Fusion protein comprising an anti-CD73 antibody and IL-2 and uses thereof

Abstract

The present invention relates to an anti-CD73 antibody or an antigen-binding fragment thereof; and a fusion protein bound to IL-2 and uses thereof. The anti-CD73 antibody or an antigen-binding fragment thereof according to the present invention; and the fusion protein containing IL-2 or a dimer thereof bind to CD73 of cancer cells to inhibit adenosine production, thereby controlling the microtumor environment by blocking the A2AR and A2BR signaling pathways. In addition, immunosuppressive cells such as regulatory T cells (Tregs) can be less activated while activating immune cells (CD8+ T cells and CD4+ T cells). Moreover, IL-2 and variants thereof in the fusion protein of the present invention can activate immune cells. Therefore, the fusion protein of the present invention and a dimer thereof can be usefully used for the prevention or treatment of cancer.

Description

Technical Field

[0001] The present invention relates to an anti-CD73 antibody or an antigen-binding fragment thereof; and a fusion protein to which IL-2 is bound and uses thereof.

Background Art

[0002] Cancer immunotherapy is a method of treating cancer by utilizing the body's immune function. Cancer immunotherapy can induce the immune system to attack cancer cells by using antigens such as cancer cell surface proteins, and can control the tumor microenvironment that causes cancer cell immune escape to enhance the activity of immune cells.

[0003] The tumor microenvironment is an environment in which cancer cells, including fibroblasts, blood vessels, lymphatic vessels, immune cells, extracellular matrix, adipocytes, etc. existing in cancer tissue, grow and evolve in addition to cancer cells. In relation to this, research results have been reported that the ATP-AMP-adenosine-A2AR / A2BR signaling pathway can regulate the activity of immune cells in the tumor microenvironment (S. Vigano et al., Front Immunol (2019) 10:925). Specifically, when adenosine binds to the A2AR / A2BR receptor on the surface of tumor cells or immune cells, immune suppressor cells such as regulatory T cells and TAM (tumor associated macrophage) are activated, and the activities of cytotoxic T cells and NK cells are suppressed. At this time, a method of activating the immunity in the tumor microenvironment has been studied by treating a substance that targets CD39, an ectonucleotidase that converts ATP to AMP, CD73, an ectonucleotidase that converts AMP to adenosine, or the A2AR / A2BR receptor that binds to adenosine to suppress the signaling pathway.

[0004] On the one hand, interleukin-2 (IL-2), also known as T-cell growth factor (TCGF), is a globular glycoprotein that plays a central role in lymphocyte production, survival, and homeostasis. IL-2 mediates various immune functions by binding to the IL-2 receptor, which is composed of three individual subunits.

[0005] In addition, IL-2 is mainly synthesized by activated T cells, particularly CD4+ helper T cells. IL-2 stimulates the proliferation and differentiation of T cells, and induces the generation of cytotoxic T lymphocytes (CTLs) and the differentiation of peripheral blood lymphocytes into cytotoxic cells and lymphokine-activated killer cells (LAK cells).

[0006] Throughout this specification, numerous papers and patent documents are referenced and their citations are indicated. The disclosures of the cited papers and patent documents are hereby incorporated by reference in their entirety to more clearly explain the state of the art in the technical field to which the present invention pertains and the content of the present invention.

Summary of the Invention

Problems to be Solved by the Invention

[0007] Therefore, the present inventors have conducted research to develop a new combination of fusion proteins that control the tumor microenvironment and enhance the activity of immune cells. As a result, it was confirmed that a fusion protein dimer containing an anti-CD73 antibody or its antigen-binding fragment and IL-2 controls the tumor microenvironment and effectively activates immune cells. Based on this, it was confirmed that the fusion protein dimer is effective as an anticancer agent, and the present invention has been completed.

Means for Solving the Problems

[0008] To achieve the above object, one aspect of the present invention provides an antibody or a fragment thereof that specifically binds to CD73; and a fusion protein containing IL-2.

[0009] Another aspect of the present invention provides a fusion protein dimer in which two of the above fusion proteins are bound.

[0010] Still another aspect of the present invention provides a polynucleotide encoding the above fusion protein, an expression vector loaded with the polynucleotide, and a transformed cell into which the expression vector has been introduced.

[0011] Still another aspect of the present invention provides a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to CD73; and a fusion protein dimer containing IL-2, the method including culturing the above transformed cell; and obtaining a fusion protein dimer.

[0012] Still another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, containing the above fusion protein, or the fusion protein dimer as an active ingredient.

[0013] Still another aspect of the present invention provides a method for preventing or treating cancer, including administering the above fusion protein, or the fusion protein dimer to a subject.

[0014] Still another aspect of the present invention provides the use of the above fusion protein, or the fusion protein dimer for preventing or treating cancer.

Advantages of the Invention

[0015] The anti-CD73 antibody or antigen-binding fragment thereof according to the present invention; and the fusion protein or dimer thereof containing IL-2 bind to CD73 of cancer cells to inhibit adenosine production (ATP→AMP→adenosine), thereby controlling the microtumor environment by blocking the A2AR and A2BR signaling pathways. In addition, immunosuppressive cells such as regulatory T cells (Tregs) can be less activated while activating immune cells (CD8+ T cells and CD4+ T cells). Moreover, IL-2 or its variant in the fusion protein of the present invention can activate immune cells. Therefore, the fusion protein and dimer thereof of the present invention can be usefully used for the prevention or treatment of cancer.

Brief Description of Drawings

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Mode for Carrying Out the Invention

[0017] An antibody or antigen-binding fragment thereof that specifically binds to CD73; and a fusion protein containing IL-2 One aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CD73; and a fusion protein containing IL-2.

[0018] CD73 and an antibody or antigen-binding fragment thereof that specifically binds to CD73 As used herein, the term "CD73 (Cluster of Differentiation 73)" is an ectonucleotidase that catalyzes the dephosphorylation of 5'-nucleotides and mainly plays a role in converting AMP (adenosine monophosphate) to adenosine. CD73 exists in the form of a homodimer on the cell membrane via a GPI anchor. The monomer is 65 kDa, and the N-terminal and C-terminal domains are linked by a flexible helical linker.

[0019] CD73 is known to be involved in adenosine production, overexpressed in cancer cells, and induce immunosuppressive effects. It is expressed in many tumor cells including leukemia, bladder cancer, glioma, glioblastoma, ovarian cancer, melanoma, prostate cancer, thyroid cancer, esophageal cancer, and breast cancer. Also, CD73 is known to be expressed on the surface of immunosuppressive cells (including regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC)). In particular, overexpression of CD73 has been reported to be related to angiogenesis, invasion, resistance to chemotherapy, tumor metastasis, and short survival of cancer patients in various tumors including breast cancer and melanoma.

[0020] In the present invention, the CD73 is inclusively and unrestrictedly any mammalian CD73, and preferably may be human CD73. Also, in the present invention, the CD73 protein includes all natural or mutant CD73 proteins, but is not limited thereto. The natural CD73 protein generally refers to a polypeptide containing the amino acid sequence of the natural CD73 protein, and the amino acid sequence of the natural CD73 protein generally refers to the amino acid sequence found in naturally occurring CD73. Information regarding the CD73 can be obtained from known databases such as GenBank of the National Institutes of Health of the United States, and can have, for example, the amino acid sequence (SEQ ID NO: 41) of Genbank accession number NP_002517.1, but is not limited thereto.

[0021] As used herein, the term "antigen" means a molecule capable of selectively binding to an antibody. The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or other naturally occurring compound, or a synthesized compound. Specifically, the antigen is a polypeptide and may be a protein present on the cell surface or intracellularly.

[0022] As used herein, the term "specifically binds" means a binding that is measurably different from non-specific interactions. Specific binding is determined by competing with a control group molecule similar to the target that does not have binding activity.

[0023] The antibody or antigen-binding fragment thereof that specifically binds to the CD73 can be collectively referred to as a molecule capable of specifically antigen-antibody binding to the CD73. Further, the antibody or antigen-binding fragment thereof can be used in any form as long as it contains an antigen-binding site capable of specifically binding to the CD73. The antibody or antigen-binding fragment thereof can contain other amino acids not directly involved in the binding, or amino acids whose effects are blocked by the amino acid residues of the antigen-binding site.

[0024] As used herein, the term "antibody" refers to a molecule containing an antigen-binding site and an immunologically active fragment of an immunoglobulin molecule containing an antigen-binding site. The immunoglobulin molecule may be an immunoglobulin molecule of IgG, IgE, IgM, IgD, IgA, IgY or its subclass. The heavy and light chains of the immunoglobulin can each contain a constant region and a variable region. The light and heavy chain variable regions of the immunoglobulin contain three hypervariable regions called complementarity determining regions (CDRs) and four framework regions (FRs). The CDRs are antigen-binding sites that mainly bind to the epitope of the antigen.

[0025] The antibody or antigen-binding fragment thereof of the present invention can include not only monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies, and any fragments thereof, but also immunoconjugates.

[0026] As used herein, the term "antigen-binding fragment of an antibody" means a part of a polypeptide to which an antigen can bind in the overall structure of an immunoglobulin, for example, Fab fragment, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv protein ("scFv"), bis-scFv (scFv)2, minibody, diabody, triabody, tetrabody, disulfide-stabilized Fv protein ("dsFv"), single-domain antibody (sdAb), and parts of full-length antibodies responsible for antigen binding, but not limited thereto. The fragments of the antibody can bind to the same antigen recognized by the intact antibody regardless of the structure.

[0027] The single-domain antibody, also called a nanobody, means an antibody fragment composed of a single monomeric variable antibody domain. The single-domain antibody can specifically bind to a specific antigen identically to a whole antibody. The single-domain antibody has a molecular weight of 12 to 15 kDa and is smaller than a general antibody (150 to 160 kDa), Fab fragment (~50 kDa), or scFv (~25 kDa). The single-domain antibody may be, for example, a monomeric form of the dimeric variable domain of a general immunoglobulin G (IgG) of humans or mice, or may be VHH of a heavy-chain antibody of Camelids or a VNAR fragment obtained from IgNAR of Cartilaginous fishes.

[0028] IL-2 or a variant thereof As used herein, the term "IL-2" or "interleukin-2" means any wild-type IL-2 obtained from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The protein size of IL-2 is 15.5 kDa to 16 kDa and it consists of 133 amino acids. The IL-2 may be obtained from animal cells, but also includes those obtained from recombinant cells capable of producing IL-2. Further, the IL-2 may be wild-type IL-2 or a variant thereof.

[0029] In this specification, IL-2 or a variant thereof is collectively referred to by the terms "IL-2 protein" or "IL-2 polypeptide". IL-2, IL-2 protein, IL-2 polypeptide, and IL-2 variant specifically bind to, for example, the IL-2 receptor. This specific binding can be confirmed by methods known to those skilled in the art.

[0030] The IL-2 may be in a mature form. Specifically, the mature IL-2 may not contain a signal sequence or may have the amino acid sequence of SEQ ID NO: 40. At this time, the IL-2 is used in the concept including a truncated fragment in which a part of the N-terminus or C-terminus of wild-type IL-2 is deleted.

