KR20210080227A - Monoclonal antibodies against dcr1 - Google Patents

Abstract

The present invention relates to a monoclonal antibody specific for human DCR1 and to anticancer activity of an expression inhibitor thereof, wherein the monoclonal antibodies and DCR1 expression inhibitors that specifically bind to a human DCR1 antigen with high binding ability according to the present invention inhibit growth and migration of cancer cells in vitro, promote apoptosis of the cancer cells, improve immune responses to the cancer cells by inducing activation of T cells and also show immune response promotion and tumor suppression effects on cancer tissues in vivo, and thus can be usefully used for prevention or treatment of cancer.

Description

Monoclonal antibody specific for DCR1 {MONOCLONAL ANTIBODIES AGAINST DCR1}

The present invention relates to the anticancer activity of a monoclonal antibody specific for human DCR1.

The smallest unit constituting the human body is called a cell. Normal cells divide and grow, die and disappear due to intracellular regulatory functions, maintaining the balance of cell numbers. If cells are damaged for some reason, they function as normal cells through treatment, or if they do not recover, they die on their own. However, if a cell's gene is changed for various reasons, the cell changes abnormally and matures incompletely, and the cell cycle is not regulated, so cell division continues. This is defined as cancer. In addition, cancer has the characteristic of not only invading and destroying the tissues and organs of the state, but also spreading to other organs. Cancer mortality is the number one cause of death in Korea, and the number is increasing every year. Among them, the incidence of liver cancer is increasing as modern people's eating habits change to high-protein and high-fat diets as the industrialized society progresses. Innovative cancer research focusing on a deeper understanding of the underlying mechanisms of DNA damage/repair, apoptosis, tumorigenesis, and treatment in molecular terms is strongly needed to conquer cancer (Varmus, 2006; Alberts, 2008, 2011) ), which could eventually be a breakthrough in cancer therapy.

In current anticancer chemotherapy, many patients suffer from the side effects of anticancer drugs, and in particular, limited administration is made due to the toxicity of anticancer drugs. Anticancer substances used in clinical practice affect not only cancer cells but also normal cells, and have problems such as side effects and resistance to anticancer drugs, such as failure of treatment as the number of administrations is repeated. Research on a new type of anticancer drug treatment substance that can overcome the side effects of existing anticancer drugs caused by the diversity of cancer itself and the diversification of pathogenesis is in progress.

On the other hand, antibody drugs are growing the fastest in the biopharmaceutical field due to their high therapeutic effect and targeted therapeutic properties. The global market for antibody drugs is growing at an average annual rate of 10-15%, and it is known to have formed a market of $44.7 billion in 2010. Most of the diseases targeted by antibody drugs are concentrated on specific disease indications, such as intractable cancer (49%) and immune diseases (35%), and competition among products for similar indications is fierce. Nevertheless, as there are subdivided therapeutic targets within diseases, the field of anticancer antibody therapy development is expected to continue to grow in the future.

Antibody fragment display technologies, such as phage display, yeast display, and ribosomal display, are interlocked with humanized antibody development research, and are currently approached with various strategies to discover antibody candidates. to be. Human antibody phage library technology has been developed since the late 1990s and has been used in various antibody development studies until now. Among them, phage display is a technology for screening antibodies by expressing antibody fragments on the surface of bacteriophages, and conventional antibody development technology (chimeric/humanized antibody development using hybridomas, antibody development using transgenic mice) Compared to that, it has the advantage of being able to identify antigen-specific antibodies in a short time. Although there is a disadvantage that an effective antibody can be identified only when a library of high diversity is secured, the securing of the library is largely solved due to the recent development of gene amplification and cloning technology. Recently, studies of single-chain fragment variable (scFv) in which only the antigen-binding sites of the light and heavy chains of the antibody are linked and expressed with various chains are being actively conducted. This is because, unlike IgG, single-chain antibodies are composed of one polypeptide chain, so that they can be expressed in large quantities in E. coli, are easy to engineer, and have the advantages of being a suitable system for large-scale screening. Since one antigen has several antibody binding sites, it is very important to develop many antibodies that can recognize a specific antigen. In addition, the antibodies developed in this way can be used importantly in industries related to various pathogen sensors, pathogen detection kits, and drug development.

An object of the present invention is to provide an antibody or a fragment having immunological activity thereof for use in the prevention or treatment of cancer, and a DCR1 expression inhibitor.

In order to achieve the above object, the present invention provides a monoclonal antibody specific for human DCR1 (decoy receptor 1) or a fragment having immunological activity thereof.

The present invention also provides an isolated nucleic acid molecule encoding the antibody or fragment having immunological activity thereof.

The present invention also provides a vector comprising the isolated nucleic acid molecule.

In addition, the present invention provides a host cell transformed with the vector.

In addition, the present invention provides a method for producing a human DCR1-specific antibody or a fragment having immunological activity thereof.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer containing the monoclonal antibody of the present invention or a fragment having immunological activity thereof.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising an expression inhibitor specific to human DCR1 of the present invention.

The human DCR1 expression inhibitor according to the present invention and the monoclonal antibodies that specifically bind to the human DCR1 antigen with high binding affinity inhibit the growth and migration of cancer cells in vitro, promote cancer cell apoptosis, and T By inducing the activation of cells, the immune response to cancer cells was improved, and as it showed the effect of promoting immune response and tumor suppression on cancer tissues even in vivo, it can be usefully used for the prevention or treatment of cancer.

1 is a diagram confirming the change of NF-kB signaling by DCR1-Fc treatment:
hi: heat inactivated.
2 is a diagram showing the evaluation results of binding affinity between the DCR1 antigen and the anti-DCR1 monoclonal antibody.
3 is a diagram confirming the cancer cell proliferation inhibitory effect of the anti-DCR1 antibody.
4 is a diagram confirming the change in the amount of IL-2 produced in cancer cells by the anti-DCR1 antibody.
5 is a diagram confirming the effect of promoting the differentiation of monocytes into M1 macrophages by the anti-DCR1 antibody.
6 is a view confirming the cancer growth inhibitory effect by the anti-DCR1 antibody in vivo .
7 is a diagram schematically illustrating the death of tumor cells by inhibiting the immune response evasion of cancer cells by the anti-DCR1 antibody or siRNA of the present invention.

Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and when it is determined that detailed descriptions of well-known techniques or configurations known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted, and , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of equivalents interpreted therefrom and the description of the claims to be described later.

In addition, the terms used in this specification are terms used to properly express the preferred embodiment of the present invention, which may vary according to the intention of a user or operator, or customs in the field to which the present invention belongs. Accordingly, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

All technical terms used in the present invention, unless otherwise defined, have the meaning as commonly understood by one of ordinary skill in the art of the present invention. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention. The contents of all publications herein incorporated by reference are incorporated herein by reference.

Throughout this specification, common one-letter and three-letter codes for naturally occurring amino acids are used as well as generally accepted for other amino acids such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc. A three-character code is used. Amino acids referred to by abbreviation in the present invention are also described according to the IUPAC-IUB nomenclature as follows:

Alanine: A, Arginine: R, Asparagine: N, Aspartic Acid: D, Cysteine: C, Glutamic Acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine : M, phenylalanine: F, proline: P, serine: S, threonine: T, tryptophan: W, tyrosine: Y and valine: V.

In one aspect, the present invention relates to a monoclonal antibody specific for human DCR1 (decoy receptor 1) or a fragment having immunological activity thereof.

In one embodiment, the fragment with immunological activity is Fab, Fd, Fab', dAb, F(ab'), F(ab') ₂ , scFv, Fv, single chain antibody, Fv dimer, complementarity determining region fragment , it may be any one or more selected from the group consisting of humanized antibodies, chimeric antibodies, and diabodies.

In one embodiment, the human DCR1 (decoy receptor 1) specific monoclonal antibody or fragment having immunological activity thereof is CDRH (Complementarity) comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 1 to 9 determining regions Heavy chain) 1, CDRH2 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 10 to 18, and CDRH3 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 19 to 27; It may comprise a VH domain comprising a.

In one embodiment, the human DCR1 (decoy receptor 1) specific monoclonal antibody or fragment having immunological activity thereof is CDRL1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 28 to 36, the sequence A VL domain comprising a CDRL2 comprising any one selected from the group consisting of an amino acid sequence of SEQ ID NOs: 37 to 45, and a CDRL3 comprising any one selected from the group consisting of an amino acid sequence of SEQ ID NOs: 46 to 54. have.

In one embodiment, the fragment having immunological activity may include an scFv comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 55 to 63.

In the present invention, the monoclonal antibody or fragment having immunological activity thereof may be selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, a human antibody, and a fragment having immunological activity thereof. The antibody may be recombinantly or synthetically produced.

An animal-derived antibody produced by immunizing an animal to be immunized with a desired antigen may generally cause an immune rejection reaction when administered to a human for therapeutic purposes, and a chimeric antibody has been developed to suppress this immune rejection reaction. A chimeric antibody is one in which the constant region of an animal-derived antibody causing an anti-isotype reaction is substituted with that of a human antibody using a genetic engineering method. Chimeric antibodies have significantly improved anti-isotype response compared to animal-derived antibodies, but still contain animal-derived amino acids in the variable region, potentially causing side effects on anti-idiotypic responses. are doing A humanized antibody was developed to improve these side effects. This is produced by grafting complementarity determining regions (CDRs), which play an important role in antigen binding, into a human antibody framework among variable regions of a chimeric antibody.

