JP2015196667A - Anti-lr11 monoclonal antibody having neutralizing activity and pharmaceutical comprising the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel monoclonal antibody that specifically binds to LR11 and has excellent neutralizing activity, and a pharmaceutical agent comprising the same for preventing and/or treating malignant tumors.SOLUTION: The invention provides an anti-LR11 monoclonal antibody having at least one amino acid sequence selected from the group of specified sequences as the complementarity determining region (CDR), which the antibody can: (a) recognizes LR11; (b) specifically binds to a protein comprising the amino acid sequence of LR11 Vps10p domain; and (c) in a cell adhesion assay, significantly inhibits the cell adhesion promoted by a protein comprising an amino acid sequence of human LR11 Vps10p domain. Also provided is a pharmaceutical agent comprising the anti-LR11 monoclonal antibody for preventing and/or treating malignant tumors. Further, the anti-LR11 monoclonal antibody can inhibit enhanced cellular migration as well as the cellular infiltration of malignant tumor cells.

Description

  The present invention specifically binds to cell surface LR11 and soluble LR11 (hereinafter referred to as “sLR11”) by an epitope encoded by a monoclonal antibody, particularly the Vps10p domain that is the extracellular domain of LR11, and has many preferable features. It relates to a monoclonal antibody having The present invention also relates to a monoclonal antibody having an activity of inhibiting and neutralizing the biological activity of cell surface LR11 and sLR11. The present invention also provides a pharmaceutical composition comprising the monoclonal antibody or the monoclonal antibody for use in a method for treating various cancers. The invention further relates to the use of said monoclonal antibody in the manufacture of a medicament for the treatment of cancer.

  LR11 (Low-density lipoprotein (LDL) receptor relative with 11 ligand-binding repeats) is an LDL receptor family protein (Patent Document 1), and its structure is a type I membrane protein containing a transmembrane region once. The ligand binding region (clusters of type A ligand-binding repeats or complement-type repeats of complement-type repeats), which is a common structure of the receptor family, has a structure that repeats 11 times (Non-patent Document 1). . LR11, such as human, rabbit, mouse, rat, chicken, etc., has a highly conserved amino acid sequence across species differences, and a Vps10p (Vacuolar protein sorting 10 protein) -like domain is present on the N-terminal side of the repeating structure of the ligand binding region. It has a structure such as a fibronectin type III domain on the C-terminal side, and further has a transmembrane region and a cytoplasmic tail C-terminal on the C-terminal side. LR11 is highly expressed in nerve cells, and in humans, it is highly expressed in brain and kidney. Moreover, distribution is seen not only on the cell surface but also in intracellular endosomes. SorLA (sorting protein-related receptor containing LDL receptor class A repeats) identified from brain tissue and LR11 are the same protein, and the human gene name is SORL1 (Non-patent Document 2). Furthermore, LR11 present on the cell surface is cleaved by protease TACE (tumor necrosis factor-alpha converting enzyme) directly above the transmembrane region, and the extracellular portion of the fraction containing Vps10p as sLR11 goes out of the cell. Released. LR11 is not expressed in normal blood vessels, but in arteriosclerotic lesions, it is known that expression is specifically increased at the site of intimal thickening formed by smooth muscle cell migration and proliferation. (Non-Patent Documents 3 and 4). LR11 binds to a urokinase (hereinafter referred to as “uPA”) receptor (hereinafter referred to as “uPAR”) on the cell surface in both membrane-bound and release-soluble forms (Non-patent Document 5). uPAR also forms a complex on the cell surface with the cell adhesion molecule integrin. Soluble LR11 degrades the extracellular matrix by activating the plasmin cascade via the uPA-uPAR system and activates the matrix metalloprotease to degrade the extracellular matrix surrounding the cell. Furthermore, uPAR bound to uPA induces integrin-mediated intracellular signals, but released sLR11 binds to uPAR, thereby activating the integrin / FAK / Rac1 pathway and altering cytoskeletal structure to smooth blood vessels. It is considered to enhance the migration ability of muscle cells (Non-patent Document 6).

