Antibody specifically binding to CD73 and use thereof

Abstract

The present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to CD73 and a pharmaceutical composition for the prevention or treatment of cancer, comprising same. The antibody of the present invention binds not only to human CD73 but also to non-human (e.g., Cynomolgus monkey, Mouse) CD73, can be stably produced with high purity, and exhibits the ability to bind to and inhibit CD73, which is equal or greater than that of oleclumab, which is a commercially available anti-CD73 antibody. In particular, an anti-CD73 single domain antibody of the present invention exhibits about 1.5-5 times higher CD73 enzyme inhibitory activity than oleclumab, and thus can function as an excellent alternative therapeutic agent for patients who exhibit low reactivity to oleclumab.

Description

Antibodies specifically binding to CD73 and uses thereof

[0001] The present invention relates to a novel antibody or antigen-binding fragment thereof that specifically binds to CD73 and a method for treating or diagnosing cancer using the same.

[0002]

[0003] Cancer immunotherapy is a method of treating cancer by leveraging the body's immune system. Cancer immunotherapy can trigger the immune system to attack cancer cells by targeting antigens, such as proteins on their surface. It can also enhance immune cell activity by controlling the tumor microenvironment, which allows cancer cells to evade immune defenses.

[0004] The tumor microenvironment is an environment in which cancer cells proliferate and evolve, including fibroblasts, blood vessels, lymphatic vessels, immune cells, extracellular matrix, and adipocytes within the cancer tissue. In this regard, research has reported that the ATP-AMP-adenosine-A2AR / A2BR signaling pathway can regulate immune cell activity in the tumor microenvironment (S. Vigano et al., Front Immunol (2019) 10:925). Specifically, when adenosine binds to A2AR / A2BR receptors on the surfaces of tumor cells or immune cells, immune suppressor cells such as regulatory T cells and tumor-associated macrophages (TAMs) are activated, and the activity of cytotoxic T cells and NK cells is suppressed. At this time, a method of activating immunity within the tumor microenvironment by inhibiting the signaling pathway by treating with a substance that targets CD39, an ectonucleotidase that converts ATP to AMP, CD73, an ectonucleotidase that converts AMP to adenosine, or A2AR / A2BR receptors that bind to adenosine is being studied.

[0005] Meanwhile, CD73 is known to be involved in the production of adenosine, is highly expressed in cancer cells, and induces immunosuppressive effects. It is expressed on many tumor cells, including leukemia, bladder cancer, glioma, glioblastoma, ovarian cancer, melanoma, prostate cancer, thyroid cancer, esophageal cancer, and breast cancer. CD73 is also known to be expressed on the surface of immunosuppressive cells (including regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). In particular, high expression of CD73 has been reported to be associated with angiogenesis, invasion, chemotherapy resistance, tumor metastasis, and shorter survival in various tumors, including breast cancer and melanoma.

[0006]

[0007] Numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the present invention.

[0008]

[0009] The present inventors have devoted extensive research efforts to develop an effective anti-CD73 antibody that significantly inhibits tumor survival and growth and provides reliable information for tumor diagnosis by binding with high affinity to CD73, a protein highly expressed in cancer cells within the tumor microenvironment and a key regulator of angiogenesis and immune evasion mechanisms. As a result, the present invention was completed by discovering numerous antibodies that recognize CD73 on the surface of cancer cells with high affinity and specificity and by clearly elucidating the structure of their antigen recognition sites.

[0010] Accordingly, the purpose of the present invention is to provide an antibody or an antigen-binding fragment thereof that specifically binds to CD73 and a nucleic acid molecule encoding the same.

[0011] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

[0012]

[0013] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0014]

[0015] To achieve the above object, one aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to a CD73 protein, comprising a heavy chain variable region comprising an HCDR1 region selected from the group consisting of sequences 1 to 9 and sequences 78 to 80 of the sequence listing; an HCDR2 region selected from the group consisting of sequences 10 to 18 and sequences 81 to 83 of the sequence listing; and an HCDR3 region selected from the group consisting of sequences 19 to 28 and sequences 84 to 86 of the sequence listing.

[0016] The term “antibody” as used herein refers to a peptide that recognizes and specifically binds to a specific epitope of the CD73 protein, and includes not only a complete antibody form but also an antigen-binding fragment (antibody fragment) of a full-length antibody molecule. A complete antibody has a structure having two full-length light chains and two full-length heavy chains, each light chain being linked to a heavy chain by a disulfide bond. The heavy chain constant region is of the gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and has subclasses of gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1), and alpha2 (α2). The light chain constant region is of the kappa (κ) and lambda (λ) types.

[0017] The term “antigen-binding fragment of an antibody” as used herein means a fragment having significant antigen-antibody binding function within a full-length antibody molecule, and includes Fab, F(ab'), F(ab')2, Fv, and nanobody.

[0018] Among antibody fragments, Fab has a structure with variable regions of the light and heavy chains, constant regions of the light chain, and the first constant region (CH1) of the heavy chain, and has one antigen-binding site.

[0019] F(ab') differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are generated when the cysteine ​​residues in the hinge region of F(ab') form a disulfide bond. Fv is the smallest antibody fragment that has only a heavy chain variable region and a light chain variable region. A two-chain Fv has a heavy chain variable region and a light chain variable region linked non-covalently, and a single-chain Fv has a heavy chain variable region and a single chain variable region covalently linked through a peptide linker or directly linked at the C-terminus, so that it can form a dimer-like structure like a two-chain Fv.

[0020] The term “heavy chain” as used herein encompasses both full-length heavy chains and fragments thereof, which comprise a variable region domain (VH) comprising an amino acid sequence having sufficient variable region sequence to confer specificity for an antigen, and three constant region domains (CH1, CH2, and CH3).

[0021] As used herein, the term “CDR (complementarity determining region)” refers to the amino acid sequence of the hypervariable region of the immunoglobulin heavy and light chains. The heavy chain (HCDR1, HCDR2, and HCDR3) and the light chain (LCDR1, LCDR2, and LCDR3) each contain three CDRs, which provide key contact residues for antibody binding to an antigen or epitope.

[0022] The scope of the antibodies or antigen-binding fragments of the present invention includes variants having conservative amino acid substitutions in the CDR regions. In addition, the antibodies or antigen-binding fragments of the present invention may include variants of the amino acid sequences set forth in the attached sequence listing, as long as they can specifically recognize CD73. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody, and are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Based on these considerations, arginine, lysine, and histidine; Alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine can be considered biologically functional equivalents.

[0023] Furthermore, amino acid substitutions in proteins that do not alter the overall activity of the molecule are well known in the art (H. Neurathet et al., The Proteins, Academic Press, New York, 1979). The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0024] Considering the mutations having the above-described biological equivalent activity, the amino acid sequence constituting the antibody of the present invention is interpreted to also include sequences showing substantial identity with the sequences listed in the sequence listing. The substantial identity means a sequence showing at least 61% homology, in one specific example 70% homology, in another specific example 80% homology, and in yet another specific example 90% homology, when the sequence of the present invention and any other sequence are aligned to the greatest extent possible and the aligned sequences are analyzed using an algorithm commonly used in the art. Alignment methods and algorithms for sequence comparison are disclosed in Huang et al. Comp. Appl. BioSci. (1992) 8:155-65 and Pearson et al. Meth. Mol. Biol. (1994) 24:307-31, etc.

