CD73 antibody cocktail therapy

Abstract

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, and further providing a pharmaceutical composition comprising the pharmaceutical combination and its use.

Description

Technical Field

[0001] The present application relates to the field of biopharmaceuticals, and specifically to CD73 antibody cocktail therapy.

Background Art

[0002] CD73, also known as NT5E, is an extracellular 5-nucleotidase (NT5E) with a molecular weight of approximately 70 Kd. It is anchored to the cell surface by glycosylphosphatidylinositol (GPI) and mainly exists as a dimer. CD73 can be expressed in various tissues or organs under normal physiological conditions, such as tissues like the liver, large intestine, kidney, spleen, lung, ovary, lymph nodes, etc.

[0003] Results of a large number of preclinical studies show 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. Moreover, clinical data indicate that the high expression of CD73 is closely related to poor prognosis of tumor patients. Therefore, CD73 can be used as a potential target for clinical treatment and prognosis of various tumors.

[0004] Therefore, in order to provide more new ideas for the prevention and / or treatment of diseases caused by abnormal expression of CD73, the development of multiple therapies targeting CD73 is urgently needed.

Summary of the Invention

[0005] 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 in the present application has one or more of the following properties: (1) it can specifically bind to the CD73 protein; (2) it has a high affinity for the CD73 protein; (3) it can inhibit the growth and / or proliferation of tumor cells.

[0006] In some embodiments, the first antigen-binding protein and the second antigen-binding protein bind non-competitively to the CD73 protein. In some embodiments, the first antigen-binding protein and the second antigen-binding protein are present in the form of a mixture. In some embodiments, the first antigen-binding protein comprises at least one CDR of the variable heavy chain of an antibody VH comprising the amino acid sequence shown in SEQ ID NO: 8.

[0007] In some embodiments, the first antigen-binding protein comprises an HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the first antigen-binding protein comprises an HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the first antigen-binding protein comprises an HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 3.

[0008] In some embodiments, the first antigen-binding protein comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the first antigen-binding protein comprises an antibody heavy chain constant region. In some embodiments, the antibody heavy chain constant region is derived from an IgG heavy chain constant region.

[0009] In some embodiments, the antibody heavy chain constant region is derived from a human IgG heavy chain constant region. In some embodiments, the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 9. In some embodiments, the first antigen-binding protein comprises an antibody heavy chain comprising the amino acid sequence shown in SEQ ID NO: 10.

[0010] In some embodiments, the first antigen-binding protein comprises at least one CDR of the variable light chain region VL comprising the amino acid sequence shown in SEQ ID NO: 18. In some embodiments, the first antigen-binding protein comprises LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the first antigen-binding protein comprises LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 12 (WAS).

[0011] In some embodiments, the first antigen-binding protein comprises LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the first antigen-binding protein comprises VL comprising the amino acid sequence shown in SEQ ID NO: 18. In some embodiments, the first antigen-binding protein comprises a constant light chain region of an antibody.

[0012] In some embodiments, the constant light chain region of the antibody is derived from a human Igκ constant region. In some embodiments, the constant light chain region of the antibody comprises the amino acid sequence shown in SEQ ID NO: 19. In some embodiments, the first antigen-binding protein comprises a light chain of an antibody comprising the amino acid sequence shown in SEQ ID NO: 20.

[0013] In some embodiments, the second antigen-binding protein comprises at least one CDR of the variable heavy chain region VH comprising the amino acid sequence shown in SEQ ID NO: 27. In some embodiments, the second antigen-binding protein comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 21. In some embodiments, the second antigen-binding protein comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 22.

[0014] In some embodiments, the second antigen-binding protein comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, the second antigen-binding protein comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 27. In some embodiments, the second antigen-binding protein comprises an antibody heavy chain constant region.

[0015] In some embodiments, the antibody heavy chain constant region is derived from an IgG heavy chain constant region. In some embodiments, the antibody heavy chain constant region is derived from a human IgG heavy chain constant region. In some embodiments, the second antigen-binding protein comprises an antibody heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 9.

[0016] In some embodiments, the second antigen-binding protein comprises an antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, the second antigen-binding protein comprises at least one CDR of an antibody light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, the second antigen-binding protein comprises an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 29.

[0017] In some embodiments, the second antigen-binding protein comprises an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30 (LAS). In some embodiments, the second antigen-binding protein comprises an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, the second antigen-binding protein comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0018] In some embodiments, the second antigen-binding protein comprises an antibody light chain constant region. In some embodiments, the antibody light chain constant region is derived from a human Igκ constant region. In some embodiments, the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 19.

[0019] In some embodiments, the second antigen-binding protein comprises an antibody light chain comprising the amino acid sequence shown in SEQ ID NO: 36. In some 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. In some embodiments, the mass ratio of the first antigen-binding protein to the second antigen-binding protein in the pharmaceutical combination is about 1:5.

[0020] In some 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. In some embodiments, the mass ratio of the first antigen-binding protein to the second antigen-binding protein in the pharmaceutical combination is about 1:1. In some 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.

[0021] In some embodiments, the mass ratio of the first antigen-binding protein to the second antigen-binding protein in the pharmaceutical combination is about 5:1. In another aspect, the present application further provides a pharmaceutical composition comprising the pharmaceutical combination and optionally one or more pharmaceutically acceptable carriers. In some embodiments, the concentration of the pharmaceutical combination is about 3 to 300 mg / mL.

[0022] In some embodiments, the concentration of the pharmaceutical combination is about 3 to 150 mg / mL. In some embodiments, the concentration of the pharmaceutical combination is about 5 to 100 mg / mL. In some embodiments, the concentration of the pharmaceutical combination is about 10 to 100 mg / mL.

[0023] In some embodiments, the concentration of the pharmaceutical combination is about 10 to 50 mg / mL. In some embodiments, the concentration of the pharmaceutical combination is 30 ± 5 mg / mL. In some embodiments, the concentration of the pharmaceutical combination is 30 ± 3 mg / mL.

[0024] In some embodiments, the pharmaceutically acceptable carrier includes a buffer solution. In some embodiments, the buffer solution includes an acidic buffer solution. In some embodiments, the buffer solution includes a neutral buffer solution.

[0025] In some embodiments, the buffer solution is selected from the group consisting of acetate - acetic acid buffer, histidine - histidine hydrochloride buffer, citrate - citric acid buffer, phosphate buffer, histidine - acetic acid buffer, tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer, phosphate - citrate, or combinations thereof. In some embodiments, the buffer solution 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 combinations thereof. In some embodiments, the buffer solution 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.

[0026] In some embodiments, the concentration of the buffer solution is about 5 to 100 mM. In some embodiments, the concentration of the buffer solution is about 5 to 60 mM. In some embodiments, the concentration of the buffer is about 5 to 50 mM. In some embodiments, the concentration of the buffer is about 10 to 40 mM. In some embodiments, the concentration of the buffer is 20 ± 5 mM. In some embodiments, the concentration of the buffer is 20 ± 2 mM. In some embodiments, the pH value of the pharmaceutical composition is about 5 to 7.

[0027] In some embodiments, the pH value of the pharmaceutical composition is about 6 ± 0.5. In some embodiments, the pH value of the pharmaceutical composition is about 6 ± 0.3. In some embodiments, the pH value of the pharmaceutical composition is about 6 ± 0.1. In some embodiments, the pH value of the pharmaceutical composition is about 6.5 ± 0.3. In some embodiments, the pH value of the pharmaceutical composition is about 7 ± 0.3. In some embodiments, the pharmaceutically acceptable carrier includes a stabilizer.

[0028] In some embodiments, the stabilizer is selected from the group consisting of sodium chloride, amino acids, sugar alcohols, and combinations thereof. In some embodiments, the amino acid is selected from the group consisting of proline, arginine, glycine, histidine, lysine, methionine, and combinations thereof. In some embodiments, the sugar alcohol is selected from the group consisting of sucrose, mannitol, trehalose, sorbitol, and combinations thereof.

[0029] In some 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. In some embodiments, the content of the stabilizer is about 0.15 wt.% to 20 wt.%. In some embodiments, the content of the stabilizer is about 0.15 wt.% to 10 wt.%.

[0030] In some embodiments, the content of the stabilizer is about 0.15 wt.% to 8 wt.%. In some embodiments, the content of the stabilizer is about 4 ± 1 wt.% or 8 ± 1 wt.%. In some embodiments, the content of the stabilizer is about 4 ± 0.5 wt.% or 8 ± 0.5 wt.%.

[0031] In some embodiments, the content of the stabilizer is about 4 wt.%, 5 wt.%, 7 wt.% or 8 wt.%. In some embodiments, the content of the stabilizer is about 8 wt.%. In some embodiments, the pharmaceutically acceptable carrier includes a surfactant.

[0032] In some embodiments, the surfactant is selected from the group consisting of polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, glycerin fatty acid esters, polysorbates, poloxamers, and combinations thereof. In some 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). In some embodiments, the surfactant is selected from the group consisting of PS-20 (Tween-20) and PS-80 (Tween-80).

[0033] In some embodiments, the concentration of the surfactant is about 0.001% to 0.5%. In some embodiments, the concentration of the surfactant is about 0.005% to 0.1%. In some embodiments, the concentration of the surfactant is about 0.005%, 0.01%, 0.03%, 0.05% or 0.1%.

