CD39 / CD73 bispecific antigen binding proteins and uses thereof

Abstract

An isolated antigen binding protein is provided comprising a first binding domain capable of binding to a CD39 protein and a second binding domain capable of binding to a CD73 protein. Also provided are nucleic acid molecules encoding the isolated antigen-binding proteins, vectors comprising the nucleic acid molecules, cells, pharmaceutical compositions comprising the isolated antigen-binding proteins, methods of preparing the isolated antigen-binding proteins, and uses of the isolated antigen-binding proteins.

Description

CD39 / CD73 bispecific antigen binding proteins and uses thereof Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a CD39 / CD73 bispecific antigen binding protein and its use. Background Art

[0002] CD39, also known as ectonucleoside triphosphate diphosphohydrolase 1 (ENTPDase1), is an integral membrane protein that hydrolyzes ATP phosphate to produce ADP and AMP. CD39 is expressed in many solid tumor cells and can participate in tumor immune escape by inhibiting the activation, clonal expansion, and homing of tumor-specific T cells, thereby impairing tumor cell killing by effector T lymphocytes. CD39 directly regulates cancer cell growth, differentiation, invasion, migration, metastasis, and angiogenesis. CD39 is important for both the initiation and progression of angiogenesis.

[0003] CD73, also known as NT5E, is an extracellular 5-nuclease (NT5E) with a molecular weight of approximately 70 kDa. It is anchored to the cell surface via glycophosphatidylinositol (GPI) and exists primarily as a dimer. Under normal physiological conditions, CD73 is expressed in various tissues and organs, including the liver, large intestine, kidneys, spleen, lungs, ovaries, and lymph nodes.

[0004] Functionally, CD39 can bind to extracellular ATP (eATP) and hydrolyze it into AMP; CD73 hydrolyzes AMP into ADO. In the ATP-ADO pathway, CD39 is the rate-limiting enzyme for eATP hydrolysis, while CD73 is the rate-limiting enzyme for AMP hydrolysis. CD39 and CD73 control the generation of purine nucleotides, thereby regulating nucleotide signaling. CD39 can hydrolyze eATP, which can effectively activate immune cell activity, thereby causing tumor immune escape; while the ADO produced by CD73 hydrolysis can activate immune cells and reduce the immune function of T cells, thereby leading to tumor immune escape.

[0005] Therefore, there is an urgent need to develop drugs targeting CD39 / CD73 to provide new directions for tumor immunotherapy.

[0006] Summary of the Invention

[0007] The present application provides an isolated antigen-binding protein having one or more of the following properties: (1) -9 M or lower K D The value of binding to CD39 protein; (2) 1×10 -9 M or lower K D(3) has an inhibitory effect on the enzymatic activity of CD39 antigen; (4) has an inhibitory effect on the enzymatic activity of CD73 antigen; (5) has thermal stability; (6) can bind to human CD39 and / or cynomolgus monkey CD73; (7) can bind to human CD73 and / or cynomolgus monkey CD73; (8) can inhibit the growth of tumor cells.

[0008] In one aspect, the present application provides an isolated antigen-binding protein comprising a first binding domain and a second binding domain, wherein the first binding domain is capable of binding to CD39 protein, and the second binding domain is capable of binding to CD73 protein.

[0009] In certain embodiments, the first binding domain of the isolated antigen binding protein comprises an antibody or an antigen-binding fragment thereof.

[0010] In certain embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a single-chain antibody, a chimeric antibody, a multispecific antibody, a humanized antibody, and a fully human antibody.

[0011] In certain embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv, F(ab')2, scFv, VHH, di-scFv, and dAb fragments.

[0012] In certain embodiments, the first binding domain of the isolated antigen binding protein is a VHH.

[0013] In certain embodiments, the first binding domain of the isolated antigen-binding protein comprises at least one CDR in an antibody heavy chain variable region VHH, wherein the VHH comprises the amino acid sequence shown in SEQ ID NO:8.

[0014] In certain embodiments, the first binding domain of the isolated antigen binding protein comprises CDR3, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:1.

[0015] In certain embodiments, the first binding domain of the isolated antigen binding protein comprises CDR2, and the CDR2 comprises the amino acid sequence shown in SEQ ID NO:2.

[0016] In certain embodiments, the first binding domain of the isolated antigen binding protein comprises CDR1, and the CDR1 comprises the amino acid sequence shown in SEQ ID NO:3.

[0017] In certain embodiments, the first binding domain of the isolated antigen-binding protein comprises FR1, the C-terminus of FR1 is directly or indirectly connected to the N-terminus of CDR1, and the FR1 comprises the amino acid sequence shown in SEQ ID NO:4.

[0018] In certain embodiments, the first binding domain of the isolated antigen binding protein comprises FR2, wherein the FR2 is located between the CDR1 and the CDR2, and the FR2 comprises the amino acid sequence shown in SEQ ID NO:5.

[0019] In certain embodiments, the first binding domain of the isolated antigen binding protein comprises FR3, wherein the FR3 is located between the CDR2 and the CDR3, and the FR3 comprises the amino acid sequence shown in SEQ ID NO:6.

[0020] In certain embodiments, the first binding domain of the isolated antigen-binding protein comprises FR4, the N-terminus of the FR4 is directly or indirectly connected to the C-terminus of the CDR3, and the FR4 comprises the amino acid sequence shown in SEQ ID NO:7.

[0021] In certain embodiments, the first binding domain of the isolated antigen-binding protein comprises a VHH, and the VHH comprises the amino acid sequence shown in SEQ ID NO:8.

[0022] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises an antibody or an antigen-binding fragment thereof.

[0023] In certain embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a single-chain antibody, a chimeric antibody, a multispecific antibody, a humanized antibody, and a fully human antibody.

[0024] In certain embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv, F(ab')2, scFv, VHH, di-scFv, and dAb fragments.

[0025] In certain embodiments, the second binding domain of the isolated antigen-binding protein comprises at least one CDR in the antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 33.

[0026] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:9 or SEQ ID NO:27.

[0027] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 28.

[0028] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:29.

[0029] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are selected from the group consisting of:

[0030] (1) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 9;

[0031] (2) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 29, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 28, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27.

[0032] In certain embodiments, the second binding domain of the isolated antigen-binding protein comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 30.

[0033] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 31.

[0034] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 32.

[0035] In certain embodiments, the second binding domain of the isolated antigen-binding protein comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:15.

[0036] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1, H-FR2, H-FR3 and H-FR4 comprise an amino acid sequence selected from the group consisting of:

[0037] (1) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 12, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 13, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 14, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 15;

[0038] (2) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 30, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 31, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 32, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 15.

[0039] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 33.

[0040] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises an antibody heavy chain constant region.

[0041] In certain embodiments, the antibody heavy chain constant region is derived from an IgG heavy chain constant region.

[0042] In certain embodiments, the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

[0043] In certain embodiments, the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region.

[0044] In certain embodiments, the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:43.

[0045] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 41.

[0046] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises at least one CDR in the light chain variable region VL of an antibody, wherein the VL comprises the amino acid sequence shown in SEQ ID NO: 24 or SEQ ID NO: 40.

[0047] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 34.

[0048] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 18 (WAS) or SEQ ID NO: 35 (LAS).

[0049] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 36.

[0050] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise an amino acid sequence selected from the group consisting of:

[0051] (1) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 19, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 18 (WAS), and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 17;

[0052] (2) The LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 36, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 35 (LAS), and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 34.

[0053] In certain embodiments, the second binding domain of the isolated antigen-binding protein comprises L-FR1, the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the L-CDR1, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 20 or SEQ ID NO: 37.

[0054] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises L-FR2, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:21 or SEQ ID NO:38.

[0055] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises L-FR3, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:22 or SEQ ID NO:39.

[0056] In certain embodiments, the second binding domain of the isolated antigen-binding protein comprises L-FR4, the N-terminus of the L-FR4 is directly or indirectly connected to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:23.

[0057] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR1, L-FR2, L-FR3 and L-FR4 comprise an amino acid sequence selected from the group consisting of:

[0058] (1) the L-FR1 comprises the amino acid sequence of SEQ ID NO: 20, the L-FR2 comprises the amino acid sequence of SEQ ID NO: 21, the L-FR3 comprises the amino acid sequence of SEQ ID NO: 22, and the L-FR4 comprises the amino acid sequence of SEQ ID NO: 23;

[0059] (2) The L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 37, the L-FR2 comprises the amino acid sequence shown in SEQ ID NO: 39, the L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 39, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 23.

[0060] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises a VL comprising the amino acid sequence shown in SEQ ID NO: 24 or SEQ ID NO: 40.

[0061] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises an antibody light chain constant region.

[0062] In certain embodiments, the antibody light chain constant region is derived from an Ig kappa constant region.

[0063] In certain embodiments, the antibody light chain constant region is derived from a human Ig kappa constant region.

[0064] In certain embodiments, the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO:44.

[0065] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises an antibody light chain comprising the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 42.

[0066] In certain embodiments, the second domain of the isolated antigen binding protein comprises VH and VL, and the VH and VL comprise an amino acid sequence selected from the group consisting of:

[0067] (1) the VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the VL comprises the amino acid sequence shown in SEQ ID NO: 24;

[0068] (2) The VH comprises the amino acid sequence shown in SEQ ID NO: 33, and the VL comprises the amino acid sequence shown in SEQ ID NO: 40.

[0069] In certain embodiments, the second binding domain of the isolated antigen binding protein comprises an antibody heavy chain and an antibody light chain, and the antibody heavy chain and the antibody light chain comprise an amino acid sequence selected from the group consisting of:

[0070] (1) The antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 25, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 26;

[0071] (2) The antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO:41, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO:42.

[0072] In certain embodiments, the first binding domain and the second binding domain of the isolated antigen binding protein are directly or indirectly linked.

[0073] In certain embodiments, the C-terminus of the first binding domain of the isolated antigen binding protein is linked to the N-terminus of the second binding domain.

[0074] In certain embodiments, the C-terminus of the first binding domain of the isolated antigen binding protein is linked to the N-terminus of the heavy chain variable region of the second binding domain.

[0075] In certain embodiments, the first binding domain and the second binding domain of the isolated antigen binding protein are connected by a linker.

[0076] In certain embodiments, the linker comprises a peptide linker.

[0077] In certain embodiments, the linker comprises an amino acid sequence of (GGGGS)n, wherein n is any positive integer between 0 and 10.

[0078] In certain embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO:47.

[0079] In certain embodiments, the isolated antigen binding protein comprises two of the first binding domains.

[0080] In certain embodiments, the two first binding domains of the isolated antigen binding protein are directly or indirectly linked to the N-terminus of the second binding domain, respectively.

[0081] In certain embodiments, the two first binding domains of the isolated antigen binding protein are directly or indirectly linked to the N-termini of the two heavy chains of the second binding domain, respectively.

[0082] In certain embodiments, the isolated antigen binding protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the heavy chain of the first binding domain and the second binding domain.

[0083] In certain embodiments, the N-termini of the heavy chains of the first binding domain and the second binding domain of the isolated antigen binding protein are directly or indirectly linked.

[0084] In certain embodiments, the N-termini of the heavy chains of the first binding domain and the second binding domain of the isolated antigen binding protein are connected by a linker.

[0085] In certain embodiments, the first polypeptide chain of the isolated antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:45 or SEQ ID NO:46.

[0086] In certain embodiments, the second polypeptide chain of the isolated antigen binding protein comprises the light chain of the second binding domain.

[0087] In certain embodiments, the second polypeptide chain of the isolated antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:26 or SEQ ID NO:42.

[0088] In certain embodiments, the isolated antigen binding protein comprises two of the first polypeptide chains and two of the second polypeptide chains.

[0089] On the other hand, the present application also provides a drug combination, which comprises an antigen binding protein capable of binding to CD39 and an antigen binding protein capable of binding to CD73.

[0090] In certain embodiments, the antigen binding protein capable of binding to CD39 has one or more of the following properties:

[0091] (1) binds to human CD39 with an EC50 of less than about 0.5 μg / mL as determined by FACS assay;

[0092] (2) binds to cynomolgus macaque (cyno) CD39 with an EC50 of less than about 0.7 μg / mL as determined by FACS assay;

[0093] (3) As determined by Biacore assay, the -8 The KD of M binds to human CD39;

[0094] (4) As determined by Biacore assay, the -7 The KD of M binds to cynomolgus macaque (cyno) CD39;

[0095] (5) inhibiting ATPase activity in cells expressing CD39 with an EC50 of less than about 0.8 μg / mL as measured by an ATPase activity assay;

[0096] (6) Inhibit CD39 function in tumor cells.

[0097] In certain embodiments, the antigen binding protein capable of binding to CD39 comprises at least one CDR in the heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:8.

[0098] In certain embodiments, the antigen binding protein capable of binding to CD39 comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 1.

[0099] In certain embodiments, the antigen binding protein capable of binding to CD39 comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2.

[0100] In certain embodiments, the antigen binding protein capable of binding to CD39 comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3.

[0101] In certain embodiments, the antigen binding protein capable of binding to CD39 comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:8.