The most important thing in the CDR grafting technology for producing a humanized antibody is to select an optimized human antibody that can best accept the CDR region of an animal-derived antibody. structure analysis, molecular modeling technology, etc. are used. However, even when the CDR regions of an animal-derived antibody are transplanted into an optimized human antibody framework, there are cases in which amino acids that affect antigen binding are present while being located in the framework of an animal-derived antibody, so that antigen-binding ability is not preserved in many cases. Therefore, it can be said that the application of additional antibody engineering technology to restore antigen binding force is essential.

The monoclonal antibody or fragment having immunological activity thereof may be isolated from the living body (not present in the living body) or non-naturally occurring, for example, synthetically or recombinantly produced. it may have been

As used herein, the term "antibody" refers to a substance produced by stimulation of an antigen within the immune system, the type is not particularly limited, and can be obtained naturally or non-naturally (eg, synthetically or recombinantly). can Antibodies are advantageous for mass expression and production because they are very stable and have a long half-life not only in vitro but also in vivo. In addition, since the antibody essentially has a dimer structure, the adhesion (avidity) is very high. A complete antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. The constant region of an antibody is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has the kappa (κ) and lambda (λ) types.

_{As used herein, the term "heavy chain" refers to a variable region domain V H} and three constant region domains C _H1 , C _H2 and C _H3 comprising an amino acid sequence having a variable region sequence sufficient to confer specificity to an antigen. and a full-length heavy chain including a hinge and a fragment thereof. Further, the term "light chain (light chain)" Both the full-length light chain and fragments thereof containing the variable region domain, V _L, and a constant region domain, C _L comprises an amino acid sequence having sufficient variable region sequence to impart specificity to the antigen interpreted as including

As used herein, the term “variable region or variable domain” refers to a portion of an antibody molecule that exhibits many variations in sequence while performing a function of specifically binding to an antigen, and the variable region has complementarity There are determining regions CDR1, CDR2 and CDR3. Between the CDRs, a framework region (FR) portion exists to support the CDR ring. The "complementarity determining region" is a ring-shaped region involved in antigen recognition, and the specificity of the antibody for the antigen is determined as the sequence of this region changes.

As used herein, the term "scFv (single chain fragment variable)" refers to a single-chain antibody made by expressing only the variable region of an antibody through genetic recombination, and a single-chain form in which the VH region and the VL region of the antibody are connected by a short peptide chain. refers to the antibody of The term "scFv" is intended to include scFv fragments, including antigen-binding fragments, unless otherwise specified or otherwise understood by context. This is obvious to a person skilled in the art.

As used herein, the term "complementarity determining region (CDR)" refers to the amino acid sequence of the hypervariable region of the heavy chain and light chain of an immunoglobulin. The heavy and light chains may each comprise three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide key contact residues for antibody binding to an antigen or epitope.

In the present invention, the terms "specifically binding" or "specifically recognized" have the same meaning as commonly known to those skilled in the art, and mean that an antigen and an antibody specifically interact to undergo an immunological reaction. .

As used herein, the term “antigen-binding fragment” refers to a fragment of the entire immunoglobulin structure, and refers to a portion of a polypeptide including an antigen-binding portion. For example, it may be scFv, (scFv) 2 , scFv-Fc, Fab, Fab' or F(ab') 2 , but is not limited thereto. Among the antigen-binding fragments, Fab has a structure having a light chain and heavy chain variable regions, a light chain constant region and a heavy chain first constant region (C _H1 ), and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region comprising one or more cysteine residues at the C-terminus of the heavy chain C _{H1 domain.} The F(ab') 2 antibody is produced by forming a disulfide bond with a cysteine residue in the hinge region of Fab'. Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region, and a recombinant technique for generating an Fv fragment is well known in the art. In a double-chain Fv (two-chain Fv), the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, and in single-chain Fv (single-chain Fv), the heavy chain variable region and the single chain variable region are generally shared through a peptide linker. It is linked by a bond or is directly linked at the C-terminus, so that it can form a dimer-like structure like a double-stranded Fv. The linker may be a peptide linker consisting of any amino acids from 1 to 100 or 2 to 50 amino acids, and an appropriate sequence is known in the art. The antigen-binding fragment can be obtained using a proteolytic enzyme (for example, Fab can be obtained by restriction digestion of the whole antibody with papain, and F(ab') 2 fragment can be obtained by digestion with pepsin), It can be produced through genetic recombination technology.

In the present invention, the term "hinge region" is a region included in the heavy chain of an antibody, _{which exists between the C H1} and C _H2 regions, and functions to provide flexibility of the antigen-binding site in the antibody. means area. For example, the hinge may be derived from a human antibody, and specifically, it may be derived from IgA, IgE, or IgG, such as IgG1, IgG2, IgG3, or IgG4.

As used herein, the term "panning" refers to a peptide having a property of binding to a target molecule (antibody, enzyme, cell surface receptor, etc.) from a phage library that expresses a peptide on the outer wall of the phage. It refers to the process of selecting only phages expressed on the surface. In general, when biopanning through the phage display technique is performed, the number of phage populations having strong binding ability with the target target increases as the number of pannings increases. Therefore, the increase in phage titer according to the number of panning is an experiment in the phage display experiment. This is known as the basic parameter that determines whether this is going in the intended direction.

In one aspect, the invention relates to an isolated nucleic acid molecule encoding a monoclonal antibody of the invention or a fragment having immunological activity thereof.

In one embodiment, the nucleic acid molecule may include any one or more selected from the group consisting of nucleotide sequences of SEQ ID NOs: 64 to 72.

An isolated nucleic acid molecule encoding a monoclonal antibody of the present invention or a fragment having immunological activity thereof has a coding region due to codon degeneracy or in consideration of codons preferred in the organism in which the antibody is to be expressed. Various modifications can be made to the coding region within the range that does not change the amino acid sequence of the antibody expressed from It will be appreciated by those skilled in the art that such modified genes are also included within the scope of the present invention. That is, as long as the nucleic acid molecule of the present invention encodes a protein having an equivalent activity, one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included in the scope of the present invention. The sequence of such a nucleic acid molecule may be single-stranded or double-stranded, and may be a DNA molecule or an RNA (mRNA) molecule.

In one aspect, the invention relates to a vector comprising an isolated nucleic acid molecule of the invention.

In one embodiment, when constructing the vector, expression control sequences such as promoter, terminator, enhancer, etc., membrane targeting or secretion according to the type of host cell in which the antibody is to be produced. Sequences and the like can be appropriately selected and combined in various ways depending on the purpose.

The vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector. Suitable vectors include a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as promoter, operator, start codon, stop codon, polyadenylation signal and enhancer, and may be prepared in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. The signal sequence includes a PhoA signal sequence and an OmpA signal sequence when the host is Escherichia sp., and an α-amylase signal sequence and a subtilisin signal when the host is a Bacillus sp. When the host is yeast, the MFα signal sequence, SUC2 signal sequence, etc., and when the host is an animal cell, an insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. can be used, It is not limited thereto. The vector may also contain a selection marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, an origin of replication.

In one aspect, the invention relates to a host cell transformed with a vector of the invention.

In one embodiment, the host cell may be a bacterium or an animal cell, the animal cell line may be a CHO cell, a HEK cell or an NSO cell, and the bacterium may be E. coli.

In one aspect, the invention provides a method comprising: culturing a host cell comprising a vector comprising an isolated nucleic acid molecule of the invention; And it relates to a method for producing a human DCR1-specific antibody or fragment having immunological activity, comprising the step of recovering the antibody or immunologically active fragment thereof from the host cell culture.

After the vector according to the present invention is transformed into an appropriate host cell, for example, E. coli or yeast cells, the antibody according to the present invention can be mass-produced by culturing the transformed host cell. Appropriate culture methods and medium conditions according to the type of host cells can be easily selected by those skilled in the art from known techniques known to those skilled in the art. The host cell may be a prokaryotic organism such as E. coli or Bacillus subtilis. It may also be a eukaryotic cell derived from yeast, insect cells, plant cells, animal cells, such as Saccharomyces cerevisiae. More preferably, the animal cells may be autologous or allogeneic animal cells. A transformant prepared by introducing an autologous or allogeneic animal cell can be administered to an individual and used in cell therapy to treat cancer. Any method known to those skilled in the art may be used for the vector introduction method into the host cell.

Antibodies of the present invention or fragments having immunological activity thereof can be generated using various phage display methods known in the art [Brinkman et al., 1995, J. Immunol. Methods, 182:41-50]; [Ames et al., 1995, J. Immunol. Methods, 184, 177-186]; [Kettleborough et al. 1994, Eur. J. Immunol, 24, 952-958]; [Persic et al., 1997, Gene, 187, 9-18]; and Burton et al., 1994, Advances in Immunol, 57, 191-280; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; and WO 95/20401; and US 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743; and 5,969,108.

Other organisms, including transgenic (eg, genetically engineered) mice, or other mammals, may be used to produce antibodies of the invention or fragments with immunological activity thereof (see, eg, US 6,300,129). For example, mice engineered by replacing only the variable regions (heavy chain V, D, and J segments, and light chain V and J segments) of a mouse immune gene with the corresponding human variable sequences can generate high-affinity antibodies with human variable sequences. It is known that it can be used for mass production (see, for example, US Pat. No. 6,586,251; US Pat. No. 6,596,541, and US Pat. No. 7,105,348).

In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating cancer containing a monoclonal antibody specific for human DCR1 (decoy receptor 1) or a fragment having immunological activity thereof of the present invention.

In one embodiment, the cancer is brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colorectal cancer, Small intestine cancer, rectal cancer, fallopian tube carcinoma, perianal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, Soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma and It may be any one selected from the group consisting of pituitary adenoma.

