Pharmaceutical composition and its use

Abstract

The present invention relates to a pharmaceutical composition comprising an anti-CD73 (e.g., human CD73) antibody or an antigen-binding fragment thereof, and an anti-PD-1-anti-VEGFA bispecific antibody or an antigen-binding fragment thereof. Specifically, the heavy chain variable region of the anti-CD73 antibody comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 15 to 17; and the light chain variable region of the antibody comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 18 to 20.

Description

Technical Field

[0001] The present invention relates to the field of immunology, and in particular, to a pharmaceutical composition comprising an anti-CD73 (e.g., human CD73) antibody or an antigen-binding fragment thereof, and an anti-PD-1-anti-VEGFA bispecific antibody or an antigen-binding fragment thereof.

Background Art

[0002] Ecto-5'-nucleotidase, namely CD73 protein, is a multifunctional glycoprotein encoded by the NT5E gene and having a molecular weight of 70 KD, and is anchored on the cell membrane by glycosylphosphatidylinositol (GPI) (Zimmermann H., Biochem J., 1992; 285:345-365).

[0003] CD73 is widely distributed on the surface of human tissue cells. In research, CD73 has been found to be highly expressed in the tumor microenvironment in various solid tumors, particularly in cancer cells, dendritic cells, regulatory T cells (Treg), natural killer cells (NK cells), myeloid-derived suppressor cells (MDSC), tumor-associated macrophages (TAM), etc. Hypoxia induces the upregulation of molecules such as hypoxia-inducible factor-1 (HIF-1), thereby leading to the widespread expression of CD73 in the tumor microenvironment (Synnestvedt K, et al. J Clin Invest. 2002; 110:993-1002). Analysis of clinical tumor samples has shown that high expression of CD73 is a potential biomarker and is closely related to the poor prognosis of various types of tumors, including breast cancer, lung cancer, ovarian cancer, kidney cancer, gastric cancer, head and neck cancer, etc.

[0004] CD73 has both hydrolytic enzyme activity and non-hydrolytic enzyme activity. The enzymatic and non-enzymatic functions of CD73 act simultaneously in related processes in tumors, mutually promoting and maintaining tumor progression. CD73 is an important regulatory molecule for tumor cell proliferation, metastasis and invasion in vitro, as well as for tumor angiogenesis and tumor immune escape mechanisms in vivo. It is becoming increasingly clear that an important immunosuppressive mechanism is mediated by the CD73-adenosine metabolism signaling pathway. CD39 upstream of CD73 catalyzes ATP to generate adenosine monophosphate (AMP), and the generated AMP is converted to adenosine by CD73. Adenosine binds to the downstream adenosine receptor (A2AR). A2AR inhibits a series of signaling pathways related to immune activation such as LCK, MAPK, and PKC, and inhibits the immune killing effect of T cells by activating protein kinase A (PKA) and Csk kinase, thereby playing a role in immunosuppression (Antonioli L, et al. Nat Rev Cancer. 2013; 13:842-857).

[0005] The transmembrane receptor PD-1 (programmed cell death protein-1) is a member of the CD28 family and is expressed in activated T cells, B cells, and myeloid cells. Both ligands, PDL1 (programmed cell death 1 ligand 1 or PDL-1) and PDL2 (programmed cell death 1 ligand 2 or PDL-2), are members of the B7 superfamily. PDL1 is expressed in various cells including T cells, B cells, endothelial cells and epithelial cells, and PDL2 is expressed only in antigen-presenting cells such as dendritic cells and macrophages.

[0006] The PD-1 / PDL1 signaling pathway plays an important role in the regulation of immune tolerance, microbial infection, and tumor immune escape. PD-1 is mainly expressed in immune cells such as T cells, and the ligand of PD-1, PDL1, is highly expressed in multiple human tumor tissues. Blocking the PD-1 / PDL1 signaling pathway can activate suppressed T cells, which may attack cancer cells. By blocking PD-1 / PDL1 signaling, the proliferation of tumor antigen-specific T cells can be promoted, the tumor cell killing process can be activated, and local tumor growth can be further inhibited (Julie R et al., 2012, N Engl J Med., 366:2455-2465). Furthermore, tumors with high PDL1 expression are associated with cancers that are difficult to detect (Hamanishi et al., 2007, Proc. Natl. Acad. Sci. USA, 104:3360-5). An effective method is to administer anti-PD-1 antibodies to regulate the expression of PD-1. Due to the broad anti-tumor prospects and surprising efficacy of PD-1 antibodies, antibodies targeting the PD-1 pathway have been widely accepted in the industry for bringing breakthroughs in various tumors, such as non-small cell lung cancer, renal cell cancer, ovarian cancer, melanoma (Homet M. B., Parisi G., et al., 2015, Semin Oncol., 42(3):466-473), leukemia, and anemia (Held SA, Heine A, et al., 2013, Curr Cancer Drug Targets., 13(7):768-74).

[0007] Vascular endothelial growth factor (VEGF) is a growth factor that promotes the division and proliferation of endothelial cells, promotes the formation of new blood vessels, and improves vascular permeability. It plays a role by binding to vascular endothelial growth factor receptors on the cell surface and activating the tyrosine kinase signaling pathway. In tumor tissues, tumor cells, and macrophages and mast cells that invade tumors secrete high levels of VEGF, which paracrinely stimulates tumor vascular endothelial cells, promotes the proliferation and migration of endothelial cells, induces angiogenesis, promotes the continuous growth of tumors, improves vascular permeability, causes fibrin deposition in surrounding tissues, promotes the infiltration of monocytes, fibroblasts, and endothelial cells, which promotes the formation of tumor stroma and the invasion of tumor cells into new blood vessels, and promotes tumor metastasis. Therefore, inhibition of tumor angiogenesis is considered one of the most promising tumor treatment methods currently. The VEGF family includes VEGFA, VEGFB, VEGFC, VEGFD, and PIGF. Vascular endothelial growth factor receptors (VEGFRs) include VEGFR1 (also known as Flt1), VEGFR2 (also known as KDR or Flk1), VEGFR3 (also known as Flt4), and Neuropilin-1 (NRP-1). The first three receptors are structurally similar, belong to the tyrosine kinase superfamily, and are composed of an extracellular region, a transmembrane segment, and an intracellular region. The extracellular region is composed of immunoglobulin-like domains, and the intracellular region is a tyrosine kinase region. VEGFR1 and VEGFR2 are mainly found on the surface of vascular endothelial cells, and VEGFR3 is mainly found on the surface of lymphatic endothelial cells.

[0008] Molecules of the VEGF family have different affinities for these receptors. VEGFA mainly acts in combination with VEGFR1, VEGFR2, and NRP-1. VEGFR1 was the first receptor recognized and has a higher affinity for VEGFR2 than VEGFR2 under normal physiological conditions, but its tyrosine kinase activity in the intracellular segment is lower than that of VEGFR2 ((Ma Li, Chinese Journal of Birth Health and Heredity, 24(5) (2016):146-148).

[0009] VEGFR2 is a major regulator of angiogenesis and vascular engineering and has much higher tyrosine kinase activity than VEGFR1. After binding to the ligand VEGFA, VEGFR2 mediates the proliferation and differentiation of vascular endothelial cells, as well as the processes of blood vessel formation and vascular permeability (Roskoski R Jr. et al., Crit Rev Oncol Hematol, 62(3) (2007):179-213). After binding to VEGFR2, VEGFA mediates the transcriptional expression of intracellular related protein genes through the downstream PLC-γ-PKC-Raf-MEK-MAPK signaling pathway, thus promoting the proliferation of vascular endothelial cells (Takahashi T et al., Oncogene, 18(13) (1999):2221-2230).

[0010] VEGFR3 is a member of the tyrosine kinase family and is mainly expressed in fetal vascular endothelial cells and adult lymphatic endothelial cells. VEGFC and VEGFD bind to VEGFR3 to stimulate the proliferation and migration of lymphatic endothelial cells and promote lymphangiogenesis; NRP-1 is a non-tyrosine kinase transmembrane protein that cannot transmit biological signals independently and can only mediate signal transduction after forming a complex with the VEGF tyrosine kinase receptor (Ma Li, Chinese Journal of Birth Health and Heredity, 24(5) (2016):146-148).

[0011] VEGFA and VEGFR2 are mainly involved in the regulation of angiogenesis. Before and after the binding of VEGFA to VEGFR2, cascade reactions of a number of intermediate signals in the upstream and downstream pathways are formed, and finally, physiological functions change due to proliferation, survival, migration, increased permeability, invasion of peripheral tissues, and other patterns of endothelial cells (Dong Hongchao et al., Sep. 2014, Journal of Modern Oncology, 22(9):2231-3).

[0012] Currently, there are several humanized monoclonal antibodies targeting human VEGF, particularly VEGFA, such as bevacizumab, which was approved by the US Food and Drug Administration in 2004 for the treatment of various tumors, such as non-small cell lung cancer, renal cell carcinoma, cervical cancer, and metastatic colorectal cancer.

[0013] In summary, it is very important to develop a treatment or combination therapy with higher efficacy.

Summary of the Invention

[0014] After intensive research and creative efforts, the inventors used a mammalian cell expression system to express recombinant human CD73 as an antigen for immunizing mice, and obtained hybridoma cells by fusing mouse spleen cells and myeloma cells. The inventors obtained the hybridoma cell line LT014 (Accession No.: CCTCC NO: C2018137) by screening a large number of samples.

[0015] The inventors surprisingly found that the hybridoma cell line LT014 can secrete a specific monoclonal antibody (designated 19F3) that specifically binds to human CD73, and the monoclonal antibody can effectively inhibit the enzymatic activity reaction of CD73 in a non-substrate competitive mode, reduce the production of adenosine, promote the activity of T cells, and exert the effect of inhibiting tumor growth.

[0016] Furthermore, the inventors creatively prepared humanized antibodies against human CD73 (designated 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM), 19F3H2L3, and 19F3H2L3 (hG1DM)), and further introduced amino acid mutations into the anti-CD73 antibody to eliminate the ADCC and CDC effects, avoiding the undesirable toxicity mediated by the antibody.

[0017] The inventors have also surprisingly found that an antibody in combination with the anti-PD-1 / VEGFA bispecific antibody of the present invention has a pharmacological effect of more effectively inhibiting tumor growth than either the anti-PD-1 / VEGFA bispecific antibody or the anti-CD73 antibody alone.

[0018] Another aspect of the present invention further relates to an antibody. Here, the anti-CD73 antibody comprises the following.