[0102] In certain embodiments, the antigen binding protein capable of binding to CD39 is a VHH.

[0103] In certain embodiments, the antigen binding protein capable of binding to CD73 has one or more of the following properties:

[0104] (1) Ability to specifically bind to CD73 or its functionally active fragments;

[0105] (2) can inhibit the enzymatic activity of CD73;

[0106] (3) can induce the internalization of CD73 on the cell surface;

[0107] (4) Ability to inhibit the growth and / or proliferation of tumor cells.

[0108] In certain embodiments, the antigen binding protein capable of binding to CD73 comprises at least one CDR in the antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 33.

[0109] In certain embodiments, the antigen binding protein capable of binding to CD73 comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:9 or SEQ ID NO:27.

[0110] In certain embodiments, the antigen binding protein capable of binding to CD73 comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 28.

[0111] In certain embodiments, the antigen binding protein capable of binding to CD73 comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:29.

[0112] In certain embodiments, the antigen binding protein capable of binding to CD73 comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 33.

[0113] In certain embodiments, the antigen binding protein capable of binding to CD73 comprises an antibody heavy chain constant region.

[0114] In certain embodiments, the antibody heavy chain constant region is derived from an IgG heavy chain constant region.

[0115] In certain embodiments, the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

[0116] In certain embodiments, the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region.

[0117] In certain embodiments, the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:43.

[0118] In certain embodiments, the antigen binding protein capable of binding to CD73 comprises an antibody heavy chain comprising the amino acid sequence shown in SEQ ID NO: 25 or SEQ ID NO: 41.

[0119] In certain embodiments, the antigen binding protein capable of binding to CD73 comprises at least one CDR in the antibody light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO: 24 or SEQ ID NO: 40.

[0120] In certain embodiments, the antigen binding protein capable of binding to CD73 comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 34.

[0121] In certain embodiments, the antigen binding protein capable of binding to CD73 comprises LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 18 (WAS) or SEQ ID NO: 35 (LAS).

[0122] In certain embodiments, the antigen binding protein capable of binding to CD73 comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 36.

[0123] In certain embodiments, the antigen binding protein capable of binding to CD73 comprises a VL comprising the amino acid sequence shown in SEQ ID NO: 24 or SEQ ID NO: 40.

[0124] In certain embodiments, the antigen binding protein capable of binding to CD73 comprises an antibody light chain constant region.

[0125] In certain embodiments, the antibody light chain constant region is derived from a human Ig kappa constant region.

[0126] In certain embodiments, the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO:44.

[0127] In certain embodiments, the antigen binding protein capable of binding to CD73 comprises an antibody light chain comprising the amino acid sequence shown in SEQ ID NO: 26 or SEQ ID NO: 42.

[0128] In certain embodiments, the antigen binding protein capable of binding to CD73 comprises VH and VL, wherein VH and VL comprise an amino acid sequence selected from the group consisting of:

[0129] (1) the VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the VL comprises the amino acid sequence shown in SEQ ID NO: 24;

[0130] (2) The VH comprises the amino acid sequence shown in SEQ ID NO: 33, and the VL comprises the amino acid sequence shown in SEQ ID NO: 40.

[0131] In certain embodiments, the antigen binding protein capable of binding to CD73 comprises an antibody heavy chain and an antibody light chain, wherein the antibody heavy chain and the antibody light chain comprise an amino acid sequence selected from the group consisting of:

[0132] (1) The antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 25, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 26;

[0133] (2) The antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO:41, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO:42.

[0134] In another aspect, the present application provides isolated one or more nucleic acid molecules encoding the isolated antigen binding protein.

[0135] In another aspect, the present application provides a vector comprising the nucleic acid molecule.

[0136] In another aspect, the present application provides a cell comprising the nucleic acid molecule or the vector.

[0137] In another aspect, the present application provides a pharmaceutical composition comprising the isolated antigen-binding protein, the pharmaceutical combination, the nucleic acid molecule, the vector and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0138] In another aspect, the present application provides a method for preparing the isolated antigen-binding protein, which comprises culturing the cell under conditions allowing the isolated antigen-binding protein to be expressed.

[0139] On the other hand, the present application provides the use of the isolated antigen-binding protein, the drug combination, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease and / or condition.

[0140] In certain embodiments, the disease and / or condition is mediated by CD39 and / or CD73.

[0141] In certain embodiments, the disease and / or condition comprises a tumor.

[0142] In certain embodiments, the tumor comprises a solid tumor and / or a hematological tumor.

[0143] In certain embodiments, the disease and / or condition comprises pancreatic cancer and / or colorectal cancer.

[0144] On the other hand, the present application provides a method for detecting CD39 and / or CD73 in a sample, which comprises administering the isolated antigen-binding protein, the pharmaceutical combination, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition.

[0145] In certain embodiments, the method is an in vitro method and / or an ex vivo method.

[0146] On the other hand, the present application provides the use of the isolated antigen-binding protein, the drug combination, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition in preparing a kit for detecting the presence of CD39 and / or CD73 in a sample.

[0147] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0148] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0149] Figure 1 shows a schematic diagram of the structures of CD39 / CD73 bispecific antibodies 700035 and 700038.

[0150] FIG2 shows the reducing SDS-PAGE image of 700035.

[0151] FIG3 shows the reducing SDS-PAGE image of 700038.

[0152] FIG4 shows a non-reducing SDS-PAGE image of 700035.

[0153] FIG5 shows a non-reducing SDS-PAGE image of 700038.

[0154] FIG6 shows the inhibition curves of the bispecific antibodies 700035 and 700038 on CD39 enzyme activity.

[0155] Figure 7 shows the inhibition curve of bispecific antibody 700035 on cell surface CD73 enzyme activity.

[0156] Figure 8 shows the inhibition curve of bispecific antibody 700038 on the enzymatic activity of soluble CD73 antigen.

[0157] FIG9 shows the anti-tumor effect of the antigen binding protein described in the present application in a pancreatic cancer mouse model.

[0158] FIG10 shows the anti-tumor effect of the antigen binding protein described in the present application in a colorectal cancer mouse model. DETAILED DESCRIPTION

[0159] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0160] Definition of terms

[0161] In this application, the term "CD39" generally refers to the extracellular nucleoside triphosphate diphosphohydrolase 1 polypeptide encoded by the ENTPD1 gene in humans. Other names for CD39 include ENTPD1, ​​ATPDase, NTPDase-1, and SPG64. CD39 catalyzes the hydrolysis of the gamma- and beta-phosphate residues of extracellular nucleoside triphosphates (NTPs; e.g., adenosine triphosphate or ATP) and nucleoside diphosphates (NDPs; e.g., adenosine diphosphate or ADP), converting these molecules into nucleoside monophosphate (NMPs; e.g., adenosine monophosphate or AMP) derivatives. An exemplary amino acid sequence of CD39 is shown in NCBI Reference Sequence: NP_001767.3.

[0162] In this application, the term "CD73" is also referred to as "NT5E," "cluster of differentiation 73," "5'-nucleotidase (5'-NT)" or "ecto-5'-nucleotidase." The term encompasses "full-length," unprocessed CD73 as well as any form of CD73 produced by cellular processing. In this application, the term "CD73" includes full-length wild-type CD73 and mutants, fragments, variants, isoforms and homologs thereof. In certain embodiments, the CD73 may include human CD73. For example, the sequence for human CD73 can be found under Uniprot Accession No. P21589.

[0163] In this application, the term "isolated" generally refers to a substance obtained artificially from its natural state. If a substance or component is "isolated" in nature, it may be that its natural environment has been altered, or that the substance has been separated from its natural environment, or both. For example, a polynucleotide or polypeptide that is naturally present in a living animal and has not been separated is considered isolated. The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0164] In the present application, the term "isolated antigen-binding protein" generally refers to a protein with antigen-binding ability obtained from a natural state by artificial means. The "isolated antigen-binding protein" may comprise a portion that binds to an antigen and, optionally, a framework or framework portion that allows the antigen-binding portion to adopt a conformation that promotes the binding of the antigen-binding portion to the antigen. The antigen-binding protein may comprise, for example, a protein framework region (FR) derived from an antibody or an alternative protein framework region or an artificial framework region having a transplanted CDR or CDR derivative. Such frameworks include, but are not limited to, antibody-derived framework regions that contain mutations introduced, for example, to stabilize the three-dimensional structure of the antigen-binding protein and fully synthetic framework regions that contain, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20: 639-654 (2004). Examples of antigen binding proteins include, but are not limited to, human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single-chain antibodies; bifunctional antibodies; trifunctional antibodies; tetrafunctional antibodies; Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, dAb, IgD antibody; IgE antibody; IgM antibody; IgG1 antibody; IgG2 antibody; IgG3 antibody; or IgG4 antibody and fragments thereof.

[0165] In the present application, the isolated antigen-binding proteins may comprise more than one binding domain. In the present application, the binding domains may target different antigens. In the present application, the binding domains may target different epitopes of the same antigen.

[0166] In this application, the terms "variable domain" and "variable region" are used interchangeably and generally refer to a portion of an antibody heavy chain and / or light chain. The variable domains of the heavy and light chains may be referred to as "V H ” and “V L ” (or “VH” and “VL”, respectively). These domains are generally the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen-binding site.

[0167] In this application, the term "variable" generally refers to the fact that certain segments of the variable domain may differ significantly in sequence between antibodies. The variable domain mediates antigen binding and determines the specificity of a particular antibody for its specific antigen. However, variability is not evenly distributed throughout the variable domain. It is generally concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains. The more highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of natural heavy and light chains each contain four FR regions, most of which adopt a β-pleated sheet configuration and are connected by three CDRs, which form a loop connection and, in some cases, form part of the β-pleated sheet structure. The CDRs in each chain are held together in close proximity by the FR regions, and the CDRs from the other chain together contribute to the formation of the antibody's antigen binding site (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).

[0168] In this application, the term "antibody" generally refers to an immunoglobulin or a fragment or derivative thereof, encompassing any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and transplanted antibodies. Unless otherwise modified by the term "complete," as in "complete antibody," for the purposes of the present invention, the term "antibody" also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen binding function (e.g., specific binding to CLDN18.2). Typically, such fragments should include an antigen binding domain. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies are composed of five basic heterotetrameric units and an additional polypeptide called the J chain, containing 10 antigen-binding sites. IgA antibodies, on the other hand, consist of two to five basic four-chain units that can combine with the J chain to form multivalent combinations. For IgG, the four-chain unit is generally approximately 150,000 daltons. Each L chain is linked to an H chain by a covalent disulfide bond, while the two H chains are interconnected by one or more disulfide bonds that depend on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus and a constant domain at its other end. The VL corresponds to the VH, and the CL corresponds to the first constant domain (CH1) of the heavy chain. Specific amino acid residues are believed to form the interface between the light and heavy chain variable domains. VH and VL pair together to form a single antigen binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from any vertebrate species can be divided into one of two distinct types, called κ and λ, based on the amino acid sequence of its constant domain. According to the amino acid sequence of the heavy chain (CH) constant domain, immunoglobulins can be divided into different classes or isotypes. There are currently five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, with heavy chains designated α, δ, ε, γ and μ, respectively.The gamma and alpha classes are further divided into subclasses based on relatively small differences in CH sequence and function, eg, humans express the following subclasses: IgGl, IgG2A, IgG2B, IgG3, IgG4, IgAl, and IgKl.

[0169] In this application, the term "CDR", also known as "complementarity determining region", generally refers to a region in an antibody variable domain whose sequence is highly variable and / or forms a structurally defined loop. For example, an antibody may include six CDRs; three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3). In certain embodiments, naturally occurring camelid antibodies composed only of heavy chains can function normally and stably in the absence of light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al, Nature Struct. Biol. 3:733-736 (1996).

[0170] In this application, the term "FR" generally refers to the more highly conserved portion of an antibody variable domain, which is referred to as a framework region. Typically, the variable domains of a native heavy chain and / or light chain each comprise four FR regions, i.e., four in VH (H-FR1, H-FR2, H-FR3, and H-FR4), and / or four in VL (L-FR1, L-FR2, L-FR3, and L-FR4).

[0171] In this application, the term "antigen-binding fragment" generally refers to one or more fragments that have the ability to specifically bind to an antigen. In this application, the antigen-binding fragment can include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and / or dAb.

[0172] In this application, the term "Fab" generally refers to the antigen-binding fragment of an antibody. As mentioned above, papain can be used to digest intact antibodies. Papain digestion of an antibody produces two identical antigen-binding fragments, namely the "Fab" fragment, and a residual "Fc" fragment (i.e., the Fc region, as described above). The Fab fragment can be composed of a complete L chain with the variable region of a heavy chain and the H chain (V H ) of the first constant region (C H 1) Composition.

[0173] As used herein, the term "Fab' fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody that is slightly larger than a Fab fragment. For example, a Fab' fragment may include all of the light chain, all of the heavy chain variable regions, and all or part of the first and second constant regions of the heavy chain. For example, a Fab' fragment may also include part or all of the 220-330 amino acid residues of the heavy chain.