[0025] According to a specific embodiment of the present invention, the antibody or antigen-binding fragment thereof additionally comprises a light chain variable region comprising an LCDR1 region selected from the group consisting of sequences 39 to 48 of the sequence listing; an LCDR2 region selected from the group consisting of sequences 49 to 57 of the sequence listing; and an LCDR3 region selected from the group consisting of sequences 58 to 67 of the sequence listing.

[0026] The term “light chain” as used herein encompasses both full-length light chains and fragments thereof, which comprise a variable domain (VL) and a constant domain (CL) comprising an amino acid sequence having sufficient variable domain sequence to confer specificity for an antigen.

[0027] According to a specific embodiment of the present invention, the antibody or antigen-binding fragment thereof is any one selected from the following (a) to (j):

[0028] (a) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1 of sequence 1, HCDR2 of sequence 10, and HCDR3 of sequence 19; and a light chain variable region comprising LCDR1 of sequence 39, LCDR2 of sequence 49, and LCDR3 of sequence 58;

[0029] (b) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1 of sequence 2, HCDR2 of sequence 11, and HCDR3 of sequence 20; and a light chain variable region comprising LCDR1 of sequence 40, LCDR2 of sequence 50, and LCDR3 of sequence 59;

[0030] (c) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1 of sequence 3, HCDR2 of sequence 12, and HCDR3 of sequence 21; and a light chain variable region comprising LCDR1 of sequence 41, LCDR2 of sequence 51, and LCDR3 of sequence 60;

[0031] (d) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1 of sequence 4, HCDR2 of sequence 13, and HCDR3 of sequence 22; and a light chain variable region comprising LCDR1 of sequence 42, LCDR2 of sequence 52, and LCDR3 of sequence 61;

[0032] (e) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1 of sequence 5, HCDR2 of sequence 14, and HCDR3 of sequence 23; and a light chain variable region comprising LCDR1 of sequence 43, LCDR2 of sequence 53, and LCDR3 of sequence 62;

[0033] (f) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1 of sequence 6, HCDR2 of sequence 15, and HCDR3 of sequence 24; and a light chain variable region comprising LCDR1 of sequence 44, LCDR2 of sequence 54, and LCDR3 of sequence 63;

[0034] (g) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1 of sequence 7, HCDR2 of sequence 16, and HCDR3 of sequence 25; and a light chain variable region comprising LCDR1 of sequence 45, LCDR2 of sequence 49, and LCDR3 of sequence 64;

[0035] (h) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1 of sequence 8, HCDR2 of sequence 17, and HCDR3 of sequence 26; and a light chain variable region comprising LCDR1 of sequence 46, LCDR2 of sequence 55, and LCDR3 of sequence 65;

[0036] (i) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1 of sequence 3, HCDR2 of sequence 12, and HCDR3 of sequence 27; and a light chain variable region comprising LCDR1 of sequence 47, LCDR2 of sequence 56, and LCDR3 of sequence 66; and

[0037] (j) An antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1 of sequence 9, HCDR2 of sequence 18, and HCDR3 of sequence 28; and a light chain variable region comprising LCDR1 of sequence 48, LCDR2 of sequence 57, and LCDR3 of sequence 67.

[0038]

[0039] According to a specific embodiment of the present invention, (a) includes a heavy chain variable region of sequence number 29 and a light chain variable region of sequence number 68.

[0040] According to a specific embodiment of the present invention, the above (b) includes a heavy chain variable region of sequence number 30 and a light chain variable region of sequence number 69.

[0041] According to a specific embodiment of the present invention, the above (c) includes a heavy chain variable region of sequence number 31 and a light chain variable region of sequence number 70.

[0042] According to a specific embodiment of the present invention, the above (d) includes a heavy chain variable region of sequence number 32 and a light chain variable region of sequence number 71.

[0043] According to a specific embodiment of the present invention, the above (e) includes a heavy chain variable region of sequence number 33 and a light chain variable region of sequence number 72.

[0044] According to a specific embodiment of the present invention, the above (f) includes a heavy chain variable region of sequence number 34 and a light chain variable region of sequence number 73.

[0045] According to a specific embodiment of the present invention, the (g) includes a heavy chain variable region of sequence number 35 and a light chain variable region of sequence number 74.

[0046] According to a specific embodiment of the present invention, the above (h) includes a heavy chain variable region of sequence number 36 and a light chain variable region of sequence number 75.

[0047] According to a specific embodiment of the present invention, the above (i) includes a heavy chain variable region of sequence number 37 and a light chain variable region of sequence number 76.

[0048] According to a specific embodiment of the present invention, the above (j) includes a heavy chain variable region of sequence number 38 and a light chain variable region of sequence number 77.

[0049]

[0050] According to a specific embodiment of the present invention, the antigen-binding fragment is a single domain antibody.

[0051] As used herein, the term "single-domain antibody", also called a nanobody, refers to a fragment of a full-length antibody that contains a single variable domain of the antibody in monomeric form. Single-domain antibodies typically have a molecular weight of about 12 to 15 kDa, which is much smaller than the molecular weight of a fully functional full-length antibody of about 150 to 160 kDa. However, they selectively bind to a target antigen like full-length antibodies.

[0052] More specifically, the single domain antibody comprises an HCDR1 region selected from the group consisting of sequences 78 to 80 of the sequence listing; an HCDR2 region selected from the group consisting of sequences 81 to 83 of the sequence listing; and an HCDR3 region selected from the group consisting of sequences 84 to 86 of the sequence listing.

[0053] According to a specific embodiment of the present invention, the single domain antibody is any one selected from the following (i) to (iii):

[0054] (i) a single domain antibody comprising HCDR1 of sequence 78 of the sequence listing, HCDR2 of sequence 81 of the sequence listing, and HCDR3 of sequence 84 of the sequence listing;

[0055] (ii) a single domain antibody comprising HCDR1 of sequence 79, HCDR2 of sequence 82, and HCDR3 of sequence 85; and

[0056] (iii) A single domain antibody comprising HCDR1 of sequence 80, HCDR2 of sequence 83, and HCDR3 of sequence 86.

[0057] Most specifically, the single domain antibody comprises an amino acid sequence selected from the group consisting of sequences 87 to 89 of the sequence listing.

[0058] According to a specific embodiment of the present invention, the above (i) includes the amino acid sequence of sequence number 87 of the sequence listing.

[0059] According to a specific embodiment of the present invention, the above (ii) includes the amino acid sequence of sequence number 88 of the sequence listing.

[0060] According to a specific embodiment of the present invention, the above (iii) includes the amino acid sequence of sequence number 89.

[0061]

[0062] According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof that specifically binds to the CD73 protein of the present invention as described above.

[0063] The term “nucleic acid molecule” as used herein encompasses DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base moieties are modified (Uhlman et al., Chemical Reviews (1990) 90: 543-584). The sequence of a nucleic acid molecule encoding a full-length antibody of the present invention, or a heavy chain or light chain variable region, may be modified, and the modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0064] The nucleic acid molecule of the present invention is also interpreted to include a sequence that exhibits substantial identity to the nucleotide sequence of the present invention. The substantial identity refers to a nucleotide sequence that exhibits at least 80% homology, in one specific example at least 90% homology, and in another specific example at least 95% homology, when the nucleotide sequence of the present invention is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0065] According to a specific embodiment of the present invention, the nucleic acid molecule used in the present invention may be an mRNA molecule, and more specifically, may be an in vitrotranscribed (IVT) mRNA.