[0031] As used herein, the term "IL-2 variant" means a form in which a part of the amino acids of full-length IL-2 or the above-described fragment of IL-2 is substituted. That is, the IL-2 variant can have an amino acid sequence different from that of wild-type IL-2 or its fragment. However, the IL-2 variant can have an activity equivalent to or similar to that of wild-type IL-2. Here, "IL-2 activity" can mean, for example, specifically binding to the IL-2 receptor, and the specific binding can be measured by methods known to those skilled in the art.

[0032] Specifically, the IL-2 variant may be one in which some of the amino acids of wild-type IL-2 are substituted. As a specific example of an IL-2 variant by amino acid substitution, at least one of the 38th, 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 40 may be substituted.

[0033] Specifically, the IL-2 variant may be one in which at least any one of the 38th, 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 40 is substituted with another amino acid. Moreover, in the case where IL-2 is in a form in which a part of the N-terminus of the amino acid sequence of SEQ ID NO: 40 is deleted, the amino acids at positions corresponding complementarily in the amino acid sequence of SEQ ID NO: 40 may be substituted with other amino acids. According to one specific example, as long as IL-2 activity is maintained, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids may be substituted. According to another specific example, 1 to 5 amino acids may be substituted.

[0034] As a specific example, the IL-2 variant may be in a form in which two amino acids are substituted. Specifically, the IL-2 variant may be one in which the 38th and 42nd amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Further, as a specific example, the IL-2 variant may be one in which the 38th and 45th amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Further, as a specific example, the IL-2 variant may be one in which the 38th and 61st amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Further, as a specific example, the IL-2 variant may be one in which the 38th and 72nd amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Furthermore, as a specific example, the IL-2 variant may be one in which the 42nd and 45th amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Further, as a specific example, the IL-2 variant may be one in which the 42nd and 61st amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Further, as a specific example, the IL-2 variant may be one in which the 42nd and 72nd amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Further, as a specific example, the IL-2 variant may be one in which the 45th and 61st amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Furthermore, as a specific example, the IL-2 variant may be one in which the 45th and 72nd amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Further, as a specific example, the IL-2 variant may be one in which the 61st and 72nd amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted.

[0035] Furthermore, the IL-2 variant may be in a form in which three amino acids are substituted. Specifically, the IL-2 variant may be one in which the 38th, 42nd, and 45th amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 38th, 42nd, and 61st amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 38th, 42nd, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 38th, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Further, as a specific example, the IL-2 variant may be one in which the 38th, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 38th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 42nd, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 42nd, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted. Further, as a specific example, the IL-2 variant may be one in which the 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 40 are substituted.

[0036] At this time, the "other amino acid" introduced by the above substitution may be any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenyl alanine, proline, serine, threonine, tryptophan, tyrosine and valine. However, in the amino acid substitution of the IL-2 variant, the 38th amino acid in the amino acid sequence of SEQ ID NO: 40 is not substituted with arginine, the 42nd is not substituted with phenylalanine, the 45th is not substituted with tyrosine, the 61st is not substituted with glutamic acid, and the 72nd is not substituted with leucine.

[0037] In the amino acid substitution of the IL-2 variant, the arginine, which is the 38th amino acid in the amino acid sequence of SEQ ID NO: 40, may be substituted with another amino acid excluding arginine. Preferably, in the amino acid substitution of the IL-2 variant, the arginine, which is the 38th amino acid in the amino acid sequence of SEQ ID NO: 40, may be substituted with alanine (R38A).

[0038] In the amino acid substitution of the IL-2 variant, the phenylalanine, which is the 42nd amino acid in the amino acid sequence of SEQ ID NO: 40, may be substituted with another amino acid excluding phenylalanine. Preferably, in the amino acid substitution of the IL-2 variant, the phenylalanine, which is the 42nd amino acid in the amino acid sequence of SEQ ID NO: 40, may be substituted with alanine (F42A).

[0039] In the amino acid substitution of the IL-2 variant, tyrosine, which is the 45th amino acid in the amino acid sequence of SEQ ID NO: 40, may be substituted with another amino acid other than tyrosine. Preferably, in the amino acid substitution of the IL-2 variant, tyrosine, which is the 45th amino acid in the amino acid sequence of SEQ ID NO: 40, may be substituted with alanine (Y45A).

[0040] In the amino acid substitution of the IL-2 variant, glutamic acid, which is the 61st amino acid in the amino acid sequence of SEQ ID NO: 40, may be substituted with another amino acid other than glutamic acid. Preferably, in the amino acid substitution of the IL-2 variant, glutamic acid, which is the 61st amino acid in the amino acid sequence of SEQ ID NO: 40, may be substituted with arginine (E61R).

[0041] In the amino acid substitution of the IL-2 variant, leucine, which is the 72nd amino acid in the amino acid sequence of SEQ ID NO: 40, may be substituted with another amino acid other than leucine. Preferably, in the amino acid substitution of the IL-2 variant, leucine, which is the 72nd amino acid in the amino acid sequence of SEQ ID NO: 40, may be substituted with glycine (L72G).

[0042] Specifically, the IL-2 variant may have at least one substitution selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G in the amino acid sequence of SEQ ID NO: 40.

[0043] Specifically, the IL-2 variant has amino acid substitutions at two or three positions selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G.

[0044] In addition, the IL-2 variant may be in a form where two amino acids are substituted. Specifically, the IL-2 variant may be one substituted with R38A and F42A. Also, as a specific example, the IL-2 variant may be one substituted with R38A and Y45A. Also, as a specific example, the IL-2 variant may be one substituted with R38A and E61R. Also, as a specific example, the IL-2 variant may be one substituted with R38A and L72G. Also, as a specific example, the IL-2 variant may be one substituted with F42A and Y45A. Also, as a specific example, the IL-2 variant may be one substituted with F42A and E61R. Further, as a specific example, the IL-2 variant may be one substituted with F42A and L72G. Also, as a specific example, the IL-2 variant may be one substituted with E61R and L72G.

[0045] Furthermore, the IL-2 variant may be in a form in which three amino acids are substituted. Specifically, the IL-2 variant may be substituted with R38A, F42A, and Y45A. Also, as a specific example, the IL-2 variant may be substituted with R38A, F42A, and E61R. Also, as a specific example, the IL-2 variant may be substituted with R38A, F42A, and L72G. Also, as a specific example, the IL-2 variant may be substituted with R38A, Y45A, and E61R. Also, as a specific example, the IL-2 variant may be substituted with R38A, Y45A, and L72G. Further, as a specific example, the IL-2 variant may be substituted with F42A, Y45A, and E61R. Also, as a specific example, the IL-2 variant may be substituted with F42A, Y45A, and L72G. Also, as a specific example, the IL-2 variant may be substituted with F42A, E61R, and L72G. Also, as a specific example, the IL-2 variant may be substituted with Y45A, E61R, and L72G.

[0046] Also, the IL-2 variant may be in a form in which four amino acids are substituted. Specifically, the IL-2 variant may be substituted with R38A, F42A, Y45A, and E61R. Also, as a specific example, the IL-2 variant may be substituted with R38A, F42A, Y45A, and L72G. Also, as a specific example, the IL-2 variant may be substituted with R38A, F42A, E61R, and L72G. Further, as a specific example, the IL-2 variant may be substituted with R38A, Y45A, E61R, and L72G. Also, as a specific example, the IL-2 variant may be substituted with F42A, Y45A, E61R, and L72G.

[0047] Alternatively, the IL-2 variant may be substituted with R38A, F42A, Y45A, E61R, and L72G.

[0048] Preferably, the IL-2 variant of the present invention may be one in which any one or more substitutions selected from the group consisting of R38A, F42A, and E61R in the amino acid sequence of SEQ ID NO: 40 are made. More preferably, the IL-2 variant of the present invention may be one in which substitutions are made with R38A, F42A, and E61R in the amino acid sequence of SEQ ID NO: 40. Specifically, the IL-2 variant of the present invention may have the amino acid sequence of SEQ ID NO: 8.

[0049] Further, the IL-2 variant may be characterized by having low toxicity in vivo. At this time, the low toxicity in vivo refers to side effects induced by the binding of IL-2 to the alpha form of the IL-2 receptor (IL-2Rα). The IL-2 variant described in the present application has a low binding affinity for the alpha form of the IL-2 receptor (IL-2Rα) and has lower in vivo toxicity than wild-type IL-2.

[0050] Structure of a fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds to CD73 and IL-2 or a variant thereof The fusion protein of the present invention may comprise a single-domain antibody that specifically binds to CD73, an Fc region, and IL-2 or a variant thereof. At this time, the Fc region and IL-2 or a variant thereof can be bound via a linker.

[0051] Specifically, the fusion protein may comprise the following structural formula (I). N’-X-[Linker(1)]o-Fc region fragment or variant thereof-[Linker(2)]p-Y-C’ (I) At this time, in the structural formula (I), the N’ is the N-terminus, the C’ is the C-terminus, Said X is an antibody or an antigen-binding fragment thereof that specifically binds to CD73, said Y is IL-2, said linker (1) and linker (2) are peptide linkers, said o and p are each independently 0 or 1.

[0052] At this time, the antibody or fragment thereof that specifically binds to CD73; IL-2 is as described above.

[0053] In one embodiment, said antigen-binding fragment comprises a single-domain antibody, and in this case, said antibody or its antigen-binding fragment, specifically the single-domain antibody, comprises a CDR1 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 18, and 21; a CDR2 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 19, and 22; and a CDR3 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 20, and 23.

[0054] Specifically, said antigen-binding fragment can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 17; a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 20; or a CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 23.

[0055] In one embodiment, said antigen-binding fragment can comprise any one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, and 4.

[0056] The peptide linker (1) may consist of 1 to 50 consecutive amino acids, or 3 to 30 consecutive amino acids, or 5 to 15 amino acids. As a specific example, the peptide linker (1) may consist of 12 amino acids. Further, the peptide linker (1) can contain at least one cysteine. Specifically, it can contain 1, 2 or 3 cysteines. Further, the peptide linker (1) may be derived from the hinge of an immunoglobulin. For example, the hinge can be selected from the hinge regions of various IgG sub-class antibodies. Further, the hinge may be in a form in which some amino acids in the hinge region derived from an immunoglobulin are substituted with other amino acids, or may be a sequence in which some amino acid sequences are added. In one specific example, the peptide linker (1) may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 5.

[0057] The peptide linker (2) may consist of 1 to 30 consecutive amino acids, or 2 to 20 consecutive amino acids, or 2 to 10 amino acids. As a specific example, the peptide linker (2) may be (G4S)n (where n is an integer from 1 to 10). At this time, n in (G4S)n may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. As one embodiment, the peptide linker (2) may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 7.

[0058] The immunoglobulin Fc region means a protein that includes the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of an immunoglobulin, and does not include the variable regions of the heavy and light chains of the immunoglobulin and the light chain constant region (CL). The immunoglobulin Fc region may be derived from IgG, IgA, IgE, IgD or IgM. Specifically, the immunoglobulin Fc region may be IgG1, IgG2, IgG3 or IgG4, which are subclasses of IgG, or preferably may be derived from IgG4.

