WO2023246247A1 - 药物组合物及其用途 - Google Patents

药物组合物及其用途 Download PDF

Info

Publication number
WO2023246247A1
WO2023246247A1 PCT/CN2023/087998 CN2023087998W WO2023246247A1 WO 2023246247 A1 WO2023246247 A1 WO 2023246247A1 CN 2023087998 W CN2023087998 W CN 2023087998W WO 2023246247 A1 WO2023246247 A1 WO 2023246247A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
seq
acid sequence
cancer
amino acid
Prior art date
Application number
PCT/CN2023/087998
Other languages
English (en)
French (fr)
Inventor
李百勇
夏瑜
王忠民
Original Assignee
中山康方生物医药有限公司
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
Application filed by 中山康方生物医药有限公司 filed Critical 中山康方生物医药有限公司
Priority to EP23745372.5A priority Critical patent/EP4321537A1/en
Priority to CA3208473A priority patent/CA3208473A1/en
Publication of WO2023246247A1 publication Critical patent/WO2023246247A1/zh

Links

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
    • 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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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 [IGs], 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], 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
    • 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
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • 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

Definitions

  • the present invention relates to the field of immunology. Specifically, it relates to a pharmaceutical composition comprising an anti-CD73 (eg, human CD73) antibody or antigen-binding fragment thereof, and an anti-PD-1-anti-VEGFA bispecific antibody or antigen-binding fragment thereof.
  • an anti-CD73 eg, human CD73
  • an anti-PD-1-anti-VEGFA bispecific antibody or antigen-binding fragment thereof e.g, human CD73
  • Extracellular-5'-nucleotidase also known as CD73 protein
  • CD73 protein Extracellular-5'-nucleotidase
  • 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 research has 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) in the tumor microenvironment, Suppressor cells of myeloid origin (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.).
  • HIF-1 hypoxia-inducible factor-1
  • Analysis of clinical tumor samples showed that high CD73 expression is a potential biomarker that is closely related to poor prognosis in many types of tumors, including breast cancer, lung cancer, ovarian cancer, kidney cancer, gastric cancer, head and neck cancer, etc.
  • CD73 has both hydrolase activity and non-hydrolase activity.
  • the enzymatic and non-enzymatic functions of CD73 exist simultaneously in tumor-related processes and promote each other to maintain tumor evolution. More and more studies have found that CD73 is a key regulatory molecule for tumor cell proliferation, metastasis and invasion in vitro, tumor angiogenesis and tumor immune escape mechanism in vivo.
  • CD73-adenosine Mediated by the metabolic signaling pathway, CD39 upstream of CD73 can catalyze ATP to produce adenosine monophosphate (AMP). The generated AMP is converted into adenosine by CD73, and adenosine binds to the downstream adenosine receptor (A2AR).
  • A2AR adenosine receptor
  • PKA protein kinase A
  • Csk kinase By activating protein kinase A (PKA) and Csk kinase, inhibiting a series of signaling pathways related to immune activation such as LCK, MAPK, and PKC, inhibiting 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. It is expressed on activated T cells, B cells and myeloid cells.
  • the PD-1/PDL1 signaling pathway plays an important role in regulating immune tolerance, microbial infection, and tumor immune evasion.
  • PD-1 is mainly expressed in T cells and other immune cells, while the PD-1 ligand PDL1 is mainly highly expressed in many human tumor tissues.
  • Blocking the PD-1/PDL1 signaling pathway can activate suppressed T cells to attack cancer cells. Blocking PD-1/PDL1 signaling can promote the proliferation of tumor antigen-specific T cells, kill tumor cells, and thereby inhibit local tumor growth (Julie R et al., 2012, N Engl J Med. 366:2455-2465 ).
  • tumors with high PDL1 expression are accompanied by cancers that are difficult to detect (Hamanishi et al., 2007, Proc. Natl. Acad. Sci. USA 104:3360-5).
  • An effective method is to regulate the expression of PD-1 by injecting anti-PD-1 antibodies 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 cells 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).
  • VEGF Vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • Kinase signaling pathways function.
  • tumor cells, tumor-invading macrophages and mast cells can secrete high levels of VEGF, which stimulates tumor vascular endothelial cells in a paracrine form, promotes endothelial cell proliferation and migration, induces blood vessel formation, and promotes continued tumor growth.
  • VEGF family includes: VEGFA, VEGFB, VEGFC, VEGFD and PIGF.
  • VEGFR Vascular endothelial growth factor receptors
  • VEGFR1 also known as Flt1
  • VEGFR2 also known as KDR or Flk1
  • VEGFR3 also known as Flt4
  • NPP-1 Neuropilin-1
  • the first three receptors are similar in structure. They all belong to the tyrosine kinase superfamily and consist of an extramembrane region, a transmembrane segment and an intramembrane region. It consists of three parts, of which the extramembrane region is composed of an immunoglobulin-like domain and the intramembrane region is a tyrosine kinase region.
  • VEGFR1 and VEGFR2 are mainly located on the surface of vascular endothelial cells, and VEGFR3 is mainly located on the surface of lymphatic endothelial cells.
  • VEGFA mainly binds to VEGFR1, VEGFR2 and NRP-1 to play its role.
  • VEGFR1 is the earliest discovered receptor. Under normal physiological conditions, the affinity of VEGFR1 to VEGFA is higher than that of VEGFR2 to VEGFA, but its intracellular tyrosinase activity is lower than that of VEGFR2 (Ma Li, Chinese Journal of Eugenics and Genetics, 24( 5)(2016):146-148).
  • VEGFR2 is a major regulator of angiogenesis and construction and has high tyrosine kinase activity compared with VEGFR1.
  • VEGFR2 binds to the ligand VEGFA and mediates the proliferation, differentiation and other behaviors of vascular endothelial cells, as well as the formation process of blood vessels and the permeability of blood vessels (Roskoski R Jr.
  • VEGFA binds to VEGFR2, mediates the transcription and expression of intracellular related protein genes through the downstream PLC- ⁇ -PKC-Raf-MEK-MAPK signaling pathway, and promotes the proliferation of vascular endothelial cells (Takahashi T et al., Oncogene , 18(13)(1999):2221-2230).
  • VEGFR3 is a member of the tyrosine kinase family and is mainly expressed in vascular endothelial cells during the embryonic period and lymphatic endothelial cells in adulthood. VEGFC and VEGFD combine with VEGFR3 to stimulate the proliferation and migration of lymphatic endothelial cells and promote the development of lymphatic vessels. Xinsheng; NRP-1 is a non-tyrosine kinase transmembrane protein that cannot independently transduce biological signals. It must form a complex with the VEGF tyrosine kinase receptor to mediate signal transduction (Ma Li, Chinese Journal of Eugenics and Genetics , 24(5)(2016):146-148).
  • VEGFA and VEGFR2 are mainly involved in regulating angiogenesis.
  • the combination of VEGFA and VEGFR2 will trigger many intermediate signals in the upstream and downstream pathways to form a cascade reaction, which will ultimately take the form of endothelial cell proliferation, survival, migration, increased permeability, and infiltration into surrounding tissues. Change its physiological function (Dong Hongchao et al., "Modern Oncology Medicine", Volume 22, Issue 9, September 2014, Pages 2231-3).
  • VEGFA humanized monoclonal antibodies targeting human VEGF, especially VEGFA, such as bevacizumab, which were successively approved by the U.S. Food and Drug Administration during 2004. It is approved by the FDA for the treatment of non-small cell lung cancer, renal cell carcinoma, cervical cancer, metastatic colorectal cancer and other tumors.
  • the inventor used the mammalian cell expression system to express recombinant human CD73 as an antigen to immunize mice, and obtained hybridoma cells through the fusion of mouse spleen cells and myeloma cells.
  • the inventor obtained the hybridoma cell line LT014 (deposit number: CCTCC NO: C2018137) through screening a large number of samples.
  • the hybridoma cell line LT014 can secrete and produce a specific monoclonal antibody (named 19F3) that specifically binds to human CD73, and this monoclonal antibody can inhibit very effectively in a non-substrate competitive manner.
  • the enzymatic reaction of CD73 reduces the production of adenosine, promotes T cell activity, 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)).
  • the anti-CD73 antibodies introduced amino acid mutations to eliminate ADCC and CDC effects avoid unnecessary 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/VEGFA bispecific antibody has a pharmacological effect of effectively inhibiting tumor growth, which is better than the anti-PD-1/VEGFA bispecific antibody alone or Anti-CD73 antibody single agent.
  • Another aspect of the invention also relates to an antibody, wherein the anti-CD73 antibody comprises:
  • the anti-CD73 antibody comprises
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 15 and has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, At least 86%, at least 87%, at least 88%, at least 89%, at least 90%, preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least A sequence with 98% or at least 99% sequence identity, or an amino acid sequence with one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or consisting of,
  • HCDR2 which includes the amino acid sequence shown in SEQ ID NO: 16 and has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87% similarity with said sequence. %, at least 88%, at least 89%, at least 90%, preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% a sequence of sequence identity, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or consisting of it,
  • HCDR3 which includes the amino acid sequence shown in SEQ ID NO: 17 and has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87% similarity with said sequence. %, at least 88%, at least 89%, at least 90%, preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% a sequence of sequence identity, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or consisting of it,
  • LCDR1 which includes the amino acid sequence shown in SEQ ID NO: 18, has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87% similarity with said sequence. %, at least 88%, at least 89%, at least 90%, preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% a sequence of sequence identity, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or consisting of it,
  • LCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 19, has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87% similarity with said sequence. %, at least 88%, at least 89%, at least 90%, preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, to A sequence with less than 98% or at least 99% sequence identity, or an amino acid sequence with one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence , or consisting of, and
  • LCDR3 which comprises the amino acid sequence shown in SEQ ID NO:20, has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87% similarity with said sequence. %, at least 88%, at least 89%, at least 90%, preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% A sequence of sequence identity, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, or consisting of it.
  • the heavy chain variable region of the antibody comprises or consists of the following sequence:
  • SEQ ID NO:2 SEQ ID NO:6 or SEQ ID NO:10 is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95 %, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, or compared to the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:10.
  • the amino acid sequence of one or more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
  • conservative amino acid mutations preferably substitutions, insertions or deletions
  • the light chain variable region of the antibody comprises or consists of the following sequence:
  • 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 have at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, preferably at least 91%, at least 92 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, or with SEQ ID NO:4, SEQ ID NO:8, SEQ The amino acid sequence shown in ID NO:12 or SEQ ID NO:14 has one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitution, insertion or deletion) of the amino acid sequence.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:2 (preferably the nucleic acid sequence is shown in SEQ ID NO:1), and the light chain of the antibody
  • amino acid sequence of the variable region is shown in SEQ ID NO:4 (the preferred nucleic acid sequence is shown in SEQ ID NO:3);
  • amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 6 (the preferred nucleic acid sequence is as shown in SEQ ID NO: 5), and the amino acid sequence of the light chain variable region of the antibody is as SEQ ID Shown in NO:8 (the preferred nucleic acid sequence is shown in SEQ ID NO:7);
  • amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 10 (the preferred nucleic acid sequence is as shown in SEQ ID NO: 9), and the amino acid sequence of the light chain variable region of the antibody is as SEQ ID Shown in NO:12 (the preferred nucleic acid sequence is shown in SEQ ID NO:11); or
  • amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 10 (the preferred nucleic acid sequence is as shown in SEQ ID NO: 9), and the amino acid sequence of the light chain variable region of the antibody is as SEQ ID Shown in NO:14 (the preferred nucleic acid sequence is shown in SEQ ID NO:13).
  • 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. More preferably, according to the EU numbering system, the heavy chain constant region of the anti-CD73 antibody has the following mutations based on the sequence shown in ACCESSION: P01857:
  • amino acid sequence of the heavy chain constant region of the anti-CD73 antibody is shown in SEQ ID NO:21
  • amino acid sequence of the light chain constant region is shown in SEQ ID NO:22.
  • the heavy chain constant region of the antibody is based on the Ig gamma-1 chain C region, ACCESSION: P01857, with the introduction of leucine to alanine at position No. 234.
  • Mutation (L234A) a point mutation from leucine to alanine (L235A) was introduced at position 235.
  • the amino acid sequence is shown in SEQ ID NO: 21; the light chain constant region is the Ig kappa chain C region, ACCESSION: P01834, the amino acid sequence is shown in SEQ ID NO:22.
  • variable regions of the light chain and heavy chain determine the binding of antigens; the variable region of each chain contains three hypervariable regions, called complementarity-determining regions (CDRs) (the CDRs of the heavy chain (H) include HCDR1, HCDR2, and HCDR3 , the CDRs of the light chain (L) include LCDR1, LCDR2, and LCDR3; they are 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
  • CDRs may 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.
  • the amino acid sequence of the CDR region of the monoclonal antibody sequence is analyzed according to the IMGT definition through technical means well known to those skilled in the art, such as through the VBASE2 database.
  • the antibodies 19F3, 19F3H1L1(hG1DM), 19F3H2L2(hG1DM), and 19F3H2L3(hG1DM) involved in the present invention have the same CDR:
  • amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:
  • HCDR3 ARSEYRYGGDYFDY(SEQ ID NO:17);
  • amino acid sequences of the three CDR regions of its light chain variable region are as follows:
  • 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 (e.g., scFv ), humanized antibodies, chimeric antibodies or bispecific antibodies.
  • a conjugate which includes an antibody and a coupling part, wherein the antibody is an antibody or an antigen-binding fragment thereof according to any one of the present invention, and the coupling part is a purification tag. (such as His tag), detectable label; preferably, the coupling part is a radioactive isotope, a fluorescent substance, a chemiluminescent substance, a colored substance, polyethylene glycol or an enzyme.
  • the antibody is an antibody or an antigen-binding fragment thereof according to any one of the present invention
  • the coupling part is a purification tag. (such as His tag), detectable label; preferably, the coupling part is a radioactive isotope, 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 an antibody or an antigen-binding fragment thereof according to any one of the present invention.
  • kits which includes the antibody or antigen-binding fragment thereof according to 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 also includes a detectable label, such as a radioactive isotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or an enzyme.
  • Another aspect of the present invention relates to the use of the antibody or antigen-binding fragment thereof, the conjugate, fusion protein or multispecific antibody of the present invention in preparing a kit for The presence or level of CD73 in the sample is detected.
  • compositions comprising the antibody or antigen-binding fragment thereof, the conjugate, fusion protein or multispecific antibody of any one of the present invention; optionally, the
  • the pharmaceutical composition also includes pharmaceutically acceptable carriers and/or excipients.
  • the pharmaceutical composition is in a form suitable for administration by subcutaneous, intradermal, intravenous, intramuscular or intralesional injection.
  • Another aspect of the present invention relates to the use of the antibody or antigen-binding fragment thereof, the conjugate, fusion protein or multispecific antibody of the present invention for the treatment and/or prevention of tumors (such as solid tumors, preferably Non-small cell lung cancer, prostate cancer (including metastatic castrated Resistant prostate cancer (mCRPC)), triple-negative breast cancer, ovarian cancer, colorectal cancer (including microsatellite stable (MSS) and mismatch repair deficient/microsatellite unstable high (dMMR/MSI-H) type), gastric cancer (including microsatellite stable (MSS) and mismatch repair deficient/microsatellite highly unstable (dMMR/MSI-H) types), melanoma, head and neck cancer, renal cell carcinoma or pancreatic duct adenocarcinoma, Or use in the preparation of drugs for diagnosing tumors.
  • tumors such as solid tumors, preferably Non-small cell lung cancer, prostate cancer (including metastatic castrated Resistant prostate cancer (m