LR11, which is strongly expressed in the brain, is localized in the intracellular Golgi-endoplasmic reticulum network in neurons and is involved in protein transport between vesicles in neurons (Non-patent Document 7). LR11 is also involved in amyloid precursor transport, retains the amyloid precursor protein in the Golgi apparatus, returns the amyloid precursor protein transferred to the secretory vesicle to the Golgi apparatus again, and functions of β-secretase to generate amyloid from the amyloid precursor protein By inhibiting the production of amyloid β, the association between LR11 and Alzheimer's disease has been reported (Non-patent Document 7). That is, it has been pointed out that the decrease in LR11 expression in the brain of Alzheimer's disease patients (Non-Patent Documents 8 and 9) and the possibility that an LR11 gene polymorphism is a risk of developing Alzheimer's disease (Non-Patent Document 10).
Furthermore, the sLR11 concentration in the cerebrospinal fluid becomes high in Alzheimer's disease (Non-patent Document 11), and there is a report that the LR11 gene polymorphism in patients associated with the risk of developing Alzheimer's disease correlates with the LR11 concentration in cerebrospinal fluid ( Non-patent literature 12-14).

  Recently, LR11 was found to be one of the genes highly expressed in blood stem / progenitor cells, that is, CD34 (+) CD38 (−) undifferentiated bone marrow stem cells (Non-patent Document 15). Furthermore, the expression of LR11 on the surface of leukemia cells of acute leukemia patients such as acute myeloid leukemia (hereinafter referred to as “AML”) and acute lymphocytic leukemia (hereinafter referred to as “ALL”) is increased, and in the serum of patients. It has been reported that the sLR11 concentration of the drug becomes high, and the value decreases during remission by chemotherapy (Non-patent Document 16). It has also been reported that sLR11 concentration is high in patient serum in non-Hodgkin's lymphoma (diffuse large B-cell lymphoma, follicular lymphoma), and that the prognosis is poor if the initial high level is high (non Patent Document 17). Thus, sLR11 shows an abnormally high value in malignant tumors, particularly hematopoietic tumor diseases such as leukemia and malignant lymphoma, and can be one of important biomarkers (Patent Document 2).

  Acute leukemias such as AML and ALL are highly sensitive to chemotherapy, and anticancer agents often provide a “remission” in which the number of leukemia cells is significantly reduced and symptoms are improved. However, a high recurrence rate even in patients who have achieved remission is a challenge for leukemia treatment. The mechanism of recurrence in this leukemia is that the leukemia stem cells located in the border between the bone marrow and the endosteum, called the “niche” in the bone marrow, survive because they are resistant to anticancer drugs, and self-replicate It is considered to supply a cell population (Non-patent Document 18). Leukemia stem cells are resistant to anticancer drugs because leukemia stem cells specifically arrest the cell cycle in a niche environment, and anticancer drugs target cancer cells with high proliferative activity. It is considered to have resistance (Non-patent Document 18). In this way, leukemia stem cells can maintain a quiescent phase in an intraosseous microenvironment called a niche because leukemia stem cells maintain their local adhesion to the extracellular matrix (matrix) and stromal cells. Is considered important. On the other hand, recently, under hypoxic conditions such as bone marrow niche, LR11 expression of hematopoietic stem cells is enhanced and regulates adhesion to surrounding extracellular matrix and stromal cells via uPAR (Non-patent Document 19). Furthermore, U937 cells with suppressed LR11 expression in an AML model in which human monocyte lymphoma cell line U937 cells highly expressing LR11 were transplanted into immunodeficient mice that had been irradiated and administered anti-asialo GM1 antibody ( When LR11-KD) was transplanted, bone marrow infiltration and engraftment decreased, survival was increased, and LR11-KD U937 cells also had reduced adhesion to human myeloid stromal cells. (Non-Patent Document 20). These results suggest that LR11 is involved as an important modifier in the pathogenesis of AML.

  The anti-LR11 antibody has been suggested to be used as a diagnostic marker for the disease and applied to treatment (Patent Documents 2 and 3, Non-Patent Document 21). Moreover, although the effect which anti-LR11 antibody suppresses the adhesion promotion effect | action with respect to a rat smooth muscle cell is also known (nonpatent literature 22), the effect | action intensity | strength is not enough.

