CD73 antibody cocktail therapy

Abstract

The invention provides a medicine combination which comprises a first antigen binding protein specifically binding to CD73 and a second antigen binding protein specifically binding to CD73, the first antigen binding protein and the second antigen binding protein have different CD73 antigen binding epitopes, and also provides a medicine composition comprising the medicine combination and application of the medicine composition.

Description

CD73 antibody cocktail therapy Technical Field

[0001] This application relates to the field of biomedicine, and specifically to a CD73 antibody cocktail therapy. Background Art

[0002] 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.

[0003] A large number of preclinical study results have shown that CD73 is abnormally highly expressed on the surface of various tumor cells, including glioma, breast cancer, melanoma, non-small cell lung cancer, bladder cancer, ovarian cancer, colorectal cancer, etc., and clinical data have shown that high expression of CD73 is closely related to the poor prognosis of tumor patients. Therefore, CD73 can be used as a potential target for the clinical treatment and prognosis of various tumors.

[0004] Therefore, there is an urgent need to develop multiple therapies targeting CD73 to provide more new ideas for the prevention and / or treatment of diseases caused by abnormal CD73 expression.

[0005] Summary of the Invention

[0006] The present application provides a pharmaceutical combination comprising a first antigen-binding protein that specifically binds to CD73, and a second antigen-binding protein that specifically binds to CD73, wherein the first antigen-binding protein and the second antigen-binding protein have different CD73 antigen-binding epitopes. The pharmaceutical combination described herein has one or more of the following properties: (1) the ability to specifically bind to the CD73 protein; (2) having a high affinity for the CD73 protein; and (3) the ability to inhibit the growth and / or proliferation of tumor cells.

[0007] In certain embodiments, the first antigen binding protein and the second antigen binding protein bind to the CD73 protein in a non-competitive manner.

[0008] In certain embodiments, the first antigen binding protein and the second antigen binding protein are present in the form of a mixture.

[0009] In certain embodiments, the first antigen-binding protein comprises at least one CDR in the variable region VH of an antibody heavy chain, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:8.

[0010] In certain embodiments, the first antigen binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 1.

[0011] In certain embodiments, the first antigen-binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2.

[0012] In certain embodiments, the first antigen binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3.

[0013] In certain embodiments, the first antigen-binding protein comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:8.

[0014] In certain embodiments, the first antigen binding protein comprises an antibody heavy chain constant region.

[0015] In certain embodiments, the antibody heavy chain constant region is derived from an IgG heavy chain constant region.

[0016] In certain embodiments, the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

[0017] In certain embodiments, the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:9.

[0018] In certain embodiments, the first antigen-binding protein comprises an antibody heavy chain comprising the amino acid sequence shown in SEQ ID NO:10.

[0019] In certain embodiments, the first 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:18.

[0020] In certain embodiments, the first antigen binding protein comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11.

[0021] In certain embodiments, the first antigen binding protein comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 12 (WAS).

[0022] In certain embodiments, the first antigen binding protein comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:13.

[0023] In certain embodiments, the first antigen binding protein comprises a VL comprising the amino acid sequence shown in SEQ ID NO:18.

[0024] In certain embodiments, the first antigen binding protein comprises an antibody light chain constant region.

[0025] In certain embodiments, the antibody light chain constant region is derived from a human Ig kappa constant region.

[0026] In certain embodiments, the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO:19.

[0027] In certain embodiments, the first antigen-binding protein comprises an antibody light chain comprising the amino acid sequence shown in SEQ ID NO:20.

[0028] In certain embodiments, the second antigen-binding protein comprises at least one CDR in the antibody heavy chain variable region VH, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:27.

[0029] In certain embodiments, the second antigen-binding protein comprises a HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 21.

[0030] In certain embodiments, the second antigen-binding protein comprises HCDR2, wherein the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 22.

[0031] In certain embodiments, the second antigen binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:23.

[0032] In certain embodiments, the second antigen-binding protein comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:27.

[0033] In certain embodiments, the second antigen binding protein comprises an antibody heavy chain constant region.

[0034] In certain embodiments, the antibody heavy chain constant region is derived from an IgG heavy chain constant region.

[0035] In certain embodiments, the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

[0036] In certain embodiments, the antibody heavy chain constant region of the second antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:9.

[0037] In certain embodiments, the second antigen-binding protein comprises an antibody heavy chain comprising the amino acid sequence shown in SEQ ID NO:28.

[0038] In certain embodiments, the second antigen-binding protein comprises at least one CDR in the antibody light chain variable region VL, wherein the VL comprises the amino acid sequence shown in SEQ ID NO:35.

[0039] In certain embodiments, the second antigen binding protein comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:29.

[0040] In certain embodiments, the second antigen binding protein comprises LCDR2, wherein the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 30 (LAS).

[0041] In certain embodiments, the second antigen binding protein comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:31.

[0042] In certain embodiments, the second antigen-binding protein comprises a VL comprising the amino acid sequence shown in SEQ ID NO:35.

[0043] In certain embodiments, the second antigen binding protein comprises an antibody light chain constant region.

[0044] In certain embodiments, the antibody light chain constant region is derived from a human Ig kappa constant region.

[0045] In certain embodiments, the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO:19.

[0046] In certain embodiments, the second antigen-binding protein comprises an antibody light chain comprising the amino acid sequence shown in SEQ ID NO:36.

[0047] In certain embodiments, the mass ratio of the first antigen-binding protein to the second antigen-binding protein in the pharmaceutical combination is about 1:5 to 5:1.

[0048] In certain embodiments, the mass ratio of the first antigen-binding protein to the second antigen-binding protein in the pharmaceutical combination is about 1:5.

[0049] In certain embodiments, the mass ratio of the first antigen-binding protein to the second antigen-binding protein in the pharmaceutical combination is about 1:2.5.

[0050] In certain embodiments, the mass ratio of the first antigen-binding protein to the second antigen-binding protein in the pharmaceutical combination is about 1:1.

[0051] In certain embodiments, the mass ratio of the first antigen-binding protein to the second antigen-binding protein in the pharmaceutical combination is about 2.5:1.

[0052] In certain embodiments, the mass ratio of the first antigen-binding protein to the second antigen-binding protein in the pharmaceutical combination is about 5:1.

[0053] On the other hand, the present application also provides a pharmaceutical composition comprising the drug combination, and optionally one or more pharmaceutically acceptable carriers.

[0054] In certain embodiments, the concentration of the drug combination is about 3-300 mg / mL.

[0055] In certain embodiments, the concentration of the drug combination is about 3-150 mg / mL.

[0056] In certain embodiments, the concentration of the drug combination is about 5-100 mg / mL.

[0057] In certain embodiments, the concentration of the drug combination is about 10-100 mg / mL.

[0058] In certain embodiments, the concentration of the drug combination is about 10-50 mg / mL.

[0059] In certain embodiments, the concentration of the drug combination is 30±5 mg / mL.

[0060] In certain embodiments, the concentration of the drug combination is 30±3 mg / mL.

[0061] In certain embodiments, the pharmaceutically acceptable carrier comprises a buffer.

[0062] In certain embodiments, the buffer comprises an acidic buffer.

[0063] In certain embodiments, the buffer comprises a neutral buffer.

[0064] In certain embodiments, the buffer is selected from the group consisting of acetate-acetate buffer, histidine-histidine hydrochloride buffer, citrate-citrate buffer, phosphate buffer, histidine-acetate buffer, Tris-hydrochloride buffer, phosphate-citrate, or a combination thereof.

[0065] In certain embodiments, the buffer is selected from the group consisting of acetate-acetic acid, citrate-citric acid, histidine-histidine hydrochloride, phosphate, Tris-hydrochloride, histidine-acetate, phosphate-citrate, or a combination thereof.

[0066] In certain embodiments, the buffer is selected from the group consisting of sodium acetate-acetic acid, sodium citrate-citric acid, histidine-histidine hydrochloride, sodium dihydrogen phosphate-disodium hydrogen phosphate, Tris-hydrochloride, histidine-acetic acid, and phosphate-sodium citrate.

[0067] In certain embodiments, the concentration of the buffer is about 5-100 mM.

[0068] In certain embodiments, the concentration of the buffer is about 5-60 mM.

[0069] In certain embodiments, the concentration of the buffer is about 5-50 mM.

[0070] In certain embodiments, the concentration of the buffer is about 10-40 mM.

[0071] In certain embodiments, the concentration of the buffer is 20±5 mM.

[0072] In certain embodiments, the concentration of the buffer is 20±2 mM.

[0073] In certain embodiments, the pH of the pharmaceutical composition is about 5-7.

[0074] In certain embodiments, the pH of the pharmaceutical composition is about 6±0.5.

[0075] In certain embodiments, the pH of the pharmaceutical composition is about 6 ± 0.3.

[0076] In certain embodiments, the pH of the pharmaceutical composition is about 6 ± 0.1.

[0077] In certain embodiments, the pH of the pharmaceutical composition is about 6.5 ± 0.3.