[0034] In some embodiments, the concentration of the surfactant is about 0.01%. In some embodiments, the concentration of the surfactant is about 0.03%. In some embodiments, the pharmaceutically acceptable carrier is selected from the group consisting of: (1) 20 mM histidine - acetate, 8 wt.% sucrose, and 0.03 wt.% Tween - 80; (2) 20 mM sodium acetate - acetate, 8 wt.% trehalose, and 0.03 wt.% Tween - 80; (3) 20 mM histidine - acetate, 1.5 wt.% proline, 5 wt.% sucrose, and 0.03 wt.% Tween - 80; (4) 20 mM sodium acetate - acetate, 0.15 wt.% methionine, 8 wt.% trehalose, and 0.03 wt.% Tween - 80; (5) 20 mM histidine - acetate, 8 wt.% sucrose, and 0.01 wt.% Tween - 80; (6) 20 mM sodium acetate - acetate, 8 wt.% trehalose, and 0.01 wt.% Tween - 80; (7) 20 mM histidine - acetate, 1.5 wt.% proline, 5 wt.% sucrose, and 0.01 wt.% Tween - 80; (8) 20 mM sodium acetate - acetate, 0.15 wt.% methionine, 8 wt.% trehalose, and 0.01 wt.% Tween - 80.

[0035] In another aspect, the present application provides the use of the above pharmaceutical combination and / or the above pharmaceutical composition in the preparation of a drug for preventing and / or treating a disease and / or a medical condition. In some embodiments, the disease and / or medical condition includes a CD73 - mediated disease and / or medical condition. In some embodiments, the disease and / or medical condition includes a tumor.

[0036] In some embodiments, the tumor includes a solid tumor and / or a hematological tumor. In some embodiments, the disease and / or condition is selected from the group consisting of pancreatic cancer, breast cancer, and idiopathic pulmonary fibrosis (IPF). In some embodiments, the breast cancer includes triple-negative breast cancer.

[0037] In another aspect, the present application further provides the use of the pharmaceutical combination and / or the pharmaceutical composition in the preparation of a detection reagent kit for detecting the presence and / or content of CD73 protein.

[0038] Those skilled in the art can easily understand other aspects and advantages of the present application from the following detailed description. The following detailed description shows and describes only exemplary embodiments of the present application. As will be apparent to those skilled in the art, based on the content of the present application, those skilled in the art can modify the disclosed specific embodiments without departing from the spirit and scope of the invention according to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are not restrictive but merely exemplary.

Brief Description of the Drawings

[0039] The specific features of the invention according to the present application are as described in the appended claims. By referring to the exemplary embodiments and the drawings described in detail below, the features and advantages of the invention according to the present application can be better understood. The brief description of the drawings is as follows.

[0040]

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Modes for Carrying Out the Invention

[0041] Hereinafter, embodiments of the invention according to the present application will be described by specific specific examples, and those skilled in the art can easily understand other advantages and effects of the invention according to the present application from the content disclosed in this specification.

[0042] Definition of Terms In the present application, the term "pharmaceutical combination" generally refers to a combination containing at least two active ingredients / therapeutic agents. In some embodiments, each active ingredient / therapeutic agent can be prepared into independent formulations (solids, liquids, gels, etc.), in some embodiments, each active ingredient / therapeutic agent can be present in different containers, and, if necessary, can be prepared into the desired formulation simultaneously or each with an appropriate carrier, in some embodiments, each active ingredient / therapeutic agent can be derived from different sources, and in some embodiments, each active ingredient / therapeutic agent can be present in the form of a mixture.

[0043] In the present application, the term "CD73" is also referred to as "NT5E", "cluster of differentiation 73", "5'-nucleotidase (5'-NT)", or "ecto-5'-nucleotidase". The above term includes "full-length", untreated CD73, and any form of CD73 produced by cell treatment. In the present application, the term "CD73" includes full-length wild-type CD73 and its mutants, fragments, variants, isotypes, and homologs. In some embodiments, the above CD73 may include human CD73. For example, the sequence for human CD73 may refer to the Uniprot accession number P21589.

[0044] In the present application, the term "antigen-binding protein" generally refers to a protein having antigen-binding ability, which is obtained artificially from its natural state. The "antigen-binding protein" may include a portion that binds to an antigen, and optionally, a framework or framework portion that permits 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 include, for example, an antibody-derived protein framework region (FR), or a candidate protein framework region or artificial framework region having a transplanted CDR or CDR derivative. Such frameworks include, but are not limited to, antibody-derived framework regions containing mutations introduced to stabilize the three-dimensional structure of the antigen-binding protein, and fully synthetic framework regions containing, 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 fragments, F(ab’)2, F(ab)2, scFv, di-scFv, dAb, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies, and fragments thereof.

[0045] In the present application, the terms "variable domain" and "variable region" may be used interchangeably and generally refer to a part of an antibody heavy chain and / or light chain. The variable domains of the heavy chain and light chain can be referred to as "V H " and "V L " (or "VH" and "VL", respectively). These domains are usually the most variable parts of the antibody (compared to other antibodies of the same type) and contain the antigen-binding site.

[0046] In the present application, the term "variable" generally refers to the fact that certain segments of the variable domain can vary relatively greatly in sequence among antibodies. The variable domain mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the extent of the variable region. It typically concentrates in three segments, usually called the hypervariable regions (CDRs or HVRs) of the light and heavy chain variable domains. The more highly conserved portions of the variable domain are called the framework regions (FRs). The variable domains of native heavy and light chains each contain four FR regions, mostly adopt a β-sheet conformation, are linked by three CDRs that form loop connections, and in some situations, form part of the β-sheet structure. The CDRs in each chain are held in close proximity to each other by the FR regions and, together with the CDRs from another chain, facilitate the formation of the antigen-binding site of the antibody (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).

[0047] In the present application, the term "antibody" generally refers to an immunoglobulin or a fragment or derivative thereof that contains an antigen-binding site, regardless of whether it is 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, mutant, and transplanted antibodies. Unless otherwise modified by terms such as "complete" (such as "complete antibody"), the term "antibody" also includes, for the purposes of the present invention, antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain the antigen-binding function (e.g., specifically bind to human CLDN18.2). Generally, such fragments should contain an antigen-binding domain. The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units together with another polypeptide called the J chain and contain ten antigen-binding sites, while IgA antibodies contain 2 - 5 basic four-chain units that can bind to the J chain and polymerize to form a multivalent combination. For IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the heavy-chain isotype. Each H chain and L chain further have equidistant intra-chain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus and a constant domain at the other end. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. Specific amino acid residues are thought to form an interface between the light-chain variable domain and the heavy-chain variable domain. VH and VL pair to form a single antigen-binding site.For the structures 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. Light chains derived from any vertebrate species can be classified into one of two distinct types, called κ and λ, based on the amino acid sequence of their constant domains. Immunoglobulins can be classified into different classes or isotypes according to the amino acid sequence of their heavy chain (CH) constant domains. Currently, there are five immunoglobulins, IgA, IgD, IgE, IgG and IgM, which have heavy chains named α, δ, ε, γ and μ, respectively. The γ and α classes are further classified into subclasses, such as the human-expressed subclasses IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2, based on relatively small differences in CH sequence and function.

[0048] In the present application, the term "CDR" is also referred to as "complementary determining region" and usually refers to a region in the 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 some embodiments, naturally occurring camel antibodies consisting only of heavy chains are functional and stable even 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).

[0049] In the present application, the term "FR" usually refers to a more highly conserved portion of the antibody variable domain, which is called the framework region. Usually, the variable domains of the native heavy and / or light chains each contain 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).

[0050] In the present application, the term "antigen-binding fragment" usually refers to one or more fragments having the ability to specifically bind to an antigen. In the present application, the antigen-binding fragment may include Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv, and / or dAb.

[0051] In the present application, the term "Fab" usually refers to the antigen-binding fragment of an antibody. As described above, a complete antibody can be digested using papain. The antibody digested by papain produces two identical antigen-binding fragments, namely, the "Fab" fragment and the remaining "Fc" fragment (i.e., the Fc region, the same as above). The Fab fragment may be composed of the complete L chain, the variable region of the heavy chain, and the first constant region (C H ) of the H chain (V H 1).

[0052] In the present application, the term "Fab’ fragment" usually refers to a monovalent antigen-binding fragment of a human monoclonal antibody that is slightly larger than the Fab fragment. For example, the Fab’ fragment may include all of the light chain, all of the heavy chain variable region, and all or part of the first and second constant regions of the heavy chain. For example, the Fab’ fragment may further include some or all of 220-330 amino acid residues of the heavy chain.

[0053] In the present application, the term "F(ab’)2" usually refers to an antibody fragment produced by a complete antibody digested by pepsin. The F(ab’)2 fragment includes two Fab fragments maintained together by a disulfide bond and a part of the hinge region. The F(ab’)2 fragment has divalent antigen-binding activity and can cross-link antigens.

[0054] In the present application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody that includes all or a part of the heavy-chain variable region and the light-chain variable region and lacks 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 includes all or a part of the amino-terminal variable region of about 110 amino acids of the heavy chain and the light chain.