[0174] As used herein, the term "F(ab')2" generally refers to an antibody fragment produced by pepsin digestion of an intact antibody. The F(ab')2 fragment contains two Fab fragments held together by disulfide bonds and a portion of the hinge region. The F(ab')2 fragment has divalent antigen-binding activity and is capable of cross-linking antigens.

[0175] In this application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, comprising all or part of the heavy chain variable region and the light chain variable region, and lacking the heavy chain constant region and the light chain constant region. The heavy chain variable region and the light chain variable region include, for example, CDRs. For example, an Fv fragment comprises all or part of the amino-terminal variable region of approximately 110 amino acids of the heavy and light chains.

[0176] In this application, the term "scFv" generally refers to a fusion protein comprising at least one variable region antibody fragment comprising a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are adjacent (e.g., via a synthetic linker such as a short flexible polypeptide linker) and can be expressed in the form of a single-chain polypeptide, and wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, as used in this application, scFv can have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv can include VL-linker-VH or can include VH-linker-VL.

[0177] In this application, the term "dAb" generally refers to an antigen-binding fragment having a VH domain, a VL domain, or having a VH domain or a VL domain, with reference to, for example, Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6), with reference to Holt et al., Trends Biotechnol., 2003, 21(11): 484-490; and with reference to, for example, WO 06 / 030220, WO 06 / 003388 and other published patent applications of Domantis Ltd.

[0178] In this application, the term "monoclonal antibody" generally refers to an antibody molecule preparation of single molecule composition. Monoclonal antibodies are generally highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (generally having different antibodies for different determinants), each monoclonal antibody is for a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without being contaminated by other immunoglobulins. The modifier "monoclonal" represents the characteristic of the antibody obtained from a substantially homogeneous antibody population, and is not to be construed as needing to produce antibodies by any ad hoc method. For example, the monoclonal antibody used in this application can be prepared in hybridoma cells, or can be prepared by recombinant DNA methods.

[0179] In this application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody of an experimental animal such as a rodent ("parent antibody"), and the constant region is derived from a human antibody, so that the resulting chimeric antibody is less likely to induce an adverse immune response in a human individual than the parent (e.g., mouse-derived) antibody.

[0180] In this application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody (e.g., a mouse antibody) are replaced by corresponding amino acids derived from human immunoglobulins. In the CDR region, small additions, deletions, insertions, replacements, or modifications of amino acids may also be permitted, as long as they still retain the ability of the antibody to bind to a specific antigen. A humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region. A "humanized antibody" retains antigenic specificity similar to that of the original antibody. The "humanized" form of a non-human (e.g., mouse) antibody may minimally comprise a chimeric antibody derived from a sequence of a non-human immunoglobulin. In some cases, the CDR region residues in a human immunoglobulin (recipient antibody) may be replaced with CDR region residues of a non-human species (donor antibody) (such as mouse, rat, rabbit, or non-human primate) with desired properties, affinity, and / or ability. In some cases, the FR region residues of a human immunoglobulin may be replaced with corresponding non-human residues. In addition, humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made to further improve antibody performance, such as binding affinity.

[0181] The term "fully human antibody" generally refers to an antibody that contains only human immunoglobulin protein sequences. If it is produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell, then the fully human antibody may contain mouse sugar chains. Similarly, a "mouse antibody" or a "rat antibody" refers to an antibody that contains only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be generated in humans and in transgenic animals with human immunoglobulin germline sequences by phage display or other molecular biology methods. Exemplary techniques that can be used to make antibodies are described in U.S. Patents: 6,150,584, 6,458,592, and 6,420,140. Other techniques, such as the use of libraries, are known in the art.

[0182] In the present application, the term "directly connected" is contrasted with the term "indirectly connected", and the term "directly connected" generally refers to direct connection. For example, the direct connection can be a situation where the substances are directly connected without a spacer. The spacer can be a linker. For example, the linker can be a peptide linker. The term "indirectly connected" generally refers to a situation where the substances are not directly connected. For example, the indirect connection can be a situation where the substances are connected through a spacer. For example, in the isolated antigen-binding protein described in the present application, the C-terminus of the L-FR1 and the N-terminus of the LCDR1 can be directly or indirectly connected.

[0183] In this application, the term "pharmaceutical combination" generally refers to a combination comprising at least two active ingredients / therapeutic agents. In some embodiments, each active ingredient / therapeutic agent can be prepared as an independent formulation (solid, liquid, gel, etc.), in some embodiments, each active ingredient / therapeutic agent can be present in different containers and can be formulated into a desired formulation simultaneously or separately with a suitable carrier when needed; in some embodiments, each active ingredient / therapeutic agent can be from different sources; in some embodiments, each active ingredient / therapeutic agent can be mixed to form a pharmaceutical composition.

[0184] As used herein, the term "isolated nucleic acid molecule" generally refers to isolated forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, separated from their natural environment or artificially synthesized.

[0185] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. A vector can be used to transform, transduce, or transfect a host cell, allowing the genetic material elements it carries to be expressed in the host cell. For example, vectors may include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. Types of animal viruses used as vectors may include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain a replication initiation site. Vectors may also include components that assist in their entry into cells, such as viral particles, liposomes, or protein coats, but are not limited to these substances.

[0186] In the present application, the term "cell" generally refers to a single cell, cell line or cell culture that can be or has been a recipient of a subject's plasmid or vector, including nucleic acid molecules of the present invention or vectors of the present invention. Cells can include the offspring of single cells. Due to natural, accidental or intentional mutations, offspring may not necessarily be identical to the original mother cell (in the form of total DNA complement or on the genome). Cells can include cells transfected in vitro with the vectors described herein. Cells can be bacterial cells (e.g., Escherichia coli), yeast cells or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells. In certain embodiments, cells are mammalian cells.

[0187] In this application, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or condition. The pharmaceutical composition may comprise the isolated antigen-binding protein described herein, the nucleic acid molecule described herein, the carrier described herein and / or the cell described herein, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may also comprise a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention includes, but is not limited to, liquid, frozen and lyophilized compositions.

[0188] In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. Physiologically acceptable carriers may include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions, such as sodium; and / or nonionic surfactants.

[0189] In this application, the term "specific binding" or "specific" generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that can determine the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which may be an epitope) may be one that binds to that target with greater affinity, avidity, more readily, and / or for a greater duration than it binds to other targets. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins of different species. In certain embodiments, specific binding may include, but does not require, exclusive binding.

[0190] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0191] In this application, the term "tumor" generally refers to a neoplasm or solid lesion formed by abnormal cell growth. In this application, a tumor can be a solid tumor or a non-solid tumor.

[0192] In the present application, the proteins, polypeptides and / or amino acid sequences involved should also be understood to include at least the following scope: variants or homologs that have the same or similar functions as the proteins or polypeptides.

[0193] In the present application, the variant may be, for example, a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to CD73 and / or CD39 protein). For example, the functional variant may comprise a protein or polypeptide having an amino acid change by at least 1, for example, 1-30, 1-20 or 1-10, for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions and / or insertions. The functional variant may substantially retain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide before the change. For example, the substitution may be a conservative substitution.

[0194] In the present application, the homolog can be a protein or polypeptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to CD73 and / or CD39 protein).

[0195] In the present application, described homology generally refers to the similarity, similarity or association between two or more sequences.Can calculate " sequence homology per-cent " in the following manner: two sequences to be compared are compared in comparison window, determine that there is identical nucleic acid base (for example, A, T, C, G, I) or identical amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) number in the position to obtain the number of matching positions, with the number of matching positions divided by the total number of positions (that is, window size) in the comparison window, and result is multiplied by 100, to produce sequence homology per-cent.Comparison carried out in order to determine the sequence homology per-cent, can realize by several ways known in the art, for example, use publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared or within a region of interest. Homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in W. R. Earson and D. J. Lipman, "Improved tools for biological sequence comparison," Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and D. J. Lipman and W. R. Earson, "Rapid and sensitive protein similarity search," Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E. W. Myers, and D. Lipman, "A basic local alignment search tool," J. Mol. Biol., 215: 403-410, 1990.

[0196] In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "to be", "to be composed of..."

[0197] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0198] Detailed Description of the Invention

[0199] In one aspect, the present application provides an isolated antigen-binding protein comprising a first binding domain and a second binding domain, wherein the first binding domain is capable of binding to a CD39 protein, and the second binding domain is capable of binding to a CD73 protein.

[0200] In the present application, the isolated antigen-binding protein may have one or more of the following properties: (1) - 9 M or lower K D The value of binding to CD39 protein; (2) 1×10 -9 M or lower K D (3) has an inhibitory effect on the enzymatic activity of CD39 antigen; (4) has an inhibitory effect on the enzymatic activity of CD73 antigen; (5) has thermal stability; (6) can bind to human CD39 and / or cynomolgus monkey CD73; (7) can bind to human CD73 and / or cynomolgus monkey CD73; (8) can inhibit the growth of tumor cells.

[0201] For example, the tumor may comprise a CD39 positive tumor. For example, the tumor may comprise a CD73 positive tumor. For example, the tumor may comprise a solid tumor. For example, the tumor may comprise a non-solid tumor.

[0202] In the present application, the isolated antigen binding protein can be 1×10 -9 M or lower K D For example, the isolated antigen binding protein may be less than about 1×10 -9 M, less than about 8×10 -10 M, less than about 6×10 -10 M, less than about 4×10 -10 M, less than about 2×10 -10 M, less than about 1×10 -10 M or lower K D The value is bound to CD39 protein.

[0203] In the present application, the isolated antigen binding protein can inhibit ATPase activity in cells expressing CD39 with an EC50 of less than about 0.8 μg / mL as determined by an ATPase activity assay; and / or,

[0204] For example, the EC50 can be less than about 0.7 μg / mL, less than about 0.6 μg / mL, less than about 0.5 μg / mL, less than about 0.4 μg / mL, less than about 0.3 μg / mL, less than about 0.2 μg / mL, less than about 0.1 μg / mL, less than about 0.09 μg / mL, less than about 0.08 μg / mL, less than about 0.07 μg / mL, less than about 0.06 μg / mL, less than about 0.05 μg / mL, less than about 0.04 μg / mL, less than about 0.03 μg / mL, less than about 0.02 μg / mL, less than about 0.01 μg / mL, or less.

[0205] The CDR of an antibody, also known as the complementarity determining region, is part of the variable region. The amino acid residues in this region can contact the antigen or antigenic epitope. Antibody CDRs can be determined using a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, and a combination of Kabat / Chothia. These coding systems are known in the art, and for details, see http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region based on the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR region. In this application, the CDR covers CDR sequences obtained by any CDR division method; it also covers variants thereof, wherein the variant includes the amino acid sequence of the CDR being substituted, deleted, and / or having one or more amino acids added. For example, 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions; homologs thereof are also encompassed, and the homologs can be amino acid sequences having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence identity with the amino acid sequence of the CDR.

[0206] In the present application, for example, the CDRs may be divided according to the IMGT method.

[0207] In addition, it should be noted that the isolated antigen-binding proteins described herein may include heavy chain and / or light chain sequences that have one or more conservative sequence modifications thereto. The so-called "conservative sequence modifications" refer to amino acid modifications that do not significantly affect or change the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the isolated antigen-binding proteins described herein by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions are the replacement of amino acid residues with amino acid residues having similar side chains. Groups of amino acid residues with similar side chains are known in the art. These amino acid residue groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In the present application, one or more amino acid residues in the CDR region of the isolated antigen-binding protein described herein can be replaced with other amino acid residues from the same side chain group. Those skilled in the art will appreciate that some conservative sequence modifications will not abolish antigen binding. For details, see, for example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissrov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0208] The CD39 antigen binding proteins described herein can be identified, screened, or characterized by various assays known in the art.

[0209] For example, the antigen binding activity of the antigen-binding protein or fusion protein of the present invention can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., Western blot), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, etc.

[0210] In the present application, the first binding domain and the second binding domain of the isolated antigen binding protein are directly or indirectly linked.

[0211] In the present application, the C-terminus of the first binding domain of the isolated antigen binding protein may be connected to the N-terminus of the second binding domain.

[0212] In the present application, the C-terminus of the first binding domain of the isolated antigen-binding protein may be linked to the N-terminus of the heavy chain variable region of the second binding domain.

[0213] In the present application, the first binding domain and the second binding domain of the isolated antigen-binding protein are connected by a linker. In the present application, the linker may comprise a peptide linker. In the present application, the linker may comprise an amino acid sequence of (GGGGS)n, wherein n is any positive integer between 0 and 10. For example, the linker may comprise the amino acid sequence set forth in SEQ ID NO:47.

[0214] In the present application, the isolated antigen binding protein comprises two first binding domains.

[0215] In the present application, the two first binding domains of the isolated antigen binding protein can be directly or indirectly connected to the N-terminus of the second binding domain, respectively.

[0216] In the present application, the two first binding domains of the isolated antigen binding protein can be directly or indirectly connected to the N-termini of the two heavy chains of the second binding domain, respectively.

[0217] In the present application, the isolated antigen binding protein may comprise a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the heavy chains of the first binding domain and the second binding domain.

[0218] In the present application, the N-termini of the heavy chains of the first binding domain and the second binding domain of the isolated antigen binding protein may be directly or indirectly linked.

