Asymmetric antibodies with improved efficacy in killing cancer cells

Abstract

The present invention relates to an anti-CD20 asymmetric antibody, which comprises a single-chain variable fragment (scFv) on one side and a fragment antigen-binding fragment (Fab) on the other side. [Solution] The asymmetric antibody of the present invention effectively activates complement and exhibits significantly improved complement dependent cytotoxicity (CDC) compared to antibodies with a symmetric structure, and can therefore be usefully used as an efficient cancer therapeutic agent or therapeutic adjuvant.

Description

[Technical field]

[0001] The present invention relates to a novel anti-CD20 asymmetric antibody or an antigen-binding fragment thereof with improved complement dependent cytotoxicity (CDC), and / or a composition for preventing, treating and / or diagnosing cancer using the same. [Background technology]

[0002] The global anticancer drug market has shown an average annual growth rate of 10% to 13% over the past 10 years, and is expected to reach a total of $200 billion by 2022. As of 2020, 1 in 5 men and 1 in 6 women worldwide have experienced cancer at least once in their lifetime, and the prevalence and mortality rates of cancer are continually increasing, with 1 in 8 men and 1 in 11 women dying from cancer.

[0003] The global anti-cancer drug market is undergoing a paradigm shift, from first-generation chemotherapy to second-generation targeted anti-cancer drugs and third-generation immune anti-cancer drugs. Deviating from early, simple treatment methods aimed at shrinking and suppressing cancer, targeted anti-cancer treatment technologies that selectively attack only cancer cells without damaging normal, rapidly dividing cells are being actively researched, and the development of such targeted anti-cancer treatment technologies involves two steps: selecting receptors that are specifically expressed in cancer cells, which allows selective targeting of cancer cells, and developing targeting compounds that bind to the receptors.

[0004] Rituximab is a chimeric anti-CD20 monoclonal antibody that was commercialized for tumor treatment and was the first monoclonal antibody therapeutic approved by the US FDA. In the early 1980s, CD20 antigen antibodies were monoclonal antibodies made from mice, which had excellent reactivity with antigens but had the disadvantages of a short biological half-life and the induction of antigenicity due to foreign antigens (HAMA, human anti-mouse antibody). To compensate for these shortcomings of mouse-derived antibodies and increase their biological effectiveness, rituximab, a chimeric antibody in which the variable region where antigen recognition occurs is mouse-derived and the constant region of the antibody is human-derived, was synthesized through genetic recombination technology.

[0005] The CD20 antigen is known to be involved in cell cycle entry of B cell lineage and differentiation into B cells. The CD20 antigen has the property of not easily detaching from the cell surface, deforming or invaginating, making it suitable for use as a target molecule. In normal cells, the CD20 antigen is distributed widely from the pre-B cell stage to the activated B cell stage, but is not distributed in cells of a different lineage than hematopoietic stem cells and plasma cells. In lymphomas, the CD20 antigen is expressed in most B cell lymphomas and chronic lymphocytic leukemias and 50% of pre-B cell acute lymphocytic leukemias, and can be a suitable target for inducing tumor-specific immune responses using monoclonal antibodies (Maloney DG et al., IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed lowgrade non-Hodgkin's lymphoma. Blood, 90: 2188-2195, 1997; McLaughlin P et al., Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program. J Clin Oncol,16:2825-2833,1998).

[0006] However, in the case of rituximab, about half of patients with low-grade lymphoma and about 60% to 70% of patients with high-grade lymphoma show resistance to it, and although some reports have suggested that complement-mediated cytotoxicity decreases when expression of CD55 or CD59 is high, the reason why a considerable number of patients do not respond to it remains unclear.

[0007] Throughout this specification, a number of papers and patent documents are referred to and citations are displayed, the disclosure contents of which are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention pertains and the contents of the present invention. Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, the present inventors have made extensive research and efforts to develop a therapeutic agent that can effectively kill cancer cells that are unresponsive to or resistant to rituximab. As a result, they have discovered that when an anti-CD20 asymmetric antibody containing scFv on one side and Fab on the other side is used, it has significantly improved complement dependent cytotoxicity (CDC), which has led to the completion of the present invention.

[0009] Therefore, an object of the present invention is to provide an anti-CD20 asymmetric antibody or an antigen-binding fragment thereof, which has improved complement-dependent cytotoxicity (CDC), including a single-chain variable fragment (scFv) and a fragment antigen-binding (Fab).

[0010] Another object of the present invention is to provide a nucleic acid molecule encoding the anti-CD20 asymmetric antibody or an antigen-binding fragment thereof.

[0011] It is yet another object of the present invention to provide a vector comprising the nucleic acid molecule.

[0012] It is yet another object of the present invention to provide a host cell transformed with said vector.

[0013] It is yet another object of the present invention to provide a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient the anti-CD20 asymmetric antibody or antigen-binding fragment thereof, a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, or a vector containing the nucleic acid molecule.

[0014] It is yet another object of the present invention to provide a method for preventing or treating cancer, comprising administering to a subject a pharma- ceutical effective amount of the anti-CD20 asymmetric antibody or antigen-binding fragment thereof, a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, or a vector containing the nucleic acid molecule.

[0015] It is yet another object of the present invention to provide a method for the treatment of a disease comprising administering to a patient a therapeutically effective amount of said anti-CD20 asymmetric antibody or antigen-binding fragment thereof, a nucleic acid molecule encoding said antibody or antigen-binding fragment thereof, or a vector comprising said nucleic acid molecule.