  • Another aspect of the present invention relates to the hybridoma cell line LT014, which is deposited in the China Type Culture Collection Center (CCTCC) with the deposit number CCTCC NO: C2018137.
  • CTCC China Type Culture Collection Center
  • a further aspect of the invention relates to a combination product (eg a kit) comprising a first product and a second product individually packaged, wherein,
  • the first product includes the anti-CD73 antibody or antigen-binding fragment thereof according to any one of the present invention, the conjugate of the present invention or the pharmaceutical composition according to any one of the present invention;
  • the second product includes an anti-PD-1-anti-VEGFA bispecific antibody
  • the combination product further comprises an independently packaged third product, the third product comprising one or more chemotherapy drugs,
  • the first product and the second product also independently contain one or more pharmaceutically acceptable excipients;
  • the combination product also contains product instructions.
  • the instructions state that the single dosage of anti-CD73 antibody and/or anti-PD-1-anti-VEGFA bispecific antibody is 0.1-100 mg per kilogram of body weight. , preferably 1-10 mg; alternatively, the single dosage of anti-CD73 antibody and/or anti-PD-1-anti-VEGFA bispecific antibody is 10-1000 mg per subject, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg or 200 mg.
  • the instructions state that the anti-CD73 antibody and/or the anti-PD-1-anti-VEGFA bispecific antibody is administered twice a day to about once every other day, or every 3 Anti-CD73 antibody and/or anti-PD-1-anti-VEGFA bispecific administered once every day, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks Antibody.
  • the mass ratio is (1:5)-(5:1), for example: 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1 or 5:1.
  • 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 according to the invention,
  • drug A comprises the antibody or antigen-binding fragment thereof according to the invention
  • the conjugate of the present invention or the fusion protein or multispecific antibody of the present invention, drug B contains an anti-PD-1-anti-VEGFA bispecific antibody, preferably, drug A and drug B are administered simultaneously or sequentially, The sequential administration is drug A first or drug B first.
  • the heavy chain amino acid sequence of the anti-PD-1-anti-VEGFA bispecific antibody is shown in SEQ ID NO: 23, and the light chain amino acid sequence is shown in SEQ ID NO: 25.
  • the present invention relates to a method for preventing and/or treating tumors (especially malignant tumors), comprising administering to a subject a therapeutically effective amount of an anti-CD73 antibody in combination with an anti-PD-1-anti-VEGFA bispecific antibody, more preferably
  • one or more chemotherapeutic drugs preferably the chemotherapeutic drugs are chemotherapeutic agents or growth inhibitors, targeted therapeutic agents (such as antibody-drug conjugates, antibodies or antigen-binding fragments thereof)
  • chemotherapeutic drugs are chemotherapeutic agents or growth inhibitors, targeted therapeutic agents (such as antibody-drug conjugates, antibodies or antigen-binding fragments thereof)
  • chemotherapeutic drugs are chemotherapeutic agents or growth inhibitors, targeted therapeutic agents (such as antibody-drug conjugates, antibodies or antigen-binding fragments thereof)
  • chemotherapeutic drugs are chemotherapeutic agents or growth inhibitors, targeted therapeutic agents (such as antibody-drug conjugates, antibodies or antigen-binding fragments thereof
  • the chemotherapeutic agent or growth inhibitory agent is selected from the group consisting of alkylating agents, anthracyclines, antihormonal agents (eg, antiandrogens), aromatase inhibitors, protein kinase inhibitors (e.g., tyrosine kinase inhibitors), lipid kinase inhibitors, antisense oligonucleotides, ribozymes, antimetabolites, topoisomerase inhibitors, cytotoxic agents or antineoplastic antibiotics, proteasome inhibitors, Antimicrotubule agents, EGFR antagonists, VEGF antagonists, PD-1 antagonists, angiopoietin 2 antagonists, retinoids, histone deacetylase inhibitors, and combinations thereof.
  • alkylating agents e.g., anthracyclines, antihormonal agents (eg, antiandrogens), aromatase inhibitors, protein kinase inhibitors (e.g., t
  • the targeted therapeutic agent is selected from the group consisting of B-raf inhibitors, MEK inhibitors, K-ras inhibitors, c-Met inhibitors, Alk inhibitors, phosphatidylinositol 3-kinase inhibitor, Akt inhibitor, mTOR inhibitor, VEGF inhibitor, CD73 inhibitors, PARP inhibitors, PD-1 inhibitors, bisphosphatidylinositol 3-kinase/mTOR inhibitors and combinations thereof.
  • the antibody-drug conjugate comprises a drug selected from the group consisting of maytansine, monomethyl auristatin E, calicheamicin, esperamicin and radioisotope chelates mixture.
  • the CD73 inhibitors include but are not limited to one or more of BMS-986179, MEDI9447, NZV930, CPI-006, AB680, LY-3475070, TJ004309[3], ORIC-533, IPH5301AB680, and LY-3475070.
  • the PARP inhibitor is selected from etoposide, olaparib, rucaparib, niraparib, talazoparib, fluzoparib, veliparib ER, ABT-472, ABT-767, Stenoparib, AST-6828, AG-PD, ANG-2864, ANG-3038, ANG-3186, AZD-5305, AZ-0108, AZD-2461, AMXI-5001, AMXI-2001, AMXI-3001, AMXI-7001, AMXI- 9001, pamiparib, ZYTP-1, CK-102, -207, DPS-102, E-7016, iobenguane I 131, MK-2512, HCX-014, HWH-340, IDX-1197, IDX-1197, senaparib, IMP-04100, IMP-04111, IMP-04149, IMP -04249, IMP-04307, IMP-04356, JPI-2
  • the tumor is selected from one or more of the following:
  • Cervical cancer such as metastatic cervical cancer
  • lung cancer such as small cell lung cancer and non-small cell lung cancer (such as squamous non-small cell lung cancer or non-squamous non-small cell lung cancer)
  • throat cancer such as esophageal cancer, esophageal cancer Squamous cell carcinoma, thyroid cancer, mesothelioma
  • gastrointestinal cancer such as gastric cancer (including microsatellite Stable (MSS) and mismatch repair deficient/microsatellite instability high (dMMR/MSI-H) type), such as advanced gastric cancer, gastric adenocarcinoma, or gastroesophageal junction adenocarcinoma) and intestinal cancer (such as rectal cancer, colon cancer, colorectal cancer (including nodal satellite stable (MSS) and mismatch repair defective/microsatellite unstable high (dMMR/MSI-H) type), liver cancer (such as hepatocellular carcinoma, hepatobiliary cancer),
  • the anti-PD-1-anti-VEGFA bispecific antibody includes:
  • the first protein functional region is a single chain antibody, and the second protein functional region is an immunoglobulin; or the first protein functional region is an immunoglobulin, and the second protein functional region is a single chain antibody.
  • the heavy chain variable region of the immunoglobulin includes: HCDR1-HCDR3 contained in the heavy chain variable region whose amino acid sequence is as shown in SEQ ID NO:27 (preferably according to the IMGT numbering system, respectively as SEQ ID NOs:31- HCDR1-HCDR3 shown in 33), which is light
  • the chain variable region includes: LCDR1-LCDR3 contained in the light chain variable region with an amino acid sequence as shown in SEQ ID NO: 29 (preferably LCDR1-LCDR3 as shown in SEQ ID NOs: 34-36 respectively according to the IMGT numbering system) ;
  • the heavy chain variable region of the single-chain antibody includes: HCDR1-HCDR3 contained in the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:37 (preferably according to the IMGT numbering system, such as SEQ ID NOs:41- HCDR1-HCDR3 shown in 43) and the light chain variable region thereof includes the LCDR1-LCDR3 included in the light chain variable region shown in SEQ ID NO: 39 (preferably according to the IMGT numbering system, respectively, such as SEQ ID NOs: LCDR1-LCDR3 shown in 44-46);
  • the heavy chain variable region of the immunoglobulin includes: HCDR1-HCDR3 contained in the heavy chain variable region whose amino acid sequence is as shown in SEQ ID NO:37 (preferably according to the IMGT numbering system, respectively as SEQ ID NOs:41- HCDR1-HCDR3 shown in 43) and the light chain variable region thereof includes the LCDR1-LCDR3 included in the light chain variable region shown in SEQ ID NO: 39 (preferably according to the IMGT numbering system, respectively, such as SEQ ID NOs: LCDR1-LCDR3 shown in 44-46);
  • the heavy chain variable region of the single chain antibody includes: HCDR1-HCDR3 contained in the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 27 (preferably according to the IMGT numbering system, respectively as SEQ ID NOs: 31 HCDR1-HCDR3 shown in -33), whose light chain variable region includes: LCDR1-LCDR3 contained in the light chain variable region whose amino acid sequence is as shown in SEQ ID NO:29 (preferably according to the IMGT numbering system, such as SEQ ID LCDR1-LCDR3 shown in NOs: 34-36);
  • the immunoglobulin is of the human IgG1 subtype.
  • the bispecific antibody wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin has the following mutations:
  • the letters before the site represent the amino acid before mutation
  • the letters after the site represent the amino acid after mutation
  • the bispecific antibody wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin has or further has one or more mutations selected from the following :
  • the bispecific antibody wherein,
  • the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 27; and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 29; and, The amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO: 37; and the amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO: 39.
  • the bispecific antibody has a heavy chain amino acid sequence as shown in SEQ ID NO:23, and a light chain amino acid sequence as shown in SEQ ID NO:25.
  • the heavy chain of the bispecific antibody is encoded by the nucleotide sequence shown in SEQ ID NO:24, and the light chain is encoded by the amino acid sequence shown in SEQ ID NO:26.
  • the bispecific antibody wherein the single chain antibody is connected to the C-terminus of the heavy chain of an immunoglobulin. Since immunoglobulins consist of two heavy chains, one immunoglobulin molecule has two single-chain antibody molecules linked to it. Preferably, the two single chain antibody molecules are identical.
  • the bispecific antibody has two single-chain antibodies, and one end of each single-chain antibody is respectively connected to the C-terminal end of the two heavy chains of immunoglobulin or N terminus.
  • a disulfide bond exists between VH and VL of the single chain antibody.
  • Methods for introducing disulfide bonds between VH and VL of antibodies are well known in the art. For example, see U.S. Pat. No. 5,747,654; Rajagopal et.al, Prot. Engin. 10 (1997) 1453-1459; Reiter et.al, Nat. Biotechnol.
  • the bispecific antibody wherein the first protein functional region is directly connected to the second protein functional region or connected through a connecting fragment; and/or the The heavy chain variable region of the single chain antibody is connected to the light chain variable region of the single chain antibody directly or through a linking fragment.
  • the connecting fragment in the bispecific antibody is (GGGGS)n, n is a positive integer; preferably, n is 1, 2, 3, 4, 5 or 6.
  • the first protein functional region and the second protein functional region are independently one or more than two.
  • the bispecific antibody wherein the single chain antibody is connected to the C-terminus of the heavy chain of an immunoglobulin.
  • the first protein functional region and the second protein functional region are connected through a first connecting fragment; and the heavy chain variable region of the single chain antibody is connected to the single chain variable region.
  • the light chain variable regions of the chain antibodies are connected through a second connecting fragment; the first connecting fragment and the second connecting fragment are the same or different;
  • amino acid sequences of the first connection fragment and the second connection fragment are independently selected from SEQ ID NO: 47 and SEQ ID NO: 48;
  • amino acid sequences of the first connection fragment and the second connection fragment are as shown in SEQ ID NO: 48.
  • the bispecific antibody is a monoclonal antibody.
  • the bispecific antibody is a humanized antibody.
  • the unit preparation described in contains 1 to 10000 mg (preferably 10 to 1000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg or 200 mg) of the anti-CD73 antibody according to any aspect of the present invention and 1 to 10000 mg.
  • the anti-PD-1-anti-VEGFA bispecific antibody preferably 1-1000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg or 100 mg
  • one or more chemotherapeutic drugs of the present invention such as platinum drugs and/or fluorouracil anti-tumor drugs
  • the anti-CD73 antibody, the anti-PD-1-anti-VEGFA bispecific antibody and chemotherapy drugs are packaged separately.
  • the present invention relates to a method for preventing or treating cancer or tumors, wherein one or more unit preparations according to the invention are administered to a subject in need thereof, preferably, anti-PD-1 in said unit preparations -Anti-VEGFA bispecific antibody, anti-CD73 antibody and chemotherapeutic agents are each administered separately.
  • a single pharmaceutical dosage unit preferably for the treatment of tumors, comprising 0.1-10000 mg (preferably 1-1000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg or 100 mg) of the anti-CD73 antibody of any one of the present invention and 0.1-10000 mg (preferably 1-1000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg or 100 mg) of any of the present invention
  • the anti-PD-1-anti-VEGFA bispecific antibody of one item comprising 0.1-10000 mg (preferably 1-1000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg or 100 mg) of any of the present invention.
  • the anti-CD73 antibody, the VEGFA-anti-PD-1 bispecific antibody and/or the chemotherapeutic agent is in a form suitable for intravenous injection or intravenous infusion
  • Liquid form is preferred.
  • an effective amount of the anti-CD73 antibody according to any one of the invention and/or the anti-PD-1-anti-VEGFA bispecific according to any one of the invention is administered to the subject.
  • the step of inducing antibodies is before or after surgical treatment, and/or before or after radiation therapy.
  • a single administration of the anti-CD73 antibody according to any one of the invention and/or the anti-PD-1-anti-VEGFA bispecific antibody according to any one of the invention is 0.1-100 mg per kilogram of body weight, preferably 1-10 mg; or, a single dose of the anti-CD73 antibody according to any one of the present invention and/or the anti-PD-1-anti-VEGFA bispecific antibody according to any one of the present invention.
  • the dosage is 10-1000 mg per subject, preferably 50-500 mg, 100-400mg, 150-300mg, 150-250mg or 200mg,
  • administration is given twice daily to about every other day, or every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks. medicine once;
  • the administration method is intravenous drip or intravenous injection.
  • EC50 refers to the concentration for 50% of maximal effect, the concentration that causes 50% of the maximal effect.
  • antibody refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having a "light” (L) chain and a “heavy” (H) chain.
  • Antibody light chains can be classified into 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 connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 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 (V L ) and a light chain constant region ( CL ).
  • the light chain constant region consists of one domain, CL .
  • the constant region of an antibody may mediate 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 conservative regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions ( VH and VL ) of each heavy chain/light chain pair respectively form the antigen-binding site.
  • 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 cell receptors, MHC, IgSF and MhcSF.
  • antibody is not limited to any particular method of producing the antibody. For example, this includes, inter alia, recombinant antibodies, monoclonal antibodies and polyclonal antibodies.
  • the antibodies may be of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3 or IgG4 subtypes
  • IgA1, IgA2, IgD, IgE or IgM antibodies may be of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtypes), IgA1, 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 group of highly homologous antibody molecules, that is, except for natural mutations that may occur spontaneously. A group of identical antibody molecules. Monoclonal antibodies are highly specific for a single epitope on the antigen. Polyclonal antibodies are relative to monoclonal antibodies, which usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen. Monoclonal antibodies can usually be obtained using 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 using recombinant DNA technology (eg, see US Patent 4,816,567).
  • humanized antibody refers to a product in which all or part of the CDR regions of a human immunoglobulin (recipient antibody) are replaced by the CDR regions of a non-human antibody (donor antibody).
  • some amino acid residues in the framework region (FR) of the recipient antibody can also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further improve or optimize the performance of the antibody.
  • the term “isolated” or “isolated” means obtained from the natural state by artificial means. If an "isolated" substance or ingredient occurs in nature, it may be that the natural environment in which it is located has changed, or that the substance has been separated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the high purity of the same polynucleotide or polypeptide isolated from this natural state is called isolation. of.
  • isolation a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the high purity of the same polynucleotide or polypeptide isolated from this natural state is called isolation. of.
  • the term “isolated” or “isolated” does not exclude the admixture of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.
  • 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 the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyomavacuolating viruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses poxviruses