In one embodiment, the composition of the present invention can inhibit cancer proliferation, survival, metastasis, recurrence, or resistance to anticancer drugs.

In one embodiment, the composition of the present invention may further comprise one or more selected from the group consisting of a cytotoxic substance and a labeling substance.

In one embodiment, the labeling material may be at least one selected from the group consisting of a radioisotope, a fluorescent material, a chromogen, a dyeing material, and the like. The fluorescent material may be any commonly used fluorescent material, for example, fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), or It may be at least one selected from the group consisting of biotin and the like, but is not limited thereto. The labeling material may be bound (linked) to the antibody or fragment having immunological activity by a conventional method (eg, a chemical bond such as a covalent bond, a coordination bond, or an ionic bond). The binding of the antibody or a fragment having immunological activity thereof to a label may be performed according to a technique well known in the art to which the present invention pertains.

In one embodiment, the cytotoxic substance is ricin, saporin, gelonin, morodin, debouganin, diphtheria toxin, pseudomonas toxin, radiation Isotopes, duocarmycin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), N2'-diacetyl-N2'-(3- Mercapto-1-oxopropyl) maytansine (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl) maytansine; DM1), and PBD (Pyrrolobenzodiazepine) at least one selected from the group consisting of dimers can

In one embodiment, the composition of the present invention may further include an immuno-cancer agent or an anti-cancer agent, and the immuno-cancer agent may be an immune checkpoint inhibitor, programmed cell death 1 (PD-1) or PD-L1. (programmed cell death ligand 1) It is more preferable that it is an inhibitor.

In one embodiment, the immuno-cancer agent is an anti-PD1 antibody, an anti-PDL1 antibody, an anti-CTLA4 antibody, an anti-LAG3 antibody, an anti-VISTA antibody, an anti-BTLA antibody, an anti-TIM3 antibody, an anti-HVEM antibody, an anti -CD27 antibody, anti-CD137 antibody, anti-OX40 antibody, anti-CD28 antibody, anti-PDL2 antibody, anti-GITR antibody, anti-ICOS antibody, anti-SIRPα antibody, anti-ILT2 antibody, anti-ILT3 antibody, anti It may be any one or more selected from the group consisting of -ILT4 antibody, anti-ILT5 antibody, anti-EGFR antibody, anti-CD19 antibody, and anti-TIGIT antibody.

In one embodiment, the anticancer agent doxorubicin (doxorubicin), cisplatin (cisplatin), paclitaxel (paclitaxel), vincristine (vincristine), topotecan (topotecan), docetaxel (docetaxel), 5-fluorouracil (5-FU) , Gleevec (gleevec), carboplatin (carboplatin), duanorubicin (daunorubicin), valrubicin (valrubicin), flutamide (flutamide), it may be any one selected from the group consisting of gemcitabine (gemcitabine) .

In one embodiment, it may further comprise a human DCR1 expression inhibitor that specifically inhibits human DCR1 expression, and the human DCR1 expression inhibitor may be siRNA, shRNA or miRNA directed against a human DCR1 gene.

In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating cancer comprising a human DCR1 expression inhibitor.

In one embodiment, the human DCR1 expression inhibitor may be an siRNA, shRNA or miRNA directed against the human DCR1 gene.

As used herein, the term “small interfering RNA (siRNA)” refers to a short double-stranded RNA capable of inducing an RNA interference (RNAi) phenomenon through cleavage of a specific mRNA.

As used herein, the term “repression of expression” means to cause a decrease in expression (to mRNA) or translation (to protein) of a target gene, preferably, whereby the expression of the target gene becomes undetectable or insignificant. means to exist as

The composition of the present invention further contains a monoclonal antibody or fragment having immunological activity specific for human DCR1 of the present invention, or a human DCR1 expression inhibitor, as well as other active ingredients having the same or similar function, or By additionally containing other active ingredients having different functions from the components, it can be prepared as a pharmaceutical composition for the prevention or treatment of cancer.

In the present invention, the term "prevention" refers to the inhibition or recurrence of cancer by administration of a monoclonal antibody or a fragment having immunological activity according to the present invention, an inhibitor of human DCR1 expression, or a composition comprising the same. Any action that delays.

As used herein, the term "treatment" refers to the apoptosis of cancer cells or improvement of symptoms of cancer by administration of a monoclonal antibody or fragment having immunological activity according to the present invention, an inhibitor of human DCR1 expression, or a composition comprising the same. or any act that changes it for the better. Those of ordinary skill in the art to which the present invention pertains, with reference to the data presented in the Korean Medical Association, etc., know the exact criteria of the disease for which the composition of the present application is effective, and can determine the degree of improvement, improvement and treatment will be.

The therapeutically effective amount of the composition of the present invention may vary depending on several factors, for example, the administration method, the target site, the condition of the patient, and the like. Therefore, when used in the human body, the dosage should be determined as an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These considerations in determining effective amounts are found, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects, and the effective dose level is determined by the patient's Health status, type of cancer, severity, drug activity, sensitivity to drug, administration method, administration time, administration route and excretion rate, treatment period, factors including drugs used in combination or concurrently, and other factors well known in the medical field can be determined according to The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group comprising oral formulations, topical formulations, suppositories, sterile injection solutions and sprays.

The compositions of the present invention may also include carriers, diluents, excipients or combinations of two or more commonly used in biological agents. The pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. listed compounds, saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be mixed and used, and if necessary, antioxidants, buffers, bacteriostats Other conventional additives may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar function. The composition of the present invention comprises 0.0001 to 10% by weight of the protein, preferably 0.001 to 1% by weight, based on the total weight of the composition.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive includes starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate. , lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, sucrose, dextrose, sorbitol and talc and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

The composition of the present invention may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally according to a desired method, and the dosage may vary depending on the patient's weight, age, sex, health status, The range varies depending on the diet, administration time, administration method, excretion rate, and the severity of the disease. The daily dose of the composition according to the present invention is 0.0001 to 10 mg/ml, preferably 0.0001 to 5 mg/ml, and it is more preferable to divide and administer once to several times a day.

Liquid preparations for oral administration of the composition of the present invention include suspensions, internal solutions, emulsions, syrups, etc., and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. and the like may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

The present invention will be described in more detail through the following examples. However, the following examples are only for specifying the contents of the present invention, and the present invention is not limited thereto.

Example 1. Confirmation of immune system stimulating effect of DCR1-Fc

1-1. Expression and purification of DCR1-Fc

In order to analyze the function of DCR1 in cancer cells and the immune system, DCR1-Fc in which the Fc portion of the heavy chain region of the antibody is bound to DCR1 was prepared. Specifically, a gene encoding amino acids 26-236 of the human DCR1 gene was inserted into the pcDNA3.1 vector, and this was transformed into a HEK293 cell line. Thereafter, for DCR1-Fc production, the transformed cell line and a polymer that increases the intracellular gene transfer efficiency were added to RPMI medium supplemented with fetal bovine serum with ultra-low IgG content. and cultured in a cell incubator for 4 days. After the culture is complete, the supernatant containing DCR1-Fc is separated using a centrifuge, filtered once using a 0.22 μm filter, and then DCR1-Fc is purified using a recombinant Protein-A Sepharose column (GE healthcare). Isolation and purification. Purified DCR1-Fc is measured by absorbance to determine the concentration, and 2 μg of purified DCR1-Fc is added to reducing sample buffer and non-reducing sample buffer, respectively, and then pre-made SDS-PAGE gel (Invitrogen) is used to determine the concentration. electrophoresis.

As a result, it was confirmed that the recombinant DCR1-Fc protein was expressed and purified at an excellent level (purity of 90% or more). Through this, a high-quality protein with a concentration and physical properties that can be treated with a cell line was secured.

1-2. Confirmation of immune system stimulating effect of DCR1-Fc

It was confirmed how the DCR1-Fc produced in Example 1-1 had an effect on immune cell activity. Specifically, THP-1 blue cells (Invivogen), which are monocytes capable of confirming NF-kB signaling, were cultured in a 96-well plate at 5000 cells/well. After stabilizing the cells for 12 hours, DCR1-Fc was treated and LPS was immediately added to each corresponding well. _{After culturing for 48 hours in a CO 2} incubator at 37° C., the culture supernatant was separated, developed with Seap coloring reagent (Invivogen) for 1 hour, and absorbance at 650 nm was measured using a microplate reader (Molecular device). measured.

As a result, the NF-kB signal was about 0.4 in the group treated with LPS alone, and about 0.6 in the group treated with DCR1-Fc alone. On the other hand, in the experimental group treated with LPS and DCR1-Fc at the same time, the signal was as high as 1.2, indicating that DCR1-Fc effectively activated THP-1 monocytes/macrophages, which are immune cells (Fig. 1). . Through the above results, it was confirmed that DCR1-Fc activates the signaling mechanism of NF-kB, activates immune cells, THP-1 monocytes, and promotes differentiation into macrophages, thereby activating the immune system efficiently.