[0019] The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 10; and the LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, or SEQ ID NO: 14. Preferably, according to the IMGT numbering system, the anti-CD73 antibody comprises the following: the amino acid sequence shown in SEQ ID NO: 15, a sequence having 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 with the sequence, 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 the sequence, and HCDR1 comprising or consisting of such an amino acid sequence, the amino acid sequence shown in SEQ ID NO: 16, a sequence having 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 with the sequence, 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 the sequence, and HCDR2 comprising or consisting of such an amino acid sequence, the amino acid sequence shown in SEQ ID NO: 17, a sequence having 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 with the sequence, or an amino acid sequence having one or more (preferably 1,An HCDR3 comprising or consisting of an amino acid sequence having a conservative amino acid mutation (preferably a substitution, insertion or deletion) of 2 or 3), the amino acid sequence shown in SEQ ID NO: 18, 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 with the sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably a substitution, insertion or deletion) compared to the sequence; an LCDR1 comprising or consisting of an amino acid sequence having a conservative amino acid mutation (preferably a substitution, insertion or deletion) of 2 or 3), the amino acid sequence shown in SEQ ID NO: 19, 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 with the sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably a substitution, insertion or deletion) compared to the sequence; an LCDR2 comprising or consisting of an amino acid sequence having a conservative amino acid mutation (preferably a substitution, insertion or deletion) of 2 or 3), the amino acid sequence shown in SEQ ID NO: 20, 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 with the sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably a substitution, insertion or deletion) compared to the sequence; and an LCDR3 comprising or consisting of an amino acid sequence having a conservative amino acid mutation (preferably a substitution, insertion or deletion) of 2 or 3), the amino acid sequence shown in SEQ ID NO: 20, 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 with the sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably a substitution, insertion or deletion) compared to the sequence.

[0020] In some embodiments of the present invention, the heavy chain variable region of the antibody 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 98% or at least 99% sequence identity with the following sequences: SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 10, or has one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 10, and the light chain variable region of the antibody 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 98% or at least 99% sequence identity with the following sequences: SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12 or SEQ ID NO: 14, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12 or SEQ ID NO: 14, or has one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12 or SEQ ID NO: 14, and consists of or comprises an amino acid sequence having such mutations.

[0021] In some embodiments of the present invention, the 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), the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 4 (preferably, the nucleic acid sequence is described in SEQ ID NO: 3), the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 6 (preferably, the nucleic acid sequence is shown in SEQ ID NO: 5), the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 8 (preferably, the nucleic acid sequence is described in SEQ ID NO: 7), the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 10 (preferably, the nucleic acid sequence is shown in SEQ ID NO: 9), the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 12 (preferably, the nucleic acid sequence is described in SEQ ID NO: 11), or the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 10 (preferably, the nucleic acid sequence is shown in SEQ ID NO: 9), and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 14 (preferably, the nucleic acid sequence is described in SEQ ID NO: 13).

[0022] In some embodiments of the present invention, the heavy chain constant region of the antibody is the Igγ-1 chain C region, accession: P01857; the light chain constant region is the Ig kappa chain C region, accession: P01834. More preferably, the heavy chain constant region of the anti-CD73 antibody has the following mutations based on the sequence described in accession: P01857 according to the EU numbering system: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A and G237A; or It has one or more mutations selected from 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 and K320A. Most preferably, the amino acid sequence of the heavy chain constant region of the anti-CD73 antibody is shown in SEQ ID NO: 21, and the amino acid sequence of the light chain constant region of the anti-CD73 antibody is shown in SEQ ID NO: 22.

[0023] In some embodiments of the invention, the heavy chain constant region of the antibody has a leucine-alanine point mutation introduced at position 234 (L234A) and a leucine-alanine point mutation introduced at position 235 (L235A), and has an Ig gamma-1 chain C region having the amino acid sequence set forth in SEQ ID NO: 21, accession: P01857; the light chain constant region has an Ig kappa chain C region having the amino acid sequence set forth in SEQ ID NO: 22, accession: P01834.

[0024] The variable regions of the light and heavy chains determine antigen binding, and the variable region of each chain contains three hypervariable regions (CDRs) called complementarity-determining regions (the CDRs of the heavy chain (H) include HCDR1, HCDR2 and HCDR3, and the CDRs of the light chain (L) include LCDR1, LCDR2 and LCDR3, which are named by Kabat et al., Bethesda M.d., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 1991; 1-3:91-3242).

[0025] Preferably, the CDRs can also be defined by the IMGT numbering system. See Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc., IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF, and MhcSF. Nucleic acids research, 2009; 38(suppl_1): D301-D307.

[0026] The amino acid sequences of the CDRs of the monoclonal antibody sequences are analyzed according to the IMGT definition using technical means well-known to those skilled in the art, such as the VBASE2 database.

[0027] The antibodies 19F3, 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM) and 19F3H2L3 (hG1DM) involved in the present invention have the same CDRs.

[0028] The three CDRs of the heavy chain variable region have the following amino acid sequences: HCDR1: GYSFTGYT (SEQ ID NO: 15), HCDR2: INPYNAGT (SEQ ID NO: 16), and HCDR3: ARSEYRYGGDYFDY (SEQ ID NO: 17) and The three CDRs of the light chain variable region have the following amino acid sequences: LCDR1: QSLLNSSNQKNY (SEQ ID NO: 18), LCDR2: FAS (SEQ ID NO: 19), and LCDR3: QQHYDTPYT (SEQ ID NO: 20) and

[0029] In some embodiments of the present invention, the antibody is a monoclonal antibody.

[0030] In some embodiments of the present invention, the antibody is a humanized antibody, a chimeric antibody or a multispecific antibody (e.g., a bispecific antibody).

[0031] In some embodiments of the present invention, the antigen-binding fragment is selected from Fab, Fab’, F(ab’)2, Fd, Fv, dAb, Fab / c, complementarity determining region fragment, single-chain antibody (e.g., scFv), humanized antibody, chimeric antibody and bispecific antibody.

[0032] Another aspect of the present invention relates to a conjugate comprising an antibody and a conjugate moiety, wherein the antibody is an antibody or an antigen-binding fragment thereof according to any one of the aspects of the present invention, and the conjugate moiety is a purification tag (e.g., His tag), a detectable label; preferably, the conjugate moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, a coloring substance, polyethylene glycol or an enzyme.

[0033] 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 aspect of the present invention.

[0034] Another aspect of the present invention relates to a kit, conjugate, fusion protein or multispecific antibody of the present invention comprising an antibody or an antigen-binding fragment thereof according to any one of the aspects of the present invention. Preferably, the kit further comprises a secondary antibody that specifically recognizes the antibody, and optionally, the secondary antibody further comprises a detectable label such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a coloring substance or an enzyme.

[0035] Another aspect of the present invention relates to the use of an antibody or an antigen-binding fragment thereof according to any one of the aspects, conjugates, fusion proteins, or multispecific antibodies of the present invention in preparing a kit used to detect the presence or level of CD73 in a sample.

[0036] Yet another aspect of the present invention relates to a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof that follows any one of the aspects of the present invention, a conjugate, a fusion protein, or a multispecific antibody of the present invention; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient. Preferably, the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, or intralesional injection.

[0037] Yet another aspect of the present invention relates to the use of an antibody or an antigen-binding fragment thereof, a conjugate of the present invention, a fusion protein, or a multispecific antibody according to any one of the aspects of the present invention in the manufacture of a medicament for the treatment and / or prevention of tumors (solid tumors, preferably including non-small cell lung cancer, prostate cancer (including metastatic castration-resistant prostate cancer (mCRPC), etc.), triple-negative breast cancer, ovarian cancer, colorectal cancer (including microsatellite stability (MSS) and mismatch repair deficiency / microsatellite instability-high (dMMR / MSI-H) types), gastric cancer (including microsatellite stability (MSS) and mismatch repair deficiency / microsatellite instability-high (dMMR / MSI-H) types), melanoma, head and neck cancer, renal cell cancer, or pancreatic ductal adenocarcinoma), or in the manufacture of a medicament for the diagnosis of tumors.

[0038] Yet another aspect of the present invention relates to the hybridoma cell line LT014 deposited with the China Center for Type Culture Collection (CCTCC) under the accession number CCTCC NO: C2018137.

[0039] Yet another aspect of the present invention relates to a combination product (e.g., a kit) comprising a first product and a second product in separate packages, wherein the first product comprises an anti-CD73 antibody or an antigen-binding fragment thereof according to any one of the aspects of the present invention, a conjugate according to the present invention, or a pharmaceutical composition according to any one of the aspects of the present invention, the second product comprises an anti-PD-1-anti-VEGFA bispecific antibody, Preferably, the combination product further comprises a third product in a separate package containing one or more chemotherapeutic agents, Preferably, the first product and the second product further independently comprise one or more pharmaceutically acceptable excipients, Preferably, the combination product further comprises a product insert, and preferably, the insert states that the unit dosage of the anti-CD73 antibody and / or the anti-PD-1-anti-VEGFA bispecific antibody is 0.1 to 100 mg, preferably 1 to 10 mg / kg body weight; alternatively, the unit dosage of the anti-CD73 antibody and / or the anti-PD-1-anti-VEGFA bispecific antibody is 10 to 1000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg or 200 mg in each subject, and preferably, the insert states that the anti-CD73-1 antibody and / or the anti-PD-1-anti-VEGFA bispecific antibody is administered twice a day to about once every other day, or once every 3 days, 4 days, 5 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks.

[0040] In one or more embodiments of the present invention, the mass ratio of the anti-CD73 antibody or its antigen-binding fragment to the anti-PD-1-anti-VEGFA bispecific antibody in the kit or pharmaceutical composition is (1:5)-(1:5)-(1:5) based on the mass of the antibody, for example, 1:5, 1:4, 1:3, 1:2, 1:2, 1:3, 1:4 or 5:1.

[0041] Yet another aspect of the present invention relates to a method of treating and / or preventing a tumor, comprising administering to a patient a therapeutically effective amount of drug A and a therapeutically effective amount of drug B, wherein drug A comprises an antibody or its antigen-binding fragment according to the present invention, a conjugate according to the present invention, or a fusion protein or multispecific antibody according to the present invention, drug B comprises an anti-PD-1-anti-VEGFA bispecific antibody, and preferably, drug A and drug B are administered simultaneously or sequentially, where sequential administration means administering drug A first or administering drug B first.

[0042] In some embodiments of the present invention, the heavy chain amino acid sequence of the anti-PD-1-anti-VEGFA bispecific antibody is set forth in SEQ ID NO: 23, and its light chain amino acid sequence is set forth in SEQ ID NO: 25.

[0043] The present invention relates to a method for preventing and / or treating a tumor (particularly a malignant tumor), which comprises 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 agents (preferably a chemotherapeutic agent or a growth inhibitor, a targeted therapeutic agent (e.g., an antibody-drug conjugate, an antibody or an antigen-binding fragment thereof), a T cell expressing a chimeric antigen receptor, an angiogenesis inhibitor, an anti-tumor agent, a cancer vaccine, an adjuvant and combinations thereof, an antimetabolite, an antibiotic, a plant-based and / or hormonal drug, preferably cyclophosphamide, cyclophosphamide, platinum, carboplatin, oxaliplatin, doxorubicin, paclitaxel, vinca alkaloid, tamoxifen, megestrol, goserelin, asparaginase and / or fluorouracil anti-tumor agents), an anti-CD73 antibody, an anti-PD-1-anti-VEGFA bispecific antibody and a tumor chemotherapeutic agent are administered simultaneously or sequentially.