[0219] In the present application, the N-termini of the heavy chains of the first binding domain and the second binding domain of the isolated antigen binding protein are connected via a linker.

[0220] In the present application, the first polypeptide chain of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 45 or SEQ ID NO: 46.

[0221] In the present application, the second polypeptide chain of the isolated antigen binding protein may comprise the light chain of the second binding domain.

[0222] In the present application, the second polypeptide chain of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 26 or SEQ ID NO: 42.

[0223] In the present application, the isolated antigen-binding protein may comprise two first polypeptide chains and two second polypeptide chains.

[0224] Drug combinations

[0225] The present application also provides a drug combination, which comprises an antigen binding protein capable of binding to CD39 and an antigen binding protein capable of binding to CD73.

[0226] In the present application, the antigen binding protein capable of binding to CD39 and the antigen binding protein capable of binding to CD73 in the drug combination can be administered together or separately; can be administered in the same manner or in different manners; can be administered simultaneously or sequentially (the order of administration is not limited).

[0227] In the present application, the antigen binding protein capable of binding to CD39 and the antigen binding protein capable of binding to CD73 in the drug combination may be present in the same container or in different containers; they may be packaged separately or as a mixture.

[0228] In the present application, the antigen binding protein capable of binding to CD39 has one or more of the following properties:

[0229] (1) binds to human CD39 with an EC50 of less than about 0.5 μg / mL as determined by FACS assay; for example, the EC50 can be less than about 0.4 μg / mL, less than about 0.3 μg / mL, less than about 0.2 μg / mL, less than about 0.1 μg / mL, less than about 0.09 μg / mL, less than about 0.08 μg / mL, less than about 0.07 μg / mL, less than about 0.06 μg / mL, less than about 0.05 μg / mL, less than about 0.04 μg / mL, less than about 0.03 μg / mL, less than about 0.02 μg / mL, less than about 0.01 μg / mL, or less.

[0230] (2) binds to cynomolgus macaque (cyno) CD39 with an EC50 of less than about 0.7 μg / mL as determined by FACS assay; for example, the EC50 can be less than about 0.6 μg / mL, less than about 0.5 μg / mL, less than about 0.4 μg / mL, less than about 0.3 μg / mL, less than about 0.2 μg / mL, less than about 0.1 μg / mL, less than about 0.09 μg / mL, less than about 0.08 μg / mL, less than about 0.07 μg / mL, less than about 0.06 μg / mL, less than about 0.05 μg / mL, less than about 0.04 μg / mL, less than about 0.03 μg / mL, less than about 0.02 μg / mL, less than about 0.01 μg / mL, or less.

[0231] (3) As determined by Biacore assay, the -8 M binds to human CD39 with a KD of less than about 1×10 -8 M, less than about 9×10 -9 M, less than about 8×10 -9 M, less than about 7×10 - 9 M, less than about 6×10 -9 M, less than about 5×10 -9 M, less than about 4×10 -9 M, less than about 3×10 -9 M, less than about 2×10 -9 M, less than about 1×10 -9 M, less than about 8×10 -10 M, less than about 6×10 -10 M, less than about 4×10 - 10 M, less than about 2×10 -10 M, less than about 1×10 -10 M or lower.

[0232] (4) As determined by Biacore assay, the -7 M binds to cynomolgus macaque (cyno) CD39; for example, the KD can be less than about 1×10 -7 M, less than about 9×10 -8 M, less than about 8×10 - 8 M, less than about 7×10 -8 M, less than about 6×10 -8 M, less than about 5×10 -8 M, less than about 4×10 -8 M, less than about 3×10 -8M, less than about 2×10 -8 M, less than about 1×10 -8 M, less than about 8×10 -9 M, less than about 6×10 - 9 M, less than about 4×10 -9 M, less than about 2×10 -9 M, less than about 1×10 -9 M or lower.

[0233] (5) inhibits ATPase activity in cells expressing CD39 with an EC50 of less than about 0.8 μg / mL as measured by an ATPase activity assay; for example, the EC50 can be less than about 0.7 μg / mL, less than about 0.6 μg / mL, less than about 0.5 μg / mL, less than about 0.4 μg / mL, less than about 0.3 μg / mL, less than about 0.2 μg / mL, less than about 0.1 μg / mL, less than about 0.09 μg / mL, less than about 0.08 μg / mL, less than about 0.07 μg / mL, less than about 0.06 μg / mL, less than about 0.05 μg / mL, less than about 0.04 μg / mL, less than about 0.03 μg / mL, less than about 0.02 μg / mL, less than about 0.01 μg / mL, or less.

[0234] (6) Inhibit CD39 function in tumor cells.

[0235] In the present application, the antigen binding protein capable of binding to CD73 has one or more of the following properties:

[0236] (1) Ability to specifically bind to CD73 or its functionally active fragments;

[0237] (2) can inhibit the enzymatic activity of CD73;

[0238] (3) can induce the internalization of CD73 on the cell surface;

[0239] (4) Ability to inhibit the growth and / or proliferation of tumor cells.

[0240] First binding domain

[0241] In the present application, the first binding domain is capable of binding to CD39 protein. For example, the first binding domain may comprise an antigen binding protein capable of binding to CD39.

[0242] In the present application, the first binding domain may comprise an antibody or an antigen-binding fragment thereof.

[0243] In the present application, the antibody may be selected from the group consisting of a monoclonal antibody, a single-chain antibody, a chimeric antibody, a multispecific antibody, a humanized antibody, and a fully human antibody. In the present application, the antigen-binding fragment may be selected from the group consisting of a Fab, a Fab', a F(ab)2, a Fv, a F(ab')2, a scFv, a VHH, a di-scFv, and a dAb fragment.

[0244] In the present application, the first binding domain of the isolated antigen binding protein may be VHH.

[0245] In the present application, the first binding domain of the isolated antigen-binding protein comprises at least one CDR in the antibody heavy chain variable region VHH, and the VHH comprises the amino acid sequence shown in SEQ ID NO:8.

[0246] In the present application, the first binding domain may comprise CDR3, and the CDR3 may comprise the amino acid sequence shown in SEQ ID NO:1.

[0247] In the present application, the first binding domain may comprise CDR2, and the CDR2 may comprise the amino acid sequence shown in SEQ ID NO:2.

[0248] In the present application, the first binding domain may comprise CDR1, and the CDR1 may comprise the amino acid sequence shown in SEQ ID NO:3.

[0249] In the present application, the first binding domain may comprise CDR1, CDR2 and CDR3, the CDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, the CDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the CDR3 may comprise the amino acid sequence shown in SEQ ID NO:1.

[0250] In the present application, the first binding domain may comprise FR1, the C-terminus of the FR1 is directly or indirectly connected to the N-terminus of the CDR1, and the FR1 comprises the amino acid sequence shown in SEQ ID NO:4.

[0251] In the present application, the first binding domain may comprise FR2, wherein the FR2 is located between the CDR1 and the CDR2, and the FR2 may comprise the amino acid sequence shown in SEQ ID NO:5.

[0252] In the present application, the first binding domain may comprise FR3, wherein the FR3 is located between the CDR2 and the CDR3, and the FR3 may comprise the amino acid sequence shown in SEQ ID NO:6.

[0253] In the present application, the first binding domain may comprise FR4, the N-terminus of the FR4 is directly or indirectly connected to the C-terminus of the CDR3, and the FR4 comprises the amino acid sequence shown in SEQ ID NO:7.

[0254] In the present application, the first binding domain may comprise VHH, and the VHH comprises the amino acid sequence shown in SEQ ID NO:8.

[0255] Second binding domain

[0256] In the present application, the second binding domain is capable of binding to the CD73 protein. In the present application, the second binding domain can comprise an antigen binding protein capable of binding to CD73.

[0257] In the present application, the second binding domain may comprise an antibody or an antigen-binding fragment thereof.

[0258] In the present application, the antibody can be selected from the following group: a monoclonal antibody, a single-chain antibody, a chimeric antibody, a multispecific antibody, a humanized antibody and a fully human antibody.

[0259] In the present application, the antigen-binding fragment may be selected from the following group: Fab, Fab', F(ab)2, Fv, F(ab')2, scFv, VHH, di-scFv and dAb fragment.

[0260] In the present application, the second binding domain may comprise at least one CDR in the antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 33.

[0261] In the present application, the second binding domain may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 27.

[0262] In the present application, the second binding domain may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 28.

[0263] In the present application, the second binding domain may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 29.

[0264] In the present application, the second binding domain may comprise HCDR1, HCDR2, and HCDR3, wherein the HCDR1 may comprise the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 29, the HCDR2 may comprise the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 28, and the HCDR3 may comprise the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 27. For example, the HCDR1 may comprise the amino acid sequence of SEQ ID NO: 11, the HCDR2 may comprise the amino acid sequence of SEQ ID NO: 10, and the HCDR3 may comprise the amino acid sequence of SEQ ID NO: 9. For example, the HCDR1 may comprise the amino acid sequence of SEQ ID NO: 29, the HCDR2 may comprise the amino acid sequence of SEQ ID NO: 28, and the HCDR3 may comprise the amino acid sequence of SEQ ID NO: 27.

[0265] In the present application, the second binding domain may comprise H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 30.

[0266] In the present application, the second binding domain may comprise H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 31.

[0267] In the present application, the second binding domain may comprise H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 32.

[0268] In the present application, the second binding domain may comprise H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:15.

[0269] In the present application, the second binding domain may comprise H-FR1, H-FR2, H-FR3, and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 30, H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 31, H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 32, and H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 15. For example, H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 12, H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 13, H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 14, and H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 15. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO:30, the H-FR2 may include the amino acid sequence shown in SEQ ID NO:31, the H-FR3 may include the amino acid sequence shown in SEQ ID NO:32, and the H-FR4 may include the amino acid sequence shown in SEQ ID NO:15.

[0270] In the present application, the second binding domain may comprise VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 33.

[0271] In the present application, the second binding domain may comprise an antibody heavy chain constant region.

[0272] In the present application, the antibody heavy chain constant region is derived from an IgG heavy chain constant region. For example, the antibody heavy chain constant region is derived from a human IgG heavy chain constant region. For example, the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region. For example, the antibody heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 43.

[0273] In the present application, the second binding domain may comprise an antibody heavy chain, and the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 25 or SEQ ID NO: 41.

[0274] In the present application, the second binding domain may comprise at least one CDR in the antibody heavy chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO: 24 or SEQ ID NO: 40.

[0275] In the present application, the second binding domain may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 34.

[0276] In the present application, the second binding domain may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 18 (WAS) or SEQ ID NO: 35 (LAS).

[0277] In the present application, the second binding domain may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 36.

[0278] In the present application, the second binding domain may comprise LCDR1, LCDR2, and LCDR3, wherein the LCDR1 may comprise the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 36, the LCDR2 may comprise the amino acid sequence of SEQ ID NO: 18 (WAS) or SEQ ID NO: 35 (LAS), and the LCDR3 may comprise the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 34. For example, the LCDR1 may comprise the amino acid sequence of SEQ ID NO: 19, the LCDR2 may comprise the amino acid sequence of SEQ ID NO: 18 (WAS), and the LCDR3 may comprise the amino acid sequence of SEQ ID NO: 17. For example, the LCDR1 may comprise the amino acid sequence of SEQ ID NO: 36, the LCDR2 may comprise the amino acid sequence of SEQ ID NO: 35 (LAS), and the LCDR3 may comprise the amino acid sequence of SEQ ID NO: 34.

[0279] In the present application, the second binding domain may comprise L-FR1, the C-terminus of the L-FR1 being directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 20 or SEQ ID NO: 37.

[0280] In the present application, the second binding domain may comprise L-FR2, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 38.

[0281] In the present application, the second binding domain may comprise L-FR3, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 22 or SEQ ID NO: 39.

[0282] In the present application, the second binding domain may comprise L-FR4, the N-terminus of the L-FR4 being directly or indirectly connected to the C-terminus of the L-FR3, and the L-FR4 comprising the amino acid sequence shown in SEQ ID NO:23.

[0283] In the present application, the second binding domain may comprise L-FR1, L-FR2, L-FR3, and L-FR4, wherein L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 20 or SEQ ID NO: 37, L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 38, L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 22 or SEQ ID NO: 39, and L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 23. For example, L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 20, L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 21, L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 22, and L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 23. For example, the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 37, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 38, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 39, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 23.

[0284] In the present application, the second binding domain may comprise VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 24 or SEQ ID NO: 40.

[0285] In the present application, the second binding domain may comprise an antibody light chain constant region. For example, the antibody light chain constant region is derived from an Igκ constant region. For example, the antibody light chain constant region is derived from a human Igκ constant region. For example, the antibody light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 44.

[0286] In the present application, the second binding domain may comprise an antibody light chain, and the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 26 or SEQ ID NO: 42.

[0287] In the present application, the second domain may comprise VH and VL, wherein the VH may comprise the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 33, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 24 or SEQ ID NO: 40. For example, the VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the VL comprises the amino acid sequence shown in SEQ ID NO: 24. For example, the VH comprises the amino acid sequence shown in SEQ ID NO: 33, and the VL comprises the amino acid sequence shown in SEQ ID NO: 40.