[0016] It is yet another object of the present invention to provide a composition for diagnosing cancer, comprising as an active ingredient the anti-CD20 asymmetric antibody or antigen-binding fragment thereof, a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, or a vector containing the nucleic acid molecule.

[0017] It is yet another object of the present invention to provide a method for producing an anti-CD20 asymmetric antibody or an antigen-binding fragment thereof with improved complement-dependent cytotoxicity, comprising the steps of producing an scFv and producing a Fab.

[0018] It is yet another object of the present invention to provide a method for producing a nucleic acid molecule encoding an anti-CD20 asymmetric antibody or an antigen-binding fragment thereof with improved complement-dependent cytotoxicity, comprising the steps of producing a nucleic acid molecule encoding an scFv and producing a nucleic acid molecule encoding a Fab.

[0019] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, the claims and the drawings. [Means for solving the problem]

[0020] According to one aspect of the present invention, there is provided a method for preparing a nucleic acid molecule encoding an anti-CD20 asymmetric antibody or an antigen-binding fragment thereof having improved Complement Dependent Cytotoxicity (CDC), comprising the steps of:

[0021] (a) preparing a nucleic acid molecule encoding a single-chain variable fragment (scFv) comprising an LCDR1 of SEQ ID NO: 16, an LCDR2 of SEQ ID NO: 18, an LCDR3 of SEQ ID NO: 20, an HCDR1 of SEQ ID NO: 24, an HCDR2 of SEQ ID NO: 26, and an HCDR3 of SEQ ID NO: 28; and

[0022] (b) preparing a nucleic acid molecule encoding a Fab (Fragment antigen-binding) comprising an LCDR1 of SEQ ID NO: 33, an LCDR2 of SEQ ID NO: 35, an LCDR3 of SEQ ID NO: 37, an HCDR1 of SEQ ID NO: 42, an HCDR2 of SEQ ID NO: 44 and an HCDR3 of SEQ ID NO: 46.

[0023] The present inventors have conducted extensive research and efforts to develop a therapeutic agent that can effectively kill cancer cells that are unresponsive to or resistant to rituximab. As a result, they discovered that when an anti-CD20 asymmetric antibody containing scFv on one side and Fab on the other side is used, it has a significantly increased complement-dependent cytotoxicity.

[0024] As used herein, the term "antibody" refers to a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen, and includes both polyclonal and monoclonal antibodies.

[0025] Antibodies include not only complete full-length antibody forms, but also antigen-binding fragments (antibody fragments) of antibody molecules. Complete antibodies have a structure with two full-length light chains and two full-length heavy chains, with each light chain linked to a heavy chain by a disulfide bond. Heavy chain constant regions have gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, with gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2) subclasses. Light chain constant regions have kappa (κ) and lambda (λ) types.

[0026] As used herein, the term "antigen-binding fragment of an antibody" refers to a fragment that specifically recognizes an antigen within the whole antibody molecule and retains antigen-antibody binding function, and includes single domain antibodies (sdAb), single chain antibodies (scFv), Fab, F(ab'), F(ab')2, and Fv. Examples of fragments include a monovalent fragment (Fab fragment) composed of the VL, VH, Cκ (or CL), and CH1 domains; a bivalent fragment (F(ab')2 fragment) containing two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment composed of the VH and CH1 domains; an Fv fragment composed of the VL and VH domains of one arm of an antibody and a disulfide-linked Fv (sdFv); a dAb fragment composed of the VH domain; and an isolated complementarity determining region (CDR) or a combination of two or more isolated CDRs that can be selectively linked by a linker. In addition, scFvs may be linked by a linker to form a single protein chain in which the VL and VH regions are paired to form a monovalent molecule. Such single-chain antibodies are also included in the antibody fragments. The antibody or antibody fragment also includes a tetrameric antibody containing two heavy chain molecules and two light chain molecules; an antibody light chain monomer; an antibody heavy chain monomer; an antibody light chain dimer, an antibody heavy chain dimer; an intrabody; a monovalent antibody; a camelid antibody; and a single domain antibody (sdAb).

[0027] Fv is the smallest antibody fragment having only a heavy chain variable region and a light chain variable region, and recombinant techniques for producing Fv fragments are disclosed in PCT International Patent Publications WO88 / 10649, WO88 / 106630, WO88 / 07085, WO88 / 07086, and WO88 / 09344. In a two-chain Fv, the heavy chain variable region and the light chain variable region are linked by a non-covalent bond. In a single-chain Fv, the heavy chain variable region and the single chain variable region are generally linked by a covalent bond via a peptide linker or directly at the C-terminus, and can form a dimer-like structure like the two-chain Fv. Such antibody fragments can be obtained using protease (for example, Fab can be obtained by restricting and cleaving a whole antibody with papain, and F(ab')2 fragment can be obtained by cleaving with pepsin), or can be produced through gene recombination techniques.

[0028] As used herein, the term "heavy chain" refers to a full-length heavy chain and a fragment thereof that includes a variable region domain VH containing an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and three constant region domains CH1, CH2, and CH3. Additionally, as used herein, the term "light chain" refers to a full-length light chain and a fragment thereof that includes a variable region domain VL containing an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and a constant region domain Cκ(CL).