[0078] In certain embodiments, the pH of the pharmaceutical composition is about 7±0.3.

[0079] In certain embodiments, the pharmaceutically acceptable carrier comprises a stabilizer.

[0080] In certain embodiments, the stabilizer is selected from the group consisting of sodium chloride, amino acids, sugar alcohols, or combinations thereof.

[0081] In certain embodiments, the amino acid is selected from the group consisting of proline, arginine, glycine, histidine, lysine, methionine, or a combination thereof.

[0082] In certain embodiments, the sugar alcohol is selected from the group consisting of sucrose, mannitol, trehalose, sorbitol, or a combination thereof.

[0083] In certain embodiments, the stabilizer is selected from the group consisting of sucrose, trehalose, sorbitol, mannitol, proline / sucrose, methionine / trehalose, glycine / mannitol, sodium chloride / sucrose, arginine / sucrose, and lysine / trehalose.

[0084] In certain embodiments, the stabilizer is present in an amount of about 0.15 wt.% to 20 wt.%.

[0085] In certain embodiments, the stabilizer is present in an amount of about 0.15 wt.% to about 10 wt.%.

[0086] In certain embodiments, the stabilizer is present in an amount of about 0.15 wt.% to about 8 wt.%.

[0087] In certain embodiments, the stabilizer is present in an amount of about 4±1 wt.% or 8±1 wt.%.

[0088] In certain embodiments, the stabilizer is present in an amount of about 4±0.5 wt. % or 8±0.5 wt. %.

[0089] In certain embodiments, the stabilizer is present in an amount of about 4 wt.%, 5 wt.%, 7 wt.%, or 8 wt.%.

[0090] In certain embodiments, the stabilizer is present in an amount of about 8 wt.%.

[0091] In certain embodiments, the pharmaceutically acceptable carrier comprises a surfactant.

[0092] In certain embodiments, the surfactant is selected from the group consisting of polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, glycerol fatty acid esters, polysorbates, poloxamers, or combinations thereof.

[0093] In certain embodiments, the polysorbate is selected from the group consisting of polysorbate 20 (PS-20, Tween-20), polysorbate 40 (PS-40), polysorbate 60 (PS-60), and polysorbate 80 (PS-80).

[0094] In certain embodiments, the surfactant is selected from the group consisting of PS-20 (Tween-20) and PS-80 (Tween-80).

[0095] In certain embodiments, the concentration of the surfactant is about 0.001%-0.5%.

[0096] In certain embodiments, the concentration of the surfactant is about 0.005%-0.1%.

[0097] In certain embodiments, the concentration of the surfactant is about 0.005%, 0.01%, 0.03%, 0.05%, or 0.1%.

[0098] In certain embodiments, the concentration of the surfactant is about 0.01%.

[0099] In certain embodiments, the concentration of the surfactant is about 0.03%.

[0100] In certain embodiments, the pharmaceutically acceptable carrier is selected from the group consisting of: (1) 20 mM histidine-acetate, 8 wt.% sucrose, 0.03 wt.% Tween-80; (2) 20 mM sodium acetate-acetic acid, 8 wt.% trehalose, 0.03 wt.% Tween-80; (3) 20 mM histidine-acetate, 1.5 wt.% proline, 5 wt.% sucrose, 0.03 wt.% Tween-80; (4) 20 mM sodium acetate-acetic acid, 0.15 wt.% methionine, 8 wt.% trehalose, 0 .03wt.% Tween-80; (5) 20mM histidine-acetate, 8wt.% sucrose, 0.01wt.% Tween-80; (6) 20mM sodium acetate-acetic acid, 8wt.% trehalose, 0.01wt.% Tween-80; (7) 20mM histidine-acetate, 1.5wt.% proline, 5wt.% sucrose, 0.01wt.% Tween-80; (8) 20mM sodium acetate-acetic acid, 0.15wt.% methionine, 8wt.% trehalose, 0.01wt.% Tween-80.

[0101] In another aspect, the present application provides use of the pharmaceutical combination and / or the pharmaceutical composition in preparing a medicament for preventing and / or treating a disease and / or condition.

[0102] In certain embodiments, the disease and / or condition comprises a CD73-mediated disease and / or condition.

[0103] In certain embodiments, the disease and / or condition comprises a tumor.

[0104] In certain embodiments, the tumor comprises a solid tumor and / or a hematological tumor.

[0105] In certain embodiments, the disease and / or condition is selected from the group consisting of pancreatic cancer, breast cancer, and idiopathic pulmonary fibrosis (IPF).

[0106] In certain embodiments, the breast cancer comprises triple-negative breast cancer.

[0107] On the other hand, the present application also provides the use of the drug combination and / or the pharmaceutical composition in preparing a detection kit for detecting the presence and / or content of CD73 protein.

[0108] 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

[0109] 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:

[0110] Figure 1 shows the effect of the cocktail antibody therapy described in this application on inhibiting CD73 enzyme activity.

[0111] Figure 2 shows the binding affinity curves and response values ​​of 900698 and 900725 to human CD73 protein under different loading conditions.

[0112] Figure 3 shows the anti-tumor effect of anti-CD73 cocktail antibodies in pancreatic cancer animal models.

[0113] Figure 4 shows the anti-tumor effect of anti-CD73 cocktail antibodies in a triple-negative breast cancer animal model.

[0114] FIG5 is a schematic diagram showing the dosing regimen for the idiopathic pulmonary fibrosis (IPF) mouse model.

[0115] FIG6 shows the body weight of mice in each group of the idiopathic pulmonary fibrosis (IPF) mouse model.

[0116] FIG7 shows the results of airway hyperresponsiveness test in mice (area under the curve of Penh change rate-Mch concentration).

[0117] FIG8 shows a categorical bar graph of the total number of inflammatory-related cells and lymphocyte counts in the bronchoalveolar lavage fluid.

[0118] FIG9 shows the pathological scoring results of idiopathic pulmonary fibrosis.

[0119] FIG10 shows representative images of lung tissue pathology sections showing the pathological scoring results in the experiment. DETAILED DESCRIPTION

[0120] 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.

[0121] Definition of terms

[0122] In this application, the term "drug 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 also 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 present in the form of a mixture.

[0123] 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.

[0124] In this application, the term "antigen binding protein" generally refers to a protein with antigen binding ability obtained from a natural state by artificial means. The "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.

[0125] 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.

[0126] 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)).

[0127] 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.

[0128] 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).

[0129] 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).

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] In this application, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or condition. The pharmaceutical composition may comprise one or more active ingredients, and optionally a pharmaceutically acceptable carrier. For example, 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.

[0143] In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients and / or stabilizers that are non-toxic to cells or mammals exposed thereto at the doses 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] In the present application, the variant may be, for example, a protein or polypeptide in which one or more amino acids have been 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). 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.

[0148] 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).

[0149] 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.

[0150] 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..."

[0151] 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.

[0152] Detailed Description of the Invention

[0153] In one aspect, the present application provides a pharmaceutical combination comprising a first antigen binding protein that specifically binds to CD73, and a second antigen binding protein that specifically binds to CD73.

[0154] In the present application, the first antigen-binding protein has one or more of the following properties:

[0155] (1) Ability to specifically bind to CD73 or its functionally active fragments;

[0156] (2) can inhibit the enzymatic activity of CD73;

[0157] (3) can induce the internalization of CD73 on the cell surface;

[0158] (4) Ability to inhibit the growth and / or proliferation of tumor cells.

[0159] In the present application, the first antigen-binding protein and the second antigen-binding protein may have different CD73 antigen-binding epitopes.

[0160] In the present application, the first antigen-binding protein and the second antigen-binding protein can bind to the CD73 protein in a non-competitive manner.

[0161] In the present application, the first antigen-binding protein and the second antigen-binding protein may be present in the form of a mixture. For example, the first antigen-binding protein and the second antigen-binding protein may be present in the same container and mixed.

[0162] In the present application, the first antigen-binding protein may comprise at least one CDR in the antibody heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:8.

[0163] In the present application, the first antigen-binding protein may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 1.

[0164] In the present application, the first antigen-binding protein may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2.

[0165] In the present application, the first antigen-binding protein may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3.

[0166] In the present application, the first antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 1.

[0167] In the present application, the first antigen-binding protein 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:4.

[0168] In the present application, the first antigen-binding protein 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:5.

[0169] In the present application, the first antigen-binding protein may comprise H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6.

[0170] In the present application, the first antigen-binding protein may comprise H-FR4, wherein the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the H-FR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7.

[0171] In the present application, the first antigen-binding protein may comprise H-FR1, H-FR2, H-FR3, and H-FR4. For example, H-FR1 comprises the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:24, H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:25, H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:26, and H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:7.

[0172] In the present application, the first antigen-binding protein comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:8.

[0173] In the present application, the first antigen binding protein may comprise an antibody heavy chain constant region.

[0174] 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:9.

[0175] In the present application, the first antigen-binding protein may comprise an antibody heavy chain, and the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO:10.

