Novel antibodies against CD55 and uses thereof

Abstract

The present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to CD55; and a composition for preventing, treating and / or diagnosing cancer comprising the same. The antibody of the present invention exhibits high binding and inhibitory power against CD55 protein, which suppresses the complement immune mechanism and promotes tumor growth, and can therefore be used as an efficient therapeutic composition for various diseases mediated by CD55. In addition, the antibody of the present invention can be usefully used as an efficient therapeutic adjuvant that fundamentally removes drug resistance and significantly improves therapeutic responsiveness in various diseases in which resistance to therapeutic agents that have CDC (complement dependent cytotoxicity) as an action mechanism is induced due to overexpression of CD55.

Description

[Technical field]

[0001] The present invention relates to a novel antibody or an antigen-binding fragment thereof that specifically recognizes CD55, and a composition for preventing, treating and / or diagnosing cancer using the same. [Background technology]

[0002] The global anticancer drug market has grown at an average annual 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 will experience cancer at least once in their lifetime, and the prevalence and mortality rates of cancer are continuously 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] Meanwhile, CD55 (Decay-accelerating factor, DAF) is a receptor that suppresses the complement immune mechanism and is highly expressed in various solid cancers such as colorectal cancer, lung cancer, gastric cancer, breast cancer, ovarian cancer, leukemia, and head and neck cancer, and promotes the proliferation of cancer cells by suppressing the complement immune system in the body from killing cancer cells. Therefore, CD55 is being actively studied as a target molecule for targeted anti-cancer therapy.

[0005] In particular, solid tumors with high CD55 expression show low therapeutic response to various anticancer drugs that have complement dependent cytotoxicity (CDC) as their mechanism of action, and therefore there is a demand for the development of superior antibody therapeutics that can bind to CD55 with higher affinity and specificity and efficiently inhibit its activity.

[0006] 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 explain the state of the art to which the present invention pertains and the contents of the present invention. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Republic of Korea Patent Registration No. 10-1800774 Summary of the Invention [Problem to be solved by the invention]

[0008] The present inventors have made extensive research and efforts to develop an excellent CD55 inhibitor that can specifically recognize CD55 protein, a receptor that inhibits the complement immune mechanism, and specifically inhibit its activity, thereby efficiently restoring the immune mechanism inhibited by CD55. As a result, they discovered that when an antibody in which some amino acids in the core antigen recognition site in the variable region are substituted is used, the complement-mediated cell lysis activity against cancer cells and the like is significantly restored due to the significantly increased CD55 binding and inhibition, which led to the completion of the present invention.

[0009] Therefore, an object of the present invention is to provide an antibody or an antigen-binding fragment thereof that specifically binds to CD55; and a composition comprising the same for preventing, treating, and / or diagnosing cancer.

[0010] It is another object of the present invention to provide a bispecific antibody comprising an antigen-binding fragment of an antibody that specifically binds to CD55 and an antigen-binding fragment of an antibody that specifically binds to CD20.

[0011] 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]

[0012] According to one aspect of the present invention, there is provided an antibody or antigen-binding fragment thereof that specifically binds to CD55, comprising a heavy chain variable region including an HCDR1 region having an amino acid sequence represented by the following general formula 1; an HCDR2 region having an amino acid sequence of the first sequence in the sequence listing; and an HCDR3 region having an amino acid sequence represented by the following general formula 2:

[0013] General formula 1

[0014] DRGM-X1;

[0015] General formula 2

[0016] NA-X2-AGGWHAAYIDA,

[0017] X1 in the general formula 1 is Ala or Val, and X2 in the general formula 2 is selected from the group consisting of Val, Ala, Ile, Leu, Phe and Met.

[0018] The present inventors have made extensive research and efforts to develop an excellent CD55 inhibitor that can specifically recognize CD55 protein, a receptor that inhibits the complement immune mechanism, and can effectively restore the immune mechanism inhibited by CD55 by specifically inhibiting its activity. As a result, it was discovered that when an antibody in which some amino acids in the core antigen recognition site in the variable region are substituted is used, the complement-mediated cell lysis activity against cancer cells and the like is significantly restored due to the significantly increased CD55 binding and inhibition power.

[0019] 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.

[0020] 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.

[0021] 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, 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).

[0022] Fv is the smallest antibody fragment that has 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, a heavy chain variable region and a light chain variable region are linked by a non-covalent bond. In a single-chain Fv, a heavy chain variable region and a 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 a 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.

[0023] As used herein, the term "heavy chain" refers to a variable region domain V that contains an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. H , and three constant region domains C H1 , C H2 and C H3 As used herein, the term "light chain" refers to a full-length heavy chain and fragments thereof, including a variable region domain V that includes an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. L and constant region domain C L The term "light chain" refers to any full-length light chain and fragments thereof.

[0024] As used herein, the term "CDR (complementarity determining region)" refers to the amino acid sequences of the hypervariable regions of the heavy and light chains of immunoglobulins (Kabat et al. Sequences of Proteins of Immunological Interest, 4th Ed., USDepartment of Health and Human Services, National Institutes of Health (1987)). Each of the heavy chain (HCDR11, HCDR2, and HCDR3) and the light chain (LCDR1, LCDR2, and LCDR3) contains three CDRs, which provide the main contact residues when an antibody binds to an antigen or epitope.

[0025] The scope of the antibody or antibody fragment of the present invention includes variants having conservative amino acid substitutions in the CDR regions, and can include variants of the amino acid sequences set forth in the attached sequence listing, as long as they can specifically recognize CD55. For example, additional changes can 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 interpreted 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.

[0026] 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."

[0027] According to a specific embodiment of the present invention, X1 in the general formula 1 is Ala.

[0028] According to a specific embodiment of the present invention, X2 in the general formula 2 is Val.

[0029] More specifically, the heavy chain variable region of the present invention comprises one or more framework regions (FR) selected from the group consisting of HFR1 to HFR4:

[0030] HFR1: EVQLY1ESGGGLVQPGGSLRLSCY2AY3GFTFS (Y1 is Val or Ala, Y2 is Val or Ala, and Y3 is Ser or Arg.)

[0031] HFR2: WVRQY4PGKGLEWVS (Y4 is Ala or Ser).

[0032] HFR3: RY5TISRDNSKNTLYLQMNY6LRAEDTAVYYCAK (Y5 is Ala or Thr, and Y6 is Ser or Asn.)

[0033] HFR4:WGQGTTVTVSS

[0034] More specifically, the heavy chain variable region of the present invention has an amino acid sequence selected from the group consisting of SEQ ID NO:19 to SEQ ID NO:22.