Japanese Patent Laid-Open No. 9-163988 Japanese Patent No. 4955836 WO2008 / 155891 pamphlet WO2009 / 116268 pamphlet

Yamazaki H. et al., J Biol Chem., 271 (40): 24761-8, 1996. Jacobsen L. et al., J Biol Chem., 271 (49): 31379-83, 1996. Kanaki T. et al., Arterioscler Thromb Vasc Biol., 19 (11): 2687-95, 1999. Zhu Y. et al., Circ Res., 94 (6): 752-8, 2004. Bujo H. et al., Arterioscler Thromb Vasc Biol., 26 (6): 1246-52, 2006. Jiang M. et al., J Clin Invest., 118 (8): 2733-46, 2008. Ryuji Yajima et al., The Lipid, 23 (4): 390-95, 2012. Scherzer CR. Et al., Arch Neurol., 61 (8): 1200-5, 2004. Dodson SE. Et al., J Neuropathol Exp Neurol., 65 (9): 866-72, 2006. Reitz C. et al., Arch Neurol., 68 (1): 99-106, 2011. Ikeuchi T. et al., Dement Geriatr Cogn Disord., 30 (1): 28-32, 2010. Rogaeva E. et al., Nat Genet., 39 (2): 168-77, 2007. Guo LH. Et al., Eur Arch Psychiatry Clin Neurosci., 262 (6): 529-34, 2012. Caglayan S. et al., Arch Neurol., 69 (3): 373-9, 2012. Zhang X. et al., Exp Hematol., 28 (11): 1286-96,2000. Sakai S. et al., Clin Chim Acta., 413 (19-20): 1542-8, 2012. Kawaguchi T. et al., Br J Haematol., 163 (2): 277-291, 2013. Ishikawa Fumihiko, Cell Engineering, 31 (1): 48-52, 2012. Nishii K. et al., J Biol Chem., 288 (17): 11877-86, 2013. Keigo Nishii, The 75th Annual Meeting of the Japanese Society of Hematology (October 11-13, 2013, Sapporo), "LR11 plays a critical role in leukemogenesis of AML by regulating cell adhesion and homing ability." Matsuo M., et al., Clin. Chem., 55 (10): 1801-1808, 2009. Kenji O., et al., Arterioscler Thromb Vasc Biol., 27 (5): 1050-1056, 2007.

  An object of the present invention is to provide a novel monoclonal antibody that specifically binds to LR11, has an excellent neutralizing activity, and has an excellent prophylactic and therapeutic effect on malignant tumors such as hematopoietic tumors and epithelial malignant tumors, and It is in providing the pharmaceutical composition which contains.

  The present inventors have not reported clearly with existing monoclonal antibodies recognizing LR11, and have a novel activity that acts as a neutralizing antibody that inhibits the cell adhesion enhancing action and cell migration enhancing action that are biological activities of sLR11. We have been investigating the development of anti-LR11 monoclonal antibodies, and by screening antibodies produced using specific antigens by adhesion assays, they are excellent as neutralizing antibodies that inhibit cell adhesion enhancement and cell migration enhancement. It was found that an anti-LR11 monoclonal antibody having an action can be obtained. The antibody thus obtained not only has an excellent adhesion inhibitory action and migration inhibitory action, but also suppresses the cell invasion action of cancer cells, and this antibody prevents and malignant tumors. The inventors have found that the present invention has a therapeutic effect and have completed the present invention.

That is, the present invention is an antibody produced using the Vps10p domain of LR11 as an antigen, and can significantly suppress cell adhesion enhanced by a protein comprising the Vps10p domain of human LR11 in an adhesion assay. The present invention relates to a monoclonal antibody and a method for producing the same.
The present invention also relates to a pharmaceutical composition comprising the anti-LR11 monoclonal antibody of the present invention.

Furthermore, the present invention relates to a method for treating a malignant tumor or a method for preventing a malignant tumor by administering an effective amount of the anti-LR11 monoclonal antibody of the present invention to a patient with a malignant tumor or a patient suspected of having a malignant tumor. .
The present invention also relates to an anti-LR11 monoclonal antibody for use in a pharmaceutical composition for preventing or treating malignant tumors.