[0288] In the present application, the second domain may comprise an antibody heavy chain and an antibody light chain. For example, the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 25, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 26. For example, the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 41, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 42.

[0289] Antigen binding protein capable of binding to CD39

[0290] In the present application, the antigen-binding protein capable of binding to CD39 may comprise an antibody or an antigen-binding fragment thereof.

[0291] In the present application, the antibody may be selected from the group consisting of a monoclonal antibody, a single-chain antibody, a chimeric antibody, a multispecific antibody, a humanized antibody, and a fully human antibody. In the present application, the antigen-binding fragment may be selected from the group consisting of a Fab, a Fab', a F(ab)2, a Fv, a F(ab')2, a scFv, a VHH, a di-scFv, and a dAb fragment.

[0292] In the present application, the isolated antigen-binding protein capable of binding to CD39 may be a VHH.

[0293] In the present application, the isolated antigen-binding protein capable of binding to CD39 may comprise at least one CDR in an antibody heavy chain variable region VHH, wherein the VHH comprises the amino acid sequence shown in SEQ ID NO:8.

[0294] In the present application, the antigen binding protein capable of binding to CD39 may comprise CDR3, and the CDR3 may comprise the amino acid sequence shown in SEQ ID NO: 1.

[0295] In the present application, the antigen binding protein capable of binding to CD39 may comprise CDR2, and the CDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2.

[0296] In the present application, the antigen binding protein capable of binding to CD39 may comprise CDR1, and the CDR1 may comprise the amino acid sequence shown in SEQ ID NO:3.

[0297] In the present application, the antigen binding protein capable of binding to CD39 may comprise CDR1, CDR2 and CDR3, the CDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, the CDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the CDR3 may comprise the amino acid sequence shown in SEQ ID NO:1.

[0298] In the present application, the antigen binding protein capable of binding to CD39 may comprise FR1, the C-terminus of the FR1 is directly or indirectly connected to the N-terminus of the CDR1, and the FR1 comprises the amino acid sequence shown in SEQ ID NO:4.

[0299] In the present application, the antigen binding protein capable of binding to CD39 may comprise FR2, wherein the FR2 is located between the CDR1 and the CDR2, and the FR2 may comprise the amino acid sequence shown in SEQ ID NO:5.

[0300] In the present application, the antigen binding protein capable of binding to CD39 may comprise FR3, wherein the FR3 is located between the CDR2 and the CDR3, and the FR3 may comprise the amino acid sequence shown in SEQ ID NO:6.

[0301] In the present application, the antigen binding protein capable of binding to CD39 may comprise FR4, the N-terminus of the FR4 is directly or indirectly connected to the C-terminus of the CDR3, and the FR4 comprises the amino acid sequence shown in SEQ ID NO:7.

[0302] In the present application, the antigen binding protein capable of binding to CD39 may comprise VHH, and the VHH comprises the amino acid sequence shown in SEQ ID NO:8.

[0303] Antigen binding protein capable of binding to CD73

[0304] In the present application, the antigen binding protein capable of binding to CD73 may comprise an antibody or an antigen binding fragment thereof.

[0305] In the present application, the antibody can be selected from the following group: a monoclonal antibody, a single-chain antibody, a chimeric antibody, a multispecific antibody, a humanized antibody and a fully human antibody.

[0306] In the present application, the antigen-binding fragment may be selected from the following group: Fab, Fab', F(ab)2, Fv, F(ab')2, scFv, VHH, di-scFv and dAb fragment.

[0307] In the present application, the antigen-binding protein capable of binding to CD73 may comprise at least one CDR in the antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 33.

[0308] In the present application, the antigen binding protein capable of binding to CD73 may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 27.

[0309] In the present application, the antigen binding protein capable of binding to CD73 may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 28.

[0310] In the present application, the antigen binding protein capable of binding to CD73 may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 29.

[0311] In the present application, the antigen-binding protein capable of binding to CD73 may comprise HCDR1, HCDR2 and HCDR3, wherein the HCDR1 may comprise the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 29, the HCDR2 may comprise the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 28, and the HCDR3 may comprise the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 27. For example, the HCDR1 may comprise the amino acid sequence of SEQ ID NO: 11, the HCDR2 may comprise the amino acid sequence of SEQ ID NO: 10, and the HCDR3 may comprise the amino acid sequence of SEQ ID NO: 9. For example, the HCDR1 may comprise the amino acid sequence of SEQ ID NO: 29, the HCDR2 may comprise the amino acid sequence of SEQ ID NO: 28, and the HCDR3 may comprise the amino acid sequence of SEQ ID NO: 27.

[0312] In the present application, the antigen binding protein capable of binding to CD73 may comprise H-FR1, the C-terminus of the H-FR1 being directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 30.

[0313] In the present application, the antigen binding protein capable of binding to CD73 may comprise H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 31.

[0314] In the present application, the antigen binding protein capable of binding to CD73 may comprise H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 32.

[0315] In the present application, the antigen binding protein capable of binding to CD73 may comprise H-FR4, the N-terminus of the H-FR4 being directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprising the amino acid sequence shown in SEQ ID NO: 15.

[0316] In the present application, the antigen binding protein capable of binding to CD73 may comprise H-FR1, H-FR2, H-FR3 and H-FR4, wherein the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 30, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 31, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 32, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 15. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 12, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 13, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 14, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 15. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO:30, the H-FR2 may include the amino acid sequence shown in SEQ ID NO:31, the H-FR3 may include the amino acid sequence shown in SEQ ID NO:32, and the H-FR4 may include the amino acid sequence shown in SEQ ID NO:15.

[0317] In the present application, the antigen binding protein capable of binding to CD73 may comprise VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 33.

[0318] In the present application, the antigen binding protein capable of binding to CD73 may comprise an antibody heavy chain constant region.

[0319] In the present application, the antibody heavy chain constant region is derived from an IgG heavy chain constant region. For example, the antibody heavy chain constant region is derived from a human IgG heavy chain constant region. For example, the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region. For example, the antibody heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 43.

[0320] In the present application, the antigen binding protein capable of binding to CD73 may comprise an antibody heavy chain, and the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 25 or SEQ ID NO: 41.

[0321] In the present application, the antigen binding protein capable of binding to CD73 may comprise at least one CDR in the antibody heavy chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO: 24 or SEQ ID NO: 40.

[0322] In the present application, the antigen binding protein capable of binding to CD73 may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 34.

[0323] In the present application, the antigen binding protein capable of binding to CD73 may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 18 (WAS) or SEQ ID NO: 35 (LAS).

[0324] In the present application, the antigen binding protein capable of binding to CD73 may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 36.

[0325] In the present application, the antigen binding protein capable of binding to CD73 may comprise LCDR1, LCDR2 and LCDR3, wherein the LCDR1 may comprise the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 36, the LCDR2 may comprise the amino acid sequence of SEQ ID NO: 18 (WAS) or SEQ ID NO: 35 (LAS), and the LCDR3 may comprise the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 34. For example, the LCDR1 may comprise the amino acid sequence of SEQ ID NO: 19, the LCDR2 may comprise the amino acid sequence of SEQ ID NO: 18 (WAS), and the LCDR3 may comprise the amino acid sequence of SEQ ID NO: 17. For example, the LCDR1 may comprise the amino acid sequence of SEQ ID NO: 36, the LCDR2 may comprise the amino acid sequence of SEQ ID NO: 35 (LAS), and the LCDR3 may comprise the amino acid sequence of SEQ ID NO: 34.

[0326] In the present application, the antigen binding protein capable of binding to CD73 may comprise L-FR1, the C-terminus of the L-FR1 being directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 20 or SEQ ID NO: 37.

[0327] In the present application, the antigen binding protein capable of binding to CD73 may comprise L-FR2, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 38.

[0328] In the present application, the antigen binding protein capable of binding to CD73 may comprise L-FR3, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 22 or SEQ ID NO: 39.

[0329] In the present application, the antigen binding protein capable of binding to CD73 may comprise L-FR4, the N-terminus of the L-FR4 being directly or indirectly connected to the C-terminus of the L-FR3, and the L-FR4 comprising the amino acid sequence shown in SEQ ID NO: 23.

[0330] In the present application, the antigen binding protein capable of binding to CD73 may comprise L-FR1, L-FR2, L-FR3 and L-FR4, wherein the L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 20 or SEQ ID NO: 37, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 38, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 22 or SEQ ID NO: 39, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 23. For example, the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 20, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 21, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 22, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 23. For example, the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 37, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 38, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 39, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 23.

[0331] In the present application, the antigen binding protein capable of binding to CD73 may comprise a VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 24 or SEQ ID NO: 40.

[0332] In the present application, the antigen binding protein capable of binding to CD73 may comprise an antibody light chain constant region. For example, the antibody light chain constant region is derived from an Igκ constant region. For example, the antibody light chain constant region is derived from a human Igκ constant region. For example, the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 44.

[0333] In the present application, the antigen binding protein capable of binding to CD73 may comprise an antibody light chain, and the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 26 or SEQ ID NO: 42.

[0334] In the present application, the second domain may comprise VH and VL, wherein the VH may comprise the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 33, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 24 or SEQ ID NO: 40. For example, the VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the VL comprises the amino acid sequence shown in SEQ ID NO: 24. For example, the VH comprises the amino acid sequence shown in SEQ ID NO: 33, and the VL comprises the amino acid sequence shown in SEQ ID NO: 40.

[0335] In the present application, the second domain may comprise an antibody heavy chain and an antibody light chain. For example, the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 25, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 26. For example, the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 41, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 42.

[0336] Nucleic acid molecules, vectors, cells and pharmaceutical compositions

[0337] In another aspect, the present application provides isolated nucleic acid molecules that can encode the isolated antigen-binding proteins described herein. For example, they can be produced or synthesized by: (i) in vitro amplification, such as by polymerase chain reaction (PCR) amplification; (ii) cloning and recombination; (iii) purification, such as by enzyme digestion and gel electrophoresis fractionation; or (iv) synthesis, such as by chemical synthesis.

[0338] On the other hand, the present application provides a vector that can contain the nucleic acid molecules described herein. In addition, the vector can also contain other genes, such as marker genes that allow the vector to be selected in appropriate host cells and under appropriate conditions. In addition, the vector can also contain expression control elements that allow the coding region to be correctly expressed in an appropriate host. Such control elements are well known to those skilled in the art and can include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. The vector can be transformed, transduced, or transfected into host cells so that the genetic material elements it carries are expressed in the host cells. The vector can include, for example, a plasmid, a cosmid, a virus, a bacteriophage, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector. In addition, the vector can also include components that assist its entry into cells, such as viral particles, liposomes, or protein coats, but is not limited to these substances.

[0339] On the other hand, the application provides a kind of cell, it can comprise nucleic acid molecules described in the application and / or vectors described in the application.In certain embodiments, each kind or each host cell comprises one or a kind of nucleic acid molecules or vectors described in the application.In certain embodiments, each kind or each host cell comprises multiple (for example, 2 or more) or multiple (for example, 2 or more) nucleic acid molecules or vectors described in the application.For example, the vector described in the application can be introduced into the host cell, for example, eukaryotic cells, such as cells from plants, fungi or yeast cells, etc.In certain embodiments, the cell is a bacterial cell (for example, Escherichia coli), yeast cell or other eukaryotic cell, for example, COS cell, Chinese hamster ovary (CHO) cell, CHO-K1 cell, LNCAP cell, HeLa cell, 293T cell, COS-1 cell, SP2 / 0 cell, NS0 cell or myeloma cell.The vector described in the application can be introduced into the host cell by methods known in the art, for example, electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0340] On the other hand, the present application also provides a pharmaceutical composition, which may comprise the isolated antigen-binding protein described herein, the nucleic acid molecule described herein, the vector described herein and / or the cell described herein, and optionally a pharmaceutically acceptable carrier.

[0341] In the present application, the pharmaceutical composition may also include one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or suitable formulations of preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention includes but is not limited to liquid, frozen and lyophilized compositions.

[0342] In the present application, the pharmaceutical composition may also contain more than one active compound, generally those having complementary activities that do not adversely affect each other. The type and effective amount of such drugs may depend, for example, on the amount and type of antagonist present in the formulation, and the clinical parameters of the subject.

[0343] In this application, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents that are compatible with pharmaceutical administration and are generally safe and non-toxic.

[0344] In the present application, the pharmaceutical composition can include parenteral, percutaneous, intracavitary, intra-arterial, intrathecal and / or intranasal administration or direct injection into tissue. For example, the pharmaceutical composition can be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition can be carried out in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In certain embodiments, the pharmaceutical composition can be administered uninterruptedly. The uninterrupted (or continuous) administration can be achieved by a small pump system worn by the patient to measure the therapeutic agent flowing into the patient's body, as described in WO2015 / 036583.

[0345] Preparation method

[0346] In another aspect, the present application provides a method for preparing the antigen-binding protein. The method may include culturing the host cell described herein under conditions that allow for expression of the antigen-binding protein. For example, the method may be performed using an appropriate culture medium, at an appropriate temperature and for an appropriate time, as is known to those skilled in the art.

