Anti-CD73 antibody and its use

Abstract

The present invention relates to novel antibodies, antigen-binding fragments thereof, and uses of the antibodies and fragments, which antibodies and fragments comprise specific complementarity-determining regions (CDRs) and / or specifically bind to human CD73 at specific epitopes.

Description

Technical Field

[0001] The present invention relates to novel antibodies, antigen-binding fragments of antibodies, and uses of antibodies and fragments, which antibodies and fragments contain specific complementarity-determining regions (CDRs) and / or specifically bind to human CD73 at a specific epitope.

Background Art

[0002] The immunosuppressive effect in the tumor microenvironment is closely related to the progression of tumor growth. Tumor cells can change the tumor microenvironment and escape from immune surveillance. In recent studies, several tumor-induced immune escape mechanisms have been found, and co-inhibitory receptors expressed on the surface of T cells (also called immune checkpoints, such as CTLA-4, PD-1, etc.) have been targeted for immunotherapy and successfully developed as immune checkpoint inhibitors.

[0003] During inflammation, adenosine triphosphate (ATP) is released from inflammatory cells, and extracellular ATP attracts these immune cells through binding to purinergic receptors (such as P2X receptors and P2Y receptors) expressed on the surface of dendritic cells, macrophages or neutrophils, triggering an immune response. Furthermore, extracellular ATP can also act as a source of immunosuppressive adenosine through hydrolysis via CD39 and CD73. When inflammation subsides, the membrane protein CD39 converts ATP into adenosine diphosphate (ADP) or adenosine monophosphate (AMP), and the membrane protein CD73 converts AMP into adenosine. Adenosine can bind to receptors on immune cells (especially A2A and A2B receptors) and inhibit the immune response by promoting the synthesis of cAMP, which protects tissues from excessive inflammation. Beyond its role in regulating the immune response, the immunosuppressive effect of adenosine has also been recently reported as a significant factor promoting tumor growth. In the tumor microenvironment, the release of ATP, as well as the expression of CD39 and CD73, are increased by hypoxia-induced tumor cell necrosis, which causes the accumulation of adenosine. Furthermore, the hypoxic environment around the tumor also increases the expression of A2A and A2B receptors in immune cells. It has been demonstrated that CD73 can promote tumor growth, angiogenesis, and adenosine-induced immunosuppression, which are different from other known mechanisms (such as immune response suppression via immunoreceptor tyrosine-based inhibitory motif (ITIM)). High expression of CD73 and adenosine are both highly correlated with tumor progression and metastasis. Some adenosine signaling inhibitors targeting CD73 or adenosine A2A receptor are expected to have a synergistic effect when used in combination with other immunotherapeutic agents for cancer treatment. Therefore, for the purpose of cancer treatment, drugs or methods targeting CD73 are being actively developed to inhibit the production of adenosine and / or adenosine-induced immunosuppression.

[0004] Several types of anti-CD73 antibodies are being used in Phase 1 or Phase 2 clinical trials, either alone or in combination with other agents such as anti-PD-1, anti-PDL1, anti-CTLA4, EGFR inhibitors, and A2AR inhibitors (e.g., ipilimumab and nivolumab). For example, oleclumab (MEDI9447), a human anti-CD73 antibody, is currently being evaluated in clinical trials. Nevertheless, there remains a need and an urgency to develop novel anti-CD73 antibodies that can provide better inhibitory effects against CD73.

Summary of the Invention

Means for Solving the Problems

[0005] The inventors of the present invention have found novel antibodies and antigen-binding fragments thereof. The novel antibodies and antigen-binding fragments contain specific complementarity-determining regions (CDRs) and can specifically bind to human CD73 at specific epitopes (e.g., the 296th glutamine residue and the 297th arginine residue), thereby inhibiting CD73 activity and inhibiting the consumption of AMP. Furthermore, compared with a positive control (e.g., oleclumab), the antibodies and antigen-binding fragments of the present invention have a better and longer-lasting effective period of inhibiting AMP consumption and have better effects on activating immune cells and inhibiting tumor growth.

[0006] Accordingly, an object of the present invention is to provide an antibody or an antigen-binding fragment thereof comprising: (i) a heavy-chain variable domain comprising an HCDR1 region, an HCDR2 region, and an HCDR3 region, wherein the HCDR1 region comprises the amino acid sequence of SEQ ID NO: 4 having at most one mutation, the HCDR2 region comprises the amino acid sequence of SEQ ID NO: 5 having at most one mutation, and the HCDR3 region comprises the amino acid sequence of SEQ ID NO: 6 having at most one mutation; and (ii) a light-chain variable domain comprising an LCDR1 region, an LCDR2 region, and an LCDR3 region, wherein the LCDR1 region comprises the amino acid sequence of SEQ ID NO: 7 having at most one mutation, the LCDR2 region comprises the amino acid sequence of SEQ ID NO: 8 having at most one mutation, and the LCDR3 region comprises the amino acid sequence of SEQ ID NO: 9 having at most one mutation.

[0007] Preferably, in the antibody or an antigen-binding fragment thereof according to the present invention described above, the HCDR1 region comprises the amino acid sequence of SEQ ID NO: 4 having at most one mutation, the HCDR2 region comprises the amino acid sequence of SEQ ID NO: 5 having at most one mutation, the HCDR3 region comprises the amino acid sequence of SEQ ID NO: 6 having at most one mutation, the LCDR1 region comprises the amino acid sequence of SEQ ID NO: 7, the LCDR2 region comprises the amino acid sequence of SEQ ID NO: 8, and the LCDR3 region comprises the amino acid sequence of SEQ ID NO: 9 having at most one mutation.

[0008] In certain embodiments of the antibody or antigen-binding fragment thereof according to the present invention described above, the HCDR1 region comprises the amino acid sequence of SEQ ID NO: 4 having a mutation at the 8th position, the HCDR2 region comprises the amino acid sequence of SEQ ID NO: 5 having a mutation at the 1st position, the HCDR3 region comprises the amino acid sequence of SEQ ID NO: 6 having a mutation at the 1st position, the LCDR1 region comprises the amino acid sequence of SEQ ID NO: 7, the LCDR2 region comprises the amino acid sequence of SEQ ID NO: 8, and the LCDR3 region comprises the amino acid sequence of SEQ ID NO: 9 having a mutation at the 8th position. Preferably, the HCDR1 region comprises the amino acid sequence of SEQ ID NO: 4 in which the 8th tryptophan residue is substituted with threonine, the HCDR2 region comprises the amino acid sequence of SEQ ID NO: 5 in which the 1st glutamine residue is substituted with serine, arginine, threonine, or histidine, the HCDR3 region comprises the amino acid sequence of SEQ ID NO: 6 in which the 1st phenylalanine residue is substituted with leucine, tyrosine, or isoleucine, and the LCDR3 region comprises the amino acid sequence of SEQ ID NO: 9 in which the 8th leucine residue is substituted with methionine, glycine, histidine, arginine, glutamine, or isoleucine.

[0009] In certain embodiments of the antibody or antigen-binding fragment thereof according to the present invention described above, the HCDR1 region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 15, the HCDR2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NOs: 16-19, the HCDR3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6 (FAD), SEQ ID NO: 20 (LAD), SEQ ID NO: 21 (YAD), and SEQ ID NO: 22 (IAD). In certain embodiments of the antibody or antigen-binding fragment thereof according to the present invention described above, the LCDR1 region comprises the amino acid sequence of SEQ ID NO: 7, the LCDR2 region comprises the amino acid sequence of SEQ ID NO: 8, and the LCDR3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NOs: 23-28.

[0010] In certain embodiments of the antibody or antigen-binding fragment thereof according to the present invention described above, the heavy chain variable domain comprises an amino acid sequence selected from the group consisting of: (i) an amino acid sequence having at least 95% homology with SEQ ID NO: 2 (i.e., 10H5 chimeric heavy chain); (ii) an amino acid sequence having at least 95% homology with SEQ ID NO: 10 (i.e., 10H5 humanized heavy chain HuB9); and (iii) an amino acid sequence having at least 95% homology with SEQ ID NO: 11 (i.e., 10H5 humanized heavy chain HuB10). More preferably, the heavy chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NOs: 29-38.

[0011] In certain embodiments of the antibody or antigen-binding fragment thereof according to the present invention described above, the light chain variable domain comprises an amino acid sequence selected from the group consisting of: (i) an amino acid sequence having at least 95% homology with SEQ ID NO: 3 (i.e., 10H5 chimeric light chain); (ii) an amino acid sequence having at least 95% homology with SEQ ID NO: 12 (i.e., 10H5 humanized light chain HdB6); and (iii) an amino acid sequence having at least 95% homology with SEQ ID NO: 13 (i.e., 10H5 humanized light chain HdB7). More preferably, the light chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NOs: 39-44.

[0012] In certain embodiments of the present invention, the antibody or antigen-binding fragment thereof described above further comprises one or more of: (1) a linker peptide between the heavy chain variable domain and the light chain variable domain; (2) a heavy chain constant region; (3) a light chain constant region; and (4) an Fc region.

[0013] Preferably, the antibody or antigen-binding fragment thereof according to the present invention described above is a single-chain antibody fragment, bispecific antibody, single-domain antibody, nanobody, chimeric antibody, or partially or fully humanized antibody. More preferably, the antibody or antigen-binding fragment thereof is further linked to a drug conjugate to form an antibody-drug conjugate (ADC), or is further linked to a second antibody or second antigen-binding fragment to form a bispecific antibody.

[0014] In various embodiments of the present invention, the heavy chain constant region or fragment thereof is an IgG constant region including, for example, an IgG1 constant region, an IgG2 constant region, an IgG3 constant region, or an IgG4 constant region. In one embodiment, the antibody or antigen-binding fragment thereof further includes a fragment obtained from IgG4.

[0015] In various embodiments of any aspect described herein, the IgG constant region has one or more amino acid substitutions relative to the wild-type IgG constant region, and the modified IgG has a longer half-life compared to the half-life of IgG having the wild-type IgG constant region.

[0016] In various embodiments of any aspect described herein, the IgG constant region has one or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, and the numbering is according to the Kabat EU index.

[0017] In various embodiments of any aspect described herein, the antigen-binding fragment is Fv, Fab, F(ab’)2, Fab’, dsFv, scFv, or sc(Fv)2.

[0018] In one embodiment of the present invention, the antibody or antigen-binding fragment thereof described above further includes a fragment obtained from IgG, IgM, IgA, IgE, or IgD.

[0019] In certain embodiments of the present invention, the above-described antibody or antigen-binding fragment thereof specifically binds to CD73. Preferably, the antibody or antigen-binding fragment thereof specifically binds to CD73 comprising the amino acid sequence of SEQ ID NO: 14 (i.e., human CD73 269-304). More preferably, the antibody or antigen-binding fragment thereof specifically binds to CD73 comprising the amino acid sequence of SEQ ID NO: 1 (i.e., human CD73).

[0020] In certain embodiments of the present invention, the above-described antibody or antigen-binding fragment thereof binds to at least one of the 296th glutamic acid residue and the 297th arginine residue of CD73. Preferably, the antibody or antigen-binding fragment thereof binds to both the 296th glutamic acid residue and the 297th arginine residue of CD73.

[0021] Another object of the present invention is to provide an antibody or antigen-binding fragment thereof that specifically binds to at least one of the 296th glutamic acid residue and the 297th arginine residue of human CD73. Preferably, the antibody or antigen-binding fragment thereof binds to both the 296th glutamic acid residue and the 297th arginine residue of human CD73.