[0176] In the present application, the first antigen-binding protein may comprise at least one CDR in the antibody light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:18.

[0177] In the present application, the first antigen-binding protein may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:11.

[0178] In the present application, the first antigen-binding protein may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 12 (WAS).

[0179] In the present application, the first antigen-binding protein may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:13.

[0180] In the present application, the first antigen-binding protein may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 13, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 12 (WAS), and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 11.

[0181] In the present application, the first antigen-binding protein 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:14.

[0182] In the present application, the first antigen-binding protein 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:15.

[0183] In the present application, the first antigen-binding protein 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:16.

[0184] In the present application, the first antigen-binding protein may comprise L-FR4, wherein the N-terminus of the L-FR4 is directly or indirectly connected to the C-terminus of the L-FR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:17.

[0185] In the present application, the first antigen-binding protein may comprise L-FR1, L-FR2, L-FR3, and L-FR4. For example, L-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 14, L-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 15, L-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 16, and L-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 17.

[0186] In the present application, the first antigen-binding protein comprises VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:18.

[0187] In the present application, the first antigen-binding protein may comprise an antibody light chain constant region.

[0188] In the present application, the antibody light chain constant region may be derived from a human Igκ constant region.

[0189] In the present application, the antibody light chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 19.

[0190] In the present application, the first antigen-binding protein may comprise an antibody light chain, and the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 20.

[0191] In the present application, the first antigen-binding protein may comprise VH and VL, the VH may comprise the amino acid sequence shown in SEQ ID NO: 8, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 18.

[0192] In the present application, the first antigen-binding protein may comprise a heavy chain and a light chain, the heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 10, and the light chain may comprise the amino acid sequence shown in SEQ ID NO: 20.

[0193] In the present application, the second antigen-binding protein may comprise at least one CDR in the antibody heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 27.

[0194] In the present application, the second antigen-binding protein may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 21.

[0195] In the present application, the second antigen-binding protein may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 22.

[0196] In the present application, the second antigen-binding protein may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 23.

[0197] In the present application, the second antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 22, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 21.

[0198] In the present application, the second antigen-binding protein 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:24.

[0199] In the present application, the second antigen-binding protein 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: 25.

[0200] In the present application, the second antigen-binding protein may comprise H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 26.

[0201] In the present application, the second antigen-binding protein may comprise H-FR4, the N-terminus of the H-FR4 being directly or indirectly connected to the C-terminus of the H-FR3, and the H-FR4 comprising the amino acid sequence shown in SEQ ID NO:7.

[0202] In the present application, the second antigen-binding protein may comprise H-FR1, H-FR2, H-FR3, and H-FR4. For example, H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 24, H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 25, H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 26, and H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 7.

[0203] In the present application, the second antigen-binding protein comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:8.

[0204] In the present application, the second antigen-binding protein may comprise an antibody heavy chain constant region.

[0205] 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:9.

[0206] In the present application, the second antigen-binding protein may comprise an antibody heavy chain, and the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 28.

[0207] In the present application, the second antigen-binding protein may comprise at least one CDR in the antibody light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 35.

[0208] In the present application, the second antigen-binding protein may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 29.

[0209] In the present application, the second antigen-binding protein may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 30 (LAS).

[0210] In the present application, the second antigen-binding protein may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:31.

[0211] In the present application, the second antigen-binding protein may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:31, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:30 (LAS), and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:29.

[0212] In the present application, the second antigen-binding protein 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:32.

[0213] In the present application, the second antigen-binding protein 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:33.

[0214] In the present application, the second antigen-binding protein 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: 34.

[0215] In the present application, the second antigen-binding protein 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:17.

[0216] In the present application, the second antigen-binding protein may comprise L-FR1, L-FR2, L-FR3, and L-FR4. For example, L-FR1 may comprise the amino acid sequence set forth in SEQ ID NO:32, L-FR2 may comprise the amino acid sequence set forth in SEQ ID NO:33, L-FR3 may comprise the amino acid sequence set forth in SEQ ID NO:34, and L-FR4 may comprise the amino acid sequence set forth in SEQ ID NO:17.

[0217] In the present application, the second antigen-binding protein comprises a VL, and the VL comprises the amino acid sequence shown in SEQ ID NO: 35.

[0218] In the present application, the second antigen-binding protein may comprise an antibody light chain constant region.

[0219] In the present application, the antibody light chain constant region may be derived from a human Igκ constant region.

[0220] In the present application, the antibody light chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 19.

[0221] In the present application, the second antigen-binding protein may comprise an antibody light chain, and the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 36.

[0222] In the present application, the second antigen-binding protein may comprise VH and VL, the VH may comprise the amino acid sequence shown in SEQ ID NO: 27, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 35.

[0223] In the present application, the second antigen-binding protein may comprise a heavy chain and a light chain, the heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 28, and the light chain may comprise the amino acid sequence shown in SEQ ID NO: 36.

[0224] In the present application, the first antigen-binding protein and the second antigen-binding protein in the pharmaceutical combination can be mixed at a certain mass ratio. For example, they can be mixed at a mass ratio of about 1:5 to 5:1. For example, the mass ratio of the first antigen-binding protein to the second antigen-binding protein is about 1:5. For example, the mass ratio of the first antigen-binding protein to the second antigen-binding protein is about 1:2.5. For example, the mass ratio of the first antigen-binding protein to the second antigen-binding protein is about 1:1. For example, the mass ratio of the first antigen-binding protein to the second antigen-binding protein is about 2.5:1. For example, the mass ratio of the first antigen-binding protein to the second antigen-binding protein is about 5:1.

[0225] In the present application, the pharmaceutical combination may be formulated for administration by intravenous injection.

[0226] Pharmaceutical composition

[0227] On the other hand, the present application also provides a pharmaceutical composition comprising the pharmaceutical combination, and optionally one or more pharmaceutically acceptable carriers.

[0228] In the present application, the pharmaceutical composition may be a mixture comprising the first antigen-binding protein and the second antigen-binding protein. The mixture may further comprise inactive ingredients to be formulated into a pharmaceutically acceptable preparation.

[0229] In the present application, the concentration of the drug combination is about 3-300 mg / mL, alternatively about 3-150 mg / mL, alternatively about 5-100 mg / mL, alternatively about 10-100 mg / mL, alternatively about 10-50 mg / mL, for example, about 30±5 mg / mL, for example, about 30±3 mg / mL.

[0230] In the present application, the pharmaceutical composition may comprise a buffer. For example, the buffer may be an acidic buffer. For example, the buffer may be a neutral buffer.

[0231] For example, the buffer can be selected from the group consisting of acetate-acetate buffer, histidine-histidine hydrochloride buffer, citrate-citrate buffer, phosphate buffer, histidine-acetate buffer, Tris-hydrochloride buffer, phosphate-citrate, or a combination thereof.

[0232] For example, the buffer may be selected from the group consisting of acetate-acetic acid, citrate-citric acid, histidine-histidine hydrochloride, phosphate, Tris-hydrochloride, histidine-acetate, phosphate-citrate, or a combination thereof.

[0233] For example, the buffer can be selected from one or more of the following groups: sodium acetate-acetic acid, sodium citrate-citric acid, histidine-histidine hydrochloric acid, sodium dihydrogen phosphate-disodium hydrogen phosphate, Tris-hydrochloride, histidine-acetic acid and phosphate-sodium citrate. For example, the buffer can be a histidine-acetate buffer.

[0234] In the present application, the concentration of the buffer may be about 5-100 mM, alternatively about 5-60 mM, alternatively about 5-50 mM, alternatively about 10-40 mM, alternatively about 20±5 mM, alternatively about 20±2 mM.

[0235] In the present application, the pH of the pharmaceutical composition can be about 5-7. For example, the pH of the pharmaceutical composition is about 6 ± 0.5. For example, the pH of the pharmaceutical composition is about 6 ± 0.3. For example, the pH of the pharmaceutical composition is about 6 ± 0.1. For example, the pH of the pharmaceutical composition is about 6.5 ± 0.3. For example, the pH of the pharmaceutical composition is about 7 ± 0.3.

[0236] In the present application, the pharmaceutical composition may contain a stabilizer. In the present application, the stabilizer may be selected from the following group: sodium chloride, amino acids, sugar alcohols, or a combination thereof. For example, the amino acids may be selected from the following group: proline, arginine, glycine, histidine, lysine, methionine, or a combination thereof. For example, the sugar alcohol may be selected from the following group: sucrose, mannitol, trehalose, sorbitol, or a combination thereof.

[0237] For example, the stabilizer can be selected from one or more of the following groups: sucrose, trehalose, sorbitol, mannitol, proline / sucrose, methionine / trehalose, glycine / mannitol, sodium chloride / sucrose, arginine / sucrose, and lysine / trehalose. For example, the stabilizer of the pharmaceutical composition can be sucrose.