[0055] In the present application, the term "scFv" generally refers to a fusion protein that includes at least one antibody fragment including a light-chain variable region and at least one antibody fragment including a heavy-chain variable region, wherein the light-chain and heavy-chain variable regions are adjacent (e.g., via a synthetic linker such as a short flexible peptide linker), and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, the scFv may have the above VL and VH variable regions in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), and the scFv may include VL-linker-VH, or may include VH-linker-VL.

[0056] In the present 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. See, for example, Ward et al. (Nature, Oct 12, 1989, 341(6242):544-6), Holt et al. (Trends Biotechnol., 2003, 21(11):484-490), and other published patent applications, such as WO06 / 030220, WO06 / 003388, and Domantis Ltd.

[0057] In the present application, the term "monoclonal antibody" generally refers to a preparation of antibody molecules consisting of a single molecule. Monoclonal antibodies generally have high specificity for a single antigenic site. Also, unlike conventional polyclonal antibody preparations (which generally have different antibodies to different determinants), each monoclonal antibody is directed against a single determinant in the antigen. In addition to their specificity, the advantages of monoclonal antibodies are that they can be synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not construed to require that the antibody be produced by any particular method. For example, the monoclonal antibodies used in the present application may be prepared in hybridoma cells or by recombinant DNA methods.

[0058] In the present 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. Generally, 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, whereby the resulting chimeric antibody has a reduced potential to induce a harmful immune response in a human individual compared to the parent (e.g., mouse-derived) antibody.

[0059] As used herein, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids other than the CDR regions of a non-human antibody (e.g., a mouse antibody) are replaced with the corresponding amino acids from a human immunoglobulin. In the CDR regions, small additions, deletions, insertions, substitutions or modifications of amino acids may be tolerated as long as the antibody retains its ability to bind to a particular antigen. A humanized antibody may optionally include at least a portion of a human immunoglobulin constant region. A "humanized antibody" retains the antigen specificity similar to the original antibody. The "humanized" form of a non-human (e.g., mouse) antibody may minimally include a chimeric antibody derived from the sequence of a non-human immunoglobulin. In some cases, the CDR region residues in a human immunoglobulin (receptor antibody) may be replaced with the CDR region residues of a non-human species (donor antibody) (e.g., mouse, rat, rabbit or non-human primate) having the desired properties, affinity and / or ability. In some cases, the FR region residues of a human immunoglobulin may be replaced with the corresponding non-human residues. Also, a humanized antibody may include amino acid modifications that are not present in either the receptor antibody or the donor antibody. These modifications can be made to further improve the performance of the antibody, such as its binding affinity.

[0060] The term "fully humanized antibody" generally refers to an antibody that contains only human immunoglobulin protein sequences. A fully humanized antibody may contain mouse sugar chains if produced in a mouse, mouse cells, or a hybridoma derived from mouse cells. Similarly, a "mouse antibody" or a "rat antibody" refers to an antibody that contains only mouse or rat immunoglobulin sequences, respectively. A fully humanized antibody can be generated in the body of a human or a transgenic animal having human immunoglobulin germline sequences by phage display or other molecular biology methods. Exemplary techniques useful for manufacturing antibodies are described in U.S. Patents 6,150,584, 6,458,592, 6,420,140. Other techniques, such as using libraries, are known in the art.

[0061] In the present application, the term "direct connection" is in contrast to the term "indirect connection", and the term "direct connection" usually means being directly connected. For example, the above direct connection may be a situation where there is no spacer between substances and they are directly connected. The above spacer may be a linker. For example, the above linker may be a peptide linker. The term "indirect connection" usually refers to a situation where substances are not directly connected. For example, the above indirect connection may be a situation where they are connected via a spacer. For example, in the isolated antigen-binding protein described in the present application, the C-terminus of the above L-FR1 and the N-terminus of the above LCDR1 may be directly or indirectly connected.

[0062] In the present application, the term "pharmaceutical composition" usually refers to a composition for preventing / treating a disease or medical condition. The above pharmaceutical composition may contain one or more active ingredients and optionally a pharmaceutically acceptable carrier. For example, the above pharmaceutical composition may further contain an appropriate formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizing agents, surfactants, emulsifiers and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the dosages and concentrations used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

[0063] In the present application, the term "pharmaceutically acceptable carrier" usually includes pharmaceutically acceptable carriers, excipients and / or stabilizers that are non-toxic at the dosages and concentrations used for the cells or mammals to which they are exposed. 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.

[0064] In the present application, the term "subject" usually refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys.

[0065] In the present application, the term "tumor" generally refers to a neoplasm or solid lesion formed by abnormal cell growth. In the present application, the tumor may be a solid tumor or a non-solid tumor.

[0066] In the present application, such proteins, polypeptides and / or amino acid sequences should also be understood to include at least variants or homologs having the same or similar functions as the above proteins or polypeptides.

[0067] In the present application, the above variants may be, for example, proteins or polypeptides in which one or more amino acids are substituted, deleted or added in the amino acid sequences of the above proteins and / or the above polypeptides (for example, an antibody specifically binding to CD73 or a fragment thereof). For example, the above functional variants may include proteins or polypeptides having amino acid modifications by substitution, deletion and / or insertion of at least one, for example, 1 to 30, 1 to 20 or 1 to 10, and also for example 1, 2, 3, 4 or 5 amino acids. The above functional variants can basically retain the biological properties of the above proteins or polypeptides before modification (for example, substitution, deletion or addition). For example, the above functional variants can retain at least 60%, 70%, 80%, 90% or 100% of the biological activity (for example, antigen-binding ability) of the above proteins or polypeptides before modification. For example, the above substitutions may be conservative substitutions.

[0068] In the present application, the above homologs may be proteins or polypeptides having at least about 85% (for example, 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 sequences of the above proteins and / or the above polypeptides (for example, an antibody specifically binding to CD73 or a fragment thereof).

[0069] As used herein, the above homology generally refers to similarity, resemblance or relatedness between two or more sequences. The "percent sequence homology" can be calculated by the following method: Compare two aligned sequences in a comparison window, determine the number of positions where the same nucleobase (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) exists in the two sequences, to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (i.e., the size of the window), and multiply the result by 100 to generate the percent sequence homology. The alignment for determining the percent sequence homology can be achieved by using various methods known in the art, such as publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for the alignment of sequences, including any algorithms necessary to achieve the maximum alignment within the range of the full-length sequences being compared or within the region of the target sequence. The above homology can also be measured by the FASTA and BLAST methods. For an explanation of the FASTA algorithm, reference can be made to "Improved Tools for Biological Sequence Comparison" by W.R. Pearson and D.J. Lipman, Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci.), 85:2444-2448, 1988, and "Rapid and Sensitive Protein Similarity Searches" by D.J. Lipman and W.R. Pearson, Science, 227:1435-1441, 1989. For an explanation of the BLAST algorithm, reference can be made to "Basic Local Alignment Search Tool" by S. Altschul, W. Gish, W. Miller, E.W. Myers and D. Lipman, Journal of Molecular Biology, 215:403-410, 1990.

[0070] In the present application, the term "comprising" generally refers to including, aggregating, containing, or encompassing. In some situations, it also represents the meaning of "being" or "consisting of".

[0071] In the present application, the term "about" generally refers to a variation within the range of 0.5% to 10% or more or less of the specified value, for example, a variation within the 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% or more or less of the specified value. [Detailed Description of the Invention]

[0072] 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.

[0073] In the present application, the first antigen-binding protein (1) can specifically bind to CD73 or a functionally active fragment thereof, (2) can inhibit the enzymatic activity of CD73, (3) can induce endocytosis of cell surface CD73, (4) has one or more of the properties of being able to inhibit the growth and / or proliferation of tumor cells.

[0074] In the present application, the first antigen-binding protein and the second antigen-binding protein may have different CD73 antigen-binding epitopes. In the present application, the first antigen-binding protein and the second antigen-binding protein can bind to the CD73 protein non-competitively. In the present application, the first antigen-binding protein may exist in the form of a mixture with the second antigen-binding protein. For example, the first antigen-binding protein may be present and mixed in the same container as the second antigen-binding protein.

[0075] In the present application, the first antigen-binding protein may include at least one CDR in the variable region VH of the antibody heavy chain that may include the amino acid sequence shown in SEQ ID NO: 8. In the present application, the first antigen-binding protein may include an HCDR3 that may include the amino acid sequence shown in SEQ ID NO: 1. In the present application, the first antigen-binding protein may include an HCDR2 that may include the amino acid sequence shown in SEQ ID NO: 2.

[0076] In the present application, the first antigen-binding protein may also include an HCDR1 that may include the amino acid sequence shown in SEQ ID NO: 3. In the present application, the first antigen-binding protein may include HCDR1, HCDR2, and HCDR3. The HCDR1 may include the amino acid sequence shown in SEQ ID NO: 3, the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 1. In the present application, the first antigen-binding protein may include H-FR1. The C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 4.

[0077] In the present application, the first antigen-binding protein may include H-FR2. The H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 5. In the present application, the first antigen-binding protein may include H-FR3. The H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 6. In the present application, the first antigen-binding protein may include H-FR4. The N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the H-FR3, and the H-FR4 includes the amino acid sequence shown in SEQ ID NO: 7.