[0022] Preferably, in the antibody or antigen-binding fragment thereof according to the present invention that specifically binds to at least one of the 296th glutamic acid residue and the 297th arginine residue of human CD73, CD73 comprises the amino acid sequence of SEQ ID NO: 14 (i.e., human CD73 269-304). More preferably, CD73 comprises the amino acid sequence of SEQ ID NO: 1 (i.e., human CD73).

[0023] Preferably, the antibody or antigen-binding fragment thereof according to the present invention that specifically binds to at least one of the 296th glutamic acid residue and the 297th arginine residue of human CD73 is a single-chain antibody fragment, bispecific antibody, single-domain antibody, nanobody, chimeric antibody, or partially or fully humanized antibody. More preferably, the antibody or antigen-binding fragment thereof that specifically binds to at least one of the 296th glutamic acid residue and the 297th arginine residue of human CD73 is further conjugated to a drug conjugate to form an antibody-drug conjugate (ADC), or is further conjugated to a second antibody or a second antigen-binding fragment to form a bispecific antibody.

[0024] In certain embodiments of the antibody or antigen-binding fragment thereof according to the present invention that specifically binds to at least one of the 296th glutamic acid residue and the 297th arginine residue of human CD73, the antibody or antigen-binding fragment further comprises a fragment obtained from IgG1, IgG2, IgG3, or IgG4. In certain embodiments of the antibody or antigen-binding fragment thereof that specifically binds to at least one of the 296th glutamic acid residue and the 297th arginine residue of human CD73, the antibody or antigen-binding fragment further comprises a fragment obtained from IgG4.

[0025] Yet another object of the present invention is to provide a nucleic acid molecule encoding the above-described antibody or antigen-binding fragment thereof.

[0026] Yet another object of the present invention is to provide a vector containing the above-described nucleic acid molecule.

[0027] Yet another object of the present invention is to provide a recombinant host cell containing the above-described nucleic acid molecule and / or vector.

[0028] Yet another object of the present invention is to provide a pharmaceutical composition comprising (i) the above-described antibody or antigen-binding fragment thereof, a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, a vector comprising the nucleic acid molecule, a recombinant host cell comprising the nucleic acid molecule, or a recombinant host cell comprising the vector, and (ii) a pharmaceutically acceptable carrier. In certain embodiments of the present invention, the pharmaceutical composition is used to inhibit CD73. In certain embodiments of the present invention, the pharmaceutical composition further comprises one or more other immunotherapeutic agents.

[0029] Yet another object of the present invention is to provide a pharmaceutical composition for use in inhibiting CD73 in a subject in need thereof, the pharmaceutical composition comprising (i) the above-described antibody or antigen-binding fragment thereof, a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, a vector comprising the nucleic acid molecule, a recombinant host cell comprising the nucleic acid molecule, or a recombinant host cell comprising the vector, and (ii) a pharmaceutically acceptable carrier. In certain embodiments of the pharmaceutical composition for use in inhibiting CD73 of the present invention, the pharmaceutical composition is administered in combination with one or more other immunotherapeutic agents.

[0030] Preferably, the other immunotherapeutic agent used in the present invention is a PD-1 antagonist, a PD-L1 antagonist, or a CTLA-4 antagonist.

[0031] Preferably, the pharmaceutical composition according to the present invention is used to activate T cells, activate B cells, activate NK cells, and / or inhibit cancer cells.

[0032] Preferably, the pharmaceutical composition according to the present invention is used to treat, ameliorate, and / or prevent cancer. More preferably, the cancer is selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, gallbladder cancer, prostate cancer, ovarian cancer, chronic or acute lymphocytic leukemia, bladder cancer, brain tumor, kidney carcinoma, head and neck squamous cell carcinoma, glioblastoma, esophageal cancer, pancreatic cancer, renal carcinoma, oral cancer, lung cancer, colon adenocarcinoma, malignant melanoma, and lymphoma.

[0033] Yet another object of the present invention is to provide a method for inhibiting CD73, which comprises administering an effective amount of the above-described pharmaceutical composition to a subject in need of CD73 inhibition. Preferably, the method according to the present invention further comprises administering one or more other immunotherapeutic agents to a subject in need of CD73 inhibition. More preferably, the other immunotherapeutic agent is a PD-1 antagonist, a PD-L1 antagonist, or a CTLA-4 antagonist.

[0034] Preferably, the method according to the present invention is for activating T cells, activating B cells, activating NK cells, and / or inhibiting cancer cells.

[0035] Preferably, the method according to the present invention is for treating, ameliorating, and / or preventing cancer. More preferably, the cancer is selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, gallbladder cancer, prostate cancer, ovarian cancer, chronic or acute lymphocytic leukemia, bladder cancer, brain tumor, kidney carcinoma, head and neck squamous cell carcinoma, glioblastoma, esophageal cancer, pancreatic cancer, renal carcinoma, oral cancer, lung cancer, colon adenocarcinoma, malignant melanoma, and lymphoma.

[0036] Yet another object of the present invention is to provide the use of the above-described antibody or its antigen-binding fragment in the manufacture of a medicament, wherein the medicament is for inhibiting CD73. Preferably, the medicament is used in combination with one or more other immunotherapeutic agents. More preferably, the other immunotherapeutic agent is a PD-1 antagonist, a PD-L1 antagonist, or a CTLA-4 antagonist.

[0037] Preferably, the medicament is used for activating T cells, activating B cells, activating NK cells, and / or inhibiting cancer cells.

[0038] Preferably, the medicament is used for treating, ameliorating and / or preventing cancer. More preferably, the cancer is selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, gallbladder cancer, prostate cancer, ovarian cancer, chronic or acute lymphocytic leukemia, bladder cancer, brain tumor, kidney carcinoma, head and neck squamous cell carcinoma, glioblastoma, esophageal cancer, pancreatic cancer, renal carcinoma, oral cancer, lung cancer, colon adenocarcinoma, malignant melanoma, and lymphoma.

[0039] Yet another object of the present invention is to provide the use of the above-described antibody or antigen-binding fragment thereof for inhibiting CD73. Preferably, the antibody or antigen-binding fragment thereof is used in combination with one or more other immunotherapeutic agents. More preferably, the other immunotherapeutic agents are PD-1 antagonists, PD-L1 antagonists, or CTLA-4 antagonists.

[0040] Preferably, the antibody or antigen-binding fragment thereof is used for activating T cells, activating B cells, activating NK cells, and / or inhibiting cancer cells.

[0041] Preferably, the antibody or antigen-binding fragment thereof is used for treating, ameliorating and / or preventing cancer. More preferably, the cancer is selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, gallbladder cancer, prostate cancer, ovarian cancer, chronic or acute lymphocytic leukemia, bladder cancer, brain tumor, kidney carcinoma, head and neck squamous cell carcinoma, glioblastoma, esophageal cancer, pancreatic cancer, renal carcinoma, oral cancer, lung cancer, colon adenocarcinoma, malignant melanoma, and lymphoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The patent application includes at least one color drawing. Copies of this patent including the color drawing are provided by the Patent and Trademark Office upon request and payment of the necessary fees.

[0043]

Figure 1A

Figure 1B

Figure 2A

Figure 2B

Figure 3A

Figure 3B

Figure 3C

Figure 3D

Figure 4A

Figure 4B

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14A

Figure 14B

Figure 14C

Figure 15

Figure 16

Figure 17A

Figure 17B

Mode for Carrying Out the Invention

[0044] In the following paragraphs, several embodiments of the present invention will be described in detail. However, the present invention can be implemented in various embodiments without departing from the spirit of the present invention, and should not be limited to the embodiments described in the specification or the embodiments defined in the appended claims.

[0045] Unless otherwise specified in this specification, expressions such as "a", "an", and "the" described in the specification of the present invention (especially the claims) are intended to include both singular and plural forms; the term "subject" described in this specification refers to humans or non-human mammals (e.g., dogs, cats).

[0046] The term "CD73" described in this specification refers to primate CD73 such as human CD73. Human CD73 contains the amino acid sequence of SEQ ID NO: 1, and the amino acid sequence at positions 269-304 of human CD73 is SEQ ID NO: 14.

[0047] CD73 is known to be closely related to the progression and prognosis of many types of cancer. The relationship between CD73 and cancers such as breast cancer (e.g., triple-negative breast cancer and invasive lobular breast cancer), gastric cancer, colorectal cancer, gallbladder cancer, prostate cancer, ovarian cancer, chronic or acute lymphocytic leukemia, bladder cancer, brain tumors, kidney carcinoma, head and neck squamous cell carcinoma, glioblastoma, esophageal cancer, pancreatic cancer, renal carcinoma, oral cancer (e.g., oral squamous cell carcinoma), lung cancer (e.g., non-small cell lung cancer, large cell lung cancer, and lung adenocarcinoma), colon adenocarcinoma, malignant melanoma, and lymphoma can be confirmed in the publications listed below.

[0048]

Table 1-1

Table 1-2

[0049] The term "antibody" described in this specification includes polyclonal antibodies, monoclonal antibodies, single-chain antibody fragments, bispecific antibodies, single-domain antibodies, nanobodies, chimeric antibodies, or partially or fully humanized antibodies. Methods for generating antibodies that react with specific antigens are known in the art. For example, antibodies can be generated by recombinant methods or by immunizing animals with an antigen or a nucleic acid encoding the antigen.

[0050] A typical IgG antibody comprises two heavy chains and two light chains interconnected by disulfide bonds. Each heavy chain has a constant domain (i.e., "C H " or "CH") and a variable domain (i.e., "V H " or "VH"). Each light chain has a constant domain (i.e., "C L " or "CL") and a variable domain (i.e., "V L " or "VL"). Each V H and V L contains three complementarity-determining regions (CDRs) and four framework regions (FRs), the CDRs are hypervariable, the FRs are more conserved, and are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDRs mainly play a role in binding to the epitope of the antigen. In certain embodiments of the present invention, the antibody comprises a fragment obtained from IgG1, IgG2, IgG3, or IgG4. For example, in certain embodiments of the present invention, the antibody comprises a fragment of the variable domain of IgG1, IgG2, IgG3, or IgG4.

[0051] The term "epitope" is the site on an antigen to which an antibody binds. U.S. Patent No. 9,938,356 describes antibodies that can bind to epitopes such as the 206th and 211th positions of CD73 (the 206th and 211th positions are the same as the 180th and 185th positions described in U.S. Patent No. 9,938,356 because the signal peptide sequence containing 26 amino acids of the CD73 sequence has been removed in U.S. Patent No. 9,938,356). Different from U.S. Patent No. 9,938,356, the antibodies of the present invention bind to at least one of the 296th glutamic acid residue and the 297th arginine residue of CD73.

[0052] The positive control (i.e., ocrelizumab) used in the embodiments described herein was synthesized by the inventors and has the same amino acid sequence as MEDI9447 disclosed in U.S. Patent No. 9,938,356.

[0053] As used herein, terms such as "antigen-binding fragment" of an antibody include any polypeptide or glycoprotein that is naturally occurring, enzymatically obtained, synthetically made, or genetically engineered and that specifically binds to an antigen to form a complex. Antigen-binding fragments of an antibody can be obtained from whole antibody molecules using any suitable standard techniques, such as proteolysis or recombinant techniques including manipulation and expression of DNA encoding antibody variable domains and optionally constant domains. Such DNA is known and / or can be readily obtained, for example, from commercially available DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA is sequenced and manipulated chemically or by molecular biological techniques, for example, by arranging one or more variable and / or constant domains in a suitable configuration, or by introducing codons to generate cysteine residues, or by changing, adding, or deleting amino acids.