[0066] When mRNA is used as the nucleic acid molecule of the present invention, various modifications may be made, such as changing the length of the poly(A) tail or substituting some adenine bases; modifying the 5'cap; applying one or more modified nucleosides, in order to improve the expression (translation) efficiency of the antibody or antigen-binding fragment of the present invention. Modified nucleosides that may be applied include, but are not limited to, N1-methylpseudouridine, pseudouridine, 2-thiouridine, 5-methyluridine, 5-methylcytidine, and 5-methoxyuridine, and any modified nucleoside known in the art to be capable of reducing the immunogenicity of the mRNA molecule may be applied.

[0067] According to another aspect of the present invention, the present invention provides a gene delivery vehicle comprising the nucleic acid molecule of the present invention as described above.

[0068] According to the present invention, the antibody of the present invention or an antigen-binding fragment thereof may be recombinantly obtained in vitro by expressing a nucleic acid molecule encoding the antibody of the present invention or an antigen-binding fragment thereof in a host cell, or the nucleic acid molecule itself may be delivered into a patient's body as a pharmacological ingredient to produce the antibody of the present invention or an antigen-binding fragment thereof in the body through the patient's expression system.

[0069] As used herein, the term “express” means artificially introducing a gene using a gene vector to cause a target cell to express an exogenous gene or to increase the natural expression level of an endogenous gene, thereby making the gene replicable as an extrachromosomal element or by completion of chromosomal integration within the target cell. Therefore, the term “expression” has the same meaning as “transformation,” “transfection,” or “transduction.” More specifically, in the present invention, “express” means causing a target cell to artificially express an exogenous gene.

[0070] As used herein, the term "gene carrier" refers to any means of transporting a gene into a cell. Gene transfer is synonymous with transduction of a gene into a cell, and gene transfer at the tissue level is synonymous with spread of a gene. Accordingly, the gene delivery system of the present invention may be described as a gene transduction system and a gene spread system.

[0071] The gene delivery system of the present invention may be comprised in the form of an expression cassette, which is a polynucleotide structure containing all elements necessary for the autonomous expression of a gene to be introduced. The expression cassette typically includes a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, which are operably linked to the gene. The expression cassette may be in the form of a self-replicating expression vector. As used herein, the term “operably linked” means a functional linkage between a nucleic acid expression control sequence, such as a promoter, a signal sequence, or an array of transcription factor binding sites, and another nucleic acid sequence, whereby the expression control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0072] The recombinant vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001).

[0073] The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when the vector of the present invention is an expression vector and uses a prokaryotic cell as a host, it generally includes a strong promoter capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. When E. coli (e.g., HB101, BL21, DH5α, etc.) is used as a host cell, E. The promoter and operator region of the tryptophan biosynthetic pathway of E. coli (Yanofsky, CJ Bacteriol. 158:1018-1024 (1984)) and the left-hand promoter of phage λ (pLλ promoter, Herskowitz, I. et al., Ann. Rev. Genet. 14:399-445 (1980)) can be used as regulatory regions. When Bacillus is used as the host cell, the promoter of the toxin protein gene of Bacillus thuringiensis (Appl. Environ. Microbiol. 64:3932-3938 (1998); Mol. Gen. Genet. 250:734-741 (1996)) or any promoter that can be expressed in Bacillus can be used as regulatory regions.

[0074] The vector expressing the antibody of the present invention can be a vector system in which the light chain and the heavy chain are simultaneously expressed from a single vector, or a system in which the light chain and the heavy chain are each expressed from separate vectors. In the latter case, the two vectors are introduced into a host cell through co-transformation and targeted transformation. Co-transformation is a method in which vector DNA encoding the light chain and the heavy chain are simultaneously introduced into a host cell, and then cells expressing both the light chain and the heavy chain are selected. Targeted transformation is a method in which cells transformed with a vector containing a light chain (or heavy chain) are selected, and the selected cells expressing the light chain are transformed again with a vector containing a heavy chain (or light chain), thereby finally selecting cells expressing both the light chain and the heavy chain.

[0075] According to another aspect of the present invention, the present invention provides a host cell transformed with the gene delivery vehicle of the present invention described above.

[0076] Any host cell known in the art can be used as the host cell capable of stably and continuously cloning and expressing the gene delivery system of the present invention, and for example, in the case of prokaryotic cells, includes, but is not limited to, Bacillus strains such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis or Staphylococcus (e.g., Staphylococcus carnosus).

[0077] Suitable eukaryotic host cells that can be used in the present invention may be, for example, fungi such as Aspergillus species, yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, insect-derived cells, plant-derived cells, or mammalian-derived cells such as CHO (Chinese hamster ovary) cells.

[0078] Host cell culture for the production of antibodies or antigen-binding fragments thereof can be performed using appropriate media and culture conditions known in the art. Those skilled in the art can easily adjust this culture process to suit the selected strain, and specific examples of culture methods are disclosed in various references (e.g., James M. Lee, Biochemical Engineering, Prentice-Hall International Editions, 138-176). Antibodies obtained by culturing host cells can be used in an unpurified state, or they can be further purified to high purity using various conventional methods, such as dialysis, salt precipitation, and chromatography. When utilizing chromatography, the type and order of columns can be selected from ion exchange chromatography, size exclusion chromatography, and affinity chromatography, depending on the characteristics of the antibody and the host cell culture method.

[0079]

[0080] According to another aspect of the present invention, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CD73 protein via at least one epitope selected from the group consisting of (a) an epitope comprising an amino acid sequence of Sequence Listing No. 92; (b) an epitope comprising an amino acid sequence of Sequence Listing No. 93; and (c) an epitope comprising an amino acid sequence of Sequence Listing No. 94.

[0081] According to the present invention, the antibody of the present invention or an antigen-binding fragment thereof recognizes an epitope different from the epitopes (YLPYKVLPVGDEV and KLKTLNVN) of Oleclumab, a commercially available anti-CD73 monoclonal antibody, and thus can function as an excellent alternative treatment for patients showing resistance or low responsiveness to Oleclumab.

[0082] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the antibody or antigen-binding fragment thereof, nucleic acid molecule or gene delivery system of the present invention as an active ingredient.

[0083] The term “prevention” as used herein means inhibiting the occurrence of a disease or condition in a subject who has not been diagnosed as having the disease or condition but is susceptible to such disease or condition.

[0084] As used herein, the term “treatment” means (a) suppressing the development of a disease, condition, or symptom; (b) alleviating a disease, condition, or symptom; or (c) eliminating a disease, condition, or symptom. When the composition of the present invention is administered to a subject, it inhibits adenosine production by cancer cells that specifically and highly express CD73 on their surface, suppresses angiogenesis, and blocks immune evasion mechanisms, thereby inhibiting the progression of, eliminating, or alleviating symptoms caused by cancer. Therefore, the composition of the present invention may be a composition for treating these diseases on its own, or may be administered together with other pharmacological ingredients and used as an adjuvant treatment for the diseases. Accordingly, the terms “treatment” or “therapeutic agent” as used herein include the meaning of “adjuvant treatment” or “adjuvant treatment agent.”