[0078] In the present application, the first antigen-binding protein may include H-FR1, H-FR2, H-FR3, and H-FR4. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 24, the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 25, the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 26, and the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 7.

[0079] In the present application, the first antigen-binding protein includes a VH including the amino acid sequence shown in SEQ ID NO: 8. In the present application, the first antigen-binding protein may include an antibody heavy chain constant region. 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 includes the amino acid sequence shown in SEQ ID NO: 9.

[0080] In the present application, the first antigen-binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 10. In the present application, the first antigen-binding protein may include at least one CDR of the antibody light chain variable region VL including the amino acid sequence shown in SEQ ID NO: 18. In the present application, the first antigen-binding protein may include an LCDR3 including the amino acid sequence shown in SEQ ID NO: 11.

[0081] In the present application, the first antigen-binding protein may include an LCDR2 including the amino acid sequence shown in SEQ ID NO: 12 (WAS). In the present application, the first antigen-binding protein may include an LCDR1 that may include the amino acid sequence shown in SEQ ID NO: 13. In the present application, the first antigen-binding protein may include LCDR1, LCDR2, and LCDR3. The LCDR1 may include the amino acid sequence shown in SEQ ID NO: 13. The LCDR2 may include the amino acid sequence shown in SEQ ID NO: 12 (WAS). And the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 11.

[0082] In the present application, the first antigen-binding protein may include L-FR1. The C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1. And the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 14. In the present application, the first antigen-binding protein may include L-FR2. The L-FR2 is located between the LCDR1 and the LCDR2. And the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 15. In the present application, the first antigen-binding protein may include L-FR3. The L-FR3 is located between the LCDR2 and the LCDR3. And the L-FR3 includes the amino acid sequence shown in SEQ ID NO: 16.

[0083] In the present application, the first antigen-binding protein may include L-FR4. The N-terminus of the L-FR4 is directly or indirectly linked to the C-terminus of the L-FR3. And the L-FR4 includes the amino acid sequence shown in SEQ ID NO: 17. In the present application, the first antigen-binding protein may include L-FR1, L-FR2, L-FR3, and L-FR4. For example, the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 14. The L-FR2 may include the amino acid sequence shown in SEQ ID NO: 15. The L-FR3 may include the amino acid sequence shown in SEQ ID NO: 16. And the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 17.

[0084] In the present application, the first antigen-binding protein includes a VL containing the amino acid sequence shown in SEQ ID NO: 18. In the present application, the first antigen-binding protein may include an antibody light chain constant region. In the present application, the antibody light chain constant region may be derived from a human Igκ constant region.

[0085] In the present application, the antibody light chain constant region may include the amino acid sequence shown in SEQ ID NO: 19. In the present application, the first antigen-binding protein may include an antibody light chain that may include the amino acid sequence shown in SEQ ID NO: 20.

[0086] In the present application, the first antigen-binding protein may include VH and VL, the VH may include the amino acid sequence shown in SEQ ID NO: 8, and the VL may include the amino acid sequence shown in SEQ ID NO: 18. In the present application, the first antigen-binding protein may include a heavy chain and a light chain, the heavy chain may include the amino acid sequence shown in SEQ ID NO: 10, and the light chain may include the amino acid sequence shown in SEQ ID NO: 20.

[0087] In the present application, the second antigen-binding protein may include at least one CDR in the antibody heavy chain variable region VH that may include the amino acid sequence shown in SEQ ID NO: 27. In the present application, the second antigen-binding protein may include an HCDR3 that may include the amino acid sequence shown in SEQ ID NO: 21. In the present application, the second antigen-binding protein may include an HCDR2 that may include the amino acid sequence shown in SEQ ID NO: 22.

[0088] In the present application, the second antigen-binding protein may include an HCDR1 that may include the amino acid sequence shown in SEQ ID NO: 23. In the present application, the second antigen-binding protein may include HCDR1, HCDR2, and HCDR3. The HCDR1 may include the amino acid sequence shown in SEQ ID NO: 23. The HCDR2 may include the amino acid sequence shown in SEQ ID NO: 22. And the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 21. In the present application, the second antigen-binding protein may include H-FR1. The C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1. And the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 24.

[0089] In the present application, the second antigen-binding protein may include H-FR2. The H-FR2 is located between the HCDR1 and the HCDR2. And the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 25. In the present application, the second antigen-binding protein may include H-FR3. The H-FR3 is located between the HCDR2 and the HCDR3. And the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 26. In the present application, the second antigen-binding protein may include H-FR4. The N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the H-FR3. And the H-FR4 includes the amino acid sequence shown in SEQ ID NO: 7.

[0090] In the present application, the second antigen-binding protein may include H-FR1, H-FR2, H-FR3, and H-FR4. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 24, the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 25, the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 26, and the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 7. In the present application, the second antigen-binding protein includes a VH including the amino acid sequence shown in SEQ ID NO: 8. In the present application, the second antigen-binding protein may include an antibody heavy chain constant region.

[0091] In the present application, the above-mentioned antibody heavy chain constant region is derived from an IgG heavy chain constant region. For example, the above-mentioned antibody heavy chain constant region is derived from a human IgG heavy chain constant region. For example, the above-mentioned 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 above-mentioned antibody heavy chain constant region contains the amino acid sequence shown in SEQ ID NO: 9. In the present application, the above-mentioned second antigen-binding protein may contain an antibody heavy chain, and the above-mentioned antibody heavy chain may contain the amino acid sequence shown in SEQ ID NO: 28. In the present application, the above-mentioned second antigen-binding protein may contain at least one CDR of the antibody light chain variable region VL containing the amino acid sequence shown in SEQ ID NO: 35.

[0092] In the present application, the above-mentioned second antigen-binding protein may contain an LCDR3 containing the amino acid sequence shown in SEQ ID NO: 29. In the present application, the above-mentioned second antigen-binding protein may contain an LCDR2 containing the amino acid sequence shown in SEQ ID NO: 30 (LAS). In the present application, the above-mentioned second antigen-binding protein may contain an LCDR1 containing the amino acid sequence shown in SEQ ID NO: 31.

[0093] In the present application, the above-mentioned second antigen-binding protein may contain LCDR1, LCDR2, and LCDR3. The above-mentioned LCDR1 may contain the amino acid sequence shown in SEQ ID NO: 31, the above-mentioned LCDR2 may contain the amino acid sequence shown in SEQ ID NO: 30 (LAS), and the above-mentioned LCDR3 may contain the amino acid sequence shown in SEQ ID NO: 29. In the present application, the above-mentioned second antigen-binding protein may contain L-FR1. The C-terminus of the above-mentioned L-FR1 is directly or indirectly linked to the N-terminus of the above-mentioned LCDR1, and the above-mentioned L-FR1 may contain the amino acid sequence shown in SEQ ID NO: 32. In the present application, the second antigen-binding protein may include L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 33.

[0094] In the present application, the second antigen-binding protein may include L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 includes the amino acid sequence shown in SEQ ID NO: 34. In the present application, the second antigen-binding protein may include L-FR4, the N-terminus of the L-FR4 is directly or indirectly linked to the C-terminus of the L-FR3, and the L-FR4 includes the amino acid sequence shown in SEQ ID NO: 17. In the present application, the second antigen-binding protein may include L-FR1, L-FR2, L-FR3, and L-FR4. For example, the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 32, the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 33, the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 34, and the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 17.

[0095] In the present application, the second antigen-binding protein includes a VL including the amino acid sequence shown in SEQ ID NO: 35. In the present application, the second antigen-binding protein may include an antibody light chain constant region. In the present application, the antibody light chain constant region may be derived from a human Igκ constant region.

[0096] In the present application, the antibody light chain constant region may include the amino acid sequence shown in SEQ ID NO: 19. In the present application, the second antigen-binding protein may include an antibody light chain, and the antibody light chain may include the amino acid sequence shown in SEQ ID NO: 36. In the present application, the second antigen-binding protein may include VH and VL. The VH may include the amino acid sequence shown in SEQ ID NO: 27, and the VL may include the amino acid sequence shown in SEQ ID NO: 35.

[0097] In the present application, the second antigen-binding protein may include a heavy chain and a light chain. The heavy chain may include the amino acid sequence shown in SEQ ID NO: 28, and the light chain may include the amino acid sequence shown in SEQ ID NO: 36. In the present application, the first antigen-binding protein in the pharmaceutical combination may be mixed with the second antigen-binding protein at a certain mass ratio. For example, it may 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. In the present application, the pharmaceutical combination may be formulated for administration by intravenous injection.

[0098] Pharmaceutical composition In another aspect, the present application further provides a pharmaceutical composition comprising the pharmaceutical combination and optionally one or more pharmaceutically acceptable carriers. In the present application, the pharmaceutical composition may be a mixture comprising the first antigen-binding protein and the second antigen-binding protein, and the mixture may further comprise an inert ingredient formulated as a pharmaceutically acceptable preparation. In the present application, the concentration of the above pharmaceutical combination is about 3 to 300 mg / mL, optionally about 3 to 150 mg / mL, optionally about 5 to 100 mg / mL, optionally about 10 to 100 mg / mL, optionally about 10 to 50 mg / mL, for example about 30 ± 5 mg / mL, for example about 30 ± 3 mg / mL.