[0029] As used herein, the term "complementarity determining region (CDR)" refers to the amino acid sequences of the hypervariable regions of immunoglobulin heavy and light chains (Kabat et al. Sequences of Proteins of Immunological Interest, 4th Ed., USDepartment of Health and Human Services, National Institutes of Health (1987)). The heavy chain (HCDR1, HCDR2, and HCDR3) and the light chain (LCDR1, LCDR2, and LCDR3) each contain three CDRs, which provide the main contact residues when the antibody binds to an antigen or epitope. The CDRs are interspersed with further conserved regions named framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, which are arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0030] The scope of the antibody or antibody fragment of the present invention includes variants having conservative amino acid substitutions in the CDR regions, and may include variants of the amino acid sequences set forth in the attached sequence listing, as long as they are capable of specifically recognizing CD20. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the half-life, biocompatibility, and other biological properties of the antibody in addition to the binding affinity. In consideration of such variations having bioequivalent activity, the antibody of the present invention or the nucleic acid molecule encoding the same is construed as including sequences that show substantial identity with the sequences set forth in the sequence listing. The substantial identity means a sequence that shows at least 61% homology, in one particular example, 70% homology, in one particular example, 80% homology, and in yet another particular example, 90% homology, when the sequence of the present invention is aligned with any other sequence to maximize its correspondence and the aligned sequences are analyzed using an algorithm commonly used in the art. Alignment methods for comparing sequences are well known in the art. Various methods and algorithms for alignment are disclosed in Smith and Waterman, Adv. Appl. Math. (1981) 2:482 Needleman and Wunsch, J. Mol. Bio. (1970) 48:443; Pearson and Lipman, Methods in Mol. Biol. (1988) 24:307-31; Higgins and Sharp, Gene (1988) 73:237-44; Higgins and Sharp, CABIOS (1989) 5:151-3; Corpet et al. Nuc. Acids Res. (1988) 16:10881-90; Huang et al. Comp. Appl. BioSci. (1992) 8:155-65 and Pearson et al. Meth. Mol. Biol. (1994) 24:307-31.

[0031] As used herein, the term "symmetric antibody" refers to an antibody having two light chains and two heavy chains, each of which has a symmetric structure (e.g., an antibody having the same light chain variable region and the same heavy chain variable region on each side).

[0032] As used herein, the term "asymmetric antibody" refers to an antibody having an asymmetric structure that is not the "symmetric antibody". For example, one side may contain a fragment comprising VL-linker-VH (i.e., scFv), and the other side may contain a fragment comprising VL-Cκ (or CL)-linker-VH-CH1 (i.e., Fab). Alternatively, one side may contain a fragment comprising Fab, and the other side may contain a fragment comprising Fab and scFv. The asymmetric antibody may further contain an Fc site. For example, an antibody having an asymmetric structure may be produced by including a fragment comprising VL-linker-VH-CH2-CH3 on one side, and a fragment comprising VL-Cκ (or CL)-linker-VH-CH1-CH2-CH3 on the other side.

[0033] As used herein, the term "affinity" refers to the binding strength between an antibody or an antigen-binding fragment thereof and an antigen, which may also be expressed as "avidity."

[0034] According to a specific embodiment of the present invention, the scFv comprises a light chain variable region (VL) sequence of SEQ ID NO:51.

[0035] According to a specific embodiment of the present invention, the scFv comprises a heavy chain variable region (VH) sequence of SEQ ID NO:52.

[0036] According to a specific embodiment of the present invention, the scFv comprises an array selected from the group consisting of LFR1 of SEQ ID NO: 15, LFR2 of SEQ ID NO: 17, LFR3 of SEQ ID NO: 19, LFR4 of SEQ ID NO: 21, HFR1 of SEQ ID NO: 23, HFR2 of SEQ ID NO: 25, HFR3 of SEQ ID NO: 27 and HFR4 of SEQ ID NO: 29.

[0037] According to a specific embodiment of the present invention, the scFv is in the form of VL-linker-VH.

[0038] According to a specific embodiment of the present invention, the linker may be a combination of Gly and Ser.

[0039] According to a specific embodiment of the present invention, the Fab comprises a light chain variable region (VL) sequence of SEQ ID NO:53.

[0040] According to a specific embodiment of the present invention, the Fab comprises a heavy chain variable region (VH) sequence of SEQ ID NO:54.

[0041] According to a specific embodiment of the present invention, the Fab comprises an array selected from the group consisting of LFR1 of SEQ ID NO: 32, LFR2 of SEQ ID NO: 34, LFR3 of SEQ ID NO: 36, LFR4 of SEQ ID NO: 38, HFR1 of SEQ ID NO: 41, HFR2 of SEQ ID NO: 43, HFR3 of SEQ ID NO: 45 and HFR4 of SEQ ID NO: 47.

[0042] According to a specific embodiment of the present invention, the Fab comprises a Cκ (C kappa) sequence of SEQ ID NO:39.

[0043] According to a specific embodiment of the present invention, the Fab comprises the CH1 sequence of SEQ ID NO:48.

[0044] According to a specific embodiment of the present invention, the Fab has the form VL-Cκ-linker-VH-CH1.

[0045] According to a specific embodiment of the present invention, the linker may be a combination of Gly and Ser.

[0046] According to a specific embodiment of the present invention, the asymmetric antibody of the present invention may use a linker consisting of 10 to 100, more specifically 15 to 60, Gly and Ser residues to prevent undesired pairing of heavy chains.

[0047] Specifically, the bispecific antibody of the present invention may be in a form in which CD20-scFv-knob (VL-linker20-VH-CH2-CH3) and CD20-Fab-hole (VL-Cκ-linker40-VH-CH1-CH2-CH3) are linked together.

[0048] Linker 20 contains 20 of the Gly or Ser, and examples thereof may include, but are not limited to, Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Ser.

[0049] Linker 40 contains 40 of the Gly or Ser, and examples thereof may include, but are not limited to, Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Gly Ser Ser.