[0085] As used herein, the term “administration” or “administer” refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the body of the subject.

[0086] In the present invention, the term “therapeutically effective amount” means the content of a composition containing a pharmacological ingredient in the composition sufficient to provide a therapeutic or preventive effect to a subject to whom the pharmaceutical composition of the present invention is to be administered, and includes a “prophylactically effective amount”.

[0087] The term “subject” as used herein includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus macaque. Specifically, the subject of the present invention is a human.

[0088] According to a specific embodiment of the present invention, the cancer that can be prevented or treated with the composition of the present invention is a CD73-related cancer. As used herein, the term "CD73-related cancer" refers to a malignant tumor that specifically overexpresses CD73 on the surface of cancer cells, and whose symptoms can be improved or eliminated by inhibiting the expression or activity of CD73.

[0089] The term “inhibition of expression or activity” as used herein means causing a decrease in the activity or expression of the CD73 protein, such that the activity or expression of CD73 becomes undetectable or is present at an insignificant level, as well as reducing the activity or expression of CD73 to such an extent that the conversion of AMP to adenosine and the immunosuppressive action by CD73 are significantly inhibited.

[0090] More specifically, cancers that can be prevented or treated with the composition of the present invention are selected from the group consisting of leukemia, bladder cancer, brain tumor, melanoma, thyroid cancer, esophageal cancer, lung cancer, prostate cancer, breast cancer, colon cancer, stomach cancer, head and neck cancer, kidney cancer, pancreatic cancer, liver cancer, neuroblastoma, retinoblastoma, salivary gland cancer, lymphoma, cervical cancer, ovarian cancer, and testicular cancer.

[0091]

[0092] According to another aspect of the present invention, the present invention provides a composition for detecting CD73 expressing cells, comprising the antibody of the present invention or an antigen-binding fragment thereof as an active ingredient.

[0093] According to the present invention, the antibody of the present invention specifically recognizes cells, particularly cancer cells, that specifically express CD73 on their surface, and thus can be usefully utilized to selectively isolate, detect, and quantify CD73-expressing cells within a biological sample containing various cell populations. Accordingly, the term "detection" is used synonymously with "isolation," "sorting," and "quantification."

[0094]

[0095] According to another aspect of the present invention, the present invention provides a composition for diagnosing cancer comprising the antibody of the present invention or an antigen-binding fragment thereof as an active ingredient.

[0096] The term “diagnosis” as used herein includes determining an individual’s susceptibility to a particular disease, determining whether an individual currently has a particular disease, and determining a prognosis for an individual with a particular disease.

[0097] The term “diagnostic composition” as used herein means an integrated mixture or device including a means for measuring the expression level of CD73 protein to determine whether or not a pathological condition predictable by the expression level of CD73, such as the occurrence of CD73-related cancer, or to predict the possibility of occurrence, and may also be expressed as a “diagnostic kit.”

[0098] According to another aspect of the present invention, the present invention provides a method for preventing or treating cancer, comprising administering to a subject an antibody of the present invention or an antigen-binding fragment thereof as described above.

[0099] According to another aspect of the present invention, the present invention provides a use of the antibody of the present invention or an antigen-binding fragment thereof described above for the prevention or treatment of cancer.

[0100]

[0101] The features and advantages of the present invention are summarized as follows:

[0102] (a) The present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to CD73 and a nucleic acid molecule encoding the same.

[0103] (b) The antibody of the present invention binds with high affinity to CD73, a glycoprotein specifically highly expressed on the surface of tumor cells, thereby suppressing excessive angiogenesis in the tumor microenvironment, inhibiting adenosine production, and reducing the activity of immunosuppressive cells such as regulatory T cells (Treg) while enhancing the activity of immune cells such as T cells, thereby efficiently inducing the death of tumor cells.

[0104] (c) The antibody of the present invention exhibits CD73 binding affinity and inhibitory activity equivalent to or greater than that of Oleclumab, a commercially available anti-CD73 antibody, and in particular, the anti-CD73 single domain antibody of the present invention exhibits CD73 inhibitory activity that is approximately 1.5-5 times superior to that of Oleclumab, thereby functioning as an excellent alternative treatment for patients who exhibit low responsiveness to Oleclumab.

[0105]

[0106] Figure 1 is a graph showing the results of confirming the soluble CD73 enzyme activity inhibition ability of the produced monoclonal antibodies CSA0055 to CSA0074 and oleclumab.

[0107] Figure 2 is a graph showing the results of confirming the soluble CD73 enzyme activity inhibition ability of the produced monoclonal antibodies CSA0075 to CSA0135 and oleclumab.

[0108] Figure 3 is a graph showing the results of confirming the Soluble CD73 enzyme activity inhibition ability of the produced monoclonal antibodies CSA0160 to CSA0247 and oleclumab.

[0109] Figure 4 is a graph showing the results of confirming the Soluble CD73 enzyme activity inhibition ability of 10 single-domain antibodies produced and oleclumab.

[0110] Figure 5a is a diagram showing the epitope mapping results of a monoclonal antibody (CSA0060) and a single domain antibody (AHF10240, AHP04167).

[0111] Figure 5b is a diagram showing the epitope of oleclumab.

[0112] Figure 6 is a graph showing the proliferation of CD8+ T cells after treatment with vehicle (hIgG4), monoclonal antibody (CSA0060), and oleculumab in CD8+ T cells whose activity was inhibited by CD73-adenosine.

[0113] Figure 7 is a graph showing the proliferation of CD4+ T cells after treatment with vehicle (hIgG4), monoclonal antibody (CSA0060), and oleculumab in CD4+ T cells whose activity was inhibited by CD73-adenosine.

[0114]

[0115] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0116]

[0117] Example 1. Screening of anti-CD73 antibodies (mAb)

[0118] Monoclonal anti-CD73 antibody is a human antibody library Ymax of Y Biologics Co., Ltd. ⓡ-Custom-made using an antibody screening service selected from ABL. The human antibody screening process used human CD73 antigen (CD73-His, CD73-Fc) and a human single-chain variable fragment (scFv) phage library, and a total of 24 rounds of panning were performed using immunotube panning, bead panning, and cell panning methods, and 10,600 colonies were selected. Monophage ELISA was performed on the 10,600 selected colonies to select 1,475 hit clones, and 227 independent clones were selected from the 1,475 hit clones through base sequence analysis. Subsequently, Phage FACS was performed to screen 61 monoclonal phage antibodies that bind to CD73-positive cells among the 227 independent clones.

[0119] Afterwards, ELISA binding affinity test, Cell FACS, and soluble CD73 blockade assay were performed for 61 monoclonal phage antibodies after IgG conversion.

[0120]

[0121] Example 2. Production of anti-CD73 antibody (mAb)

[0122] In order to convert the monoclonal phage antibody screened in Example 1 from scFv form to IgG form, an IgG conversion process was additionally requested from Y Biologics Co., Ltd. For the IgG conversion process, the base sequence of the 61 heavy chain variable region was cloned into pNATVH (Y Biologics Co., Ltd.) using the restriction enzyme SfiI / NheI sites to produce the N293F HC vector, and the base sequence of the light chain variable region was cloned into pNATVL (Y Biologics Co., Ltd.) using the restriction enzyme SfiI / BglⅡ sites to produce the N293F LC vector.