[0099] In the present application, the above pharmaceutical composition may contain a buffer. For example, the above buffer may be an acidic buffer. For example, the above buffer may be a neutral buffer. For example, the above buffer may be selected from the group consisting of acetate - acetic acid buffer, histidine - histidine hydrochloride buffer, citrate - citric acid buffer, phosphate buffer, histidine - acetic acid buffer, tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer, phosphate - citrate, or a combination thereof. For example, the above buffer may be 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. For example, the above buffer may be one or more 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 sodium phosphate - citrate. For example, the above buffer may be a histidine - acetic acid buffer.

[0100] In the present application, the concentration of the above buffer may be about 5 to 100 mM, optionally about 5 to 60 mM, optionally about 5 to 50 mM, optionally about 10 to 40 mM, optionally about 20 ± 5 mM, optionally about 20 ± 2 mM. In the present application, the pH of the above pharmaceutical composition may be about 5 to 7. For example, the pH value of the above pharmaceutical composition is about 6 ± 0.5. For example, the pH value of the above pharmaceutical composition is about 6 ± 0.3. For example, the pH value of the above pharmaceutical composition is about 6 ± 0.1. For example, the pH value of the above pharmaceutical composition is about 6.5 ± 0.3. For example, the pH value of the above pharmaceutical composition is about 7 ± 0.3. In the present application, the pharmaceutical composition may contain a stabilizer. In the present application, the stabilizer may be selected from the group consisting of sodium chloride, amino acids, sugar alcohols, and combinations thereof. For example, the amino acid may be selected from the group consisting of proline, arginine, glycine, histidine, lysine, methionine, and combinations thereof. For example, the sugar alcohol may be selected from the group consisting of sucrose, mannitol, trehalose, sorbitol, and combinations thereof.

[0101] For example, the stabilizer may be one or more selected from the group consisting of 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 may be sucrose.

[0102] In the present application, the content of the stabilizer in the pharmaceutical composition is about 0.15 - 20 wt.%, optionally the content of the stabilizer is about 0.15 wt.% - 10 wt.%, optionally the content of the stabilizer is about 0.15 wt.% - 8 wt.%, optionally the content of the stabilizer is about 4 ± 1 wt.% or 8 ± 1 wt.%, optionally the content of the stabilizer is about 4 ± 0.5 wt.%, 8 ± 0.5 wt.%, and optionally the content of the stabilizer is about 4 wt.%, 5 wt.%, 7 wt.% or 8 wt.%. For example, the content of the stabilizer is about 0.15 wt.%, about 0.2 wt.%, about 0.3 wt.%, about 0.4 wt.%, about 0.5 wt.%, about 0.6 wt.%, about 0.8 wt.%, about 1 wt.%, about 1.5 wt.%, about 2 wt.%, about 2.5 wt.%, about 3 wt.%, about 3.5 wt.%, about 4 wt.%, about 4.5 wt.%, about 5 wt.%, about 5.5 wt.%, about 6 wt.%, about 7 wt.%, about 8 wt.%.

[0103] In the present application, the pharmaceutical composition may contain a surfactant. In the present application, the surfactant may be selected from the group consisting of polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, glycerin fatty acid esters, polysorbates, poloxamers, and combinations thereof. In the present application, the polysorbate may 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 may contain Tween-20 (PS-20) and / or Tween-80 (PS-80). For example, the concentration of the surfactant is about 0.001% to 0.5%, optionally about 0.005% to 0.1%, optionally about 0.005%, 0.01%, 0.03%, 0.05% or 0.1%, optionally 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%.

[0104] In the present application, the buffer system of the pharmaceutical composition may contain 20 mM histidine-acetic acid, 8 wt.% sucrose, and 0.03 wt.% Tween-80. In the present application, the buffer system of the pharmaceutical composition may contain 20 mM sodium acetate-acetic acid, 8 wt.% trehalose, and 0.03 wt.% Tween-80. In the present application, the buffer system of the pharmaceutical composition may contain 20 mM histidine-acetic acid, 1.5 wt.% proline, 5 wt.% sucrose, and 0.03 wt.% Tween-80.

[0105] In the present application, the buffer system of the pharmaceutical composition may contain 20 mM sodium acetate-acetic acid, 0.15 wt.% methionine, 8 wt.% trehalose, and 0.03 wt.% Tween-80. In the present application, the buffer system of the pharmaceutical composition may contain 20 mM histidine-acetic acid, 8 wt.% sucrose, and 0.01 wt.% Tween-80. In the present application, the buffer system of the above pharmaceutical composition may contain 20 mM sodium acetate - acetic acid, 8 wt.% trehalose, and 0.01 wt.% Tween - 80.

[0106] In the present application, the buffer system of the above pharmaceutical composition may contain 20 mM histidine - acetic acid, 1.5 wt.% proline, 5 wt.% sucrose, and 0.01 wt.% Tween - 80. In the present application, the buffer system of the above pharmaceutical composition may contain 20 mM sodium acetate - acetic acid, 0.15 wt.% methionine, 8 wt.% trehalose, and 0.01 wt.% Tween - 80.

[0107] Use In another aspect, the present application provides the use of the above pharmaceutical combination and / or the above pharmaceutical composition in the preparation of a drug for preventing and / or treating a disease and / or a medical condition. In another aspect, the present application further provides a method for preventing and / or treating a disease and / or a medical condition, which method may include administering the above pharmaceutical combination and / or the above pharmaceutical composition of the present application to a subject in need thereof. In the present application, the above administration can be carried out by different means such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0108] In another aspect, the above pharmaceutical combination and / or the above pharmaceutical composition of the present application can be used for preventing and / or treating a disease and / or a medical condition. In the present application, the above disease and / or medical condition may be a disease and / or medical condition mediated by abnormal expression of CD73. In the present application, the above disease and / or medical condition may include a tumor.

[0109] In the present application, the above tumor may include a solid tumor and / or a hematological tumor. In the present application, the above-mentioned diseases and / or medical conditions may include pancreatic cancer, breast cancer and / or idiopathic pulmonary fibrosis (IPF). For example, the above-mentioned breast cancer may include triple-negative breast cancer. In another aspect, the present application further provides a method for detecting CD73 in a sample, the method comprising administering the above-mentioned pharmaceutical combination and / or the above-mentioned pharmaceutical composition.

[0110] In the present application, the above method for detecting CD73 in a sample may be an in vitro method. In the present application, the above method for detecting CD73 in a sample may be for non-therapeutic purposes. In the present application, the above method for detecting CD73 in a sample is not a diagnostic method. In another aspect, the present application further provides a reagent or reagent kit for detecting CD73 in a sample, the reagent or reagent kit comprising the above-mentioned pharmaceutical combination and / or the above-mentioned pharmaceutical composition.

[0111] In another aspect, the present application further provides the use of the above-mentioned pharmaceutical combination and / or the above-mentioned pharmaceutical composition in the preparation of a reagent kit for detecting the presence and / or content of CD73 in a sample. Without being limited to any theory, the following examples are only for explaining the pharmaceutical combination, preparation method and uses according to the present application, and do not limit the scope of the invention of the present application.

Examples

[0112] General measurement method SEC-UPLC: Using an ACQUITY UPLC(R) Protein BEH SEC analytical column (4.6 mm×150 mm), measured by a Waters ultra-high performance liquid chromatography apparatus (Acquity H Class) with reference to the high performance liquid chromatography in General Rule 0514 of the Pharmacopoeia of the People's Republic of China (2015 Edition, Part III), and the purity was calculated by the area normalization method.

[0113] CEX-HPLC: ProPac TMUsing a WCX-10 analytical column (4 mm × 250 mm), measurements were carried out with a Waters high-performance liquid chromatography apparatus (E2695) with reference to the high-performance liquid chromatography in the general rules of the "Pharmacopoeia of the People's Republic of China" (2015 Edition, Part III), and the purity was calculated by the area normalization method.

[0114] Measurement of Tm value (IF, full-spectrum fluorescence): Using Uncle (Unchained Labs), heating was carried out at 0.3 °C / min in the range of 25 °C to 95 °C, and the melting temperature Tm of different formulations was calculated using the full-spectrum fluorescence module.

[0115] Measurement of Tagg value (SLS, static light scattering): Using the Uncle instrument, heating was carried out at 0.3 °C / min from 25 °C to 95 °C, and the aggregation onset temperature Tagg of antibody molecules with different formulations was calculated using the static light scattering module.