Example 2. Construction of DCR1-specific recombinant monoclonal antibody

2-1. Anti-DCR1 monoclonal screening

To construct a recombinant monoclonal antibody that specifically binds to human decoy receptor 1 (DCR1) antigen, lambda, kappa and VH3VL1 phage systems were used. Biopanning was performed to select antibodies with strong binding force among the phage libraries expressing the human DCR1 antibody. Specifically, for antigen immobilization, DCR1 antigen was added at a concentration of 5 μg/mL to an immune test tube, and then treated for 1 hour to allow adsorption on the surface of the test tube. Thereafter, the remaining antigen was removed and washed with 3% skim milk solution to suppress non-specific reactions. After adding 3% skim milk solution to ^{the lambda, kappa and VH3VL1 phage library (10 12} PFU), which expressed only the single-chain variable fragment (scFv) of the human DCR1 antibody on the surface, in each immune test tube Antigen and antibody were bound. After binding, the remaining antigen-specific antibody and the non-specifically bound antibody were washed three times with TBST (tris buffered saline-Tween20) solution, and then the antibody specifically bound to the human DCR1 antigen was mixed with 100 mM triethyl liberated in the amine solution. The human DCR1 antibody gene obtained through phage was neutralized using 1.0M Tris-HCl buffer (pH 7.8), treated with E. coli ER2537, and infected at 37°C for 1 hour. The gene product was obtained by culturing the infected E. coli on LB agar medium containing carbenicillin at 37° C. for 16 hours. After suspending the formed E. coli colonies in SB (super broth)-Carbenicili culture medium, 15% glycerol was added and stored at -80° C. to secure the selected antibody library pool. The lambda, kappa, and VH3VL1 antibody library genes that specifically bind to the primary selected human DCR1 antigen were re-inoculated in SB-carbenicillin-2% glucose solution and incubated at 37° C., and then the supernatant containing phages was prepared above. As a method, the specificity was improved by secondary biopanning. A total of 4 times of biopanning was performed to secure and enrich the antibody pool specific for the DCR1 antigen.

2-2. Anti-DCR1 monoclonal antibody screening

In order to obtain a single clone that specifically binds to the human DCR1 antigen from the gene pool obtained through biopanning in the above, lambda, kappa and VH3VL1 antibody libraries containing antibody genes specific to the human DCR1 antigen of clones were cloned into a single clone. E. coli infected with a phage having a human DCR1 antibody gene was smeared on LB agar medium containing carbenicillin and cultured for 16 hours at 37 ° C. Each colony of E. coli formed as a monoclonal reevaluation and final selection of specificity for future antigens used for work. Specifically, each monoclonal was re-inoculated in SB-carbenicillin-2% glucose solution and incubated at 37°C _{until the absorbance (OD 600nm} ) became 0.5, then IPTG was added and added at 30°C for 16 hours. Antibody molecules were expressed by incubation. Thereafter, periplasmic extraction was performed to secure the protein of the single antibody clone, and the evaluation of binding affinity to the DCR1 antigen and verification of specificity were confirmed by ELISA. Specifically, DCR1 antigen was prepared at a concentration of 5 μg/mL per well in a coating buffer (0.1M sodium carbonate, pH 9.0), and then dispensed on an ELISA plate and reacted for 3 hours at room temperature to plate the antigen. and then washed 3 times with PBST (phosphate buffered saline-Tween20) to remove unbound antigen, and then 2% bovine serum albumin (BSA) was added to each well to remove non-specific binding. The reaction was carried out at room temperature for 1 hour. After washing with 2% bovine serum albumin (BSA) using PBST, a cytoplasmic extract containing the secured E. coli monoclonal, that is, scFv, was added to each well, and reacted for 1 hour at room temperature to bind antigen. After 1 hour of binding reaction, unbound scFv was removed through a washing process 3 times using PBST. After adding 4 μg/mL of a detection antibody and reacting at room temperature for 1 hour, it was washed with PBST, and after adding a TMB solution and reacting for 10 minutes to develop color, absorbance at 450 nm was measured and analyzed to interact with DCR1 antigen. Binding and specificity were confirmed, and the final 30 positive anti-DCR1 monoclonal antibodies were obtained.

2-3. Gene sequence confirmation of selected individual clones

DNA sequences were confirmed for characterization of the 30 anti-DCR1 monoclonal antibodies selected and secured in the above Examples. Specifically, after culturing each of the selected antibody clones, plasmid DNA was extracted, and the gene sequence of the anti-DCR1 monoclonal antibody contained in the plasmid DNA was confirmed using a DNA sequencing method. Thereafter, as a result of analyzing and comparing the sequences of complementarity-determining regions (CDR) amino acids, individual clones of anti-DCR1 monoclonal antibodies having a total of 9 different amino acid sequences (3B11: SEQ ID NO: 1, 3D7: SEQ ID NO: 2, 3F2) : SEQ ID NO: 3, 5C4: SEQ ID NO: 4, 5B1: SEQ ID NO: 5, 5D9: SEQ ID NO: 6, 5F9: SEQ ID NO: 7, 4F10: SEQ ID NO: 8 and 4F11 (FS): SEQ ID NO: 9) were obtained.

2.4. Construction and Purification of Anti-DCR1 Monoclonal Antibodies

Using the nucleotide sequences of the nine anti-DCR1 monoclonal antibodies identified in Example 2-3, an expression vector was prepared in which heavy and light chains capable of expressing the antibody were separated. Specifically, genes were inserted so that the heavy and light chains could be expressed in the pCIW3.3 vector using the analyzed CDR sequences, respectively. The prepared vector for expression of heavy and light chains was mixed with PEI (polyethylenimine) in a mass ratio of 1:1 and transfected into 293T cells to induce antibody expression. On the 8th day, the culture medium was centrifuged to remove the cells, and the culture medium was washed. obtained. The obtained culture solution was filtered _{and resuspended using a solution in which 0.1 M NaH 2} PO ₄ and 0.1 M Na ₂ HPO ₄ (pH 7.0) were mixed. The resuspended solution was purified through affinity chromatography using protein A beads (GE healthcare), and finally eluted using an Elution buffer (Thermofisher). In order to confirm the prepared antibody, reducing sample buffer and non-reducing sample buffer were added to 5 μg of purified antibody, respectively, and electrophoresis was performed using pre-made SDS-PAGE (Invitrogen), after which Kuma The protein was stained using sea blue. As a result, it was confirmed that an anti-DCR1 monoclonal antibody having a purity of 90% or more was produced and purified.

Example 3. Evaluation of binding affinity between DCR1 antigen and anti-DCR1 monoclonal antibody

Binding ELISA was performed to test whether the selected monoclonal antibody specifically binds to the DCR1 antigen. Specifically, DCR1 antigen diluted in a coating buffer (0.1M sodium carbonate, pH 9.0) at a concentration of 8 μg/mL was reacted at room temperature for 3 hours, and then the unbound antigen was treated with PBST (phosphate buffered saline- Tween 20) was used to wash 3 times. The non-specific reaction was eliminated by reacting with 300 μL of PBST supplemented with 5% bovine serum albumin (BSA) at room temperature for 1 hour and washing again with PBST. Thereafter, the selected anti-DCR1 monoclonal antibody was diluted by concentration on an ELISA plate coated with the DCR1 antigen and allowed to bind to the antigen at room temperature for 1 hour. The unbound anti-DCR1 monoclonal antibody was removed by washing 3 times using PBST, and the detection antibody (HRP conjugated anti-Fc IgG) was reacted at a concentration of 4 μg/mL at room temperature for 1 hour, followed by PBST. Thus, the unbound detection antibody was removed. For the color reaction, after treatment with TMB solution for 10 minutes, 2 N sulfuric acid solution was added to terminate the reaction, and absorbance was measured at 450 nm.

As a result, it was confirmed that the anti-DCR1 monoclonal antibody discovered in the present invention binds to the DCR1 antigen with strong binding force from the results of the Binding ELISA shown by the sigmoid curve (FIG. 2). In addition, the effective concentration of drug that causes 50% of the maxium response (EC50) of the anti-DCR1 monoclonal antibody is 0.2 νg/mL or less in all 6 clones (5C4, 5B1, 5D9, 5F9, 4F10, 4F11 (FS)) , confirming that it has a very high binding affinity.

Example 4. DCR1 monoclonal antibody in vitro Anti-cancer activity confirmation

4-1. Confirmation of expression of DCR1 in cancer cells

In order to determine whether the obtained anti-DCR1 antibody can be applied as an anticancer agent, it was confirmed whether the DCR1 protein was expressed in various cancer cells. For this purpose, MKN45 (human gastric cancer cell line), IM95 (human gastric cancer cell line), HT-29 (human colon cancer cell line), A549 (human lung cancer cell line), CT-26 (human colorectal cancer cell line), HCT116 (human colorectal cancer cell line) ), MDA-MB-231 (human breast cancer cell line) and HepG2 (human liver cancer cell line) were cultured to evaluate the expression of DCR1 at mRNA and protein levels. Specifically, the presence or absence of mRNA expression of DCR1 protein in each cancer cell line was confirmed by real-time qPCR using a primer set that specifically binds to the DCR1 gene. Western blot analysis was performed using the

As a result, it was confirmed that the DCR1 antigen was expressed at the mRNA and protein level in the various cancer cells, and it was confirmed that the anti-DCR1 monoclonal antibody of the present invention can be utilized as an anticancer agent.

4-2. Confirmation of cancer cell growth inhibitory effect

MKN45 (human gastric cancer cell line), IM95 (human gastric cancer cell line), HT-29 (human colorectal cancer cell line), A549 (human lung cancer cell line), CT-26 (human colorectal cancer cell line), HCT116 (human colorectal cancer cell line), MDA -MB-231 (human breast cancer cell line) and HepG2 (human liver cancer cell line) were analyzed for changes in proliferation of each cancer cell line according to the presence or absence of treatment with anti-DCR1 monoclonal antibody. Specifically, each cancer cell line was ^{inoculated at 1x10 4} cells/well in a 96-well plate and cultured for 18 hours to stabilize. After that, the anti-DCR1 monoclonal antibody DCR_mAb_1 of the present invention selected in the above example was treated for 8 hours at 0.1, 1, or 10 μg/ml, and then further cultured for 3 days, and then CCK-8 (Dojindo) was added to each well. The growth degree of the cancer cell line was confirmed by measuring the absorbance at 450 nm with a microplate reader 4 hours after addition of 10 μl.