[0044] In one or more embodiments of the present invention, the chemotherapeutic agent or growth inhibitor is selected from alkylating agents, anthracyclines, antihormonal agents (e.g., antiandrogen agents), aromatase inhibitors, protein kinase inhibitors (e.g., tyrosine kinase inhibitors), lipid kinase inhibitors, antisense oligonucleotides, ribozymes, antimetabolites, topoisomerase inhibitors, cytotoxic agents or antitumor antibiotics, proteasome inhibitors, antimicrotubule inhibitors, EGFR antagonists, VEGFR antagonists, PD-1 antagonists, angiopoietin 2 antagonists, retinoids, histone deacetylase inhibitors, and combinations thereof.

[0045] In one or more embodiments of the present invention, the targeted therapeutic agent is selected from a B-raf inhibitor, a MEK inhibitor, a K-ras inhibitor, a c-Met inhibitor, an Alk inhibitor, a phosphatidylinositol 3-kinase inhibitor, an Akt inhibitor, an mTOR inhibitor, a VEGF inhibitor, a CD73 inhibitor, a PARP inhibitor, a PD-1 inhibitor, a diphosphatidylglycerol 3-kinase / mTOR inhibitor, and combinations thereof.

[0046] In one or more embodiments of the present invention, the antibody-drug conjugate comprises a drug selected from the group consisting of maytansine, monomethyl auristatin E, calicheamicin, esperamicin, and a radioisotope chelating agent.

[0047] The CD73 inhibitor includes, but is not limited to, one or more of BMS-986179, MEDI9447, NZV930, CPI-006, AB680, LY-3475070, TJ004309[3], ORIC-533, IPH5301AB680, and LY-3475070.

[0048] The PARP inhibitor is selected from one or more of 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, XZ-120312, YHP-743, iobenguane I 131, rucaparib camsilate, 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, and WB-1340.

[0049] In one or more embodiments of the present invention, the tumor is selected from one or more of the following: cervical cancer (metastatic uterine cancer, lung cancer, endometrial cancer, non-squamous cell cancer, endometrial cancer, esophageal cancer, esophageal squamous cell cancer, gastrointestinal cancer (including microsatellite stability (MSS) and mismatch repair dysfunction / microsatellite high instability (dMMR / MSI-H) types), such as advanced gastric cancer, gastric adenocarcinoma, or gastroesophageal junction adenocarcinoma), intestinal cancer (e.g., rectal cancer, colon cancer, colorectal cancer (including microsatellite stability (MSS) and mismatch repair dysfunction / microsatellite high instability (dMMR / MSI-H) types), liver cancer (e.g., hepatocellular carcinoma, hepatobiliary cancer), cholangiocarcinoma, pancreatic cancer, ovarian cancer (e.g., advanced ovarian cancer), fallopian tube cancer, glioma, recurrent glioma, melanoma, hematological malignancy (e.g., acute myeloid leukemia), lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma), multiple myeloma, sarcoma (e.g., leiomyosarcoma, rhabdomyosarcoma), osteosarcoma, neuroblastoma (e.g., multiple myeloma), large cell neuroendocrine cancer, urothelial cancer (e.g., upper urothelial cancer or bladder cancer), prostate cancer (metastatic castration-resistant prostate cancer (mCRPC)), testicular cancer, triple-negative breast cancer, peripheral T cell lymphoma, nasopharyngeal cancer, microsatellite high instability (MSI-H) or mismatch repair dysfunction (dMMR) type solid tumors, head and neck cancer, brain cancer (e.g., invasive brain cancer), squamous cell cancer, basal cell cancer, adenoma (e.g., breast cancer, thymic cancer, ileocecal adenocarcinoma, ampulla adenocarcinoma, pancreatic duct adenocarcinoma, mucinous or serous cystadenocarcinoma), choriocarcinoma, malignant cystic teratoma, malignant cystic stromal cell tumor, malignant granuloma, undifferentiated embryonal cell tumor, glioblastoma, mycosis, intrahepatic cholangiocarcinoma, Merkel cell carcinoma, Merkel cell carcinoma, squamous cell cancer, tongue squamous cell cancer, head and neck squamous cell cancer, and other hematological malignancies.

[0050] In one or more embodiments of the present invention, the anti-PD-1-anti-VEGFA bispecific antibody has a first protein functional region that targets PD-1, and a second protein functional region that targets VEGFA and contains Here, 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 contains HCDR1-HCDR3 (preferably, according to the IMGT numbering system, HCDR1-HCDR3 shown in SEQ ID NOs: 31-33, respectively) contained in the heavy-chain variable region having the amino acid sequence shown in SEQ ID NO: 27, and its light-chain variable region contains LCDR1-LCDR3 (preferably, according to the IMGT numbering system, LCDR1-LCDR3 shown in SEQ ID NOs: 34-36, respectively) contained in the light-chain variable region having the amino acid sequence shown in SEQ ID NO: 29; The heavy-chain variable region of the single-chain antibody contains HCDR1-HCDR3 (preferably, according to the IMGT numbering system, HCDR1-HCDR3 described in SEQ ID NOs: 41-43, respectively) contained in the heavy-chain variable region having the amino acid sequence shown in SEQ ID NO: 37, and its light-chain variable region contains LCDR1-LCDR3 (preferably, according to the IMGT numbering system, LCDR1-LCDR3 described in SEQ ID NOs: 44-46, respectively) contained in the light-chain variable region having the amino acid sequence shown in SEQ ID NO: 39; or The heavy-chain variable region of the immunoglobulin contains HCDR1-HCDR3 (preferably, according to the IMGT numbering system, HCDR1-HCDR3 described in SEQ ID NOs: 41-43, respectively) contained in the heavy-chain variable region having the amino acid sequence shown in SEQ ID NO: 37, and its light-chain variable region contains LCDR1-LCDR3 (preferably, according to the IMGT numbering system, LCDR1-LCDR3 described in SEQ ID NOs: 44-46, respectively) contained in the light-chain variable region having the amino acid sequence shown in SEQ ID NO: 39; The heavy chain variable region of the single-chain antibody comprises HCDR1 to HCDR3 (preferably, according to the IMGT numbering system, HCDR1 to HCDR3 shown in SEQ ID NOs: 31 to 33, respectively) contained in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 27, and its light chain variable region comprises LCDR1 to LCDR3 (preferably, according to the IMGT numbering system, LCDR1 to LCDR3 shown in SEQ ID NOs: 34 to 36, respectively) contained in the light chain variable region having the amino acid sequence shown in SEQ ID NO: 29; and Preferably, the immunoglobulin is of the human IgG1 subtype.

[0051] In one or more embodiments of the present invention, for bispecific antibodies, according to the EU numbering system, the heavy chain constant region of the immunoglobulin has the following mutations: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A and G237A and has.

[0052] In the present invention, the letter before the position number represents the amino acid before the mutation, and the letter after the position number represents the amino acid after the mutation unless otherwise specified.

[0053] In one or more embodiments of the present invention, for bispecific antibodies, according to the EU numbering system, the heavy chain constant region of the immunoglobulin has one or more mutations selected from 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 and K320A or further has the same.

[0054] In one or more embodiments of the present invention, for the bispecific antibody, the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 27, the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 29, 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.

[0055] In one or more embodiments of the present invention, the bispecific antibody has a heavy chain amino acid sequence shown in SEQ ID NO: 23 and a light chain amino acid sequence shown in SEQ ID NO: 25. Preferably, the heavy chain of the bispecific antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 24, and its light chain is encoded by the amino acid sequence shown in SEQ ID NO: 26.

[0056] In some embodiments of the present invention, for the bispecific antibody, the single-chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin. Since the immunoglobulin has two heavy chains, two single-chain antibody molecules are bound to one immunoglobulin molecule. Preferably, the two single-chain antibody molecules are identical.

[0057] In some embodiments of the present invention, for the bispecific antibody, there are two single-chain antibodies, and one end of each single-chain antibody is linked to the C-terminus or N-terminus of one of the two heavy chains of the immunoglobulin.

[0058] In some embodiments of the present invention, a disulfide bond exists between the VH and VL of a single-chain antibody. Methods for introducing a disulfide bond between the VH and VL of an antibody are well known in the art, for example, U.S. Patent No. 5,747,654, which is incorporated herein by reference; 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., Immunity 2(1995)281-287; Reiter et al., JBC 269(1994)18327-18331; Reiter et al., Inter. J. of Cancer 58(1994)142-149; or Reiter et al., Cancer Res. 54(1994)2714-2718.

[0059] In one or more embodiments of the present invention, for a bispecific antibody, the first protein functional region is linked to the second protein functional region directly or via a linker fragment; and / or the heavy chain variable region of a single-chain antibody is linked to the light chain variable region of the single-chain antibody directly or via a linker fragment.

[0060] In one or more embodiments of the present invention, for a bispecific antibody, the linker fragment is (GGGGS)n, where n is a positive integer, preferably n is 1, 2, 3, 4, 5 or 6.

[0061] In one or more embodiments of the present invention, in a bispecific antibody, the number of the first protein functional region and the second protein functional region is each independently 1, 2 or more.

[0062] In one or more embodiments of the present invention, for bispecific antibodies, the single-chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin.

[0063] In one or more embodiments of the present invention, the first protein functional region is linked to the second protein functional region via a first linker fragment; the heavy chain variable region of the single-chain antibody is linked to the light chain variable region of the single-chain antibody via a second linker fragment; the first linker fragment and the second linker fragment are the same or different; Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are independently selected from SEQ ID NO: 47 and SEQ ID NO: 48; Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are shown in SEQ ID NO: 48.

[0064] In one or more embodiments of the present invention, the bispecific antibody is a monoclonal antibody. In one or more embodiments of the present invention, the bispecific antibody is a humanized antibody.

[0065] Another aspect of the present invention relates to unit dosage forms, preferably used for treating tumors, including 1 to 10,000 mg (preferably 10 to 1,000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg or 200 mg) of an anti-CD73 antibody according to any one of the aspects of the present invention, and 1 to 10,000 mg (preferably 1 to 1,000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, 200 mg or 100 mg) of an anti-PD-1-anti-VEGFA bispecific antibody according to any one of the aspects of the present invention, optionally including one or more of the chemotherapeutic agents (such as platinum-based drugs and / or fluorouracil anti-tumor drugs) according to the present invention, and the anti-CD73 antibody, the anti-PD-1-anti-VEGFA bispecific antibody and the chemotherapeutic agent are packaged separately.

[0066] The present invention relates to a method for preventing or treating cancer or a tumor, which comprises separately administering to a subject in need thereof, preferably, an anti-PD-1-anti-VEGFA bispecific antibody, an anti-CD73 antibody, and a chemotherapeutic agent in a unit dosage form, respectively.

[0067] Another aspect of the present invention relates to a single-dose unit comprising 0.1 to 10,000 mg (preferably 1 to 1,000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, 200 mg or 100 mg) of an anti-CD73 antibody according to any aspect of the present invention, preferably used for treating a tumor, and 0.1 to 10,000 mg (preferably 1 to 1,000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, 200 mg or 100 mg) of an anti-PD-1 anti-VEGFA bispecific antibody according to any aspect of the present invention.