[0050] According to another aspect of the invention, there is provided a nucleic acid molecule encoding the above-described antibody or antigen-binding fragment thereof of the invention.

[0051] As used herein, the term "nucleic acid molecule" is intended to comprehensively include DNA (gDNA and cDNA) and RNA molecules, and the basic building block of a nucleic acid molecule is a nucleotide, which includes not only natural nucleotides but also analogues in which the sugar or base moiety is modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, (1990) 90:543-584). The sequences of the nucleic acid molecules encoding the heavy and light chain variable regions of the present invention can be modified. Such modifications include addition, deletion, non-conservative substitution, or conservative substitution of nucleotides.

[0052] The nucleic acid molecule of the present invention is also understood to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence, where substantial identity means a nucleotide sequence that exhibits at least 80% homology, in a particular example at least 90% homology, and in a particular example at least 95% homology, when the nucleotide sequence of the present invention is aligned with any other sequence for maximum correspondence and the aligned sequences are analyzed using algorithms commonly used in the art.

[0053] In yet another aspect, the present invention provides a vector comprising a nucleic acid molecule encoding the above-described antibody or antigen-binding fragment thereof of the present invention.

[0054] As used herein, the term "vector" refers to a vehicle into which a polynucleotide sequence can be inserted for introduction into a cell capable of replicating said polynucleotide sequence. The polynucleotide sequence can be exogenous or heterologous. Vectors can include, but are not limited to, plasmids, cosmid vectors, and viral vectors (such as retroviruses, adenoviruses, and adeno-associated virus vectors). Those skilled in the art can construct vectors by standard recombinant techniques (e.g., Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994).

[0055] As used herein, the term "expression vector" refers to a vector that contains a nucleotide sequence that codes for at least a portion of a gene product to be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. Expression vectors can contain a variety of regulatory sequences. Along with regulatory sequences that control transcription and translation, vectors and expression vectors can also contain nucleic acid sequences that provide other functions.

[0056] According to yet another aspect, the present invention provides a host cell transformed with the above-mentioned vector.

[0057] As used herein, the term "cell" refers to any transformable cell, including eukaryotic and prokaryotic cells, that can replicate the vector or express a gene encoded by the vector. A cell may be transfected, transduced, or transformed by the vector, which refers to the process by which an exogenous polynucleotide (nucleic acid molecule) is transferred or introduced into a host cell. As used herein, the term "transformation" is used to include both transfection and transduction.

[0058] The (host) cell of the present invention is preferably, but not limited to, an insect cell or a mammalian cell, more preferably, in the case of an insect cell, Sf9, and in the case of a mammalian cell, HEK293 cell, HeLa cell, ARPE-19 cell, RPE-1 cell, HepG2 cell, Hep3B cell, Huh-7 cell, C8D1a cell, Neuro2A cell, CHO cell, MES13 cell, BHK-21 cell, COS7 cell, COP5 cell, A549 cell, MCF-7 cell, HC70 cell, HCC1428 cell, BT-549 cell, PC3 cell, LNCaP cell, Capan-1 cell, Panc-1 cell, MIA cell, or the like. PaCa-2 cells, SW480 cells, HCT166 cells, LoVo cells, A172 cells, MKN-45 cells, MKN-74 cells, Kato-III cells, NCI-N87 cells, HT-144 cells, SK-MEL-2 cells, SH-SY5Y cells, C6 cells, HT-22 cells, PC-12 cells, NIH3T3 cells, etc. can be used.

[0059] The host cell of the present invention is preferably an isolated host cell.

[0060] In yet another aspect, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient the above-mentioned antibody or antigen-binding fragment thereof of the present invention, a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, or a vector containing the nucleic acid molecule.

[0061] According to yet another aspect of the present invention, there is provided a method for preventing or treating cancer, comprising the step of administering to a subject a pharma- ceutical effective amount of the above-described antibody or antigen-binding fragment thereof of the present invention, a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, or a vector containing the nucleic acid molecule.

[0062] According to yet another aspect of the present invention, there is provided a therapeutic use of the above-mentioned antibody or antigen-binding fragment thereof of the present invention, a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, or a vector comprising the nucleic acid molecule.

[0063] As used herein, the term "prevention" refers to 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.

[0064] As used herein, the term "treatment" means (a) inhibiting the development of a disease, illness, or condition; (b) alleviating a disease, illness, or condition; or (c) eliminating a disease, illness, or condition. When the composition of the present invention is administered to a subject, it exhibits a binding strength to CD20 at a level equivalent to that of rituximab, and inhibits the proliferation of cancer cells by a significant complement activation ability and / or CDC effect due to the same, as compared to rituximab, thereby inhibiting the progression of cancer or eliminating or alleviating symptoms due to the same. Thus, the composition of the present invention may be a composition for treating cancer itself, or may be administered together with other pharmacological ingredients and applied as a therapeutic adjuvant to improve the therapeutic response. Thus, as used herein, the term "treatment" or "therapeutic agent" includes the meaning of "therapeutic adjuvant" or "therapeutic adjuvant".

[0065] As used herein, the term "administration" or "administering" means administering a therapeutically effective amount of a composition of the present invention directly to a subject, thereby causing the same to be formed in the subject's body.

[0066] In the present invention, the term "therapeutically effective amount" means the content of the composition in which the pharmacological component in the composition is contained to an extent sufficient to provide a therapeutic or prophylactic effect to an individual to whom the pharmaceutical composition of the present invention is to be administered, and this includes a "prophylactically effective amount."

[0067] As used herein, the term "subject" includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus monkey. Specifically, the subject of the present invention is a human.