[0035] According to a specific embodiment of the present invention, the antibody or antigen-binding fragment thereof of the present invention further comprises a light chain variable region including: an LCDR1 region having the amino acid sequence of the second sequence in the sequence listing; an LCDR2 region having the amino acid sequence represented by the following general formula 3; and an LCDR3 region having the amino acid sequence of the third sequence in the sequence listing:

[0036] General formula 3

[0037] WN-X3-KRPS,

[0038] X3 in the general formula 3 is Asn or Asp.

[0039] More specifically, said X3 is Asp.

[0040] More specifically, the light chain variable region of the present invention comprises one or more framework regions (FR) selected from the group consisting of LFR1 to LFR4:

[0041] LFR1:SYELTQPPSVSVSPGQTASITC

[0042] LFR2: WY7QQKPGQSPVTVIY (Y7 is Phe or Tyr) (F or Y)

[0043] HFR3:GIPERFSGSKSGNTATLTISGTQAMDEADYYC

[0044] HFR4:FGGGTKLTVL

[0045] More specifically, the light chain variable region of the present invention has the amino acid sequence of SEQ ID NO:4 or 18.

[0046] According to another aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CD55, comprising the following light chain variable region and heavy chain variable region:

[0047] a) a light chain variable region of the fourth sequence in the sequence listing; or a light chain variable region of the fourth sequence in the sequence listing, the light chain variable region including one or more substitutions selected from the group consisting of a substitution of Y (Tyr) at the 32nd amino acid sequence with F (Phe) and a substitution of D (Asp) at the 48th amino acid sequence with N (Asn), and

[0048] b) a heavy chain variable region of sequence 5 of the sequence listing; or a heavy chain variable region of sequence 5 of the sequence listing, comprising one or more substitutions selected from the group consisting of a substitution of V (Val) at the 5th amino acid position with A (Ala), a substitution of A (Ala) at the 23rd amino acid position with V (Val), a substitution of S (Ser) at the 25th amino acid position with R (Arg), a substitution of A (Ala) at the 35th amino acid position with V (Val), a substitution of A (Ala) at the 40th amino acid position with S (Ser), a substitution of A (Ala) at the 68th amino acid position with T (Thr), a substitution of S (Ser) at the 85th amino acid position with N (Asn), and a substitution of G (Gly) at the 101st amino acid position with V (Val), L (Leu), I (Ile), F (Phe), M (Met), or A (Ala).

[0049] 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.

[0050] 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.

[0051] 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.

[0052] In yet another aspect, the present invention provides a composition for preventing or treating cancer, comprising the above-mentioned antibody or antigen-binding fragment thereof of the present invention as an active ingredient.

[0053] In yet another aspect, the present invention provides a method for preventing or treating cancer, comprising administering to a subject a pharma- ceutically effective amount of the above-mentioned antibody or antigen-binding fragment thereof of the present invention.

[0054] According to yet another aspect of the present invention, there is provided a method for treating a disease comprising administering to a patient an antibody or an antigen-binding fragment thereof, comprising administering to said patient a therapeutic agent in a therapeutic setting.

[0055] 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.

[0056] As used herein, the term "treatment" refers to (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 serves to restore the complement immune mechanism by suppressing the activity of the CD55 receptor, inhibit the progression of cancer by suppressing the proliferation of cancer cells, and eliminate or alleviate the symptoms caused by the cancer. Thus, the composition of the present invention may be a composition for treating cancer itself, or may be administered together with other pharmacological components, for example, a therapeutic agent whose mechanism of action is complement dependent cytotoxicity (CDC), and may be used 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".

[0057] 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.

[0058] 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."

[0059] 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.

[0060] According to a specific embodiment of the present invention, the composition further comprises one or more pharmacological ingredients selected from the group consisting of an anti-CD20 antibody and a tyrosine kinase inhibitor.

[0061] More specifically, said anti-CD20 antibody is Rituximab.

[0062] More specifically, the tyrosine kinase inhibitor is selected from the group consisting of Imatinib and Gefitinib.

[0063] According to a specific embodiment, the cancer that can be prevented or treated by the composition of the present invention is a CD55-positive cancer.

[0064] As used herein, the term "CD55-positive cancer" refers to a carcinoma composed of cancer cells expressing the CD55 receptor, specifically, a cancer in which CD55 is expressed to such an extent that complement immune activity in the subject is measurably inhibited. Thus, the term "CD55-positive cancer" includes "CD55-overexpressing cancer". According to the present invention, the composition of the present invention can eliminate drug resistance and enhance therapeutic sensitivity in various carcinomas in which resistance to therapeutic agents is induced due to overexpression of CD55.

[0065] CD55 positive cancers include, for example, but are not limited to, melanoma, lymphoma, 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, leukemia, bone marrow cancer, ovarian cancer, cervical cancer, sarcoma, cholangiocarcinoma, and endometrial cancer, and include all solid cancers and blood cancers that express CD55.

[0066] In still another aspect, the present invention provides a composition for diagnosing cancer, comprising the above-mentioned antibody of the present invention or an antigen-binding fragment thereof as an active ingredient.

[0067] 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.

[0068] 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.

[0069] The CD55 receptor is a typical cancer diagnostic marker that shows a higher expression level in cancer tissues than in normal tissues, and the antibody of the present invention that specifically binds to CD55 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.

[0070] 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 CD55 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."

[0071] As used herein, the term "biological sample" includes, but is not limited to, tissues, cells, whole blood, serum, plasma, saliva, urine, lymph fluid, spinal fluid, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatants, ruptured eukaryotic cells, and bacterial expression systems.

[0072] In biological samples, detection of CD55 protein can be performed 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.

[0073] 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.

[0074] 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.

[0075] According to yet another aspect of the present invention, there is provided a bispecific antibody or a functional fragment thereof comprising the above-mentioned antigen-binding fragment of the present invention and an antigen-binding fragment of an antibody that specifically binds to CD20.

[0076] As used herein, the term "bispecific antibody" refers to an antibody that contains two distinct antigen-binding regions that are defined by different amino acid sequences. Typically, the two antigen-binding regions contained in a bispecific antibody are each specific for a different antigen or epitope.

[0077] The bispecific antibody of the present invention is a single antibody (CD20 x CD55) molecule in which the above-mentioned CD55-specific antigen-binding fragment of the present invention is combined with another antigen-binding fragment that specifically recognizes CD20, and according to the present invention, shows a more excellent therapeutic effect against rituximab-resistant tumors than the combined administration of each anti-CD55 antibody (e.g., EP-10H in the Examples described below) and an anti-CD20 antibody (e.g., rituximab).