[0123] The cloned N293F HC vector and N293F LC vector were co-transfected into HEK293F cells, and the supernatant was collected on the 7th day. After removing cells and floating substances through centrifugation and a 0.22 ㎛ top filter, the supernatant was collected and purified with protein A beads.

[0124] Afterwards, protein A chromatography and SDS-PAGE were performed on the above 61 monoclonal antibodies to analyze productivity such as concentration, purity, and production amount (Table 1).

[0125] No.ID농도(㎎ / ㎖)단백질량(㎎)생산량(㎎ / L)1CSA00550.060.163.892CSA00560.651.5538.803CSA00570.420.8521.224CSA00580.481.2631.525CSA00591.483.5488.556CSA00601.002.4060.067CSA00610.541.2330.778CSA00621.904.19104.659CSA00631.053.6691.5910CSA00641.513.4686.5811CSA00651.665.32132.9012CSA00660.140.348.5913CSA00670.561.2932.3514CSA00681.012.3257.9615CSA00691.715.98149.4616CSA00701.072.6766.6317CSA00710.621.5438.5918CSA00720.702.4561.3219CSA00730.712.2856.9220CSA00741.122.5964.6721CSA00750.792.7669.0722CSA00761.202.8771.8223CSA00771.876.55163.6424CSA00780.020.061.4025CSA00791.293.8896.9826CSA00800.973.3884.4927CSA00810.280.5714.1528CSA00821.524.72118.0929CSA00831.353.2380.7130CSA00840.040.092.3131CSA00851.786.05151.3132CSA00860.431.0425.9633CSA01270.080.204.9134CSA01281.223.6791.8135CSA01292.365.91147.6736CSA01300.250.6215.4137CSA01312.321.1628.9638CSA01321.935.80145.0539CSA01331.745.23130.8540CSA01341.924.79119.7741CSA01351.634.07101.6842CSA01601.043.9699.1143CSA01610.301.1628.9344CSA01620.953.5989.8645CSA01630.431.3032.5446CSA01641.013.8495.9647CSA01651.274.81120.3048CSA01661.033.9398.2149 CSA01671.294.89122.2950CSA02360.130.358.6751CSA02370.481.2932.2552CSA02380.180.4912.3753CSA02 390.110.317.7554CSA02400.371.0025.0655CSA02410.170.4611.5756CSA02420.010.030.8257CSA02430.270 .7217.9358CSA02440.080.215.1459CSA02450.010.030.8760CSA02460.20.5513.8161CSA02470.280.7619.11.

[0126]

[0127] Example 3. Characterization of anti-CD73 (mAb) antibody

[0128] Example 3-1. ELISA binding affinity test

[0129] His-tagged CD73 was added to 100 ng of Pierce™ Nickel Coated Plates (#15142, Thermo Scientific™) wells and coated at room temperature for 1 hour. Human CD73-His (Y-Biologics Co., Ltd.) was used as an antigen. After washing three times with washing solution (PBS containing 0.05% tween-20 (#P9416, Sigma-Aldrich)), 300 ㎕ of 4% skim milk (#232120, BD Difco, USA) / PBS blocking solution was added according to the conditions and reacted at room temperature for 1 hour to block nonspecific binding.

[0130] Each well was added with 61 monoclonal antibodies serially diluted from 100 nM to 0.5 pM and incubated at room temperature for 1 hour. After washing, HRP-labeled anti-human Kappa antibody (#A7164, Sigma) was added and incubated at room temperature for 1 hour. At this time, the anti-CD73 antibody Olecluamb was used as a control antibody. After washing five times with washing solution, TMB substrate solution (#T0440-1L, Sigma) was added and incubated at room temperature for more than 3 minutes under light protection to confirm color development. The reaction was stopped by adding sulfuric acid solution (#S1478, Samchun). The absorbance was measured at a wavelength of 450 nm using a spectrophotometer (#GM3000, Promega or SpectraMaxM5, Molecular devices). The values ​​of the binding affinity of the above 61 monoclonal antibodies and control antibodies to CD73-His are shown in Tables 2 to 4 below.

[0131] Binding affinity KDR squareIgG-Oleclumab 1.2×10 -11 0.9912CSA00551.8×10 -8 0.6369CSA00564.2×10 -11 0.9152CSA00573.6×10 -9 0.9771CSA00583.3×10 -9 0.9106CSA00597.8×10 -9 0.9355CSA00605.6×10 -11 0.9943CSA00612.2×10 -9 0.8108CSA00621.5×10 -9 0.9779CSA00632.2×10 -9 0.9810CSA00641.5×10 -10 0.9800CSA00651.0×10 -9 0.9674CSA00664.1×10 -10 0.9646CSA00676.4×10 -9 0.7846CSA00682.1×10 -80.8236CSA00697.4×10 -10 0.9810CSA00701.5×10 -9 0.9831CSA00712.0×10 -9 0.9846CSA00723.6×10 -10 0.9669CSA00734.2×10 -10 0.9857CSA00743.2×10 -8 0.7176CSA00751.5×10 -9 0.9677CSA00761.6×10 -9 0.9750CSA00771.2×10 -9 0.9832CSA00783.0×10 -9 0.9842CSA00791.7×10 -10 0.9921CSA00803.0×10 -9 0.9501CSA00815.9×10 -11 0.9981CSA00822.5×10 -9 0.7756CSA00834.2×10 -9 0.6213CSA00846.2×10 -9 0.9531CSA00851.2×10 -9 0.9755CSA00862.5×10 -9 0.9688CSA0127-0.09492CSA01281.0×10 -8 0.8201CSA01291.0×10 -8 0.5881CSA01303.9×10 -11 0.9979CSA01311.0×10 -8 0.09028CSA01322.0×10 -11 0.9903CSA01331.9×10 -9 0.9352CSA01341.0×10 -8 0.1965CSA01357.5×10 -10 0.9272

[0132]

[0133] 구분결합 친화도KDR squareIgG-Oleclumab1.0×10 -11 0.9926CSA01609.6×10 -110.9980CSA01616.4×10 -10 0.9909CSA01623.1×10 -9 0.9894CSA01635.6×10 -9 0.8355CSA01649.5×10 -10 0.9932CSA01653.4×10 -9 0.9758CSA01669.5×10 -10 0.9753CSA01675.1×10 -11 0.9933

[0134] Binding affinity KDR squareIgG-Oleclumab4.4×10 -12 0.9970CSA02364.1×10 -9 0.9795CSA02372.9×10 -9 0.9910CSA02383.6×10 -10 0.9746CSA02394.3×10 -10 0.9910CSA02401.1×10 -9 0.9928CSA02411.6×10 -9 0.9961CSA0242--CSA02439.4×10 -10 0.9615CSA0244--CSA0245--CSA02462.8×10 -9 0.7182CSA02471.9×10 -11 0.9929

[0135]

[0136] Example 3-2. CELL FACS

[0137] FACS analysis was performed to determine whether monoclonal antibodies (CSA0055 to CSA0086, CSA0127 to CSA0136, CSA0236 to CSA0247) bind to CD73-positive cells (MDA-MB-231) and CD73-negative cells (Jurkat). 0.5 × 10 of CD73-positive cells (MDA-MB-231) and CD73-negative cells (Jurkat) were sampled. 6Cells were prepared and reacted with the 53 produced monoclonal antibodies prepared at a concentration of 2 μg / mL at 4°C for 30 minutes. After that, the cells were washed three times with PBS (#LB001-02, Welgene) containing 2% fetal bovine serum (#26140-079, Thermo), reacted with anti-human IgG antibody (#FI-3000, Vectorlabs) or PE-anti-hIgG antibody (#555787, BD) conjugated with fluorescein isothiocyanate (FITC) fluorescein, and washed in the same manner as above. The cells were suspended in 0.5 mL of PBS containing 2% FBS and analyzed using a FACSCanto Ⅱ flow cytometer (BD Biosciences, USA).