[0347] Methods and uses

[0348] On the other hand, the present application provides the use of the isolated antigen-binding protein, the drug combination, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease and / or condition.

[0349] On the other hand, the present application also provides a method for preventing and / or treating a disease and / or condition, which may comprise administering the isolated antigen-binding protein, the drug combination, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition described herein to a subject in need thereof.

[0350] In the present application, the use can be carried out by different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0351] On the other hand, the isolated antigen-binding protein, the pharmaceutical combination, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition described herein can be used to prevent and / or treat diseases and / or disorders.

[0352] In the present application, the disease and / or disorder may be a disease and / or disorder mediated by CD39 and / or CD73.

[0353] In the present application, the disease and / or disorder may include a tumor.

[0354] In the present application, the tumor may include solid tumors and / or hematological tumors.

[0355] In the present application, the disease and / or condition may include pancreatic cancer and / or colorectal cancer.

[0356] On the other hand, the present application also provides a method for detecting CD39 and / or CD73 in a sample, which comprises administering the isolated antigen-binding protein, the drug combination, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition.

[0357] In the present application, the method for detecting CD39 and / or CD73 in a sample may be an in vitro method. In the present application, the method for detecting CD39 and / or CD73 in a sample may be for non-therapeutic purposes. In the present application, the method for detecting CD39 and / or CD73 in a sample is not a diagnostic method.

[0358] On the other hand, the present application also provides a reagent or kit for detecting CD39 and / or CD73 in a sample, which comprises the isolated antigen-binding protein, the drug combination, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition.

[0359] On the other hand, the present application also provides the use of the isolated antigen-binding protein, the drug combination, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition in preparing a kit for detecting the presence and / or content of CD39 and / or CD73 in a sample.

[0360] Without intending to be bound by any theory, the following examples are merely intended to illustrate the antigen-binding proteins, preparation methods, and uses of the present application, and are not intended to limit the scope of the present invention.

[0361] Example

[0362] Example 1 Sequence Design of the Heavy and Light Chains of the CD39-CD73 Bispecific Antibody

[0363] The structure of the CD39-CD73 bispecific antibody in this application belongs to VHH-IgG, that is, the N-terminus of the two heavy chains of an IgG antibody is connected to the VHH fragment of another nanobody, and its configuration is shown in Figure 1. This form of bispecific antibody structure is abbreviated as CD39-VHH-CD73-IgG, and the main components of its heavy chain and light chain are designed as shown in Table 1 below.

[0364] Table 1 Main component design of CD39-VHH-CD73-IgG heavy chain and light chain

[0365] The heavy chain sequences of the bispecific antibodies 700035 and 700038 in the CD39-VHH-CD73-IgG format are shown in Table 2 below;

[0366] Table 2 Heavy chain sequences of CD39-VHH-CD73-IgG bispecific antibodies

[0367] The light chain sequences of the bispecific antibodies 700035 and 700038 in the CD39-VHH-CD73-IgG format were derived from the light chain of 900725 (SEQ ID NO: 42) and the light chain of 900698 (SEQ ID NO: 26), respectively.

[0368] Example 2 Expression and purification of CD39-CD73 bispecific antibody

[0369] The expression vector obtained in Example 1 was amplified in E. coli, and sufficient plasmid was prepared using an endotoxin-free plasmid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., #DP117) for transient transfection to express the bispecific antibody. The host cells used for expression were CHO-S cells (Thermo Fisher Scientific, #R80007). The two heavy chain vectors and the light chain vector were mixed with polyetherimide (PEI, Polysciences, #24765-1) to form liposome complexes. The complexes were then transfected into CHO-S cells and cultured in an incubator for 5-7 days. The cell culture supernatant was collected by centrifugation and purified using a Protein A affinity chromatography column to obtain the bispecific antibody. Protein aggregation was further assessed by size exclusion chromatography.

[0370] The expression, purification, and testing of the bispecific antibodies are shown in Figures 2-5 and Table 3. The results demonstrate that for the CD39-VHH-CD73-IgG bispecific antibody, IgG derived from different parental monoclonal antibodies exhibited high SEC purity, reaching approximately 95%. Reducing SDS-PAGE images revealed no degradation of either the heavy or light chains of 700035 and 700038, demonstrating the excellent stability of 700035 and 700038.

[0371] Table 3 SEC purity data of CD39-CD73 bispecific antibody

[0372] Example 3 Determination of Kinetic Parameters of CD39-CD73 Bispecific Antibody

[0373] This experiment used the SPR method to determine the binding kinetics and affinity of CD39-CD73 bispecific antibodies 700035 and 700038, as well as parental control antibodies 700053 (CD39 nanobody), 900698 (CD73 parent monoclonal antibody corresponding to 700038) and 900725 (CD73 parent monoclonal antibody corresponding to 700035) to the antigen.

[0374] The test method is as follows:

[0375] Anti-Human Capture-CM5 chip (GE, #BR-1005-30) was prepared according to the coupling method of Human Antibody Captrue Kit (GE, #BR-1008-39) and amino coupling kit (GE, BR-1000-50). The chip was equilibrated at room temperature for 20-30 minutes and loaded into the Biacore 8K instrument; the antigen and antibody were released to the experimental working concentration using the equilibration buffer; the antigen was diluted to 50 nM using the equilibration buffer, and then diluted 3-fold into 7 concentration gradients, and 2 zero concentrations (i.e., equilibration buffer) and 1 repeat concentration (generally the lowest concentration repeat) were set; in the order of antibody, antigen, and regeneration, 10 antigen concentrations (2 zero concentrations, 7 gradient concentrations, and 1 repeat concentration) were analyzed repeatedly, with an antigen injection flow rate of 30 μL / min, a binding time of 120 seconds, and a dissociation time of 600 seconds; after the analysis was completed, the corresponding analysis program was used to analyze the data, confirming that there was no obvious reference binding, and Kinetics, a 1:1 binding model, was used to fit the data to obtain the kinetic-related parameters Ka, Kd, ​​and KD values ​​of the bispecific antibody and the parental control antibody.

[0376] The test results are shown in Tables 4 and 5 below. The results show that for the human CD39 antigen, the affinities of 700035 and 700038 are comparable, and significantly better than the affinity of the parental nanobody 700053 for the CD39 antigen. For the human CD73 antigen, the affinity of the 700035 bispecific antibody is comparable to that of the parental 700025 monoclonal antibody, and the affinity of the 700038 bispecific antibody is comparable to that of the 900698 monoclonal antibody.

[0377] Table 4 Binding affinity test of candidate antibodies to human CD39 antigen

[0378] Table 5 Binding affinity test of candidate antibodies to CD73 antigen

[0379] Example 4 Biological Activity Determination of CD39-CD73 Bispecific Antibody

[0380] 4.1 Inhibitory effect of CD39-CD73 bispecific antibody on CD39 function

[0381] The experimental steps for detecting the inhibitory effect of CD39-CD73 bispecific antibody on CD39 function are as follows: dilute the antibody to 30μg / mL with TM buffer working solution (TM buffer stock solution is mixed with DI Water in a ratio of 1:1), and then dilute it 3-fold in 10 gradients. Count the cells CHOK1-huCD39-10B6 overexpressing CD39, resuspend them in TM buffer working solution to 1*10E5 / mL, and plate 50μL per well in a 96-well U-bottom plate. Add 50μL per well of the diluted antibody to the 96-well U-bottom plate with cells and incubate for 1h. Dilute ATP to 60μM with TM Buffer working solution, add 50μL per well to the 96-well U-bottom plate after the cell-antibody incubation, and incubate for 1h. After the incubation, centrifuge the culture plate (400G 2min), and take 50μL of supernatant from each well and transfer it to the corresponding well of the 96-well black board. Substrate joins Buffer, mix well and equilibrate to room temperature, then add 50 μL per well to the above 96-well black plate, react at room temperature in the dark for 10 min, and finally read the signal intensity with MD i3x microplate reader.

[0382] The test results are shown in Figure 6 and Table 6. The results show that the inhibitory ability of bispecific antibodies 700035 and 700038 on CD39 antigen enzymatic activity is about 3 times stronger than that of parent CD39 nanobody 700053, and the two bispecific antibodies have comparable enzymatic activity inhibition abilities.

[0383] Table 6 Inhibitory effects of bispecific antibodies 700035 and 700038 on CD39 function

[0384] 4.2 Inhibitory effect of CD39-CD73 bispecific antibody on CD73 function

[0385] 4.2.1 The experimental steps for detecting the inhibitory effect of CD39-CD73 bispecific antibody 700035 on the enzymatic activity of CD73 on the membrane surface are as follows:

[0386] The antibody was diluted to 30 μg / mL in PBS and then diluted five-fold in eight steps. CHOK1-huCD73-3F4 cells overexpressing CD73 were counted and resuspended in culture medium to 4 × 105 / mL. 100 μL was plated per well in a 96-well U-bottom plate and centrifuged (1500 rpm for 5 minutes). The supernatant was discarded. The cells were resuspended in the diluted antibody of the series, 100 μL per well, and incubated at 37°C for 20 minutes. After incubation, 200 μL of TM Buffer (a 1:1 mixture of TM Buffer stock solution and DI water) was added to each well for washing. The cells were centrifuged (1500 rpm for 5 minutes) and the supernatant was discarded. The cells were washed again with 200 μL of TM Buffer per well, centrifuged, and the supernatant was discarded. AMP was diluted to 180 μM in TM Buffer and the cells were resuspended in 100 μL per well of the plate. The cells were incubated at 37°C for 1 hour. After incubation, centrifuge the culture plate (1500 rpm for 5 minutes) and transfer 50 μL of supernatant from each well to the corresponding well of the 96-well black plate. Dilute ATP to 60 μM with TM Buffer working solution and add 50 μL of supernatant from each well to the 96-well black plate. Incubate at 37°C for 15 minutes. Substrate joins Buffer, mix well and equilibrate to room temperature, then add 100 μL per well to the above 96-well black plate, react at room temperature in the dark for 10 min, and finally read the signal intensity with MD i3x microplate reader.

[0387] The results of bispecific antibody testing are shown in Figure 7 and Table 7. The results show that the inhibitory effect of bispecific antibody 700035 on CD73 function is only about 2 times weaker than that of CD73 parent monoclonal antibody 900725.

[0388] Table 7 Inhibitory effect of bispecific antibody 700035 on membrane-expressed CD73 enzyme activity

[0389] 4.2.2 The experimental steps for detecting the inhibitory effect of CD39-CD73 bispecific antibody 700038 on the enzymatic activity of soluble recombinant protein CD73 are as follows:

[0390] Dilute the antibody to 128 μg / mL with TM buffer working solution (mix TM buffer stock solution with DI Water in a ratio of 1:1), and then dilute it 2-fold in 10 gradients; dilute CD73 soluble protein to 0.3 μg / mL with TM buffer working solution, and then add the diluted bispecific antibody (25 μL per well) and CD73 soluble protein (25 μL per well) to the 96-well black board in sequence. Dilute AMP and ATP to 240 μM and 80 μM respectively with TM Buffer working solution, and mix them in a ratio of 1:1. Then, add 50 μL of the mixture to each well of the 96-well black board containing the antibody-soluble CD73 protein and react for 10 minutes. Substrate joins Buffer, mix well and equilibrate to room temperature, then add 100 μL per well to the above 96-well black plate, react at room temperature in the dark for 10 min, and finally read the signal intensity with MD i3x microplate reader.

[0391] The results of bispecific antibody testing are shown in Figure 8 and Table 8. The results show that the bispecific antibody 700038 has a comparable inhibitory effect on CD73 function as the CD73 parent monoclonal antibody 900698.

[0392] Table 8 Inhibitory effect of bispecific antibody 700038 on the enzymatic activity of soluble protein CD73

[0393] Example 5 Detection of thermal stability of CD39-CD73 bispecific antibody

[0394] The thermal stability of CD39-CD73 bispecific antibodies 700035 and 700038 was tested as follows: The antibodies were concentrated to 20 mg / mL in a buffer system consisting of 20 mM acetic acid-sodium acetate, 8% w / v sucrose, pH 5.7. The melting temperature (Tm) and aggregation temperature (Tag) of bispecific antibodies 700035 and 700038 were measured using a protein stability analyzer (UNcle, UNCHAINED LABS, US). The Tm and Tag values ​​were measured over a temperature range of 25°C to 95°C, with a ramp rate of 0.3°C.

[0395] The thermal stability test results of the bispecific antibodies are shown in Table 9. The results show that the thermal stability data Tm and Tagg values ​​of this form of bispecific antibody are both in the range of 69°C to 80°C, and the thermal stability performance is very excellent. Among them, the thermal stability of 700035 is better than that of 700038.