[0068] In one or more embodiments of the present invention, the anti-CD73 antibody, the anti-PD-1-anti-VEGFA bispecific antibody and / or the chemotherapeutic agent are in a form suitable for intravenous injection or intravenous infusion, preferably in a liquid form.

[0069] In one or more embodiments of the present invention, an effective amount of the anti-CD73 antibody according to any one of the aspects of the present invention and / or the anti-PD-1-anti-VEGFA bispecific antibody according to any one of the aspects of the present invention is administered to the subject before and / or after surgical treatment and / or before and / or after radiotherapy.

[0070] In one or more embodiments of the present invention, the unit dosage of the anti-CD73 antibody according to any one of the aspects of the present invention and / or the anti-PD-1-anti-VEGFA bispecific antibody according to any one of the aspects of the present invention is 0.1 to 100 mg, preferably 1 to 10 mg / kg body weight; alternatively, the unit dosage of the anti-CD73 antibody according to any one of the aspects of the present invention and / or the anti-PD-1-anti-VEGFA bispecific antibody according to any one of the aspects of the present invention is 10 to 1000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg or 200 mg in each subject, Preferably, the dosage is administered about once every other day to twice a day, or once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks; Preferably, the administration route is intravenous infusion or intravenous injection.

[0071] In the present invention, unless otherwise defined, scientific and technical terms used herein have the meanings generally understood by those skilled in the art. Furthermore, the experimental operations of cell culture, molecular genetics, nucleic acid chemistry and immunology used herein are routine procedures widely used in the corresponding fields. On the other hand, to better understand the present invention, the definitions and explanations of related terms are provided below.

[0072] As used herein, the term "EC50" refers to the concentration at 50% of the maximum effect, i.e., the concentration that can cause 50% of the maximum effect.

[0073] As used herein, the term "antibody" generally refers to an immunoglobulin molecule consisting of two pairs of polypeptide chains (each pair consisting of one "light" (L) chain and one "heavy" (H) chain). Antibody light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, and epsilon. The isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE. In the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain further contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including binding to the first component (C1q) of the classical complement system of various cells of the immune system (e.g., effector cells). The VH and VL regions can be further subdivided into hypervariable regions (complementarity-determining regions, or CDRs) and conserved regions called framework regions (FRs) that are distributed between the CDRs. Each VH and VL consists of three CDRs and four FRs arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus toward the carboxyl terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form an antigen-binding site, respectively.The assignment of amino acid regions or domains is based on the Kabat sequences of the proteins of immunological interest (National Institutes of Health, Bethesda, M.d. (1987 and 1991), or Chothia & Lesk, J. Mol. Biol., 1987; 196:901-917; Chothia et al., Nature, 1989; 342:878-883), or the definitions of 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 term “antibody” is not limited by any particular method for producing an antibody. For example, antibodies include, inter alia, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be antibodies of different isotypes, such as IgG (e.g., subtypes IgG1, IgG2, IgG3 or IgG4), IgA1, IgA2, IgD, IgE or IgM.

[0074] Given the known sequences of the variable regions of the heavy and light chains of an antibody, there are several methods for determining the CDRs of the antibody, including the Kabat, IMGT, Chothia, and AbM numbering systems. However, the application of all definitions of CDRs to an antibody or its variants shall fall within the scope of the terms defined and used herein. When the amino acid sequence of the variable region of an antibody is known, one of ordinary skill in the art can generally determine the specific CDRs without relying on experimental data beyond the sequence itself.

[0075] As used herein, the terms "mAb" and "monoclonal antibody" refer to a group of highly homologous antibodies, i.e., antibodies or antibody fragments derived from a group of identical antibody molecules, excluding naturally occurring mutations that may occur. Monoclonal antibodies are highly specific for a single epitope on an antigen. Polyclonal antibodies against monoclonal antibodies generally contain at least two or more different antibodies that generally recognize different epitopes on an antigen. Monoclonal antibodies can generally be obtained using the hybridoma technology first reported by Kohler et al. (Kohler G, Milstein C., Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256(5517):495).

[0076] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained when all or part of the CDRs of a human immunoglobulin (receptor antibody) are replaced with the CDRs of a non-human antibody (donor antibody), where the donor antibody can be a non-human (e.g., mouse, rat, or rabbit) antibody having the expected specificity, affinity, or reactivity. Furthermore, some amino acid residues in the framework region (FR) of the receptor antibody can also be replaced with the amino acid residues of the corresponding non-human antibody, or the amino acid residues of another antibody, to further improve or optimize the performance of the antibody. For details of humanized antibodies, see, for example, 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; and Clark M., Antibody humanization: a case of the “Emperor’s new clothes” [J]. Immunol. Today, 2000; 21(8):397-402.

[0077] As used herein, the term "isolated" refers to obtaining by artificial means from a natural state. When an "isolated" substance or component exists in nature, there may be a change in its natural environment, isolation from its natural environment, or both. For example, a particular non-isolated polynucleotide or polypeptide exists naturally in a living animal, and the same polynucleotide or polypeptide of high purity isolated from such a natural state is called an isolated polynucleotide or polypeptide. The term "isolated" does not exclude the presence of artificial substances, synthetic substances, or other impurities that do not affect the activity of the substance.

[0078] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, and as a result, the genetic material element carried by the vector can be expressed within the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages such as λ phage or M13 phage; and animal viruses. 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, and papovaviruses (such as SV40). A vector can contain various elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Further, a vector can further contain an origin of replication.

[0079] As used herein, the term "host cell" refers to a cell into which a vector can be introduced, and includes, but is not limited to, 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, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, GS cells, BHK cells, HEK293 cells, or human cells.

[0080] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. In some embodiments, an antibody that specifically binds to an antigen (or an antibody specific to the antigen) means that the antibody binds to the antigen with an affinity (KD) of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less.

[0081] As used herein, the term "KD" refers to the dissociation equilibrium constant for a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. A smaller dissociation equilibrium constant indicates a stronger antibody-antigen binding and a higher affinity between the antibody and the antigen. Generally, an antibody binds to an antigen (e.g., PD-1 protein) with a dissociation equilibrium constant (KD) of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less. KD can be determined by methods known to those skilled in the art, such as using a Fortebio molecular interaction device.

[0082] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and can be used interchangeably; the terms "polyclonal antibody" and "pAb" have the same meaning and can be used interchangeably; the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. Also, as used herein, amino acids are generally represented by the one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala. As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient. Such carriers and / or excipients are well known in the art and include, but are not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers (see, for example, Remington’s Pharmaceutical Sciences, edited by Gennaro AR, 19th Ed., Pennsylvania: Mack Publishing Company, 1995). For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants such as Tween®-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0083] As used herein, the term "effective amount" refers to an amount sufficient to obtain the desired effect, or at least partially sufficient to obtain the desired effect. For example, a prophylactically effective amount against a disease (e.g., a tumor) refers to an amount sufficient to prevent, halt, or delay the onset of the disease (e.g., a tumor), and a therapeutically effective amount refers to an amount sufficient to cure or at least partially halt the disease and its complications in a patient suffering from the disease.

[0084] The term "single administration unit" means a single pharmaceutical administration form, such as an injection placed in an ampoule, containing the anti-CD73 antibody and the anti-PD-1-anti-VEGFA bispecific antibody of the present invention, at the time of the regimen, preferably per kg body weight of the subject. In a specific embodiment of the present invention, the regimen includes administration of single administration units according to an administration cycle, for example, from twice a day to about once 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.

[0085] In the present invention, the terms "first" (e.g., the first protein functional region or the first linker fragment) and "second" (e.g., the second protein functional region or the second linker fragment) are used for the purpose of distinguishing expression or clarity and do not have a typical sequential meaning unless otherwise specified.

[0086] 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 a disease or promotes the regression of a disease as evidenced by a decrease in the severity of the disease, an increase in the frequency and duration of asymptomatic periods, or the prevention of damage or impairment caused by the disease. The ability of a therapeutic agent to promote the regression of a disease can be evaluated by various methods known to those skilled in the art, such as in human subjects in clinical trials, in animal model systems for predicting efficacy in humans, or by determining the activity of the drug in in vitro assays.

[0087] A "preventively effective amount" of a drug refers to any amount of the drug that, when administered alone or in combination with an anti-tumor agent to a subject at risk of developing cancer (e.g., a subject with a pre-cancerous condition) or a subject at risk of cancer recurrence, inhibits the occurrence or recurrence of cancer. In some embodiments, the preventively effective amount completely prevents the occurrence 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.

[0088] Advantageous effects: The monoclonal antibody of the present invention can bind specifically enough to CD73, can effectively inhibit the enzymatic activity reaction of CD73 in a non-substrate competition mode, can reduce the production of adenosine, and can promote the activity of T cells and the tumor suppression effect. On the other hand, the antibody combined with the anti-PD-1-anti-VEGFA bispecific antibody of the present invention has a pharmacological effect of effectively inhibiting tumor growth superior to either the anti-VEGFA / PD-1 bispecific antibody or the anti-CD73 antibody alone. Brief description of the drawings

[0089]

Figure 1

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Figure 3

Figure 4

Figure 5

Figure 6

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Figure 8

Figure 9

Figure 10

Mode for Carrying Out the Invention

[0090] Hereinafter, with reference to the examples, the embodiments of the present invention will be described in detail. Those skilled in the art will understand that the following examples are merely illustrative of the present invention and should not be construed as a limitation on the scope of the present invention. Examples where specific techniques or conditions are not specified are carried out according to the techniques or conditions described in the publications of the relevant technology (for example, refer to the Guide to Molecular Cloning Experiments written by J. Sambrook et al. and translated by Huang Peitang et al., 3rd edition, Science Press), or according to the product manuals. The reagents or instruments used are conventional commercially available products when the manufacturer is not specified.

[0091] In the following examples of the present invention, the BALB / c mice used were purchased from the Guangdong Medical Experimental Animal Center.

[0092] In the following examples of the present invention, the positive control antibody MEDI9447 (Oleclumab) used was manufactured by Akeso Biopharma, Inc., and its sequence was identical to the antibody sequence described in the International Nonproprietary Name (INN) for pharmaceutical substances published by Melmumune Limited on the WHO website (World Health Organization (2016). “International Nonproprietary Names for Pharmaceutical Substances (INN). Proposed INN: List 116” (PDF). WHO Drug Information. 30(4), P661-662).