  • baculoviruses papillomaviruses
  • papillomaviruses papillomaviruses
  • Polyomavacuolating viruses such as SV40.
  • a vector can contain a variety of expression-controlling elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is 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
  • 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 to an antigen refers to an antibody that binds to an antigen at a concentration of 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 of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen.
  • antibodies dissociate with a dissociation equilibrium constant (K D ) of less than about 10 ⁇ 5 M, such as less than about 10 ⁇ 6 M, 10 ⁇ 7 M, 10 ⁇ 8 M, 10 ⁇ 9 M, or 10 ⁇ 10 M or less.
  • Binds to an antigen e.g., PD-1 protein).
  • KD can be determined using methods known to those skilled in the art, such as using the Fortebio Molecular Interaction Instrument.
  • 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” means a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, They are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers agent.
  • pH adjusters include but are not limited to phosphate buffer; 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.
  • an effective amount refers to an amount sufficient to obtain, at least in part, the desired effect.
  • an effective amount to prevent a disease is an amount that is sufficient to prevent, prevent, or delay the occurrence of a disease (such as a tumor);
  • an effective amount to treat a disease is an amount that is sufficient to cure or at least partially prevent the occurrence of a disease in a patient who already has the disease. The amount of disease and its complications.
  • single pharmaceutical dosage unit means a drug containing an anti-CD73 antibody and anti-PD-1-anti-VEGFA according to the invention to be administered to a subject at the time of the dosing regimen (preferably per kilogram of body weight of the subject).
  • dosing regimens include, for example, administration of a single pharmaceutical dosage unit according to a dosing cycle from twice daily to about once every other day, or every 3 days, 4 days, 5 days, 6 days , administered once every 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks.
  • first such as the first protein functional region, the first connecting fragment
  • second such as the second protein functional region, the second connecting fragment
  • a “therapeutically effective amount” or “therapeutically effective dose” of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of disease or promotes regression of disease, Disease regression is evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free phases, or the prevention of impairment or disability caused by disease afflictions.
  • the ability of therapeutic agents to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by in vitro assays to determine the activity of the agent.
  • a “prophylactically effective amount" of a drug means that when administered alone or in combination with an antineoplastic agent to a subject at risk of developing cancer (e.g., a subject with a premalignant condition) or a subject at risk of recurrence of cancer, An amount of any drug that inhibits the occurrence or recurrence of cancer. In certain embodiments, the prophylactically effective amount completely prevents the development or recurrence of cancer. "Inhibiting" the occurrence or recurrence of cancer means reducing the likelihood of the occurrence or recurrence of cancer, or completely preventing the occurrence or recurrence of cancer.
  • the monoclonal antibody of the present invention can specifically bind to CD73 very well, and can very effectively inhibit the enzymatic reaction of CD73 in a non-substrate competition manner, reduce the production of adenosine, and promote T cell activity and tumor suppression effects.
  • the combination of the antibody of the present invention and the anti-PD-1-anti-VEGFA bispecific antibody has the pharmacological effect of effectively inhibiting tumor growth. Better than anti-VEGFA/PD-1 bispecific antibody single agent or anti-CD73 antibody single agent.
  • Anti-CD73 antibodies effectively inhibit CD73 enzymatic activity.
  • FIG. 8 Effect of 19F3H2L3 (hG1DM) antibody combined with anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) on B6-hPD1/hPDL1/hCD73 mouse MC38-hPDL1/hCD73 tumor model.
  • Anti-CD73 specific antibody combined with anti-PD-1-anti-VEGFA bispecific antibody promotes the secretion of cytokine IFN- ⁇ .
  • the BALB/c mice used were purchased from Guangdong Medical Experimental Animal Center.
  • the positive control antibody MEDI9447 (Oleclumab) used is produced by Zhongshan Kangfang Biopharmaceutical Co., Ltd., and its sequence is consistent with the International Nonproprietary Names for Pharmaceutical Substances (INN) published publicly on the WHO website by Medlmmune Limited. The antibody sequences recorded in (World Health Organization (2016). "International Nonproprietary Names for Pharmaceutical Substances (INN). Proposed INN: List 116" (PDF). WHO Drug Information. 30 (4), P661-662).
  • the combined anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) used was produced by Zhongshan Kangfang Biopharmaceutical Co., Ltd., and its sequence and preparation can be found in the published patent CN112830972A.
  • the above-mentioned antibody VP101 (hG1DM) wherein the full-length amino acid sequence of the heavy chain of VP101 (hG1DM) is shown in SEQ ID NOs: 23, and the full-length amino acid sequence of the light chain is shown in SEQ ID NOs: 25.
  • the structure of VP101 (hG1DM) is IgG-scFv, where the IgG part is an anti-VEGFA antibody and the scFv part is an anti-PD-1 antibody.
  • the HCDR1 sequence of the anti-VEGFA antibody is shown in SEQ ID NO:31
  • the HCDR2 sequence is shown in SEQ ID NO:32
  • the HCDR3 sequence is shown in SEQ ID NO:33
  • the VH sequence is shown in SEQ ID NO:27
  • the anti-VEGFA The LCDR1 sequence of the antibody is shown in SEQ ID NO:34
  • the LCDR2 sequence is shown in SEQ ID NO:35
  • the LCDR3 sequence is shown in SEQ ID NO:36
  • the VL sequence is shown in SEQ ID NO:29.
  • the HCDR1 sequence of the anti-PD1 antibody is shown in SEQ ID NO:41
  • the HCDR2 sequence is shown in SEQ ID NO:42
  • the HCDR3 sequence is shown in SEQ ID NO:43
  • the VH sequence is shown in SEQ ID NO:37.
  • the LCDR1 sequence of the PD1 antibody is shown in SEQ ID NO:44
  • the LCDR2 sequence is shown in SEQ ID NO:45
  • the LCDR3 sequence is shown in SEQ ID NO:46
  • the VL sequence is shown in SEQ ID NO:39.
  • the heavy chain constant region of the positive control antibody wild IgG1 control antibody is Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region is Ig kappa chain C region, ACCESSION: P01834.
  • the C1q used was purchased from fizgerald, product number: A16050201;
  • the Fc ⁇ RIIIa-bio used was purchased from Sino Biological, product number: LC09JA0407;
  • the CD73 (5′-nuclease) specific inhibitor APCP (alpha, beta-methylene adenosine-5′-diphosphate, 5′- ⁇ , ⁇ - Methylene-adenosine diphosphate) from sigma, Cat. No.: M3763-10MG.
  • the isotype control antibody used is human anti-Hen Egg Lysozyme IgG (anti-HEL antibody, or human IgG, hIgG for short, or isotype control), the sequence Variable region sequence from the Fab F10.6.6 sequence published by Acierno et al. J Mol Biol. 2007; 374(1): 130-146.).
  • the antigen used to prepare anti-CD73 antibodies is human NT5E-His (NT5E is Genbank ID: NP 002517.1, position: 1-552, prepared by Zhongshan Kangfang Biopharmaceutical Co., Ltd.).
  • the spleen cells of the immunized mice were fused with mouse myeloma cells to produce hybridoma cells.
  • NT5E is Genbank ID: NP 002517.1, position: 1-552
  • -Biotin N5E-Biotin, prepared by Zhongshan Kangfang Biopharmaceutical Co., Ltd.
  • indirect ELISA screening of hybridoma cells was performed to obtain cells capable of secreting Hybridoma cells with antibodies that specifically bind to CD73.
  • stable hybridoma cell lines were obtained through limiting dilution method.
  • the above hybridoma cell line was named hybridoma cell line LT014, and the monoclonal antibody secreted by it was named 19F3.
  • the hybridoma cell line LT014 (also known as CD73-19F3) was deposited in the China Type Culture Collection (CCTCC) on June 21, 2018.
  • the deposit number is CCTCC NO:C2018137, and the deposit address is Wuhan University, Wuhan, China. , Postal code: 430072.
  • the LT014 cell lines prepared above were cultured in CD medium (Chemical Defined Medium) (CD medium, containing 1% penicillin and streptomycin, cultured in a 5% CO 2 , 37°C cell culture incubator). After 7 days, the cell culture supernatant was collected, subjected to high-speed centrifugation, vacuum filtration with a microporous membrane, and purified using a HiTrap protein A HP column to prepare antibody 19F3.
  • CD medium Chemical Defined Medium
  • PCR amplification products are directly subjected to TA cloning.
  • TA cloning For specific operations, refer to the pEASY-T1 Cloning Kit (Transgen CT101) kit instructions.
  • the TA cloned product was directly sequenced, and the sequence analysis and sequencing results of the anti-CD73 antibody 19F3 were as follows:
  • the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, with a length of 363bp.
  • the encoded amino acid sequence is shown in SEQ ID NO:2, with a length of 121aa;
  • 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, with a length of 339bp.
  • the encoded amino acid sequence is shown in SEQ ID NO:4, with a length of 113aa;
  • 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
  • the sequence of light chain CDR3 is shown in SEQ ID NO:20.
  • Example 3 Design and preparation of light and heavy chains of humanized antibodies against human CD73
  • the designed variable region sequence of the above-mentioned humanized antibody is as follows:
  • the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:5, with a length of 363bp.
  • the encoded amino acid sequence is shown in SEQ ID NO: 6, and the length is 121aa.
  • 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 sequence of heavy chain CDR3 is shown in SEQ ID NO: 16.
  • 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, with a length of 339bp.
  • the encoded amino acid sequence is shown in SEQ ID NO:8 and is 113aa in length.
  • 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
  • the sequence of light chain CDR3 is shown in SEQ ID NO:19.
  • 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, with a length of 363 bp.
  • the encoded amino acid sequence is shown in SEQ ID NO:10, with a length of 121aa.
  • 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:16.
  • 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, with a length of 339bp.
  • the encoded amino acid sequence is shown in SEQ ID NO:12 and is 113aa in length.
  • 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
  • the sequence of light chain CDR3 is shown in SEQ ID NO:19.
  • 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, with a length of 363 bp.
  • the encoded amino acid sequence is shown in SEQ ID NO:10, with a length of 121aa.
  • 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:16.
  • 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, with a length of 339bp.
  • the encoded amino acid sequence is shown in SEQ ID NO:14, with a length of 113aa.
  • 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
  • the sequence of light chain CDR3 is shown in SEQ ID NO:19.
  • the sequence is shown in SEQ ID NO:20.
  • the light chain constant region of the antibodies 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 introduces a point mutation from leucine to alanine (L234A) at position 234, position 235 A point mutation (L235A) from leucine to alanine (SEQ ID NO: 21) was introduced at position No., and humanized antibodies named 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM) and 19F3H2L3 (hG1DM) were obtained.
  • the heavy chain cDNA and light chain cDNA of 19F3H1L1 (hG1DM), the heavy chain cDNA and light chain cDNA of 19F3H2L2 (hG1DM), and the heavy chain cDNA and light chain cDNA of 19F3H2L3 (hG1DM) were cloned into pUC57simple (Genscript (provided by the company) vector, pUC57simple-19F3H1 (hG1DM), pUC57simple-19F3L1; pUC57simple-19F3H2 (hG1DM), pUC57simple-19F3L2 and pUC57simple-19F3L3 were obtained respectively.
  • the designed gene combinations containing the corresponding light and heavy chain recombinant plasmids (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 fluid was collected for purification. After sequencing and verification, an endotoxin-free expression plasmid was prepared and the plasmid was transiently transfected into HEK293 cells for antibody expression. After 7 days of culture Collect the cell culture fluid and use Protein A column for affinity purification to obtain humanized antibodies.
  • Example 4 Dynamic affinity determination of anti-CD73 antibodies with Clq and Fc ⁇ RIIIa.
  • the sample dilution buffer is PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 50 ⁇ g/mL antibody is fixed on the FAB2G sensor with a fixed height of about 2.0 nm.
  • the sensor is equilibrated in the buffer for 60 seconds and fixed on the sensor.
  • the antibody on the antibody binds to the antigen C1q.
  • the antigen concentration is 0.63-10nM (two-fold gradient dilution) for 60 seconds.
  • the antigen-antibody dissociates in the buffer for 60 seconds.
  • the sensor was regenerated using 10mM glycine, pH 1.7, time 5s, repeated 4 times.
  • the vibration rate of the sample plate is 1000rpm, the detection temperature is 30 degrees, and the detection frequency is 0.6Hz.
  • Data are modeled 1:1 Fitting analysis was performed to obtain the affinity constant.
  • the data acquisition software is Fortebio Data Acq ⁇ isition7.0, and the data analysis software is Fortebio Data Analysis 7.0.
  • the sample dilution buffer is PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4.
  • 0.5 ⁇ g/mL Fc ⁇ RIIIa (from Sino Biological) is fixed on the SA sensor for 120s, and the sensor is equilibrated in the buffer for 60s.
  • CD16a fixed on the sensor binds to each antibody.
  • the antibody concentration is 31.3-500nM (two-fold gradient dilution) for 60 seconds.
  • the antibody antigen is dissociated in the buffer for 60 seconds.
  • the sensor was regenerated using 10mM NaOH.
  • the detection temperature is 30°C and the frequency is 0.6Hz.
  • the data were fit and analyzed using a 1:1 model to obtain the affinity constants.
  • Example 5 Detection of anti-CD73 antibody inhibiting endogenous expression of CD73 enzyme activity in cells
  • the experimental steps are as follows: Take the logarithmic phase MDA-MB-231 cells (derived from ATCC, HTB-26) in good condition, resuspend the cells in serum-free RPMI-1640 culture medium, and count; Inoculate into a 96-well plate, 3*10 4 cells/100 ⁇ l/well; dilute the antibody with serum-free RPMI-1640 culture medium, with a starting concentration of 200 ⁇ g/ml, and carry out gradient dilution by 2.5 times; add the antibody to the 96-well plate, each time 50 ⁇ l into each well and incubate at 37°C for 1 hour.
  • Example 6 Pharmacodynamic evaluation of anti-CD73 specific antibody combined with anti-PD-1-anti-VEGFA bispecific antibody in mouse tumor cell subcutaneous transplantation model
  • the day of grouping was defined as day D0, and administration started on day D0 of grouping.
  • the administration method of the combined administration group is: the drugs are prepared separately, first Anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) was administered, followed 2 hours later by anti-CD73 specific antibody 19F3H2L3 (hG1DM).
  • the modeling and specific administration methods are shown in Table 3. After administration, the length and width of the tumors in each group were measured, and the tumor volume was calculated.
  • tumor-bearing mice had good tolerance to the test drugs 19F3H2L3 (hG1DM) and VP101 (hG1DM) alone and in combination, and each group had no effect on the body weight of tumor-bearing mice.
  • Example 7 Mixed lymphocyte reaction detection of anti-CD73 specific antibody combined with anti-PD-1-anti-VEGFA bispecific antibody to promote cytokine secretion
  • the GenBank registration number of PDL1 is NP_054862.1, and PDL1FL represents the full length of the PDL1 gene.
  • the human PD-L1 overexpression lentiviral vector plenti6.3/V5-PDL1FL-BSD was purchased from Invitrogen, product number: K5315-20) was packaged by the virus and infected Raji cells.
  • BSD Bacillus subtilis, Gibco product number: R210-01
  • the Raji-PDL1 cell line was obtained.
  • PBMC normal human PBMC according to the operating instructions of the separation liquid Ficoll-Paque TM Plus. Two days before the experiment, PBMC were resuscitated and cultured in complete medium (1640+10% FBS) in a 37°C, 5% carbon dioxide incubator. After 2 hours, when the PBMC state recovered, SEB (final concentration 0.5 ⁇ g/mL, Toxin technology, product number: BT202) was added to stimulate for two days.
  • SEB final concentration 0.5 ⁇ g/mL, Toxin technology, product number: BT202
  • PBMC Collect PBMC after two days of SEB stimulation and Raji-PDL1 cells after MMC treatment, and seed them into a 96-well U-shaped plate at PBMC 1*10 5 /well, Raji-PDL1 1*10 5 /well, and A549 1*10 4 /well.
  • AMP adenosine-5'-monophosphate, TCI, Cat. No.: A0158
  • PBMC+Raji-PDL1+A549+AMP isotype Control
  • hlgG1 and hIgGDM isotype Control
  • the results are shown in Figure 10.
  • the results show that compared with the isotype control antibody, the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) can effectively promote the secretion of cytokine IFN- ⁇ in the mixed lymphoid system, and the combination of 19F3H2L3 (hG1DM) + VP101 (hG1DM)
  • the group showed combined anti-tumor efficacy, and its combination to promote the secretion of cytokine IFN- ⁇ was better than that of each single drug group, and the combined activity of 150nM 19F3H2L3(hG1DM)+VP101(hG1DM) was better than that of 300nM 19F3H2L3(hG1DM) Or the single-agent activity of VP101(hG1DM).
  • Amino acid sequence of 19F3 light chain variable region (SEQ ID NO: 4)
  • Nucleic acid sequence of 19F3H1L1 (hG1DM) heavy chain variable region (SEQ ID NO: 5)
  • Bevacizumab-Hv Bevacizumab heavy chain variable region
  • Bevacizumab-Lv Amino acid sequence of Bevacizumab light chain variable region (Bevacizumab-Lv): (107aa)
  • amino acid sequences of the three CDR regions of the Bevacizumab heavy chain variable region are as follows:
  • amino acid sequences of the three CDR regions of the Bevacizumab light chain variable region are as follows:
  • the amino acid sequence of the 14C12H1L1 (M) heavy chain variable region is shown in SEQ ID NO: 37.