[0054] Antigen-binding fragments of an antibody typically contain at least one variable domain. The variable domain can be of any size and typically contains at least one CDR adjacent to or in-frame with one or more framework sequences. In an antigen-binding fragment containing a VH domain associated with a VL domain, the VH domain and the VL domain can be arranged relative to each other in any suitable arrangement. For example, the variable regions can be dimeric and can contain VH-VH, VH-VL, or VL-VL dimers. Linkers can be composed of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, thereby providing a flexible or semi-flexible linkage between adjacent VH and / or VL domains in a single polypeptide molecule. Alternatively, an antigen-binding fragment of an antibody can contain a monomeric VH or VL domain.

[0055] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently attached to at least one constant domain, and the variable and constant domains may be directly linked to each other or may be linked by a linker. The linker may be composed of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, thereby providing a flexible or semi-flexible linkage between adjacent variable and / or constant domains within a single polypeptide molecule. Further, the antigen-binding fragment of an antibody may include homodimers or heterodimers (or other multimers) of either variable or constant domain constructs, non-covalently associated with each other and / or with one or more monomeric VH or VL domains (e.g., by disulfide bonds).

[0056] In certain embodiments, an antibody or antigen-binding fragment thereof according to the invention may be linked to a drug conjugate to form an antibody-drug conjugate (ADC), or may be further linked to a second antibody or second antigen-binding fragment to form a bispecific antibody.

[0057] In another embodiment, an antibody according to the invention is a humanized antibody. A "humanized antibody" refers to a recombinant protein in which the CDRs of an antibody of one species (e.g., a murine antibody) are converted from the heavy and light chains of that species (murine antibody) to human heavy and light chain domains and which includes human framework region (FR) sequences. The constant domains of the antibody molecule are obtained from human antibody constant domains.

[0058] As used herein, the term "% identity" is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that match the amino acid residues or nucleotides in a reference polypeptide or nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to maximize sequence identity, and no conservative substitutions are considered part of sequence identity.

[0059] As described above, the inventors of the present invention have found a novel anti-CD73 antibody that includes specific complementarity-determining regions (CDRs) and is effective in inhibiting CD73. Compared with a positive control (i.e., ocrelizumab), the antibody and antigen-binding fragment according to the present invention have better CD73 binding activity, a longer effective period of inhibiting AMP consumption, and better effects on inhibiting activation of immune cells and tumor growth.

[0060] Accordingly, the present invention relates to a novel antibody or its antigen-binding fragment, comprising: (i) a heavy-chain variable domain comprising an HCDR1 region, an HCDR2 region, and an HCDR3 region, wherein the HCDR1 region comprises the amino acid sequence of SEQ ID NO: 4 having at most one mutation, the HCDR2 region comprises the amino acid sequence of SEQ ID NO: 5 having at most one mutation, and the HCDR3 region comprises the amino acid sequence of SEQ ID NO: 6 having at most one mutation; and (ii) a light-chain variable domain comprising an LCDR1 region, an LCDR2 region, and an LCDR3 region, wherein the LCDR1 region comprises the amino acid sequence of SEQ ID NO: 7 having at most one mutation, the LCDR2 region comprises the amino acid sequence of SEQ ID NO: 8 having at most one mutation, and the LCDR3 region comprises the amino acid sequence of SEQ ID NO: 9 having at most one mutation.

[0061] Furthermore, the inventors of the present invention have found that the antibody and antigen-binding fragment according to the present invention can specifically bind to at least one of the 296th glutamate residue and the 297th arginine residue of human CD73. Accordingly, the present invention also relates to a novel antibody or its antigen-binding fragment that specifically binds to at least one of the 296th glutamate residue and the 297th arginine residue of human CD73.

[0062] Furthermore, the present invention relates to a nucleic acid molecule encoding the above-described antibody or an antigen-binding fragment thereof; a vector containing the above-described nucleic acid molecule; a recombinant host cell containing the above-described nucleic acid molecule and / or vector; (i) the above-described antibody or an antigen-binding fragment thereof, the nucleic acid molecule encoding the antibody or an antigen-binding fragment thereof, the vector containing the nucleic acid molecule, the recombinant host cell containing the nucleic acid molecule, or the recombinant host cell containing the vector, and (ii) a pharmaceutically acceptable carrier, a pharmaceutical composition; a method for inhibiting CD73, comprising administering an effective amount of the above-described pharmaceutical composition to a subject in need of CD73 inhibition; use of the above-described antibody or an antigen-binding fragment thereof in the manufacture of a medicament for inhibiting CD73; and use of the above-described antibody or an antigen-binding fragment thereof for inhibiting CD73.

[0063] The pharmaceutical composition or medicament according to the present invention is used for systemic or local administration and can be delivered by various drug delivery systems (DDS) such as an oral drug delivery system, a transdermal drug delivery system, a transmucosal drug delivery system, or an injection drug delivery system. For example, in order to enhance bioavailability, control the drug release rate, accurately target the lesion site, and / or reduce side effects, the pharmaceutical composition or medicament can be delivered by, but not limited to, liposomes, microcapsules, nanoparticles, or microneedles.

[0064] Depending on the desired purpose, the pharmaceutical composition or medicament according to the present invention can be provided in any suitable form without particular limitation. For example, the pharmaceutical composition or medicament can be provided in a form for oral administration, intravenous injection (including drip and bolus injection), intramuscular injection, subcutaneous injection, intra-arterial injection, intraperitoneal injection, transdermal administration (such as patches, etc.), or transmucosal administration (such as spray-type nasal drops, nasal drops, and suppositories, etc.), but is not limited thereto. Depending on the form and purpose, an appropriate carrier known in the field of pharmaceutical engineering is selected and used to provide the pharmaceutical composition or medicament. Examples of carriers include, but are not limited to, excipients, diluents, fillers, buffers, adjuvants, stabilizers, absorption promoters, disintegrants, hydrotropes, antioxidants, adhesives, binders, tackifiers, dispersants, suspending agents, lubricants, and humectants.

[0065] As a form for oral administration, the pharmaceutical composition or medicament according to the present invention can be provided by any suitable method in any suitable form for oral administration. Liquid forms suitable for oral administration include syrups, oral solutions, suspensions, and elixirs. Solid forms suitable for oral administration include powders, granules, troches, sugar-coated tablets, enteric-coated tablets, chewable tablets, effervescent tablets, film-coated tablets, capsules, and long-acting sustained-release tablets. The pharmaceutical composition or medicament provided by the present invention may contain any pharmaceutically acceptable carrier that does not adversely affect the desired effect of the active ingredient (i.e., the antibody of the present invention or its antigen-binding fragment). For example, pharmaceutically acceptable carriers for the above liquid forms include, but are not limited to, water, physiological saline, dextrose, glycerol, ethanol or its analogs, oils (such as olive oil, castor oil, cottonseed oil, peanut oil, corn oil, germ oil), glycerol, polyethylene glycol, and combinations thereof; pharmaceutically acceptable carriers for the above solid forms include, but are not limited to, cellulose, starch, kaolin, bentonite, sodium citrate, gelatin, agar, carboxymethylcellulose, gum arabic, tragacanth, seaweed gel, glyceryl monostearate, calcium stearate, colloidal silicon dioxide, and combinations thereof.

[0066] As a form for transdermal administration, the pharmaceutical composition or medicine according to the present invention may also include any pharmaceutically acceptable carrier such as water, mineral oil, propylene glycol, polyethylene oxide, liquid petrolatum, sorbitan monostearate, and polysorbate 60, which does not adversely affect the desired effect of the active ingredient (i.e., the antibody of the present invention or its antigen-binding fragment). The pharmaceutical composition or medicine can be provided by any suitable method in any suitable form for transdermal administration, such as in the form of a patch (such as a microneedle patch), but is not limited thereto.

[0067] As a form for injection or infusion, the pharmaceutical composition or medicine includes one or more components such as isotonic solutions, salt buffer physiological saline (e.g., phosphate buffer physiological saline or citrate buffer physiological saline), hydrotropes, emulsifiers, 5% dextrose solution, and other carriers to provide the pharmaceutical composition or medicine as an intravenous injection, emulsified intravenous injection, injection powder, injection suspension, or injection powder suspension. Alternatively, the pharmaceutical composition or medicine can be prepared as a solid before injection. The desired injection solution is provided by dissolving or emulsifying the solid before injection in other solutions or suspensions before administration to the subject in need.

[0068] As a form for transmucosal administration, the pharmaceutical composition or medicine includes one or more components such as penetration enhancers, surfactants, viscosity modifiers, pH adjusters, preservatives, stabilizers, osmotic pressure adjusters, and other carriers to provide the pharmaceutical composition or medicine as eye drops, ointments, orally disintegrating tablets, spray-type nasal drops, nasal drops, or suppositories.

[0069] Optionally, the pharmaceutical composition or medicine according to the present invention may also include an appropriate amount of additives such as toners or colorants to enhance the visibility of the pharmaceutical composition or medicine, and / or buffers, preservatives, antiseptics, antibacterial agents, or antifungal agents to improve the stability and storage properties of the pharmaceutical composition or medicine.

[0070] The pharmaceutical composition or medicament according to the present invention may further optionally contain one or more other active ingredients (such as PD-1 antagonist, PD-L1 antagonist, CTLA-4 antagonist, EGFR inhibitor, and A2AR inhibitor, etc.) in order to further enhance the effect of the pharmaceutical composition or medicament, or to enhance the application flexibility and adaptability of the formulation thus provided, as long as the other active ingredients do not adversely affect the desired effect of the active ingredient of the present invention (i.e., the antibody of the present invention or its antigen-binding fragment).

[0071] The present invention also provides a method for inhibiting CD73, which comprises administering an effective amount of the above-described antibody or its antigen-binding fragment to a subject in need of inhibition of CD73. The term "subject in need of inhibition of CD73" refers to a subject with a weak immune system and / or suffering from cancer, particularly cancer with high expression of CD73. Preferably, the cancer is selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, gallbladder cancer, prostate cancer, ovarian cancer, chronic or acute lymphocytic leukemia, bladder cancer, brain tumor, kidney carcinoma, head and neck squamous cell carcinoma, glioblastoma, esophageal cancer, pancreatic cancer, renal carcinoma, oral cancer, lung cancer, colon adenocarcinoma, malignant melanoma, and lymphoma. In the method according to the present invention, the antibody or its antigen-binding fragment can be administered to the subject in need thereof in the form of the above-described pharmaceutical composition or medicament, and the administration type, administration route, administration form, administration frequency and use of the pharmaceutical composition and medicament are also all in accordance with the above description.

[0072] The present invention will be described in more detail in the following specific examples. However, the following examples are provided only for the purpose of explaining the present invention, and the scope of the present invention is not limited thereby. The scope of the present invention is shown in the appended claims.

[0073] (Example)

[0074] Example 1. Preparation of anti-CD73 antibody

[0075] 1-1. Construction of scFv antibody library

[0076] Using recombinant human CD73-6×His having the amino acid sequence of Accession No. 1 as an antigen, mice were immunized once a week for 6 to 9 times. After immunization, the mice were sacrificed and spleens and their lymph nodes were obtained. Total RNA of the spleen and its lymph nodes was extracted and reverse transcribed by RT-PCR using primers for constructing antibody fragments containing VH and VL. Antibody fragments were assembled into scFv fragments by polymerase chain reaction (PCR) to construct an scFv library.