[0238] In the present application, the content of the stabilizer in the pharmaceutical composition is about 0.15wt.%-20wt.%, optionally the content of the stabilizer is about 0.15wt.%-10wt.%, optionally the content of the stabilizer is about 0.15wt.%-8wt.%, optionally the content of the stabilizer is about 4±1wt.% or 8±1wt.%, optionally the content of the stabilizer is about 4±0.5wt.%, 8±0.5wt.%, optionally the content of the stabilizer is about 4wt.%, 5wt.%, 7wt.% or 8wt.%. For example, the content of the stabilizer is about 0.15wt.%, about 0.2wt.%, about 0.3wt.%, about 0.4wt.%, 0.5wt.%, about 0.6wt.%, about 0.8wt.%, about 1wt.%, about 1.5wt.%, about 2wt.%, about 2.5wt.%, about 3wt.%, about 3.5wt.%, about 4wt.%, about 4.5wt.%, about 5wt.%, about 5.5wt.%, about 6wt.%, about 7wt.%, about 8wt.%.

[0239] In the present application, the pharmaceutical composition may comprise a surfactant.

[0240] In the present application, the surfactant can be selected from the group consisting of polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, glycerol fatty acid esters, polysorbates, poloxamer, or combinations thereof. In the present application, the polysorbate can be selected from the group consisting of polysorbate 20 (PS-20, Tween-20), polysorbate 40 (PS-40), polysorbate 60 (PS-60), and polysorbate 80 (PS-80). For example, the surfactant can include Tween-20 (PS-20) and / or Tween-80 (PS-80). For example, the concentration of the surfactant is about 0.001%-0.5%, alternatively about 0.005%-0.1%, alternatively about 0.005%, 0.01%, 0.03%, 0.05%, or 0.1%, alternatively about 0.01% or 0.03%. For example, the concentration of the surfactant is about 0.005%, about 0.01%, 0.03%, about 0.05%, about 0.1%.

[0241] In the present application, the buffer system of the pharmaceutical composition may comprise 20 mM histidine-acetate, 8 wt.% sucrose, and 0.03 wt.% Tween-80.

[0242] In the present application, the buffer system of the pharmaceutical composition may comprise 20 mM sodium acetate-acetic acid, 8 wt.% trehalose, and 0.03 wt.% Tween-80.

[0243] In the present application, the buffer system of the pharmaceutical composition may comprise 20 mM histidine-acetate, 1.5 wt.% proline, 5 wt.% sucrose, and 0.03 wt.% Tween-80.

[0244] In the present application, the buffer system of the pharmaceutical composition may comprise 20 mM sodium acetate-acetic acid, 0.15 wt.% methionine, 8 wt.% trehalose, and 0.03 wt.% Tween-80.

[0245] In the present application, the buffer system of the pharmaceutical composition may comprise 20 mM histidine-acetate, 8 wt.% sucrose, and 0.01 wt.% Tween-80.

[0246] In the present application, the buffer system of the pharmaceutical composition may comprise 20 mM sodium acetate-acetic acid, 8 wt.% trehalose, and 0.01 wt.% Tween-80.

[0247] In the present application, the buffer system of the pharmaceutical composition may comprise 20 mM histidine-acetate, 1.5 wt.% proline, 5 wt.% sucrose, and 0.01 wt.% Tween-80.

[0248] In the present application, the buffer system of the pharmaceutical composition may comprise 20 mM sodium acetate-acetic acid, 0.15 wt.% methionine, 8 wt.% trehalose, and 0.01 wt.% Tween-80.

[0249] use

[0250] In another aspect, the present application provides use of the pharmaceutical combination and / or the pharmaceutical composition in preparing a medicament for preventing and / or treating a disease and / or condition.

[0251] 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 pharmaceutical combination and / or the pharmaceutical composition described herein to a subject in need thereof.

[0252] In the present application, the administration can be performed by different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0253] On the other hand, the pharmaceutical combination and / or the pharmaceutical composition described herein can be used to prevent and / or treat diseases and / or disorders.

[0254] In the present application, the disease and / or disorder may be a disease and / or disorder mediated by abnormal expression of CD73.

[0255] In the present application, the disease and / or disorder may include a tumor.

[0256] In the present application, the tumor may include solid tumors and / or hematological tumors.

[0257] In the present application, the disease and / or condition may include pancreatic cancer, breast cancer and / or idiopathic pulmonary fibrosis (IPF).For example, the breast cancer may include triple-negative breast cancer.

[0258] On the other hand, the present application also provides a method for detecting CD73 in a sample, which comprises administering the drug combination and / or the pharmaceutical composition.

[0259] In the present application, the method for detecting CD73 in a sample may be an in vitro method. In the present application, the method for detecting CD73 in a sample may be for non-therapeutic purposes. In the present application, the method for detecting CD73 in a sample is not a diagnostic method.

[0260] On the other hand, the present application also provides a reagent or kit for detecting CD73 in a sample, which comprises the drug combination and / or the pharmaceutical composition.

[0261] On the other hand, the present application also provides the use of the drug combination and / or the pharmaceutical composition in preparing a kit for detecting the presence and / or content of CD73 in a sample.

[0262] Without intending to be bound by any theory, the following examples are merely intended to illustrate the drug combination, preparation method and use of the present application, and are not intended to limit the scope of the invention of the present application.

[0263] Example

[0264] General determination method

[0265] SEC-UPLC: Using ACQUITY UPLC Protein BEH SEC analytical column (4.6 mm × 150 mm) was used with a Waters ultra-high performance liquid chromatography instrument (Acquity H Class) according to the general high performance liquid chromatography method specified in 0514 of the Pharmacopoeia of the People's Republic of China (2015 edition, Volume III), and the purity was calculated by the area normalization method.

[0266] CEX-HPLC: with ProPac TM The purity was determined by high performance liquid chromatography (HPLC) using a WCX-10 analytical column (4 mm × 250 mm) and a Water high performance liquid chromatography instrument (E2695) according to the general high performance liquid chromatography method of the Pharmacopoeia of the People's Republic of China (2015 edition, Volume III). The purity was calculated by the area normalization method.

[0267] Tm value determination (IF, full spectrum fluorescence): Uncle (Unchained Labs) was used to calculate the melting temperature (Tm) of different formulations using the full spectrum fluorescence module with heating from 25°C to 95°C at 0.3°C / min.

[0268] Tagg value determination (SLS, static light scattering): using Uncle instrument at 25°C to 95°C, heating at 0.3°C / min, and using static light scattering module to calculate the initial aggregation temperature Tagg of antibody molecules with different formulations.

[0269] Example 1 CD73 enzyme activity inhibition experiment of components 900698 and 900725 in the anti-CD73 cocktail antibody

[0270] 900698 (heavy chain sequence as shown in SEQ ID NO:10, light chain sequence as shown in SEQ ID NO:20) and 900725 (heavy chain sequence as shown in SEQ ID NO:28, light chain sequence as shown in SEQ ID NO:36) were diluted to 30 μg / ml with PBS and then mixed at a ratio of 1:5, 1:2.5, 1:1, 2.5:1, and 5:1, and then diluted 5-fold in eight steps. A suspension of human CD73-expressing cells (CHOK1-huCD73-3F4, Huabo Bio) in logarithmic growth phase was prepared in culture medium to a cell suspension of 4 × 105 / ml. 100 μl (approximately 40,000 cells) was added to each well of a 96-well U-shaped plate and centrifuged at 1500 rpm for 5 minutes. The supernatant was removed. The cells were then resuspended in serially diluted antibodies, 100 μl was added to each well, mixed, and incubated at 37°C for 20 minutes. After the incubation, centrifuge at 1500 rpm for 5 minutes, remove the supernatant, wash once with 200 ul / well PBS, and then resuspend with 100 ul / well 180 uM AMP and incubate at 37°C for 60 minutes. After the incubation, centrifuge at 1500 rpm for 5 minutes, take 50 ul / well of the supernatant, transfer to a 96-well black plate, add 50 ul / well of the prepared ATP, mix well, and react at 37°C for 15 minutes. Substrate joins Buffer was added, mixed, and equilibrated to room temperature. Then, 100 μL / well was added to the test wells. Incubation was performed at room temperature for 10 minutes, and full-spectrum detection was performed. The percentage of enzyme activity was estimated as follows: residual CD73 activity was calculated as follows: (CHOK1-huCD73-3F4 cells + Ab + ATP + AMP) - (ATP + AMP) / (CHOK1-huCD73-3F4 cells + ATP + AMP) - (ATP + AMP) * 100.

[0271] The inhibitory effects of 900698, 900725, and mixtures of 900698 and 900725 in different ratios on the enzyme activity of CD73 expressed on the cell membrane (CHOK1-huCD73-3F4, Huabo Bio) are shown in Figure 1. The results show that when the antibody concentration is between 0.1 and 1 ug / ml, the ability of the mixture of 900698 and 900725 to inhibit enzyme activity is stronger than that of either 900698 or 900725 alone under the same concentration conditions (as indicated by the black dotted arrow, the lower the lower platform value of the enzyme activity inhibition curve, the stronger the maximum inhibitory ability of the antibody on enzyme activity). This indicates that the mixture of 900698 and 900725 has a synergistic inhibitory effect, and the effect appears to be optimal when the ratio of 900698 to 900725 is 1:1.