Landscapes

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

Abstract

涉及药物组合物，其包含抗CD73(例如人CD73)抗体或其抗原结合片段，和抗PD-1-抗VEGFA双特异性抗体或其抗原结合片段。具体地，所述抗CD73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

药物组合物及其用途 技术领域
本发明涉及免疫学领域。具体地，涉及药物组合物，其包含抗CD73(例如人CD73)抗体或其抗原结合片段，和抗PD-1-抗VEGFA双特异性抗体或其抗原结合片段。
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 1ligand 1，亦简称为PDL-1)和PDL2(Programmed cell death 1ligand 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)。
血管内皮生长因子(VEGF)是一类能促进内皮细胞分裂增殖、促进新生血管的形成、提高血管通透性的生长因子，它与细胞表面的血管内皮生长因子受体结合，通过激活酪氨酸激酶信号转导途径发挥功能。在肿瘤组织中，肿瘤细胞、肿瘤侵入的巨噬细胞和肥大细胞能分泌高水平的VEGF，以旁分泌的形式刺激肿瘤血管内皮细胞，促进内皮细胞增殖、迁移，诱导血管形成，促进肿瘤持续生长，并提高血管通透性，引起周围组织纤维蛋白沉着，促进单核细胞、成纤维细胞、内皮细胞侵润，有利于肿瘤基质形成和肿瘤细胞进入新生血管，促进肿瘤转移。因而抑制肿瘤血管生成被认为是当前最具有前途的肿瘤治疗方法之一。VEGF家族包括：VEGFA、VEGFB、VEGFC、VEGFD和PIGF。血管内皮生长因子受体(VEGFR)包括VEGFR1(又称Flt1)、VEGFR2(又称KDR或Flk1)、 VEGFR3(又称Flt4)和Neuropilin-1(NRP-1)，其中，前三种受体在结构上类似，都属于酪氨酸激酶超家族，均由膜外区、跨膜片段及膜内区三部分构成，其中膜外区由免疫球蛋白样结构域组成，膜内区属酪氨酸激酶区。VEGFR1和VEGFR2主要位于血管内皮细胞表面，VEGFR3主要位于淋巴管内皮细胞表面。
VEGF家族分子对于几种受体有不同的亲和力。VEGFA主要与VEGFR1、VEGFR2和NRP-1结合发挥作用。VEGFR1是最早发现的受体，在正常生理情况下VEGFR1与VEGFA的亲和力比VEGFR2与VEGFA的亲和力高，但是其胞内段酪氨酸酶活性比VEGFR2低(马莉，中国优生与遗传杂志，24(5)(2016)：146-148)。
VEGFR2是血管发生和构建的主要调节者，与VEGFR1相比VEGFR2具有很高的酪氨酸激酶活性。VEGFR2与配体VEGFA结合后介导血管内皮细胞的增殖、分化等行为，以及血管的形成过程和血管的通透性(Roskoski R Jr.等人，Crit Rev Oncol Hematol，62(3)(2007)：179-213.)，VEGFA与VEGFR2结合后通过下游PLC-γ-PKC-Raf-MEK-MAPK信号传导通路介导胞内相关蛋白基因转录表达，促进血管内皮细胞增殖(Takahashi T等人，Oncogene，18(13)(1999)：2221-2230)。
VEGFR3属于酪氨酸激酶家族成员之一，主要表达在胚胎时期的血管内皮细胞和成年期的淋巴管内皮细胞，VEGFC和VEGFD与VEGFR3结合以刺激淋巴管内皮细胞的增殖与迁移，促进淋巴管的新生；NRP-1是一种非酪氨酸激酶跨膜蛋白，不能独立转导生物信号，而与VEGF酪氨酸激酶受体形成复合物后才能介导信号传导(马莉，中国优生与遗传杂志，24(5)(2016)：146-148)。
VEGFA与VEGFR2主要参与调控血管生成，VEGFA与VEGFR2结合前后会引发上下游通路中众多中间信号形成级联反应，最终以内皮细胞增殖、存活、迁移、通透性增加及浸润到周围组织等不同形式改变其生理功能(董宏超等人，《现代肿瘤医学》，第22卷，第9期2014年9月，第2231-3页)。
目前已经有多种靶向人VEGF特别是VEGFA的人源化单克隆抗体，如贝伐单抗(Bevacizumab)，其于2004年期间陆续被美国食品药品管理 局批准用于治疗非小细胞肺癌、肾细胞癌、宫颈癌、转移性结直肠癌等多种肿瘤。
综上所述，开发更有效的治疗手段以及联合给药治疗方案具有巨大的临床意义。
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/VEGFA双特异性抗体联用具有有效抑制肿瘤生长的药理学效果，优于抗PD-1/VEGFA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％、至少81％、至少82％、至少83％、至少84％、至少85％、 至少86％、至少87％、至少88％、至少89％、至少90％，优选至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少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％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％，优选至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少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％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％，优选至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少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％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％，优选至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少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％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％，优选至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至 少98％或至少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％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％，优选至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少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％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％，优选至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少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％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％，优选至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少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(优选核酸序列如SEQ ID NO:1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(优选核酸序列如SEQ ID NO:3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(优选核酸序列如SEQ ID NO:5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(优选核酸序列如SEQ ID NO:7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(优选核酸序列如SEQ ID NO:9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(优选核酸序列如SEQ ID NO:11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(优选核酸序列如SEQ ID NO:9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(优选核酸序列如SEQ ID NO:13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。更优选地，按照EU编号系统，所述抗CD73抗体的重链恒定区在ACCESSION:P01857所示序列的基础上具有如下突变：
L234A和L235A；或者
L234A和G237A；或者
L235A和G237A；
或者
L234A、L235A、G237A；
或选自如下的一个或多个突变：
N297A、D265A、D270A、P238D、L328E、E233D、H268D、P271G、A330R、C226S、C229S、E233P、P331S、S267E、L328F、A330L、M252Y、S254T、T256E、N297Q、P238S、P238A、A327Q、A327G、P329A、K322A、T394D、G236R、G236A、L328R、A330S、P331S、 H268A、E318A和K320A。最优选地，所述抗CD73抗体的重链恒定区氨基酸序列如SEQ ID NO:21所示，轻链恒定区氨基酸序列如SEQ ID NO:22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)，氨基酸序列如SEQ ID NO:21所示；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))、三阴性乳腺癌、卵巢癌、结直肠癌(包括微卫星稳定(MSS)和错配修复功能缺陷/微卫星高度不稳定(dMMR/MSI-H)型)、胃癌(包括微卫星稳定(MSS)和错配修复功能缺陷/微卫星高度不稳定(dMMR/MSI-H)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组合产品(例如试剂盒)，其包含独立包装的第一产品和第二产品，其中，
所述第一产品包含本发明中任一项所述的抗CD73抗体或其抗原结合片段、本发明的偶联物或者本发明中任一项所述的药物组合物；
所述第二产品包含抗PD-1-抗VEGFA双特异性抗体；
优选地，所述组合产品还包含独立包装的第三产品，所述第三产品包含一种或多种化疗药物，
优选地，所述第一产品和所述第二产品还独立地包含一种或多种药学上可接受的辅料；
优选地，所述组合产品还包含产品说明书，优选地，所述说明书中记载抗CD73抗体和/或抗PD-1-抗VEGFA双特异性抗体的单次给药剂量为每千克体重0.1-100mg，优选1-10mg；或者，抗CD73抗体和/或抗PD-1-抗VEGFA双特异性抗体的单次给药剂量为每位受试者10-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg或200mg，优选地，所述说明书中记载每天两次至约每隔一天一次给药抗CD73抗体和/或抗PD-1-抗VEGFA双特异性抗体，或每3天、4天、5天、6天、10天、1周、2周、3周、4周、5周或6周给药一次抗CD73抗体和/或抗PD-1-抗VEGFA双特异性抗体。
在本发明的一个或多个实施方案中，其中，按照抗体的质量计算，所述的试剂盒或药物组合物中抗CD73抗体或其抗原结合片段与抗PD-1-抗VEGFA双特异性抗体的质量比为(1：5)-(5：1)，例如：1：5、1：4、1：3、1：2、1：1、2：1、3：1、4：1或5：1。
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-抗VEGFA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-抗VEGFA双特异性抗体的重链氨基酸序列如SEQ ID NO:23所示，轻链氨基酸序列如SEQ ID NO:25所示。
本发明涉及一种预防和/或治疗肿瘤(特别是恶性肿瘤)的方法，包括给受试者施用治疗有效量的抗CD73抗体，联合施用抗PD-1-抗VEGFA双特异性抗体，更优选地，进一步联合施用一种或多种化疗药物(优选所述化疗药物为化疗剂或生长抑制剂，靶向治疗剂(例如抗体-药物缀合物、抗体或其抗原结合片段)，表达嵌合抗原受体的T细胞，血管生成抑制剂，抗肿瘤剂，癌症疫苗，佐剂及其组合，抗代谢药物，抗生素，植物类药物和/或激素类药物，优选环磷酰胺，培美曲塞，铂类药物如顺铂、卡铂、奥沙利铂，阿霉素类，紫杉醇，长春碱类，他莫昔芬，甲地孕酮，戈舍瑞林，门冬酰胺酶和/或氟尿嘧啶类抗肿瘤药)，优选地，所述抗CD73抗体、抗PD-1-抗VEGFA双特异性抗体和肿瘤化疗药物同时或顺序施用。
在本发明的一个或多个实施方案中，所述化疗剂或生长抑制剂选自烷化剂、蒽环类、抗激素剂(例如抗雄激素剂)、芳香酶抑制剂、蛋白激酶抑制剂(例如酪氨酸激酶抑制剂)、脂质激酶抑制剂、反义寡核苷酸、核酶、抗代谢物、拓扑异构酶抑制剂、细胞毒性剂或抗肿瘤抗生素、蛋白酶体抑制剂、抗微管剂、EGFR拮抗剂、VEGF拮抗剂、PD-1拮抗剂、血管生成素2拮抗剂、类视色素、组蛋白脱乙酰酶抑制剂及其组合。
在本发明的一个或多个实施方案中，所述靶向治疗剂选自B-raf抑制剂、MEK抑制剂、K-ras抑制剂、c-Met抑制剂、Alk抑制剂、磷脂酰肌醇3-激酶抑制剂、Akt抑制剂、mTOR抑制剂、VEGF抑制剂、 CD73抑制剂、PARP抑制剂、PD-1抑制剂、双磷脂酰肌醇3-激酶/mTOR抑制剂及其组合。
在本发明的一个或多个实施方案中，所述抗体-药物缀合物包含选自下组的药物：美登新碱、单甲基auristatin E、加利车霉素、esperamicin和放射性同位素螯合剂。
所述CD73抑制剂包括但不限于BMS-986179、MEDI9447、NZV930、CPI-006、AB680、LY-3475070、TJ004309[3]、ORIC-533、IPH5301AB680、LY-3475070中的一种或多种。
所述PARP抑制剂选自依托泊苷、奥拉帕利、芦卡帕利、尼拉帕利、他拉唑帕利、氟唑帕利、veliparib ER、ABT-472、ABT-767、Stenoparib、AST-6828、AG-PD、ANG-2864、ANG-3038、ANG-3186、AZD-5305、AZ-0108、AZD-2461、AMXI-5001、AMXI-2001、AMXI-3001、AMXI-7001、AMXI-9001、pamiparib、ZYTP-1、CK-102、XZ-120312、YHP-743、iobenguane I 131、rucaparib camsylate、CVL-218、CPH-101、CPH-102、CBX-11、CBX-15、minocycline、DB-207、DPS-102、E-7016、iobenguane I 131、MK-2512、HCX-014、HWH-340、IDX-1197、IDX-1197、senaparib、IMP-04100、IMP-04111、IMP-04149、IMP-04249、IMP-04307、IMP-04356、JPI-289、JPI-547、JPI-283、fluzoparib、GT-1620、iobenguane I 131、DR-2313、MP-124、H-10、NT-125、BGP-15、NMSP-293、NMSP-293、NMSP-118、NMSP-648、NMSP-914、DB-207、NUV-1156、NUV-1176、JPI-289、Stenoparib、OX-401、NU-1025、NU-1085、PLX-376、R-554、RBN-2397、RBN-012759、PJ-34、INO-1001、WW-46、BSI-401、iniparib、SOMCL-9112、SC-10914、HTMC-0435、SRX-3128、TSL-1502、PJ-34、CEP-8983、CK-102、THG-009、talazoparib SR、L-2286、mitoparib和WB-1340中的一种或多种。
在本发明的一个或多个实施方案中，所述肿瘤选自如下的一种或多种：