As a result, it was found that cell proliferation was inhibited in a concentration-dependent manner of the anti-DCR1 antibody in the A549 lung cancer cell line and the CT-26 colorectal cancer cell line ( FIG. 3 ), and markedly in various cancer cell lines treated with the anti-DCR1 monoclonal antibody. It was confirmed that the growth of cancer cells was inhibited.

4-3. Confirmation of cancer cell migration inhibitory effect

MKN45 (human gastric cancer cell line), IM95 (human gastric cancer cell line), HT-29 (human colorectal cancer cell line), A549 (human lung cancer cell line), CT-26 (human colorectal cancer cell line), HCT116 (human colorectal cancer cell line), MDA -MB-231 (human breast cancer cell line) and HepG2 (human liver cancer cell line) were treated with anti-DCR1 monoclonal antibody, and the migration of cancer cells was confirmed by a wound healing assay. ^{Specifically, 1x10 5} cells/well of each of the cancer cell lines were inoculated into a 6-well plate and cultured for 16 hours. After incubation for 16 hours, the bottom of the plate is scraped using a pipette tip to create an artificial gap, and the detached cells are washed with a medium and then reacted for 8 hours by adding anti-DCR1 monoclonal antibody. Then, for 24 hours, cancer cells were monitored to fill this gap. Based on the area of the artificially created gap by scraping with a pipette tip, the distance traveled by cancer cells was measured, and this was calculated as a percentage (%) to determine the degree of inhibition of cancer cell migration.

As a result, it was confirmed that the migration of cancer cells was significantly inhibited in various cancer cell lines treated with anti-DCR1 monoclonal antibody.

4-4. Confirmation of the apoptosis promoting effect of cancer cells

To determine whether the ability to resist apoptosis, a characteristic of cancer cells, is affected by anti-DCR1 monoclonal antibodies, MKN45 (human gastric cancer cell line), IM95 (human gastric cancer cell line), HT-29 (human colon cancer cell line ), A549 (human lung cancer cell line), CT-26 (human colon cancer cell line), HCT116 (human colorectal cancer cell line), MDA-MB-231 (human breast cancer cell line) and HepG2 (human liver cancer cell line) in 96-well plates Each of 1x10 ⁴ pieces/well was dispensed and cultured for 16 hours. After that, the anti-DCR1 antibody was treated for 8 hours, rapamycin was treated for 48 hours, and the survival degree of cancer cells was analyzed by fluorescence using annexin V and PI (propidium iodide). was confirmed as

As a result, it was confirmed that the ability to resist apoptosis of cancer cells was significantly inhibited in cancer cell lines treated with anti-DCR1 monoclonal antibody.

4-5. Confirmation of the effect of inhibiting the signaling pathway of cancer cells

To determine the effect of anti-DCR1 monoclonal antibody on signaling important for the proliferation of cancer cells, MKN45 (human gastric cancer cell line), IM95 (human gastric cancer cell line), HT-29 (human colorectal cancer cell line), A549 (human lung cancer cell line) cell lines), CT-26 (human colon cancer cell line), HCT116 (human colorectal cancer cell line), MDA-MB-231 (human breast cancer cell line) and HepG2 (human liver cancer cell line) cell lines at various concentrations (0.5 , 01, and 0.005 μM), and phosphorylation of AKT and ERK, which are activators of downstream signaling pathways, was confirmed by Western blot analysis.

As a result, it was confirmed that the phosphorylation of AKT and ERK proteins was inhibited in a concentration-dependent manner in cancer cells treated with the anti-DCR1 monoclonal antibody. Through this, the anti-DCR1 monoclonal antibody effectively inhibited the growth signaling mechanism of cancer cells. It was confirmed that it can inhibit the growth of cancer cells.

4-6. Confirmation of T cell activation-inducing effect

In order to confirm whether the anti-DCR1 monoclonal antibody exhibits an anticancer effect by activating T cells, the production of IL-2 (Interleukin-2), an immune factor that helps the growth, proliferation and differentiation of T cells, was confirmed. An increase in the production amount of IL-2 means that T cells are activated by increasing the stimulation that induces the differentiation, proliferation, and growth of T cells to increase. Specifically, after adding anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody to a 96-well plate at a concentration of 2 μg/well, respectively, and fixing for 24 hours, 1x10 ⁵ cells/well of Jurkat T cells (human T lymphocyte cell line) and 10 μg/well of anti-DCR1 monoclonal antibody were co-treated. Thereafter, the production amount of IL-2 was measured using an ELISA kit (IL-2 Quantikine kit, R&D systems), and compared with a control group not treated with anti-DCR1 monoclonal antibody.

As a result, as shown in FIG. 4, when the anti-DCR1 monoclonal antibody was treated to the Jurkat T cells activated by treatment with the anti-CD3 monoclonal antibody and the anti-CD28 monoclonal antibody, the production amount of IL-2 was increased. , and through the above results, it was confirmed that the anti-DCR1 monoclonal antibody of the present invention can activate T cells and, through this, can inhibit the growth of cancer tissues by inducing anticancer immune action.

4.7. Confirmation of M1 macrophage differentiation ability of anti-DCR1 monoclonal antibody

To determine whether anti-DCR1 monoclonal antibody induces monocyte differentiation into M1 macrophage, 1.5x10 ⁴ cells/well of THP-1 (human monocyte cell line) was dispensed in a 96-well plate, and 10 μg/mL of anti-DCR1 monoclonal antibody and/or 100 ng/mL of LPS were treated. After reaction for 48 hours, the production amount of TNF-a (Tumor necrosis factor-a), a differentiation marker of M1 macrophages, was measured using an ELISA kit (Human TNF-a Quantikine kit, R&D systems).

As a result, as shown in FIG. 5 , it was confirmed that the production amount of TNF-a was increased by about 2 times or more in the group treated with the anti-DCR1 monoclonal antibody compared to the control group (Con) treated with LPS alone.

4-8. Confirmation of cancer cell proliferation inhibitory effect by suppressing DCR1 protein expression

To determine whether the inhibition of DCR1 protein expression exhibits an effect similar to that of the anti-DCR1 monoclonal antibody, siRNA of a DCR1-specific sequence was used to inhibit the expression of DCR1 protein and compared with the treatment with the anti-DCR1 monoclonal antibody. Specifically, cells were transfected with DCR1 siRNA (sc-40235, SantaCruz) using Lipofectamine RNAiMax (life Technologies) and cultured for 30 hours. As a control, scrambled RNA was used. Cell lysis solution (20mM Tris-Hcl(pH7.5), 150mM NaCl, 1mM Na ₂ EDTA, 1mM EGTA, 1% Triton, 2.5mM sodium pyrophosphate, 1mM glycerophosphate, 1mM Na ₃ V0 ₄ , 1 μg/mL of leupeptin and 1 mM PMSF) were treated to recover cell lysates. The amount of the protein contained in the cell lysate was quantified using the BCA method, and Western blot analysis was performed using the same amount of an antibody that specifically binds to DCR1 (sc-365279, SantaCruz) to determine the amount of DCR1 siRNA in cancer cells. It was confirmed that the inhibition of DCR1 expression in Thereafter, in order to determine whether inhibition of DCR1 expression through DCR1 siRNA treatment can inhibit the proliferation of cancer cells, various cancer cell lines were inoculated in a 96-well plate to 10,000 cells per well and then cultured for 16 hours. DCR1 siRNA was treated and cultured for 5 days, and the degree of proliferation of cancer cells was measured using CCK-8.

As a result, it was confirmed that DCR1 siRNA effectively inhibited the proliferation of cancer cells.

Example 5. DCR1 monoclonal antibody in vivo Anti-cancer activity confirmation

5-1. Anti-DCR1 monoclonal antibody in vivo Confirmation of cancer growth inhibitory effect in

In order to confirm the anti-cancer effect of anti-DCR1 monoclonal antibody in vivo ^{, 2x10 5} lung cancer cell line (A549) was subcutaneously injected into 8-week-old BALB/c mice by subcutaneous injection to form an allograft mouse tumor. A model (Syngenic mouse model) was prepared. All animal breeding and experiments were conducted in SPF (specific pathogen free) facilities. 12 days after transplantation of the lung cancer cell line, when the tumor size reached 100 mm ³ or more, anti-DCR1 monoclonal antibody was injected into the mice at 10, 1, and 0.1 ug/ml concentrations, respectively, and as a control, phosphate buffer solution ( After the same amount of phosphate buffered saline (PBS) was injected, the size of the cancer was measured for 25 days.

As a result, as shown in FIG. 6 , it was confirmed that cancer growth was effectively inhibited in the experimental group administered with the anti-DCR1 monoclonal antibody compared to the control group.

5-2. Confirmation of immune-boosting effect against cancer

To determine whether inhibition of DCR1 function and/or expression is associated with immune anticancer effects, 10 μg of anti-DCR1 monoclonal antibody was administered to BALB/c mice transplanted with cancer cells by intraperitoneal injection on days 0.1 and 5 After euthanasia on day 7, the bone marrow of mice was isolated. Myeloid-derived suppressor cells (MDSC), lymphocytes and cytotoxic T lymphocytes were identified from the isolated bone marrow using a flow cytometer (FACS).