[0272] Example 2 SPR competition binding experiment of anti-CD73 cocktail antibody

[0273] To investigate whether antibodies 900698 and 900725 compete for CD73 binding, we performed an SPR competition binding experiment. First, human CD73 antigen protein (ACRO Biosystems, Catalog No. CD3-H52H7) was captured using a BIAcore 8k, and then the antibodies were used as analytes. Antibodies were added in the following order: 900698 + 900725 (900698 added first, then 900725) or 900725 + 900698 (900725 added first, then 900698). Controls were performed using 900698 + Buffer and 900725 + Buffer.

[0274] As shown in Table 1 and Figure 2 below, whether 900698 or 900725 is added first, it does not affect the binding of the other antibody to the CD73 antigen, indicating that the two components of the anti-CD73 cocktail antibody, 900698 and 900725, have different binding epitopes on the CD73 antigen and can bind to the CD73 antigen simultaneously.

[0275] Table 1 Binding affinity test of 900698 and 900725 to human CD73 protein under different loading conditions

[0276] Example 3 Detection of the anti-tumor effect of anti-CD73 cocktail antibody in pancreatic cancer animal model

[0277] In this experiment, NPG mice were used to establish a BxPC-3 pancreatic cancer animal model and test the efficacy of the test antibodies. The human pancreatic cancer cells BxPC-3 used in this experiment were cultured in RPMI-1640 medium supplemented with 10% FBS in a 37°C incubator containing 5% CO2. Before the cells were cultured for ten consecutive generations, 100 μL of PBS containing approximately 1×107 BxPC-3 cells and an equal volume of Matrigel were mixed and inoculated subcutaneously into 36 NPG mice on the right side of the back near the armpit, with an inoculation volume of about 200 μL. The mice were anesthetized with 2-5% isoflurane before inoculation. On the day of inoculation, 1.0X10 7PBMC (100 μL). When tumors grow to an average of approximately 50-80 mm³, 36 tumor-bearing mice are randomly divided into 6 groups of 6 mice each based on tumor volume and body weight. Dosing is performed on the day of grouping. The specific dosing schedule is shown in the table below. 900201 is a negative control antibody that does not bind to the CD73 antigen. The amino acid sequence of the heavy chain (HC) of 900201 is shown in SEQ ID NO:37, and the amino acid sequence of the light chain (LC) of 900201 is shown in SEQ ID NO:38. Oleclumab is an anti-CD73 monoclonal antibody developed by AstraZeneca and is currently in Phase 3 clinical trials. Uliledlimab is an anti-CD73 monoclonal antibody developed by I-Mab Biopharmaceuticals and is currently in Phase 1 clinical trials.

[0278] The results are shown in Table 2 and Figure 3 below. Compared with the negative control group 900201, the positive drugs oleclumab, uliledlimab and the test drugs 900698, 900725, and anti-CD73 cocktail antibodies can significantly inhibit the growth of BxPC-3 pancreatic cancer, and the same dose of anti-CD73 cocktail antibodies has a better tumor inhibition effect than the positive control oleclumab, uliledlimab, 900698 and 900725, proving that the anti-CD73 antibody cocktail antibody composed of an equal proportion of 900698 and 900725 has a good synergistic anti-pancreatic cancer tumor effect.

[0279] Table 2 Dosage regimen for pancreatic cancer model

[0280] Example 4 Detection of the anti-tumor effect of anti-CD73 cocktail antibody in triple-negative breast cancer animal model

[0281] This study used NPG mice to establish an MDA-MB-231 triple-negative breast cancer model and to test the efficacy of the test antibodies. The human breast cancer MDA-MB-231 cells used in this experiment were cultured in L-15 medium supplemented with 10% FBS in a 37°C incubator without CO2. Before the tenth generation of cell culture, a medium containing approximately 5.0X10 6 MDA-MB-231 cells were suspended in 100 μL PBS and mixed with an equal volume of Matrigel. The cells were then inoculated subcutaneously into the right side of the back of NPG humanized mice near the armpit. The inoculation volume was approximately 200 μL. The mice were anesthetized with 2-5% isoflurane before inoculation. On the day of inoculation, 1.0×10 7 PBMC (100uL), when the tumor grows to an average of about 50-80mm 3Around 6:00 p.m., 30 tumor-bearing mice were randomly divided into five groups of six mice each based on tumor volume and body weight. Dosing was performed on the day of grouping. The specific dosing schedule is shown in the table below. 900201 is a negative control antibody that does not bind to the CD73 antigen. Oleclumab is an anti-CD73 monoclonal antibody developed by AstraZeneca and is currently in Phase 3 clinical trials.

[0282] The results are shown in Table 3 and Figure 4 below. Compared with the negative control group 900201, the positive drug oleclumab and the test drugs 900698, 900725, and the anti-CD73 cocktail antibody can significantly inhibit the growth of MDA-MB-231 triple-negative breast cancer, and the same dose of anti-CD73 cocktail antibody has a better tumor inhibition effect than the positive control oleclumab and 900698 and 900725, proving that the anti-CD73 antibody cocktail antibody composed of an equal proportion of 900698 and 900725 has a good synergistic anti-triple-negative breast cancer tumor effect.

[0283] Table 3 Dosage regimen for triple-negative breast cancer model

[0284] Example 5 Therapeutic effect of anti-CD73 cocktail antibody in an animal model of idiopathic pulmonary fibrosis (IPF)

[0285] Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by progressive and irreversible pulmonary fibrosis, with no effective treatment. Most patients die from progressive respiratory failure within 3-8 years of symptom onset. Although the pathogenesis of IPF is poorly understood, hallmark pathological features include inflammatory changes in the lungs, excessive fibroblast proliferation, and abnormal deposition of extracellular matrix (ECM). Bleomycin (BLM)-induced fibrosis is widely used to study the pathogenesis of IPF.

[0286] The specific operation of the bleomycin-induced CD73 humanized transgenic mouse idiopathic pulmonary fibrosis model experiment is as follows: CD73 humanized mice are grouped according to the following Table 4, that is, except for 3 randomly selected animals directly included in the G1 normal group, all other groups of animals receive tracheal instillation of bleomycin for model induction. The specific induction method is: on the first day, the animals are anesthetized by inhalation of 2-5% isoflurane. Based on body weight, the animals will be given bleomycin (2U / kg, the specific administration volume will be calculated and recorded based on the animal weight) through the tracheal route. Three days after bleomycin instillation, according to the animal weight and animal status, animals with excessive weight loss and slight weight changes are removed, and the 48 animals are divided into the remaining 6 groups by the BioBook random allocation function to achieve approximate average weight of each group and reduce inter-group deviation. All animals will start dosing on the 4th day until the end of the experiment. The specific dosing schedule is shown in Figure 5 and Table 4. The weight of the animals will be weighed and recorded three times a week throughout the experimental period. The results of mouse weight are shown in Figure 6. During the entire experimental period, except for the normal group, the animals gradually lost weight after modeling, and their weight gradually and continuously increased with the progress of the experiment and self-recovery. There was no significant difference among the groups.

[0287] Table 4. Grouping and dosing regimen of idiopathic pulmonary fibrosis (IPF) mouse model

[0288] 5.1 Airway Responsiveness Measurement:

[0289] On day 21, all animals underwent airway hyperresponsiveness measurements to assess lung function. Airway hyperresponsiveness was measured in experimental mice using a WBP system. First, mice inhaled a PBS solution by nebulization. Subsequently, continuous nebulization of methacholine (Mch) at concentrations of 1.5625, 3.125, 6.25, 12.5, 25, and 50 mg / mL was performed. The enhanced expiratory interval (Penh) at the corresponding concentrations was measured. Each concentration was stimulated for 90 seconds. The rate of change of Penh relative to baseline versus Mch concentration was plotted, and the area under the curve was calculated.

[0290] The results, as shown in Figure 7, showed a significant increase in the area under the Penh curve (AUC) in the model group compared to the normal animals. Compared to the model group, the high-dose group showed a trend of improvement, with a significant difference (P=0.024 Kruskal-Wallis test). No improvement was observed in the positive control group, nintedanib, demonstrating that the anti-CD73 cocktail antibody is superior to the positive control, nintedanib, in improving lung function in idiopathic pulmonary fibrosis.

[0291] 5.2 Determination of the number of inflammatory cells in bronchoalveolar lavage fluid

[0292] After pulmonary function testing, animals were anesthetized with Zoltrate (intraperitoneal injection, 25-50 mg / kg Zoltrate, 5-10 mg / kg xylazine) and intubated. The lungs were lavaged for the first time with 0.4 mL of PBS (containing 1% FBS). Separate lavages were performed for the second and third lung lavages with 0.4 mL of PBS (containing 1% FBS). 100 μL of the lavage fluid was suspended and counted. The lavage fluid was centrifuged at 300 g for 5 minutes at 4°C. The cell pellet obtained after centrifugation was resuspended and used for smear preparation. The cells were stained with Wright-Giemsa solution to distinguish eosinophils, neutrophils, macrophages, and lymphocytes. Cells were counted under a light microscope. Data are expressed as mean ± standard error (SEM). Data were analyzed using SPSS or Graphpad Prism. Specific analytical methods are described in the figure legends and table footnotes. P < 0.05 was considered statistically significant.