宫颈癌(如转移性宫颈癌)、子宫内膜癌、肺癌如小细胞肺癌和非小细胞肺癌(例如鳞状非小细胞肺癌或非鳞状非小细胞肺癌)、咽喉癌、食管癌、食管鳞癌、甲状腺癌、间皮瘤、胃肠道癌如胃癌((包括微卫星 稳定(MSS)和错配修复功能缺陷/微卫星高度不稳定(dMMR/MSI-H)型)，例如晚期胃癌、、胃腺癌或胃食管结合部腺癌)和肠癌(如直肠癌、结肠癌、结肠直肠癌(包括结卫星稳定(MSS)和错配修复功能缺陷/微卫星高度不稳定(dMMR/MSI-H)型))、肝癌(如肝细胞癌、肝胆癌)、胆道癌(如胆管癌和胆囊癌)、胰腺癌、胰脏癌、肾癌、卵巢癌(如晚期卵巢癌)、输卵管癌、肛管表皮样癌、腹膜癌、胶质瘤、神经胶质瘤、复发性胶质瘤、皮肤癌、黑色素瘤、血液恶性肿瘤(如白血病(如急性髓系白血病)、淋巴瘤(如霍奇金淋巴瘤、非霍奇金淋巴瘤)、多发性骨髓瘤、B淋巴细胞瘤(如浆细胞癌)、骨癌、肉瘤(如平滑肌肉瘤、横纹肌肉瘤)、骨肉瘤、软骨肉瘤、神经母细胞瘤、骨髓瘤、(如多发性骨髓瘤)大细胞神经内分泌癌、尿路上皮癌(例如上尿路上皮癌或膀胱癌)、前列腺癌(包括转移性去势抵抗前列腺癌(mCRPC))、睾丸癌、三阴性乳腺癌、外周T细胞淋巴瘤、鼻咽癌，高度微卫星不稳定型(MSI-H)或错配修复缺陷型(dMMR)实体瘤、头和颈癌、脑癌(如侵袭性脑癌)、鳞状细胞癌、基底细胞癌、腺瘤(如乳腺癌、胸腺癌、回盲部腺癌、壶腹部腺癌、胰管腺癌、粘液性或浆液性囊腺癌)、绒毛膜上皮癌、恶性葡萄胎、恶性支持细胞-间质细胞瘤、恶性颗粒细胞瘤、无性细胞瘤、胶质母细胞瘤、真菌病、默克尔细胞癌、肝内胆管癌、梅克尔细胞癌、鳞状细胞肛肠癌、舌鳞状细胞癌、头颈鳞状细胞癌和其他血液系统恶性肿瘤。
在本发明的一个或多个实施方案中，所述抗PD-1-抗-VEGFA双特异性抗体包括：
靶向PD-1的第一蛋白功能区，和
靶向VEGFA的第二蛋白功能区；
其中，所述第一蛋白功能区为单链抗体，所述第二蛋白功能区为免疫球蛋白；或者所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为单链抗体，
其中，
所述的免疫球蛋白的重链可变区包含：氨基酸序列如SEQ ID NO:27所示的重链可变区所包含的HCDR1-HCDR3(优选按照IMGT编号系统分别如SEQ ID NOs:31-33所示的HCDR1-HCDR3)，其轻 链可变区包含：氨基酸序列如SEQ ID NO:29所示的轻链可变区所包含的LCDR1-LCDR3(优选按照IMGT编号系统分别如SEQ ID NOs:34-36所示的LCDR1-LCDR3)；
所述的单链抗体的重链可变区包含：氨基酸序列如SEQ ID NO:37所示的重链可变区所包含的HCDR1-HCDR3(优选按照IMGT编号系统分别如SEQ ID NOs:41-43所示的HCDR1-HCDR3)并且其轻链可变区包含氨基酸序列如SEQ ID NO:39所示的轻链可变区所包含的LCDR1-LCDR3(优选按照IMGT编号系统分别如SEQ ID NOs:44-46所示的LCDR1-LCDR3)；
或者
所述的免疫球蛋白的重链可变区包含：氨基酸序列如SEQ ID NO:37所示的重链可变区所包含的HCDR1-HCDR3(优选按照IMGT编号系统分别如SEQ ID NOs:41-43所示的HCDR1-HCDR3)并且其轻链可变区包含氨基酸序列如SEQ ID NO:39所示的轻链可变区所包含的LCDR1-LCDR3(优选按照IMGT编号系统分别如SEQ ID NOs:44-46所示的LCDR1-LCDR3)；
所述的单链抗体的的重链可变区包含：氨基酸序列如SEQ ID NO:27所示的重链可变区所包含的HCDR1-HCDR3(优选按照IMGT编号系统分别如SEQ ID NOs:31-33所示的HCDR1-HCDR3)，其轻链可变区包含：氨基酸序列如SEQ ID NO:29所示的轻链可变区所包含的LCDR1-LCDR3(优选按照IMGT编号系统分别如SEQ ID NOs:34-36所示的LCDR1-LCDR3)；
优选地，所述免疫球蛋白为人IgG1亚型。
在本发明的一个或多个实施方案中，所述的双特异性抗体，其中，按照EU编号系统，所述免疫球蛋白的重链恒定区具有如下突变：
L234A和L235A；或者
L234A和G237A；或者
L235A和G237A；
或者
L234A、L235A、G237A。
本发明中，如果没有特别说明，位点之前的字母表示突变前的氨基酸，位点之后的字母表示突变后的氨基酸。
在本发明的一个或多个实施方案中，所述的双特异性抗体，其中，按照EU编号系统，所述免疫球蛋白的重链恒定区具有或还具有选自如下的一个或多个突变：
N297A、D265A、D270A、P238D、L328E、E233D、H268D、P271G、A330R、C226S、C229S、E233P、P331S、S267E、L328F、A330L、M252Y、S254T、T256E、N297Q、P238S、P238A、A327Q、A327G、P329A、K322A、T394D、G236R、G236A、L328R、A330S、P331S、H268A、E318A和K320A。
在本发明的一个或多个实施方案中，所述的双特异性抗体，其中，
所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:27所示；并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:29所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:37所示；并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:39所示。
在本发明的一个或多个实施方案中，所述双特异性抗体具有如SEQ ID NO:23所示的重链氨基酸序列，和如SEQ ID NO:25所示的轻链氨基酸序列。优选地，所述双特异性抗体的重链由SEQ ID NO:24所示的核苷酸序列编码，轻链由SEQ ID NO:26所示的氨基酸序列编码。
在本发明的一些实施方案中，所述的双特异性抗体，其中，所述单链抗体连接在免疫球蛋白的重链的C末端。由于免疫球蛋白由两条重链，因此，一个免疫球蛋白分子连接有两个单链抗体分子。优选地，两个单链抗体分子相同。
在本发明的一些实施方案中，所述的双特异性抗体，其中，所述单链抗体为两条，每条单链抗体的一端分别连接在免疫球蛋白的两条重链的C末端或N末端。
在本发明的一些实施方案中，所述单链抗体的VH与VL之间存在二硫键。在抗体的VH和VL之间引入二硫键的方法是本领域熟知的，例如可参见美国专利US5,747,654；Rajagopal et.al，Prot.Engin. 10(1997)1453-1459；Reiter et.al，Nat.Biotechnol.14(1996)1239-1245；Reiter et.al，Protein Engineering 8(1995)1323-1331；Webber et.al，Molecular Immunology 32(1995)249-258；Reiter et.al，Immunity2(1995)281-287；Reiter et.al，JBC 269(1994)18327-18331；Reiter et.al，Inter.J.of Cancer 58(1994)142-149；或者Reiter et.al，Cancer Res.54(1994)2714-2718；其通过引用并入本文。
在本发明的一个或多个实施方案中，所述的双特异性抗体，其中，所述第一蛋白功能区与所述第二蛋白功能区直接连接或者通过连接片段连接；和/或所述单链抗体的重链可变区与所述单链抗体的轻链可变区直接连接或者通过连接片段连接。
在本发明的一个或多个实施方案中，所述的双特异性抗体中的所述连接片段为(GGGGS)n，n为正整数；优选地，n为1、2、3、4、5或6。
在本发明的一个或多个实施方案中，在所述的双特异性抗体中，所述第一蛋白功能区和第二蛋白功能区独立地为1个或者2个以上。
在本发明的一个或多个实施方案中，所述的双特异性抗体，其中，所述单链抗体连接在免疫球蛋白的重链的C末端。
在本发明的一个或多个实施方案中，所述第一蛋白功能区与所述第二蛋白功能区通过第一连接片段连接；并且所述单链抗体的重链可变区与所述单链抗体的轻链可变区通过第二连接片段连接；所述第一连接片段和所述第二连接片段相同或不同；
优选地，所述第一连接片段和所述第二连接片段的氨基酸序列独立地选自SEQ ID NO:47和SEQ ID NO:48；
优选地，所述第一连接片段和所述第二连接片段的氨基酸序列均如SEQ ID NO:48所示。
在本发明的一个或多个实施方案中，所述双特异性抗体为单克隆抗体。
在本发明的一个或多个实施方案中，所述双特异性抗体为人源化抗体。
在本发明的另一个方面，涉及单位制剂，优选用于治疗肿瘤，其 中所述单位制剂包含1～10000mg(优选10-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg或200mg)的本发明任一方面所述的抗CD73抗体和1～10000mg(优选1-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg、200mg或100mg)的本发明任一方面所述的抗PD-1-抗VEGFA双特异性抗体，和任选地一种或多种本发明所述的化疗药(如铂类药物和/或氟尿嘧啶类抗肿瘤药)；其中，所述抗CD73抗体、所述抗PD-1-抗VEGFA双特异性抗体和化疗药分别单独包装。
本发明涉及用于预防或治疗癌症或肿瘤的方法，其中，向有需要的受试者给予一份或多份本发明所述的单位制剂，优选地，所述单位制剂中的抗PD-1-抗VEGFA双特异性抗体，抗CD73抗体和化疗药各自分开施用。
在本发明的另一个方面，涉及单次药物剂量单元，优选用于治疗肿瘤，其包含0.1-10000mg(优选1-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg、200mg或100mg)的本发明任一项所述的抗CD73抗体和0.1-10000mg(优选1-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg、200mg或100mg)的本发明任一项所述的抗PD-1-抗VEGFA双特异性抗体。
在本发明的一个或多个实施方案中，其中所述抗CD73抗体，所述VEGFA-抗PD-1双特异性抗体和/或所述化疗药为适于静脉注射或静脉滴注的形式，优选液体的形式。
在本发明的一个或多个实施方案中，其中给受试者施用有效量的本发明任一项所述抗CD73抗体和/或本发明任一项所述抗PD-1-抗VEGFA双特异性抗体的步骤为在手术治疗之前或之后，和/或在放射治疗之前或之后。
在本发明的一个或多个实施方案中，其中，本发明任一项所述抗CD73抗体和/或本发明任一项所述抗PD-1-抗VEGFA双特异性抗体的单次给药剂量为每千克体重0.1-100mg，优选1-10mg；或者，本发明任一项所述抗CD73抗体和/或本发明任一项所述抗PD-1-抗VEGFA双特异性抗体的单次给药剂量为每位受试者10-1000mg，优选50-500mg、 100-400mg、150-300mg、150-250mg或200mg，
优选地，每天两次至约每隔一天一次给药，或每3天、4天、5天、6天、10天、1周、2周、3周、4周、5周或6周给药一次；
优选地，给药方式为静脉滴注或静脉注射。
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EC50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(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL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。VH和VL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VH和VL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(VH和VL)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。
在已知抗体重链和轻链可变区序列的情况下，目前有几种确定抗体CDR区的方法，包括Kabat，IMGT，Chothia和AbM编号系统。然而，每种关于抗体或其变体的CDR的定义的应用都将在本文定义和使用的术语的范围内。如果给定该抗体的可变区氨基酸序列，则本领域技术人员通常可确定特定CDR，而不依赖于该序列自身之外的任何实验数据。
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(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(KD)结合该抗原。
如本文中所使用的，术语“KD”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(KD)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KD，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。
术语“单次药物剂量单元”表示在给药方案时刻，(优选按照受试者每kg体重)待给药于受试者的包含本发明所述的抗CD73抗体和抗PD-1-抗VEGFA双特异性抗体的单次药物剂型，如注射剂，例如置于安瓿瓶中。在本发明的具体实施方案中，给药方案例如包括根据每天两次至约每隔一天一次的给药周期来给药单次药物剂量单元，或者每3天、4天、5天、6天、10天、1周、2周、3周、4周、5周或6周给药一次。
在本发明中，如果没有特别说明，所述“第一”(例如第一蛋白功能区、第一连接片段)和“第二”(例如第二蛋白功能区、第二连接片段)是为了指代上的区分或表述上的清楚，并不具有典型的次序上的含义。
药物或治疗剂的“治疗有效量”或“治疗上有效的剂量”是当单独使用或与另一种治疗剂联合使用时保护主体免于疾病发作或促进疾病消退的药物的任何量，所述疾病消退通过疾病征状的严重程度的降低、无疾病征状阶段的频率和持续时间的增加、或由疾病折磨引起的损伤或失能的预防来证明。使用熟练的从业人员已知的多种方法可以评价治疗剂的促进疾病消退的能力，诸如在临床试验期间在人主体中，在预测对于人类的效力的动物模型系统中，或通过在体外测定法中测定所述药剂的活性。
药物的“预防有效量”是指，其为当单独地或与抗肿瘤剂联合施用给处于发生癌症的风险的主体(例如，具有恶化前病症的主体)或具有癌症复发的风险的主体时，抑制癌症的发生或复发的任何药物量。在某些实施方案中，预防有效量完全阻止癌症的发生或复发。“抑制”癌症的发生或复发是指减少癌症的发生或复发的可能性，或完全阻止癌症的发生或复发。
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-抗VEGFA双特异性抗体联用具有有效抑制肿瘤生长的药理学效果， 优于抗VEGFA/PD-1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.抗CD73抗体有效抑制CD73酶学活性。
图8. 19F3H2L3(hG1DM)抗体与抗PD-1-抗VEGFA双特异抗体VP101(hG1DM)联用对B6-hPD1/hPDL1/hCD73小鼠MC38-hPDL1/hCD73肿瘤模型效果。
图9. 19F3H2L3(hG1DM)抗体与抗PD-1-抗VEGFA双特异抗体VP101(hG1DM)联用对B6-hPD1/hPDL1/hCD73小鼠MC38-hPDL1/hCD73肿瘤模型体重变化。
图10.抗CD73特异性抗体联合抗PD-1-抗VEGFA双特异性抗体促细胞因子IFN-γ的分泌。
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，所用的联用抗PD-1-抗VEGFA双特异性抗体VP101(hG1DM)，生产自中山康方生物医药有限公司，其序列和制备参见公开专利CN112830972A中所述的抗体VP101(hG1DM)，其中VP101(hG1DM)重链氨基酸全长序列如SEQ ID NOs:23所示，轻链氨基酸全长序列如SEQ ID NOs:25所示。VP101(hG1DM)结构为IgG-scFv，其中IgG部分为抗VEGFA抗体，scFv部分为抗PD-1抗体，
抗VEGFA抗体的HCDR1序列如SEQ ID NO:31所示，HCDR2序列如SEQ ID NO:32所示，HCDR3序列如SEQ ID NO:33所示，VH序列如SEQ ID NO:27所示，抗VEGFA抗体的LCDR1序列如SEQ ID NO:34所示，LCDR2序列如SEQ ID NO:35所示，LCDR3序列如SEQ ID NO:36所示，VL序列如SEQ ID NO:29所示，
其中抗PD1抗体的HCDR1序列如SEQ ID NO:41所示，HCDR2序列如SEQ ID NO:42所示，HCDR3序列如SEQ ID NO:43所示，VH序列如SEQ ID NO:37所示，抗PD1抗体的LCDR1序列如SEQ ID NO:44所示，LCDR2序列如SEQ ID NO:45所示，LCDR3序列如SEQ ID NO:46所示，VL序列如SEQ ID NO:39所示。
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)，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％CO2，37℃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 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 Acqμisition7.0，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。