[0078] According to a specific embodiment of the present invention, the antigen-binding fragment of the anti-CD55 antibody contained in the bispecific antibody is in the form of an scFv.

[0079] According to a specific embodiment of the present invention, the antigen-binding fragment of an antibody that specifically binds to CD55 comprises an scFv.

[0080] According to a specific embodiment of the present invention, the antigen-binding fragment of an antibody that specifically binds to CD55 has a VL-linker-VH-CH2-CH3 format.

[0081] According to a specific embodiment of the present invention, the antigen-binding fragment of the anti-CD20 antibody contained in the bispecific antibody is in the form of Fab.

[0082] According to a specific embodiment, the antigen-binding fragment of an antibody that specifically binds to CD20 comprises Fab.

[0083] According to a specific embodiment of the present invention, the antigen-binding fragment of an antibody that specifically binds to CD20 has the form VL-CK-linker-VH-CH1-CH2-CH3.

[0084] According to a specific embodiment of the present invention, the antibody that specifically binds to CD20 is rituximab.

[0085] The bispecific antibodies of the 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 bispecific antibodies of the 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 bispecific antibodies of the present invention can use the knob-into-hole approach to prevent undesired pairing of CD20 XCD55 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 bispecific antibody of the present invention may be in a form in which an antigen-binding fragment of an anti-CD55 antibody in the form of VL-linker-VH-CH2-CH3 is combined with an antigen-binding fragment of an anti-CD20 antibody in the form of VL-CK-linker-VH-CH1-CH2-CH3.

[0088] According to a specific embodiment of the present invention, the bispecific 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 CD20 x CD55 heavy chains.

[0089] Specifically, the bispecific antibody of the present invention may be in a form in which CD55-scFv-knob (VL-linker20-VH-CH2-CH3) and CD20-Fab-hole (VL-CK-linker40-VH-CH1-CH2-CH3) are linked together.

[0090] 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.

[0091] Linker 40 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 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Gly Ser Ser. Effect of the Invention

[0092] The features and advantages of the present invention can be summarized as follows:

[0093] (a) The present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to CD55; and a composition for preventing, treating and / or diagnosing cancer comprising the same.

[0094] (b) The antibody of the present invention exhibits high binding and inhibitory activity against CD55 protein, which suppresses complement immune mechanisms and promotes tumor growth, and can therefore be used as an effective therapeutic composition for various diseases mediated by CD55.

[0095] (c) Furthermore, the antibody of the present invention can be usefully used as an efficient therapeutic adjuvant that fundamentally eliminates drug resistance and significantly improves therapeutic response in various diseases in which resistance to therapeutic agents having complement dependent cytotoxicity (CDC) as a mechanism of action is induced due to overexpression of CD55. [Brief description of the drawings]

[0096] [Figure 1] FIG. 1 shows the results of ELISA analysis measuring the affinity of 4-1H and G4-1H for CD55. [Diagram 2] FIG. 1 shows the results of ELISA analysis measuring the affinity for CD55 of antibodies prepared by screening from a library in which random mutations were introduced into the genes of the G4-1H variable region. [Diagram 3] FIG. 1 shows the results of ELISA analysis measuring the affinity for CD55 of antibodies prepared by substituting the binding-enhancing core site with hydrophobic amino acids. [Figure 4] FIG. 1 shows the results of a FACS assay analyzing the killing effect of BJAB cells by combined treatment with rituximab and EP-10H. [Figure 5a] FIG. 1 shows the results of a FACS assay comparing and analyzing the expression levels of CD20 and CD55 in Ramos and Ramos-RR. [Figure 5b] FIG. 1 shows the results of a FACS assay analyzing the killing effect of Ramos-RR cells by combined treatment with rituximab and EP-10H antibody. [Figure 6a] FIG. 1 shows the results of a FACS assay comparing and analyzing the expression levels of CD55 in K562 and K562-IR. [Figure 6b] FIG. 1 shows the results of a FACS assay analyzing the killing effect of 562-IR cells by combined treatment with imatinib and EP-10H antibody. [Figure 7a] FIG. 1 shows the results of a FACS assay comparing and analyzing the levels of CD55 expression in PC9 and PC9-GR. [Figure 7b] FIG. 1 shows the results of a FACS assay analyzing the killing effect of PC9-GR cells by combined treatment with imatinib and EP-10H antibody. [Figure 8] FIG. 1 is a schematic diagram of an antibody comparing the CD20×CD55 bibody of the present invention, SBU, with the bibody form reported by Macor. [Figure 9] FIG. 1 shows the results of SDS-PAGE analysis of the CD20X CD55 bibody of the present invention in the SBU format. [Figure 10a] FIG. 10 shows the results of ELISA analysis measuring the affinity of CD20×CD55 double antibodies for CD20 (FIG. 10a) and CD55 (FIG. 10b), respectively. [Figure 10b] FIG. 10 shows the results of ELISA analysis measuring the affinity of CD20×CD55 double antibodies for CD20 (FIG. 10a) and CD55 (FIG. 10b), respectively. [Figure 11a] FIG. 1 shows the results of a FACS assay analyzing the killing effect of BJAB cells by CD20×CD55 double antibody. [Figure 11b] FIG. 1 shows the results of comparing the death rates of BJAB cells following treatment with rituximab alone, combined treatment with rituximab and EP-10H antibody, and treatment with CD20 XCD55 double antibody. [Figure 12a] FIG. 1 shows the results of a FACS assay analyzing the killing effect of Ramos cells by CD20×CD55 double antibody. [Figure 12b] FIG. 1 shows the results of comparing the death rates of Ramos cells following treatment with rituximab alone, combined treatment with rituximab and EP-10H antibody, and treatment with CD20 XCD55 double antibody. [Figure 13a] FIG. 1 shows the results of a FACS assay analyzing the killing effect of Ramos-RR cells by CD20 x CD55 double antibody. [Figure 13b] FIG. 1 shows the results of comparing the death rates of Ramos-RR cells following treatment with rituximab alone, combined treatment with rituximab and EP-10H antibody, and treatment with CD20 XCD55 double antibody. [Figure 14] FIG. 1 shows the results of confirming the amount of C4d produced in the supernatant obtained by treating Ramos-RR cells with rituximab and a CD20X CD55 double antibody in the form of an SBU. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0097] 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 explaining the present invention in more detail, and that the scope of the present invention is not limited to these examples according to the gist of the present invention.