[0059] In addition, the Fc region of the immunoglobulin may be not only the wild-type Fc region but also a variant of the Fc region. Furthermore, the term "variant of the Fc region" as used herein may be different from the glycosylation pattern of the wild-type Fc region, or may have an increased sugar chain, a decreased sugar chain, or a deglycosylated form compared to the wild-type Fc region. Also, an aglycosylated Fc region is included. The Fc region or the variant may have a modified number of sialic acids, fucosylation, or glycosylation adjusted by culture conditions or genetic manipulation of the host.

[0060] Moreover, the sugar chain of the Fc region of the immunoglobulin can be modified by ordinary methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Also, the variant of the Fc region may be in a form in which the Fc regions of immunoglobulins IgG, IgA, IgE, IgD, or IgM are mixed. Furthermore, the variant of the Fc region may be in a form in which some amino acids of the Fc region are substituted with other amino acids. In one embodiment, the Fc region can include the amino acid sequence of SEQ ID NO: 6.

[0061] In addition, the fusion protein of the present invention may be a polypeptide containing the variable heavy chain region (VH), the constant heavy chain region 1 (CH1), the Fc region, and IL-2 or a variant thereof of an anti-CD73 antibody. At this time, the Fc region and IL-2 or a variant thereof can be bound via a linker.

[0062] Furthermore, the fusion protein may include the following structural formulas (II) and (III): N’-X-[Linker(3)]q-Fc region fragment or a variant thereof-[Linker(4)]r-Y-C’ (II) and N’-X’-C’ (III) At this time, in the structural formulas (II) and (III), Said N’ is the N-terminus, Said C’ is the C-terminus, Said X is an antibody or an antigen-binding fragment thereof that specifically binds to CD73, and comprises a heavy chain variable region (VH) and a heavy chain constant region 1 (CH1), Said X’ is an antibody or an antigen-binding fragment thereof that specifically binds to CD73, and comprises a light chain variable region (VL) and a light chain constant region (CL), Said Y is IL-2, Said linker (3) and linker (4) are peptide linkers, Said q and r are each independently 0 or 1.

[0063] In one embodiment, said antibody or its antigen-binding fragment comprises a heavy chain variable region comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 24, an HCDR2 having the amino acid sequence of SEQ ID NO: 25, and an HCDR3 having the amino acid sequence of SEQ ID NO: 26; and a light chain variable region comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 27, an LCDR2 having the amino acid sequence of SEQ ID NO: 28 (Asp-Ala-Ser, DAS), and an LCDR3 having the amino acid sequence of SEQ ID NO: 29.

[0064] In one embodiment, said heavy chain variable region can comprise the amino acid sequence of SEQ ID NO: 9. In one embodiment, said light chain variable region can comprise the amino acid sequence of SEQ ID NO: 13.

[0065] The peptide linker (3) may consist of 1 to 50 consecutive amino acids, or 3 to 30 consecutive amino acids, or 5 to 15 amino acids. As a specific example, the peptide linker (3) may consist of 12 amino acids. Further, the peptide linker (3) can contain at least one cysteine. Specifically, it can contain 1, 2 or 3 cysteines. Also, the peptide linker (3) may be derived from the hinge of an immunoglobulin. For example, the hinge can be selected from the hinge regions of various IgG subclass antibodies. Furthermore, the hinge may be in a form in which some amino acids in the hinge region derived from an immunoglobulin are substituted with other amino acids, or may be a sequence with some amino acid sequences added. In one specific example, the peptide linker (3) may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 11.

[0066] The peptide linker (4) may consist of 1 to 30 consecutive amino acids, or 2 to 20 consecutive amino acids, or 2 to 10 amino acids. As a specific example, the peptide linker (4) may be (G4S)n (where n is an integer from 1 to 10). At this time, in (G4S)n, n may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. As an example, the peptide linker (4) may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 12.

[0067] The "Fc region fragment or its variant" used in this example is as described above.

[0068] Fusion protein dimer Another aspect of the present invention provides a dimer in which two fusion proteins are bound, one of which is an antibody or an antigen-binding fragment thereof that specifically binds to CD73, and the other is IL-2 or a variant thereof. The antibody or antigen-binding fragment thereof that specifically binds to CD73, and IL-2 are as described above. At this time, the binding between the fusion proteins constituting the dimer may be a disulfide bond via a cysteine present in the linker, but is not limited thereto. The fusion proteins constituting the dimer may be a homodimer composed of the same ones.

[0069] Specifically, in one embodiment, the dimer may be a dimer in which two fusion proteins, each containing a single-domain antibody that specifically binds to CD73, an Fc region, and IL-2 or a variant thereof, are disulfide-bonded by cysteine. Also, in one embodiment, the dimer may be a dimer in which two fusion proteins are disulfide-bonded by cysteine, one of which contains a polypeptide containing a variable heavy chain region (VH), a constant heavy chain region 1 (CH1), an Fc region, and IL-2 or a variant thereof of an anti-CD73 antibody, and the other contains a polypeptide containing a variable light chain region (VL) and a constant light chain region (CL) of the anti-CD73 antibody.

[0070] Polynucleotide encoding a fusion protein Still another aspect of the present invention provides a polynucleotide encoding a fusion protein containing an antibody or an antigen-binding fragment thereof that specifically binds to CD73, and IL-2 or a variant thereof. At this time, the antibody or antigen-binding fragment thereof that specifically binds to CD73, and IL-2 or a variant thereof are as described above.

[0071] Specifically, the polynucleotide encoding a fusion protein containing a single-domain antibody that specifically binds to CD73, an Fc region, and IL-2 or a variant thereof may contain any one of the base sequences selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 33, and SEQ ID NO: 35.

[0072] In addition, a fusion protein comprising the heavy chain variable region (VH), heavy chain constant region 1 (CH1), Fc region of the anti-CD73 antibody, and a polypeptide comprising IL-2 or a variant thereof, and a polypeptide comprising the light chain variable region (VL) and light chain constant region (CL) of the anti-CD73 antibody can be encoded by the nucleotide sequences of SEQ ID NO: 37 and SEQ ID NO: 39, respectively.

[0073] Furthermore, if the polynucleotide encodes the same polypeptide, one or more bases are mutated by substitution, deletion, insertion, or a combination thereof. When chemically synthesizing and manufacturing a polynucleotide sequence, synthetic methods widely known in the art can be used, such as the methods described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988), including triester, phosphite, phosphoramidite, and H-phosphate methods, PCR and other autoligation methods, oligonucleotide synthesis methods on solid supports, and the like.

[0074] According to one specific example, the polynucleotide can include a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to the nucleotide sequences of SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 39, respectively.

[0075] The polynucleotide can additionally include a signal sequence or a leader sequence. As used herein, the term "signal sequence" means a nucleic acid encoding a signal peptide that directs the secretion of the target protein. The signal peptide is cleaved after being translated in the host cell. Specifically, the signal sequence of the present invention is a polynucleotide encoding an amino acid sequence that initiates the movement of a protein that penetrates the endoplasmic reticulum (ER) membrane.

[0076] Signal sequences are well-characterized in the art and typically contain 16 to 30 amino acid residues, although they can contain more or fewer amino acid residues. A typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region contains 4 to 12 hydrophobic residues that anchor the signal sequence through the membrane lipid bilayer during the movement of the immature polypeptide.

[0077] After initiation, the signal sequence is cleaved within the lumen of the ER by cellular enzymes commonly known as signal peptidases. At this time, the signal sequence may be the signal sequence of tPa (tissue Plasminogen Activation), HSV gDs (signal sequence of Herpes simplex virus glycoprotein D), IgG signal sequence, or growth hormone. Preferably, a secretory signal sequence used in higher eukaryotic cells including mammals can be used. Signal sequences useful in the present invention include the light chain signal sequence of an antibody, such as the antibody 14.18 (Gillies et al., J. Immunol. Meth 1989. 125: 191 - 202), the heavy chain signal sequence of an antibody, such as the heavy chain signal sequence of the MOPC141 antibody (Sakano et al., Nature, 1980. 286: 676 - 683), and other signal sequences known in the art (see, e.g., Watson et al., Nucleic Acid Research, 1984. 12: 5145 - 5164). As a specific example, the signal sequence can include the amino acid sequence of SEQ ID NO: 1.

[0078] A vector loaded with a polynucleotide Still another aspect of the present invention provides an expression vector loaded with a polynucleotide encoding a fusion protein comprising an antibody or an antigen - binding fragment thereof that specifically binds to the CD73; and IL - 2 or a variant thereof. At this time, the antibody or an antigen - binding fragment thereof that specifically binds to the CD73, the antigen - binding site, IL - 2, and its variants are as described above.

[0079] Specifically, the polynucleotide encoding the fusion protein comprising the anti - CD73 single - domain antibody and IL - 2 or a variant thereof can include any one of the base sequences selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO: 34.

[0080] In addition, the polynucleotide encoding the fusion protein comprising the anti-CD73 antibody and IL-2 or its variant can include the nucleotide sequence of SEQ ID NO: 37 encoding the heavy chain region of the anti-CD73 antibody and the nucleotide sequence of SEQ ID NO: 39 encoding the light chain region. Further, a specific example including a signal sequence in each nucleotide sequence may be the nucleotide sequence of SEQ ID NO: 36 encoding the heavy chain region and the nucleotide sequence of SEQ ID NO: 38 encoding the light chain region. At this time, the polynucleotide may be loaded onto each expression vector or may be loaded as one onto a bicistronic expression vector.

[0081] As used herein, the term "vector" is introduced into a host cell and recombined and inserted into the host cell genome. Alternatively, the vector is understood as a nucleic acid means containing a nucleotide sequence that can replicate spontaneously as an episome. The vector includes linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, mini-chromosomes and the like. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses and adeno-associated viruses.

[0082] Specifically, the vector may be plasmid DNA, phage DNA, etc., and may be a commercially developed plasmid (such as pUC18, pBAD, pIDTSAMRT-AMP, etc.), an E. coli-derived plasmid (such as pYG601BR322, pBR325, pUC118, pUC119, etc.), a Bacillus subtilis-derived plasmid (such as pUB110, pTP5, etc.), a yeast-derived plasmid (such as YEp13, YEp24, YCp50, etc.), phage DNA (such as Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), an animal virus vector (such as retrovirus, adenovirus, vaccinia virus, etc.), an insect virus vector (such as baculovirus, etc.). Since the expression level and modification of proteins vary depending on the host cell, it is preferable to select and use the host cell most suitable for the purpose. In addition, the plasmid may contain a selection marker such as an antibiotic resistance gene, and the host cell maintaining the plasmid can be cultured under selective conditions.

[0083] As used herein, the term "gene expression" or "expression" of the target protein is understood to mean the transcription of a DNA sequence, the translation of an mRNA transcript, and the secretion of a fusion protein product or a fragment thereof. A useful expression vector may be RcCMV (Invitrogen, Carlsbad) or a variant thereof. The expression vector may contain a human cytomegalovirus promoter for promoting continuous transcription of the target gene in mammalian cells and a bovine growth hormone polyadenylation signal sequence for enhancing the steady-state level of post-transcriptional RNA.

[0084] transformed cell Still another aspect of the present invention provides a transformed cell into which an expression vector loaded with a polynucleotide encoding a fusion protein comprising an antibody or an antigen-binding fragment thereof that specifically binds to the CD73 and IL-2 or a variant thereof has been introduced. At this time, the expression vector loaded with the polynucleotide is as described above.