The present invention will be described in more detail as follows.
(1) An anti-LR11 monoclonal antibody having, as a complementarity determining region (CDR), at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5 and 6,
(A) can recognize LR11,
(B) has a specific binding ability to a protein consisting of the amino acid sequence of the Vps10p domain of LR11, and
(C) significantly suppresses cell adhesion enhanced by a protein consisting of the amino acid sequence of the Vps10p domain of human LR11 in the adhesion assay,
An anti-LR11 monoclonal antibody characterized by that.
(2) The anti-LR11 monoclonal antibody according to (1), which has the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6 as complementarity determining regions (CDRs).

(3) SEQ ID NOs: 1, 2 and 3 are H chain complementarity determining regions (CDR), and SEQ ID NOs: 4, 5 and 6 are L chain complementarity determining regions (CDR), The anti-LR11 monoclonal antibody according to (2).
(4) The sequence of the variable region of the H chain is an amino acid sequence represented by SEQ ID NO: 7, and the sequence of the variable region of the L chain is an amino acid sequence represented by SEQ ID NO: 8, The anti-LR11 monoclonal antibody according to any one of the above.
(5) The adhesion assay is an assay using a human monocyte lymphoma cell line U937 cell and a container coated with vitronectin, according to any one of (1) to (4) above Anti-LR11 monoclonal antibody.
(6) In the adhesion assay, when the value enhanced by a protein consisting of the amino acid sequence of the Vps10p domain of human LR11 is defined as 100%, the value can be suppressed to 80% or less. The anti-LR11 monoclonal antibody according to any one of 5).

(7) In the adhesion assay, the concentration of the protein comprising the amino acid sequence of the Vps10p domain of human LR11 is 1 μg / mL to 100 μg / mL, and the antibody concentration is 0.1 μg / mL to 100 μg / mL, The anti-LR11 monoclonal antibody according to any one of 1) to (6).
(8) Human LR11 has a specific binding ability to a protein consisting of the amino acid sequence of the Vps10p domain of human LR11, compared to a protein consisting of the protein consisting of the amino acid sequence of human sortilin Vps10p domain at a concentration of antibody of at least 1 μg / mL. The anti-LR11 monoclonal antibody according to any one of (1) to (7) above, which has the ability to bind strongly to a protein comprising the amino acid sequence of the Vps10p domain.
(9) The anti-LR11 monoclonal antibody according to any one of (1) to (8), wherein the antibody further has an inhibitory action on a cell migration enhancing action.
(10) The anti-LR11 monoclonal antibody according to any one of (1) to (9), wherein the antibody further has an ability to suppress cell invasion action of malignant tumor cells.

(11) A preventive and / or therapeutic agent for malignant tumor, comprising the anti-LR11 monoclonal antibody according to any one of (1) to (10).
(12) The preventive and / or therapeutic agent for malignant tumor according to (11), wherein the malignant tumor is a hematopoietic tumor or an epithelial malignant tumor.
(13) The preventive and / or therapeutic agent for malignant tumor according to (12), wherein the hematopoietic tumor is leukemia or malignant lymphoma.
(14) A tumor cell adhesion inhibitory, migration inhibitory, or invasion inhibitor comprising the anti-LR11 monoclonal antibody according to any one of (1) to (10).
(15) The tumor cell adhesion suppression, migration suppression, or infiltration suppression agent according to (14) above, wherein the tumor cell is a hematopoietic tumor or an epithelial malignant tumor.

The anti-LR11 monoclonal antibody of the present invention is produced using a specific antigen and has an excellent neutralizing activity against LR11 by screening by a cell adhesion assay.
And, by using the anti-LR11 monoclonal antibody of the present invention, the migration (homing) and engraftment (establishment) of hematopoietic stem cells to the bone marrow is suppressed, and an excellent anti-malignant tumor action, tumor cell adhesion suppression action, tumor cell The migration inhibitory effect of and / or tumor cell invasion inhibitory effect is shown. The anti-LR11 monoclonal antibody of the present invention is useful for the prevention and / or treatment of malignant tumors, particularly hematopoietic tumors.

FIG. 1 shows the inhibitory action of mouse anti-LR11 monoclonal antibody on the vitronectin adhesion enhancing action of U937 cells by sLR11. FIG. 2 shows the inhibitory effect of mouse anti-LR11 monoclonal antibody on the cell migration enhancement effect of U937 cells by partially purified human sLR11 protein. FIG. 3 shows the inhibitory action of mouse anti-LR11 monoclonal antibody on cell invasion of lymphoma cell lines. FIG. 4 shows the specificity for LR11 by a mouse anti-LR11 monoclonal antibody.