[0098] EXAMPLES

[0099] Example 1: Humanization of 4-1H

[0100] In order to reduce the immunogenicity of 4-1H disclosed in Korean Patent Application No. 10-2016-0132333, humanization was carried out in the following manner:

[0101] For humanization of 4-1H, three FRs (LFR1, LFR2, and LFR3) in the light chain variable region of 4-1H were cloned into IGLV3-1 * 01 FR, and three FRs (HFR1, HFR2, and HFR3) in the heavy chain variable region were replaced with IGHV3-23D *02 FR. After that, taking into consideration the similarity or dissimilarity of the physicochemical properties of the amino acids, specific amino acids were replaced again with amino acids of the parent antibody (4-1H). The amino acids replaced again in the parent antibody (4-1H) were L46T and N66K in the light chain variable region and F68A in the heavy chain variable region. The amino acid residue numbers of the antibody domains were numbered according to the Kabat EU numbering system (Kabat EU numbering system, Kabat et al., "Sequences of Proteins of Immunological Interest", 5th Ed., USDepartment of Health and Human Services, NIH Publication No. 91-3242, 1991) commonly used in the art. The J gene of the light chain variable region was fixed to FGGGTKLTVL with reference to US2010-0056386, and the J gene of the heavy chain variable region was fixed to WGQGTTVTVSS with reference to US2014-0206849. The humanized 4-1H antibody was named G4-1H, and its nucleotide sequence is shown in Table 1.

[0102] [Table 1]

[0103] Amino acid sequences of the heavy and light chain variable regions of G4-1H

[0104] In the table, the underlined letters indicate the CDR regions and the bold type indicates the amino acids that were substituted back into the parent antibody (4-1H).

[0105] Example 2: Conversion and expression / purification of complete antibody of G4-1H clone

[0106] The DNA of the variable region of the G4-1H antibody prepared in Example 1 was synthesized in the form of scFv (Cosmogenetech, Korea) and converted to a full-length antibody (full-length IgG) through PCR. First, fragments of the variable and constant regions of the heavy and light chains were extracted from a pUC vector (Cosmogenetech, Korea) containing scFv, and the VFv fragments shown in Table 2 below were extracted. H , C H and V L , C K The heavy chain was treated with EcoRI and NotI (New England Biolab, UK) enzymes and ligated to a pCMV vector (ThermoFisher Scientific, USA), an animal cell expression vector, similarly treated with the same restriction enzymes. The light chain was treated with XbaI (New England Biolab, UK) enzymes and ligated to a pCMV vector 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.

[0107] [Table 2]

[0108] Primers used for G4-1H complete antibody cloning

[0109] The heavy and light chain plasmids converted to complete antibodies were transfected into Expi293F cells (Invitrogen, USA) using polyethyleneimine (PEI) (Polysciences, USA) and 150 mM NaCl, and the cells were cultured in suspension in Freestyle 293 expression medium (Invitrogen, USA) at 37°C, 8% CO2, and 55% humidity in Erlenmeyer flasks for 7 days. The expression cell culture was centrifuged at 4,000 rpm for 10 minutes, and the supernatant was collected and then filtered through a 0.22 μm filter. The filtered supernatant was induced to bind to 1 ml of MabSelect sure (GE Healthcare, USA) resin at 4°C. The bound resin was washed with 10 cv (column volume) of 20 mM dibasic sodium phosphate and 500 mM sodium chloride (pH 7.0) solution, and the bound antibody was eluted with 100 mM citric acid (pH 3.0) solution, followed by neutralization with 1 M Tris-HCL (pH 9.0). After buffer exchange with PBS (pH 7.2-7.4) using a Slide-A-Lyzer dialysis cassette (ThermoFisher Scientific, USA), the size and purity of the purified antibody light and heavy chains were finally confirmed through SDS-PAGE, and the molecular weight and high purity were confirmed to be consistent with the theoretical calculation.

[0110] Example 3: Affinity analysis of 4-1H and G4-1H for CD55

[0111] The binding ability of the G4-1H monoclonal antibody prepared in Example 2 to CD55 was confirmed by indirect ELISA. Indirect ELISA was performed by diluting recombinant human CD55 (R&D Systems, USA) to 1 μg / ml in 50 μl of PBS, placing it in a 96-well immunoplate (Corning, USA), storing it at 4°C, and adsorbing it overnight. After reacting with a buffer solution containing 3% bovine serum albumin (Millipore, USA) at room temperature for 1 hour, the plate was washed three times with a buffer solution containing 0.5% Tween 20 (Amresco, USA), and treating each antibody diluted at successive concentrations (0.0001, 0.0003, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10 nM) with 50 μl 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 and treated with 50 μl per well and reacted at room temperature for 1 hour. After the reaction was completed, the plate was washed three times with a buffer solution containing 0.5% Tween 20 (Amresco, USA) and colored for 10 minutes with 50 μl of 3,3',5,5'-tetramethylbenzidine (TMB) (ThermoFisher Scientific, USA). The absorbance was measured at 450 nm using a spectrophotometer (Biotek, USA), and G4-1H showed a lower affinity for CD55 than 4-1H (Figure 1).

[0112] Example 4: Construction of a library in which random mutations are introduced into variable region genes

[0113] To improve the affinity of G4-1H for CD55, a library was constructed in which random mutations were introduced into the variable region genes of the G4-1H antibody through an error-prone PCR process. Specifically, the pComb3x vector containing G4-1H scFv was loaded with scFv sequencing primer 1 and primer 2, polymerase without proofreading function (Takara, Japan), polymerase buffer, dATP, dTTP, dCTP, and dGTP, and the mutation frequency was adjusted by adjusting the salt concentration and manganese amount. Error-prone PCR consisted of 30 cycles of preheating at 94°C for 5 minutes, double helix separation at 91°C for 1 minute, primer binding at 55°C for 1 minute, and DNA synthesis reaction at 72°C for 3 minutes, followed by DNA synthesis reaction at 72°C for 5 minutes. The purified PCR product and the phagemid vector pComb3x vector were cleaved with the SfiI restriction enzyme (New England Biolab, UK), then ligated together, transformed into ER2738 E. coli, and cultured.

[0114] [Table 3]

[0115] Primers used for error-prone PCR

[0116] Example 5: Selection of clones with improved affinity to CD55 compared to 4-1H

[0117] Through biopanning using the library prepared in Example 4, clones having improved affinity to CD55 compared to G4-1H and 4-1H were selected.

