuticals Inc
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Akeso Biopharma Inc
Akeso Pharmaceuticals Inc
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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
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    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
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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.

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WO2024040194A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
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