[0077] 1-2. Preparation of scFv Phage for Biopanning

[0078] The scFv library provided in Example 1-1 was inoculated into 2×YT medium (2YTAG) containing 100 μg / ml ampicillin and 2% glucose, shaken at 37°C, and incubated until the OD at 600 nm reached 0.5. The culture was infected with helper phage and cultured in a 37°C water bath for 30 minutes without shaking. Cells in the culture were collected, suspended in 2×YT medium (2YTAK) containing 100 μg / ml ampicillin and 25 μg / ml kanamycin, shaken at 30°C, and incubated overnight. The supernatant of the culture was collected, mixed with 1 / 5 volume of PEG / NaCl (20% polyethylene glycol 8000, 2.5 M NaCl), and placed at 4°C for at least 1 hour. After centrifugation, the pellet was collected, suspended in PBS, and spun again to collect the supernatant.

[0079] 1-3. Biopanning of scFv Phage Using ELISA

[0080] ELISA plates (Nunc) were coated with 5 - 25 μg / 100 μl of antigen per well and incubated overnight at 4°C in sodium bicarbonate buffer (pH 9.6). The wells were washed three times with PBS and blocked with 300 μl of 5% skim milk in PBS (MPBS) per well for 2 hours at 37°C. After washing three times with PBS, 100 μl of phage in 5% MPBS and a fusion protein containing a histidine tag were added and incubated at 37°C for 90 minutes. The wells were washed 4 - 10 times with 0.05% Tween 20 in PBS (PBST) and 4 - 10 times with PBS, then 100 μl of 100 mM triethylamine (TEA) was added to elute the phage and reacted at 37°C for 20 minutes. The 100 μl of eluted phage was neutralized with 50 μl of 1 M Tris, pH 7.4. 3 mL of TG1 in the exponential growth phase was added to the eluted phage. For infection, the culture was incubated at 37°C for 30 minutes without shaking. 20 mL of 2×YT - AG was added to the infected TG1 bacteria and then incubated overnight at 37°C.

[0081] 1 - 4. Preparation of phage for the next round

[0082] The culture obtained in Examples 1 - 3 was spun down and collected, and suspended in 0.5 mL of 2×YT - AG, 15% glycerol. Then, 10 μl of bacteria was added to 10 mL of 2×YT - AG and shaken at 37°C to grow the bacteria until the OD at 600 nm reached 0.5. Helper phage was added to the 10 mL culture at a ratio of 1:20 (M13KO7 helper phage: culture) to infect with M13KO7 helper phage, and the infected culture was incubated in a 37°C water bath for 30 minutes without shaking. The culture was spun down to collect the pellet, the pellet was suspended in 25 mL of 2×YT - AK, and then cultured overnight at 30°C. Furthermore, the 25 mL of the overnight - cultured culture was spun at 10,000 rpm for 20 minutes to collect the supernatant, and 1 / 5 volume (5 mL) of PEG / NaCl was added to the supernatant to obtain a mixture. The mixture was spun at 10,000 rpm for 20 minutes, the pellet was collected and suspended in 0.5 mL of PBS.

[0083] 1-5. Screening of human CD73-positive phage clones by ELISA

[0084] The suspensions obtained in Examples 1-4 were spread on plates and cultured to obtain individual colonies. The obtained individual colonies were inoculated into a 96-well plate containing 200 μl of 2×YT-AG, shaken overnight at 37 °C for growth, and then 10 μl of the culture solution was transferred to a second 96-well plate containing 180 μl of 2×YT-A per well and shaken at 37 °C for 2 hours. Then, 50 μl of 2×YT-A containing 10 2.4×10 3 pfu / mL of M13KO7 helper phage was added to each well of the second plate to obtain a mixture. The mixture was shaken at 37 °C for 2 hours. 50 μl of 2×YT-AK (the concentration of kanamycin was 300 μg / mL) was added to the mixture, and then it was shaken overnight at 30 °C for growth. 50 μl of MPBS was added to the culture solution to obtain a phage mixture, and 100 μl of the phage mixture was taken for phage ELISA.

[0085] The ELISA plate was coated with 1 μg / mL of protein antigen per well, then rinsed three times with PBS, and blocked with 300 μl of 5% MPBS per well at 37 °C for 2 hours. After rinsing three more times with PBS, 100 μl of the phage mixture described above was added and incubated at 37 °C for 90 minutes. The phage solution was discarded, and the wells were washed 6 times with PBST and 6 times with PBS. Then, HRP-anti-M13 antibody appropriately diluted with 5% MPBS was added to obtain a mixture. The mixture was incubated at 37 °C for 60 minutes and then washed 6 times with PBST. The wells were developed with the substrate solution (TMB), and 100 μl of 1 M hydrochloric acid was added to stop the reaction. When the color turned yellow, the OD at 650 nm and 450 nm was measured.

[0086] After screening, a total of 317 clones with different CDR sequences were identified.

[0087] 1-6. Preparation of monoclonal phages

[0088] The 317 clones obtained in Examples 1-5 were subjected to the following procedure. The bacteria were cultured overnight at 37°C. Then, 2×YT-AG was added to 100 μl of the bacteria, and the bacteria were grown by shaking at 37°C until the OD at 600 nm reached 0.5. Helper phage was added to 10 mL of the culture solution at a ratio of 1:20 (M13KO7 helper phage: culture solution) to infect the M13KO7 helper phage, and the infected culture solution was cultured in a 37°C water bath without shaking for 30 minutes. The culture solution was spun to collect the pellet, the pellet was suspended in 25 mL of 2×YT-AK, and then cultured overnight at 30°C. Further, the 25 mL of the overnight-cultured culture solution was spun at 10,000 rpm for 20 minutes to collect the supernatant, and 1 / 5 volume (5 ml) of PEG / NaCl was added to the supernatant to obtain a mixture. The mixture was spun at 10,000 rpm for 20 minutes, the pellet was collected, and suspended in 0.5 mL of PBS.

[0089] 1-7. Screening of CD73 phages that bind to KLM-1 cells

[0090] To screen for phage clones that can bind to CD73-expressing cells, KLM-1 cells were mixed with the anti-CD73 phages obtained in Examples 1-6 (10 11 virions / tube, 100 μl). After incubation at 4°C for 1 hour, the cells were washed and centrifuged, and reacted with anti-M13-FITC (1:500) for each tube. After washing and centrifuging, 0.3 mL of Flow Cytometry Staining Buffer was added to each tube. Gently mixed and analyzed with a BD FACSVerse (trademark) Flow Cytometer.

[0091] After analysis by flow cytometry, 27 phage clones were shown to have the function of binding to CD73-expressing cells, KLM-1.

[0092] Thereafter, as described in Example 1-8 below, full-length antibodies were expressed by constructing the VH and VL chains of these CD73-binding phage clones into a full-length antibody expression vector.

[0093] 1 - 8. Expression of full - length antibody

[0094] The VH and VL chains derived from the scFv phage clones obtained in Examples 1 - 7 were inserted into expression vectors containing the CH and CL chains, respectively, to construct genes encoding anti - human CD73 antibodies. The constructed vectors were transfected into 293 free - style cells. The antibodies were purified using Protein A Sepharose Fast Flow (GE Healthcare, 17 - 1279 - 02). After purification, the OD at 280 nm was measured to quantify the antibodies, and confirmed by reducing and non - reducing PAGE.

[0095] 1 - 9. Binding affinity assay using ELISA

[0096] ELISA plates were coated with 100 μl of human CD73 per well at 4°C overnight, then washed three times with PBS, and blocked with 300 μl of 5% MPBS per well at 37°C for 2 hours. The wells were washed three times with PBS, 100 μl of anti - human CD73 antibody (in 2 - fold serial dilution) was added, and incubated at 37°C for 90 minutes. The test solution was discarded, and the wells were washed three times with PBS. The antibodies obtained in Examples 1 - 8 appropriately diluted with 5% MPBS (1:1000) were added to the wells, and the wells were incubated at 37°C for 60 minutes, then the wells were washed three times with PBS. The wells were developed with 100 μl of substrate solution TMB, and 50 μl of 1 M sulfuric acid was added to stop the reaction. When the color turned yellow, the OD at 650 nm and 450 nm was measured.

[0097] The binding affinity values (K D ) of the antibodies obtained in Examples 1 - 8 were calculated according to the one - site binding equation (hyperbola) of GraphPad Prism software. For the following functional assay of cell CD73 activity inhibition, antibodies with K D less than 10 -9 M were selected.

[0098] Example 2: Selection of Antibodies with Better CD73 Inhibitory Functional Effects

[0099] To examine the inhibitory effect of the selected antibodies on CD73 activity in CD73-high expressing cells, the following experiment was conducted using the AMP consumption assay as the method of Hay et al. (Oncoimmunology 2016, 5, 1208875). AMP is known to inhibit the luminescence ability of ATP. Therefore, the consumption of AMP can be determined by measuring the luminescence level. Furthermore, since AMP can be converted to adenosine by the membrane protein CD73, the lower the luminescence level, the less the AMP consumption, and the higher the inhibitory effect of the antibody against CD73. MDA-MB-231 was seeded (2.5×10 3 cells / well) in a 96-well plate in advance every 16 hours, and then the cells in each well were treated under the following conditions: The cells were cultured in L-15 medium containing 100 μM AMP and antibodies of each group including isotype IgG, olaratumab, and the selected antibodies (8 concentration steps in serial dilution) in an incubator at 37 °C for 24 hours. After collecting the supernatant of each group in a round-bottom 96-well plate, ATP was added at a final concentration of 100 μM. There are several control treatments: The "cells + ATP" group means that the cells were only cultured in L-15 medium without antibodies and AMP, and then ATP was added. "Cells + AMP + ATP" means that the cells were cultured in L-15 medium containing 100 μM AMP, and then ATP was added. "ATP only" means that L-15 medium without cells was incubated, and then ATP was added. "AMP + ATP" means that the medium containing 100 μM AMP without cells was incubated, and then ATP was added.

[0100] After the above procedure was completed, the medium of each group was mixed with Celltiter-glo reagent at a ratio of 1:1 and incubated in the dark for 10 minutes. A luminometer was used to measure the luminescence of each group. The CD73 enzyme activity (i.e., the percentage of AMP consumption) of each group was calculated by the following formula 1, and the results are shown in Figure 1.

[0101]

Number

[0102] Identification of antibodies having an inhibitory function of better CD73 activity

[0103] After selecting antibodies by both cell-based (flow assay) and antigen (ELISA) binding affinity assays, the next step was to confirm the inhibitory effect of these antibodies on CD73 activity using the AMP consumption assay described above. None of the selected antibodies had a significantly better effect on the inhibition of CD73 activity than the reference antibody, ocrelizumab. However, only 10H5 significantly inhibits CD73 compared to ocrelizumab. As shown in Figure 1A, after culturing MDA-MB-231 and 100 μM AMP with the reference antibody ocrelizumab for 24 hours, almost all of the AMP was consumed. However, the "10H5" group could significantly inhibit the consumption of AMP, and when the concentration of 10H5 was higher than 10 nM, the consumption of AMP decreased to the lowest level (<20%). These results indicate that 10H5 has a better and more excellent inhibitory effect on CD73 activity (after 24 hours) in MDA-MB-231 cancer cells compared to ocrelizumab.

[0104] 2-2.Confirmation of the novel CD73 inhibitory function of 10H5 by NSCLC Calu-1 cells

[0105] To confirm the excellent inhibitory effect of 10H5 on CD73, CD73-high expressing NSCLC Calu-1 was used to test whether it functions similarly in MDA-MB-231.

[0106] NSCLC Calu-1 cells were seeded in 96-well plates (500 cells / well) and cultured at 37 °C, 5% CO in McCoy's 5a medium containing 100 μM AMP and antibodies (serial dilutions of 8 concentrations) including isotype IgG, ocrelizumab, and 10H5. 2They were cultured in an incubator for 24 hours and then 100 μM ATP was added. The other control groups and the following procedures were the same as described above, and the results are shown in Figure 1B.