[0138] As a result, it was confirmed that 32 monoclonal antibodies (CSA0055 to CSA0086) first bound to CD73-positive cells (MDA-MB-231), and it was confirmed that CSA0071, CSA0073, CSA0075, and CSA0079 monoclonal anti-CD73 antibodies also bound to CD73-negative cells (Jurkat).

[0139] Additionally, among the nine monoclonal antibodies (CSA0127 to CSA0135), it was confirmed that eight antibodies, excluding the CSA0127 monoclonal antibody, bound to CD73-positive cells. In addition, it was confirmed that the control antibody (Oleclumab) and the CSA0133 monoclonal antibody nonspecifically bound to CD73-negative cells.

[0140] Among the 12 monoclonal antibodies (CSA0236 to CSA0247), it was confirmed that the remaining 10 antibodies, excluding the CSA0244 and CSA0245 monoclonal antibodies, bound to CD73-positive cells.

[0141]

[0142] Example 3-3. Soluble CD73 blockade assay

[0143] To evaluate the inhibitory ability of the 61 monoclonal antibodies above to inhibit soluble CD73 enzyme activity, experiments were conducted according to the manufacturer's manual using the AMP-Glo™ Assay kit (Promega, V5012). Specifically, the 61 monoclonal antibodies and the control antibody, Oleclumab, were each diluted to 0.05 ng / mL and treated with 2.5 μL per well of a 96-well plate, and 12.5 μL of 10 μM AMP was added to each well. Subsequently, 10 μL of 2.5 ng / mL recombinant human CD73 protein was added to each well and incubated at room temperature for 10 minutes.

[0144] The reaction was terminated by treating each well with 25 μl of AMP-Glo™ Reagent (Promega), shaking for 2 minutes, and incubating for 1 hour at room temperature. The AMP Detection solution was treated to each well, shaking for 2 minutes, and incubating for 1 hour at room temperature. The luminescence was measured using the GloMax®-Multi Detection System (Promega).

[0145] As a result, among 61 monoclonal antibodies, 10 clones (CSA0060, CSA0065, CSA0073, CSA0074, CSA0081, CSA0132, CSA0135, CSA0238, CSA0240, CSA0247) that effectively inhibit Soluble CD73 enzyme activity were selected (Figs. 1 to 3).

[0146] The sequence numbers and specific amino acid sequences of the 10 selected clones are shown in Tables 5 and 6, respectively.

[0147] ID sequence number HCDR1HCDR2HCDR3All VHLCDR1LCDR2LCDR3All VLCSA0060110192939495868CSA0065211203040505969CSA0073312213141516070CSA0074413223242526171CSA0081514233343536272CSA0132615243444546373CSA0135716253545496474CSA0238817263646556575CSA0240312273747566676CSA0247918283848576777Constant regionHeavy chain constant region: 90 Light chain constant region: 91

[0148]

[0149]

[0150] Example 4. Screening of anti-CD73 single-domain antibodies (sdAb)

[0151] Single-domain anti-CD73 antibodies were custom-designed using Genscript Probio's Single Domain Antibody Discovery Services. The single-domain antibody screening process involved immunizing non-immunized alpacas with human CD73 antigen at approximately two-week intervals for four to six rounds, according to Genscript Probio's protocol. Blood samples were collected during each immunization and the immune response was observed by ELISA and FACS. To construct a single-domain antibody library, total RNA was extracted from lymphocytes obtained from immunized alpacas, and PCR was performed using primers (forward and reverse primers) to amplify the VHH fragment. The PCR products were inserted into the Llama library vector to construct a single-domain antibody library.

[0152] Solid-phase panning was performed using the constructed single-domain antibody library to select colonies. Monophage ELISA and Phage FACS were performed on the selected colonies to screen for 10 purified CD73 single-domain antibodies with high binding affinity to human CD73.

[0153]

[0154] Example 5. Production of anti-CD73 single domain antibody (sdAb)

[0155] The base sequence of the single-domain antibody screened in Example 4 was cloned into the pTT5 vector (Genscript) and co-transfected into Expi293F™ cells, followed by culture. The culture medium was then collected and purified using protein A beads.

[0156] Afterwards, protein A chromatography and SDS-PAGE were performed on the above 10 single domain antibodies to analyze productivity such as concentration, purity, and production amount (Table 7).

[0157] Classification Concentration (mg / ml) Volume (ml) Protein mass (mg) Purity (%) AHF102353.7234.215.63799% AHF102391.4024.25.88899% AHF102403.1833.812.09599% AHF102463.4514.013.80499% AHF102941.0 554.24.43199%AHF102952.2634.09.05299%AHP040481.7774.07.10895%AHP0414 62.6074.210.94999%AHP041482.9994.212.59699%AHP041673.0924.012.36899%

[0158]

[0159] Example 6. Characterization of anti-CD73 single-domain antibody (sdAb)

[0160] Example 6-1. ELISA:EC 50

[0161] 1 μg / ml human CD73 (ACRO Biosystems) and 1 μg / ml mouse CD73 (ACRO Biosystems) were added to each well of a 96-well plate (100 μl each) and coated overnight at 4°C. After washing, a blocking solution of 3% skim milk / PBS was added and reacted at room temperature for 1 hour to block nonspecific binding.

[0162] Each well was filled with 100 μl of 10 single-domain antibodies serially diluted at a certain dilution factor and incubated at room temperature for 1 hour and 30 minutes. A washing process was performed and HRP-labeled MonoRab TM Rabbit anti-camelid VHH Cocktail (A02016-200, GenScript) was added and reacted at room temperature for 45 minutes. After washing, TMB substrate solution was added and reacted at room temperature for more than 3 minutes in a light-shielded manner to confirm color development. The absorbance was measured at a wavelength of 450 nm using a spectrophotometer. The EC50 of the above 10 single-domain antibodies was used to determine the degree of binding to human CD73 and mouse CD73. 50 The values ​​were calculated and shown in Table 8 below.

[0163] Classification hCD73 EC 50 (㎍ / ㎖)mCD73 EC 50 (㎍ / ㎖)AHF102350.0016440.006139AHF102390.0020460.002623AHF102400. 0023770.006455AHF102460.0012660.001944AHF102940.0020450.002094AH F102950.0028010.01015AHP040480.52863.075AHP041460.0019660.002108AHP041480.0016420.002070AHP041670.0017210.004032NCNANABlankNANA

[0164]

[0165] Example 6-2. Soluble CD73 blockade assay

[0166] To evaluate the inhibitory ability of the above 10 single-domain antibodies to soluble CD73 enzyme activity, an experiment was conducted using the AMP-Glo™ Assay kit (Promega, V5012) according to the manufacturer's manual. Specifically, the 10 single-domain antibodies and the control, oleculumab, were diluted to 0.05 ng / mL and treated with 2.5 μL per well of a 96-well plate, and 12.5 μL of 10 μM AMP was added to each well. Subsequently, 10 μL of 2.5 ng / mL recombinant human CD73 protein was added to each well and incubated at room temperature for 10 minutes.