[0116] Example 1, CD73 enzyme activity inhibition experiment of components 900698 and 900725 in anti-CD73 cocktail antibody 900698 (heavy chain sequence is shown in SEQ ID NO: 10, light chain sequence is shown in SEQ ID NO: 20) and 900725 (heavy chain sequence is shown in SEQ ID NO: 28, light chain sequence is shown in SEQ ID NO: 36) were diluted to 30 μg / mL with PBS, and further mixed according to 1:5, 1:2.5, 1:1, 2.5:1, 5:1, and further serially diluted 5-fold into eight gradients. A suspension of cells expressing human CD73 (CHOK1-huCD73-3F4, Huabo Biotech) during the logarithmic growth phase was taken, and the cells were prepared into a cell suspension of 4×105 / mL using the medium. 100 μL (about 40,000 cells) was added to each well of a 96-well U-bottom plate, centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed. Then, the cells were resuspended with the serially diluted antibody, 100 μL was added to each well, mixed uniformly, and incubated at 37°C for 20 minutes. After the incubation, it was centrifuged at 1500 rpm for 5 minutes, the supernatant was removed, washed once with 200 μL / well of PBS, then resuspended with 100 μL / well of 180 μM AMP, and incubated at 37°C for 60 minutes. After the incubation, it was centrifuged at 1500 rpm for 5 minutes, 50 μL / well of the supernatant was collected, transferred to a 96-well black plate, and further 50 μL / well of ATP prepared was added, mixed uniformly, and reacted at 37°C for 15 minutes. Finally, CellTiter-Glo® substrate was added to CellTiter-Glo® buffer, mixed uniformly and equilibrated to room temperature, then 100 μL / well was added to the wells waiting to be measured, reacted at room temperature for 10 minutes, and detected at full wavelength. The percentage of enzyme activity was evaluated as follows: The activity of residual CD73 is (CHOK1-huCD73-3F4 cells + Ab + ATP + AMP) - (ATP + AMP) / (CHOK1-huCD73-3F4 cells + ATP + AMP) - (ATP + AMP) * 100.

[0117] Figure 1 shows the effects of 900698, 900725, and mixtures of 900698 and 900725 at different ratios on the inhibition of CD73 (CHOK1-huCD73-3F4, Huabo Biologics) enzyme activity expressed on the cell membrane. As can be seen from the results, when the antibody concentration is 0.1 - 1 μg / mL, under the same concentration conditions, the mixture of 900698 and 900725 has a higher inhibitory ability against enzyme activity than 900698 and 900725 alone (as indicated by the black dashed arrow, the lower the plateau value under the enzyme activity inhibition curve, the higher the maximum inhibitory ability of the antibody against enzyme activity), indicating that there is a synergistic inhibitory effect after mixing 900698 and 900725, and it seems that the effect is optimal when the ratio of 900698 and 900725 is 1:1.

[0118] Example 2, SPR competitive binding experiment of anti-CD73 cocktail antibody The inventors conducted an SPR competitive binding experiment to investigate whether there is a competitive effect when 900698 and 900725 antibodies bind to CD73. First, human CD73 antigen protein (manufacturer: ACRO Biosystems, product number: CD3-H52H7) was captured as a ligand using BIAcore 8k, and then the antibody was used as the analyte. The order of adding the antibodies was sequentially injected in the manner of 900698 + 900725 (first add 900698, and then add 900725 further) or 900725 + 900698 (first add 900725, and then add 900698 further), and at the same time, it was controlled in the manner of 900698 + buffer and 900725 + buffer.

[0119] As shown in Table 1 and Figure 2 below, adding either 900698 or 900725 first has no effect on the binding of another antibody to the CD73 antigen, indicating that the two components of the anti-CD73 cocktail antibody, namely 900698 and 900725, have different binding epitopes on the CD73 antigen and can bind to the CD73 antigen simultaneously.

[0120]

Table 1

[0121] Example 3, Detection of antitumor effect of anti-CD73 cocktail antibody in pancreatic cancer animal model In this experiment, a BxPC-3 pancreatic cancer animal model was established using NPG mice, and the efficacy of the test antibody was tested. The human pancreatic cancer cell BxPC-3 used in this experiment was cultured in RPMI-1640 medium supplemented with 10% FBS in an incubator at 37 °C containing 5% CO2. Before culturing the cells continuously for 10 generations, about 1×10 7 BxPC-3 cells were uniformly mixed with 100 μL of PBS and an equal volume of Matrigel, and then inoculated subcutaneously at an inoculation volume of about 200 μL near the lower right side of the back of 36 NPG mice. The mice were anesthetized with 2-5% isoflurane before inoculation. On the day of inoculation, 1.0×10 7 PBMC (100 μL) was injected via the tail vein. When the tumors grew to an average of about 50-80 mm3, the 36 tumor-bearing mice were randomly divided into 6 groups of 6 mice each according to tumor volume and body weight. Administration was carried out on the day of grouping, and the specific dosing regimen is shown in the following table. Among them, 900201 is a negative control antibody that does not bind to the CD73 antigen. The heavy chain (HC) amino acid sequence of 900201 is shown in SEQ ID NO: 37, and the light chain (LC) amino acid sequence of 900201 is shown in SEQ ID NO: 38. Oleclumab is an anti-CD73 monoclonal antibody developed by AstraZeneca that is currently in Phase III clinical trials. Uliledlimab is an anti-CD73 monoclonal antibody developed by Tianjing Biopharma that is currently in Phase I clinical trials.

[0122] The results are shown in Table 2 and Figure 3 below. Compared with 900201 in the negative control group, the positive drugs oleclumab, uliledlimab, and the test drugs 900698, 900725, and the anti-CD73 cocktail antibody can all significantly inhibit the growth of BxPC-3 pancreatic cancer, and the tumor inhibitory effect of the anti-CD73 cocktail antibody at the same dose is superior to that of the positive controls oleclumab, uliledlimab, and 900698 and 900725. It was demonstrated that the anti-CD73 antibody cocktail antibody prepared by mixing 900698 and 900725 in an equal ratio has a good synergistic anti-pancreatic cancer tumor effect.

[0123]

Table 2

[0124] Example 4, Detection of antitumor effect of anti-CD73 cocktail antibody in triple negative breast cancer animal model In this experiment, an animal model of MDA-MB-231 triple-negative breast cancer was established using NPG mice, and the efficacy of the test antibody was tested. The human breast cancer cell MDA-MB-231 used in this experiment was cultured in L-15 medium supplemented with 10% FBS in a 37 °C incubator without CO2. Before culturing the cells continuously for 10 generations, approximately 5.0×10 6 MDA-MB-231 cells were suspended in 100 μL of PBS, uniformly mixed with an equal volume of Matrigel, and then inoculated subcutaneously at an inoculation volume of approximately 200 μL near the lower right side of the back of NPG humanized mice. The mice were anesthetized with 2-5% isoflurane before inoculation. On the day of inoculation, 1.0×10 7 PBMC (100 μL) was injected via the tail vein. When the tumors grew to an average of approximately 50-80 mm 3 in size, 30 tumor-bearing mice were randomly divided into 5 groups of 6 mice each according to tumor volume and body weight. Administration was carried out on the day of grouping, and the specific dosing regimen is shown in the following table. Among them, 900201 is a negative control antibody that does not bind to the CD73 antigen. Oleclumab is an anti-CD73 monoclonal antibody developed by AstraZeneca that is currently in clinical phase III.

[0125] The results are shown in Table 3 and Figure 4 below. Compared with 900201 in the negative control group, the positive drug oleclumab and the test drugs 900698, 900725, and the anti-CD73 cocktail antibody can all significantly inhibit the growth of MDA-MB-231 triple-negative breast cancer. Moreover, the tumor inhibitory effect of the anti-CD73 cocktail antibody at the same dose is superior to that of the positive control oleclumab, as well as 900698 and 900725. It was demonstrated that the anti-CD73 antibody cocktail antibody mixed with 900698 and 900725 in an equal ratio has a good synergistic anti-triple-negative breast cancer tumor effect.

[0126]

Table 3

[0127] Example 5, Therapeutic effect of anti-CD73 cocktail antibody in animal model of idiopathic pulmonary fibrosis (IPF) Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by progressive and irreversible pulmonary fibrosis, and there has been no effective treatment method so far. Most patients die of progressive respiratory failure within 3 to 8 years after the onset of symptoms. Although the pathogenesis of IPF is mostly unknown, the characteristic pathological features include changes in pulmonary inflammation, excessive proliferation of fibroblasts, and abnormal deposition of extracellular matrix (ECM). Bleomycin (BLM)-induced fibrosis is widely used in the study of the pathogenesis of idiopathic pulmonary fibrosis.

[0128] The specific operations of the bleomycin-induced CD73 humanized transgenic mouse idiopathic pulmonary fibrosis model experiment are as follows: CD73 humanized mice are grouped according to Table 4 below. That is, except for directly putting 3 randomly selected animals into the G1 normal group, all other animals in the groups received intratracheal instillation of bleomycin for model induction. The specific induction method is as follows: On the first day, the animals are anesthetized by inhaling 2 - 5% isoflurane, and based on the body weight, bleomycin is administered to the animals via the intratracheal administration route (2 U / kg, and the specific administration volume is calculated and recorded based on the body weight of the animals). Three days after the drip injection of bleomycin, according to the body weight and the state of the animals, animals with too much weight loss and animals with minor weight changes are removed. To achieve the approximate average body weight of each group and reduce the deviation between groups, 48 animals are divided into the remaining 6 groups by the BioBook random distribution function. Until the end of the experiment, administration to all animals starts on the fourth day, and the specific administration regimen is shown in Figure 5 and Table 4. Throughout the experimental period, the body weight of the animals is weighed and recorded three times a week. The results of the mouse body weight are shown in Figure 6. Throughout the experimental period, except for the normal group, after modeling the body weight of the animals, the body weight gradually decreased, and with the progress of the test and self-recovery, the body weight gradually and continuously increased, and there was no significant difference between groups.