[0093] In the following examples of the present invention, the combined anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) used was manufactured by Akeso Biopharma, Inc., and its sequence and preparation are referred to as the antibody VP101 (hG1DM) described in the published patent CN112830972A. Here, the full-length amino acid sequence of the heavy chain of VP101 (hG1DM) is described in SEQ ID NO: 23, and the full-length amino acid sequence of its light chain is described in SEQ ID NO: 25. VP101 (hG1DM) has an IgG-scFv structure, the IgG part is an anti-VEGFA antibody, and the scFv part is an anti-PD-1 antibody. The anti-VEGFA antibody has an HCDR1 sequence shown in SEQ ID NO: 31, an HCDR2 sequence shown in SEQ ID NO: 32, an HCDR3 sequence shown in SEQ ID NO: 33, and a VH sequence shown in SEQ ID NO: 27. The anti-VEGFA antibody has an LCDR1 sequence shown in SEQ ID NO: 34, an LCDR2 sequence shown in SEQ ID NO: 35, an LCDR3 sequence shown in SEQ ID NO: 36, and a VL sequence shown in SEQ ID NO: 29; and The anti-PD1 antibody has an HCDR1 sequence shown in SEQ ID NO: 41, an HCDR2 sequence shown in SEQ ID NO: 42, an HCDR3 sequence shown in SEQ ID NO: 43, and a VH sequence shown in SEQ ID NO: 37. The anti-PD1 antibody has an LCDR1 sequence shown in SEQ ID NO: 44, an LCDR2 sequence shown in SEQ ID NO: 45, an LCDR3 sequence shown in SEQ ID NO: 46, and a VL sequence shown in SEQ ID NO: 39.

[0094] In the following examples of the present invention, the heavy chain constant region of the positive control antibody wild IgG1 control antibody used was the Igγ-1 chain C region, accession: P01857, and the light chain constant region was the Igκ chain C region, accession: P01834.

[0095] In the following examples of the present invention, the C1q used was purchased from Fitzgerald, Cat No A16050201; In the following examples of the present invention, the FcγRIIIa-bio used was purchased from Sino Biological, Cat No Lc09ja0407; In the following examples of the present invention, the CD73 (5'-nuclease) specific inhibitor APCP (α,β-methylene adenosine-5'-diphosphate, 5'-α,β-methylene-adenosine diphosphate) was derived from Sigma, Cat No M3763-10MG.

[0096] In the following examples of the present invention, the sequence of the isotype control antibody, human anti-hen egg lysozyme IgG (i.e., anti-HEL antibody or human IgG abbreviated as hIgG, or isotype control) was derived from the variable region sequence of the Fab F10.6.6 sequence reported in the study by Acierno et al., titled "Affinity maturation increases the stability and plasticity of the Fv domain of an anti-protein antibody" (Acierno et al., J Mol Biol., 2007; 374(1):130-146).

Example

[0097] Example 1: Preparation of anti-CD73 antibody 19F3 1. Preparation of Hybridoma Cell Line LT014 The antigen used to prepare the anti-CD73 antibody was human NT5E-His (for NT5E, Genbank ID: NP 002517.1, position: 1-552, prepared by Akeso Biopharma, Inc.). Spleen cells from immunized mice were fused with mouse myeloma cells to prepare hybridoma cells. The hybridoma cells were screened by indirect ELISA using human NT5E-biotin (for NT5E, Genbank ID: NP 002517.1, position: 1-552, prepared by Akeso Biopharma, Inc.) as the antigen, and hybridoma cells that could secrete antibodies specifically binding to CD73 were obtained. The hybridoma cells obtained by screening were subjected to limiting dilution to obtain stable hybridoma cell lines. The hybridoma cell line was named hybridoma cell line LT014, and the monoclonal antibody secreted therefrom was named 19F3.

[0098] The hybridoma cell line LT014 (also called CD73-19F3) was deposited with the China Center for Type Culture Collection (CCTCC) on June 21, 2018, with the accession number CCTCC NO: C2018137. This deposit address is Wuhan University, Wuhan City, China, postal code 430072.

[0099] 2. Preparation of Anti-CD73 Antibody 19F3 The LT014 cell line prepared above was cultured at 37 °C using CD medium containing 5% penicillin-streptomycin in a 5% CO2 cell incubator. After 7 days, the cell culture supernatant was collected, filtered by high-speed centrifugation and vacuum filtration using a precision filtration membrane, and purified using a Hi Trap Protein A column to obtain antibody 19F3.

[0100] Example 2: Sequence Analysis of Anti-CD73 Antibody 19F3 mRNA was extracted from the cell line LT014 cultured in Example 1 according to the method described in the manual of the RNAprep Pure Cell / Bacteria Kit (Tiangen, Cat. No. DP430).

[0101] cDNA was synthesized according to the manual of Invitrogen SuperScript® III First-Strand System for RT-PCR and amplified by PCR.

[0102] The PCR amplification product was directly subjected to TA cloning according to the manual of the pEASY-T1 cloning kit (Transgen CT101).

[0103] The TA cloning product was directly sequenced, and the sequencing results of the anti-CD73 antibody 19F3 are shown below. The nucleic acid sequence of the heavy chain variable region is 363 bp in length and is shown in SEQ ID NO: 1.

[0104] The encoded amino acid sequence is 121 aa in length and is described in SEQ ID NO: 2. Here, the sequences of heavy chain CDR1, CDR2, and CDR3 are described in SEQ ID NOs: 15, 16, and 17, respectively.

[0105] The nucleic acid sequence of the light chain variable region is 339 bp in length and is shown in SEQ ID NO: 3.

[0106] The encoded amino acid sequence is 113 aa in length and is described in SEQ ID NO: 4. Here, the sequences of light chain CDR1, CDR2, and CDR3 are described in SEQ ID NOs: 18, 19, and 20, respectively.

[0107] Example 3. Design and Preparation of the Light and Heavy Chains of a Humanized Anti-Human CD73 Antibody The variable region sequences of 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM), and 19F3H2L3 (hG1DM) were obtained by computer modeling of antibody models by model design mutations based on the sequence of antibody 19F3 obtained in Example 2 and based on the three-dimensional crystal structure of human CD73 protein (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). The designed variable region sequences of the humanized antibodies were as follows: (1) Heavy and light chain variable region sequences of humanized monoclonal antibody 19F3H1L1 (hG1DM) The nucleic acid sequence of the heavy chain variable region is 363 bp in length and is shown in SEQ ID NO: 5. The encoded amino acid sequence is 121a in length and is described in SEQ ID NO: 6, while the sequences of heavy chain CDR1, CDR2, and CDR3 are described in SEQ ID NOs: 15, 16, and 17, respectively. The nucleic acid sequence of the light chain variable region is 339 bp in length and is shown in SEQ ID NO: 7. The encoded amino acid sequence is 113aa in length and is described in SEQ ID NO: 8, while the sequences of light chain CDR1, CDR2, and CDR3 are described in SEQ ID NOs: 18, 19, and 20, respectively.

[0108] (2) Heavy and light chain variable region sequences of humanized monoclonal antibody 19F3H2L2 (hG1DM) The nucleic acid sequence of the heavy chain variable region is 363 bp in length and is shown in SEQ ID NO: 9. The encoded amino acid sequence is 121a in length and is described in SEQ ID NO: 10, while the sequences of heavy chain CDR1, CDR2, and CDR3 are described in SEQ ID NOs: 15, 16, and 17, respectively. The nucleic acid sequence of the light chain variable region is 339 bp in length and is shown in SEQ ID NO: 11. The encoded amino acid sequence is 113aa in length and is described in SEQ ID NO: 12, while the sequences of light chain CDR1, CDR2, and CDR3 are described in SEQ ID NOs: 18, 19, and 20, respectively.

[0109] (3) Nucleotide sequences of the heavy and light chain variable regions of the humanized monoclonal antibody 19F3H2L3 (hG1DM) The nucleotide sequence of the heavy chain variable region is 363 bp in length and is shown in SEQ ID NO: 9. The encoded amino acid sequence is 121a in length and is described in SEQ ID NO: 10, while the sequences of heavy chain CDR1, CDR2 and CDR3 are described in SEQ ID NOs: 15, 16 and 17, respectively. The nucleotide sequence of the light chain variable region is 339 bp in length and is shown in SEQ ID NO: 13. The encoded amino acid sequence is 113aa in length and is described in SEQ ID NO: 14, while the sequences of light chain CDR1, CDR2 and CDR3 are described in SEQ ID NOs: 18, 19 and 20, respectively.

[0110] 3. Preparation of humanized 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM) and 19F3H2L3 (hG1DM) The light chain constant regions of 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM) and 19F3H2L3 (hG1DM) are the Igκ chain C region, accession: P01834.

[0111] Based on the Ig gamma-1 chain C region, a humanized antibody was obtained by introducing a point mutation from leucine to alanine at position 234 (L234A) and a point mutation from leucine to alanine at position 235 (L235A) in the heavy chain constant region (SEQ ID NO: 21) in accession: P01857, and named 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM) and 19F3H2L3 (hG1DM).

[0112] The heavy-chain cDNA and light-chain cDNA of 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM), as well as the heavy-chain cDNA and light-chain cDNA of 19F3H2L3 (hG1DM) and the heavy-chain cDNA and light-chain cDNA of 19F3H2L3 (hG1DM) were isolated and cloned into the pUC57 simple vector (provided by Genscript) to obtain pUC57 simple-19F3H1 (hG1DM) and pUC57 simple-19F3L1; pUC57 simple-19F3H2 (hG1DM), pUC57 simple-19F3L2 and pUC57 simple-19F3L3. Referring to the standard techniques described in Molecular Cloning: Laboratory Manual (2nd Edition), the full-length genes of the heavy chain and light chain synthesized by EcoRI & HindIII digestion were subcloned into the expression vector pcDNA3.1 by digestion with restriction enzymes (EcoRI & HindIII) to obtain the expression plasmids pcDNA3.1-19F3H1 (hG1DM), pcDNA3.1-19F3L1, pcDNA3.1-19F3H2 (hG1DM), pcDNA3.1-19F3L2 and pcDNA3.1-19F3L3, and the heavy chain / light chain genes of the recombinant expression plasmids were further subjected to sequence analysis. Subsequently, the designed gene combinations containing the corresponding light chain and heavy chain recombinant plasmids (pcDNA3.1-19F3H1 (hG1DM) / pcDNA3.1-19F3L1, pcDNA3.1-19F3H2 (hG1DM) / pcDNA3.1-19F3L2, and pcDNA3.1-19F3H2 (hG1DM) / pcDNA3.1-19F3H2 (hG1DM) / pcDNA3.1-19F3L3) were separately co-transfected into 293F cells, and the culture solutions were collected and purified. After confirming the sequences, endotoxin-free expression plasmids were prepared and transiently transfected into HEK293 cells for antibody expression. The culture broth was collected after 7 days and affinity purified with a Protein A column to obtain humanized antibodies.

[0113] Example 4: Kinetic Affinity Assay of Anti-CD73 Antibody with C1q and FcγRIIIa (1) Kinetic Affinity Assay of Anti-CD73 Antibody with C1q The sample dilution buffer was PBS (0.02% Tween®-20, 0.1% BSA, pH 7.4). An antibody at 50 μg / mL was immobilized on the FAB2G sensor at a fixed height of approximately 2.0 nm. The sensor was equilibrated in the buffer for 60 seconds for blocking, and the binding (serial two-fold dilution) of the immobilized antibody on the sensor to antigen C1q at a concentration of 0.63 - 10 nM was assayed for 60 seconds. The antigen-antibody was dissociated in the buffer for 60 seconds. The sensor was refreshed 4 times with 10 mM glycine pH 1.7, 5 seconds each. The shaking speed of the sample plate was 1000 rpm, the temperature was 30 °C, and the frequency was 0.6 Hz. The data was analyzed by 1:1 model fitting to obtain the affinity constant. The data acquisition software was Fortebio Data Acquisition 7.0, and the data analysis software was Fortebio Data Analysis 7.0.