[0107] As shown in Figure 1B, after culturing for 24 hours, almost all of the AMP was consumed in the "Oleclumab" group. However, the "10H5" group was still able to significantly inhibit the consumption of AMP, and when the concentration of 10H5 was higher than 10 nM, the consumption of AMP decreased to the lowest level (10%). These results indicate that compared with Oleclumab, the antibody 10H5 of the present invention has a better inhibitory effect on CD73 activity (after 24 hours) in Calu-1 cancer cells.

[0108] Example 3: Humanization of the antibody

[0109] 3-1. CDR grafting

[0110] Since it was found that the 10H5 antibody has a novel and specific inhibitory effect on CD73 activity, we humanized the 10H5 antibody for further development as a therapeutic antibody. As described above, the variable regions of the 10H5 antibody were obtained from the mouse scFv sequence. To generate the humanized 10H5 antibody, the framework of the 10H5 antibody was replaced with a human framework selected from the IMGT database (http: / / www.imgt.org / ) (heavy chain: IGHV1-46*01F; light chain: IGKV1-12*01). Then, 21 amino acids of the heavy chain and 26 amino acids of the light chain were mutated in order to replace the entire framework of the 10H5 antibody with a human framework. Therefore, the mutations in the heavy chain and light chain were 25.6% (21 mutations / 82 amino acids of the heavy chain) and 34.2% (26 mutations / 76 amino acids of the light chain), respectively.

[0111] 3-2. Reverse mutation

[0112] This is because the framework of the humanized antibody can directly or indirectly affect the binding affinity for the antigen or the inhibitory effect on CD73 activity. Therefore, it is necessary to introduce mutations that revert some specific amino acid residues of the human framework to those of the mouse framework.

[0113] 3-3. Inhibitory Effect of the Humanized Antibody on CD73 Activity

[0114] The humanized antibodies were selected based on three criteria: binding affinity, CD73 inhibitory activity, and the minimum number of back mutations. There are three antibodies, HuB9 / HdB7, HuB10 / HdB6, and HuB10 / HdB7, that meet these three conditions, which are paired with two heavy chains: HuVHB9 (HuB9) and HuVHB10 (HuB10), and two light chains: HdVLB6 (HdB6) and HdVLB7 (HdB7). The amino acid sequences of HuB9, HuB10, HdB6, and HdB7 are shown in SEQ ID NO: 10 (HuB9), SEQ ID NO: 11 (HuB10), SEQ ID NO: 12 (HdB6), and SEQ ID NO: 13 (HdB7). To further investigate the characteristics of the humanized antibodies of the present invention, the ability of HuB9 / HdB7, HuB10 / HdB6, and HuB10 / HdB7 to inhibit the consumption of AMP (i.e., inhibit CD73 activity) was analyzed by the AMP consumption assay described in Example 2. The results are shown in FIGS. 2A and 2B.

[0115] As shown in FIGS. 2A and 2B, HuB9 / HdB7, HuB10 / HdB6, and HuB10 / HdB7 showed an inhibitory effect on AMP consumption similar to that of the 10H5 antibody in MDA-MB231 (FIG. 2A) and Calu-1 (FIG. 2B) cells.

[0116] Example 4: Binding Affinity of the Antibodies of the Present Invention

[0117] To investigate the characteristics of the antibodies of the present invention, including 10H5 and its humanized derivatives, HuB9 / HdB7, HuB10 / HdB6, and HuB10 / HdB7, the binding affinity of these antibodies was analyzed by Biacore SPR.

[0118] The analysis conditions were a flow rate of 30 μL / min, an association time of 180 seconds, and a dissociation time of 600 seconds. The concentrations of the 10H5 antibody were 0.625 nM, 1.25 nM, 2.5 nM, 5 nM, and 10 nM. The binding affinities of 10H5 and its humanized derivatives, HuB9 / HdB7, HuB10 / HdB6, and HuB10 / HdB7 are shown in Figures 3A, 3B, 3C, and 3D, respectively.

[0119] As shown in Figure 3A, the KD value of the 10H5 antibody measured by Biacore SPR was 6.835×10 -10 M. As shown in Figures 3B, 3C, and 3D, the KD values of the HuB9 / HdB7, HuB10 / HdB6, and HuB10 / HdB7 antibodies measured by Biacore SPR were 6.227, 8.028, and 6.781×10 -10 M, respectively. All three humanized antibodies have the same binding affinity to human CD73 as the parental 10H5 antibody.

[0120] Example 5: Sustained inhibitory effect of the antibody of the present invention on CD73 activity

[0121] To further confirm that the antibodies of the present invention have a CD73 inhibitory effect that lasts longer than ocrelizumab, the antibodies, 10H5 and its humanized derivatives, were treated with both MDA-MB-31 and Calu-1 cells. After 6 and 24 hours of treatment, the inhibitory effect was analyzed by an AMP consumption assay. The experimental methods and processes were the same as in Example 2, but the antibody concentration was only 10 nM and the incubation times were 6 hours and 24 hours. The results of CD73 activity are shown in Figures 4A and 4B.

[0122] As shown in Figure 4A, after treating MDA-MB-231 cells with AMP and antibodies for 6 hours, AMP consumption was clearly observed in the "isotype IgG" group (about 60%). However, it was inhibited by all antibody treatments including "oleclumab" (less than 20%), "10H5" and its derivatives (less than 10%), and AMP consumption was low. However, after 24 hours, the AMP of "oleclumab" was almost completely consumed (more than 90%). However, "10H5" and its derivatives showed significantly less AMP consumption than "oleclumab" (less than 50%) in MDA-MB-231 cancer cells. As shown in Figure 4B, similar trends were observed in Calu-1 cells. After 6 hours, AMP consumption was low in "oleclumab" and a series of 10H5, but after 24 hours, the AMP of "oleclumab" was almost consumed (more than 90%). However, "10H5" and its derivatives showed significantly less AMP consumption than "oleclumab" (<20%) in Calu-1 NSCLC cells. These results indicate that the antibodies of the present invention, 10H5 and its humanized derivatives, have inhibitory properties against CD73 activity that are significantly better and longer-lasting than oleclumab.

[0123] Example 6: Analysis of the inhibitory effect of the antibodies of the present invention on CD73 activity by measuring extracellular AMP concentration

[0124] The AMP consumption measured in Examples 2, 3, and 5 was estimated from the luminescence level. To examine the actual change in AMP concentration due to the reaction between intracellular CD73 and the antibody, the following experiment was conducted by measuring the AMP concentration.

[0125] Seed MDA-MB-231 cells in a 12-well plate (2.5×10 4Cells / well), the cells in each well were cultured in 1.5 ml of serum-free medium containing 400 μM AMP and 10 nM antibody containing ocrelizumab, 10H5 or its derivative. The following conditions were 6 hours or 24 hours at 37°C. There were two control groups as follows. The "AMP only" group means 1.5 ml of medium containing 400 μM AMP (without cells), and the "cells + AMP" group means that the cells were cultured in 1.5 ml of serum-free medium containing 400 μM AMP and no antibody. After completion of the above procedure, the AMP concentration in the supernatant was measured by LC / MS. The results are shown in Figure 5.

[0126] As shown in Fig. 5, after 6 hours of culture, the AMP concentration in the "AMP only" group was 385 μM, while that in the "cells + AMP" group was 239 μM, indicating that MDA-MB-231 cells could consume AMP in 6 hours. However, the "cells + AMP + olaratumab" group (i.e., with olaratumab added) could maintain an AMP concentration of 324 μM, and the "cells + AMP + 10H5" group (i.e., with 10H5 antibody added) could maintain an AMP concentration of 347 μM. The humanized antibodies HuB9 / HdB7, HuB10 / HdB6, and HuB10 / HdB7 could maintain AMP to a similar extent as 10H5, at 345, 336, and 331 μM, respectively. These results indicate that olaratumab, 10H5, and its derivatives slightly and equally inhibit the AMP consumption of MDA-MB-231 cells for 6 hours. After 24 hours of culture, the AMP concentration in the "AMP only" group was still 385 μM, while in the "cells + AMP" group it was only 0.5 μM, indicating that MDA-MB-231 cells could consume all AMP after 24 hours. The "cells + AMP + olaratumab" group (i.e., with olaratumab added) could only maintain an AMP concentration of 147 μM. However, the "cells + AMP + 10H5" group (i.e., with the 10H5 antibody of the present invention added) could maintain an AMP concentration of 290 μM. Also, the treatment with the humanized antibodies HuB9 / HdB7, HuB10 / HdB6, and HuB10 / HdB7 also maintained an AMP concentration similar to that of 10H5 after 24 hours, at 298, 286, and 287 μM, respectively. These results indicate that the antibody of the present invention (e.g., 10H5) has a significantly better and longer-term inhibitory effect on the CD73 activity of MDA-MB-231 cells compared to olaratumab.

[0127] Example 7: Activation effect of the antibody of the present invention on T cells

[0128] CD3 / CD28 Dynabeads can induce cell division of CD4 + T cells and is known. Furthermore, CD4 +By staining T cells with CFSE and analyzing the fluorescence levels of the stained CD4+ T cells, the status of cell division can be determined (the more cell division occurs, the weaker the fluorescence becomes, and the peak on the flow cytometry chart shifts to the left). To examine whether the 10H5 antibody of the present invention can activate immune cells (CD4 + T cells, etc.), the following experiments were conducted.

[0129] First, 20 mL of human blood was collected from a donor and mixed with 20 mL of PBS. A small amount of the blood mixture was added to a 50 mL tube containing 15 mL of Lymphoprep (STEMCELL Tech.). Centrifugation was performed at 2000 rpm for 30 minutes without reducing the speed. The white thin layer between the serum and Lymphoprep was PBMC. The PBMC was collected and placed in a new 50 mL tube. The tube was washed 3 times with PBS, and then CD4 in PBMC was isolated using a CD4 isolation kit. + T cells were separated and counted.

[0130] An appropriate number of CD4 + T cells were collected and centrifuged. Then, the CD4 + T cells were stained with 1 μM CFSE and made uniform so that the cell concentration became 1×10 6 cells / mL. Then, the cells were cultured in an incubator at 37°C for 20 minutes and washed 2 times with the medium. Then, an appropriate number of cells were collected and centrifuged. The cells were resuspended so that the cell concentration became approximately 1×10 6 cells / mL. Cultured in a medium containing 30 IU / mL of IL-2 and CD3 / CD28 Dynabeads equivalent to the number of cells, 10 nM of hIgG, ocrelizumab, 10H5 or its humanized derivative, HuB9 / HdB7, HuB10 / HdB6, or HuB10 / HdB7 were added, and after culturing the cells in an incubator at 37°C for 60 minutes, 400 μM AMP was added and seeded in a 96-well plate (100 μL / well), and cultured in an incubator at 37°C for 3 to 4 days.

[0131] The "control" group means that no CD3 / CD28 Dynabeads and IL-2, or antibodies or AMPs were added. The "CD3 / CD28" group means that the cells were activated with IL-2 and CD3 / CD28 Dynabeads, but no subsequent antibodies or AMPs were added. The "CD3 / CD28 / AMP" group means that the cells were activated with IL-2 and CD3 / CD28 Dynabeads, then the cells were cultured for 60 minutes and seeded into 96-well plates, and then 400 μM AMP was further added and cultured in an incubator at 37°C for 3 - 4 days. After the above procedures were completed, the cells of each group were collected and analyzed by flow cytometry. The results are shown in Figure 6.