[0068] According to a specific embodiment, the cancer that can be prevented or treated by the composition of the present invention is a CD20-positive cancer.

[0069] CD20-positive cancers include, for example, blood cancers such as lymphoma and leukemia, and solid cancers such as melanoma, lung cancer, thyroid cancer, breast cancer, colon cancer, prostate cancer, head and neck cancer, gastric cancer, liver cancer, bladder cancer, kidney cancer, cervical cancer, pancreatic cancer, ovarian cancer, cervical cancer, and endometrial cell cancer, and are preferably B-cell non-Hodgkin's lymphomas (NHL), follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma (SLL), marginal zone lymphoma (MZL), lymphoplasmacytic These include, but are not limited to, primary macroglobulinemia (Waldenstrom macroglobulinemia (WM)), and the like.

[0070] According to yet another aspect of the present invention, there is provided a composition for diagnosing cancer, comprising as an active ingredient the above-mentioned antibody or antigen-binding fragment thereof of the present invention, a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, or a vector containing the nucleic acid molecule.

[0071] The antibodies or antigen-binding fragments thereof used in the present invention and the target cancers have already been described in detail, so descriptions thereof will be omitted to avoid excessive duplication.

[0072] As used herein, the term "diagnosis" includes determining an individual's susceptibility to a particular disease, determining whether an individual currently has a particular disease, and determining the prognosis of a subject with a particular disease.

[0073] CD20 is a typical cancer diagnostic marker that shows a higher expression level than the normal level in cancer tissues such as B cell lymphoma and chronic lymphocytic leukemia, and the antibody of the present invention that specifically binds to CD20 can accurately determine the presence of a tumor in a biological sample. In addition, the antibody of the present invention can be useful in early establishment of a customized treatment strategy according to the characteristics of the cancer by accurately measuring the expression level of CD55 in the tumor through various immunoassay methods and predicting the therapeutic sensitivity of the tumor to a CDC drug.

[0074] As used herein, the term "diagnostic composition" refers to an integrated mixture or device containing the antibody of the present invention as a means for measuring the expression level of CD20 protein in order to determine the presence or absence of cancer in a subject or to predict therapeutic sensitivity to a CDC drug, which may also be expressed as a "diagnostic kit."

[0075] As used herein, the term "biological sample" includes, but is not limited to, tissues, cells, whole blood, serum, plasma, saliva, urine, lymphatic fluid, spinal fluid, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatants, ruptured eukaryotic cells, and bacterial expression systems.

[0076] In biological samples, CD20 protein can be detected through detection of antigen-antibody complex formation using colorimetric methods, electrochemical methods, fluorimetric methods, luminometry, particle counting methods, visual assessment, or scintillation counting methods. As used herein, the term "detection" refers to a series of processes for determining whether or not an antigen-antibody complex is formed. Detection can be performed using a variety of labels, including enzymes, fluorescent substances, ligands, luminescent substances, microparticles, or radioisotopes.

[0077] Enzymes that can be used as detection labels include, for example, acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase, and β-lactamase, and fluorescent substances include fluorescein, Eu 3+ ,EU 3+ Examples of the luminescent material include acridinium esters and isoluminol derivatives. Examples of the fine particles include gold colloids and colored latex. Examples of the radioisotopes include 57 Co, 3 H, 125 I and 125 It may include I-Bolton Hunter reagent and the like.

[0078] According to one embodiment of the present invention, the antigen-antibody complex can be detected using an enzyme-linked immunosorbent assay (ELISA). The antibody of the present invention can have a detectable label, and if it does not have a detectable label, the antibody of the present invention can be captured and further treated with another antibody having a detectable label to confirm it.

[0079] According to yet another aspect of the present invention, there is provided a method for producing an anti-CD20 asymmetric antibody or an antigen-binding fragment thereof having improved Complement Dependent Cytotoxicity (CDC), the method comprising the steps of:

[0080] (a) producing an scFv (Single-chain variable fragment) comprising an LCDR1 of SEQ ID NO: 16, an LCDR2 of SEQ ID NO: 18, an LCDR3 of SEQ ID NO: 20, an HCDR1 of SEQ ID NO: 24, an HCDR2 of SEQ ID NO: 26, and an HCDR3 of SEQ ID NO: 28; and

[0081] (b) producing a Fab (fragment antigen-binding) comprising an LCDR1 of SEQ ID NO: 33, an LCDR2 of SEQ ID NO: 35, an LCDR3 of SEQ ID NO: 37, an HCDR1 of SEQ ID NO: 42, an HCDR2 of SEQ ID NO: 44 and an HCDR3 of SEQ ID NO: 46.

[0082] In yet another aspect, the present invention provides a method for preparing a nucleic acid molecule encoding an anti-CD20 asymmetric antibody or an antigen-binding fragment thereof having improved Complement Dependent Cytotoxicity (CDC), comprising the steps of:

[0083] (a) preparing a nucleic acid molecule encoding a single-chain variable fragment (scFv) comprising an LCDR1 of SEQ ID NO: 16, an LCDR2 of SEQ ID NO: 18, an LCDR3 of SEQ ID NO: 20, an HCDR1 of SEQ ID NO: 24, an HCDR2 of SEQ ID NO: 26, and an HCDR3 of SEQ ID NO: 28; and

[0084] (b) preparing a nucleic acid molecule encoding a Fab (Fragment antigen-binding) comprising an LCDR1 of SEQ ID NO: 33, an LCDR2 of SEQ ID NO: 35, an LCDR3 of SEQ ID NO: 37, an HCDR1 of SEQ ID NO: 42, an HCDR2 of SEQ ID NO: 44 and an HCDR3 of SEQ ID NO: 46.