[0118] First, 2.5μg of recombinant human CD55 (R&D Systems 2009-CD / CF, USA) was conjugated to magnetic beads (Invitrogen, USA). The phage library was reacted with CD55 conjugated to magnetic beads for 2 hours at room temperature to bind phages with affinity to CD55, and then the phages were reacted with 4-1H antibody for 30 minutes at room temperature to compete with 4-1H. The phages were then washed with a buffer solution containing 0.5% Tween 20 (Amresco, USA) and eluted with an acidic solution. The eluted phages were infected with E. coli ER2738 for the next round of panning, and then cultured overnight to grow. This process was repeated four times to perform biopanning. The phages were washed once in the first round, three times in the second and third rounds, and five times in the fourth round. The number of washes was increased as the number of panning rounds increased to accumulate phages with high binding strength.

[0119] 192 clones selected from the plates of the third and fourth biopanning results were placed in a 96-deep well plate with 100 μg / ml carbenicillin, 70 μg / ml kanamycin, and VCSM13 helper phage, and then cultured overnight at 37 ° C to induce proliferation of phages expressing antibodies. The culture medium was centrifuged to obtain a medium supernatant containing phages, which was placed in an ELISA plate coated with recombinant human CD55 (R&D Systems 2009-CD / CF, USA) and cultured for 2 hours at 37 ° C. Antibodies that bind to CD55 were confirmed by ELISA using HRP-conjugated anti-M13 antibody (Merck, USA) as a secondary antibody. The phage of the 4-1H clone was used as a control group, and five clones with higher binding strength were selected.

[0120] Example 6: Conversion and expression / purification of complete antibodies of selected clones

[0121] EP-1E, EP-1F, EP-9B, 3R-2-1H and EP-10H were cloned to produce full-length complete IgG forms, and have the CDR sequences shown in Table 4 below.

[0122] [Table 4]

[0123] Amino acid sequences of the light and heavy chain variable regions of the selected clones

[0124] In the table, the underlined parts indicate the CDR regions, and the bold type indicates amino acids that are different from G4-1H. The antibodies EP-1E, EP-1F, EP-9B, 3R-2-1H, and EP-10H selected in Example 5 were in the form of scFvs, and were converted to complete antibodies, and the conversion and expression / purification of the complete antibodies were performed in the same manner as in Example 2 above.

[0125] [Table 5]

[0126] Primers used for complete antibody cloning of selected clones

[0127] Example 7: Analysis of affinity of selected and produced complete antibodies to CD55

[0128] The affinity of the antibody prepared in Example 6 for CD55 was confirmed by indirect ELISA. The indirect ELISA was performed in the same manner as in Example 3 above.

[0129] As a result of ELISA, the antibody of Example 6 showed higher affinity for CD55 than 4-1H (Figure 2). EP-1E, EP-1F, 3R-2-1H and EP-10H showed similar affinity, and EP-9B showed higher affinity than 4-1H but lower affinity than other antibodies. Each antibody with higher affinity for CD55 than 4-1H can be very useful for various cancer diagnosis and preparation of pharmaceutical therapeutic compositions.

[0130] Example 8: Amino acid substitutions at the core site to improve binding

[0131] In each of the clones listed in Table 4, G4-1H was replaced with V at the H100A position according to the Kabat EU numbering system. This portion is included in the HCDR3 region, which is important for antigen binding of the antibody. The present inventors recognized this site as a key site for improving binding strength, and performed cloning to replace this site with a hydrophobic amino acid other than V. 10H(A), 10H(I), 10H(L), 10H(F), and 10H(M), which were produced using EP-10H, which differs from G4-1H only at the H100A position, as the basic backbone, have the following CDR sequences (Table 6).

[0132] [Table 6]

[0133] In the heavy chain variable region of EP-10H, a complete IgG form cloned into the pCMV vector (ThermoFisher Scientific, USA), an animal cell expression vector, amino acids in the heavy chain complementarity determining region that had undergone mutations different from those in G4-1H were replaced with hydrophobic amino acids.

[0134] First, the heavy chain variable region and constant region were obtained by PCR from the pCMV vector containing the heavy chain variable region of EP-10H using the combination of HC1 and HC2 primers in Table 4. The heavy chain variable region and constant region obtained by PCR were subjected to overlap PCR using HC3 forward primer and HC2 reverse primer to obtain the VH-CH1-CH2-CH3 form. The heavy chain thus obtained was cleaved with EcoRI and NotI (New England Biolab, UK) restriction enzymes and ligated to the pCMV vector (ThermoFisher Scientific, USA), an animal cell expression vector similarly treated with the same restriction enzymes. The ligated plasmid was transformed into DH5α E. coli (New England Biolab, UK) by heat shock and then cultured in large quantities to obtain the plasmid.

[0135] [Table 7]

[0136] Primers used for amino acid substitutions at the core site to improve binding

[0137] After completing the cloning, expression / purification of complete antibodies using the EP-10H light chain plasmid and the heavy chain plasmid in Table 6 was performed in the same manner as in Example 2 above.

[0138] Example 9: Analysis of affinity of complete antibodies prepared by substituting core sites to improve binding to CD55

[0139] The affinity of the antibody prepared in Example 8 for CD55 was confirmed by indirect ELISA. The indirect ELISA was performed in the same manner as in Example 3 above.

[0140] ELISA analysis showed that the antibodies prepared by substituting hydrophobic amino acids at the core of binding showed higher affinity for CD55 than 4-1H (Figure 3). However, 10H(A), 10H(I), 10H(L), 10H(F) and 10H(M) showed lower affinity than EP-10H. This means that the substitution of G to V at the H100A position is the most efficient way to improve binding.