[0114] According to the results shown in Table 1 and Figures 1 - 3, neither 19F3H2L3 (hG1DM) nor MEDI9447 had binding activity to C1q.

[0115]

Table 1

[0116] (2) Dynamic affinity assay of anti-CD73 antibody using FcγRIIIa The sample dilution buffer was PBS (0.02% Tween®-20, 0.1% BSA, pH 7.4). FcγRIIIa (manufactured by Sino Biological) at 0.5 μg / mL was immobilized on the SA sensor for 120 seconds. The sensor was equilibrated in the buffer for 60 seconds, and the binding of the immobilized CD16a on the sensor to the antibody was assayed for 60 seconds at a concentration of 31.3 - 500 nM (serial two-fold dilution). The antibody-antigen was dissociated in the buffer for 60 seconds. The sensor was refreshed with 10 mM NaOH. The temperature was 30 °C and the frequency was 0.6 Hz. The data was analyzed by 1:1 model fitting to obtain the affinity constant.

[0117] According to the results shown in Table 2 and Figures 4 to 6, 19F3H2L3 (hG1DM) did not bind to FcγRIIIa, while MEDI9447 had binding activity to FcγRIIIa.

[0118]

Table 2

[0119] Example 5. Detection of Inhibition of Anti-CD73 Antibody against Enzyme Activity of CD73 Expressed Intracellularly The experimental procedure was as follows. Log-phase MDA-MB-231 cells (derived from ATCC, HTB-26) in good condition were collected, resuspended in serum-free RPMI-1640 culture solution, and then counted. MDA-MB-231 cells were seeded in a 96-well plate at 3×10 4 cells / 100 μL / well. The antibody was diluted with serum-free RPMI-1640 culture solution at an initial concentration of 200 μg / mL (serial 2.5-fold dilution). The antibody was added to the 96-well plate at 50 μL / well, and the plate was incubated at 37 °C for 1 hour. After 1 hour, 50 μL of RPMI-1640-diluted 600 μM AMP was added to each well. After 3 hours, 25 μL of the cell culture supernatant was collected, transferred to a new 96-well plate, and 25 μL of 100 μM ATP was added to each well. 50 μL of CTG (CellTiter-Glo (registered trademark) One Solution Assay, Promega, Cat. No. G8461) chromogenic solution was added to each well for color development, and the data was read by a multi-label microplate tester (PerkinElmer, Cat No. 2140-0020). The isotype control antibody and the CD73-specific inhibitor APCP were used as the negative control and the positive control, respectively.

[0120] The experimental results were as follows: All of Figure 7, 19F3, 19F3H2L3 (hG1DM), 19F3H2L3 (hG1DM), and 19F3H2L3 (hG1DM) showed dose-dependent inhibition of the activity of the endogenous expressed CD73 enzyme that catalyzes AMP against adenosine A in MDA-MB-231, thereby dose-dependently reducing the average fluorescence intensity RLU generated.

[0121] The above experimental results showed that the added AMP was catalyzed by the CD73 enzyme endogenously expressed on the cell surface by MDA-MB-231 and then converted to adenosine under the condition without CD73 antibody treatment, resulting in the reduction of the inhibition of luciferase activity. However, after the addition of the antibody, CD73 bound to the antibody, so its enzyme activity decreased and AMP could not be converted to adenosine. It was suggested that the anti-CD73 antibody effectively inhibited the enzyme activity reaction of CD73 in a non-substrate competition mode and reduced the production of adenosine.

[0122] Example 6: Pharmacodynamic evaluation of an anti-CD73 specific antibody combined with an anti-PD-1-anti-VEGFA bispecific antibody in a mouse tumor cell subcutaneous xenograft model To determine the in vivo antitumor activity of the anti-CD73 specific antibody 19F3H2L3 (hG1DM) combined with the anti-PD-1 anti-VEGFA bispecific antibody VP101 (hG1DM), MC38-hPDL1 / hCD73 cells (purchased from GemPharmatech) were subcutaneously inoculated into female B6-hPD1 / hPDL1 / hCD73 mice (purchased from GemPharmatech) aged 6.7 - 7.7 weeks. When the average tumor volume reached 80 - 120 mm3, the mice were randomly divided into 4 groups of 6 mice each based on tumor volume. The grouping day was designated as D0, and administration was started on the grouping day. The administration pattern of the combination administration group was to formulate the drugs separately, administer the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) first, and then administer the anti-CD73 specific antibody 19F3H2L3 (hG1DM) 2 hours later. The modeling and specific regimen 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.

[0123]

Table 3

[0124] The results are shown in Figure 8. As a result, both the 19F3H2L3 (hG1DM) antibody and the anti-PD-1 anti-VEGFA bispecific antibody VP101 (hG1DM) can effectively inhibit the growth of mouse tumors compared with the isotype control antibody, and the combination group of 19F3H2L3 (hG1DM) and VP101 (hG1DM) showed a combined antitumor effect on the model, and the combination group was superior in tumor inhibition to the single-use groups of the drugs tested.

[0125] Furthermore, as shown in Figure 9, tumor-bearing mice were well-tolerant to single and combined use of the tested drugs 19F3H2L3 (hG1DM) and VP101 (hG1DM), and each group had no effect on the body weight of tumor-bearing mice.

[0126] Example 7: Detection of cytokine secretion promotion by an anti-CD73 specific antibody combined with an anti-PD-1-anti-VEGFA bispecific antibody using a mixed lymphocyte reaction In the present invention, the GenBank accession number of PDL1 is NP054862.1, and PDL1FL represents the full length of the PDL1 gene.

[0127] Raji cells infected with the human PD-L1 overexpression lentiviral vector 6.3 / V5-PDL1FL-BSD (a large amount of V5-BSD purchased from Invitrogen, product number: K5315-20) after virus packaging, and the Raji-PDL1 cell line was obtained after drug screening with BSD (blasticidin, Gibco product number: R210-01).

[0128] Normal human PBMCs were isolated according to the operating instructions of Ficoll-Paque (TM) Plus. Two days before the experiment, the PBMCs were thawed and cultured in complete medium (1640 + 10% FBS) in a 5% carbon dioxide incubator at 37°C. After 2 hours, when the PBMCs had recovered, SEB (final concentration 0.5 μg / mL, Toxin technology, Cat No. BT202) was added to stimulate the cells for 2 days. On the day of the test, Raji-PDL1 cells (manufactured by Akeso Biopharma) were collected, centrifuged, resuspended (in analysis medium 1640 + 10% FBS), counted, and adjusted to a concentration of 2×10 6 cells / mL. MMC (mitomycin C, final concentration 2 μg / mL, Stressmarq, Cat No. SIH-246-10MG) was added, and the mixture was treated in a 5% carbon dioxide incubator at 37°C for 1 hour. A549 lung cancer cells (purchased from the Chinese Academy of Sciences) were routinely collected, centrifuged, resuspended, and counted. After MMC treatment, PBMCs and Raji-PDL1 cells 2 days after SEB stimulation were collected. For PBMCs, 1×10 5 cells / well, for Raji-PDL1, 1×10 5 cells / well, and for A549, 1×10 4 cells / well were seeded in a 96-well U-bottom plate (Corning, Cat No. 3799) with respect to AMP (adenosine-5'-monophosphate, TCI, Cat No). According to the experimental design, antibodies were added, and negative controls (PBMC + Raji-PDL1 + A549 + AMP) and isotype controls (hIgG1 and hIgGDM) were set. The cells were incubated for a total of 3 days (the final volume of the system was 200 μL). After 3 days, the cells were centrifuged at 250×g for 5 minutes (Beckman centrifuge), and the cell supernatant was collected. The IFN-γ content was detected using a Dakewe kit.

[0129] The results are shown in Figure 10. As a result, compared with the isotype control antibody, the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) can effectively promote the secretion of the cytokine IFN-γ in the mixed lymphocyte system, and the combination group of 19F3H2L3 (hG1DM) + VP101 (hG1DM) was shown to exhibit a combined antitumor effect. The combination group was superior to the single group in promoting the secretion of the cytokine IFN-γ, and the combined activity of 150 nM 19F3H2L3 (hG1DM) + VP101 (hG1DM) was superior to the monotherapy of 300 nM 19F3H2L3 (hG1DM) or VP101 (hG1DM).

[0130] Sequence Listing Nucleic acid sequence of the heavy chain variable region of 19F3: (SEQ ID NO: 1) GAGGTGCAGCTGCAGCAGTCCGGACCAGAGCTGGTGAAGCCTGGCGCCTCCATGCGGATGTCTTGTAAGGCCTCTGGCTACAGCTTCACCGGCTATACAATGAACTGGGTGAAGCAGTCTCACGGCAAGAATCTGGAGTGGATCGGCCTGATCAACCCTTACAATGCCGGCACCAGCTATAACCAGAAGTTTAAGGGCAAGGCCACCCTGACAGTGGACAAGAGCTCCTCTACCGCCTACATGGAGCTGCTGTCCCTGACATCTGAGGATAGCGCCGTGTACTATTGCGCCCGGTCCGAGTACAGATATGGCGGCGACTACTTTGATTATTGGGGCCAGGGCACCACACTGACAGTGAGCTCC

[0131] Amino acid sequence of the heavy chain variable region of 19F3: (SEQ ID NO: 2) EVQLQQSGPELVKPGASMRMSCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNAGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSEYRYGGDYFDYWGQGTTLTVSS

[0132] Nucleic acid sequence of the light chain variable region of 19F3: (SEQ ID NO: 3) GACATCGTGATGACCCAGTCTCCAAGCTCCCTGGCAATGAGCGTGGGACAGAAGGTGACAATGTCTTGTAAGTCTAGCCAGAGCCTGCTGAACTCCTCTAATCAGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCAGGCCAGTCTCCCAAGCTGCTGGTGTACTTTGCCAGCACCAGGGAGTCCGGAGTGCCTGACAGATTCATCGGCTCCGGCTCTGGCACAGACTTCACCCTGACAATCAGCTCCGTGCAGGCAGAGGACCTGGCAGATTATTTCTGCCAGCAGCACTACGACACCCCTTATACATTTGGCGGCGGCACCAAGCTGGAGATCAAG

[0133] Amino acid sequence of the light chain variable region of 19F3: (SEQ ID NO: 4) DIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYDTPYTFGGGTKLEIK

[0134] Nucleic acid sequence of the heavy chain variable region of 19F3H1L1 (hG1DM): (SEQ ID NO: 5) CAGGTGCAGCTGCAGCAGTCTGGAGCAGAGGTGGTGAAGCCAGGAGCCTCTATGAAGATGAGCTGTAAGGCCAGCGGCTACTCCTTCACCGGCTATACAATGAACTGGGTGAAGCAGGCCCACGGCCAGAATCTGGAGTGGATCGGCCTGATCAACCCTTACAATGCCGGCACCTCTTATAACCAGAAGTTTCAGGGCAAGGCCACCCTGACAGTGGACAAGTCCACCTCTACAGCCTACATGGAGCTGAGCTCCCTGCGGAGCGAGGATACAGCCGTGTACTATTGCGCCCGGTCCGAGTACAGATATGGCGGCGACTACTTTGATTATTGGGGCCAGGGCACCACACTGACCGTGTCTAGC

[0135] Amino acid sequence of the heavy chain variable region of 19F3H1L1 (hG1DM): (SEQ ID NO: 6) QVQLQQSGAEVVKPGASMKMSCKASGYSFTGYTMNWVKQAHGQNLEWIGLINPYNAGTSYNQKFQGKATLTVDKSTSTAYMELSSLRSEDTAVYYCARSEYRYGGDYFDYWGQGTTLTVSS

[0136] Nucleic acid sequence of the light chain variable region of 19F3H1L1 (hG1DM): (SEQ ID NO: 7) GACATCGTGATGACCCAGTCCCCAAGCTCCCTGGCAATGTCTGTGGGAGAGAGGGTGACAATGTCCTGTAAGTCTAGCCAGTCTCTGCTGAACTCCTCTAATCAGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCTAAGCTGCTGGTGTACTTTGCCTCTACCAGGGAGAGCGGAGTGCCAGACAGATTCTCTGGCAGCGGCTCCGGCACAGACTTCACCCTGACAATCAGCTCCGTGCAGGCAGAGGACCTGGCAGATTATTTCTGCCAGCAGCACTACGATACCCCCTATACATTTGGCGGCGGCACCAAGCTGGAGATCAAG

[0137] Amino acid sequence of the light chain variable region of 19F3H1L1 (hG1DM): (SEQ ID NO: 8) DIVMTQSPSSLAMSVGERVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQAPKLLVYFASTRESGVPDRFSGSGSGTDFTLTISSVQAEDLADYFCQQHYDTPYTFGGGTKLEIK

[0138] Nucleic acid sequence of the heavy chain variable regions of 19F3H2L2 (hG1DM) and 19F3H2L3 (hG1DM): (SEQ ID NO: 9) CAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGGTGAAGCCAGGAGCCTCTGTGAAGGTGAGCTGTAAGGCCAGCGGCTACTCCTTCACCGGCTATACAATGAACTGGGTGAGGCAGGCACCAGGACAGAATCTGGAGTGGATCGGCCTGATCAACCCTTACAATGCCGGCACCTCTTATAACCAGAAGTTTCAGGGCAAGGTGACCCTGACAGTGGACAAGTCCACCTCTACAGCCTACATGGAGCTGAGCTCCCTGCGGAGCGAGGATACAGCCGTGTACTATTGCGCCCGGTCCGAGTACAGATATGGCGGCGACTACTTTGATTATTGGGGCCAGGGCACCACACTGACCGTGTCTAGC

[0139] Amino acid sequences of the heavy chain variable regions of 19F3H2L2 (hG1DM) and 19F3H2L3 (hG1DM): (SEQ ID NO: 10) QVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQAPGQNLEWIGLINPYNAGTSYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYCARSEYRYGGDYFDYWGQGTTLTVSS

[0140] Nucleic acid sequence of the light chain variable region of 19F3H2L2 (hG1DM): (SEQ ID NO: 11) GACATCGTGATGACCCAGTCCCCAAGCTCCCTGGCCGTGTCTGTGGGAGAGCGGGTGACAATCTCCTGTAAGTCTAGCCAGTCTCTGCTGAACTCCTCTAATCAGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCTAAGCTGCTGATCTACTTCGCCTCTACCAGGGAGAGCGGAGTGCCAGACAGATTCTCTGGCAGCGGCTCCGGCACAGACTTCACCCTGACAATCAGCTCCGTGCAGGCAGAGGACGTGGCAGATTACTATTGCCAGCAGCACTACGATACCCCCTATACATTTGGCGGCGGCACCAAGCTGGAGATCAAG

[0141] Amino acid sequence of the light chain variable region of 19F3H2L2 (hG1DM): (SEQ ID NO: 12) DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQKPGQAPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVADYYCQQHYDTPYTFGGGTKLEIK

[0142] Nucleic acid sequence of the light chain variable region of 19F3H2L3 (hG1DM): (SEQ ID NO: 13) GACATCGTGATGACCCAGTCCCCAAGCTCCCTGGCCGTGTCTGTGGGAGAGCGGGTGACAATCTCCTGTAAGTCTAGCCAGTCTCTGCTGAACTCCTCTAATCAGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCTAAGCTGCTGATCTACTTCGCCTCTACCAGGGAGAGCGGAGTGCCAGACAGATTCTCTGGCAGCGGCTCCGGCACAGACTTCACCCTGACAATCAGCTCCCTGCAGGCAGAGGACGTGGCCGTGTACTATTGCCAGCAGCACTACGATACCCCCTATACATTTGGCGGCGGCACCAAGCTGGAGATCAAG

[0143] Amino acid sequence of the light chain variable region of 19F3H2L3 (hG1DM): (SEQ ID NO: 14) DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQKPGQAPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYDTPYTFGGGTKLEIK

[0144] CDRs of 19F3 and 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM), and 19F3H2L3 (hG1DM) HCDRr1: 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)

[0145] Sequences of the heavy chain constant regions (330 aa, mutation sites underlined) of 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM), and 19F3H2L3 (hG1DM): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 21)

[0146] Sequences of the light chain constant regions (107 aa) of 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM), and 19F3H2L3 (hG1DM): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 22)

[0147] Amino acid sequence of the heavy chain of VP101 (hG1DM) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKR (SEQ ID NO: 23)

[0148] Nucleic acid sequence of the heavy chain of VP101(hG1DM)

[0149] Amino acid sequence of the light chain of VP101(hG1DM) DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 25)

[0150] Nucleic acid sequence of the light chain of VP101(hG1DM) GATATTCAGATGACTCAGAGCCCCTCCTCCCTGTCCGCCTCTGTGGGCGACAGGGTCACCATCACATGCAGTGCTTCACAGGATATTTCCAACTACCTGAATTGGTATCAGCAGAAGCCAGGAAAAGCACCCAAGGTGCTGATCTACTTCACTAGCTCCCTGCACTCAGGAGTGCCAAGCCGGTTCAGCGGATCCGGATCTGGAACCGACTTTACTCTGACCATTTCTAGTCTGCAGCCTGAGGATTTCGCTACATACTATTGCCAGCAGTATTCTACCGTGCCATGGACATTTGGCCAGGGGACTAAAGTCGAGATCAAGCGGACCGTGGCCGCTCCCAGTGTCTTCATTTTTCCCCCTAGCGACGAACAGCTGAAATCCGGGACAGCCTCTGTGGTCTGTCTGCTGAACAACTTCTACCCTAGAGAGGCAAAAGTGCAGTGGAAGGTCGATAACGCCCTGCAGAGTGGCAATTCACAGGAGAGCGTGACAGAACAGGACTCCAAAGATTCTACTTATAGTCTGTCAAGCACACTGACTCTGAGCAAGGCTGACTACGAAAAGCATAAAGTGTATGCATGTGAGGTCACCCACCAGGGGCTGAGCAGTCCAGTCACCAAGTCATTCAACAGAGGCGAGTGC (SEQ ID NO: 26)

[0151] Amino acid sequence of the bevacizumab heavy chain variable region (bevacizumab-Hv): (123 aa) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSS (SEQ ID NO: 27)

[0152] Nucleic acid sequence encoding the bevacizumab heavy chain variable region: (369 bp) GAGGTGCAGCTGGTCGAGTCCGGGGGGGGGCTGGTGCAGCCAGGCGGGTCTCTGAGGCTGAGTTGCGCCGCTTCAGGGTACACCTTCACAAACTATGGAATGAATTGGGTGCGCCAGGCACCAGGAAAGGGACTGGAGTGGGTCGGCTGGATCAACACTTACACCGGGGAACCTACCTATGCAGCCGACTTTAAGCGGCGGTTCACCTTCAGCCTGGATACAAGCAAATCCACTGCCTACCTGCAGATGAACAGCCTGCGAGCTGAGGACACCGCAGTCTACTATTGTGCTAAATATCCCCACTACTATGGGAGCAGCCATTGGTATTTTGACGTGTGGGGGCAGGGGACTCTGGTGACAGTGAGCAGC(SEQ ID NO: 28)

[0153] Amino acid sequence of the bevacizumab light chain variable region (bevacizumab-Lv): (107aa) DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK(SEQ ID NO: 29)

[0154] Nucleic acid sequence encoding the bevacizumab light chain variable region: (321bp) GATATTCAGATGACTCAGAGCCCCTCCTCCCTGTCCGCCTCTGTGGGCGACAGGGTCACCATCACATGCAGTGCTTCACAGGATATTTCCAACTACCTGAATTGGTATCAGCAGAAGCCAGGAAAAGCACCCAAGGTGCTGATCTACTTCACTAGCTCCCTGCACTCAGGAGTGCCAAGCCGGTTCAGCGGATCCGGATCTGGAACCGACTTTACTCTGACCATTTCTAGTCTGCAGCCTGAGGATTTCGCTACATACTATTGCCAGCAGTATTCTACCGTGCCATGGACATTTGGCCAGGGGACTAAAGTCGAGATCAAG(SEQ ID NO: 30)

[0155] The amino acid sequences of the three CDRs of the heavy chain variable region of bevacizumab are as follows: HCDR1: GYTFTNYG (SEQ ID NO: 31) HCDR2: INTYTGEP (SEQ ID NO: 32) HCDR3: AKYPHYYGSSHWYFDV (SEQ ID NO: 33)

[0156] The amino acid sequences of the three CDRs of the light chain variable region of bevacizumab are as follows: LCDR1: QDISNY (SEQ ID NO: 34) LCDR2: FTS (SEQ ID NO: 35) LCDR3: QQYSTVPWT (SEQ ID NO: 36)

[0157] The amino acid sequence of the heavy chain variable region of 14C12H1L1(M) is set forth in SEQ ID NO: 37. EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS (SEQ ID NO: 37)

[0158] The nucleic acid sequence encoding the heavy chain variable region of 14C12H1L1: (354bp) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCTAGT(SEQ ID NO: 38)

[0159] Amino acid sequence of the light chain variable region of 14C12H1L1(M) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKR(SEQ ID NO: 39)