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。

KD＝kdis/kon
实施例5：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*104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(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7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。
实施例6：抗CD73特异性抗体联合抗PD-1-抗VEGFA双特异性抗体在小鼠肿瘤细胞皮下移植模型中的药效学评价
为检测抗CD73特异性抗体19F3H2L3(hG1DM)与抗PD-1-抗VEGFA双特异抗体VP101(hG1DM)联用的体内抑瘤活性，首先用MC38-hPDL1/hCD73细胞(购自江苏集萃药康生物科技股份有限公司)，接种于6.7-7.7周龄的雌性B6-hPD1/hPDL1/hCD73小鼠(购自江苏集萃药康生物科技股份有限公司)皮下，当平均肿瘤体积达到80-120mm3时，小鼠根据肿瘤体积随机分成4组，每组6只。分组当天定义为D0天，并于分组当天D0天开始给药。联合给药组给药方式为：药物单独配制，先 给药抗PD-1-抗VEGFA双特异抗体VP101(hG1DM)，2h后再给药抗CD73特异性抗体19F3H2L3(hG1DM)。造模与具体给药方式见表3。给药后测量各组肿瘤的长宽，计算肿瘤体积。
表3：19F3H2L3(hG1DM)抗体与抗PD-1-抗VEGFA双特异抗体VP101(hG1DM)联用治疗MC38-hPDL1/hCD73移植瘤
B6-hPD1/hPDL1/hCD73小鼠模型的给药方案
结果图8所示。结果表明：相比同型对照抗体，19F3H2L3(hG1DM) 抗体与抗PD-1-抗VEGFA双特异抗体VP101(hG1DM)均能有效抑制小鼠肿瘤的生长，且19F3H2L3(hG1DM)+VP101(hG1DM)联用组对此模型展现联合抗肿瘤药效，其联合对肿瘤的抑制优于受试药单用组。
此外，如图9所示，荷瘤鼠对受试药19F3H2L3(hG1DM)和VP101(hG1DM)单用和联用的耐受性均良好，各组对荷瘤小鼠体重无影响。
实施例7：混合淋巴细胞反应检测抗CD73特异性抗体联合抗PD-1-抗VEGFA双特异性抗体促细胞因子分泌
在本发明中，PDL1的GenBank登记号为NP_054862.1，PDL1FL表示PDL1基因全长。
人PD-L1过表达慢病毒载体plenti6.3/V5-PDL1FL-BSD(plenti6.3/V5-BSD购自Invitrogen，产品编号：K5315-20)经病毒包装后感染Raji细胞，经BSD(Blasticidin，Gibco货号：R210-01)加药筛选后，获得Raji-PDL1细胞株。
按分离液Ficoll-PaqueTM Plus操作说明书分离获得正常人PBMC。实验前两天，复苏PBMC，完全培养基(1640+10％FBS)培养于37℃，5％二氧化碳培养箱。2h后，待PBMC状态恢复，添加SEB(终浓度0.5μg/mL，Toxin technology，货号：BT202)刺激两天。实验当天，收集Raji-PDL1细胞(康方生物构建)，离心重悬计数(用分析培养基1640+10％FBS重悬)，调整细胞密度至2*106/mL，加入MMC(Mito-mycin C，终浓度为2μg/mL，Stressmarq，货号：SIH-246-10MG)于37℃，5％二氧化碳培养箱处理1小时，常规收集A549肺癌细胞(购自中国科学院)，离心重悬计数。收集SEB刺激两天后的PBMC以及MMC处理后的Raji-PDL1细胞，按PBMC 1*105/孔、Raji-PDL1 1*105/孔、A549 1*104/孔接种到96孔U型板中(Corning，货号：3799)；按实验设计加入AMP(腺苷-5'-单磷酸，TCI，货号：A0158)和抗体，并设置阴性对照(PBMC+Raji-PDL1+A549+AMP)和同型对照(hIgG1和hIgGDM)，共培养3天(体系终体积为200μL)。3天后，250xg离心5min(Beckman离心机)，收集细胞上清，通过达科为试剂盒检测IFN-γ含量。
结果如图10所示。结果表明：相比同型对照抗体，抗PD-1-抗VEGFA双特异抗体VP101(hG1DM)能有效促进混合淋巴体系中细胞因子IFN-γ的分泌，且19F3H2L3(hG1DM)+VP101(hG1DM)联用组对此展现联合抗肿瘤药效，其联合促细胞因子IFN-γ的分泌要优于各单药组，且150nM的19F3H2L3(hG1DM)+VP101(hG1DM)联用活性优于300nM 19F3H2L3(hG1DM)或者VP101(hG1DM)单药活性。
序列表
19F3重链可变区的核酸序列：(SEQ ID NO:1)
19F3重链可变区的氨基酸序列：(SEQ ID NO:2)
19F3轻链可变区的核酸序列：(SEQ ID NO:3)
19F3轻链可变区的氨基酸序列：(SEQ ID NO:4)
19F3H1L1(hG1DM)重链可变区的核酸序列：(SEQ ID NO:5)
19F3H1L1(hG1DM)重链可变区的氨基酸序列：(SEQ ID NO:6)
19F3H1L1(hG1DM)轻链可变区的核酸序列：(SEQ ID NO:7)
19F3H1L1(hG1DM)轻链可变区的氨基酸序列：(SEQ ID NO:8)
19F3H2L2(hG1DM)及19F3H2L3(hG1DM)重链可变区的核酸序列：(SEQ ID NO:9)

19F3H2L2(hG1DM)及19F3H2L3(hG1DM)重链可变区的氨基酸序列：(SEQ ID NO:10)
19F3H2L2(hG1DM)轻链可变区的核酸序列：(SEQ ID NO:11)
19F3H2L2(hG1DM)轻链可变区的氨基酸序列：(SEQ ID NO:12)
19F3H2L3(hG1DM)轻链可变区的核酸序列：(SEQ ID NO:13)

19F3H2L3(hG1DM)轻链可变区的氨基酸序列：(SEQ ID NO:14)
19F3和19F3H1L1(hG1DM)、19F3H2L2(hG1DM)、19F3H2L3(hG1DM)的CDR区
HCDR1:GYSFTGYT(SEQ ID NO:15)
HCDR2:INPYNAGT(SEQ ID NO:16)
HCDR3:ARSEYRYGGDYFDY(SEQ ID NO:17)
LCDR1:QSLLNSSNQKNY(SEQ ID NO:18)
LCDR2:FAS(SEQ ID NO:19)
LCDR3:QQHYDTPYT(SEQ ID NO:20)
19F3H1L1(hG1DM)、19F3H2L2(hG1DM)和19F3H2L3(hG1DM)的重链恒定区序列(330aa，突变位点用下划线标出)：
19F3H1L1(hG1DM)、19F3H2L2(hG1DM)和19F3H2L3(hG1DM)的轻链恒定区序列(107aa)：
VP101(hG1DM)的重链氨基酸序列
VP101(hG1DM)的重链核酸序列

VP101(hG1DM)的轻链氨基酸序列
VP101(hG1DM)的轻链核酸序列
Bevacizumab重链可变区(Bevacizumab-Hv)的氨基酸序列：(123aa)
编码Bevacizumab重链可变区的核酸序列：(369bp)