[0167] The reaction was terminated by treating each well with 25 μl of AMP-Glo™ Reagent (Promega), shaking for 2 minutes, and incubating for 1 hour at room temperature. Each well was treated with AMP Detection solution, shaking for 2 minutes, and incubating for 1 hour at room temperature. The luminescence was measured using the GloMax®-Multi Detection System (Promega), and IC 50 The values ​​were calculated using GraphPad Prism 8.0 software (Table 9).

[0168] Separation IC 50 (ng / mL)Oleclumab45.7AHF1023516.9AHF1023920.4AHF102405.5AHF10246Ambiguous (~ 3.661e+0.14)AHF1029429.9AHF102956.4AHP04048703AHP04146889AHP041481242AHP041677.5

[0169] As a result, it was confirmed that three single-domain antibodies (AHF10235, AHF10240, and AHP0416) significantly inhibited Soluble CD73 enzyme activity more than the control, oleclumab (Fig. 4). IC of three monoclonal antibodies (AHF10235, AHF10240, and AHP0416) 50The values ​​were confirmed to be 16.9 ng / ㎖ (0.2 nM), 5.5 ng / ㎖ (0.06 nM), and 7.5 ng / ㎖ (0.08 nM), respectively, and the IC of oleclumab 50 The values ​​were confirmed to be 45.7 ng / ㎖ (0.3 nM) respectively.

[0170] By synthesizing the results of Examples 5, 6-1, and 6-2, three single-domain antibodies (AHF10235, AHF10240, and AHP04167) were selected from ten single-domain antibodies. The sequence numbers and specific amino acid sequences of the selected single-domain antibodies are shown in Table 10.

[0171] IDVHCDR1CDR2CDR3AHP04167EVQLVESGGGLVQAGGSLLRLSCVVSGRTFSNYHMGWFRQAPGKEREFVAVIGRSGGGPTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAGDWRNSDSPSKLKPVYDYWGQ GTQVTVSS(87)GRTFSNYH(78)IGRSGGPT(81)AGDWRNSDSPSKLKPVYDY(84)AHF10235QVQVQESGGGLVQAGDSLRLSCAASGRSISRYNMGWFRQAPGKEREFVAAIISRSGGVTYYADSVKGRLTISRDNAK SAVYLQMNSLKPEDTAVYYCAADWRNSDSPSPLIIVYDYWGQGTQVTVSS(88)GRSISRYN(79)ISRSGGVT(82)AADWRNSDSPSPLIIVYDY(85)AHF10240EVQLVESGGGVVQTGGSLRLSCAASGRTFTHLAM GWFRQAPGKEREFVAAIISNSGAGTQFADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADWGTRDSPSKLNAVYDYWGRGTQVTVSS(89)GRTFTHLA(80)ISNSGAGT(83)AADWGTRDSPSKLNAVYDY(86)

[0172]

[0173] Example 7. Identification of the binding site of anti-CD73 antibodies (mAb, sdAb) to CD73

[0174] To determine the binding site for CD73 of the monoclonal antibody (CSA0060) and single domain antibody (AHF10240, AHP04167) finally selected in Examples 3-3 and 6-2 above, AbSolute™ epitope mapping analysis using peptide microarray was requested from JinOn Biotech.

[0175] Using the amino acid sequence of CD73, we generated an antigen-specific peptide array containing 160,000 spots composed of linear overlapping fragments (1,120 spots) and discontinuous structural fragments (158,880 spots). Spots containing peptides with high affinity for the test antibodies were scanned, visualized, and analyzed using a GenePix 4100A Microarray Scanner (Molecular Devices).

[0176] As a result, we confirmed that the monoclonal antibody (CSA0060) and single-domain antibodies (AHF10240, AHP04167) recognize the highly flexible α-helical linker (INKWRIK) between the NC-terminal domains of CD73. In addition, we confirmed that the single-domain antibodies (AHF10240, AHP04167) additionally recognize one epitope (IWFTVYK) and two epitopes (IWFTVYK and VVQAYAFGKYLGYLK), respectively (Fig. 5a).

[0177] The control antibody, olekulumab, was confirmed to recognize two epitopes (YLPYKVLPVGDEV and KLKTLNVN) (James C. Geoghegan et al (2016), mAbs, 8:3, 454-467) (Fig. 5b). Therefore, olekulumab has a different binding site from the monoclonal antibody and single-domain antibody of the present invention.

[0178]

[0179] Example 8. Confirmation of reinvigoration of T cells inhibited by CD73-adenosine by anti-CD73 monoclonal antibody (CSA0060)

[0180] To determine whether anti-CD73 monoclonal antibody (CSA0060) restores CD8+ T cell and CD4+ T cell activity inhibited by CD73-adenosine, human PBMCs were cultured overnight in TexMACS™ medium (Miltenyi Biotec) containing 5% human serum (Sigma-Aldrich®), 1% penicillin / streptomycin, and 5 mM β-mercaptoethanol (ThermoFisher Scientific) at 37°C in a humidified atmosphere with 5% CO2. The following day, cells were labeled with CTV and cultured at a density of 1 × 10 6 Cells were seeded into 96-well plates at a density of 10 cells / ml. Cells were stimulated using αCD3 / CD28 dynabeads (ratio 1:1).

[0181] Vehicle (hIgG4), anti-CD73 monoclonal antibody (CSA0060), and oleculumab were treated, respectively. The final concentration of the treated drugs was 50 nM, and each well was added. Subsequently, 500 μM AMP was added to each well, and the cells were cultured in a humidified atmosphere at 37°C and 5% CO2 for 3 days.

[0182] To assess T cell functional activation, cells were restimulated with αCD3 / CD28 dynabeads (ratio 1:1) and 50 nM or 500 μM AMP in the presence of Brefeldin A for 4 h at 37°C in a humidified atmosphere with 5% CO2. To analyze Teff cell proliferation by FACS, the cells were stained for surface markers using the following antibodies: anti-CD3-PerCP-Cy5.5 (Clone: ​​UCHT1, BD Biosciences), anti-CD8-Alexa Fluor700 (Clone: ​​RPA-T8, Biolegend Inc.), and Fixable Viability Dye eFluor 780 (Invitrogen Inc.). Data on the stained cells were acquired using a Symphony A3 instrument and analyzed using FlowJo software.

[0183] As a result, when AMP was treated and vehicle (hIgG4) was treated, CD8+ T cell proliferation was reduced. On the other hand, when anti-CD73 antibody (CSA0600) was treated, CD8+ T cell proliferation was confirmed to increase. Meanwhile, when oleclumab was treated alone, there was no tendency for CD8+ T cell proliferation to increase (Fig. 6).

[0184] Furthermore, when AMP was treated with vehicle (hIgG4), CD4+ T cell proliferation was reduced. Conversely, when anti-CD73 antibody was treated alone, CD4+ T cell proliferation increased. Meanwhile, when oleclumab was treated, there was no trend toward increased CD4+ T cell proliferation (Fig. 7).