[0129]

Table 4

[0130] 5.1. Measurement of airway reactivity: On the 21st day, all animals underwent measurement of airway reactivity to detect lung function. Airway hypersensitivity was measured in experimental mice using the WBP system. First, after the mice inhaled an aerosol of PBS solution, they were subjected to continuous aerosol inhalation of methacholine (Mch) at concentrations of 1.5625, 3.125, 6.25, 12.5, 25, and 50 mg / mL. The enhanced expiratory pause value (Penh) at the corresponding concentration was measured, stimulated for 90 seconds at each concentration, and a Penh change rate-Mch concentration curve relative to the baseline was plotted, and the area under the curve was calculated.

[0131] The results are shown in Figure 7. Compared with normal animals, the area under the Penh curve (AUC) of the animals in the model group increased significantly. Compared with the model animal group, the medium and high dose groups of the test substance showed an improvement trend, and the high dose group had a significant difference (P = 0.024, Kruskal-Wallis Test). No improvement was observed in the positive control group with nintedanib, and it was proved that the anti-CD73 cocktail antibody was superior to nintedanib, the positive control, in improving the lung function of idiopathic pulmonary fibrosis.

[0132] 5.2. Measurement of the number of inflammatory cells in bronchoalveolar lavage fluid After the pulmonary function test was completed, the animals were anesthetized with Zoletil (intraperitoneal injection, 25 - 50 mg / kg of Zoletil, 5 - 10 mg / kg of xylazine), then tracheally intubated, and the lungs were first washed with 0.4 mL of PBS (containing 1% FBS). Also, 0.4 mL of PBS (containing 1% FBS) was taken respectively for the second and third washes of the lungs. 100 μL was suspended to count the total number of cells in the bronchoalveolar lavage fluid. The bronchoalveolar lavage fluid was centrifuged at 4°C, 300 g for 5 minutes, the cell pellet obtained after centrifugation was resuspended to prepare a smear specimen, and eosinophils, neutrophils, macrophages and lymphocytes were distinguished by staining with Wright-Giemsa staining solution. Counting was performed under an optical microscope. The experimental data were expressed as the mean ± standard error (mean ± S.E.M). The data were analyzed using SPSS or Graphpad Prism. The specific analysis methods used were described in the annotations below the figure legends and tables. When P < 0.05, a statistical difference was considered to exist.

[0133] According to the counting results of the lung lavage fluid cells, compared with the normal group, the total number of inflammation-related cells and the percentage of lymphocytes in the bronchoalveolar lavage fluid of the animals in the model group were both significantly increased. After administration of the positive drug and test articles of anti-CD73 cocktail antibodies at different concentrations, the levels of the total number of inflammation-related cells and lymphocytes were significantly decreased, suggesting that the anti-CD73 cocktail antibody can significantly improve the degree of inflammation in idiopathic pulmonary fibrosis disease.

[0134] 5.3, Degree score of pathological fibrosis: After washing, the animals were euthanized by cervical dislocation. The left lung was collected, fixed with neutral formalin and embedded in wax blocks, sectioned transversely, and a total of 3 slices / lung lobe were taken at the upper, middle and lower segments respectively, embedded in one wax block, and stained with Masson for use in the degree score of pathological fibrosis. The scoring criteria are shown in Table 5 below.

[0135] The score results are shown in Figures 9 and 10. Compared with the animals in the normal group, the animals in the model group showed significant pulmonary fibrosis changes (P<0.001) after bleomycin induction, indicating that the modeling of the bleomycin-induced pulmonary fibrosis model was successful. In both the nintedanib group, which is a positive drug, and the anti-CD73 cocktail antibody administration group, the pathological scores were improved. Using the Kruskal-Wallis test method for examination, there was a significant difference in the medium-dose anti-CD73 cocktail antibody administration group compared with the model group. It was shown that the anti-CD73 cocktail antibody can effectively improve the pathological score of idiopathic pulmonary fibrosis.

[0136]

Table 5

[0137] Example 6, Comparison of different buffer systems The antibody was replaced with each screening buffer containing 10 mM in the WaterSep hollow fiber (Discover12) (shown in Table 6 below), and the antibody concentration was adjusted to approximately 5 mg / mL with each buffer. After sterilization and filtration, it was prepared for use.

[0138] New samples were prepared, and the stability of each buffer was evaluated by Tm and Tagg, and the results are shown in Tables 6 and 7. At the same time, the newly prepared samples were placed under various pressure conditions including a high-temperature test (40°C ± 2°C, left standing for 3 weeks) and repeated freeze-thaw cycles (5 cycles, ≤ -70°C / 5°C ± 3°C). Their stability was evaluated by detection items such as appearance and purity, and the results are shown in Tables 8 and 9.

[0139]

Table 6

[0140]

Table 7

[0141]

Table 8

[0142]

Table 9

[0143] As can be seen from Table 6 and Table 7, there were no significant differences in Tm and Tagg of each buffer system. As can be seen from Table 8 and Table 9, under the freeze-thaw pressure conditions, there were no significant differences in purity among each group, and under the high-temperature pressure conditions, there were no significant differences in appearance and purity among each buffer system group. Subsequently, histidine-acetic acid (pH 6) was selected as the buffer solution, and screening of other auxiliary materials was carried out.

[0144] Example 7, Stability comparison of different stabilizers The antibody was replaced with a histidine-acetic acid (pH 6) buffer system using WaterSep hollow fibers (Discover12), and high-concentration mother liquors of each stabilizer were added respectively to prepare each stabilizer screening group (shown in Table 10). The antibody concentration was adjusted to about 30 mg / mL, sterilized and filtered, and then prepared for use.

[0145] The newly prepared samples were placed under each pressurized condition including a high-temperature test (40°C ± 2°C, left standing for 3 weeks) and repeated freeze-thaw cycles (5 cycles, ≤ -70°C / 5°C ± 3°C). Their stability was evaluated by detection items such as appearance and purity, and some of the detection results are shown in Table 10 and Table 11 below.

[0146]

Table 10

[0147]

Table 11

[0148] As can be seen from Tables 10 and 11, under freeze-thaw pressure, there was no significant difference among the other groups except that the SEC purity of the mannitol group was slightly lower than that of the other groups. Under high-temperature pressure, there was no significant difference among the other groups 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. Subsequently, sucrose was selected as the stabilizer, and a surfactant screening experiment was conducted.

[0149] Example 8, Screening of surfactants The anti-CD73 cocktail antibody was replaced with WaterSep hollow fiber (Discover12) in a histidine-acetic acid (pH 6) buffer system containing 8 wt.% sucrose. The high-concentration stock solution of each surfactant was added, and each surfactant screening group (shown in Table 12) was prepared. The antibody concentration was adjusted to approximately 30 mg / mL, sterilized by filtration, and then prepared for use.

[0150] The newly prepared samples were placed under each shaking pressure condition 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 detection results are shown in Table 12 below.

[0151]

Table 12

[0152] As can be seen from Table 12, under shaking pressure, compared with the surfactant-free group, the addition of PS-20 and PS-80 can significantly inhibit the increase in the number of insoluble particles caused by shaking. The appearance “+” indicates the level of the opalescence phenomenon, “+++” indicates severe, “++” indicates relatively severe, and “+” indicates mild. From the appearance, the addition of PS-20 and PS-80 can improve the severity of the opalescence phenomenon of the sample, but when the concentration of PS-20 and PS-80 exceeds 0.05%, the improvement effect is inferior.

[0153] Example 9, Preparation of anti-CD73 cocktail antibody formulations with different buffer systems From Tables 6 to 12, it was found that the experimental results of each component showed no significant difference among other groups except being slightly inferior individually.

[0154] Summarizing the above, later, a buffer system of multiple groups was selected for consideration of pressurization, acceleration, and long-term stability. The buffer systems are as follows: 1) 20 mM histidine - acetic acid, 8 wt.% sucrose, and 0.03 wt.% Tween - 80, pH 6 ± 0.5 2) 20 mM sodium acetate - acetic acid, 8 wt.% trehalose, and 0.03 wt.% Tween - 80, PH 6 ± 0.5 3) 20 mM histidine - acetic acid, 1.5 wt.% proline, 5 wt.% sucrose, and 0.03 wt.% Tween - 80, pH 6 ± 0.5 4) 20 mM sodium acetate - acetic acid, 0.15 wt.% methionine, 8 wt.% trehalose, and 0.03 wt.% Tween - 80, pH 6 ± 0.5

[0155] First, using the 30KD ultrafiltration membrane package (PLCTK) of Merk Company, the anti - CD73 cocktail antibody was replaced in each buffer system, the antibody concentration was adjusted to about 30 ± 3 mg / mL, after sterilization and filtration, it was aseptically dispensed into 1.5 mL EP tubes, one tube at a time for each formulation, and 1 mL per tube.

[0156] Example 10, Stability of anti-CD73 cocktail antibody formulations Example 10 considered the stability of the buffer - system anti - CD73 cocktail antibody drug formulations of multiple groups prepared in Example 9. The stability consideration includes pressurization, acceleration, and long - term stability considerations, specifically as follows.

[0157] 10.1, Stability test under pressurized conditions The pressurized conditions include 3 weeks at high temperature (40℃ ± 2℃), 3 days of shaking at room temperature (60 rmp), repeated freeze - thawing (5 cycles, ≤ - 70℃ / 5℃ ± 3℃ freeze - thawing), etc.