[0132] As shown in Figure 6, three days after culturing, the left shift of fluorescence in the "CD3 / CD28" group was 82.7%, which was significantly more than that of the "control" group (4.91%). However, in the "CD3 / CD28 / AMP" group, due to the inhibitory effect of adenosine (adenosine converted from AMP by CD73 in CD4+ T cells) on cell division, the left shift was only 61.0%. The same was true when 10 nM IgG was added before the addition of 400 μM AMP, and the level of left shift (62.7%) was almost the same as that of the "CD3 / CD28 / AMP" group. However, when 10 nM ocrelizumab was added before the addition of 400 μM AMP, since it could prevent AMP from being converted to adenosine, the level of left shift reversed from 62.7% to 72.9%. Furthermore, with the effect of the same 10 nM dose of 10H5, the level of left shift changed from 62.7% to 82.7%, indicating that the antibody 10H5 is more effective in preventing the conversion of AMP to adenosine than ocrelizumab and has a better ability to reverse the level of left shift. These results indicate that the antibody of the present invention (for example, 10H5) is effective in activating immune cells.

[0133] In addition, the activation effect of the humanized antibody of the present invention on CD4 T cells was measured. Since HuB9 / HdB7, HuB10 / HdB6, and HuB10 / HdB7 can also prevent the conversion from AMP to adenosine, the levels of left shift are reversed (83.5%, 83.7%, and 83.4% respectively). The above-described effects were the same as those of the 10H5 antibody. These results indicate that the humanized antibody of the present invention can provide an activation effect on T cells.

[0134] Example 8: Inhibitory effect of the antibody of the present invention on tumor growth in a mouse model

[0135] Nude mice are known to be a natural mutation lacking T cells. Therefore, in the following experiments, nude mice were used as a model to test whether the antibody of the present invention can inhibit tumor growth in vivo.

[0136] Research has shown that the expression of CD73 may affect the tumorigenicity of MDA-MB-231 tumor cells (Clin Exp Metastasis (2007) 24:439-448, and Cancer Sci. 2010, vol. 101, 2561-2569). Therefore, MDA-MB-231 xenograft is a major animal model for testing the in vivo effect of anti-CD73 antibodies. From the perspective of the above facts, the following experiments were conducted to evaluate the in vivo activity of the antibody of the present invention.

[0137] 8-1. Inhibitory effect of the antibody of the present invention on tumor growth in a mouse model

[0138] To examine the inhibition of tumor growth by 10H5 and its humanized derivatives, HuB9 / HdB7, HuB10 / HdB6, and HuB10 / HdB7, a xenograft model was constructed using 25 Balb-C / Nu mice and then divided into 5 groups (5 mice / group). MDA-MB-231 cells were injected into the mice, and when the tumor size reached 170 mm 3When the tumors had grown to a certain size, mice were injected with 10H5 and three humanized antibodies, HuB9 / HdB7, HuB10 / HdB6, and HuB10 / HdB7, at 30 mpk (mg / kg) twice a week (for a total of 8 times).

[0139] The tumor sizes of the mice in each group were checked twice a week. The results are shown in Figure 7 and Table 1 below.

[0140]

Table 1

[0141] As shown in Figure 7 and Table 1, compared with the "vehicle (PBS)" group, tumor growth was inhibited in all cases with 10H5, HuB9 / HdB7, HuB10 / HdB6, and HuB10 / HdB7 treatments. The tumor growth inhibition rates (TGI) of 10H5, HuB9 / HdB7, HuB10 / HdB6, and HuB10 / HdB7 were 53.6%, 41.1%, 65.8%, and 49.5%, respectively. These results indicate that 10H5 and its humanized derivatives can all inhibit MDA-MB-231 tumor growth in the Balb-C / Nu mouse model.

[0142] 8-2. Inhibitory effect of the antibody of the present invention on CD73 activity in tumor tissue

[0143] To further analyze the ability of the 10H5 antibody to actually inhibit CD73 activity in tumor tissue, first, MDA-MB-231 cells formed tumors in Balb-C / Nu mice, and then different doses (3, 10, and 30 mpk) of 10H5 were injected into the tail veins of the mice. After collecting blood 24 hours later, the animals were sacrificed to remove the tumors, and the CD73 activity in each tumor tissue was evaluated. The concentration of the antibody in the blood was analyzed by MSD assay. The concentration of 10H5 in the blood is shown in Table 2. The concentration of 10H5 increased accurately according to the dose of the administered antibody (Table 2).

[0144]

Table 2

[0145] After freezing the tumor tissue into sections, the tumor tissue was stained in situ using the method of Mireia et al. (in situ CD73 activity assay: In Situ Identification of Ectoenzymes Involved in the Hydrolysis of Extracellular Nucleotides DOI: http: / / dx.doi.org / 10.5772 / intechopen.84495). The lighter the staining, the more inhibited the activity of CD73 by the antibody. As shown in Figure 8, the vehicle-treated tumor tissue was stained dark brown. However, as the dose of 10H5 treatment increased, the staining of the tumor gradually changed to light brown. The phenomenon of dose-dependent in situ CD73 inhibition was also observed with the humanized antibody.

[0146] Example 9: Epitope mapping

[0147] The following experiments were conducted to examine the epitope of the antibody of the present invention.

[0148] Five mouse-human domain-swapping proteins were constructed. As shown in Figure 9, the five proteins have the following sequences. 1. mCD73hF4: Exchanged amino acids 1-304 of mouse CD73 with the amino acid sequence of human CD73. 2. mCD73hF3: Exchanged amino acids 146-304 of mouse CD73 with the amino acid sequence of human CD73. 3. mCD73hF2: Exchanged amino acids 197-304 of mouse CD73 with the amino acid sequence of human CD73. 4. mCD73hF1: Exchanged amino acids 269-304 of mouse CD73 with the amino acid sequence of human CD73. 5. mCD73hF1-K206A-N211G: Not only exchanged amino acids 269-304 of mouse CD73 with the amino acid sequence of human CD73, but also substituted the 206th lysine residue and the 211th asparagine residue with alanine and glycine, respectively.

[0149] The above five mouse-human domain-swapping proteins and mouse CD73 were individually cloned into and expressed in F293 cells. Using an ELISA assay, the binding affinities of these proteins to anti-CD73 antibodies (e.g., orelumab and 10H5) were analyzed. The results are shown in Figure 10.

[0150] As shown in Figure 10, orelumab could bind to mouse CD73 and mCD73hF1 to mCD73hF4, but not to mCD73hF1-K206A-N211G (it is disclosed in U.S. Patent No. 9,938,356 that this mutation renders it unable to bind to MEDI9447). That is, orelumab can bind to the 206th lysine residue and the 211th asparagine residue of mouse CD73 and human CD73. On the other hand, 10H5 could bind to mCD73hF1 to mCD73hF4 and mCD73hF1-K206A-N211G, but not to mouse CD73. These results indicate that 10H5 has species specificity and binds to amino acids 269 to 304 of CD73. Also, it was shown that 10H5 is different from orelumab due to a binding site at positions 269 to 304 that is different from K206 and N211 recognized by orelumab.

[0151] To further clarify the positions 269 to 304 of CD73 to which the 10H5 antibody binds, the amino acid residues at positions 269, 274, 292, 296, 297, 302, and 304, which are different between human CD73 and mouse CD73, were individually mutated to confirm the binding epitope of the 10H5 antibody. Therefore, further mutations were introduced at the above positions of mCD73hF1 to construct the following eight proteins: mCD73hF1-S269A, mCD73hF1-K274Q, mCD73hF1-I292V, mCD73hF1-E296D, mCD73hF1-R297K, mCD73hF1-S302T, mCD73hF1-H304Y, and mCD73hF1-E296D-R297K. The positions of the mutation sites are shown in Figure 11.

[0152] The above eight proteins and mCD73hF1 were each cloned into and expressed in F293 cells, and then the binding affinity between the 10H5 antibody and these proteins was analyzed using ELISA. The results are shown in Figure 12.

[0153] As shown in Figure 12, 10H5 was able to completely bind to mCD73hF1, mCD73hF1-S269A, mCD73hF1-K274Q, mCD73hF1-I292V, mCD73hF1-S302T, and mCD73hF1-H304Y, but the binding affinity (KD = 2.49×10 -9 ) with mCD73hF1-R297K was only about 1 / 10 of that with mCD73hF1 (KD = 2.54×10 -10 ). When the 296th glutamic acid residue was mutated (i.e., mCD73hF1-E296D and mCD73hF1-E296D-R297K), the binding affinity between the 10H5 antibody and CD73 disappeared.

[0154] These results indicate that the antibody of the present invention (e.g., 10H5) binds to CD73 depending on the 296th glutamic acid residue and the 297th arginine residue.

[0155] Furthermore, two mutations were introduced into mouse CD73, substituting the 296th aspartic acid residue with a glutamic acid residue and the 297th lysine residue with an arginine residue (hereinafter referred to as "mouse CD73-D296E-K297R") (Figure 13). The binding affinity between 10H5 and mouse CD73 and mouse CD73-D296E-K297R was analyzed using ELISA. The results are shown in Figure 13.

[0156] As shown in Figure 13, the 10H5 antibody could not bind to mouse CD73 but could bind to mouse CD73-D296E-K297R. These results further indicate that the antibody of the present invention (e.g., 10H5) binds to CD73 depending on the 296th glutamic acid residue and the 297th arginine residue.

[0157] Example 10: Mapping the Binding Site of the Antibody of the Present Invention to CD73

[0158] 10-1. 3D Structural Model Simulation of the Interaction between CD73 and 10H5 scFv Antibody

[0159] To identify the amino acids of the 10H5 antibody regarding its interaction with E296 / R297 of the human CD73 antigen, 3D structural model simulation and logical intelligence analysis were used (Figure 14A).

[0160] As shown in Figure 14B, the 10H5 scFv antibody binds to the 296th glutamic acid residue and the 297th arginine residue of CD73. When the residues at these two positions were mutated to aspartic acid and lysine respectively, the binding between the 10H5 scFv antibody and CD73 disappeared. According to Figures 14A and 14B, E296 / R297 of human CD73 may be surrounded by four loops (i.e., CDR-H1, CDR-H2, CDR-H3, and CDR-L3) of the 10H5 antibody, and six potential amino acids on the antibody (W33 of CDR-H1, N35 of CDR-H1, Q50 of CDR-H2, F95 of CDR-H3, Y91 of CDR-L3, L96 of CDR-L3) that can generate hydrogen bonds, hydrophobic effects, and ion-ion interactions with CD73 (Figure 14C).

[0161] 10-2. Binding Site Mapping

[0162] To examine the binding site of the antibody of the present invention and confirm the prediction of the above 3D model, the following experiments were conducted.

[0163] To prove that the prediction of the above 3D model is correct, four mutant antibodies, including 10H5-CDRH1N35A, 10H5-CDRH3F95A, 10H5-CDRH1W33A, and 10H5-CDRL3Y91A, were constructed and expressed, and whether these antibodies had lost their functions was tested. The binding affinities of the above four mutant antibodies, 10H5 antibody, ocrelizumab, and IgG to mouse CD73 and mouse CD73hF1 were analyzed using an ELISA assay, respectively. The results are shown in Figure 15.