[0085] The asymmetric antibody of the present invention can comprise an Fc region consisting of a first and a second subunit capable of stably associating. As used herein, the term "Fc region" refers to the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region of a full-length antibody, including both wild-type sequence Fc regions and mutant Fc regions. The Fc region of an IgG comprises an IgG CH2 and an IgG CH3 domain. The CH3 region of the asymmetric antibody of the present invention can be a wild-type or mutant CH3 domain (e.g., a CH3 domain having an introduced "protrusion" ("knob") in one chain and a corresponding introduced "cavity" ("hole") in the other chain.

[0086] According to a specific embodiment of the present invention, the asymmetric antibody of the present invention can use the knob-into-hole approach to prevent binding of undesired pairs of heavy chains. The "knob-into-hole" approach [US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); Carter, J Immunol Meth 248, 7-15 (2001)] introduces a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide such that the protrusion can be located in the cavity to promote heterodimer formation and inhibit homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). Complementary cavities of identical or similar size to the overhang are created on the interface of the second polypeptide by replacing large amino acid side chains with smaller side chains (eg, alanine or threonine).

[0087] According to a specific embodiment of the present invention, the asymmetric antibody of the present invention may be in a form in which an scFv in the form of VL-linker-VH-CH2-CH3 is linked to an Fab in the form of VL-Cκ-linker-VH-CH1-CH2-CH3. Effect of the Invention

[0088] The features and advantages of the present invention can be summarized as follows:

[0089] (i) The present invention provides an anti-CD20 asymmetric antibody or an antigen-binding fragment thereof which comprises a single-chain variable fragment (scFv) on one side and a fragment antigen-binding fragment (Fab) on the other side and has improved complement dependent cytotoxicity (CDC).

[0090] (ii) The asymmetric antibodies of the present invention exhibit significantly improved complement dependent cytotoxicity (CDC) compared to antibodies with a symmetric structure, and can therefore be usefully used as efficient therapeutic or therapeutic adjuvant agents that fundamentally eliminate drug resistance and significantly improve therapeutic responsiveness in various diseases in which resistance has been induced to anticancer drugs such as rituximab. [Brief description of the drawings]

[0091] [Figure 1] FIG. 1 is a schematic diagram showing IgG and asymmetric antibodies.

[0092] [Diagram 2] FIG. 1 shows SDS-PAGE analysis of anti-CD20 antibodies in asymmetric form.

[0093] [Diagram 3] FIG. 1 shows the results of ELISA analysis of the affinity of asymmetric anti-CD20 antibodies to CD20.

[0094] [Figure 4] FIG. 1 shows the results of ELISA comparing the complement activation abilities of rituximab and an asymmetric anti-CD20 antibody by measuring C4d concentrations.

[0095] [Diagram 5] FIG. 1 shows the results of comparing the CDC effects of rituximab and an asymmetric anti-CD20 antibody in Ramos-RR cells that are resistant to rituximab. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0096] The present invention will be described in more detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention in more detail, and that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. EXAMPLES

[0097] Example 1. Cloning of anti-CD20 antibodies in asymmetric configurations for animal cell expression

[0098] The anti-CD20 antibody in asymmetric form used the knob-into-hole technique to prevent binding of undesired pairs of heavy chains, and a linker consisting of Gly and Ser was used to prevent incorrect binding of light chains. Cloning was carried out in the form of CD20-scFv-knob (VL-linker20-VH-CH2-CH3) and CD20-Fab-hole (VL-Cκ-linker40-VH-CH1-CH2-CH3), respectively, and in the case of CD20-scFv-knob, PCR was performed using the primer combination in Table 1 based on the Rituximab sequence listed in the Drug Data Bank. The amplified gene was treated with BssHII and XbaI (New England Biolab, UK) enzymes and ligated into the pMAZ vector, an animal cell expression vector that had been similarly treated with the same restriction enzymes. The ligated plasmid was transformed into DH5α competent cells (New England Biolab, UK) by heat shock, and the obtained colonies were cultured in large quantities to obtain the plasmid.

[0099] To generate the CD20-Fab-hole (VL-Cκ-linker40-VH-CH1-CH2-CH3) form, PCR was performed using the primer combinations in Table 2 based on the Rituximab sequence in the Drug Data Bank, as well as the CD20-scFv-knob. The amplified gene was treated with BssHII and XbaI enzymes and ligated into the pMAZ vector, an animal cell expression vector that had been treated with the same restriction enzymes. The ligated plasmid was transformed into DH5α competent cells (New England Biolab, UK) by heat shock, and the obtained colonies were cultured in large quantities to obtain the plasmid, and sequence analysis (Table 3) was completed.

[0100] [Table 1]

[0101] Primer list used during cloning of CD20-scFv-knob

[0102] [Table 2]

[0103] Primer list used during cloning of CD20-Fab-hole

[0104] [Table 3]

[0105] Amino acid sequences of CD20-scFv-knob and CD20-Fab-hole

[0106] Example 2. Expression in animal cells and purification

[0107] Asymmetric anti-CD20 antibodies were prepared by transfecting HEK293F cells (Invitrogen, USA) with the plasmids of SEQ ID NOs: 13 and 14 using polyethyleneimine (PEI) (Polysciences, USA) and 150 mM NaCl, and culturing them in Freestyle 293 expression medium (Invitrogen, USA) for 7 days at 37°C, 8% CO2, and 55% humidity. The expression cell culture was centrifuged at 4,000 rpm for 10 minutes, and the supernatant was collected and filtered through a 0.22 μm filter. The filtered supernatant was induced to bind to 1 ml of KappaSelect (GE Healthcare, USA) resin at 4°C. The bound resin was washed with 10 cv (column volume) of PBS solution, and the bound antibody was eluted using 100 mM glycine-HCl (pH 2.7) solution, followed by neutralization with 1 M Tris-HCl (pH 9.0). After buffer exchange with PBS of pH 7.2-7.4, the size and purity of the purified antibody light and heavy chains were confirmed by SDS-PAGE. The molecular weight and high purity were confirmed to be consistent with the theoretical calculation values ​​(Figure 2).