[0085] As used herein, the term "transformed cell" refers to prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. The transformed cell can be prepared by introducing and transforming a vector into a host cell. Further, the polynucleotide contained in the vector can be expressed to produce the fusion protein of the present invention.

[0086] The transformation is carried out by various methods and is not particularly limited as long as the fusion protein of the present invention can be produced. Specifically, the transformation methods include the CaCl2 precipitation method, the Hanahan method in which the efficiency is enhanced by using a reducing substance DMSO (dimethyl sulfoxide) in the CaCl2 precipitation method, the electroporation method, the calcium phosphate precipitation method, the protoplast fusion method, the stirring method using silicon carbide fibers, the Agrobacterium-mediated transformation method, the transformation method using PEG, the dextran sulfate, lipofectamine, and the drying / suppression-mediated transformation method, etc. can be used. Further, the target substance can be transmitted into the cell using virus particles by means of infection. Also, the vector can be introduced into the host cell by gene bombardment or the like.

[0087] In addition, the host cell used for producing the transformed cell is not particularly limited as long as it can produce the antibody of the present invention. Specifically, the host cell can include, but is not limited to, prokaryotic cells, eukaryotic cells, mammals, plants, insects, fungi, or cells of cellular origin. As an example of the prokaryotic cell, Escherichia coli can be used. Further, as an example of the eukaryotic cell, yeast can be used. As the mammalian cell, CHO cells, F2N cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, SP2 / 0 cells, human lymphoblastoid, NSO cells, HT-1080 cells, PERC.6 cells, HEK293 cells, or HEK293T cells, etc. can be used, but not limited thereto, and all cells that can be used as mammalian host cells known to those skilled in the art are available.

[0088] Also, for optimizing the properties of the antibody as a therapeutic agent or for other purposes, glycosylation-related genes of the host cell can be manipulated by methods known to those skilled in the art to adjust the sugar chain pattern of the antibody (e.g., sialic acid, fucosylation, glycosylation).

[0089] Method for producing a fusion protein dimer Still another aspect of the present invention provides a method for producing a fusion protein dimer comprising an antibody or an antigen-binding fragment thereof that specifically binds to the above CD73; and IL-2 or a variant thereof. At this time, the fusion protein and the fusion protein dimer are as described above.

[0090] Specifically, the method for producing the fusion protein dimer can include: i) culturing the transformed cell; and ii) obtaining the fusion protein dimer.

[0091] As used herein, the term "culturing" means a method of growing microorganisms under appropriately artificially controlled environmental conditions.

[0092] The method for culturing the transformed cells can be carried out using methods widely known in the art. Specifically, the culturing is not particularly limited as long as the antibody of the present invention can be expressed and produced. Specifically, the culturing can be continuously carried out in a batch process or an infusion batch or a repeated infusion batch process (fed batch or repeated fed batch process).

[0093] Also, the step of obtaining the antibody from the culture is carried out by methods known in the art. Specifically, the obtaining method is not particularly limited as long as the fusion protein of the present invention produced can be obtained. Preferably, the obtaining method may be methods such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (for example, ammonium sulfate precipitation), chromatography (for example, ion exchange, affinity, hydrophobicity and size exclusion).

[0094] Use of the fusion protein (dimer) Still another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises an antibody specifically binding to CD73 or an antigen-binding fragment thereof; and a fusion protein comprising IL-2 or a variant thereof, or a dimer of the fusion protein as an active ingredient. At this time, the fusion protein and the dimer of the fusion protein are as described above.

[0095] Specifically, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises a fusion protein comprising an anti-CD73 single-domain antibody and IL-2 or a variant thereof, or a dimer of the fusion protein as an active ingredient.

[0096] Also, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises a fusion protein comprising an anti-CD73 antibody and IL-2 or a variant thereof, or a dimer of the fusion protein as an active ingredient.

[0097] As used herein, the term "cancer" refers to a disease in which normal tissue cells proliferate without limit for some reason and continue to grow rapidly regardless of the living phenomena of the living body or the surrounding tissue state. The cancer in the present invention may be any one selected from the group consisting of various cancers of the human body, such as gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer and lymphoma, but is not limited thereto.

[0098] The preferred dosage of the pharmaceutical composition varies depending on the patient's condition and body weight, the degree of the disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. In the pharmaceutical composition for preventing or treating cancer of the present invention, the fusion protein can be contained in any amount (effective amount) depending on the use, dosage form, compounding purpose, etc., as long as it exhibits anti-cancer activity, particularly, as long as it can exhibit a therapeutic effect on cancer. However, the normal effective amount is determined within the range of 0.001% by weight to 20.0% by weight based on the total weight of the composition. Here, the "effective amount" refers to the amount of the active ingredient that can induce an improvement in the disease state or a therapeutic effect, particularly, an improvement in the cancer state or a therapeutic effect. Such an effective amount is determined experimentally within the normal ability range of those skilled in the art.

[0099] As used herein, the term "treatment" can be used in the sense of including all therapeutic and prophylactic treatments, and includes all applications and any form of medication for treating diseases in mammals including humans. Further, the term includes suppressing or delaying the progression of the disease; restoring or repairing damaged or defective functions to partially or completely relieve the disease; or stimulating an inefficient process; or relieving a serious disease. The "prevention" can be used in the sense of relieving or reducing the pathological state or disease of an individual.

[0100] Pharmacokinetic parameters such as bioavailability and underlying parameters such as clearance rate can also affect efficacy. Thus, "improved efficacy" (e.g., improvement in efficacy) can result from improved pharmacokinetic parameters and can be measured by comparing parameters such as clearance rate and treatment or improvement of cancer disease in test animals or human subjects.

[0101] On the other hand, the pharmaceutical composition of the present invention is administered in a "therapeutically effective amount".

[0102] As used herein, the term "administer" means introducing a predetermined substance into an individual by an appropriate method, and the administration route of the composition can be through any common route as long as the target tissue can be reached. It may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, topically, intranasally, intrapulmonary, rectally, but is not limited thereto.

[0103] As used herein, the term "therapeutically effective amount" or "pharmaceutically effective amount" means an amount of a compound or composition effective to prevent or treat a target disease, which is sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment and does not cause side effects. The level of the effective amount is determined by factors including the patient's health status, type of disease, severity, activity of the drug, sensitivity to the drug, administration method, administration time, administration route and excretion ratio, treatment period, ingredients including drugs formulated or used concomitantly, and other factors well known in the medical field. In one embodiment, the therapeutically effective amount means an amount of a drug effective to treat cancer.

[0104] At this time, the pharmaceutical composition may further contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be any carrier as long as it is a non-toxic substance suitable for transmission to a patient. Distilled water, alcohol, fats, waxes, and inert solids are included as carriers. Pharmaceutically acceptable adjuvants (buffers, dispersants) are also included in the pharmaceutical composition.

[0105] Specifically, the pharmaceutical composition can be manufactured into a parenteral dosage form by an administration route in a conventional method known in the art, including a pharmaceutically acceptable carrier. Here, the meaning of "pharmaceutically acceptable" means that it has no more toxicity than can be tolerated by the subject to which it is applied (formulated) without suppressing the activity of the active ingredient.

[0106] When the pharmaceutical composition is manufactured into a parenteral dosage form, together with a suitable carrier, it may be formulated into the form of an injection, a transdermal administration agent, a nasal inhalant, and a suppository by a method known in the art. When formulating into an injection, as a suitable carrier, sterile water, ethanol, polyols such as glycerol and propylene glycol, or a mixture thereof can be used. Preferably, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, isotonic solutions such as 5% dextrose can be used. It is known in the art regarding the formulation of pharmaceutical compositions. Specifically, reference can be made to documents such as [Remington’s Pharmaceutical Sciences (19th ed., 1995)]. The said document is regarded as a part of this specification.

[0107] The preferred dosage of the pharmaceutical composition may be in the range of 0.0001 μg / kg to 100 g / kg per day depending on the patient's condition, weight, gender, age, severity of the patient, and administration route. The administration is carried out once a day or divided into several times. Such a dosage should not be construed as limiting the scope of the present invention in any way.

[0108] The subjects to which the pharmaceutical composition can be applied (prescribed) are mammals and humans, and in particular, the case of humans is preferred. The pharmaceutical composition of the present invention can additionally contain any compound or natural extract known to have a cancer treatment effect.

[0109] Still another aspect of the present invention provides the use of an antibody specifically binding to the CD73 or an antigen-binding fragment thereof; and a fusion protein or a fusion protein dimer containing IL-2 or a variant thereof for manufacturing a medicament for preventing or treating cancer.

[0110] Still another aspect of the present invention provides a method for preventing or treating cancer, which includes the step of administering to an individual an antibody specifically binding to the CD73 or an antigen-binding fragment thereof; and a fusion protein or a fusion protein dimer containing IL-2 or a variant thereof. The individual may be a mammal, or preferably, a human. Also, the individual may be a patient suffering from cancer or an individual with a high likelihood of suffering from cancer.

[0111] The administration route, dosage, and frequency of administration of the fusion protein are administered to the subject in various ways and amounts depending on the condition of the patient and the presence or absence of side effects, and an ordinary technician can select the optimal administration method, dosage, and frequency of administration within an appropriate range. The preferred dosage of the antigen-binding site specifically binding to the CD73 and the fusion protein containing IL-2 or a variant thereof may be in the range of 0.0001 μg / kg to 100 g / kg per day depending on the condition, body weight, gender, age, severity of the patient, and administration route of the patient. Administration is carried out once a day or divided into several times a day. Such dosages should not be construed as limiting the scope of the present invention in any way.

[0112] Hereinafter, the present invention will be described in more detail through examples. These examples are merely for explaining the present invention more specifically, and it will be apparent to those with ordinary knowledge in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

Examples

[0113] I. Production of Fusion Protein and Its Dimer Example 1. Production of GI-108A1: Anti-CD73 sdAb-hIgG4 Fc-hIL2v3 To produce a fusion protein containing an anti-CD73 single-domain antibody (AHF10235, AHF10240, AHP04167) that specifically binds to CD73 and an IL-2 variant, a polynucleotide (SEQ ID NO: 30, 32, or 34) encoding a signal peptide (SEQ ID NO: 1), an anti-CD73 single-domain antibody (SEQ ID NO: 2, 3, or 4), linker (1) (SEQ ID NO: 5), an IgG4 Fc region (SEQ ID NO: 6), linker (2) (SEQ ID NO: 7), and an IL-2 variant with 3 amino acids substituted (SEQ ID NO: 8) was loaded into the pCGS3 vector (Sigma-Aldrich®) using the BioxpTM 3250 SYSTEM (Tables 1-3).

[0114] [Table 1] [Table 2] [Table 3]

[0115] Also, the vector was introduced into CHO cells (Expi-CHO (registered trademark), Thermo Fisher Scientific) to express the fusion protein. After introducing the vector, the cells were cultured under the conditions of 37°C, 125 rpm, and 8% CO2. Then, the culture broth was collected and the fusion protein dimer was purified. The three purified fusion protein dimers were named "GI-108A1 (AHF10235)", "GI-108A1 (AHF10240)", and "GI-108A1 (AHP04167)", respectively. Specifically, the three fusion protein dimers were purified using chromatography containing Protein A resin. After filtering the collected culture broth, the filtered culture broth was passed through a column and bound. Then, the fusion protein dimer was recovered with 50 mM glycine, pH 3.4.