[0141] Example 10: Therapeutic effect of EP-10H in drug-resistant cancer cell lines overexpressing CD55

[0142] Experimental Example 10-1: Combined effect of rituximab and EP-10H on Burkitt's lymphoma cell line BJAB

[0143] BJAB has complement resistance, which is due to the expression of CD55 (Golay, et al., Blood, 95(12):3900-8, 2000). For this reason, it has low responsiveness to rituximab, whose main therapeutic mechanism is CDC (complement dependent cytotoxicity). To confirm the possibility of improving the therapeutic effect of rituximab against Burkitt lymphoma, which is known as a malignant B-cell lymphoma, we confirmed the cell killing ability in combination with EP-10H in the Burkitt lymphoma cell line BJAB. 5 × 10 per sample 5For BJAB cells, rituximab was diluted at a concentration of 20 μg / ml, and 4-1H or EP-10H and rituximab were diluted at concentrations of 50 μg / ml and 20 μg / ml in RPMI medium supplemented with 50% complement sera human (Sigma, USA) to confirm the effect of combined administration, and 100 μl was finally treated and cultured for 1 hour under conditions of 37°C and 5% CO2. Then, dead cells were confirmed using the FITC Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend, USA) with the FACS analysis equipment Attune NxT (ThermoFisher Scientific, USA) and CDC was observed (Figure 4). As previously reported, BJAB cells had low reactivity to rituximab, but the effect was increased when co-administered with anti-CD55 antibodies (4-1H and EP-10H), and the extent of the increased effect was greater when rituximab was combined with EP-10H than when rituximab was combined with 4-1H. These results indicate that the expression of CD55 inhibits the CDC effect of rituximab, but the inhibited effect is restored when CD55 is suppressed using an antibody, and that such an effect is more pronounced when EP-10H, which has a higher affinity for CD55, is used.

[0144] Experimental Example 10-2a: Confirmation of CD55 expression in the rituximab-resistant cell line Ramos-RR

[0145] Ramos conducted the following experiment to confirm CD55 expression in Ramos-RR, a rituximab-resistant cell line that was created using Ramos as a rituximab-responsive B cell lymphoma cell line.

[0146] 5 x 10 per sample 5Ramos and Ramos-RR were suspended in PBS containing or not containing EP-10H monoclonal antibody at a concentration of 10 μg / ml and incubated at 4°C for 1 hour. The culture medium was centrifuged at 4,000 rpm for 5 minutes, washed with 100 μl of PBS, and centrifuged again at 4,000 rpm for 5 minutes. The cells were treated with goat anti-human IgG antibody, Alexa Fluor 488 (ThermoFisher Scientific, USA) diluted 1:100 with PBS and incubated at 4°C for 30 minutes in the dark. The fluorescently stained cells were washed with PBS, suspended in 400 μl of PBS, and analyzed using Attune NxT (ThermoFisher Scientific, USA), a FACS analysis device, and the results are shown in Figure 5a.

[0147] As a result, we confirmed that the expression of CD55 was increased in Ramos-RR compared to Ramos, indicating that CD55 is involved in drug resistance to rituximab.

[0148] Experimental Example 10-2b: Combined effect of rituximab and EP-10H on rituximab-resistant cell line Ramos-RR

[0149] In Ramos-RR, which has overexpressed CD55 and shows resistance to rituximab, the cell death ability was confirmed by co-administration of rituximab and EP-10H as in Experimental Example 10-1 (Figure 5b). Rituximab induced cell death in Ramos, while the degree of cell death induction in Ramos-RR was lower than that of Ramos. However, the combination of EP-10H and 4-1H increased cell death in Ramos-RR, and as with BJAB, the combination with EP-10H further increased the cell death ability of rituximab compared to the combination with 4-1H. These results indicate that EP-10H has a therapeutic effect not only on primary cancers in B-cell lymphoma (BJAB cell line is a primary cancer cell line) but also on cancers that have acquired resistance to existing treatments.

[0150] Experimental Example 10-3a: Confirmation of CD55 expression in imatinib-resistant cell line K562-IR

[0151] K562 is a leukemia cell line that is responsive to imatinib, a tyrosine kinase inhibitor. In K562-IR, an imatinib-resistant cell line created using K562, CD55 expression was confirmed by the same method as in Experimental Example 10-2a. As a result, it was confirmed that CD55 expression in K562-IR was increased compared to K562 (Figure 6a). This means that CD55 is involved in drug resistance to imatinib.

[0152] Experimental Example 10-3b: Confirmation of the combined effect of imatinib and EP-10H in K562-IR cell line

[0153] K562 and K562-IR, which overexpresses CD55 and shows resistance to imatinib, were cultured in 12-well cell culture plates at 2 × 10 cells per well. 5 Immediately after dispensing, imatinib was diluted to a concentration of 2 μM, and to confirm the effect of coadministration, imatinib 2 μM and 4-1H or EP-10H were diluted to a concentration of 10 μg / ml in RPMI medium, and finally 500 μl was treated and cultured for 24 hours under 37°C and 5% CO2 conditions. After that, dead cells were analyzed using 7-AAD and FITC Annexin V apoptosis detection kits with Attune NxT FACS analysis equipment (Figure 6b).

[0154] Imatinib induced cell death in K562 cell line, but had no effect on K562-IR. However, when co-administered with EP-10H or 4-1H, it induced cell death in K562-IR. In addition, the combination of EP-10H and imatinib showed a greater cell death effect than the combination of 4-1H and imatinib. These results, together with the results of Experimental Example 10-3a, prove that the cause of resistance to imatinib, a tyrosine kinase inhibitor, is CD55, and mean that inhibition of CD55 using EP-10H is an effective strategy to overcome imatinib resistance. Imatinib is a long-term drug that is widely used in the treatment of leukemia, and there is a strong need for a strategy to overcome drug resistance that can be induced by long-term administration. Therefore, the strategy to overcome imatinib resistance using EP-10H is expected to be very effective in the treatment of leukemia.

[0155] Experimental Example 10-4a: Confirmation of CD55 expression in PC9-GR, a gefitinib-resistant cell line

[0156] PC9 is a non-small cell lung cancer (NSCLC) cell line that is responsive to gefitinib, a tyrosine kinase inhibitor. In PC9-GR, a gefitinib-resistant cell line created using PC9, CD55 expression was confirmed by the method of Experimental Example 10-2a, and it was confirmed that CD55 expression in PC9-GR was increased compared to PC9 (Figure 7a). This means that CD55 is involved in drug resistance to gefitinib.

[0157] Experimental Example 10-4b: Confirmation of the combined effect of gefitinib and EP-10H in PC9-GR

[0158] NSCLC cell lines PC9 and PC9-GR, which overexpress CD55 and show resistance to gefitinib, were cultured in 12-well cell culture plates at 1 × 10 cells / well. 5After dispensing, the cells were cultured at 37℃ under 5% CO2 conditions. 24 hours after dispensing, gefitinib was added at a concentration of 5μg / ml, and gefitinib 5μg / ml and 4-1H or EP-10H were added at a concentration of 5μg / ml each in RPMI medium to confirm the effect of combined administration, and 500μl was finally treated and cultured again at 37℃ under 5% CO2 conditions for 24 hours. After that, dead cells were analyzed using 7-AAD and FITC Annexin V apoptosis detection kits with Attune NxT, a FACS analysis device (Figure 7b).