As a result, in the case of mice treated with anti-DCR1 monoclonal antibody, lymphocytes and cytotoxic T lymphocytes (CD8+) were increased, and it was confirmed that the induction of lymphocyte differentiation (CD4+) was promoted. On the other hand, it was confirmed that the number of bone marrow-derived suppressor cells was decreased.

5-3. Confirmation of tumor suppression effect when administered alone or in combination

In order to ensure that represents the anti-cancer effect in a humanized monoclonal antibody wherein -DCR1 mouse model, by intravenous administration to the human cancer cell lines 10-luciferase ^six humanized mouse model expressing (luciferase) (humanized mouse, huCD34 -NSG mouse) A human solid cancer xenogeneic model was constructed. From 3 days after cancer cell administration, 20 mpk of anti-DCR1 monoclonal antibody was divided into 10 mpk-administered and non-administered groups of pembrolizumab (anti-PD-1 antibody), and each antibody was administered intraperitoneally twice a week, alone or in combination, a total of 4 times. Then, 15 mg/kg of luciferin was administered intraperitoneally and contrasted through the IVIS spectrum in vivo imaging system (Perkin Elmer, USA) to determine the tumor suppression effect of the monoclonal antibody and the effect of co-administration with the existing immune checkpoint inhibitor. Confirmed.

As a result, it was possible to confirm the high tumor suppression rate and cancer cell killing ability even in the anti-DCR1 monoclonal antibody alone group.