[0293] The results of lung lavage fluid cell counts showed that the percentage of total inflammation-related cells and lymphocytes in the alveolar lavage fluid of the model group animals was significantly increased compared with that of the normal group; after administration of positive drugs and different concentrations of anti-CD73 cocktail antibody test substances, the levels of total inflammation-related cells and lymphocytes were significantly reduced, indicating that anti-CD73 cocktail antibodies can significantly improve the degree of inflammation in idiopathic pulmonary fibrosis.

[0294] 5.3 Pathological fibrosis score:

[0295] After lavage, the animals were euthanized by cervical dislocation. The left lung was collected, fixed with neutral formalin, and embedded in paraffin. Transverse sections were taken from the upper, middle, and lower segments, totaling three sections per lung lobe, embedded in a single paraffin block, and stained with Masson staining for pathological fibrosis scoring. The scoring criteria are shown in Table 5 below.

[0296] The scoring results are shown in Figures 9 and 10. Compared with the animals in the normal group, the animals in the model group showed significant changes in pulmonary fibrosis after bleomycin induction (P<0.001), indicating that the bleomycin pulmonary fibrosis model was successfully established. The positive drug nintedanib group and the anti-CD73 cocktail antibody group both improved the pathological scores. Using the Kruskal-Wallis test method, the medium-dose anti-CD73 cocktail antibody group had significant differences compared with the model group. This shows that anti-CD73 cocktail antibodies can effectively improve the pathological score of idiopathic pulmonary fibrosis.

[0297] Table 5. Pathological fibrosis scoring criteria

[0298] Example 6 Comparison of different buffer systems

[0299] The antibody was replaced with 10 mM of each screening buffer (as shown in Table 6 below) using a WaterSep hollow fiber (Discover 12), and the antibody concentration was adjusted to approximately 5 mg / mL using the respective buffers. The solution was sterilized and filtered before use.

[0300] The stability of each buffer was evaluated using Tm and Tagg for freshly prepared samples. The results are shown in Tables 6 and 7. Freshly prepared samples were also subjected to various stress conditions, including a high-temperature test (40°C ± 2°C for three weeks) and freeze-thaw cycles (five cycles, ≤ -70°C / 5°C ± 3°C). Stability was evaluated using various test items, including appearance and purity. The results are shown in Tables 8 and 9.

[0301] Table 6 Summary of Tm and Tagg results of 900698 antibody in different buffers

[0302] Table 7 Summary of Tm and Tagg results of 900725 antibody in different buffers

[0303] Table 8 Summary of the appearance and purity of 900698 antibody in different buffer systems under different pressure conditions

[0304] Table 9 Summary of the appearance and purity results of 900725 antibody in different buffer systems under different pressure conditions

[0305] As can be seen from Tables 6 and 7, the Tm and Tagg of each buffer system were not significantly different. As can be seen from Tables 8 and 9, under freeze-thaw stress conditions, there was no significant difference in purity among the groups; under high temperature stress conditions, there was no significant difference in appearance and purity among the buffer system groups.

[0306] Subsequently, histidine-acetate (pH 6) will be selected as a buffer to screen other excipients.

[0307] Example 7 Comparison of stability of different stabilizers

[0308] The antibody was displaced into a histidine-acetate (pH 6) buffer system using a WaterSep hollow fiber (Discover 12). Highly concentrated stock solutions of each stabilizer were added to prepare stabilizer screening panels (as shown in Table 10). The antibody concentration was adjusted to approximately 30 mg / mL, and the mixture was sterile-filtered and set aside.

[0309] Freshly prepared samples were subjected to various stress conditions, including a high-temperature test (40°C ± 2°C for three weeks) and a freeze-thaw cycle (five cycles, ≤ -70°C / 5°C ± 3°C). Their stability was evaluated through various tests, including appearance and purity. Some of the test results are shown in Tables 10 and 11 below.

[0310] Table 10 Summary of the appearance and purity results of 900698 antibody under different pressure conditions and different stabilizer systems

[0311] Table 11 Summary of the appearance and purity results of 900725 antibody under different pressure conditions and different stabilizer systems

[0312] As can be seen from Tables 10 and 11, under freeze-thaw stress, except that the SEC purity of the mannitol group was slightly lower than that of the other groups, there were no significant differences among the other groups; under high temperature stress, except that the SEC purity of the sodium chloride-sucrose group and the lysine-trehalose group was slightly lower than that of the other groups, there were no significant differences among the other groups.

[0313] Subsequently, sucrose will be used as a stabilizer to carry out surfactant screening experiments.

[0314] Example 8 Surfactant Screening

[0315] The anti-CD73 cocktail antibody was displaced into a histidine-acetate (pH 6) 8 wt.% sucrose buffer system using a WaterSep hollow fiber (Discover 12). The highly concentrated stock solutions of each surfactant were added to prepare the surfactant screening panels (as shown in Table 12). The antibody concentration was adjusted to approximately 30 mg / mL and the mixture was sterile filtered and set aside.

[0316] The freshly prepared samples were placed under various shaking and pressurizing conditions and shaken at room temperature for 3 days (200 rpm). The protective effects of the surfactants Tween-20 (PS-20) and Tween-80 (PS-80) were evaluated by appearance and the number of insoluble particles. Some of the test results are shown in Table 12 below.

[0317] Table 12 Summary of particle numbers of different surfactants under oscillating pressure conditions

[0318] As shown in Table 12, under shock pressure, the addition of PS-20 and PS-80 significantly suppressed the increase in insoluble particles caused by shock, compared to the control group without surfactant. Appearance: "+" indicates the degree of opalescence, "+++" indicates severe, "++" indicates moderately severe, and "+" indicates mild. From the appearance perspective, the addition of PS-20 and PS-80 both improved the severity of the sample's opalescence, but the improvement diminished when the PS-20 and PS-80 concentrations exceeded 0.05%.

[0319] Example 9 Preparation of anti-CD73 cocktail antibody preparations in different buffer systems

[0320] From Tables 6 to 12, it can be seen that except for a few slightly worse experimental results of each component, there is no significant difference among other groups.

[0321] In summary, several buffer systems will be selected for pressurization, acceleration, and long-term stability studies. The buffer systems are as follows:

[0322] 1) 20 mM histidine-acetate, 8 wt.% sucrose, 0.03 wt.% Tween-80, pH 6 ± 0.5

[0323] 2) 20 mM sodium acetate-acetic acid, 8 wt.% trehalose, 0.03 wt.% Tween-80, pH 6 ± 0.5

[0324] 3) 20 mM histidine-acetate, 1.5 wt.% proline, 5 wt.% sucrose, 0.03 wt.% Tween-80, pH 6 ± 0.5

[0325] 4) 20 mM sodium acetate-acetic acid, 0.15 wt.% methionine, 8 wt.% trehalose, 0.03 wt.% Tween-80, pH 6 ± 0.5

[0326] First, use Merck's 30KD ultrafiltration membrane package (PLCTK) to replace the anti-CD73 cocktail antibody into each buffer system, adjust the antibody concentration to approximately 30±3 mg / mL, sterile filter, and aseptically dispense into 1.5 ml EP tubes. For each system preparation at each time point, one tube, each 1 ml.

[0327] Example 10 Stability of anti-CD73 cocktail antibody preparation

[0328] Example 10 investigated the stability of several groups of anti-CD73 cocktail antibody pharmaceutical preparations in the buffer system prepared in Example 9. The stability investigations included pressurized, accelerated, and long-term stability investigations, as follows:

[0329] 10.1 Stability test under pressurized conditions

[0330] The pressurized conditions include: high temperature for 3 weeks (40℃±2℃), room temperature shaking for 3 days (60rmp), repeated freeze-thaw (5 cycles, ≤-70℃ / 5℃±3℃ freeze-thaw), etc.

[0331] Several groups of anti-CD73 cocktail antibody drug preparation samples prepared according to Example 9 were stored under various pressure conditions (as shown in Table 13). Samples were taken for inspection at set time points or after the number of treatments. Their stability was evaluated by appearance, number of insoluble particles, and purity. Some of the test results are shown in Table 13 below.

[0332] Table 13 Summary of stability data of anti-CD73 cocktail antibody drug formulations under various pressure conditions

[0333] Note: T0 control refers to the control when the preparation is completed.

[0334] As can be seen in Table 13, the various pressure conditions affect product quality in varying degrees. Shock and high-temperature pressure primarily increase the number of large particles within the insoluble microparticles, resulting in a slight decrease in purity, but still well above the quality standard. Overall, the pharmaceutical compositions produced using these buffer systems are highly stable, effectively protecting the active ingredient, the anti-CD73 antibody cocktail, and thus resisting the destructive effects of various pressure conditions.