[0159] Gefitinib induced cell death in PC9 cell line, which is an NSCLC cell line, but had no effect on PC9-GR. However, the combination of EP-10H or 4-1H with gefitinib induced cell death in PC9-GR. In addition, the combination of EP-10H with gefitinib showed a greater cell death effect than the combination of 4-1H with gefitinib. These results, together with the results of Experimental Example 10-4a, prove that the cause of resistance to gefitinib, a tyrosine kinase inhibitor among targeted anticancer drugs, is CD55, and mean that inhibition of CD55 using EP-10H is an effective strategy to overcome gefitinib resistance. Gefitinib is an effective drug for NSCLC, for which there are few therapeutic agents, and since it is difficult to select a second drug when drug resistance appears in a situation where there are not many other options for therapeutic agents, a strategy to overcome drug resistance is urgently needed. The strategy of overcoming gefitinib resistance using EP-10H is expected to be highly effective in treating NSCLC.

[0160] Example 11: Preparation and efficacy of double antibody using EP-10H

[0161] The combined effect of EP-10H and various drugs was confirmed in Example 10. Based on the combined effect with rituximab confirmed in Experimental Examples 10-1 and 10-2 of Example 10, in order to compare the cancer cell killing ability of a biantibody that can simultaneously target two antigens with a single antibody, a CD20 x CD55 biantibody was prepared using rituximab and EP-10H, and the cancer cell killing ability of the biantibody was confirmed in comparison with that of the combined administration.

[0162] Experimental Example 11-1. Cloning of double antibodies (CD20 xCD55) for expression in animal cells

[0163] In the present invention, we have developed a novel bi-antibody that simultaneously targets CD20 and CD55, which is different from the previously reported bi-antibody (Macor, et al., Leukemia, 29(2):406-14, 2014). In order to compare the efficacy of different forms of bi-antibodies, we cloned a bi-antibody that simultaneously targets CD20 and CD55 using the genes of rituximab and EP-10H. The bi-antibody constructed in the present invention was named SBU (Specific Bi-functional Unit) (Figure 8).

[0164] The SBU-type CD20 x CD55 bibody 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 performed in the form of CD55-scFv-knob (VL-linker20-VH-CH2-CH3) and CD20-Fab-hole (VL-CK-linker40-VH-CH1-CH2-CH3), respectively. For CD55-scFv-knob, PCR was performed using the EP-10H prepared in Example 6 as a template and the primer combinations in Table 8. 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 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.

[0165] To prepare the CD20-Fab-hole (VL-CK-linker40-VH-CH1-CH2-CH3) form, PCR was performed using the primer combination in Table 9 based on the Rituximab sequence published in the Drug Data Bank. The amplified gene was treated with BssHII and XbaI enzymes and ligated to the pMAZ vector, an animal cell expression vector 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, and sequence analysis (Table 10) was completed.

[0166] [Table 8]

[0167] Primers used for CD55-scFv-knob cloning

[0168] [Table 9]

[0169] Primers used for CD20-Fab-hole cloning

[0170] [Table 10]

[0171] Amino acid sequences of the variable regions of CD55-scFv-knob and CD20-Fab-hole in SBU format

[0172] Experimental Example 11-2. Expression in animal cells and purification

[0173] The DNA prepared in Experimental Example 11-1 was expressed in the same manner as in Example 2. For the CD20X CD55 bibody in the SBU form, each plasmid of SEQ ID NO: 47 and 48 was transfected into HEK293F cells (Invitrogen, USA) using polyethyleneimine (PEI) (Polysciences, USA) and 150 mM NaCl, and the cells were cultured in Freestyle 293 expression medium (Invitrogen, USA) at 37°C, 8% CO2 and 55% humidity for 7 days. A bibody in a form similar to SBU has been reported (Moore, et al., A robust heterodimeric Fc platform engineered for efficient development of bispecific antibodies of multiple formats. Methods, 154:38-50), but the bibody disclosed in the literature requires three types of DNA for expression. Unlike conventional methods for producing double antibodies that require three types of DNA, the SBU of the present invention uses a linker consisting of Gly and Ser to prevent erroneous binding between light chains, making it possible to express double antibodies using two types of DNA. 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 a 100 mM glycine-HCl (pH 2.7) solution and neutralized with 1 M Tris-HCL (pH 9.0). After buffer exchange with PBS at pH 7.2-7.4, the size and purity of the light and heavy chains of the purified antibody were confirmed through SDS-PAGE, and the molecular weight and high purity were confirmed to be consistent with theoretical calculation values ​​(FIG. 9). The SBU format of the bibody is a heteroform in which the CD20 binding site is a Fab and the CD55 binding site is an scFv.By purifying with Protein L using the C kappa portion of Fab, it is possible to suppress the formation of knob-knob homodimers or knob monomers (Giese, et al., Biotechnol Prog, 34(2)397-404), which are known to be the most likely to be formed during purification of double antibodies, and to obtain only the desired form, the heterodimer.

[0174] Experimental Example 11-3. Confirmation of binding strength of double antibodies through ELISA

[0175] The affinity of the CD20X CD55 bibody prepared in Experimental Example 11-2 for the target antigens CD20 and CD55 was confirmed by indirect ELISA. Indirect ELISA was performed as in Example 3 and showed excellent binding to both CD20 and CD55 (Figures 10a and 10b). In particular, the SBU-type CD20X CD55 bibody showed higher affinity to CD20 than the CD20 XCD55 bibody disclosed in the prior literature by Macor, and showed similar affinity to CD55 as the CD20 XCD55 bibody disclosed in the prior literature by Macor. As a CD20 XCD55 bibody that can be used as a therapeutic agent for blood cancer in which CD20 is overexpressed, the SBU-type CD20 XCD55 bibody, which has a higher affinity to CD20, is more effective.

[0176] Experimental Example 11-4. Confirmation of cell killing ability of CD20X CD55 double antibody in BJAB

[0177] The cell killing ability of the CD20X CD55 double antibody was confirmed by the method of Experimental Example 10-1. 5Rituximab was administered to BJAB cells at a concentration of 20 μg / ml, EP-10H and rituximab were administered at a concentration of 20 μg / ml each to confirm the effect of combined administration, and CD20X CD55 bibody in the form disclosed in the prior literature written by Macor and in the SBU form were each diluted at a concentration of 20 μg / ml in RPMI medium supplemented with 20% complement sera human (Sigma, USA), and finally 100 μl was treated. The rest was performed in the same manner as in Experimental Example 10-1.