<110> Scimmune Inc. <120> MONOCLONAL ANTIBODIES AGAINST DCR1 <130> PN <160> 72 <170> KoPatentIn 3.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> 3B11_CDRH1 <400> 1 Ser Tyr Tyr Trp Ser 1 5 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> 3D7_CDRH1 <400> 2 Ser Tyr Ala Met His 1 5 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> 3F2_CDRH1 <400> 3 Ser Tyr Ala Met Ser 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> 5C4_CDRH1 <400> 4 Gly Tyr Ser Met Ser 1 5 <210> 5 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> 5B1_CDRH1 <400> 5 Gly Tyr Ala Met Ser 1 5 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> 5D9_CDRH1 <400> 6 Asp Tyr Asp Met Ser 1 5 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> 5F9_CDRH1 <400> 7 Asn Tyr Ala Met Ser 1 5 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> 4F10_CDRH1 <400> 8 Gly Tyr Tyr Met Ser 1 5 <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> 4F11(FS)_CDRH1 <400> 9 Gly Tyr Asp Met Ser 1 5 <210> 10 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> 3B11_CDRH2 <400> 10 Ser Ile Ser Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Thr Asp Ser Val Lys 1 5 10 15 Gly <210> 11 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 3D7_CDRH2 <400> 11 Ser Ala Ile Asn Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Ser <210> 12 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 3F2_CDRH2 <400> 12 Ser Ala Ile Ser Gly Arg Gly Thr Ser Thr Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly <210> 13 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> 5C4_CDRH2 <400> 13 Gly Ile Ser Tyr Gly Asp Gly Ser Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> 5B1_CDRH2 <400> 14 Ala Ile Ser His Gly Gly Ser Ser Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 15 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> 5D9_CDRH2 <400> 15 Leu Ile Tyr Tyr Asp Ser Gly Ser Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 16 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> 5F9_CDRH2 <400> 16 Gly Ile Ser Tyr Gly Asp Ser Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 17 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> 4F10_CDRH2 <400> 17 Val Ile Ser His Asp Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 18 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> 4F11(FS)_CDRH2 <400> 18 Val Ile Tyr Pro Asp Gly Gly Asn Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 19 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> 3B11_CDRH3 <400> 19 Ala Arg Asp Thr Trp Trp Glu Gly Tyr Phe Asp Leu 1 5 10 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 3D7_CDRH3 <400> 20 Ala Arg Gly Met Trp Gly Met Asp Val 1 5 <210> 21 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 3F2_CDRH3 <400> 21 Ala Arg Ser Ser Gln Gly Ile Phe Asp Ile 1 5 10 <210> 22 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> 5C4_CDRH3 <400> 22 Ala Lys Asp Thr Arg Ser Arg Asn Pro Gly Ala Gln Ser Ser Ser Asn 1 5 10 15 Ala Met Asp Val 20 <210> 23 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> 5B1_CDRH3 <400> 23 Ala Lys Tyr Thr Val Asp Arg Lys Glu Ser Pro Ser Tyr Tyr Tyr Tyr 1 5 10 15 Ala Met Asp Val 20 <210> 24 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> 5D9_CDRH3 <400> 24 Ala Arg Ala Pro Leu Thr Cys Thr Leu Thr Gln Cys Ser Tyr Asp Asp 1 5 10 15 Ala Met Asp Val 20 <210> 25 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> 5F9_CDRH3 <400> 25 Ala Lys Asp Arg Arg Gly Cys Asp Ala Arg Ile Cys Tyr Ser Asp Asp 1 5 10 15 Ala Met Asp Val 20 <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 4F10_CDRH3 <400> 26 Ala Lys Asn Asn Trp Thr Phe Asp Tyr 1 5 <210> 27 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 4F11(FS)_CDRH3 <400> 27 Ala Arg Arg Asn Arg Ser Phe Asp Tyr 1 5 <210> 28 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 3B11_CDRL1 <400> 28 Arg Ala Ser Gln Ser Ile Ser Ser His Leu Ala 1 5 10 <210> 29 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> 3D7_CDRL1 <400> 29 Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn Tyr Val Gln 1 5 10 <210> 30 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> 3F2_CDRL1 <400> 30 Thr Gly Thr Ser Ser Asp Val Gly Asn Tyr Asn Leu Val Ser 1 5 10 <210> 31 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> 5C4_CDRL1 <400> 31 Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn Asp Val Thr 1 5 10 <210> 32 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> 5B1_CDRL1 <400> 32 Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Asp Val Asn 1 5 10 <210> 33 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> 5D9_CDRL1 <400> 33 Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Thr 1 5 10 <210> 34 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> 5F9_CDRL1 <400> 34 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Asp Val Asn 1 5 10 <210> 35 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> 4F10_CDRL1 <400> 35 Ser Gly Ser Pro Ser Asn Ile Gly Ser Asn Ser Val Thr 1 5 10 <210> 36 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> 4F11(FS)_CDRL1 <400> 36 Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Ala Val Asn 1 5 10 <210> 37 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 3B11_CDRL2 <400> 37 Asp Ala Ser Leu Arg Ala Thr 1 5 <210> 38 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 3D7_CDRL2 <400> 38 Asp Asn Asn Lys Arg Pro Ser 1 5 <210> 39 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> 3F2_CDRL2 <400> 39 Asn Thr Ser Asn Lys His 1 5 <210> 40 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 5C4_CDRL2 <400> 40 Ala Asp Ser Lys Arg Pro Ser 1 5 <210> 41 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 5B1_CDRL2 <400> 41 Ser Asn Ser His Arg Pro Ser 1 5 <210> 42 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 5D9_CDRL2 <400> 42 Asp Asn Ser Gln Arg Pro Ser 1 5 <210> 43 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 5F9_CDRL2 <400> 43 Ser Asn Asn His Arg Pro Ser 1 5 <210> 44 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 4F10_CDRL2 <400> 44 Asn Asp Asn Lys Arg Pro Ser 1 5 <210> 45 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 4F11(FS)_CDRL2 <400> 45 Tyr Asp Ser Asn Arg Pro Ser 1 5 <210> 46 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 3B11_CDRL3 <400> 46 Gln Gln Ser Tyr Ser Thr Pro Phe Thr 1 5 <210> 47 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 3D7_CDRL3 <400> 47 Asn Thr Trp Asp Asp Ser Leu Glu Gly Pro Val 1 5 10 <210> 48 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 3F2_CDRL3 <400> 48 Gln Thr Trp Asp Ser Ser Thr Asp Val Val 1 5 10 <210> 49 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 5C4_CDRL3 <400> 49 Ala Ser Trp Asp Ser Ser Leu Asn Gly Tyr Val 1 5 10 <210> 50 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 5B1_CDRL3 <400> 50 Gly Thr Trp Asp Asp Ser Leu Asn Gly Tyr Val 1 5 10 <210> 51 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 5D9_CDRL3 <400> 51 Gly Ser Trp Asp Ala Ser Leu Ser Gly Tyr Val 1 5 10 <210> 52 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 5F9_CDRL3 <400> 52 Gly Thr Trp Asp Ser Ser Leu Ser Gly Tyr Val 1 5 10 <210> 53 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 4F10_CDRL3 <400> 53 Gly Ala Trp Asp Ser Ser Leu Asn Gly Tyr Val 1 5 10 <210> 54 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 4F11(FS)_CDRL3 <400> 54 Gly Ala Trp Asp Ser Ser Leu Ser Ala Tyr Val 1 5 10 <210> 55 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> 3B11 <400> 55 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Tyr Trp Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Thr Trp Trp Glu Gly Tyr Phe Asp Leu Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr 130 135 140 Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser 145 150 155 160 Gln Ser Ile Ser Ser His Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 165 170 175 Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Leu Arg Ala Thr Gly Ile 180 185 190 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195 200 205 Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln 210 215 220 Ser Tyr Ser Thr Pro Phe Thr Phe Gly Gin Gly Thr Lys Val Glu Ile 225 230 235 240 Lys <210> 56 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> 3D7 <400> 56 Arg Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asn Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Met Trp Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr 130 135 140 Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile 145 150 155 160 Ala Ser Asn Tyr Val Gln Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro 165 170 175 Lys Leu Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Val Arg Asp 180 185 190 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 195 200 205 Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Thr Trp Asp 210 215 220 Asp Ser Leu Glu Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val 225 230 235 240 Leu <210> 57 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> 3F2 <400> 57 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Arg Gly Thr Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Gln Gly Ile Phe Asp Ile Trp Gly Gly Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Ser Ala Ser Gly 130 135 140 Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp 145 150 155 160 Val Gly Asn Tyr Asn Leu Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr 165 170 175 Ala Pro Lys Leu Leu Ile Tyr Asn Thr Ser Asn Lys His Gly Val Pro 180 185 190 Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile 195 200 205 Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Trp 210 215 220 Asp Ser Ser Thr Asp Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val 225 230 235 240 Leu <210> 58 <211> 252 <212> PRT <213> Artificial Sequence <220> <223> 5C4 <400> 58 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Tyr Gly Asp Gly Ser Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Thr Arg Ser Arg Asn Pro Gly Ala Gln Ser Ser Ser Asn 100 105 110 Ala Met Asp Val Trp Gly Gly Gly Thr Leu Val Thr Val Ser Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser 130 135 140 Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val 145 150 155 160 Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn Asp Val 165 170 175 Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr 180 185 190 Ala Asp Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 195 200 205 Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu 210 215 220 Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Ser Ser Leu Asn Gly 225 230 235 240 Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 245 250 <210> 59 <211> 252 <212> PRT <213> Artificial Sequence <220> <223> 5B1 <400> 59 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser His Gly Gly Ser Ser Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Thr Val Asp Arg Lys Glu Ser Pro Ser Tyr Tyr Tyr Tyr 100 105 110 Ala Met Asp Val Trp Gly Gly Gly Thr Leu Val Thr Val Ser Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser 130 135 140 Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val 145 150 155 160 Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Asp Val 165 170 175 Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr 180 185 190 Ser Asn Ser His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 195 200 205 Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu 210 215 220 Asp Gly Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Asp Ser Leu Asn Gly 225 230 235 240 Tyr Val Phe Gly Gly Ser Thr Lys Leu Thr Val Leu 245 250 <210> 60 <211> 252 <212> PRT <213> Artificial Sequence <220> <223> 5D9 <400> 60 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Tyr Tyr Asp Ser Gly Ser Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Pro Leu Thr Cys Thr Leu Thr Gln Cys Ser Tyr Asp Asp 100 105 110 Ala Met Asp Val Trp Gly Gly Gly Thr Leu Val Thr Val Ser Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser 130 135 140 Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val 145 150 155 160 Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val 165 170 175 Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr 180 185 190 Asp Asn Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 195 200 205 Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu 210 215 220 Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Ala Ser Leu Ser Gly 225 230 235 240 Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 245 250 <210> 61 <211> 252 <212> PRT <213> Artificial Sequence <220> <223> 5F9 <400> 61 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Tyr Gly Asp Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Arg Gly Cys Asp Ala Arg Ile Cys Tyr Ser Asp Asp 100 105 110 Ala Met Asp Val Trp Gly Gly Gly Thr Leu Val Thr Val Ser Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser 130 135 140 Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val 145 150 155 160 Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Asp Val 165 170 175 Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr 180 185 190 Ser Asn Asn His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 195 200 205 Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu 210 215 220 Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu Ser Gly 225 230 235 240 Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 245 250 <210> 62 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> 4F10 <400> 62 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser His Asp Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Asn Trp Thr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr 130 135 140 Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Pro Ser Asn Ile 145 150 155 160 Gly Ser Asn Ser Val Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro 165 170 175 Lys Leu Leu Ile Tyr Asn Asp Asn Lys Arg Pro Ser Gly Val Pro Asp 180 185 190 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 195 200 205 Gly Leu Arg Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp 210 215 220 Ser Ser Leu Asn Gly Tyr Val Phe Gly Gly Ser Thr Lys Leu Thr Val 225 230 235 240 Leu <210> 63 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> 4F11 (FS) <400> 63 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr Pro Asp Gly Gly Asn Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Gly Ser Pro Ser Pro Glu Thr Ile Pro Arg Thr Arg Cys Ile 65 70 75 80 Cys Lys Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asn Arg Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Pro Ala Ser Gly Thr 130 135 140 Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile 145 150 155 160 Gly Asn Asn Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro 165 170 175 Lys Leu Leu Ile Tyr Tyr Asp Ser Asn Arg Pro Ser Gly Val Pro Asp 180 185 190 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 195 200 205 Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp 210 215 220 Ser Ser Leu Ser Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val 225 230 235 240 Leu <210> 64 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> 3B11 <400> 64 gaggtgcagc tgctggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60 agctgcgccg ccagcggctt caccttcagc agctactact ggagctgggt gaggcaggcc 120 cccggcaagg gcctggagtg ggtgagcagc atcagcagca gcagcagcta catctactac 180 accgacagcg tgaagggcag gttcaccatc agcagggaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc cagggacacc 300 tggtgggagg gctacttcga cctgtggggc cagggcaccc tggtgaccgt gagcagcagc 360 ggcggcggca gcggcggcgg cggcagcggc ggcggcggca gcgagatcgt gctgacccag 420 agccccggca ccctgagcct gagccccggc gagagggcca ccctgagctg cagggccagc 480 cagagcatca gcagccacct ggcctggtac cagcagaagc ccggccaggc ccccaggctg 540 ctgatctacg acgccagcct gagggccacc ggcatccccg acaggttcag cggcagcggc 600 agcggcaccg acttcaccct gaccatcagc aggctggagc ccgaggactt cgccgtgtac 660 tactgccagc agagctacag cacccccttc accttcggcc agggcaccaa ggtggagatc 720 aag 723 <210> 65 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> 3D7 <400> 65 agggtgcagc tgctggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60 agctgcgccg ccagcggctt caccttcagc agctacgcca tgcactgggt gaggcaggcc 120 cccggcaagg gcctggagtg ggtgagcgcc atcaacggca gcggcggcag cacctactac 180 gccgacagcg tgaagagcag gttcaccatc agcagggaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc caggggcatg 300 tggggcatgg acgtgtgggg ccagggcacc ctggtgaccg tgagcagcag cggcggcggc 360 agcggcggcg gcggcagcgg cggcggcggc agccagagcg tgctgaccca gccccccagc 420 gccagcggca cccccggcca gagggtgacc atcagctgca ccaggagcag cggcagcatc 480 gccagcaact acgtgcagtg gtaccagcag ctgcccggca ccgcccccaa gctgctgatc 540 tacgacaaca acaagaggcc cagcggcgtg agggacaggt tcagcggcag caagagcggc 600 accagcgcca gcctggccat cagcggcctg aggagcgagg acgaggccga ctactactgc 660 aacacctggg aggacagcct ggagggcccc gtgttcggcg gcggcaccaa gctgaccgtg 720 ctg 723 <210> 66 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> 3F2 <400> 66 gaggtgcagc tgctggagag cggcggcggc ctggtgcaga gcggcggcag cctgaggctg 60 agctgcgccg ccagcggctt caccttcagc agctacgcca tgagctgggt gaggcaggcc 120 cccggcaagg gcctggagtg ggtgagcgcc atcagcggca ggggcaccag cacctactac 180 gccgacagcg tgaagggcag gttcaccatc agcagggaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc caggagcagc 300 cagggcatct tcgacatctg gggccagggc accctggtga ccgtgagcag cggcggcggc 360 ggcagcggcg gcggcggcag cggcggcggc ggcagccaga gcgtgctgac ccagcccccc 420 agcgccagcg gcacccccgg ccagagggtg accatcagct gcaccggcac cagcagcgac 480 gtgggcaact acaacctggt gagctggtac cagcagctgc ccggcaccgc ccccaagctg 540 ctgatctaca acaccagcaa caagcacggc gtgcccgaca ggttcagcgg cagcaagagc 600 ggcaccagcg ccagcctggc catcagcggc ctgaggagcg aggacgaggc cgactactac 660 tgccagacct gggacagcag caccgacgtg gtgttcggcg gcggcaccaa gctgaccgtg 720 ctg 723 <210> 67 <211> 756 <212> DNA <213> Artificial Sequence <220> <223> 5C4 <400> 67 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc ggttattcta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcaggg atctcttatg gtgatggtag taaatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaaagatact 300 cgttcgcgga atcctggggc gcagtcttct tctaatgcta tggacgtctg gggccagggt 360 acactggtca ccgtgagctc aggtggaggc ggttcaggcg gaggtggatc cggcggtggc 420 ggatcgcagt ctgtgctgac tcagccaccc tcagcgtctg ggacccccgg gcagagggtc 480 accatctctt gtactggctc ttcatctaat attggcagta atgatgtcac ctggtaccag 540 cagctcccag gaacggcccc caaactcctc atctatgctg atagtaagcg gccaagcggg 600 gtccctgacc gattctctgg ctccaagtct ggcacctcag cctccctggc catcagtggg 660 ctccggtccg aggacgaggc tgattattac tgtgcttctt gggattctag cctgaatggt 720 tatgtcttcg gcggaggcac caagctgacg gtccta 756 <210> 68 <211> 756 <212> DNA <213> Artificial Sequence <220> <223> 5B1 <400> 68 gaggtgcagc tgttggagtc tgggggaggc ttggtacaga ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc ggttatgcta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagcg atctctcatg gtggtagtag taaatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaaatatact 300 gttgatcgga aggagtcgcc ttcgtattat tattatgcta tggacgtctg gggccagggt 360 acactggtca ccgtgagctc aggtggaggc ggttcaggcg gaggtggatc cggcggtggc 420 ggatcgcagt ctgtgctgac tcagccaccc tcagcgtctg ggacccccgg gcagagggtc 480 accatctctt gtagtggctc ttcatctaat attggcaata atgatgtcaa ctggtaccag 540 cagctcccag gaacggcccc caaactcctc atctattcta atagtcatcg gccaagcggg 600 gtccctgacc gattctctgg ctccaagtct ggcacctcag cctccctggc catcagtggg 660 ctccggtccg aggatggggc tgattattac tgtggtactt gggatgatag cctgaatggt 720 tatgtcttcg gcggaagcac caagctgacg gtccta 756 <210> 69 <211> 756 <212> DNA <213> Artificial Sequence <220> <223> 5D9 <400> 69 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc gattatgata tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcattg atctattatg atagtggtag tatatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagagctcct 300 cttacttgta cgctgacgca gtgttcttat gatgatgcta tggacgtctg gggccagggt 360 acactggtca ccgtgagctc aggtggaggc ggttcaggcg gaggtggatc cggcggtggc 420 ggatcgcagt ctgtgctgac tcagccaccc tcagcgtctg ggacccccgg gcagagggtc 480 accatctctt gtactggctc ttcatctaat attggcagta atactgtcac ctggtaccag 540 cagctcccag gaacggcccc caaactcctc atctatgata atagtcagcg gccaagcggg 600 gtccctgacc gattctctgg ctccaagtct ggcacctcag cctccctggc catcagtggg 660 ctccggtccg aggatgaggc tgattattac tgtggttctt gggatgctag cctgagtggt 720 tatgtcttcg gcggaggcac caagctgacg gtccta 756 <210> 70 <211> 756 <212> DNA <213> Artificial Sequence <220> <223> 5F9 <400> 70 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc aattatgcta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcaggg atctcttatg gtgatagtag tacatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaaagatcgt 300 cgtgggtgtg atgctaggat ttgttattct gatgatgcta tggacgtctg gggccagggt 360 acactggtca ccgtgagctc aggtggaggc ggttcaggcg gaggtggatc cggcggtggc 420 ggatcgcagt ctgtgctgac tcagccaccc tcagcgtctg ggacccccgg gcagagggtc 480 accatctctt gtagtggctc ttcatctaat attggcagta atgatgtcaa ctggtaccag 540 cagctcccag gaacggcccc caaactcctc atctattcta ataatcatcg gccaagcggg 600 gtccctgacc gattctctgg ctccaagtct ggcacctcag cctccctggc catcagtggg 660 ctccggtccg aggatgaggc tgattattac tgtggtactt gggattctag cctgagtggt 720 tatgtcttcg gcggaggcac caagctgacg gtccta 756 <210> 71 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> 4F10 <400> 71 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc ggttattata tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagtg atctctcatg atggtagtag tacatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaaaaataat 300 tggactttcg actactgggg ccagggtaca ctggtcaccg tgagctcagg tggaagcggt 360 tcaggcggag gtggatccgg cggtggcgga tcgcagtctg tgctgactca gccaccctca 420 gcgtctggga cccccgggca gagggtcacc atctcttgta gtggctctcc atctaatatt 480 ggcagtaatt ctgtcacctg gtaccagcag ctcccaggaa cggcccccaa actcctcatc 540 tataatgata ataagcggcc aagcggggtc cctgaccgat tctctggctc caagtctggc 600 acctcagcct ccctggccat cagtgggctc cggcccgagg atgaggctga ttattactgt 660 ggtgcttggg attctagcct gaatggttat gtcttcggcg gaagcaccaa gctgacggtc 720 cta 723 <210> 72 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> 4F11 (FS) <400> 72 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc ggttatgata tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagtg atctatcctg atggtggtaa tatatattac 180 gctgattctg taaaaggcgg ttcaccatct ccagagacaa ttccaagaac acgctgtatc 240 tgcaaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagacggaat 300 aggtctttcg actactgggg ccagggtaca ctggtcaccg tgagctcagg tggaggcggt 360 tcaggcggag gtggatccgg cggtggcgga tcgcagtctg tgctgactca gccaccccca 420 gcgtctggga cccccgggca gagggtcacc atctcttgta gtggctcttc atctaatatt 480 ggcaataatg ctgtcaactg gtaccagcag ctcccaggaa cggcccccaa actcctcatc 540 tattatgata gtaatcggcc aagcggggtc cctgaccgat tctctggctc caagtctggc 600 acctcagcct ccctggccat cagtgggctc cggtccgagg atgaggctga ttattactgt 660 ggtgcttggg attctagcct gagtgcttat gtcttcggcg gaggcaccaa gctgacggtc 720 cta 723