Bevacizumab轻链可变区(Bevacizumab-Lv)的氨基酸序列：(107aa)
编码Bevacizumab轻链可变区的核酸序列：(321bp)
Bevacizumab重链可变区的3个CDR区的氨基酸序列如下：
HCDR1:GYTFTNYG(SEQ ID NO:31)
HCDR2:INTYTGEP(SEQ ID NO:32)
HCDR3:AKYPHYYGSSHWYFDV(SEQ ID NO:33)
Bevacizumab轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1:QDISNY(SEQ ID NO:34)
LCDR2:FTS(SEQ ID NO:35)
LCDR3:QQYSTVPWT(SEQ ID NO:36)
14C12H1L1(M)重链可变区的氨基酸序列如SEQ ID NO:37所示。
编码14C12H1L1重链可变区的核酸序列：(354bp)
14C12H1L1(M)轻链可变区的氨基酸序列
14C12H1L1(M)的轻链可变区14C12L1(M)的核酸序列：
14C12H1L1(M)的重链可变区的CDR:
HCDR1:GFAFSSYD(SEQ ID NO:41)
HCDR2:ISGGGRYT(SEQ ID NO:42)
HCDR3:ANRYGEAWFAY(SEQ ID NO:43)
14C12H1L1(M)的轻链可变区的CDR：
LCDR1:QDINTY(SEQ ID NO:44)
LCDR2:RAN(SEQ ID NO:45)
LCDR3:LQYDEFPLT(SEQ ID NO:46)
第一连接片段氨基酸序列
第二连接片段氨基酸序列

Claims (9)

  1. 药物组合物，其包含抗CD73(例如人CD73)抗体或其抗原结合片段，和抗PD-1-抗VEGFA双特异性抗体或其抗原结合片段，可选地，所述药物组合物还包含药学上可接受的载体和/或赋形剂，
    其中，所述抗CD73抗体包含：
    SEQ ID NO:2所示的重链可变区包含的HCDR1，HCDR2和HCDR3；和SEQ ID NO:4所示的轻链可变区包含的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％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％，优选至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的序列，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％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％，优选至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的序列或由其组成，
    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％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％，优选至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的序列，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％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％，优选至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的序列，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％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％，优选至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的序列，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％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％，优选至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的序列，或由其组成；
    所述抗PD-1-抗VEGFA双特异性抗体包含：
    靶向PD-1的第一蛋白功能区，和
    靶向VEGFA的第二蛋白功能区；
    其中，所述第一蛋白功能区为单链抗体，所述第二蛋白功能区为免疫球蛋白；或者所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为单链抗体，
    其中，
    所述的免疫球蛋白的重链可变区包含：氨基酸序列如SEQ ID NO:27所示的重链可变区所包含的HCDR1-HCDR3(优选按照IMGT编号系统分别如SEQ ID NOs:31-33所示的HCDR1－HCDR3)，其轻链可变区包含：氨基酸序列如SEQ ID NO:29所示的轻链可变区所包含的LCDR1-LCDR3(优选按照IMGT编号系统分别如SEQ ID NOs:34－36所示的LCDR1－LCDR3)；
    所述的单链抗体的重链可变区包含：氨基酸序列如SEQ ID NO:37所示的重链可变区所包含的HCDR1-HCDR3(优选按照IMGT编号系统分别如SEQ ID NOs:41-43所示的HCDR1－HCDR3)并且其轻链可变区包含氨基酸序列如SEQ ID NO:39所示的轻链可变区所包含的LCDR1-LCDR3(优选按照IMGT编号系统分别如SEQ ID NOs:44-46所示的LCDR1－LCDR3)；
    或者
    所述的免疫球蛋白的重链可变区包含：氨基酸序列如SEQ ID NO:37所示的重链可变区所包含的HCDR1-HCDR3(优选按照IMGT编号系统分别如SEQ ID NOs:41-43所示的HCDR1－HCDR3)并且其轻链可变区包含氨基酸序列如SEQ ID NO:39所示的轻链可变区所包含的LCDR1-LCDR3(优选按照IMGT编号系统分别如SEQ ID NOs:44-46所示的LCDR1－LCDR3)；
    所述的单链抗体的的重链可变区包含：氨基酸序列如SEQ ID NO:27所示的重链可变区所包含的HCDR1-HCDR3(优选按照IMGT编号系统分别如SEQ ID NOs:31-33所示的HCDR1－HCDR3)，其轻链可变区包含：氨基酸序列如SEQ ID NO:29所示的轻链可变区所包含的LCDR1-LCDR3(优选按照IMGT编号系统分别如SEQ ID NOs:34－36所示的LCDR1－LCDR3)。
  2. 权利要求1所述的药物组合物，其中所述抗CD73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％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％，优选至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的序列；并且
    所述抗CD73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％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％，优选至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的序列，
    优选地
    所述抗CD73抗体的重链可变区的氨基酸序列如SEQ ID NO:2所 示(优选核酸序列如SEQ ID NO:1所示)，并且所述抗CD73抗体的轻链可变区的氨基酸序列如SEQ ID NO:4所示(优选核酸序列如SEQ ID NO:3所示)；
    所述抗CD73抗体的重链可变区的氨基酸序列如SEQ ID NO:6所示(优选核酸序列如SEQ ID NO:5所示)，并且所述抗CD73抗体的轻链可变区的氨基酸序列如SEQ ID NO:8所示(优选核酸序列如SEQ ID NO:7所示)；
    所述抗CD73抗体的重链可变区的氨基酸序列如SEQ ID NO:10所示(优选核酸序列如SEQ ID NO:9所示)，并且所述抗CD73抗体的轻链可变区的氨基酸序列如SEQ ID NO:12所示(优选核酸序列如SEQ ID NO:11所示)；或
    所述抗CD73抗体的重链可变区的氨基酸序列如SEQ ID NO:10所示(优选核酸序列如SEQ ID NO:9所示)，并且所述抗CD73抗体的轻链可变区的氨基酸序列如SEQ ID NO:14所示(优选核酸序列如SEQ ID NO:13所示)，
    优选地，其中所述抗CD73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，更优选地，按照EU编号系统，所述抗CD73抗体的重链恒定区在ACCESSION:P01857所示序列基础上具有如下突变：
    L234A和L235A；或者
    L234A和G237A；或者
    L235A和G237A；
    或者
    L234A、L235A、G237A
    或选自如下的一个或多个突变：
    N297A、D265A、D270A、P238D、L328E、E233D、H268D、P271G、A330R、C226S、C229S、E233P、P331S、S267E、L328F、A330L、M252Y、S254T、T256E、N297Q、P238S、P238A、A327Q、A327G、P329A、K322A、T394D、G236R、G236A、L328R、A330S、P331S、H268A、E318A和K320A；最优选地，所述抗CD73抗体的重链恒定 区氨基酸序列如SEQ ID NO:21所示，轻链恒定区氨基酸序列如SEQ ID NO:22所示；
    优选地，其中所述抗CD73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)，
    更优选地，所述抗CD73抗体是由杂交瘤细胞株LT014产生的抗体，所述杂交瘤细胞株保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2018137。
  3. 权利要求1所述的药物组合物，其中所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:27所示；并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:29所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:37所示；并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:39所示，或者所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:37所示；并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:39所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:27所示；并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:29所示，
    优选地，所述的双特异性抗体，其中，按照EU编号系统，所述免疫球蛋白的重链恒定区具有如下突变：
    L234A和L235A；或者
    L234A和G237A；或者
    L235A和G237A；
    或者
    L234A、L235A、G237A，更优选地，所述的双特异性抗体，其中，按照EU编号系统，所述免疫球蛋白的重链恒定区具有或还具有选自如下的一个或多个突变：
    N297A、D265A、D270A、P238D、L328E、E233D、H268D、P271G、A330R、C226S、C229S、E233P、P331S、S267E、L328F、A330L、M252Y、S254T、T256E、N297Q、P238S、P238A、A327Q、A327G、P329A、K322A、T394D、G236R、G236A、L328R、A330S、P331S、H268A、E318A和K320A，
    优选地，所述免疫球蛋白为人IgG1亚型，
    优选地，所述抗PD-1-抗VEGFA双特异性抗体具有如SEQ ID NO:23所示的重链氨基酸序列，和如SEQ ID NO:25所示的轻链氨基酸序列，
    优选地，所述抗PD-1-抗VEGFA双特异性抗体的重链由SEQ ID NO:24所示的核苷酸序列编码，轻链由SEQ ID NO:26所示的氨基酸序列编码，
    优选地，在所述抗PD-1-抗VEGFA双特异性抗体中，所述单链抗体连接在免疫球蛋白的重链的C末端，优选一个免疫球蛋白分子连接有两个单链抗体分子，更优选地，两个单链抗体分子相同，
    优选地，在所述抗PD-1-抗VEGFA双特异性抗体中，所述单链抗体为两条，每条单链抗体的一端分别连接在免疫球蛋白的两条重链的C末端或N末端，更优选地，所述单链抗体连接在免疫球蛋白的重链的C末端，
    优选地，在所述抗PD-1-抗VEGFA双特异性抗体中，所述第一蛋白功能区与所述第二蛋白功能区直接连接或者通过连接片段连接；和/或所述单链抗体的重链可变区与所述单链抗体的轻链可变区直接连接或者通过连接片段连接，
    优选地，所述连接片段为(GGGGS)n，n为正整数；优选地，n为1、2、3、4、5或6，
    优选地，所述第一蛋白功能区与所述第二蛋白功能区通过第一连接片段连接；并且所述单链抗体的重链可变区与所述单链抗体的轻链可变区通过第二连接片段连接；所述第一连接片段和所述第二连接片段相同或不同；
    优选地，所述第一连接片段和所述第二连接片段的氨基酸序列独立地选自SEQ ID NO:47和SEQ ID NO:48；
    优选地，所述第一连接片段和所述第二连接片段的氨基酸序列均如SEQ ID NO:48所示，
    优选地，在所述的双特异性抗体中，所述第一蛋白功能区和第二蛋白功能区独立地为1个、或者2个以上，
    优选地，所述抗PD-1-抗VEGFA双特异性抗体为单克隆抗体或人源化抗体。
  4. 权利要求1-3任一项所述的药物组合物，其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。
  5. 组合产品(例如试剂盒)，其包含独立包装的第一产品和第二产品，其中，
    所述第一产品包含权利要求1-3任一项中所定义的抗CD73抗体或其抗原结合片段；
    所述第二产品包含权利要求1-3任一项中所定义的抗PD-1-抗VEGFA双特异性抗体；
    优选地，所述组合产品还包含独立包装的第三产品，所述第三产品包含一种或多种化疗药物，
    优选地，所述第一产品和所述第二产品还独立地包含一种或多种药学上可接受的辅料；
    优选地，所述组合产品还包含产品说明书，优选地，所述说明书中记载权利要求1-3任一项所定义的抗CD73抗体和/或抗PD-1-抗VEGFA双特异性抗体的单次给药剂量为每千克体重0.1-100mg，优选1-10mg；或者，权利要求1-3任一项所定义的抗CD73抗体和/或抗PD-1-抗VEGFA双特异性抗体的单次给药剂量为每位受试者10-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg或200mg，优选地，所述说明书中记载每天两次至约每隔一天一次给药抗CD73抗体和/或抗PD-1-抗VEGFA双特异性抗体，或每3天、4天、5天、6天、10天、1周、2周、3周、4周、5周或6周给药一次抗CD73抗体和/或抗PD-1-抗VEGFA双特异性抗体，
    优选地，在所述组合产品中，按照抗体的质量计算，抗CD73抗体或其抗原结合片段与抗PD-1-抗VEGFA双特异性抗体的质量比为(1：5)-(5：1)，例如：1：5、1：4、1：3、1：2、1：1、2：1、3：1、4：1或5：1，
    优选地，其中所述抗CD73抗体，所述抗PD-1-抗VEGFA-双特异 性抗体和/或所述化疗药物为适于皮下注射、皮内注射、静脉内注射、肌内注射或病灶内注射施用的形式，优选为适于静脉注射或静脉滴注的形式，优选液体的形式。
  6. 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1-3任一项中所定义的抗CD73抗体或其抗原结合片段，药物B包含权利要求1-3任一项中所定义的抗PD-1-抗VEGFA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，所述方法进一步联合施用一种或多种化疗药物(优选所述化疗药物为化疗剂或生长抑制剂，靶向治疗剂(例如抗体-药物缀合物、抗体或其抗原结合片段)，表达嵌合抗原受体的T细胞、血管生成抑制剂、抗肿瘤剂、癌症疫苗、佐剂及其组合、烷化剂，抗代谢药物，抗生素，植物类药物和/或激素类药物，优选环磷酰胺，培美曲塞，铂类药物如顺铂、卡铂、奥沙利铂，阿霉素类，紫杉醇，长春碱类，他莫昔芬，甲地孕酮，戈舍瑞林，门冬酰胺酶和/或氟尿嘧啶类抗肿瘤药)，优选地，所述抗CD73抗体、抗PD-1-抗VEGFA双特异性抗体和化疗药物同时或顺序施用，更优选地，所述抗CD73抗体、抗PD-1-抗VEGFA双特异性抗体为在手术治疗之前或之后，和/或在放射治疗之前或之后施用，
    优选地，所述化疗剂或生长抑制剂选自烷化剂、蒽环类、抗激素剂(如抗雄激素剂)、芳香酶抑制剂、蛋白激酶抑制剂(如酪氨酸激酶抑制剂)、脂质激酶抑制剂、反义寡核苷酸、核酶、抗代谢物、拓扑异构酶抑制剂、细胞毒性剂或抗肿瘤抗生素、蛋白酶体抑制剂、抗微管剂、EGFR拮抗剂、VEGF拮抗剂、PD-1拮抗剂、血管生成素2拮抗剂、类视色素、组蛋白脱乙酰酶抑制剂及其组合，
    优选地，所述靶向治疗剂选自B-raf抑制剂、MEK抑制剂、K-ras抑制剂、c-Met抑制剂、Alk抑制剂、磷脂酰肌醇3-激酶抑制剂、Akt抑制剂、mTOR抑制剂、VEGF抑制剂、CD73抑制剂、PARP抑制剂、PD-1抑制剂、双磷脂酰肌醇3-激酶/mTOR抑制剂及其组合，
    优选地，所述抗体-药物缀合物包含选自下组的药物：美登新碱、单甲基auristatin E、加利车霉素、esperamicin和放射性同位素螯合剂，
    优选地，所述CD73抑制剂包括但不限于BMS-986179、MEDI9447、NZV930、CPI-006、AB680、LY-3475070、TJ004309[3]、ORIC-533、IPH5301AB680、LY-3475070中的一种或多种，
    优选地，所述PARP抑制剂选自依托泊苷、奥拉帕利、芦卡帕利、尼拉帕利、他拉唑帕利、氟唑帕利、veliparib ER、ABT-472、ABT-767、Stenoparib、AST-6828、AG-PD、ANG-2864、ANG-3038、ANG-3186、AZD-5305、AZ-0108、AZD-2461、AMXI-5001、AMXI-2001、AMXI-3001、AMXI-7001、AMXI-9001、pamiparib、ZYTP-1、CK-102、XZ-120312、YHP-743、iobenguane I 131、rucaparib camsylate、CVL-218、CPH-101、CPH-102、CBX-11、CBX-15、minocycline、DB-207、DPS-102、E-7016、iobenguane I 131、MK-2512、HCX-014、HWH-340、IDX-1197、IDX-1197、senaparib、IMP-04100、IMP-04111、IMP-04149、IMP-04249、IMP-04307、IMP-04356、JPI-289、JPI-547、JPI-283、fluzoparib、GT-1620、iobenguane I 131、DR-2313、MP-124、H-10、NT-125、BGP-15、NMSP-293、NMSP-293、NMSP-118、NMSP-648、NMSP-914、DB-207、NUV-1156、NUV-1176、JPI-289、Stenoparib、OX-401、NU-1025、NU-1085、PLX-376、R-554、RBN-2397、RBN-012759、PJ-34、INO-1001、WW-46、BSI-401、iniparib、SOMCL-9112、SC-10914、HTMC-0435、SRX-3128、TSL-1502、PJ-34、CEP-8983、CK-102、THG-009、talazoparib SR、L-2286、mitoparib和WB-1340中的一种或多种。
  7. 单位制剂，优选用于治疗肿瘤，其中所述单位制剂包含1～10000mg(优选10-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg或200mg)的权利要求1-3任一项所定义的抗CD73抗体和1～10000mg(优选1-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg、200mg或100mg)的权利要求1-3任一项所定义的抗PD-1-抗VEGFA双特异性抗体，和任选地一种或多种权利要求6所定义的化疗药物(如铂类药物和/或氟尿嘧啶类抗肿瘤药)；其中，所述抗CD73抗体、所述抗PD-1-抗VEGFA双特异性抗体和化疗药物分别单独包装，
    优选地，权利要求1-3任一项所定义的抗CD73抗体和/或抗PD-1-抗VEGFA双特异性抗体的单次给药剂量为每千克体重0.1-100mg，优选1-10mg；或者，权利要求1-3任一项所定义的抗CD73抗体和/或抗PD-1-抗VEGFA双特异性抗体的单次给药剂量为每位受试者10-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg或200mg，
    优选地，每天两次至约每隔一天一次给药，或每3天、4天、5天、6天、10天、1周、2周、3周、4周、5周或6周给药一次。
  8. 单次药物剂量单元，优选用于治疗肿瘤，其包含0.1-10000mg(优选1-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg、200mg或100mg)的权利要求1-3任一项所定义的抗CD73抗体和0.1-10000mg(优选1-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg、200mg或100mg)的权利要求1-3任一项所定义的抗PD-1-抗VEGFA双特异性抗体。
  9. 权利要求6所述的方法，权利要求7所述的单位制剂或权利要求8所述的单次药物剂量单元，其中所述肿瘤选自如下的一种或多种：
    宫颈癌(如转移性宫颈癌)、子宫内膜癌、肺癌如小细胞肺癌和非小细胞肺癌(例如鳞状非小细胞肺癌或非鳞状非小细胞肺癌)、咽喉癌、食管癌、食管鳞癌、甲状腺癌、间皮瘤、胃肠道癌如胃癌((包括微卫星稳定(MSS)和错配修复功能缺陷/微卫星高度不稳定(dMMR/MSI-H)型)，例如晚期胃癌、胃腺癌或胃食管结合部腺癌)和肠癌(如直肠癌、结肠癌、结肠直肠癌(包括微卫星稳定(MSS)和错配修复功能缺陷/微卫星高度不稳定(dMMR/MSI-H)型))、肝癌(如肝细胞癌、肝胆癌)、胆道癌(如胆管癌和胆囊癌)、胰腺癌、胰脏癌、肾癌、卵巢癌(如晚期卵巢癌)、输卵管癌、肛管表皮样癌、腹膜癌、胶质瘤、神经胶质瘤、复发性胶质瘤、皮肤癌、黑色素瘤、血液恶性肿瘤(如白血病(如急性髓系白血病)、淋巴瘤(如霍奇金淋巴瘤、非霍奇金淋巴瘤)、多发性骨髓瘤、B淋巴细胞瘤(如浆细胞癌)、骨癌、肉瘤(如平滑肌肉瘤、横纹肌肉瘤)、骨肉瘤、软骨肉瘤、神经母细胞瘤、骨髓瘤(如多发性骨髓瘤)、大细胞神经内分泌癌、尿路上皮癌(例如上尿路上皮癌或膀胱癌)、前列 腺癌(包括转移性去势抵抗前列腺癌(mCRPC))、睾丸癌、三阴性乳腺癌、外周T细胞淋巴瘤、鼻咽癌，高度微卫星不稳定型(MSI-H)或错配修复缺陷型(dMMR)实体瘤、头和颈癌、脑癌(如侵袭性脑癌)、鳞状细胞癌、基底细胞癌、腺瘤(如乳腺癌、胸腺癌、回盲部腺癌、壶腹部腺癌、胰管腺癌、粘液性或浆液性囊腺癌)、绒毛膜上皮癌、恶性葡萄胎、恶性支持细胞-间质细胞瘤、恶性颗粒细胞瘤、无性细胞瘤、胶质母细胞瘤、真菌病、默克尔细胞癌、肝内胆管癌、梅克尔细胞癌、鳞状细胞肛肠癌、舌鳞状细胞癌、头颈鳞状细胞癌和其他血液系统恶性肿瘤。
PCT/CN2023/087998 2022-06-22 2023-04-13 药物组合物及其用途 WO2023246247A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP23745372.5A EP4321537A1 (en) 2022-06-22 2023-04-13 Pharmaceutical composition and use thereof
CA3208473A CA3208473A1 (en) 2022-06-22 2023-04-13 Pharmaceutical composition and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210712836 2022-06-22
CN202210712836.9 2022-06-22