[0335] 10.2 Accelerated stability test

[0336] Several groups of anti-CD73 cocktail antibody drug preparation samples in the buffer system prepared according to Example 9 were stored under accelerated conditions (25°C ± 2°C), and samples were taken for inspection at set time points. Their stability was evaluated by insoluble particulate matter and purity. Some of the test results are shown in Table 14 below.

[0337] Table 14 Summary of stability data of anti-CD73 cocktail antibody drug formulations under accelerated conditions

[0338] Note: T0 control refers to the control when the preparation is completed.

[0339] As can be seen from Table 14, accelerated conditions can cause a certain degree of decline in the quality of the pharmaceutical preparation. Among the buffer systems, 20HA6Arg1.5Suc5ps0.03 performed slightly worse. Compared with the T0 sample, the SEC molecular sieve purity of the sample accelerated for 3 months decreased by approximately 1.4%, the CEX main peak (38) content decreased by approximately 4.4%, and the CEX main peak (39) content decreased by approximately 9.6%, but still met the quality standards. In summary, the pharmaceutical compositions of these buffer systems are highly stable and can be stored stably for at least 3 months under accelerated conditions at 25°C.

[0340] 10.3 Long-term stability test

[0341] Several groups of anti-CD73 cocktail antibody drug preparation samples in the buffer system prepared according to Example 9 were stored under long-term refrigeration conditions (5°C ± 3°C). Samples were taken and tested at set time points, and their stability was evaluated by insoluble particulate matter, purity, etc. Some of the test results are shown in Table 15 below.

[0342] Table 15 Summary of stability data of anti-CD73 cocktail antibody drug preparations under long-term refrigeration conditions

[0343] Note: T0 control refers to the control when the preparation is completed.

[0344] As shown in Table 15, these buffered pharmaceutical formulations exhibit excellent stability under long-term refrigerated conditions (5°C ± 3°C). Three-month samples showed no significant change in insoluble particulate matter, and while purity decreased slightly, it remained well above the quality standard. In summary, these buffered pharmaceutical compositions exhibit high stability and can be stored stably under long-term refrigerated conditions.

[0345] In summary, it can be seen that the several groups of buffer system anti-CD73 cocktail antibody drug systems prepared in Example 9 have good stability. The anti-CD73 cocktail antibody maintains good stability under various pressure conditions (high temperature, repeated freeze-thaw and shock), and can be stably stored for at least three months under accelerated conditions (25°C±2°C). At the same time, it shows high stability under long-term refrigeration conditions and can be stably stored.

Claims

1. A pharmaceutical combination comprising a first antigen-binding protein that specifically binds to CD73, and a second antigen-binding protein that specifically binds to CD73, wherein the first antigen-binding protein and the second antigen-binding protein have different CD73 antigen-binding epitopes.

2. The pharmaceutical combination according to claim 1, wherein the first antigen binding protein and the second antigen binding protein bind to the CD73 protein in a non-competitive manner.

3. The pharmaceutical combination according to any one of claims 1-2, wherein the first antigen-binding protein and the second antigen-binding protein are present in the form of a mixture.

4. The pharmaceutical combination according to any one of claims 1 to 3, wherein the first antigen-binding protein comprises at least one CDR in an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:

8. 5 . The pharmaceutical combination according to claim 1 , wherein the first antigen-binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

1.

6. The pharmaceutical combination according to any one of claims 1 to 5, wherein the first antigen-binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:

2. 7 . The pharmaceutical combination according to any one of claims 1 to 6 , wherein the first antigen-binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:

3.

8. The pharmaceutical combination according to any one of claims 1 to 7, wherein the first antigen-binding protein comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:

8.

9. The pharmaceutical combination according to any one of claims 1 to 8, wherein the first antigen binding protein comprises an antibody heavy chain constant region.

10. The pharmaceutical combination according to claim 9, wherein the antibody heavy chain constant region is derived from an IgG heavy chain constant region.

11. The pharmaceutical combination according to any one of claims 9-10, wherein the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

12. The pharmaceutical combination according to any one of claims 9 to 11, wherein the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:

9.

13. The pharmaceutical combination according to any one of claims 1 to 12, wherein the first antigen-binding protein comprises an antibody heavy chain comprising the amino acid sequence shown in SEQ ID NO:

10.

14. The pharmaceutical combination according to any one of claims 1 to 13, wherein the first antigen-binding protein comprises at least one CDR in an antibody light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:

18.

15. The pharmaceutical combination according to any one of claims 1 to 14, wherein the first antigen-binding protein comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

11.

16. The pharmaceutical combination according to any one of claims 1 to 15, wherein the first antigen-binding protein comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 12 (WAS). 17 . The pharmaceutical combination according to any one of claims 1 to 16 , wherein the first antigen-binding protein comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:

13.

18. The pharmaceutical combination according to any one of claims 1 to 17, wherein the first antigen-binding protein comprises a VL comprising the amino acid sequence shown in SEQ ID NO:

18.

19. The pharmaceutical combination according to any one of claims 1 to 18, wherein the first antigen binding protein comprises an antibody light chain constant region.

20. The pharmaceutical combination according to claim 19, wherein the antibody light chain constant region is derived from a human Igκ constant region.

21. The pharmaceutical combination according to any one of claims 19-20, wherein the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

19.

22. The pharmaceutical combination according to any one of claims 1-21, wherein the first antigen-binding protein comprises an antibody light chain comprising the amino acid sequence shown in SEQ ID NO:

20.

23. The pharmaceutical combination according to any one of claims 1 to 22, wherein the second antigen-binding protein comprises at least one CDR in an antibody heavy chain variable region VH, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:

27.

24. The pharmaceutical combination according to any one of claims 1-23, wherein the second antigen-binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

21.

25. The pharmaceutical combination according to any one of claims 1-24, wherein the second antigen-binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:

22.

26. The pharmaceutical combination according to any one of claims 1-25, wherein the second antigen-binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:

23.

27. The pharmaceutical combination according to any one of claims 1-26, wherein the second antigen-binding protein comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:

27.

28. The pharmaceutical combination according to any one of claims 1-27, wherein the second antigen binding protein comprises an antibody heavy chain constant region.

29. The pharmaceutical combination according to claim 28, wherein the antibody heavy chain constant region is derived from an IgG heavy chain constant region.

30. The pharmaceutical combination according to any one of claims 28-29, wherein the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

31. The pharmaceutical combination according to any one of claims 28 to 30, wherein the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:

9.

32. The pharmaceutical combination according to any one of claims 1-31, wherein the second antigen-binding protein comprises an antibody heavy chain comprising the amino acid sequence shown in SEQ ID NO:

28.

33. The pharmaceutical combination according to any one of claims 1-32, wherein the second antigen-binding protein comprises at least one CDR in an antibody light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:

35.

34. The pharmaceutical combination according to any one of claims 1-33, wherein the second antigen binding protein comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

29.

35. The pharmaceutical combination according to any one of claims 1-34, wherein the second antigen binding protein comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 30 (LAS).

36. The pharmaceutical combination according to any one of claims 1-35, wherein the second antigen binding protein comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:

31.

37. The pharmaceutical combination according to any one of claims 1-36, wherein the second antigen-binding protein comprises a VL comprising the amino acid sequence shown in SEQ ID NO:

35.

38. The pharmaceutical combination of any one of claims 1-37, wherein the second antigen binding protein comprises an antibody light chain constant region.

39. The pharmaceutical combination according to claim 38, wherein the antibody light chain constant region is derived from a human Igκ constant region.

40. The pharmaceutical combination according to any one of claims 38-39, wherein the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

19.

41. The pharmaceutical combination according to any one of claims 1-40, wherein the second antigen-binding protein comprises an antibody light chain comprising the amino acid sequence shown in SEQ ID NO:

36.

42. The pharmaceutical combination according to any one of claims 1-41, wherein the mass ratio of the first antigen-binding protein to the second antigen-binding protein is about 1:5 to 5:

1.

43. The pharmaceutical combination according to any one of claims 1-42, wherein the mass ratio of the first antigen binding protein to the second antigen binding protein is about 1:

5.

44. The pharmaceutical combination according to any one of claims 1-42, wherein the mass ratio of the first antigen binding protein to the second antigen binding protein is about 1:2.

5.

45. The pharmaceutical combination according to any one of claims 1-42, wherein the mass ratio of the first antigen binding protein to the second antigen binding protein is about 1:

1.

46. ​​The pharmaceutical combination of any one of claims 1-42, wherein the mass ratio of the first antigen binding protein to the second antigen binding protein is about 2.5:

1.

47. The pharmaceutical combination according to any one of claims 1-42, wherein the mass ratio of the first antigen binding protein to the second antigen binding protein is about 5:

1.