[0116] The buffer containing the three recovered fusion protein dimers (GI-108A1 (AHF10235), GI-108A1 (AHF10240), GI-108A1 (AHP04167)) was changed to PBS (phosphate buffered saline, pH 7.4) using dialysis. Using a NanoDrop device (Thermo Fisher Scientific), it was confirmed that the fusion protein dimers were contained at concentrations of 3.86 mg / mL, 4.12 mg / mL, and 12.86 mg / mL, respectively. Also, as a result of confirming the purity by size exclusion chromatography (SEC) analysis, it was confirmed that the purities of the three separated and purified fusion protein dimers were 71.81%, 75.36%, and 93.87%, respectively (Tables 4 and 5).

[0117]

Table 4

Table 5

Example

[0118] Example 2. Production of GI-108B1: Anti-CD73 Ab-hIgG4 Fc-hIL2v3 To produce a fusion protein dimer containing an anti-CD73 antibody that specifically binds to CD73 and an IL-2 variant, a polynucleotide (SEQ ID NO: 36) encoding a signal peptide (SEQ ID NO: 1), a variable region (SEQ ID NO: 9) and a constant region (SEQ ID NO: 10) of the heavy chain of the anti-CD73 antibody, a linker (1) (SEQ ID NO: 11), an IgG4 Fc region (SEQ ID NO: 6), a linker (2) (SEQ ID NO: 12) and an IL-2 variant with 3 amino acids substituted (SEQ ID NO: 8), and a polynucleotide (SEQ ID NO: 38) encoding a signal peptide (SEQ ID NO: 1), a variable region (SEQ ID NO: 13) and a constant region (SEQ ID NO: 14) of the light chain of the anti-CD73 antibody were loaded into a pCGS3 vector (Sigma-Aldrich®) using a BioxpTM 3250 SYSTEM (Table 6).

[0119] Also, the above vector was introduced into CHO cells (Expi-CHO®, Thermo Fisher Scientific) to express the fusion protein dimer. After introducing the vector, the cells were cultured at 37°C, 125 rpm, and 8% CO2. Then, the culture broth was collected and the fusion protein dimer was purified. The purified fusion protein dimer was named "GI-108B1".

[0120] [Table 6]

[0121] Specifically, the above GI-108B1 was purified using chromatography containing Protein A resin. After filtering the collected culture broth, the filtered culture broth was passed through a column for binding. Then, the fusion protein dimer was recovered with 50 mM glycine, pH 3.4.

[0122] The buffer containing the recovered fusion protein dimer was changed to PBS (pH 7.4) using dialysis, and it was confirmed using a NanoDrop device (Thermo Fisher Scientific) that the fusion protein dimer was contained at a concentration of 4.55 mg / mL. Also, as a result of confirming the purity by size exclusion chromatography (SEC) analysis, it was confirmed that the purity of the isolated and purified fusion protein dimer was 83.63% (Tables 7 and 8).

[0123]

Table 7

Table 8

Example

[0124] Example 3. Production of anti-CD73 antibody: GI-αCD73 To produce an anti-CD73 antibody, a polynucleotide (SEQ ID NO: 42) encoding a signal peptide (SEQ ID NO: 1), the variable region (SEQ ID NO: 9) and constant region (SEQ ID NO: 10) of the heavy chain of the anti-CD73 antibody, a linker (1) (SEQ ID NO: 11), and an IgG4 Fc region (SEQ ID NO: 6), and a polynucleotide (SEQ ID NO: 38) encoding a signal peptide (SEQ ID NO: 1), the variable region (SEQ ID NO: 13) and constant region (SEQ ID NO: 14) of the light chain of the anti-CD73 antibody were loaded into a pCGS3 vector (Sigma-Aldrich (registered trademark)) using a BioXPTM 3250 SYSTEM.

[0125] Also, the vector was introduced into CHO cells (Expi-CHOTM, Thermo Fisher Scientific) to express a control antibody anti-CD73 antibody. After introducing the vector, the cells were cultured under conditions of 37°C, 125 rpm, and 8% CO2, and then the culture solution was collected and the control antibody anti-CD73 antibody was purified. The purification was performed in the same manner as in Example 1.

Example

[0126] Example 4. Production of IgG4 Fc-IL-2v A polynucleotide (SEQ ID NO: 43) containing a signal peptide (SEQ ID NO: 1), linker (1) (SEQ ID NO: 11), IgG4 Fc region (SEQ ID NO: 6), linker (2) (SEQ ID NO: 12), and an IL-2 variant with three amino acids substituted (SEQ ID NO: 8) was loaded into a pCGS3 vector (Sigma-Aldrich®) using a BioXPTM 3250 SYSTEM.

[0127] Also, the vector was introduced into CHO cells (Expi-CHOTM, Thermo Fisher Scientific) to express the fusion protein. After introducing the vector, the cells were cultured for 7 days at 37 °C, 127 rpm, 5% CO2, and 80% humidity. Then, the culture broth was collected and the IgG4 Fc-IL-2v fusion protein was purified.

Example

[0128] II. Confirmation of characteristics of fusion protein dimer Example 5. Measurement of binding affinity of each section of GI-108 to target proteins To quantitatively analyze the binding affinities of anti-CD73 antibodies that specifically bind to CD73 and IL-2 variants conjugated to GI-108A1 (AHF04167), GI-108A1 (AHP10240), and GI-108B1, and Aldesleukin (trade name: Proleukin®, Novartis) to CD73 and IL-2 receptors, the binding affinities were measured using SPR (Surface plasmon resonance). Using a Biacore® T200 instrument (Cytiva), the binding affinities of GI-108A1 (AHF10240), GI-108A1 (AHP04167), and GI-108B1 to hCD73, cyCD73, mCD73, hIL-2Rα, hIL-2Rβ, hIL-2Rβγ were analyzed. Oleclumab (Astrazeneca), ordered and prepared from Ybiologics, was used as a control group.

[0129] Specifically, for the binding force analysis of hCD73, cyCD73, and mCD73, the GI-108A1 (AHF10240), GI-108A1 (AHP04167), GI-108B1, and Oleclumab were each diluted with HBS-EP+ buffer (Cytiva, USA) and reversibly immobilized on a Sensor Chip CM5 (Cytiva, USA) treated with a human antibody capture kit (Cytiva, USA). CD73 was diluted with HBS-EP+ buffer and then analyzed for binding and dissociation at a flow rate of 30 μL / min for 5 minutes each.

[0130] For the binding force analysis with hIL-2Rα, after reversibly treating hIL-2Rα on a Sensor Chip CM5 treated with a His capture kit (Cytiva, USA), GI-108A1 (AHF10240), GI-108A1 (AHP04167), GI-108B1, and Proleukin® diluted with HBS-EP+ buffer were each treated for 1 minute to confirm binding and dissociation. Also, the binding forces with hIL-2Rβ and hIL-2Rβγ were measured by a three-step analysis method in which biotinylated CD73 was treated on a Sensor Chip SA (Cytiva, USA), then GI-108A1 (AHF10240), GI-108A1 (AHP04167), GI-108B1, and Proleukin® were bound onto CD73, and then hIL-2Rβ and hIL-2Rβγ diluted with HBS-P+ buffer (Cytiva, USA) were treated thereon. For hIL-2Rβ, binding was performed for 1 minute and dissociation for 1 minute. For hIL-2Rβγ, single cycle kinetics analysis was performed, with binding for 5 minutes for each concentration and dissociation for 2 hours. Each sensorgram was normalized and subtracted compared to a blank cell to calculate the affinity.

[0131] The measurement results of the binding affinity of the above GI-108A1 (AHF10240), GI-108A1 (AHP04167), and GI-108B1 to CD73 and the IL-2 receptor using a Biacore (registered trademark) T200 instrument (Cytiva) are shown in Table 9 below.

[0132]

Table 9

Example

[0133] Example 6. Confirmation of the ability of the IL-2 variant of GI-108 to activate the JAK-STAT pathway This experiment is to confirm the activity of the IL-2 variant site of GI-108. Specifically, the experiment was carried out using HEK-Blue (registered trademark) IL-2 reporter cells (InvivoGen Inc.) that express IL-2Rβγ. HEK-Blue (registered trademark) IL-2 reporter cells induce the production of the reporter protein SEAP (secreted embryonic alkaline phosphatase) when the JAK-STAT pathway is activated by IL-2. In this experiment, the activation ability by IL-2 was confirmed by detecting the SEAP protein.

[0134] HEK-Blue (registered trademark) IL-2 reporter cells were cultured in DMEM medium (Gibco (registered trademark)) containing 10% FBS (Gibco (registered trademark)), 100 U / mL penicillin (Welgene Inc.), 100 μg / mL streptomycin (Welgene Inc.), and 100 μg / mL Normocin (registered trademark) (cat. ant-nr-1, InvivoGen Inc.). After subculturing and stabilizing HEK-Blue (registered trademark) IL-2 reporter cells, they were harvested using trypsin (Gibco (registered trademark)). Then, they were washed with PBS to remove dead cells. The separated cells were about 2.8×10 5A cell suspension was prepared to be at the cell / mL level.

[0135] GI-108A1 (AHF10240), GI-108A1 (AHP04167), GI-108B1 and the control group Proleukin® were diluted with PBS and dispensed into a 96-well plate (cat. 30096, SPL) at 20 μL per well. The cell suspension prepared for this treated 96-well plate was added at 180 μL per well to reach approximately 3×10 4 cells per well, and then cultured in an incubator at 37 °C and 5% CO2 for 24 hours.

[0136] After 24 hours, the 96-well plate was taken out of the incubator and centrifuged at 300Xg for 5 minutes, and 20 μL of the supernatant was transferred to a new 96-well plate. To each well containing the supernatant, 180 μL of QUANTI-Blue® solution (cat. Rep-qbs, InvivoGen Inc.) dissolved at room temperature was dispensed, and then reacted in an incubator at 37 °C and 5% CO2 for 30 minutes. After the reaction, the absorbance at a wavelength of 630 nm was measured using a spectrophotometer (VersaMax® Absorbance Microplate Reader). The data was analyzed using Graphpad prism 8.0 software.

[0137] As a result, the absorbance increased in a concentration-dependent manner with GI-108A1 (AHF10240), GI-108A1 (AHP04167), GI-108B1 and Proleukin®. Thereby, it was confirmed that the JAK-STAT signaling pathway was activated by the IL-2 mutant sites of the two types of GI-108A1 and GI-108B1 (Figure 3). The EC 50 values of GI-108A1 (AHF10240) and GI-108A1 (AHP04167) were confirmed to be 2.63 pM and 3.94 pM respectively, and the EC 50 value of GI-108B1 was confirmed to be 1.28 pM. The EC 50The value was confirmed to be 10.3 pM.

Example

[0138] Example 7. Confirmation of the ability of IL-2 variants of GI-108 to induce cell proliferation The biological activities of the IL-2 variants of GI-108A1 and GI-108B were confirmed by a proliferation experiment using CTLL-2 cells expressing hIL-2Rαβγ. CTLL-2 cells were cultured in complete RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 0.2 mM L-glutamine, T-STIM® culture supplement (containing ConA (Concanavalin-A)), 0.2 mM sodium pyruvate, and 10% FBS.