[0178] As a result, the CD20 x CD55 bibody showed a significant difference in cell killing ability in BJAB cells, which have low reactivity to rituximab, compared to the control group treated with only complement serum human, and SBU showed a significant difference in cell killing ability, especially compared to rituximab (Figures 11a and 11b). These results indicate that a bibody that can simultaneously target two antigens is more effective in inducing CDC on cancer cells than the co-administration of two antibodies targeting different antigens, and that the SBU of the present invention is more suitable for inducing CDC on cancer cells than the bibody of Macor, which is a representative CD20 x CD55 bibody. In addition, the CD20 x CD55 bibody in the form of an SBU can be an effective therapeutic agent for Burkitt's lymphoma, which is known to be a malignant B-cell lymphoma.

[0179] Experimental Example 11-5. Confirmation of cell killing ability of CD20X CD55 double antibody in Ramos

[0180] The cell killing ability of CD20X CD55 bibody in Ramos cell line was confirmed using the method of Experimental Example 11-4 (FIGS. 12a and 12b). As a result, CD20X CD55 bibody in rituximab-responsive Ramos cells showed a significantly different cell killing ability compared to rituximab. In addition, SBU of the present invention showed a better cell killing ability than Macor bibody. These results indicate that CD20X CD55 bibody can show a more effective anti-cancer effect than rituximab in rituximab-responsive carcinomas, and SBU can be used as an effective therapeutic agent among CD20X CD55 bibody in blood cancers expressing CD20.

[0181] Experimental Example 11-6. Confirmation of cell killing ability of CD20X CD55 double antibody with Ramos-RR

[0182] The cell killing ability of CD20X CD55 bibody in Ramos-RR cell line was confirmed using the method of Experimental Example 11-4 (FIGS. 13a and 13b). As a result, CD20X CD55 bibody in Ramos-RR cells, which do not show reactivity to rituximab, showed a significantly different cell killing ability compared to rituximab. As with the results of BJAB cell line, the SBU of the present invention showed a far superior cell killing ability than the Macor bibody among the bibodies. These results indicate that the CD20XCD55 bibody in the form of SBU can be used as an effective therapeutic agent for rituximab-resistant carcinomas among blood cancers in which CD20 is expressed.

[0183] Experimental Example 11-7. CDC activation analysis of CD20X CD55 biantibody in SBU format

[0184] In the above Experimental Example 11-5, the supernatants treated with rituximab and SBU-type CD20X CD55 biantibody were collected before flow cytometry of Ramos-RR cell pellets, and CDC activation was analyzed (FIG. 14). The CDC mechanism is a reaction mediated by the binding of C1q to an antibody, and activation analysis is possible at an intermediate stage in the entire CDC process based on the amount of C4d produced as a by-product. When analyzed using an ELISA kit (Quidel, USA) that can confirm the amount of C4d produced in each supernatant, more C4d was produced in the supernatant treated with SBU-type CD20X CD55 biantibody than in the supernatant treated with rituximab, and the difference was significant. These results clearly indicate that the excellent cell killing ability of SBU-type CD20X CD55 biantibody is due to the CDC activation mechanism.

[0185] Although the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that such specific techniques 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. A heavy chain variable region including an HCDR1 region having an amino acid sequence represented by the following general formula 1; an HCDR2 region having an amino acid sequence of SEQ ID NO: 1 in the sequence listing; and an HCDR3 region having an amino acid sequence represented by the following general formula 2; An antibody or antigen-binding fragment thereof that specifically binds to CD55, comprising a light chain variable region including: an LCDR1 region having the amino acid sequence of SEQ ID NO:2 in the sequence listing; an LCDR2 region having the amino acid sequence represented by the following general formula 3; and an LCDR3 region having the amino acid sequence of SEQ ID NO:3 in the sequence listing: General formula 1 D-R-G-M-X １ ; General formula 2 N-A-X ２ -A-G-G-W-H-A-A-Y-I-D-A、 General formula 3 W-N-X 3 -K-R-P-S X in the above general formula 1 １ is Ala, and X in the general formula 2 is ２ is Val, and X 3 in the general formula 3 is Asp.

2. An antibody or antigen-binding fragment thereof that specifically binds to CD55, comprising the following light chain variable region and heavy chain variable region: a) a light chain variable region of SEQ ID NO: 4 in the sequence listing, and b) A heavy chain variable region of SEQ ID NO: 19 in the sequence listing.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that the antigen-binding fragment is an scFv, a Fab fragment, an F(ab') fragment, an F(ab')2 fragment or an Fv fragment.

4. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 3.

5. A pharmaceutical composition for preventing or treating cancer, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 as an active ingredient.

6. The composition according to claim 5, further comprising one or more pharmacological ingredients selected from the group consisting of an anti-CD20 antibody and a tyrosine kinase inhibitor.

7. The composition of claim 6, wherein the anti-CD20 antibody is rituximab.

8. The composition according to claim 6, wherein the tyrosine kinase inhibitor is one or more inhibitors selected from the group consisting of imatinib and gefitinib.

9. The composition according to claim 5 , wherein the cancer is a CD55-positive cancer.

10. A composition for diagnosing cancer, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 as an active ingredient.

11. The composition according to claim 10, wherein the cancer is a CD55-positive cancer.

12. A bispecific antibody or a functional fragment thereof comprising an antigen-binding fragment of an antibody that specifically binds to CD55 of any one of claims 1 to 3 and an antigen-binding fragment of an antibody that specifically binds to CD20.

13. The bispecific antibody or functional fragment thereof according to claim 12, wherein the antibody that specifically binds to CD20 is rituximab.

14. The bispecific antibody or functional fragment thereof described in claim 12, characterized in that the antigen-binding fragment of the antibody that specifically binds to CD55 comprises an scFv.

15. The bispecific antibody or functional fragment thereof according to claim 12, wherein the antigen-binding fragment of the antibody that specifically binds to CD55 is in the form of VL-linker-VH-CH2-CH3.

16. The bispecific antibody or functional fragment thereof according to claim 12, characterized in that the antigen-binding fragment of the antibody that specifically binds to CD20 comprises Fab.

17. The bispecific antibody or functional fragment thereof according to claim 12, wherein the antigen-binding fragment of the antibody that specifically binds to CD20 is in the form of VL-CK-linker-VH-CH1-CH2-CH3.

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