[0108] Asymmetric anti-CD20 antibodies are heterodimers in which one binding site is Fab and the other binding site is scFv. Purification with KappaSelect using the C kappa portion of Fab suppresses the formation of knob-knob homodimers or knob monomers (Giese, et al., Bispecific antibody process development: Assembly and purification of knob and hole bispecific antibody. Biotechnol Prog, 34(2)397-404), which are known to be most likely to be formed during purification of bispecific antibodies, and allows the production of only the desired heterodimer.

[0109] Example 3. Confirmation of CD20 binding ability of asymmetric anti-CD20 antibodies through ELISA

[0110] The affinity of the asymmetric anti-CD20 antibody prepared in Example 2 to CD20, which is the target antigen, was confirmed by indirect ELISA.

[0111] For indirect ELISA, CD20 ectodomain loop region antigen was diluted to 1 μg / ml in 50 μl of PBS, placed in a 96-well immunoplate (Corning, USA), and stored at 4°C for overnight adsorption. After reacting with a buffer solution containing 4% skim milk (BD, USA) for 1 hour at room temperature, the plate was washed three times with a buffer solution containing 0.5% Tween 20 (Amresco, USA) and treated with 50 μl of each antibody diluted at successive concentrations (0.1, 0.3, 1, 3, 11, 33, 100 nM) per well. After reacting for 2 hours at room temperature to allow the antibody to bind to the antigen, the plate was washed three times with a buffer solution containing 0.5% Tween 20 (Amresco, USA). Anti-human immunoglobulin Fc-HRP antibody (Jackson Immunoresearch, USA) was diluted 1:3,000 with a buffer solution containing 2% skim milk (BD, USA) and treated with 50 μl per well and reacted at room temperature for 1 hour. After the reaction was completed, the wells were washed three times with a buffer solution containing 0.5% Tween 20 (Amresco, USA) and then 50 μl of 3,3',5,5'-tetramethylbenzidine (TMB) (ThermoFisher Scientific, USA) was added to each well to develop color for 10 minutes. The absorbance was measured at 450 nm using a spectrophotometer (Biotek, USA) and the results are shown in Figure 3. As a result, the asymmetric anti-CD20 antibody showed the same level of binding to CD20 as rituximab.

[0112] Example 4. Confirmation of complement activation ability of asymmetric anti-CD20 antibodies through C4d ELISA

[0113] The complement activation ability of the asymmetric anti-CD20 antibody prepared in Example 2 was confirmed by C4d ELISA. C4d is a marker for activation of the classical complement pathway (Cohen, et al., Pros and cons for C4d as a biomarker. Kidney Int., 81(7)628-39), and complement activation ability can be confirmed by measuring the C4d concentration.

[0114] First, 10 μl of 100 μg / ml rituximab or asymmetric anti-CD20 antibody was reacted with 90 μl of 1 mg / ml complement (Quidel, USA) for 1 hour, then diluted 10-fold and C4d concentration was confirmed using MicroVue Complement C4d Fragment EIA Kit (Quidel, USA), and the results are shown in Figure 4. As a result, asymmetric anti-CD20 antibody increased C4d concentration compared to rituximab, whose main therapeutic mechanism is complement dependent cytotoxicity (CDC). This means that the complement activation ability of asymmetric anti-CD20 antibody is superior to rituximab.

[0115] Example 5. Confirmation of CDC effect of asymmetric anti-CD20 antibody on rituximab-resistant cell line Ramos-RR

[0116] We confirmed the CDC effect of rituximab and an asymmetric anti-CD20 antibody in Ramos-RR, which shows resistance to rituximab. 5Ramos-RR cells were treated with 100 μl of either rituximab or anti-CD20 antibody in asymmetric form, diluted to a concentration of 20 μg / ml in RPMI medium supplemented with 20% complement sera human (Sigma, USA), and then cultured for 2 hours under 37°C / 5% / CO2 conditions. After that, dead cells were confirmed using 7-AAD and FITC Annexin V apoptosis detection kits with the Attune NxT FACS analyzer (ThermoFisher Scientific, USA) and CDC was observed (Figure 5). Ramos-RR cells showed low CDC effect in response to rituximab, but anti-CD20 antibody in asymmetric form increased the effect.

[0117] These results indicate that asymmetric antibodies activate complement and induce CDC more effectively than IgG antibodies, implying that asymmetric antibodies are more advantageous for cancer treatment, in which CDC is the primary therapeutic mechanism.

[0118] Although each embodiment of the present invention has been described above, a person having ordinary knowledge in the relevant technical field can modify and change the present invention in various ways by adding, changing, deleting or adding components without departing from the concept of the present invention described in the claims, and this can also be said to be within the scope of the claims of the present invention.