[0139] GI-108A1 (AHF10240), GI-108A1 (AHP04167), GI-108B1, and Proleukin® as a control group were diluted with PBS and dispensed into a 96-well plate (cat. 30096, SPL) at 50 μL per well. CTLL-2 cells prepared in the 96-well plate treated with the drug were added at a cell number of 1.2×10 5 cells per well, and 10 μL of WST-1 was added to each well for culturing at 37 °C for 4 hours, after which the absorbance was measured.

[0140] The absorbance at wavelengths of 450 nm and 690 nm (reference) was measured using a spectrophotometer (VersaMax® Absorbance Microplate Reader). The CTLL-2 cell proliferation data were analyzed by subtracting the absorbance value at 690 nM from the absorbance value at 450 nM.

[0141] As a result, the absorbance increased in a concentration-dependent manner with GI-108A1 (AHF10240), GI-108A1 (AHP04167), GI-108B1, and Proleukin (registered trademark). Thus, it was confirmed that the growth of CTLL-2 cells was induced by the IL-2 mutant sites of the two types of GI-108A1 and GI-108B1 (Figure 4). The EC 50 values of GI-108A1 (AHF10240) and GI-108A1 (AHP04167) were confirmed to be 2.02 nM and 3.93 nM, respectively, and the EC 50 value of GI-108B1 was confirmed to be 1.70 nM. The EC 50 value of Proleukin (registered trademark), which is the positive control group, was confirmed to be 0.08 nM.

[0142] The reason why the EC 50 value of Proleukin (registered trademark) is lower than that of GI-108A1 and GI-108B1 is due to the fact that CTLL-2 cells expressed hIL-2Rαβγ, and since GI-108A1 and GI-108B1 do not bind to IL-2Rα, it was confirmed that the EC 50 value is more than 20 times higher than that of Proleukin (registered trademark).

Example

[0143] Example 8. Confirmation of the ability of GI-108 to inhibit membrane-bound CD73 enzyme activity 1×10 4 MDA-MB-231 (human breast cancer cells) were dispensed into a 96-well plate, and GI-108A1 (AHF10240), GI-108A1 (AHP04167), GI-108B1, and Oleclumab, which is the control group, were diluted from 300 μg / mL to a final concentration of 0.002 μg / mL, and 50 μL of each was added to the 96-well plate. Then, 100 μL of 200 μM AMP (Sigma-Aldrich (registered trademark)) was added to each well, and the mixture was cultured at 37°C for 6 hours.

[0144] The 96-well plate was centrifuged, and the supernatant was transferred to a new 96-well plate. Then, the adenosine concentration in the supernatant was measured using an Adenosine Assay kit (Cell Biolabs, Inc) according to the manufacturer's protocol. IC 50 values were calculated using GraphPad prism software.

[0145] As a result, the degree of luminescence decreased in a concentration-dependent manner by GI-108A1 (AHF10240), GI-108A1 (AHP04167), GI-108B1, and Oleclumab. Thereby, it was confirmed that the two types of GI-108A1 and GI-108B1 suppress the membrane-bound CD73 enzyme activity (Figure 5). The IC 50 values of GI-108A1 (AHF10240) and GI-108A1 (AHP04167) were confirmed to be 2.75 nM and 5.58 nM, respectively, and the IC 50 value of GI-108B1 was confirmed to be 3.37 nM. The IC 50 value of the control group Oleclumab was confirmed to be 1.47 nM. The IC 50 value of GI-108B1 was higher than the IC 50 values of GI-108A1 (AHF10240) and Oleclumab, but showed the maximum potential in the enzyme inhibition efficacy.

Example

[0146] Example 9. Confirmation of the ability of GI-108 to inhibit soluble CD73 enzyme activity To evaluate the soluble CD73 enzyme activity inhibitory ability of GI-108, the experiment was carried out according to the manufacturer's manual using the AMP-Glo® Assay kit (Promega, V5012). Specifically, GI-108A1 (AHF10240), GI-108A1 (AHP04167), GI-108B1 and the control group Oleclumab were diluted to 0.05 ng / mL, and 2.5 μL of each was treated per well in a 96-well plate. Then, 12.5 μL of 10 μM AMP was added to each well. Subsequently, 10 μL of 2.5 ng / mL recombinant human CD73 protein was added to each well and cultured at room temperature for 10 minutes.

[0147] 25 μL of AMP-Glo® Reagent (Promega) was added to each well to terminate the reaction, shaken for 2 minutes, and then cultured at room temperature for 1 hour. AMP Detection solution was added to each well, shaken for 2 minutes, and then cultured at room temperature for 1 hour. The luminescence degree was measured using the GloMax®-Multi Detection System (Promega), and the IC 50 value was calculated using GraphPad prism 8.0 software.

[0148] As a result, the luminescence degree decreased in a concentration-dependent manner with GI-108A1 (AHF10240), GI-108A1 (AHP04167) and GI-108B1. Thus, it was confirmed that the two types of GI-108A1 and GI-108B1 inhibited soluble CD73 enzyme activity (Figure 6). On the other hand, a hook effect appeared in which the higher the treatment concentration of Oleclumab, the lower the enzyme inhibition efficacy. The IC 50 values of GI-108A1 (AHF10240) and GI-108A1 (AHP04167) were confirmed to be 0.060 nM and 0.102 nM respectively, and the IC 50 values of GI-108B1 and Oleclumab were confirmed to be 0.126 nM and 0.03 nM respectively. The IC 50The value was higher than the IC values of the two types of GI-108A1 and Oleclumab, but showed the maximum potential in enzyme inhibitory efficacy. 50 The value was higher than the IC values of the two types of GI-108A1 and Oleclumab, but showed the maximum potential in enzyme inhibitory efficacy.

Example

[0149] Example 10. Confirmation of reinvigoration of T cell activity inhibited by CD73-adenosine of GI-108B1 To confirm whether GI-108B1 restores CD8+ T cell activity inhibited by CD73-adenosine, human PBMCs were cultured overnight at 37 °C and 5% CO2 in a humidified condition in TexMACS® medium (Miltenyi Biotec) containing 5% human serum (Sigma-Aldrich®), 1% penicillin / streptomycin and 5 mM β-mercaptoethanol (ThermoFisher Scientific). The next day, the cells were labeled with CTV and dispensed into a 96-well plate at 1×10 6 cells / ml. The cells were stimulated using αCD3 / CD28 dynabeads (ratio 1:1).

[0150] Vehicle (hIgG4), Proleukin®, GI-108B1, anti-CD73 antibody (GI-αCD73), IgG4 Fc-IL-2v and Oleclumab were treated alone, and anti-CD73 antibody (GI-αCD73) and IgG4 Fc-IL-2v, Oleclumab and IgG4 Fc-IL-2v were treated in combination. Each well was added with the final concentration of the treated drug to be 50 nM. Then, 500 μM of AMP was added to each well, and the cells were cultured for 3 days at 37 °C and 5% CO2 in a humidified condition.

[0151] To evaluate the activation of T cell function, cells were restimulated with αCD3 / CD28 dynabeads (ratio 1:1), 50 nM or 500 μM of AMP, in the presence of Brefeldin A, at 37 °C, 5% CO2 in humidified conditions for 4 hours. To analyze the proliferation of Teff cells by FACS, cell surface markers were stained using the following antibodies: anti-CD3-PerCP-Cy5.5 (Clone: UCHT1, BD Biosciences), anti-CD8-Alexa Fluor700 (Clone: RPA-T8, Biolegend Inc.) and Fixable Viability Dye eFluor780 (Invitrogen Inc.). To stain intracellular IFN-γ, cells were treated with fixation / permeabilization buffer and then stained with anti-IFNγ-APC (Clone: 4S.B3, Biolegend Inc.) antibody. Data for the stained cells were obtained using a Symphony A3 instrument and analyzed using FlowJo software.

[0152] As a result, when treated with AMP and Vehicle (hIgG4), the proliferation of CD8+ T cells decreased. In contrast, when treated with GI-108B1, the proliferation of CD8+ T cells increased significantly. It was also confirmed that the proliferation of CD8+ T cells increased when treated with anti-CD73 antibody alone or in combination with anti-CD73 antibody and IgG4 Fc-IL-2v. On the other hand, when treated with Proleukin (registered trademark), Oleclumab or IgG4 Fc-IL-2v alone, or when Oleclumab and IgG4 Fc-IL-2v were administered in combination, there was no tendency for the proliferation of CD8+ T cells to increase (Figure 7). Also, when treated with AMP and Vehicle (hIgG4), the production of IFN-γ in CD8+ T cells decreased. In contrast, when treated with GI-108B1, the production of IFN-γ in CD8+ T cells increased significantly (Figure 8).

[0153] Subsequently, when AMP was treated and Vehicle (hIgG4) was treated, the proliferation of CD4+ T cells decreased. In contrast, when treated with GI-108B1, the proliferation of CD4+ T cells increased significantly. Also, when the anti-CD73 antibody was treated alone or the anti-CD73 antibody and IgG4 Fc-IL-2v were treated in combination, the proliferation of CD4+ T cells increased. On the other hand, when Proleukin (registered trademark), Oleclumab or IgG4 Fc-IL-2v was treated alone, or Oleclumab and IgG4 Fc-IL-2v were administered in combination, there was no tendency for the proliferation of CD4+ T cells to increase (Figure 9). Also, when AMP was treated and Vehicle (hIgG4) was treated, the production of IFN-γ in CD4+ T cells decreased. In contrast, when treated with GI-108B1, it was confirmed that the production of IFN-γ in CD4+ T cells increased significantly (Figure 10). Thereby, it was confirmed that GI-108B1 restored the activities of CD8+ T cells and CD4+ T cells inhibited by CD73-adenosine.

Example

[0154] Example 11. Confirmation of the activity of GI-108B1 using pSTAT5 analysis To confirm the activity of GI-108B1, pSTAT5 in T cell populations (CD8+ T cells and Treg cells) within healthy human PBMCs was measured after treatment with Proleukin® alone, GI-108B1 alone, IgG4 Fc-IL-2v alone, and anti-CD73 antibody (GI-αCD73), or after combined treatment with anti-CD73 antibody (GI-αCD73) and IgG4 Fc-IL-2v. Specifically, human PBMCs were treated with Proleukin®, GI-108B1, IgG4 Fc-IL-2v, and anti-CD73 antibody alone, or with anti-CD73 antibody and IgG4 Fc-IL-2v at various concentrations, and cultured at 37°C for 20 minutes. For cell surface staining, cells were stained with the following antibodies: anti-CD3-BV510 (Clone SP34-2, BD Biosciences), anti-CD4-PE-Cy7 (Clone OKT-4, Biolegend Inc.), anti-CD8-AF700 (Clone RPA-T8, Biolegend Inc.), anti-CD25-BV421 (Clone M-A251, BD Biosciences), anti-CD56-BV605 (Clone NCAM16.2, BD Biosciences), anti-CD127-BV650 (Clone A019DS, Biolegend Inc.), and Fixable Viability Dye eFluor780 (Invitrogen Inc.).