[0396] Table 9 Thermal stability data of bispecific antibodies 700035 and 700038

[0397] Example 6 Species Cross-Study of CD39-CD73 Bispecific Antibody

[0398] This study used SPR to determine the binding kinetics and affinity of CD39-CD73 bispecific antibodies 700035 and 700038 to CD39 and CD73 antigens from different species (Human / Cynomolgus / Mouse). The experimental method is as follows:

[0399] Anti-Human Capture-CM5 chip (GE, #BR-1005-30) was prepared according to the coupling method of Human Antibody Captrue Kit (GE, #BR-1008-39) and amino coupling kit (GE, BR-1000-50). The chip was equilibrated at room temperature for approximately 20-30 minutes and then loaded into the Biacore 8K instrument. The antigen and antibody were released to the experimental working concentration using the equilibration buffer. The antigen was diluted to 50 nM using the equilibration buffer and then diluted three-fold into seven concentration gradients, with two zero concentrations (i.e., the equilibration buffer) and one replicate (generally the lowest replicate). Ten antigen concentrations (two zero concentrations, seven gradient concentrations, and one replicate) were analyzed repeatedly in the order of antibody, antigen, and regeneration. The antigen injection flow rate was 30 μL / min, the association time was 120 seconds, and the dissociation time was 600 seconds. After the analysis was completed, the data was analyzed using the corresponding analysis program to confirm that there was no obvious reference binding. Kinetics and a 1:1 binding model were used to fit the data to obtain the kinetic parameters Ka, Kd, ​​and KD values ​​related to the bispecific antibody.

[0400] The test results are shown in Table 10 below. The results show that for the CD39 antigen, the two bispecific antibodies 700035 and 700038 have extremely high affinities for the human CD39 antigen, reaching 7.08E-11 and 9.18E-11, respectively, and their affinities for the monkey CD39 antigen are also 2.61E-9 and 2.69E-9. For the CD73 antigen, the two bispecific antibodies 700035 and 700038 have comparable affinities for humans and monkeys, reaching around E-10. At the same time, the two bispecific antibodies do not bind to mouse CD39 or CD73. In summary, for the two targets CD39 and CD73, humans and cynomolgus macaques are relevant species, while mice are not.

[0401] Table 10 Affinity detection of CD39 and CD73 antigens in different species

[0402] Example 7 Pharmacodynamic Study of the Test Substance in Human Pancreatic Cancer Cell BxPC-3hPBMC Model

[0403] Human pancreatic cancer cells BxPC-3 were cultured in RPMI-1640 medium supplemented with 10% FBS in a 37°C incubator containing 5% CO2. Before the tenth generation of cell culture, approximately 1×10 7 100 μL of PBS containing BxPC-3 cells and an equal volume of Matrigel were mixed and inoculated subcutaneously into 30 NPG mice on the right side of the back near the armpit. The inoculation volume was approximately 200 μL. The mice were anesthetized with 2-5% isoflurane before inoculation. When the tumors grew to an average of approximately 50-80 mm 3 At about 14 days, 30 tumor-bearing mice were randomly divided into 5 groups according to tumor volume and body weight, with 6 mice in each group, and injected with 1×10 7 PBMC (100 μL). Dosing was performed on the day of grouping, with the dosing volume adjusted to 10 μL / g body weight. The grouping and dosing schedule are shown in Table 10. The appearance and behavior of each mouse were observed daily for 4 consecutive weeks starting from grouping. Body weight (BW), tumor volume (TV), relative tumor volume (RTV), and relative tumor growth inhibition (TGI) were measured.

[0404] Table 11 Grouping and Dosage Regimen

[0405] The results are shown in Figure 9. It can be seen from the results that compared with the negative control antibody 900543 (heavy chain sequence as shown in SEQ ID NO: 48, light chain sequence as shown in SEQ ID NO: 49), CD39 nanoantibody 700053 and CD73 monoclonal antibody 900725 both have good anti-pancreatic cancer tumor growth effects. The combination of 700053 and 900725 has a better anti-tumor effect than the two monoclonal antibodies. At the same time, the CD39 / CD73 dual antibody 700035 has a comparable effect to the combination of 700053 and 9000725, demonstrating that the dual antibody has a synergistic anti-tumor effect relative to the two parent antibodies.

[0406] Example 8 Pharmacodynamic Study of the Test Substance in Mouse Colorectal Cancer Model

[0407] MC38-hCD73 mouse colorectal cancer cells were cultured at a density of 5 × 10 5 The cells were inoculated into the right side of female B-hCD73 / hCD39 (v3) humanized mice at a concentration of 0.1 mL. 3Around 3:00 p.m., the mice were randomly divided into groups for drug administration (the day of grouping was recorded as D0). The grouping and dosing regimen are shown in Table 11. There were 6 mice in each group, with a total of 4 groups, namely 900543 (hIgG1 isotype control, 10 mg / kg), 700053 (CD39 nano monoclonal antibody, 5.2 mg / kg), 900698 (CD73 monoclonal antibody, 10 mg / kg) and 700038 (CD39 / CD73 dual antibody, 11.6 mg / kg). After grouping, each group of mice was intraperitoneally injected with the drug, twice a week, for 4 consecutive weeks, for a total of 8 times. The body weight and tumor volume of the mice were measured twice a week during the administration and observation period, and the measured values ​​were recorded. The experiment was terminated on the 25th day after grouping (D25), the animals were euthanized, the tumors were removed, weighed, and photographed, and the tumor volume growth inhibition rate TGITV (%) and tumor weight inhibition rate TGITW (%) were calculated and statistically analyzed. During the treatment period, mice in each group ate and drank normally, their body weights were stable, there was no drug discontinuation and no death, no obvious toxic and side effects of the drug were observed, and the mice had good drug tolerance.

[0408] Table 12 Grouping and Dosage Regimen

[0409] The results are shown in Figure 10. At the end of the experiment (D25), the average tumor volumes of the 700053 (5.2 mg / kg) group, the 900698 (10 mg / kg) group, and the 700038 (11.6 mg / kg) group were 1858±318 mm3, 1420±347 mm3, and 1213±274 mm3, respectively. The corresponding tumor volume growth inhibition rates TGITV were 21.9%, 41.4%, and 50.6%. Compared with the isotype control antibody 900543 group (2352±248mm3&2.477±0.358g), the tumor volume and tumor weight of the 700053 group, 900698 group and 700038 group were significantly reduced and decreased. The above data show that 700053, 900698 and 700038 all have the effect of inhibiting tumor growth, and the anti-tumor effect of CD39 / CD73 dual antibody has a synergistic effect compared with CD39 nano monoclonal antibody and CD73 monoclonal antibody.

Claims

1. An isolated antigen binding protein comprising a first binding domain and a second binding domain, wherein the first binding domain is capable of binding to a CD39 protein and the second binding domain is capable of binding to a CD73 protein.

2. The isolated antigen-binding protein according to claim 1, having one or more of the following properties: (1) 1×10 -9 M or lower K D The value is bound to CD39 protein; (2) 1×10 -9 M or lower K D The value is bound to CD73 protein; (3) Inhibits the enzymatic activity of CD39 antigen; (4) Inhibits the enzymatic activity of CD73 antigen; (5) Thermal stability; (6) able to bind to human CD39 and / or cynomolgus monkey CD73; (7) capable of binding to human CD73 and / or cynomolgus monkey CD73; and (8) Can inhibit the growth of tumor cells.

3. The isolated antigen binding protein of any one of claims 1-2, wherein the first binding domain comprises an antibody or an antigen-binding fragment thereof.

4. The isolated antigen-binding protein of claim 3, wherein the antibody is selected from the group consisting of a monoclonal antibody, a single-chain antibody, a chimeric antibody, a multispecific antibody, a humanized antibody, and a fully human antibody.

5. The isolated antigen binding protein according to any one of claims 3-4, wherein the antigen binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv, F(ab')2, scFv, VHH, di-scFv and dAb fragments.

6. The isolated antigen binding protein of any one of claims 1 to 5, wherein the first binding domain is a VHH.

7. The isolated antigen-binding protein according to any one of claims 1 to 6, wherein the first binding domain comprises at least one CDR in an antibody heavy chain variable region VHH, wherein the VHH comprises the amino acid sequence shown in SEQ ID NO:

8.

8. The isolated antigen-binding protein according to any one of claims 1 to 7, wherein the first binding domain comprises a CDR3 comprising the amino acid sequence shown in SEQ ID NO:

1.

9. The isolated antigen-binding protein of any one of claims 1-8, wherein the first binding domain comprises CDR2, and the CDR2 comprises the amino acid sequence shown in SEQ ID NO:

2.

10. The isolated antigen binding protein of any one of claims 1-9, wherein the first binding domain comprises CDR1 comprising the amino acid sequence shown in SEQ ID NO:

3.

11. The isolated antigen-binding protein according to any one of claims 1 to 10, wherein the first binding domain comprises FR1, the C-terminus of the FR1 is directly or indirectly connected to the N-terminus of the CDR1, and the FR1 comprises the amino acid sequence shown in SEQ ID NO:

4.

12. The isolated antigen-binding protein according to any one of claims 1 to 11, wherein the first binding domain comprises FR2, the FR2 is located between the CDR1 and the CDR2, and the FR2 comprises the amino acid sequence shown in SEQ ID NO:

5.

13. The isolated antigen-binding protein of any one of claims 1-12, wherein the first binding domain comprises a FR3, the FR3 being located between the CDR2 and the CDR3, and the FR3 comprising the amino acid sequence shown in SEQ ID NO:

6.

14. The isolated antigen-binding protein according to any one of claims 1 to 13, wherein the first binding domain comprises FR4, the N-terminus of the FR4 is directly or indirectly connected to the C-terminus of the CDR3, and the FR4 comprises the amino acid sequence shown in SEQ ID NO:

7.

15. The isolated antigen-binding protein of any one of claims 1-14, wherein the first binding domain comprises a VHH, and the VHH comprises the amino acid sequence shown in SEQ ID NO:

8.

16. The isolated antigen binding protein of any one of claims 1-15, wherein the second binding domain comprises an antibody or antigen-binding fragment thereof.

17. The isolated antigen-binding protein of claim 16, wherein the antibody is selected from the group consisting of a monoclonal antibody, a single-chain antibody, a chimeric antibody, a multispecific antibody, a humanized antibody, and a fully human antibody.

18. The isolated antigen binding protein of any one of claims 16-17, wherein the antigen binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv, F(ab')2, scFv, VHH, di-scFv and dAb fragments.

19. The isolated antigen-binding protein according to any one of claims 1 to 18, wherein the second binding domain comprises at least one CDR in the antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO:

33.

20. The isolated antigen binding protein of any one of claims 1-19, wherein the second binding domain comprises a HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO:

27.

21. The isolated antigen binding protein of any one of claims 1-20, wherein the second binding domain comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO:

28.

22. The isolated antigen binding protein of any one of claims 1-21, wherein the second binding domain comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO:

29.

23. The isolated antigen binding protein of any one of claims 1-22, wherein the second binding domain comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are selected from the group consisting of: (1) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 9; (2) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 29, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 28, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

27.

24. The isolated antigen-binding protein according to any one of claims 1 to 23, wherein the second binding domain comprises H-FR1, the C-terminus of H-FR1 is directly or indirectly connected to the N-terminus of HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO:

30.

25. The isolated antigen-binding protein of any one of claims 1-24, wherein the second binding domain comprises H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO:

31.

26. The isolated antigen-binding protein of any one of claims 1-25, wherein the second binding domain comprises H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and wherein the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO:

32.

27. The isolated antigen-binding protein of any one of claims 1-26, wherein the second binding domain comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:

15.

28. The isolated antigen binding protein of any one of claims 1-27, wherein the second binding domain comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1, H-FR2, H-FR3 and H-FR4 comprise an amino acid sequence selected from the group consisting of: (1) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 12, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 13, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 14, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 15; (2) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 30, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 31, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 32, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:

15.

29. The isolated antigen binding protein of any one of claims 1-28, wherein the second binding domain comprises a VH, and the VH comprises the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO:

33.

30. The isolated antigen binding protein of any one of claims 1-29, wherein the second binding domain comprises an antibody heavy chain constant region.

31. The isolated antigen binding protein of claim 30, wherein the antibody heavy chain constant region is derived from an IgG heavy chain constant region.

32. The isolated antigen binding protein of any one of claims 30-31, wherein the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

33. The isolated antigen binding protein of any one of claims 30-32, wherein the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region.

34. The isolated antigen-binding protein of any one of claims 30-33, wherein the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:

43.

35. The isolated antigen binding protein of any one of claims 1-34, wherein the second binding domain comprises an antibody heavy chain comprising the amino acid sequence shown in SEQ ID NO: 25 or SEQ ID NO:

41.

36. The isolated antigen-binding protein of any one of claims 1-35, wherein the second binding domain comprises at least one CDR in an antibody light chain variable region VL, wherein the VL comprises the amino acid sequence shown in SEQ ID NO: 24 or SEQ ID NO:

40.

37. The isolated antigen binding protein of any one of claims 1-36, wherein the second binding domain comprises a LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO:

34.

38. The isolated antigen binding protein of any one of claims 1-37, wherein the second binding domain comprises LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18 (WAS) or SEQ ID NO: 35 (LAS).

39. The isolated antigen binding protein of any one of claims 1-38, wherein the second binding domain comprises LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO:

36.

40. The isolated antigen binding protein of any one of claims 1-39, wherein the second binding domain comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise an amino acid sequence selected from the group consisting of: (1) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 19, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 18 (WAS), and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 17; (2) The LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 36, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 35 (LAS), and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

34.