[0185]

[0186] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to a CD73 protein, comprising a heavy chain variable region comprising an HCDR1 region selected from the group consisting of sequences 1 to 9 and sequences 78 to 80 of the sequence listing; an HCDR2 region selected from the group consisting of sequences 10 to 18 and sequences 81 to 83 of the sequence listing; and an HCDR3 region selected from the group consisting of sequences 19 to 28 and sequences 84 to 86 of the sequence listing.

2. An antibody or antigen-binding fragment thereof according to claim 1, characterized in that it further comprises a light chain variable region comprising an LCDR1 region selected from the group consisting of sequences Nos. 39 to 48 of the sequence listing; an LCDR2 region selected from the group consisting of sequences Nos. 49 to 57 of the sequence listing; and an LCDR3 region selected from the group consisting of sequences Nos. 58 to 67 of the sequence listing.

3. In the second paragraph, the antibody or antigen-binding fragment thereof is characterized by being any one selected from the following (a) to (j): (a) an antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising HCDR1 of sequence 1, HCDR2 of sequence 10 and HCDR3 of sequence 19; and a light chain variable region comprising LCDR1 of sequence 39, LCDR2 of sequence 49 and LCDR3 of sequence 58; (b) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1 of sequence 2, HCDR2 of sequence 11 and HCDR3 of sequence 20; and a light chain variable region comprising LCDR1 of sequence 40, LCDR2 of sequence 50 and LCDR3 of sequence 59; (c) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1 of sequence 3, HCDR2 of sequence 12 and HCDR3 of sequence 21; and a light chain variable region comprising LCDR1 of sequence 41, LCDR2 of sequence 51 and LCDR3 of sequence 60; (d) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1 of sequence 4, HCDR2 of sequence 13 and HCDR3 of sequence 22; and a light chain variable region comprising LCDR1 of sequence 42, LCDR2 of sequence 52 and LCDR3 of sequence 61; (e) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1 of sequence 5, HCDR2 of sequence 14 and HCDR3 of sequence 23; and a light chain variable region comprising LCDR1 of sequence 43, LCDR2 of sequence 53 and LCDR3 of sequence 62; (f) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1 of sequence 6, HCDR2 of sequence 15 and HCDR3 of sequence 24; and a light chain variable region comprising LCDR1 of sequence 44, LCDR2 of sequence 54 and LCDR3 of sequence 63; (g) an antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising HCDR1 of sequence 7, HCDR2 of sequence 16 and HCDR3 of sequence 25; and a light chain variable region comprising LCDR1 of sequence 45, LCDR2 of sequence 49 and LCDR3 of sequence 64; (h) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1 of sequence number 8, HCDR2 of sequence number 17 and HCDR3 of sequence number 26; and a light chain variable region comprising LCDR1 of sequence number 46, LCDR2 of sequence number 55 and LCDR3 of sequence number 65; (i) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1 of sequence 3, HCDR2 of sequence 12 and HCDR3 of sequence 27; and a light chain variable region comprising LCDR1 of sequence 47, LCDR2 of sequence 56 and LCDR3 of sequence 66; and (j) an antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising HCDR1 of sequence 9, HCDR2 of sequence 18, and HCDR3 of sequence 28; and a light chain variable region comprising LCDR1 of sequence 48, LCDR2 of sequence 57, and LCDR3 of sequence 67.

4. In paragraph 3, The above (a) is an antibody or an antigen-binding fragment thereof, characterized in that it comprises a heavy chain variable region of sequence No. 29 of the sequence listing and a light chain variable region of sequence No. 68 of the sequence listing; The above (b) is an antibody or an antigen-binding fragment thereof, characterized in that it comprises a heavy chain variable region of sequence number 30 of the sequence listing and a light chain variable region of sequence number 69 of the sequence listing; The above (c) is an antibody or an antigen-binding fragment thereof, characterized in that it comprises a heavy chain variable region of sequence number 31 of the sequence listing and a light chain variable region of sequence number 70 of the sequence listing; The above (d) is an antibody or an antigen-binding fragment thereof, characterized in that it comprises a heavy chain variable region of sequence number 32 of the sequence listing and a light chain variable region of sequence number 71 of the sequence listing; The above (e) is an antibody or an antigen-binding fragment thereof, characterized in that it comprises a heavy chain variable region of sequence number 33 in the sequence listing and a light chain variable region of sequence number 72 in the sequence listing; The above (f) is an antibody or an antigen-binding fragment thereof, characterized in that it comprises a heavy chain variable region of sequence No. 34 of the sequence listing and a light chain variable region of sequence No. 73 of the sequence listing; The above (g) is an antibody or an antigen-binding fragment thereof, characterized in that it comprises a heavy chain variable region of sequence No. 35 of the sequence listing and a light chain variable region of sequence No. 74 of the sequence listing; The above (h) is an antibody or an antigen-binding fragment thereof, characterized in that it comprises a heavy chain variable region of sequence No. 36 of the sequence listing and a light chain variable region of sequence No. 75 of the sequence listing; The above (i) is an antibody or an antigen-binding fragment thereof characterized by comprising a heavy chain variable region of sequence number 37 of the sequence listing and a light chain variable region of sequence number 76 of the sequence listing; or The above (j) is an antibody or an antigen-binding fragment thereof, characterized in that it comprises a heavy chain variable region of sequence number 38 of the sequence listing and a light chain variable region of sequence number 77 of the sequence listing.

5. An antibody or an antigen-binding fragment thereof, characterized in that the antigen-binding fragment in claim 1 is a single-domain antibody.

6. An antibody or antigen-binding fragment thereof, characterized in that in claim 5, the single domain antibody comprises an HCDR1 region selected from the group consisting of sequences Nos. 78 to 80 of the sequence listing; an HCDR2 region selected from the group consisting of sequences Nos. 81 to 83 of the sequence listing; and an HCDR3 region selected from the group consisting of sequences Nos. 84 to 86 of the sequence listing.

7. An antibody or antigen-binding fragment thereof, characterized in that in paragraph 5, the single domain antibody comprises an amino acid sequence selected from the group consisting of sequences 87 to 89 of the sequence listing.

8. A nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof that specifically binds to the CD73 protein of paragraph 1.

9. A gene vector comprising a nucleic acid molecule of claim 8.

10. A host cell transformed with the gene vector of clause 9.

11. An antibody or an antigen-binding fragment thereof that specifically binds to CD73 protein via at least one epitope selected from the group consisting of: (a) an epitope comprising an amino acid sequence of Sequence Listing No. 92; (b) an epitope comprising an amino acid sequence of Sequence Listing No. 93; and (c) an epitope comprising an amino acid sequence of Sequence Listing No.

94.

12. A pharmaceutical composition for preventing or treating cancer, comprising the antibody of claim 1 or claim 11 or an antigen-binding fragment thereof as an active ingredient.

13. A composition according to claim 12, wherein the cancer is selected from the group consisting of leukemia, bladder cancer, brain tumor, melanoma, thyroid cancer, esophageal cancer, lung cancer, prostate cancer, breast cancer, colon cancer, stomach cancer, head and neck cancer, kidney cancer, pancreatic cancer, liver cancer, neuroblastoma, retinoblastoma, salivary gland cancer, lymphoma, cervical cancer, ovarian cancer, and testicular cancer.

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