[0158] Multiple groups of anti-CD73 cocktail antibody formulation samples prepared according to Example 9 were stored under each pressure condition (shown in Table 13), sampled and inspected after the set time points or number of treatments, and their stability was evaluated by appearance, number of insoluble microparticles, purity, etc. Some of the detection results are shown in Table 13 below.

[0159]

Table 13

[0160] As can be seen from Table 13, the influencing items and degrees of each pressure condition on product quality are different. Vibration and high-temperature pressure mainly caused an increase in the number of large particles in the insoluble microparticles, and the purity decreased slightly but still far exceeded the quality standards. In short, these multiple groups of buffer system pharmaceutical compositions have high stability, can effectively protect the main drug component, the anti-CD73 cocktail antibody, and thereby can effectively resist the destructive effects of each pressurization condition.

[0161] 10.2, Accelerated stability test Multiple groups of buffer system anti-CD73 cocktail antibody drug formulation samples prepared according to Example 9 were stored under accelerated conditions (25°C ± 2°C), sampled and inspected at the set time points, and their stability was evaluated by insoluble microparticles, purity, etc. Some of the detection results are shown in Table 14 below.

[0162]

Table 14

[0163] As can be seen from Table 14, the acceleration conditions somewhat degraded the quality of the pharmaceutical preparation. Among the multiple groups of buffer systems, 20HA6Arg1.5Suc5ps0.03 was slightly inferior compared to the T0 sample. For the samples subjected to 3 months of acceleration, the SEC molecular sieve purity decreased by approximately 1.4%, the content of the CEX main peak (38) decreased by approximately 4.4%, and the content of the CEX main peak (39) decreased by approximately 9.6%, but still met the quality standards. In short, the pharmaceutical compositions of these multiple groups of buffer systems have high stability and can be stored stably for at least 3 months or more even under the acceleration conditions of 25°C.

[0164] 10.3, Long-Term Stability Test Samples of the multiple groups of buffer system anti-CD73 cocktail antibody pharmaceutical preparations prepared according to Example 9 were stored under long-term refrigeration conditions (5°C ± 3°C), sampled and inspected at the set time points, and their stability was evaluated by insolubles, purity, etc. Some of the detection results are shown in Table 15 below.

[0165]

Table 15

[0166] As can be seen from Table 15, under long-term refrigeration conditions (5°C ± 3°C), these multiple groups of buffer system pharmaceutical preparations have good stability. For the 3-month samples, there were no obvious changes in insolubles, and the purity decreased slightly but far exceeded the quality standards. In short, the pharmaceutical compositions of these multiple groups of buffer systems have high stability and can be stored stably under long-term refrigeration conditions.

[0167] As can be seen from the above, the multiple groups of buffer system anti-CD73 cocktail antibody pharmaceutical preparations prepared in Example 9 have excellent stability. The anti-CD73 cocktail antibody maintains relatively good stability under each pressure condition (high temperature, repeated freeze-thaw, and shaking), can be stored stably for at least 3 months or more under the acceleration conditions (25°C ± 2°C), and at the same time shows high stability under long-term refrigeration conditions and can be stored stably.

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, and the first antigen-binding protein comprises HCDR1-3 and LCDR1-3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 1, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 13, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 12 (WAS), and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

11. A combination of pharmaceuticals.

2. The first antigen-binding protein comprises VH and VL, wherein VH comprises the amino acid sequence shown in SEQ ID NO: 8, and VL comprises the amino acid sequence shown in SEQ ID NO:

18. The pharmaceutical combination described in claim 1.

3. The first antigen-binding protein comprises an antibody heavy chain and an antibody light chain, wherein the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 10, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO:

20. The pharmaceutical combination described in claim 1.

4. The second antigen-binding protein comprises HCDR1-3 and LCDR1-3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 23, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 22, HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 21, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 31, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 30 (LAS), and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

29. The pharmaceutical combination described in claim 1.

5. The second antigen-binding protein comprises VH and VL, wherein VH comprises the amino acid sequence shown in SEQ ID NO: 27, and VL comprises the amino acid sequence shown in SEQ ID NO:

35. The pharmaceutical combination described in claim 1.

6. The second antigen-binding protein comprises an antibody heavy chain and an antibody light chain, wherein the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 28, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO:

36. The pharmaceutical combination described in claim 1.

7. The pharmaceutical combination according to claim 1, wherein the mass ratio of the first antigen-binding protein to the second antigen-binding protein is approximately 1:5 to 5:

1.

8. The mass ratio of the first antigen-binding protein to the second antigen-binding protein is approximately 1:5, approximately 1:2.5, approximately 1:1, approximately 2.5:1, or approximately 5:

1. The pharmaceutical combination described in claim 1.

9. A pharmaceutical composition comprising the pharmaceutically acceptable combination described in claim 1 and one or more pharmaceutically acceptable carriers, Pharmaceutical composition.

10. The concentration of the aforementioned pharmaceutical combination is approximately 3 to 300 mg / mL. The pharmaceutical composition according to claim 9.

11. The concentration of the aforementioned pharmaceutical combination is 30 ± 3 mg / mL. The pharmaceutical composition according to claim 9.

12. The pharmaceutically acceptable carrier comprises a buffer, and the buffer comprises an acidic buffer or a neutral buffer. The pharmaceutical composition according to claim 9.

13. The buffer is selected from the group consisting of (1) acetate-acetic acid buffer, histidine-histidine hydrochloride buffer, citrate-citric acid buffer, phosphate buffer, histidine-acetic acid buffer, trishydroxymethylaminomethane (Tris)-hydrochloride buffer, phosphate-citrate, or a combination thereof; (2) acetate-acetic acid, citrate-citric acid, histidine-histidine hydrochloride, phosphate, Tris-hydrochloride, histidine-acetic acid, phosphate-citrate, or a combination thereof; and (3) sodium acetate-acetic acid, sodium citrate-citric acid, histidine-histidine hydrochloride, sodium dihydrogen phosphate-disodium hydrogen phosphate, Tris-hydrochloride, histidine-acetic acid, or phosphate-sodium citrate. The pharmaceutical composition according to claim 12.

14. The concentration of the buffer solution is 20 ± 5 mM. The pharmaceutical composition according to claim 12.

15. The pH value is approximately 6 ± 0.5 or approximately 7 ± 0.

3. The pharmaceutical composition according to claim 9.

16. The pharmaceutically acceptable carrier comprises a stabilizer, which is selected from the group consisting of sodium chloride, amino acids, sugar alcohols, and combinations thereof. The pharmaceutical composition according to claim 9.

17. The aforementioned stabilizer is selected from the group consisting of proline, arginine, glycine, histidine, lysine, methionine, sucrose, mannitol, trehalose, sorbitol, proline / sucrose, methionine / trehalose, glycine / mannitol, sodium chloride / sucrose, arginine / sucrose, and lysine / trehalose, and combinations thereof. The pharmaceutical composition according to claim 16.

18. The content of the aforementioned stabilizer is approximately 0.15 wt.% to 8 wt.%. The pharmaceutical composition according to claim 16.

19. The pharmaceutically acceptable carrier comprises a surfactant, which is selected from the group consisting of polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, glycerin fatty acid esters, polysorbates, poloxamers, and combinations thereof. The pharmaceutical composition according to claim 9.

20. 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). The pharmaceutical composition according to claim 19.

21. The concentration of the surfactant is approximately 0.005% to 0.1%. The pharmaceutical composition according to claim 19.

22. The pharmaceutically acceptable carriers are comprised of the following group: (1) 20 mM histidine acetate, 8 wt.% sucrose, and 0.03 wt.% Tween-80, (2) 20 mM sodium acetate-acetic acid, 8 wt.% trehalose, and 0.03 wt.% Tween-80, (3) 20 mM histidine acetate, 1.5 wt.% proline, 5 wt.% sucrose, and 0.03 wt.% Tween-80, (4) 20 mM sodium acetate-acetic acid, 0.15 wt.% methionine, 8 wt.% trehalose, and 0.03 wt.% Tween-80, (5) 20 mM histidine acetate, 8 wt.% sucrose, and 0.01 wt.% Tween-80, (6) 20 mM sodium acetate-acetic acid, 8 wt.% trehalose, and 0.01 wt.% Tween-80, (7) 20 mM histidine acetate, 1.5 wt.% proline, 5 wt.% sucrose, and 0.01 wt.% Tween-80, (8) 20 mM sodium acetate-acetic acid, 0.15 wt.% methionine, 8 wt.% trehalose, and 0.01 wt.% Tween-80 Selected from, The pharmaceutical composition according to claim 9.

23. A pharmaceutically acceptable combination according to claim 1 for use in preventing and / or treating diseases and / or medical conditions.

24. The aforementioned diseases and / or conditions include CD73-mediated diseases and / or conditions and / or tumors. The pharmaceutical combination according to claim 23.

25. The pharmaceutically acceptable combination according to claim 9 for use in preventing and / or treating diseases and / or medical conditions.

26. The disease and / or condition includes CD73-mediated diseases and / or conditions and / or tumors. The pharmaceutical combination according to claim 25.

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