[0164] As shown in Figure 15, ocrelizumab can bind to mouse CD73 and mCD73hF1, while 10H5 can only bind to mCD73hF1. On the other hand, 10H5-CDRH1N35A, 10H5-CDRH3F95A, 10H5-CDRH1W33A, and 10H5-CDRL3Y91A cannot bind to both mouse CD73 and mCD73hF1. These results indicate that W33 of CDR-H1, N35 of CDR-H1, F95 of CDR-H3, and Y91 of CDR-L3 are actually involved in the binding of the antibody of the present invention (for example, 10H5) to human CD73. That is, the 3D structure model established in Example 15-1 is correct. W33 of CDR-H1, N35 of CDR-H1, Q50 of CDR-H2, F95 of CDR-H3, Y91 of CDR-L3, and L96 of CDR-L3 are important binding positions in the 10H5 antibody.

[0165] 10-3. Screening and selection of antibodies having an epitope similar to 10H5

[0166] (i) To screen for antibodies that can bind to human CD73 (E296 / R297) with the same amino acids as the 10H5 antibody and (ii) can inhibit the activity of CD73, six amino acids (i.e., W33 of CDR-H1, N35 of CDR-H1, Q50 of CDR-H2, F95 of CDR-H3, Y91 of CDR-L3, and L96 of CDR-L3) were randomly mutated to construct a single-chain phage display library. By binding of the D296E-K297R mutation to the mouse CD73 antigen, 16 phage clones (i.e., 109A03, 110A06, 110B03, 111B03, 111D04, 112A05, 112B02, 113D04, 114B07, 114C01, 114C12, 114D07, 114E08, 114G05, 116C07, 116G02) were selected, and the heavy and light chain variable domains were constructed into a full-length IgG4 antibody. The mutation positions and sequence ID numbers of the variable domains of either the heavy or light chain of the 16 phage clones are shown in Figure 16. The binding affinity (K D ) of the above-mentioned 16 antibodies to the mouse CD73 with the D296E-K297R mutation was analyzed by ELISA and shown in Figure 16.

[0167] Inhibitory effect of the antibody selected from the library constructed according to the 10-4.3D model on CD73 activity

[0168] In this experiment, the method for detecting the inhibitory effect of CD73 activity was carried out in the same manner as in Example 1 using MDA-MB-231 cancer cells, except that (i) the concentration of the antibody was 10 nM and (ii) the concentration of AMP was 100 μM. After incubating the cells in each group for 6 hours and 24 hours, a luminometer was used to measure the luminescence of each group. The results are shown in Figure 17A (109A03, 110A06, 110B03, 111B03, 111D04, 112A05, 112B02, 113D04) and Figure 17B (114B07, 114C01, 114C12, 114D07, 114E08, 114G05, 116C07, 116G02).

[0169] As shown in FIGS. 17A and 17B, after 6 hours of culture, "ocrelizumab" and the antibodies of the present invention (i.e., 109A03, 110A06, 110B03, 111B03, 111D04, 112A05, 112B02, 113D04, 114B07, 114C01, 114C12, 114D07, 114E08, 114G05, 116C07, 116G02) were able to inhibit the consumption of AMP, and the antibodies of the present invention had a better inhibitory effect than ocrelizumab. On the other hand, after 24 hours of culture, "ocrelizumab" was unable to inhibit the consumption of AMP. However, the antibodies of the present invention (i.e., 109A03, 110A06, 110B03, 111B03, 111D04, 112A05, 112B02, 113D04, 114B07, 114C01, 114C12, 114D07, 114E08, 114G05, 116C07, 116G02) were still able to significantly inhibit the consumption of AMP. These results indicate that the antibodies of the present invention have a better and longer-term inhibitory effect on the CD73 activity of MDA-MB-231 cancer cells compared to ocrelizumab.

[0170] (Appendix) (Appendix 1) A heavy chain variable domain comprising an HCDR1 region, an HCDR2 region, and an HCDR3 region, wherein the HCDR1 region comprises the amino acid sequence of SEQ ID NO: 4 having at most one mutation, the HCDR2 region comprises the amino acid sequence of SEQ ID NO: 5 having at most one mutation, and the HCDR3 region comprises the amino acid sequence of SEQ ID NO: 6 having at most one mutation, and A light chain variable domain comprising an LCDR1 region, an LCDR2 region, and an LCDR3 region, wherein the LCDR1 region comprises the amino acid sequence of SEQ ID NO: 7 having at most one mutation, the LCDR2 region comprises the amino acid sequence of SEQ ID NO: 8 having at most one mutation, and the LCDR3 region comprises the amino acid sequence of SEQ ID NO: 9 having at most one mutation, and An antibody or an antigen-binding fragment thereof comprising

[0171] (Appendix 2) The LCDR1 region contains the amino acid sequence of SEQ ID NO: 7, the LCDR2 region contains the amino acid sequence of SEQ ID NO: 8, and the LCDR3 region contains the amino acid sequence of SEQ ID NO: 9 having at most one mutation, the antibody or antigen-binding fragment thereof according to Supplementary Note 1.

[0172] (Supplementary Note 3) The HCDR1 region contains the amino acid sequence of SEQ ID NO: 4 having a mutation at the 8th position, The HCDR2 region contains the amino acid sequence of SEQ ID NO: 5 having a mutation at the 1st position, The HCDR3 region contains the amino acid sequence of SEQ ID NO: 6 having a mutation at the 1st position, The LCDR3 region contains the amino acid sequence of SEQ ID NO: 9 having a mutation at the 8th position, The antibody or antigen-binding fragment thereof according to Supplementary Note 2.

[0173] (Supplementary Note 4) The HCDR1 region contains the amino acid sequence of SEQ ID NO: 4 in which the 8th tryptophan residue is substituted with threonine, The HCDR2 region contains the amino acid sequence of SEQ ID NO: 5 in which the 1st glutamine residue is substituted with serine, arginine, threonine, and histidine, The HCDR3 region contains the amino acid sequence of SEQ ID NO: 6 in which the 1st phenylalanine residue is substituted with leucine, tyrosine, or isoleucine, The LCDR3 region contains the amino acid sequence of SEQ ID NO: 9 in which the 8th leucine residue is substituted with methionine, glycine, histidine, arginine, glutamine, and isoleucine, The antibody or antigen-binding fragment thereof according to Supplementary Note 3.

[0174] (Supplementary Note 5) The HCDR1 region contains the amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 15, The HCDR2 region contains the amino acid sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NOs: 16 to 19, The HCDR3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 20 to SEQ ID NO: 22, The antibody or antigen-binding fragment thereof according to Supplementary Note 1.

[0175] (Supplementary Note 6) The LCDR1 region comprises the amino acid sequence of SEQ ID NO: 7, The LCDR2 region comprises the amino acid sequence of SEQ ID NO: 8, The LCDR3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 23 to SEQ ID NO: 28, The antibody or antigen-binding fragment thereof according to Supplementary Note 1.

[0176] (Supplementary Note 7) The heavy chain variable domain is (i) an amino acid sequence having at least 95% homology with SEQ ID NO: 2, (ii) an amino acid sequence having at least 95% homology with SEQ ID NO: 10, and (iii) an amino acid sequence having at least 95% homology with SEQ ID NO: 11, The antibody or antigen-binding fragment thereof according to Supplementary Note 1, comprising an amino acid sequence selected from the group consisting of

[0177] (Supplementary Note 8) The light chain variable domain is (i) an amino acid sequence having at least 95% homology with SEQ ID NO: 3, (ii) an amino acid sequence having at least 95% homology with SEQ ID NO: 12, and (iii) an amino acid sequence having at least 95% homology with SEQ ID NO: 13, The antibody or antigen-binding fragment thereof according to Supplementary Note 1, comprising an amino acid sequence selected from the group consisting of

[0178] (Supplementary Note 9) The heavy chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 29 to SEQ ID NO: 38, and is the antibody or antigen-binding fragment thereof according to Supplementary Note 7.

[0179] (Supplementary Note 10) The light chain variable domain contains an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 39 to SEQ ID NO: 44, the antibody or antigen-binding fragment thereof according to Supplementary Note 8.

[0180] (Supplementary Note 11) (1) A linker peptide between the heavy chain variable domain and the light chain variable domain, (2) a heavy chain constant region, (3) a light chain constant region, and (4) an Fc region, the antibody or antigen-binding fragment thereof according to Supplementary Note 1, further comprising one or more of these.

[0181] (Supplementary Note 12) A single-chain antibody fragment, a bispecific antibody, a single-domain antibody, a nanobody, a chimeric antibody, or a partially or fully humanized antibody, the antibody or antigen-binding fragment thereof according to Supplementary Note 1.

[0182] (Supplementary Note 13) Further linked to a drug conjugate to form an antibody-drug conjugate (ADC), or further linked to a second antibody or a second antigen-binding fragment to form a bispecific antibody, the antibody or antigen-binding fragment thereof according to Supplementary Note 1.

[0183] (Supplementary Note 14) Further comprising a fragment obtained from IgG1, IgG2, IgG3, or IgG4, the antibody or antigen-binding fragment thereof according to Supplementary Note 1.

[0184] (Supplementary Note 15) Specifically binds to CD73, the antibody or antigen-binding fragment thereof according to Supplementary Note 1.

[0185] (Supplementary Note 16) The CD73 contains the amino acid sequence of SEQ ID NO: 14, the antibody or antigen-binding fragment thereof according to Supplementary Note 15.

[0186] (Supplementary Note 17) The CD73 is an antibody or an antigen-binding fragment thereof as described in Appendix 15, which contains the amino acid sequence of SEQ ID NO: 1.

[0187] (Appendix 18) An antibody or an antigen-binding fragment thereof as described in Appendix 15, which binds to at least one of the 296th glutamate residue and the 297th arginine residue of the CD73.

[0188] (Appendix 19) An antibody or an antigen-binding fragment thereof as described in Appendix 15, which binds to both the 296th glutamate residue and the 297th arginine residue of the CD73.

[0189] (Appendix 20) An antibody or an antigen-binding fragment thereof that specifically binds to at least one of the 296th glutamate residue and the 297th arginine residue of human CD73.

[0190] (Appendix 21) An antibody or an antigen-binding fragment thereof as described in Appendix 20, which binds to both the 296th glutamate residue and the 297th arginine residue of the human CD73.

[0191] (Appendix 22) The CD73 is an antibody or an antigen-binding fragment thereof as described in Appendix 20, which contains the amino acid sequence of SEQ ID NO: 14.

[0192] (Appendix 23) The CD73 is an antibody or an antigen-binding fragment thereof as described in Appendix 20, which contains the amino acid sequence of SEQ ID NO: 1.

[0193] (Appendix 24) An antibody or an antigen-binding fragment thereof as described in Appendix 20, which is a single-chain antibody fragment, a single-domain antibody, a nanobody, a chimeric antibody, or a partially or fully humanized antibody.

[0194] (Appendix 25) The antibody or antigen-binding fragment thereof according to Appendix 20, which is further linked to a drug conjugate to form an antibody-drug conjugate (ADC), or is further linked to a second antibody or antigen-binding fragment to form a bispecific antibody.

[0195] (Appendix 26) The antibody or antigen-binding fragment thereof according to Appendix 20, further comprising a fragment obtained from IgG1, IgG2, IgG3, or IgG4.

[0196] (Appendix 27) (i) The antibody or antigen-binding fragment thereof according to any one of Appendices 1 to 26, the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, the vector containing the nucleic acid molecule, the recombinant host cell containing the nucleic acid molecule, or the recombinant host cell containing the vector, and (ii) a pharmaceutically acceptable carrier. A pharmaceutical composition comprising the same.

[0197] (Appendix 28) The pharmaceutical composition according to Appendix 27, which is used for inhibiting CD73.

[0198] (Appendix 29) The pharmaceutical composition according to Appendix 27, further comprising one or more other immunotherapeutic agents.