41. The isolated antigen-binding protein of any one of claims 1-40, wherein the second binding domain comprises L-FR1, the C-terminus of said L-FR1 is directly or indirectly linked to the N-terminus of said L-CDR1, and said L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 20 or SEQ ID NO:

37.

42. The isolated antigen-binding protein of any one of claims 1-41, wherein the second binding domain comprises L-FR2, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and wherein the L-FR2 comprises the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO:

38.

43. The isolated antigen binding protein of any one of claims 1-42, wherein the second binding domain comprises L-FR3, wherein the L-FR3 is positioned between the LCDR2 and the LCDR3, and wherein the L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 22 or SEQ ID NO:

39.

44. The isolated antigen-binding protein of any one of claims 1-43, wherein the second binding domain comprises L-FR4, the N-terminus of the L-FR4 is directly or indirectly linked to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:

23.

45. The isolated antigen binding protein of any one of claims 1-44, wherein the second binding domain comprises L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR1, L-FR2, L-FR3 and L-FR4 comprise an amino acid sequence selected from the group consisting of: (1) the L-FR1 comprises the amino acid sequence of SEQ ID NO: 20, the L-FR2 comprises the amino acid sequence of SEQ ID NO: 21, the L-FR3 comprises the amino acid sequence of SEQ ID NO: 22, and the L-FR4 comprises the amino acid sequence of SEQ ID NO: 23; (2) The L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 37, the L-FR2 comprises the amino acid sequence shown in SEQ ID NO: 39, the L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 39, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:

23.

46. ​​The isolated antigen binding protein of any one of claims 1-45, wherein the second binding domain comprises a VL comprising the amino acid sequence shown in SEQ ID NO: 24 or SEQ ID NO:

40.

47. The isolated antigen binding protein of any one of claims 1-46, wherein the second binding domain comprises an antibody light chain constant region.

48. The isolated antigen binding protein of claim 47, wherein the antibody light chain constant region is derived from an Ig kappa constant region.

49. The isolated antigen binding protein of any one of claims 47-48, wherein the antibody light chain constant region is derived from a human Ig kappa constant region.

50. The isolated antigen-binding protein of any one of claims 47-49, wherein the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

44.

51. The isolated antigen binding protein of any one of claims 1-50, wherein the second binding domain comprises an antibody light chain comprising the amino acid sequence shown in SEQ ID NO: 26 or SEQ ID NO:

42.

52. The isolated antigen binding protein of any one of claims 1-51, wherein the second domain comprises a VH and a VL comprising an amino acid sequence selected from the group consisting of: (1) The VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the VL comprises the amino acid sequence shown in SEQ ID NO: 24 The amino acid sequence shown; (2) The VH comprises the amino acid sequence shown in SEQ ID NO: 33, and the VL comprises the amino acid sequence shown in SEQ ID NO:

40.

53. The isolated antigen binding protein of any one of claims 1-52, wherein the second binding domain comprises an antibody heavy chain and an antibody light chain comprising an amino acid sequence selected from the group consisting of: (1) The antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 25, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 26; (2) The antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO:41, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO:

42.

54. The isolated antigen binding protein of any one of claims 1-53, wherein the first binding domain and the second binding domain are directly or indirectly linked.

55. The isolated antigen binding protein of any one of claims 1-54, wherein the C-terminus of the first binding domain is linked to the N-terminus of the second binding domain.

56. The isolated antigen binding protein of any one of claims 1-55, wherein the C-terminus of the first binding domain is linked to the N-terminus of the heavy chain variable region of the second binding domain.

57. The isolated antigen binding protein of any one of claims 1-56, wherein the first binding domain and the second binding domain are connected by a linker.

58. The isolated antigen binding protein of claim 57, wherein the linker comprises a peptide linker.

59. The isolated antigen binding protein of any one of claims 57-58, wherein the linker comprises an amino acid sequence of (GGGGS)n, wherein n is any positive integer from 0 to 10.

60. The isolated antigen binding protein of any one of claims 57-59, wherein the linker comprises the amino acid sequence shown in SEQ ID NO:

47.

61. The isolated antigen binding protein of any one of claims 1-60, comprising two of the first binding domains.

62. The isolated antigen binding protein of claim 61, wherein the two first binding domains are respectively linked directly or indirectly to the N-terminus of the second binding domain.

63. The isolated antigen binding protein according to any one of claims 61-62, wherein the two first binding domains are directly or indirectly linked to the N-termini of the two heavy chains of the second binding domain, respectively.

64. The isolated antigen binding protein of any one of claims 1-63, comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising the heavy chain of the first binding domain and the second binding domain.

65. The isolated antigen binding protein of claim 64, wherein the N-termini of the heavy chains of the first binding domain and the second binding domain are directly or indirectly linked.

66. The isolated antigen binding protein of any one of claims 64-65, wherein the N-termini of the heavy chains of the first binding domain and the second binding domain are connected by a linker.

67. The isolated antigen binding protein of any one of claims 64-66, wherein the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:45 or SEQ ID NO:

46.

68. The isolated antigen binding protein of any one of claims 64-67, wherein the second polypeptide chain comprises the light chain of the second binding domain.

69. The isolated antigen binding protein of any one of claims 64-68, wherein the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 26 or SEQ ID NO:

42.

70. The isolated antigen binding protein of any one of claims 64-69, comprising two of the first polypeptide chains and two of the second polypeptide chains.

71. A pharmaceutical combination comprising an antigen binding protein capable of binding to CD39 and an antigen binding protein capable of binding to CD73.

72. The pharmaceutical combination of claim 71, wherein the antigen binding protein capable of binding to CD39 has one or more of the following properties: (1) binds to human CD39 with an EC50 of less than about 0.5 μg / mL as determined by FACS assay; (2) binds to cynomolgus macaque (cyno) CD39 with an EC50 of less than about 0.7 μg / mL as determined by FACS assay; (3) As determined by Biacore assay, the -8 The KD of M binds to human CD39; (4) As determined by Biacore assay, the -7 The KD of M binds to cynomolgus macaque (cyno) CD39; (5) inhibiting ATPase activity in cells expressing CD39 with an EC50 of less than about 0.8 μg / mL as measured by an ATPase activity assay; (6) Inhibit CD39 function in tumor cells.

73. The pharmaceutical combination according to any one of claims 71-72, wherein the antigen binding protein capable of binding to CD39 comprises at least one CDR in the heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:

8.

74. The pharmaceutical combination according to any one of claims 71-73, wherein the antigen binding protein capable of binding to CD39 comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

1.

75. The pharmaceutical combination according to any one of claims 71-74, wherein the antigen binding protein capable of binding to CD39 comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:

2.

76. The pharmaceutical combination according to any one of claims 71-75, wherein the antigen binding protein capable of binding to CD39 comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:

3.

77. The pharmaceutical combination according to any one of claims 71-76, wherein the antigen binding protein capable of binding to CD39 comprises a VH comprising the amino acid sequence shown in SEQ ID NO:

8.

78. The pharmaceutical combination according to any one of claims 71 to 77, wherein the antigen binding protein capable of binding to CD39 is a VHH.

79. The pharmaceutical combination according to any one of claims 71-78, wherein the antigen binding protein capable of binding to CD73 has one or more of the following properties: (1) Ability to specifically bind to CD73 or its functionally active fragments; (2) can inhibit the enzymatic activity of CD73; (3) can induce the internalization of CD73 on the cell surface; (4) Ability to inhibit the growth and / or proliferation of tumor cells.

80. The pharmaceutical combination according to any one of claims 71 to 79, wherein the antigen binding protein capable of binding to CD73 comprises at least one CDR in the antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO:

33.

81. The pharmaceutical combination of any one of claims 71-80, wherein the antigen binding protein capable of binding to CD73 comprises a HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO:

27.

82. The pharmaceutical combination according to any one of claims 71-81, wherein the antigen binding protein capable of binding to CD73 comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO:

28.

83. The pharmaceutical combination according to any one of claims 71-82, wherein the antigen binding protein capable of binding to CD73 comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO:

29.

84. The pharmaceutical combination according to any one of claims 71-83, wherein the antigen binding protein capable of binding to CD73 comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO:

33.

85. The pharmaceutical combination of any one of claims 71-84, wherein the antigen binding protein capable of binding to CD73 comprises an antibody heavy chain constant region.

86. The pharmaceutical combination of claim 85, wherein the antibody heavy chain constant region is derived from an IgG heavy chain constant region.

87. The pharmaceutical combination according to any one of claims 85-86, wherein the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

88. The pharmaceutical combination according to any one of claims 85 to 87, wherein the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region.

89. The pharmaceutical combination according to any one of claims 85-88, wherein the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:

43.

90. The pharmaceutical combination of any one of claims 71-89, wherein the antigen binding protein capable of binding to CD73 comprises an antibody heavy chain comprising the amino acid sequence shown in SEQ ID NO: 25 or SEQ ID NO:

41.

91. The pharmaceutical combination according to any one of claims 71-90, wherein the antigen binding protein capable of binding to CD73 comprises at least one CDR in the antibody light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO: 24 or SEQ ID NO:

40.

92. The pharmaceutical combination of any one of claims 71-91, wherein the antigen binding protein capable of binding to CD73 comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO:

34.

93. The pharmaceutical combination of any one of claims 71-92, wherein the antigen binding protein capable of binding to CD73 comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 18 (WAS) or SEQ ID NO: 35 (LAS).

94. The pharmaceutical combination of any one of claims 71-93, wherein the antigen binding protein capable of binding to CD73 comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO:

36.

95. The pharmaceutical combination of any one of claims 71-94, wherein the antigen binding protein capable of binding to CD73 comprises a VL comprising the amino acid sequence shown in SEQ ID NO: 24 or SEQ ID NO:

40.

96. The pharmaceutical combination of any one of claims 71-95, wherein the antigen binding protein capable of binding to CD73 comprises an antibody light chain constant region.

97. The pharmaceutical combination of claim 96, wherein the antibody light chain constant region is derived from a human Igκ constant region.

98. The pharmaceutical combination according to any one of claims 96-97, wherein the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

44.

99. The pharmaceutical combination of any one of claims 71-98, wherein the antigen binding protein capable of binding to CD73 comprises an antibody light chain comprising the amino acid sequence shown in SEQ ID NO: 26 or SEQ ID NO:

42.

100. The pharmaceutical combination according to any one of claims 71-99, wherein the antigen binding protein capable of binding to CD73 comprises VH and VL, and the VH and VL comprise an amino acid sequence selected from the group consisting of: (1) The VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the VL comprises the amino acid sequence shown in SEQ ID NO: 24 The amino acid sequence shown; (2) The VH comprises the amino acid sequence shown in SEQ ID NO: 33, and the VL comprises the amino acid sequence shown in SEQ ID NO:

40.

101. The pharmaceutical combination according to any one of claims 71-100, wherein the antigen binding protein capable of binding to CD73 comprises an antibody heavy chain and an antibody light chain, and the antibody heavy chain and the antibody light chain comprise an amino acid sequence selected from the group consisting of: (1) The antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 25, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 26; (2) The antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO:41, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO:

42.

102. An isolated nucleic acid molecule or molecules encoding the isolated antigen binding protein of any one of claims 1-70.

103. A vector comprising the nucleic acid molecule of claim 102.

104. A cell comprising the nucleic acid molecule of claim 102 or the vector of claim 103.

105. A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1-70, the pharmaceutical combination of any one of claims 71-101, the nucleic acid molecule of claim 102, the vector of claim 103 and / or the cells of claims 1-4, and optionally a pharmaceutically acceptable carrier.

106. A method of making the isolated antigen binding protein of any one of claims 1-70, the method comprising culturing the cell of claim 104 under conditions such that the isolated antigen binding protein of any one of claims 1-70 is expressed.

107. Use of the isolated antigen-binding protein of any one of claims 1-70, the pharmaceutical combination of any one of claims 71-101, the nucleic acid molecule of claim 102, the vector of claim 103, the cell of claim 104, and / or the pharmaceutical composition of claim 105 in the preparation of a medicament for preventing and / or treating a disease and / or condition.

108. The use according to claim 107, wherein the disease and / or disorder is mediated by CD39 and / or CD73.

109. The use according to claims 107-108, wherein the disease and / or disorder comprises a tumor.

110. The use according to claim 109, wherein the tumor comprises a solid tumor and / or a hematological tumor.

111. The use according to any one of claims 107-110, wherein the disease and / or condition comprises pancreatic cancer and / or colorectal cancer.

112. A method for detecting CD39 and / or CD73 in a sample, the method comprising administering the isolated antigen binding protein of any one of claims 1-70, the pharmaceutical combination of any one of claims 71-101, the nucleic acid molecule of claim 102, the vector of claim 103, the cell of claim 104, and / or the pharmaceutical composition of claim 105.

113. The method according to claim 112, which is an in vitro method and / or an ex vivo method.

114. Use of the isolated antigen binding protein of any one of claims 1-70, the pharmaceutical combination of any one of claims 71-101, the nucleic acid molecule of claim 102, the vector of claim 103, the cell of claim 104, and / or the pharmaceutical composition of claim 105 in the preparation of a kit for detecting the presence of CD39 and / or CD73 in a sample.

Images