[0155] For intracellular staining, cells were fixed with TFP Fix / Perm buffer (BD Biosciences) for 12 minutes and reacted with Perm buffer III (BD Biosciences) for 30 minutes. Then, cells were stained with anti-pSTAT5-AF647 (Clone 47 / Stat5, BD Biosciences). Data for the stained cells were measured using Cytek Aurora (Cytek Biosciences) and analyzed using FlowJo software (BD Biosciences).

[0156] As a result, the EC of GI-108B1 for pSTAT5 in CD8+ T cells50 It was confirmed that the value was significantly lower than that of the group treated with Proleukin (registered trademark), IgG4 Fc-IL-2v, or anti-CD73 antibody alone, or the group treated with the combination of anti-CD73 antibody and IgG4 Fc-IL-2v. Thus, it was confirmed that GI-108B1 showed an excellent effect on the activation of STAT5 in CD8+ T cells compared to Proleukin (registered trademark) (FIGS. 11 and 12).

[0157] Also, the EC of GI-108B1 for pSTAT5 in Treg cells 50 The value was slightly higher than that of the group treated with IgG4 Fc-IL-2v or anti-CD73 antibody alone, or the group treated with the combination of anti-CD73 antibody and IgG4 Fc-IL-2v, and was significantly higher than the EC of the group treated with Proleukin (registered trademark). Thus, it was confirmed that in Treg cells, GI-108B1 had a much less effect on the activation of STAT5 compared to Proleukin (registered trademark) (FIGS. 13 and 14). 50 50

Example

[0158] Example 12. Confirmation of the T cell proliferation ability of GI-108B1 Human PBMCs were suspended in 4Cell (registered trademark) Nutri-T GMP medium (Sartorius) and dispensed into 96-well plates at 5×10 5 cells per well. Then, in the presence of αCD3 / CD28 dynabeads (ratio 1:1, Thermo Fisher Scientific), GI-108B1 or Proleukin (registered trademark) at a final concentration of 10 nM was added. The medium containing each drug was changed every 2 - 3 days. The cell count was performed on days 3, 5, 7, 10, and 12 using an ADAM (registered trademark)-MC2 Cell Counter (Nanoentek). On day 12, 2×10 cells from each group 6The cells were stained with the following antibodies: anti-CD3-BV510 (Clone SP34-2, BD Biosciences), anti-CD4 (Clone OKT-4, Biolegend Inc.), anti-CD8-AF700 (Clone RPA-T8, Biolegend Inc.), anti-CD25-BV421 (Clone M-A251, BD Biosciences), anti-CD45-APC (Clone HI30, eBioscience) and Fixable Viability Dye eFluor780 (Invitrogen Inc.). For intracellular staining, the cells were treated with fixation / permeabilization buffer (BD Biosciences) and stained with anti-Foxp3-PE (Clone pCH101, Invitrogen Inc.) antibody. Data on the stained cells were measured using a Cytek Aurora device and analyzed using FlowJo software.

[0159] As a result, the number of CD8+ T cells in the group treated with GI-108B1 was higher than that in the group treated with Proleukin®, and the number of Treg cells was approximately twice as large in the group treated with Proleukin® as in the group treated with GI-108B1. As a result of calculating the ratio of the number of CD8+ T cells to the number of Treg cells (CD8+T / Treg), it was confirmed that the group treated with GI-108B1 was approximately 2.46 times higher than the group treated with Proleukin®. Thereby, it was confirmed that GI-108B1 showed a superior effect in the proliferation of CD8+ T cells compared to the proliferation of Treg cells compared to Proleukin® (Figure 15).

Example

[0160] Example 13. cis-Binding Analysis of GI-108B1 in CD8+ T Cells Since CD8+ T cells express both CD73 and the IL-2 receptor complex, signal transduction by the binding of CD73 and IL-2βγ to GI-108B1 in the same CD8+ T cells can show a synergistic effect on the activation of CD8+ T cells. Therefore, to confirm whether GI-108B1 makes a cis-binding with CD73 and IL-2βγ in the same CD8+ T cells, cis-binding analysis was performed using CTV-CD8+ T cells (anti-CD73 antibody-treated / untreated groups) and CTV+ CD8+ T cells (Figure 16). Specifically, CD8+ T cells were isolated from human PBMCs using the EasySep® Human CD8+ T Cell Isolation Kit (STEMCELL technologies). Half of the isolated CD8+ T cells were labeled with 0.5 μM CTV (Cell Trace® Violet, Thermo Fisher Scientific). The unlabeled cells were divided into two groups again, and only one group was treated with a saturating concentration (1,000 nM) of anti-CD73 antibody and pretreated at 4°C for 2 hours to block all CD73 on the cell surface. Then, the unbound anti-CD73 antibody was washed away.

[0161] Thereafter, the anti-CD73 antibody-pretreated or untreated CTV-unlabeled cells were co-cultured with the CTV-labeled cells at a ratio of 1:1 each. The co-cultured cells were treated with 0.5 nM of GI-108B1 and reacted at 37°C for 20 minutes. Then, the cells were treated with Cytofix fixation buffer (BD Biosciences) and fixed for 12 minutes, and then treated with Phosflow Perm buffer III (BD Biosciences) and reacted for 30 minutes. Then, the cells were stained with anti-pSTAT5-AF647 (Clone47 / Stat5, BD Biosciences). The data for the stained cells were measured using Cytek Aurora and analyzed using FlowJo software.

[0162] As a result, in the case of CTV+CD8+T cells co-cultured with untreated CTV-CD8+T cells, the CTV-CD8+T cells and CTV+CD8+T cells showed similar levels of STAT5 phosphorylation. In contrast, in the case of CTV+CD8+T cells co-cultured with CTV-CD8+T cells pretreated with anti-CD73 antibody, the STAT5 phosphorylation level of CTV-CD8+T cells decreased by 40-60%, and the STAT5 phosphorylation level in CTV+CD8+T cells was maintained the same (Figure 17). From the fact that approximately 70% of hCD73+CD8+T cells expressing hCD73 in CD8+T cells purified from PBMC (Figure 18), and through the above results, it was confirmed that GI-108B1 preferentially cis-binds to CD8+T cells expressing CD73.

Example

[0163] III. Confirmation of the anti-cancer effect of the fusion protein dimer Example 14. Confirmation of the immune cell-mediated tumor killing ability of GI-108B1 To confirm the immune cell-mediated tumor killing ability of GI-108B1, the anti-cancer effect of GI-108B1 was confirmed using a co-culture system with MDA-MB-231 cells and healthy human PBMCs in the presence of anti-CD3 / CD28 dynabeads and / or AMP. Specifically, the cytotoxicity of PBMCs against MDA-MB-231 wild-type cells was evaluated using Annexin V / 7-AAD analysis based on flow cytometry analysis. pBMCs were purchased from Zen-bio Inc. or STEMCELL Technologies.

[0164] PBMCs were treated with hIgG4 (5 nM), a combination of anti-CD73 antibody (GI-αCD73, 5 nM) and IgG4 Fc-IL-2v (5 nM), or GI-108B1 (5 nM) alone, and together with αCD3 / CD28 dynabeads (ratio 1:1) or 500 μM of AMP, and cultured for 2 days before co-culture with target cells (MDA-MB-231 wild-type cells). The target cells were labeled with CTV, and 1×10 5They were dispensed into a 24-well plate to achieve the desired cell count. After overnight culture, pre-stimulated PBMCs were added to the target cells at a ratio of effector cells to target cells of 10:1, and co-cultured for 24 hours at 37°C and 5% CO2. The cells were stained with Annexin V and 7-AAD (Biolegend Inc.), and the death rate of CTV-labeled target cells was measured using a Symphony A3 instrument and analyzed using FlowJo software.

[0165] As a result, it was confirmed that the cell death rate was higher in the group co-culturing PBMCs treated with only anti-CD3 / CD28 dynabeads and hIgG4 with target cells (isotype control group (-AMP)) than in the group co-culturing PBMCs treated with anti-CD3 / CD28 dynabeads, AMP, and hIgG4 with target cells (isotype control group (+AMP)). This confirmed that treating with AMP inhibited the activity (cell killing ability) of immune cells under conditions rich in adenosine.

[0166] On the other hand, despite treatment with AMP, both the group co-culturing PBMCs treated with anti-CD3 / CD28 dynabeads, AMP, and GI-108B1 with target cells and the group co-culturing PBMCs treated with a combination of anti-CD3 / CD28 dynabeads, AMP, anti-CD73 antibody (GI-αCD73), and IgG4 Fc-IL-2v with target cells showed a higher cell death rate than the isotype control group (+AMP). In particular, the group co-culturing PBMCs treated with anti-CD3 / CD28 dynabeads, AMP, and GI-108B1 with target cells showed a significantly higher cell death rate of target cells and the highest cell death rate compared to the group co-culturing PBMCs treated with a combination of anti-CD3 / CD28 dynabeads, AMP, anti-CD73 antibody (GI-αCD73), and IgG4 Fc-IL-2v with target cells (Figure 19).

Example

[0167] Example 15. Confirmation of the anti-cancer effect by administration of GI-108B1 in mice implanted with mouse-derived colorectal cancer cells After administering the test substance GI-108B1 to a tumor model transplanted with the MC38 cell line (MC38-hCD73), which is a murine colon cancer cell line (murine colon cancer cell) in which the mouse CD73 gene has been replaced with the human CD73 gene, the tumor growth inhibitory effect was evaluated. First, in order to prepare a mouse model transplanted with the cancer cell line, a suspension of MC38-hCD73 cells (SMOC, China) was injected subcutaneously into the right dorsal region of C57BL / 6J-hCD73 female mice (6-8 weeks old, SMOC, China) at a dose of 1×10 6 cells / 100 μL each.

[0168] After transplanting the MC38-hCD73 cells, the morbidity and mortality were confirmed daily, and for mice without any abnormalities in their health condition, the tumor volume was measured. When the average tumor size reached 90-120 mm 3 at that time, 16 mice were randomly selected. The individual selection was made considering the physiological state of the individuals, body weight changes, and tumor growth rate. The selected animals were separated into groups of 8 each as evenly as possible based on the tumor volume and body weight. The test groups were constituted as shown in Table 10 below, and the test substance was administered.

[0169]

Table 10

[0170] During the test period, changes in tumors, body weight, and food intake were regularly observed, and body weight changes and external changes (hair / eyelid ruffles) were confirmed twice a week. Tumor volume measurement was performed twice a week during the observation period using a digital caliper (Mitutoyo, Japan) to measure the major axis (maximum length, L) and minor axis (perpendicular width, W) of the tumor, and substituting them into the following formula I to measure the tumor volume (tumor volume, TV) and tumor growth inhibition rate (tumor growth inhibition, TGI). <Formula I> TV (mm 3 ) = (W 2 × L) / 2 TGI = (1 - (Ti - T0) / (Vi - V0)) × 100 Ti: Tumor volume of the test substance administration group at the end of the test T0: Tumor volume of the test substance administration group at the time of the first administration Vi: Tumor volume of the negative control group at the end of the test V0: Tumor volume of the negative control group at the time of the first administration

[0171] The tumor volume before administration of each individual was set to the value measured at the time of group separation, and the anti-tumor efficacy was evaluated by comparison with the control group (vehicle (PBS), G1). As a result, the tumor volume of the GI-108B1 administration group decreased compared to the control group (vehicle (PBS)) (Figure 20). Also, looking at the tumor growth inhibition rate, in the control group (vehicle (PBS)), there was 1 mouse with a tumor growth inhibition rate of 50% or more, and no mouse with a tumor growth inhibition rate of 80% or more. In contrast, in the GI-108B1 administration group, there were 5 mice with a tumor growth inhibition rate of 30% or more and 3 mice with a tumor growth inhibition rate of 50% or more (Figure 21). Furthermore, as a result of confirming the survival rate of the mice in each group, in the case of the control group (vehicle (PBS)), 2 mice died on the 25th day, but in the case of the GI-108B1 administration group, all survived on the 25th day (Figure 22).