Claims

1. A complement dependent cytotoxicity (CDC) inhibitor comprising the following components: Anti-CD20 asymmetric antibodies with improved cytotoxicity (CDC) compared to anti-CD20 symmetric antibodies: (a) a single-chain variable fragment (scFv) comprising an LCDR1 of SEQ ID NO: 16, an LCDR2 of SEQ ID NO: 18, an LCDR3 of SEQ ID NO: 20, an HCDR1 of SEQ ID NO: 24, an HCDR2 of SEQ ID NO: 26, and an HCDR3 of SEQ ID NO: 28; and (b) Fab (fragment antigen-binding) comprising LCDR1 of SEQ ID NO: 33, LCDR2 of SEQ ID NO: 35, LCDR3 of SEQ ID NO: 37, HCDR1 of SEQ ID NO: 42, HCDR2 of SEQ ID NO: 44, and HCDR3 of SEQ ID NO:

46.

2. The anti-CD20 asymmetric antibody of claim 1, characterized in that the scFv comprises the light chain variable region (VL) sequence of SEQ ID NO:

51.

3. The anti-CD20 asymmetric antibody of claim 1, characterized in that the scFv comprises the heavy chain variable region (VH) sequence of SEQ ID NO:

52.

4. The anti-CD20 asymmetric antibody of claim 1, characterized in that the scFv comprises an array selected from the group consisting of LFR1 of SEQ ID NO: 15, LFR2 of SEQ ID NO: 17, LFR3 of SEQ ID NO: 19, LFR4 of SEQ ID NO: 21, HFR1 of SEQ ID NO: 23, HFR2 of SEQ ID NO: 25, HFR3 of SEQ ID NO: 27 and HFR4 of SEQ ID NO:

29.

5. The anti-CD20 asymmetric antibody according to claim 1, wherein the scFv is in the form of VL-linker-VH.

6. The anti-CD20 asymmetric antibody according to claim 5 , wherein the linker is a combination of Gly and Ser.

7. The anti-CD20 asymmetric antibody of claim 1, wherein the Fab comprises the light chain variable region (VL) sequence of SEQ ID NO:

53.

8. The anti-CD20 asymmetric antibody of claim 1, wherein the Fab comprises the heavy chain variable region (VH) sequence of SEQ ID NO:

54.

9. The anti-CD20 asymmetric antibody of claim 1, characterized in that the Fab comprises a sequence selected from the group consisting of LFR1 of SEQ ID NO: 32, LFR2 of SEQ ID NO: 34, LFR3 of SEQ ID NO: 36, LFR4 of SEQ ID NO: 38, HFR1 of SEQ ID NO: 41, HFR2 of SEQ ID NO: 43, HFR3 of SEQ ID NO: 45 and HFR4 of SEQ ID NO:

47.

10. The anti-CD20 asymmetric antibody of claim 1 , wherein the Fab comprises the Cκ (C kappa) sequence of SEQ ID NO:

39.

11. The anti-CD20 asymmetric antibody of claim 1, wherein the Fab comprises the CH1 sequence of SEQ ID NO:

48.

12. The anti-CD20 asymmetric antibody according to claim 1, wherein the Fab is in the form of VL-Cκ-linker-VH-CH1.

13. The anti-CD20 asymmetric antibody of claim 12, wherein the linker is a combination of Gly and Ser.

14. A nucleic acid molecule encoding the anti-CD20 asymmetric antibody of claim 1.

15. A vector comprising the nucleic acid molecule of claim 14.

16. 16. An isolated host cell transformed with the vector of claim 15.

17. A pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient the anti-CD20 asymmetric antibody of claim 1, a nucleic acid molecule encoding said antibody, or a vector containing said nucleic acid molecule.

18. A composition for diagnosing cancer, comprising as an active ingredient the anti-CD20 asymmetric antibody of claim 1, a nucleic acid molecule encoding said antibody, or a vector containing said nucleic acid molecule.

19. Complement Dependent Cytotoxicity, which includes the steps of: A method for producing an anti-CD20 asymmetric antibody having improved cytotoxicity (CDC) compared to an anti-CD20 symmetric antibody: (a) preparing a single-chain variable fragment (scFv) comprising an LCDR1 of SEQ ID NO: 16, an LCDR2 of SEQ ID NO: 18, an LCDR3 of SEQ ID NO: 20, an HCDR1 of SEQ ID NO: 24, an HCDR2 of SEQ ID NO: 26, and an HCDR3 of SEQ ID NO: 28; and (b) preparing a Fab (fragment antigen-binding) antibody comprising an LCDR1 of SEQ ID NO:33, an LCDR2 of SEQ ID NO:35, an LCDR3 of SEQ ID NO:37, an HCDR1 of SEQ ID NO:42, an HCDR2 of SEQ ID NO:44, and an HCDR3 of SEQ ID NO:

46.

20. Complement Dependent Cytotoxicity, which includes the steps of: A method for producing a nucleic acid molecule encoding an anti-CD20 asymmetric antibody having improved cytotoxicity (CDC) compared to an anti-CD20 symmetric antibody: (a) preparing a nucleic acid molecule encoding a single-chain variable fragment (scFv) comprising an LCDR1 of SEQ ID NO: 16, an LCDR2 of SEQ ID NO: 18, an LCDR3 of SEQ ID NO: 20, an HCDR1 of SEQ ID NO: 24, an HCDR2 of SEQ ID NO: 26, and an HCDR3 of SEQ ID NO: 28; and (b) preparing a nucleic acid molecule encoding a Fab (Fragment antigen-binding) comprising an LCDR1 of SEQ ID NO:33, an LCDR2 of SEQ ID NO:35, an LCDR3 of SEQ ID NO:37, an HCDR1 of SEQ ID NO:42, an HCDR2 of SEQ ID NO:44, and an HCDR3 of SEQ ID NO:46.

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