[0160] Nucleic acid sequence of the light chain variable region 14C12L1(M) of 14C12H1L1(M): GATATCCAGATGACCCAGTCCCCCTCCTCTATGTCTGCCAGCGTGGGCGACCGGGTGACCTTCACATGTAGAGCCTCCCAGGATATCAACACCTACCTGTCTTGGTTTCAGCAGAAGCCCGGCAAGAGCCCTAAGACACTGATCTATCGGGCCAATAGACTGGTGAGCGGAGTGCCTTCCCGGTTCTCCGGCTCTGGCAGCGGACAGGACTATACCCTGACAATCAGCTCCCTGCAGCCAGAGGATATGGCCACATACTATTGCCTGCAGTATGACGAGTTCCCCCTGACCTTCGGGGCTGGCACTAAGCTGGAGCTGAAAAGA(SEQ ID NO: 40)

[0161] CDRs of the heavy chain variable region of 14C12H1L1(M): HCDR: GFAFSSYD (SEQ ID NO: 41) HCDR2: ISGGGRYT (SEQ ID NO: 42) HCDR3: ANRYGEAWFAY (SEQ ID NO: 43)

[0162] CDRs of the light chain variable region of 14C12H1L1(M): LCDR1: QDINTY (SEQ ID NO: 44) LCDR2: RAN (SEQ ID NO: 45) LCDR3: LQYDEFPLT (SEQ ID NO: 46)

[0163] Amino acid sequence of the first linker fragment GGGGSGGGGSGGGGS (SEQ ID NO: 47)

[0164] Amino acid sequence of the second linker fragment GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 48)

Claims

1. A therapeutic combination agent for use in the treatment of tumors, comprising an effective amount of anti-CD73 antibody and an effective amount of anti-PD-1 / anti-VEGFA bispecific antibody, The anti-CD73 antibody comprises a heavy chain variable region including HCDR1 containing the amino acid sequence of SEQ ID NO: 15, HCDR2 containing the amino acid sequence of SEQ ID NO: 16, and HCDR3 containing the amino acid sequence of SEQ ID NO: 17; and a light chain variable region including LCDR1 containing the amino acid sequence of SEQ ID NO: 18, LCDR2 containing the amino acid sequence of SEQ ID NO: 19, and LCDR3 containing the amino acid sequence of SEQ ID NO:

20. The anti-PD-1 / anti-VEGFA bispecific antibody is The first protein functional domain targeting PD-1, and A second protein functional domain targeting VEGFA Includes, (i) The first protein functional domain is immunoglobulin, and the second protein functional domain is single-chain antibody, The heavy chain variable region of the immunoglobulin comprises HCDR1 to HCDR3, each containing the amino acid sequence shown in SEQ ID NOs. 41 to 43; the light chain variable region of the immunoglobulin comprises LCDR1 to LCDR3, each containing the amino acid sequence shown in SEQ ID NOs. 44 to 46; the heavy chain variable region of the single-chain antibody comprises HCDR1 to HCDR3, each containing the amino acid sequence shown in SEQ ID NOs. 31 to 33; the light chain variable region of the single-chain antibody comprises LCDR1 to LCDR3, each containing the amino acid sequence shown in SEQ ID NOs. 34 to 36; or (ii) The second protein functional domain is immunoglobulin, and the first protein functional domain is single-chain antibody, The heavy chain variable region of immunoglobulins includes HCDR1 to HCDR3, each containing the amino acid sequences shown in SEQ ID NOs. 31 to 33; the light chain variable region of immunoglobulins includes LCDR1 to LCDR3, each containing the amino acid sequences shown in SEQ ID NOs. 34 to 36; the heavy chain variable region of single-chain antibodies includes HCDR1 to HCDR3, each containing the amino acid sequences shown in SEQ ID NOs. 41 to 43; and the light chain variable region of single-chain antibodies includes LCDR1 to LCDR3, each containing the amino acid sequences shown in SEQ ID NOs. 44 to 46. The immunoglobulin is a human IgG1 subtype, and is used in the above-mentioned combination therapy.

2. According to the EU numbering system, the immunoglobulin of the anti-PD-1 / anti-VEGFA bispecific antibody contains a heavy chain constant region, and the heavy chain constant region has the following mutations: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A, and G237A A therapeutic combination agent for use according to claim 1, comprising one of the combinations of the following.

3. The therapeutic combination agent for use according to claim 1 or 2, wherein the heavy chain variable region of the anti-CD73 antibody comprises an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 10, and the light chain variable region of the anti-CD73 antibody comprises an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, or SEQ ID NO:

14.

4. The heavy chain variable region and light chain variable region of the anti-CD73 antibody are as follows (i) to (iv): (i) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 2, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 4; (ii) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 6, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 8; (iii) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 10, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 12; and (iv) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 10, and light chain variable region containing the amino acid sequence of SEQ ID NO: 14 A therapeutic combination agent for use according to claim 1 or 2, selected from any one of the following.

5. A therapeutic combination agent for use according to claim 1 or 2, wherein the anti-CD73 antibody comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region comprising an Ig gamma-1 chain C region, and the light chain constant region comprising an Ig kappa chain C region.

6. The heavy chain constant region of the anti-CD73 antibody has the following mutations according to the EU numbering system: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A, and G237A A therapeutic combination agent for use according to claim 5, comprising one of the combinations of the following.

7. The therapeutic combination agent for use according to claim 1 or 2, wherein the anti-CD73 antibody comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 21, and the light chain constant region of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO:

22.

8. The second protein functional region of the anti-PD-1 / anti-VEGFA bispecific antibody is immunoglobulin, and the first protein functional region of the anti-PD-1 / anti-VEGFA bispecific antibody is a single-chain antibody. A therapeutic combination for use according to claim 1 or 2, wherein the immunoglobulin and single-chain antibody comprise a heavy chain variable region of the immunoglobulin containing the amino acid sequence of SEQ ID NO: 27 and a light chain variable region of the immunoglobulin containing the amino acid sequence shown in SEQ ID NO: 29, and a heavy chain variable region of the single-chain antibody containing the amino acid sequence of SEQ ID NO: 37 and a light chain variable region of the single-chain antibody containing the amino acid sequence of SEQ ID NO:

39.

9. The first protein functional region of the anti-PD-1 / anti-VEGFA bispecific antibody is immunoglobulin, and the second protein functional region of the anti-PD-1 / anti-VEGFA bispecific antibody is a single-chain antibody. A therapeutic combination for use according to claim 1 or 2, wherein the immunoglobulin and single-chain antibody comprise a heavy chain variable region of the immunoglobulin containing the amino acid sequence of SEQ ID NO: 37 and a light chain variable region of the immunoglobulin containing the amino acid sequence shown in SEQ ID NO: 39, and a heavy chain variable region of the single-chain antibody containing the amino acid sequence of SEQ ID NO: 27 and a light chain variable region of the single-chain antibody containing the amino acid sequence of SEQ ID NO:

29.

10. A therapeutic combination agent for use according to claim 1 or 2, wherein the anti-PD-1 / anti-VEGFA bispecific antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 23 and a light chain containing the amino acid sequence of SEQ ID NO:

25.

11. Anti-PD-1 / anti-VEGFA bispecific antibody, The first protein functional domain targeting PD-1, and A second protein functional domain targeting VEGFA Includes, The second protein functional domain is immunoglobulin, and the first protein functional domain is single-chain antibody. The anti-PD-1 / anti-VEGFA comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 23 and a light chain containing the amino acid sequence of SEQ ID NO: 25; A single-chain antibody is bound to the C-terminus or N-terminus of the heavy chain of an immunoglobulin; A therapeutic combination agent for use according to claim 1 or 2, wherein a first protein functional region is linked to a second protein functional region via a first linker fragment; the heavy chain variable region of a single-chain antibody is linked to the light chain variable region of a single-chain antibody via a second linker fragment; and the first and second linker fragments are identical or different.

12. The therapeutic combination agent for use according to claim 11, wherein the first linker fragment and the second linker fragment each have an amino acid sequence independently selected from SEQ ID NO: 47 and SEQ ID NO:

48.

13. The therapeutic combination agent for use according to claim 11, wherein the amino acid sequences of the first linker fragment and the second linker fragment are shown in SEQ ID NO:

48.

14. Anti-PD-1 / anti-VEGFA bispecific antibody, A first protein functional region linked directly to a second protein functional region or via a linker fragment; and / or a heavy chain variable region of a single-chain antibody linked directly to the light chain variable region of the single-chain antibody or via a linker fragment. A therapeutic combination agent for use according to claim 1 or 2, comprising:

15. Anti-PD-1 / anti-VEGFA bispecific antibody, (GGGGS) ｎ A therapeutic combination agent for use according to claim 14, comprising a linker fragment containing (where n is 1, 2, 3, 4, 5, or 6).

16. The therapeutic combination agent for use according to claim 1 or 2, wherein a single-chain antibody is ligated to the C-terminus or N-terminus of the heavy chain of the immunoglobulin of an anti-PD-1 / anti-VEGFA bispecific antibody.

17. The therapeutic combination agent for use according to claim 1 or 2, wherein the therapeutic combination agent further comprises an effective amount of a chemotherapeutic agent.

18. A therapeutic combination agent for use according to claim 17, wherein the antitumor chemotherapy agent comprises an alkylating agent, an antimetabolite, an antitumor antibiotic, a plant-based anticancer agent, a hormone, or an immunoassay agent.

19. The therapeutic combination agent for use according to claim 1 or 2, wherein the therapeutic combination agent further comprises one or more pharmaceutically acceptable adjuvants.

20. The combination therapeutic agent for use according to claim 1 or 2, wherein the tumor is selected from one or more of the following: pancreatic cancer, ovarian cancer, colorectal cancer, head and neck cancer, brain tumor, laryngeal cancer, nasopharyngeal cancer, esophageal cancer, esophageal squamous cell carcinoma, thyroid cancer, mesothelioma, lung cancer, breast cancer, liver cancer, stomach cancer, biliary tract cancer, kidney cancer, fallopian tube cancer, endometrial cancer, cervical cancer, bladder cancer, urothelial carcinoma, prostate cancer, testicular cancer, skin cancer, melanoma, myeloma, plasma cell carcinoma, vulvar cancer, leukemia, lymphoma, bone cancer, and osteosarcoma.

21. a) Lung cancer is selected from one or more of the following: non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer; b) The gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; c) The lymphoma is Hodgkin lymphoma or non-Hodgkin lymphoma; or d) The therapeutic combination agent for use according to claim 20, wherein the tumor is a solid tumor of the MSI-H / dMMR phenotype selected from one or more of the following: colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumor.

22. The therapeutic combination agent for use according to claim 1 or 2, wherein the tumor is colorectal cancer, a solid tumor, or non-squamous non-small cell lung cancer.

23. A therapeutic combination agent for use according to claim 1 or 2, comprising an effective amount of anti-CD73 antibody approximately 30 mg / kg.

24. A therapeutic combination agent for use according to claim 1 or 2, comprising an effective amount of approximately 0.5 mg / kg of anti-PD-1 / anti-VEGFA bispecific antibody.

25. The therapeutic combination agent for use according to claim 1 or 2, comprising an anti-CD73 antibody and an anti-PD-1 / anti-VEGFA bispecific antibody in a mass ratio of 1:1 or 2:1.

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