Claims (22)

A monoclonal antibody specific for human DCR1 (decoy receptor 1) or a fragment having immunological activity thereof. According to claim 1, wherein the fragment having immunological activity is Fab, Fd, Fab', dAb, F(ab'), F(ab') ₂ , scFv (single chain fragment variable), Fv, single chain antibody, Fv A monoclonal antibody or fragment having immunological activity, which is any one selected from the group consisting of dimers, complementarity determining region fragments, humanized antibodies, chimeric antibodies, and diabodies. According to claim 1, Complementarity determining regions heavy chain (CDRH) comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 1 to 9, any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 10 to 18 A monoclonal antibody or fragment having immunological activity thereof, comprising a VH domain comprising CDRH2 comprising one, and CDRH3 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 19 to 27. The method according to claim 1, wherein the CDRL1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 28 to 36, CDRL2 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 37 to 45, and the sequence A monoclonal antibody or fragment having immunological activity thereof, comprising a VL domain comprising CDRL3 comprising any one selected from the group consisting of the amino acid sequence of numbers 46 to 54. The monoclonal antibody or fragment having immunological activity thereof according to claim 1, wherein the fragment having immunological activity is an scFv comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 55 to 63. An isolated nucleic acid molecule encoding the monoclonal antibody of claim 1 or a fragment having immunological activity thereof. The isolated nucleic acid molecule according to claim 6, comprising any one selected from the group consisting of nucleotide sequences of SEQ ID NOs: 64 to 72. A vector comprising the isolated nucleic acid molecule of claim 7. A host cell transformed with the vector of claim 8 . a) culturing a host cell comprising a vector comprising the isolated nucleic acid molecule of claim 7; and
b) a method for producing a human DCR1-specific antibody or fragment having immunological activity thereof, comprising the step of recovering the antibody or fragment having immunological activity thereof from host cell culture. A pharmaceutical composition for preventing or treating cancer, comprising the monoclonal antibody of claim 1 or a fragment having immunological activity thereof. 12. The method of claim 11, wherein the cancer is brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colorectal cancer , Small intestine cancer, rectal cancer, fallopian tube carcinoma, perianal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer , soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, renal or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma And a pharmaceutical composition for the prevention or treatment of cancer, characterized in that any one selected from the group consisting of a pituitary adenoma. The pharmaceutical composition for preventing or treating cancer according to claim 11, further comprising one or more selected from the group consisting of cytotoxic substances and markers. The method of claim 13, wherein the cytotoxic material is ricin, saporin (saporin), gelonin (gelonin), morodin (momordin), deboganin (debouganin), diphtheria toxin, Pseudomonas toxin (pseudomonas toxin), Radioisotopes, duocarmycin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), N2'-diacetyl-N2'-(3 -Mercapto-1-oxopropyl) maytansine (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl) maytansine; DM1), and PBD (Pyrrolobenzodiazepine) any one selected from the group consisting of dimers A pharmaceutical composition for the prevention or treatment of abnormal cancer. The pharmaceutical composition for preventing or treating cancer according to claim 11, further comprising an immuno-cancer agent or an anti-cancer agent. The pharmaceutical composition for preventing or treating cancer according to claim 15, wherein the immunotherapy is an immune checkpoint inhibitor. The pharmaceutical composition for preventing or treating cancer according to claim 16, wherein the immune checkpoint inhibitor is a PD-1 (programmed cell death 1) or PD-L1 (programmed cell death ligand 1) inhibitor. 16. The method of claim 15, wherein the immuno-oncology agent is an anti-PD1 antibody, an anti-PDL1 antibody, an anti-CTLA4 antibody, an anti-LAG3 antibody, an anti-VISTA antibody, an anti-BTLA antibody, an anti-TIM3 antibody, an anti-HVEM antibody, an anti -CD27 antibody, anti-CD137 antibody, anti-OX40 antibody, anti-CD28 antibody, anti-PDL2 antibody, anti-GITR antibody, anti-ICOS antibody, anti-SIRPα antibody, anti-ILT2 antibody, anti-ILT3 antibody, anti -ILT4 antibody, anti-ILT5 antibody, anti-EGFR antibody, anti-CD19 antibody, and any one or more selected from the group consisting of anti-TIGIT antibody, a pharmaceutical composition for the prevention or treatment of cancer. The method of claim 15, wherein the anticancer agent is doxorubicin (doxorubicin), cisplatin (cisplatin), paclitaxel (paclitaxel), vincristine (vincristine), topotecan (topotecan), docetaxel (docetaxel), 5-fluorouracil (5-FU) , Gleevec (gleevec), carboplatin (carboplatin), duanorubicin (daunorubicin), valrubicin (valrubicin), flutamide (flutamide), any one selected from the group consisting of gemcitabine (gemcitabine) of cancer A pharmaceutical composition for prophylaxis or treatment. The pharmaceutical composition for preventing or treating cancer according to claim 11, which inhibits cancer growth, survival, metastasis, recurrence, or resistance to anticancer drugs. A pharmaceutical composition for preventing or treating cancer comprising an expression inhibitor specific to human DCR1. The pharmaceutical composition for preventing or treating cancer according to claim 21, wherein the human DCR1 expression inhibitor is siRNA, shRNA, or miRNA.

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