[0199] (Appendix 30) The pharmaceutical composition according to Appendix 29, wherein the other immunotherapeutic agent is a PD-1 antagonist, a PD-L1 antagonist, or a CTLA-4 antagonist.

[0200] (Appendix 31) The pharmaceutical composition according to any one of Appendices 27 to 30, which is used for activating T cells, activating B cells, activating NK cells, and / or inhibiting cancer cells.

[0201] (Appendix 32) The pharmaceutical composition according to any one of Appendices 27 to 30, which is used for treating, ameliorating, and / or preventing cancer.

[0202] (Supplementary Note 33) The cancer is the pharmaceutical composition according to Supplementary Note 32, selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, gallbladder cancer, prostate cancer, ovarian cancer, chronic or acute lymphocytic leukemia, bladder cancer, brain tumor, kidney carcinoma, head and neck squamous cell carcinoma, glioblastoma, esophageal cancer, pancreatic cancer, renal carcinoma, oral cancer, lung cancer, colon adenocarcinoma, malignant melanoma, and lymphoma.

[0203] (Supplementary Note 34) The pharmaceutical composition according to Supplementary Note 27 for use in inhibiting CD73 in a subject in need of inhibition of CD73.

[0204] (Supplementary Note 35) The pharmaceutical composition for use according to Supplementary Note 34, administered in combination with one or more other immunotherapeutic agents.

[0205] (Supplementary Note 36) The pharmaceutical composition for use according to Supplementary Note 35, wherein the other immunotherapeutic agent is a PD-1 antagonist, a PD-L1 antagonist, or a CTLA-4 antagonist.

[0206] (Supplementary Note 37) The pharmaceutical composition for use according to any one of Supplementary Notes 34 to 36, used for activating T cells, activating B cells, activating NK cells, and / or inhibiting cancer cells.

[0207] (Supplementary Note 38) The pharmaceutical composition for use according to any one of Supplementary Notes 34 to 36, used for treating, ameliorating, and / or preventing cancer.

[0208] (Supplementary Note 39) The cancer is a pharmaceutical composition for use as described in Supplementary Note 38, selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, gallbladder cancer, prostate cancer, ovarian cancer, chronic or acute lymphocytic leukemia, bladder cancer, brain tumor, kidney carcinoma, head and neck squamous cell carcinoma, glioblastoma, esophageal cancer, pancreatic cancer, renal carcinoma, oral cancer, lung cancer, colon adenocarcinoma, malignant melanoma, and lymphoma.

Claims

**Claim 1** A heavy chain variable domain comprising an HCDR1 region, an HCDR2 region, and an HCDR3 region, wherein the HCDR1 region comprises the amino acid sequence of SEQ ID NO: 4 having at most one mutation, the HCDR2 region comprises the amino acid sequence of SEQ ID NO: 5 having at most one mutation, and the HCDR3 region comprises the amino acid sequence of SEQ ID NO: 6 having at most one mutation, and A light chain variable domain comprising an LCDR1 region, an LCDR2 region, and an LCDR3 region, wherein the LCDR1 region comprises the amino acid sequence of SEQ ID NO: 7 having at most one mutation, the LCDR2 region comprises the amino acid sequence of SEQ ID NO: 8 having at most one mutation, and the LCDR3 region comprises the amino acid sequence of SEQ ID NO: 9 having at most one mutation, and An antibody or an antigen-binding fragment thereof comprising the above. **Claim 2** The antibody or antigen-binding fragment thereof according to claim 1, wherein the LCDR1 region comprises the amino acid sequence of SEQ ID NO: 7, the LCDR2 region comprises the amino acid sequence of SEQ ID NO: 8, and the LCDR3 region comprises the amino acid sequence of SEQ ID NO: 9 having at most one mutation. **Claim 3** The HCDR1 region comprises the amino acid sequence of SEQ ID NO: 4 having a mutation at the 8th position, The HCDR2 region comprises the amino acid sequence of SEQ ID NO: 5 having a mutation at the 1st position, The HCDR3 region comprises the amino acid sequence of SEQ ID NO: 6 having a mutation at the 1st position, The LCDR3 region comprises the amino acid sequence of SEQ ID NO: 9 having a mutation at the 8th position, The antibody or antigen-binding fragment thereof according to claim 2. **Claim 4** The HCDR1 region comprises the amino acid sequence of SEQ ID NO: 4 in which the 8th tryptophan residue is replaced by threonine, The HCDR2 region comprises the amino acid sequence of SEQ ID NO: 5 in which the 1st glutamine residue is replaced by serine, arginine, threonine, and histidine, The HCDR3 region comprises the amino acid sequence of SEQ ID NO: 6 in which the 1st phenylalanine residue is replaced by leucine, tyrosine, or isoleucine, The LCDR3 region comprises the amino acid sequence of SEQ ID NO: 9 in which the 8th leucine residue is replaced by methionine, glycine, histidine, arginine, glutamine, and isoleucine, The antibody or antigen-binding fragment thereof according to claim 3. **Claim 5** The HCDR1 region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 15, The HCDR2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NOs: 16 to 19, The HCDR3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6 and SEQ ID NOs: 20 to 22, The antibody or antigen-binding fragment thereof according to claim 1.

6. The LCDR1 region comprises the amino acid sequence of SEQ ID NO: 7, The LCDR2 region comprises the amino acid sequence of SEQ ID NO: 8, The LCDR3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NOs: 23 to 28, The antibody or antigen-binding fragment thereof according to claim 1.

7. The heavy chain variable domain is (i) an amino acid sequence having at least 95% homology with SEQ ID NO: 2, (ii) an amino acid sequence having at least 95% homology with SEQ ID NO: 10, and (iii) an amino acid sequence having at least 95% homology with SEQ ID NO: 11, The antibody or antigen-binding fragment thereof according to claim 1, comprising an amino acid sequence selected from the group consisting of.

8. The light chain variable domain is (i) an amino acid sequence having at least 95% homology with SEQ ID NO: 3, (ii) an amino acid sequence having at least 95% homology with SEQ ID NO: 12, and (iii) an amino acid sequence having at least 95% homology with SEQ ID NO: 13, The antibody or antigen-binding fragment thereof according to claim 1, comprising an amino acid sequence selected from the group consisting of.

9. The heavy chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NOs: 29 to 38, the antibody or antigen-binding fragment thereof according to claim 7.

10. The light chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NOs: 39 to 44, the antibody or antigen-binding fragment thereof according to claim 8.

11. (1) a linker peptide between the heavy chain variable domain and the light chain variable domain, (2) a heavy chain constant region, (3) a light chain constant region, and (4) an Fc region, the antibody or antigen-binding fragment thereof according to claim 1, further comprising one or more of.

12. The antibody or antigen-binding fragment thereof according to claim 1, which is a single-chain antibody fragment, bispecific antibody, single-domain antibody, nanobody, chimeric antibody, or partially or fully humanized antibody.

13. The antibody or antigen-binding fragment thereof according to claim 1, which is further conjugated to a drug conjugate to form an antibody-drug conjugate (ADC), or further conjugated to a second antibody or a second antigen-binding fragment to form a bispecific antibody.

14. The antibody or antigen-binding fragment thereof according to claim 1, which further comprises a fragment obtained from IgG1, IgG2, IgG3, or IgG4.

15. The antibody or antigen-binding fragment thereof according to claim 1, which specifically binds to CD73.

16. The antibody or antigen-binding fragment thereof according to claim 15, wherein the CD73 comprises the amino acid sequence of SEQ ID NO:

14.

17. The antibody or antigen-binding fragment thereof according to claim 15, wherein the CD73 comprises the amino acid sequence of SEQ ID NO:

1.

18. The antibody or antigen-binding fragment thereof according to claim 15, which binds to at least one of the 296th glutamate residue and the 297th arginine residue of CD73.

19. The antibody or antigen-binding fragment thereof according to claim 15, which binds to both the 296th glutamate residue and the 297th arginine residue of CD73.

20. An antibody or antigen-binding fragment thereof that specifically binds to at least one of the 296th glutamate residue and the 297th arginine residue of human CD73.

21. The antibody or antigen-binding fragment thereof according to claim 20, which binds to both the 296th glutamate residue and the 297th arginine residue of the human CD73.

22. The antibody or antigen-binding fragment thereof according to claim 20, wherein the CD73 comprises the amino acid sequence of SEQ ID NO:

14.

23. The antibody or antigen-binding fragment thereof according to claim 20, wherein the CD73 comprises the amino acid sequence of SEQ ID NO:

1.

24. The antibody or antigen-binding fragment thereof according to claim 20, which is a single-chain antibody fragment, single-domain antibody, nanobody, chimeric antibody, or partially or fully humanized antibody.

25. The antibody or antigen-binding fragment thereof according to claim 20, which is further linked to a drug conjugate to form an antibody-drug conjugate (ADC), or is further linked to a second antibody or antigen-binding fragment to form a bispecific antibody.

26. The antibody or antigen-binding fragment thereof according to claim 20, further comprising a fragment obtained from IgG1, IgG2, IgG3, or IgG4.

27. A pharmaceutical composition comprising: (i) the antibody or antigen-binding fragment thereof according to any one of claims 1 to 26, a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, a vector comprising the nucleic acid molecule, a recombinant host cell comprising the nucleic acid molecule, or a recombinant host cell comprising the vector; and (ii) a pharmaceutically acceptable carrier.

28. The pharmaceutical composition according to claim 27, which is used for inhibiting CD73.

29. The pharmaceutical composition according to claim 27, further comprising one or more other immunotherapeutic agents.

30. The pharmaceutical composition according to claim 29, wherein the other immunotherapeutic agent is a PD-1 antagonist, a PD-L1 antagonist, or a CTLA-4 antagonist.

31. The pharmaceutical composition according to any one of claims 27 to 30, which is used for activating T cells, activating B cells, activating NK cells, and / or inhibiting cancer cells.

32. The pharmaceutical composition according to any one of claims 27 to 30, which is used for treating, ameliorating, and / or preventing cancer.

33. The cancer is selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, gallbladder cancer, prostate cancer, ovarian cancer, chronic or acute lymphocytic leukemia, bladder cancer, brain tumor, kidney carcinoma, head and neck squamous cell carcinoma, glioblastoma, esophageal cancer, pancreatic cancer, renal carcinoma, oral cancer, lung cancer, colon adenocarcinoma, malignant melanoma, and lymphoma. The pharmaceutical composition according to claim 32.

34. The pharmaceutical composition according to claim 27, which is used for inhibiting CD73 in a subject in need of CD73 inhibition.

35. The pharmaceutical composition for use according to claim 34, wherein one or more other immunotherapeutic agents are administered in combination.

36. The pharmaceutical composition for use according to claim 35, wherein the other immunotherapeutic agent is a PD-1 antagonist, a PD-L1 antagonist, or a CTLA-4 antagonist.

37. A pharmaceutical composition for use according to any one of claims 34 to 36, which is used for activating T cells, activating B cells, activating NK cells, and / or inhibiting cancer cells.

38. A pharmaceutical composition for use according to any one of claims 34 to 36, which is used for treating, ameliorating and / or preventing cancer.

39. The pharmaceutical composition for use according to claim 38, wherein the cancer is selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, gallbladder cancer, prostate cancer, ovarian cancer, chronic or acute lymphocytic leukemia, bladder cancer, brain tumor, kidney carcinoma, head and neck squamous cell carcinoma, glioblastoma, esophageal cancer, pancreatic cancer, renal carcinoma, oral cancer, lung cancer, colon adenocarcinoma, malignant melanoma, and lymphoma.

Images