Example

[0172] Example 16. Confirmation of the anti-cancer effect by administration of GI-108 in human-derived breast cancer cell-implanted mice In a tumor model in which MDA-MB-231 (human breast cancer cells) cells were transplanted into mice with a human immune system introduced, after administering the test substances GI-108A1 (AHP04167), GI-108B1, and the anti-PD-1 antibody Pembrolizumab (trade name: Keytruda, MSD), the tumor growth inhibitory effect was evaluated. First, to prepare a mouse model with a human immune system introduced, a suspension of human peripheral blood cells (Stemexpress, USA) was filled into a disposable syringe (31G, cat. 328820, BD, USA) at 1 × 10 7 cells / 200 μL per mouse and administered through the tail vein. After cell transplantation, the general symptoms were observed once a day.

[0173] NOG-B2m female mice were purchased from The Jackson Laboratory and maintained in individually ventilated cages placed in an animal biosafety level 3 facility at 19 - 25°C and 30 - 70% humidity.

[0174] The MDA-MB-231 human TNBC (triple-negative breast cancer) cell line was purchased from ATCC and cultured in DMEM medium (ThermoFisher Scientific) containing 10% FBS (ThermoFisher Scientific) and 1% antibiotic / antifungal agent (Gibco®). The cultured MDA-MB-231 cells were harvested using trypsin (Gibco®) and then suspended in PBS. To establish a xenograft mouse tumor model, on the 5th day after human peripheral blood cell transplantation, healthy mice were injected with an MDA-MB-231 cell suspension (5 × 10 6A solution prepared by mixing 0.025 mL of cells and 0.025 mL of Matrigel matrix phenol red-free (cat. 356237, BD, USA) was filled into a disposable syringe (31G, cat. 328820, BD, USA) and administered subcutaneously at a dose of 0.05 mL per animal at the right dorsal part of the animal for transplantation. After transplantation of MDA-MB-231 cells, general symptoms were observed once a day during the engraftment and growth period.

[0175] MDA-MB-231 cells were transplanted, and about 20 days later, the tumor volume was measured in mice without any abnormality in health status, and 32 mice were selected so that the average of each group reached 80 - 100 mm 3 Individual selection was carried out considering the physiological state of the individual (breathing, hair, behavior, tail, posture, body fluid, diet, morphological deformation, metabolism, etc.), weight change, FACS analysis results, and tumor growth rate. The selected animals were separated into groups of 8 animals each as evenly as possible based on tumor volume and weight. It was confirmed that the human CD45+ cells accounted for 80% or more of the total lymphocytes in the peripheral blood of the mice before group separation. The test groups were constituted as shown in Table 11, and the test substances were administered (Figure 23).

[0176]

Table 11

[0177] Once a day during the test period, general symptoms such as appearance, behavior, and excrement were observed and recorded for each individual, and dead animals were confirmed. Body weight measurement and tumor volume measurement were performed twice a week during the observation period. The long axis and short axis of the tumor were measured using calipers and substituted into Formula I of Example 15 to measure the tumor volume (TV) and tumor growth inhibition rate (TGI). The tumor volume before administration of each individual was set to the value measured at the time of group separation, and the antitumor efficacy was evaluated by comparison with the control group (vehicle, G1).

[0178] All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). The comparison of tumor volume measurements was performed by unpaired t-test. A p-value less than 0.05 was considered significant.

[0179] As a result, it was confirmed that tumor growth was inhibited in the GI-108A1 (AHP04167), GI-108B1, and anti-PD-1 antibody administration groups compared to the control group (vehicle). In particular, it was confirmed that tumor growth was significantly suppressed in the GI-108B1 administration group compared to the GI-108A1 (AHP04167) or anti-PD-1 antibody administration groups (Figures 24 and 25). In addition, as a result of measuring the tumor growth inhibition rate on the 18th day, there was only 1 mouse with a tumor growth inhibition rate of 30% or more in the control group, and no mice with 50% or more and 80% or more. There were 4 mice with a tumor growth inhibition rate of 30% or more, 2 mice with 50% or more in the anti-PD-1 antibody administration group, and no mice with 80% or more. In contrast, the GI-108A1 administration group had 5 mice with a tumor growth inhibition rate of 30% or more, 3 mice with 50% or more, and 1 mouse with 80% or more, and the GI-108B1 administration group had 5 mice with a tumor growth inhibition rate of 30% or more, 4 mice with 50% or more, and 3 mice with 80% or more (Figure 26).

[0180] On the other hand, there was no weight loss in either the control group or the test groups, and no significant difference was observed between the groups (Figure 27).

Example

[0181] Example 17. Vascular permeability analysis of GI-108B1 To analyze the vascular permeability of GI-108B1, 1×10 5 HUVEC cells (Lonza) were seeded into a trans-well coated with collagen using a trans-well insert (9321012, cellQART (registered trademark)) and cultured for 3 days to form a confluent monolayer. 1×10 5After aliquoting to obtain the desired number of samples, Proleukin (registered trademark) (5 nM) or GI-108B1 (5 nM) was added, and the cells were cultured at 37 °C and 5% CO2 for 24 hours.

[0182] The supernatant of the drug and PBMC culture was added to the HUVEC cells cultured in trans-well, and the cells were cultured at 37 °C and 5% CO2 for 24 hours. TNF-α (PeproTech) 100 ng / mL was used as a positive control. Then, Dextran-FITC (70 kDa) was added to the upper chamber and cultured for 1 hour and 30 minutes. The fluorescence of Dextran-FITC that had migrated to the lower chamber was measured at a wavelength of 485 nm / 500 - 550 nm (Ex / Em) using GloMax (registered trademark) Discover (promega). Statistical calculations were analyzed by one-way ANOVA using Prism 8.0 (Graph Pad Software Inc.) and performed with Dunnett's test based on the negative control group.

[0183] As a result, it was confirmed that the vascular permeability of the positive control group treated with TNF-α increased. In contrast, in the group treated with GI-108B1, it was confirmed that the vascular permeability was at a level similar to that of the untreated negative control group (Figure 28).

[0184] Although specific parts of the present invention have been described in detail above, such specific descriptions are merely preferred embodiments for those with ordinary knowledge in the art, and thus it is clear that the scope of the present invention is not limited. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An antibody or an antigen-binding fragment thereof that specifically binds to CD73; and a fusion protein comprising IL-2.

2. The fusion protein according to claim 1, wherein the fusion protein comprises the following structural formula (I): N'-X-[Linker(1)]o-Fc region fragment or a variant thereof-[Linker(2)]p-Y-C' (I) At this time, in the structural formula (I), The N' is the N-terminus, The C' is the C-terminus, The X is an antibody or an antigen-binding fragment thereof that specifically binds to CD73, The Y is IL-2, The Linker(1) and Linker(2) are peptide linkers, The o and p are each independently 0 or 1.

3. The fusion protein according to claim 2, wherein the antigen-binding fragment comprises a single-domain antibody.

4. The antigen-binding fragment A CDR1 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 18, and 21; a CDR2 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 19, and 22; a CDR3 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 20, and 23; The fusion protein according to claim 3, which comprises the above.

5. The fusion protein according to claim 3, wherein the antigen-binding fragment comprises any one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, SEQ ID NOs: 3, and SEQ ID NOs:

4.

6. The fusion protein according to claim 2, wherein the Linker(1) comprises the amino acid sequence of SEQ ID NO:

5.

7. The fusion protein according to claim 2, wherein the Linker(2) comprises the amino acid sequence of SEQ ID NO:

7.

8. The fusion protein according to claim 1, wherein the fusion protein comprises the following structural formula (II) and (III): N'-X-[Linker(3)]q-Fc region fragment or a variant thereof-[Linker(4)]r-Y-C' (II) and N'-X'-C' (III) At this time, in the structural formula (II) and (III), The N' is the N-terminus, The C' is the C-terminus, The X is an antibody or an antigen-binding fragment thereof that specifically binds to CD73, which comprises a heavy chain variable region (VH) and a heavy chain constant region 1 (CH1), Said X' is an antibody or an antigen-binding fragment thereof that specifically binds to CD73, and comprises a light-chain variable region (VL) and a light-chain constant region (CL), said Y is IL-2, said linker (3) and linker (4) are peptide linkers, said q and r are each independently 0 or 1. **Claim 9** The antibody or the antigen-binding fragment thereof comprises a heavy-chain variable region comprising an HCDR1 containing the amino acid sequence of SEQ ID NO: 24, an HCDR2 containing the amino acid sequence of SEQ ID NO: 25, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 26; and a light-chain variable region comprising an LCDR1 containing the amino acid sequence of SEQ ID NO: 27, an LCDR2 containing the amino acid sequence of SEQ ID NO: 28, and an LCDR3 containing the amino acid sequence of SEQ ID NO: 29, the fusion protein according to claim 8. **Claim 10** The heavy-chain variable region comprises the amino acid sequence of SEQ ID NO: 9; The light-chain variable region comprises the amino acid sequence of SEQ ID NO: 13, the fusion protein according to claim 9. **Claim 11** Said linker (3) comprises the amino acid sequence of SEQ ID NO: 11, the fusion protein according to claim 8. **Claim 12** Said linker (4) comprises the amino acid sequence of SEQ ID NO: 12, the fusion protein according to claim 8. **Claim 13** Said Fc comprises the amino acid sequence of SEQ ID NO: 6, the fusion protein according to claim 2 or 8. **Claim 14** Said IL-2 is an IL-2 variant, the fusion protein according to claim 2 or 8. **Claim 15** Said IL-2 variant is one in which at least one of the amino acids at positions 38, 42, and 61 in the amino acid sequence of SEQ ID NO: 40 is substituted, the fusion protein according to claim 14. **Claim 16** Said IL-2 variant is one in which at least one substitution selected from the group consisting of R38A, F42A, and E61R in the amino acid sequence of SEQ ID NO: 40 is made, the fusion protein according to claim 15. **Claim 17** A fusion protein dimer in which two fusion proteins according to claim 1 are bound. **Claim 18** A polynucleotide encoding the fusion protein according to claim 1. **Claim 19** An expression vector loaded with the polynucleotide according to claim 18. **Claim 20** A transformed cell into which the expression vector according to claim 19 has been introduced. **Claim 21** i) culturing the transformed cell according to claim 20; and ii) obtaining a fusion protein dimer; A method for producing an antibody or an antigen-binding fragment thereof that specifically binds to CD73; and a fusion protein dimer comprising IL-2.

22. A pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, the fusion protein according to claim 1 or the fusion protein dimer according to claim 17.

23. The cancer is any one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma. The pharmaceutical composition for preventing or treating cancer according to claim 22.

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