48. A pharmaceutical composition comprising the pharmaceutical combination of any one of claims 1 to 47, and optionally one or more pharmaceutically acceptable carriers.

49. The pharmaceutical composition of claim 48, wherein the concentration of the drug combination is about 3-300 mg / mL.

50. The pharmaceutical composition of any one of claims 48-49, wherein the concentration of the drug combination is about 3-150 mg / mL.

51. The pharmaceutical composition of any one of claims 48-50, wherein the concentration of the drug combination is about 5-100 mg / mL.

52. The pharmaceutical composition of any one of claims 48-51, wherein the concentration of the drug combination is about 10-100 mg / mL.

53. The pharmaceutical composition of any one of claims 48-52, wherein the concentration of the drug combination is about 10-50 mg / mL.

54. The pharmaceutical composition of any one of claims 48-53, wherein the concentration of the drug combination is 30±5 mg / mL.

55. The pharmaceutical composition of any one of claims 48-54, wherein the concentration of the drug combination is 30±3 mg / mL.

56. The pharmaceutical composition of any one of claims 48-55, wherein the pharmaceutically acceptable carrier comprises a buffer.

57. The pharmaceutical composition of claim 56, wherein the buffer comprises an acidic buffer.

58. The pharmaceutical composition of any one of claims 56-57, wherein the buffer comprises a neutral buffer.

59. The pharmaceutical composition of any one of claims 56-58, wherein the buffer is selected from the group consisting of acetate-acetate buffer, histidine-histidine hydrochloride buffer, citrate-citrate buffer, phosphate buffer, histidine-acetate buffer, Tris (Tris)-hydrochloride buffer, phosphate-citrate, or a combination thereof.

60. The pharmaceutical composition of any one of claims 56-59, wherein the buffer is selected from the group consisting of acetate-acetic acid, citrate-citric acid, histidine-histidine hydrochloride, phosphate, Tris-hydrochloride, histidine-acetic acid, phosphate-citrate, or a combination thereof.

61. The pharmaceutical composition of any one of claims 56-60, wherein the buffer is selected from the group consisting of sodium acetate-acetic acid, sodium citrate-citric acid, histidine-histidine hydrochloride, sodium dihydrogen phosphate-disodium hydrogen phosphate, Tris-hydrochloride, histidine-acetic acid, and phosphate-sodium citrate.

62. The pharmaceutical composition of any one of claims 56-61, wherein the concentration of the buffer is about 5-100 mM.

63. The pharmaceutical composition of any one of claims 56-62, wherein the concentration of the buffer is about 5-60 mM.

64. The pharmaceutical composition of any one of claims 56-63, wherein the concentration of the buffer is about 5-50 mM.

65. The pharmaceutical composition of any one of claims 56-64, wherein the concentration of the buffer is about 10-40 mM.

66. The pharmaceutical composition of any one of claims 56-65, wherein the concentration of the buffer is 20±5 mM.

67. The pharmaceutical composition of any one of claims 56-66, wherein the concentration of the buffer is 20±2 mM.

68. The pharmaceutical composition of any one of claims 48-67, having a pH of about 5-7.

69. The pharmaceutical composition of any one of claims 48-68, having a pH of about 6±0.

5.

70. The pharmaceutical composition of any one of claims 48-69, having a pH of about 6 ± 0.

3.

71. The pharmaceutical composition of any one of claims 48-70, having a pH of about 6 ± 0.

1.

72. The pharmaceutical composition of any one of claims 48-68, having a pH of about 6.5 ± 0.

3.

73. The pharmaceutical composition of any one of claims 48-68, having a pH of about 7 ± 0.

3.

74. The pharmaceutical composition of any one of claims 48-73, wherein the pharmaceutically acceptable carrier comprises a stabilizer.

75. The pharmaceutical composition of claim 74, wherein the stabilizer is selected from the group consisting of sodium chloride, amino acids, sugar alcohols, or combinations thereof.

76. The pharmaceutical composition of claim 75, wherein the amino acid is selected from the group consisting of proline, arginine, glycine, histidine, lysine, methionine, or a combination thereof.

77. The pharmaceutical composition according to any one of claims 75-76, wherein the sugar alcohol is selected from the group consisting of sucrose, mannitol, trehalose, sorbitol, or a combination thereof.

78. The pharmaceutical composition of any one of claims 74-77, wherein the stabilizer is selected from the group consisting of sucrose, trehalose, sorbitol, mannitol, proline / sucrose, methionine / trehalose, glycine / mannitol, sodium chloride / sucrose, arginine / sucrose, and lysine / trehalose.

79. The pharmaceutical composition according to any one of claims 74-78, wherein the stabilizer is present in an amount of about 0.15 wt.% to 20 wt.%.

80. The pharmaceutical composition of any one of claims 74-79, wherein the stabilizer is present in an amount of about 0.15 wt.% to 10 wt.%.

81. The pharmaceutical composition of any one of claims 74-80, wherein the stabilizer is present in an amount of about 0.15 wt.% to 8 wt.%.

82. The pharmaceutical composition according to any one of claims 74-81, wherein the stabilizer is present in an amount of about 4±1 wt.% or 8±1 wt.%.

83. The pharmaceutical composition of any one of claims 74-82, wherein the stabilizer is present in an amount of about 4±0.5 wt.% or 8±0.5 wt.%.

84. The pharmaceutical composition of any one of claims 74-81, wherein the stabilizer is present in an amount of about 4 wt.%, 5 wt.%, 7 wt.% or 8 wt.%.

85. The pharmaceutical composition of any one of claims 74-84, wherein the stabilizer is present in an amount of about 8 wt.%.

86. The pharmaceutical composition of any one of claims 48-85, wherein the pharmaceutically acceptable carrier comprises a surfactant.

87. The pharmaceutical composition of claim 86, wherein the surfactant is selected from the group consisting of polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, glycerol fatty acid esters, polysorbates, poloxamers, or combinations thereof.

88. The pharmaceutical composition of claim 87, wherein the polysorbate is selected from the group consisting of polysorbate 20 (PS-20, Tween-20), polysorbate 40 (PS-40), polysorbate 60 (PS-60), and polysorbate 80 (PS-80).

89. The pharmaceutical composition of any one of claims 86-88, wherein the surfactant is selected from the group consisting of PS-20 (Tween-20) and PS-80 (Tween-80).

90. The pharmaceutical composition of any one of claims 86-89, wherein the concentration of the surfactant is about 0.001%-0.5%.

91. The pharmaceutical composition of any one of claims 86-90, wherein the concentration of the surfactant is about 0.005%-0.1%.

92. The pharmaceutical composition of any one of claims 86-91, wherein the concentration of the surfactant is about 0.005%, 0.01%, 0.03%, 0.05% or 0.1%.

93. The pharmaceutical composition of any one of claims 86-92, wherein the concentration of the surfactant is about 0.01%.

94. The pharmaceutical composition of any one of claims 86-92, wherein the concentration of the surfactant is about 0.03%.

95. The pharmaceutical composition of any one of claims 48-94, wherein the pharmaceutically acceptable carrier is selected from the group consisting of: (1) 20 mM histidine-acetate, 8 wt.% sucrose, 0.03 wt.% Tween-80; (2) 20 mM sodium acetate-acetic acid, 8 wt.% trehalose, 0.03 wt.% Tween-80; (3) 20 mM histidine-acetate, 1.5 wt.% proline, 5 wt.% sucrose, 0.03 wt.% Tween-80; (4) 20 mM sodium acetate-acetic acid, 0.15 wt.% methionine, 8 wt.% trehalose, 0.03 wt.% Tween-80; (5) 20 mM histidine-acetate, 8 wt.% sucrose, 0.01 wt.% Tween-80; (6) 20 mM sodium acetate-acetic acid, 8 wt.% trehalose, 0.01 wt.% Tween-80; (7) 20 mM histidine-acetate, 1.5 wt.% proline, 5 wt.% sucrose, 0.01 wt.% Tween-80; (8) 20 mM sodium acetate-acetic acid, 0.15 wt.% methionine, 8 wt.% trehalose, 0.01 wt.% Tween-80.

96. Use of the pharmaceutical combination of any one of claims 1 to 47 and / or the pharmaceutical composition of any one of claims 48 to 95 in the preparation of a medicament for preventing and / or treating a disease and / or condition.

97. The use according to claim 96, wherein the disease and / or condition comprises a CD73-mediated disease and / or condition.

98. The use according to any one of claims 96-97, wherein the disease and / or disorder comprises a tumor.

99. The method of claim 98, wherein the tumor comprises a solid tumor and / or a hematological tumor.

100. The use according to any one of claims 96-99, wherein the disease and / or condition is selected from the group consisting of pancreatic cancer, breast cancer and idiopathic pulmonary fibrosis (IPF).

101. The use of claim 100, wherein the breast cancer comprises triple-negative breast cancer.

102. Use of the pharmaceutical combination of any one of claims 1-47 and / or the pharmaceutical composition of any one of claims 48-95 in the preparation of a detection kit for detecting the presence and / or content of CD73 protein.

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