Publications (1)

Publication Number Publication Date
WO2023246247A1 true WO2023246247A1 (zh) 2023-12-28

Family

ID=89218472

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/087998 WO2023246247A1 (zh) 2022-06-22 2023-04-13 药物组合物及其用途

Country Status (4)

Country Link
EP (1) EP4321537A1 (zh)
CN (1) CN117257934A (zh)
CA (1) CA3208473A1 (zh)
WO (1) WO2023246247A1 (zh)

Citations (11)

* 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
US5747654A (en) 1993-06-14 1998-05-05 The United States Of America As Represented By The Department Of Health And Human Services Recombinant disulfide-stabilized polypeptide fragments having binding specificity
WO2017019896A1 (en) * 2015-07-29 2017-02-02 Novartis Ag Combination therapies comprising antibody molecules to pd-1
WO2019173692A2 (en) * 2018-03-09 2019-09-12 Agenus Inc. Anti-cd73 antibodies and methods of use thereof
CN110498857A (zh) * 2019-08-09 2019-11-26 安徽瀚海博兴生物技术有限公司 一种新型结构的抗vegf-抗pd1双特异性抗体
CN110960543A (zh) * 2019-12-10 2020-04-07 安徽瀚海博兴生物技术有限公司 一种联合抗vegf-抗pd1双特异性抗体用于制备抗癌药物
CN111712522A (zh) * 2018-02-11 2020-09-25 北京韩美药品有限公司 抗pd-1/抗vegf天然抗体结构样异源二聚体形式双特异抗体及其制备
CN112830972A (zh) 2019-11-25 2021-05-25 中山康方生物医药有限公司 抗pd-1-抗vegfa的双特异性抗体、其药物组合物及其用途
CN113214400A (zh) * 2020-01-21 2021-08-06 甫康（上海）健康科技有限责任公司 一种双特异性抗pd-l1/vegf抗体及其用途
CN113527501A (zh) * 2020-04-22 2021-10-22 中山康方生物医药有限公司 抗cd73-抗pd-1双特异性抗体及其用途
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

Patent Citations (11)

* 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
US5747654A (en) 1993-06-14 1998-05-05 The United States Of America As Represented By The Department Of Health And Human Services Recombinant disulfide-stabilized polypeptide fragments having binding specificity
WO2017019896A1 (en) * 2015-07-29 2017-02-02 Novartis Ag Combination therapies comprising antibody molecules to pd-1
CN111712522A (zh) * 2018-02-11 2020-09-25 北京韩美药品有限公司 抗pd-1/抗vegf天然抗体结构样异源二聚体形式双特异抗体及其制备
WO2019173692A2 (en) * 2018-03-09 2019-09-12 Agenus Inc. Anti-cd73 antibodies and methods of use thereof
CN110498857A (zh) * 2019-08-09 2019-11-26 安徽瀚海博兴生物技术有限公司 一种新型结构的抗vegf-抗pd1双特异性抗体
CN112830972A (zh) 2019-11-25 2021-05-25 中山康方生物医药有限公司 抗pd-1-抗vegfa的双特异性抗体、其药物组合物及其用途
CN110960543A (zh) * 2019-12-10 2020-04-07 安徽瀚海博兴生物技术有限公司 一种联合抗vegf-抗pd1双特异性抗体用于制备抗癌药物
CN113214400A (zh) * 2020-01-21 2021-08-06 甫康（上海）健康科技有限责任公司 一种双特异性抗pd-l1/vegf抗体及其用途
CN113527501A (zh) * 2020-04-22 2021-10-22 中山康方生物医药有限公司 抗cd73-抗pd-1双特异性抗体及其用途
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 (31)

* Cited by examiner, † Cited by third party
Title
"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
ANTONIOLI L ET AL., NAT REV CANCER, vol. 13, 2013, pages 842 - 857
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
DONG HONGCHAO ET AL., JOURNAL OF MODERN ONCOLOGY, vol. 22, no. 9, September 2014 (2014-09-01), pages 2231 - 3
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, 2009
HAGE TREINEMER PSEBALD W.: "Crystals of a 1:1 complex between human interleukin-4 and the extracellular domain of its receptor alpha chain", EURJ BIOCHEM., 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
HOMET 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., N ENGL J MED., vol. 366, 2012, pages 2455 - 2465
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, NIH PUBLICATION
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
MA LI, CHINESE JOURNAL OF BIRTH HEALTH AND HEREDITY, vol. 24, no. 5, 2016, pages 146 - 148
PRESTA, CURR. OP. STRUCT. BIOL., vol. 2, 1992, pages 593 - 596
RAJAGOPAL ET AL., PROT. ENGIN., vol. 10, 1997, pages 1453 - 1459
REICHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 329
REITER ET AL., CANCER RES., vol. 54, 1994, pages 2714 - 2718
REITER ET AL., IMMUNITY, vol. 2, 1995, pages 281 - 287
REITER ET AL., INTER J. OF CANCER, vol. 58, 1994, pages 142 - 149
REITER ET AL., JBC, vol. 269, 1994, pages 18327 - 18331
REITER ET AL., NAT. BIOTECHNOL., vol. 14, 1996, pages 1239 - 1245
REITER ET AL., PROTEIN ENGINEERING, vol. 8, 1995, pages 1323 - 1331
ROSKOSKI R JR ET AL., CRIT REV ONCOL HEMATOL, vol. 62, no. 3, 2007, pages 179 - 213
SYNNESTVEDT K ET AL., J CLIN INVEST., vol. 110, 2002, pages 993 - 1002
TAKAHASHI T ET AL., ONCOGENE, vol. 18, no. 13, 1999, pages 2221 - 2230
WEBBER ET AL., MOLECULAR IMMUNOLOGY, vol. 32, 1995, pages 249 - 258
ZIMMERMANN H., BIOCHEM J., vol. 285, 1992, pages 345 - 365

Also Published As

Publication number Publication date
CA3208473A1 (en) 2023-12-22
CN117257934A (zh) 2023-12-22
EP4321537A1 (en) 2024-02-14

Similar Documents

Publication Publication Date Title
JP7374995B2 (ja) 抗ｐｄ－１／抗ｖｅｇｆａ二官能性抗体、その医薬組成物およびその使用
AU2016333517B2 (en) Anti-PD-1 antibodies and compositions
CN111944050B (zh) 一种抗b7-h3抗体及其应用
WO2021023117A1 (zh) 抗ctla4-抗pd-1双特异性抗体及其用途
AU2017342071A1 (en) Anti-LAG-3 antibodies and compositions
WO2021104302A1 (zh) 抗pd-1-抗vegfa的双特异性抗体、其药物组合物及其用途
CN114181310B (zh) 抗tigit抗体、其药物组合物及用途
JP2023522365A (ja) 抗ｃｄ７３抗体とその利用
WO2022188832A1 (zh) 含有抗pd-1-抗vegfa双特异性抗体的药物组合及其用途
US20240018254A1 (en) Anti-cd73 antibody and use thereof
WO2022218384A1 (zh) 抗cd47的单克隆抗体及其用途
CN115368458A (zh) 抗cd73抗体及其应用
WO2023246247A1 (zh) 药物组合物及其用途
WO2024051223A1 (zh) 药物组合及用途
EP4357367A1 (en) Pharmaceutical composition and use thereof
WO2023001303A1 (zh) 药物组合物及用途
WO2024109657A1 (zh) 抗ccr8抗体及其用途
WO2024131835A1 (en) Ligand-cytotoxicity drug conjugates and pharmaceutical uses thereof
KR20240044467A (ko) Cldn18.2-표적화 항체, 이중특이적 항체, 및 이들의 용도
CN116637182A (zh) 包含抗ctla4-抗pd-1双特异性抗体和西奥罗尼的药物组合

Legal Events

Date Code Title Description
ENP Entry into the national phase

Ref document number: 3208473

Country of ref document: CA

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2023745372

Country of ref document: EP

Effective date: 20230804