  Unless otherwise stated herein, technical terms used herein are to be interpreted as having the broadest meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Hereinafter, some technical terms will be described, but the present invention is not limited to these descriptions.

  The “monoclonal antibody” of the present invention is generally an antibody having a single binding specificity or affinity for a specific epitope, and is described in Kohler et al. (Kohler, et al, Nature, 1975, 256, 495). Hybridoma method; recombinant DNA method (see, for example, US Pat. No. 4,816,567); Claxson et al. (Clackson, et al., Nature, 1991, 352, 624-628) and Marks et al. (Marks, et al , J. Mol. Biol., 1991, 222, 581-597), and the like. In addition, the type of “monoclonal antibody” of the present invention is not particularly limited, and mouse antibody, human antibody, rat antibody, rabbit antibody, sheep antibody, guinea pig antibody, hamster antibody, avian antibody, etc. In order to avoid inducing a response, any of recombinant antibodies that have been artificially modified for the purpose of reducing the heteroantigenicity of antibodies, such as humanized antibodies, may be used. These antibodies may be either low molecular weight antibodies such as antibody fragments or modified antibodies. Examples of the antibody fragment include Fab, Fab ′, F (ab ′) 2, scFv, dsFv, and a peptide containing a variable region complementarity determining region (CDR).

  The term “human antibody” refers to an antibody that naturally exists in the human body, but a human antibody phage library and a human antibody production trans gene produced by the advancement of genetic engineering, cell engineering, and developmental engineering techniques. Also included are antibodies obtained from transgenic animals. The antibody present in the human body can be cultured by, for example, isolating human peripheral blood lymphocytes, infecting and immortalizing and cloning EB virus and the like, and culturing lymphocytes that produce the antibody. Can be purified. The human antibody phage library is a library in which antibody fragments such as Fab and scFv are expressed on the phage surface by inserting an antibody gene prepared from human B cells into the phage gene. From this library, phages expressing antibody fragments having antigen-binding activity can be recovered using the binding activity to the immobilized antigen as an index. The antibody fragment can be further converted into a human antibody molecule comprising two complete heavy chains and two complete light chains by genetic engineering techniques. A human antibody-producing transgenic animal means an animal in which a human antibody gene is incorporated into cells. Specifically, a human antibody-producing transgenic animal can be produced by introducing a human antibody gene into a mouse ES cell, and transplanting the ES cell into an early embryo of another mouse, followed by generation. A human antibody production method from a human antibody-producing transgenic animal is obtained by culturing a human antibody-producing hybridoma obtained by a hybridoma production method performed in a normal non-human mammal, and purifying the antibody from the culture supernatant. be able to.

  Examples of humanized antibodies include human chimeric antibodies and human CDR-grafted antibodies. The human chimeric antibody means an antibody comprising an antibody heavy chain variable region and antibody light chain variable region of a non-human animal, a human antibody heavy chain constant region, and a human antibody light chain constant region. Any animal other than humans can be used as long as it can produce hybridomas such as mice, rats, guinea pigs, hamsters, rabbits and the like. The non-human animal in the human chimeric antibody of the present invention is preferably a mouse. In this case, the DNA encoding the heavy and light chain variable regions of the mouse antibody is linked to the DNA encoding the heavy and light chain constant regions of the human antibody, and this is incorporated into an expression vector and introduced into the host for production. Can be obtained. The heavy chain constant region of the human chimeric antibody may be any as long as it belongs to human immunoglobulin (hIg), but is preferably of the hIgG class, and any of the subclasses of hIgG1, hIgG2, hIgG3, hIgG4 should be used. Can do. The light chain constant region of the human CDR-grafted antibody may be any as long as it belongs to hIg, and those of κ class or λ class can be used.

  The human CDR-grafted antibody is a non-human animal such as a mouse antibody heavy chain variable region and light chain variable region complementarity determining region (CDR) amino acid sequence. Means an antibody transplanted at an appropriate position. For example, a humanized antibody sequence having a framework sequence similar to the framework sequence other than the CDR of the variable region of a mouse antibody is obtained from, for example, a public database of humanized antibodies, and the selected humanized antibody. It can be created by replacing the framework sequence with the framework sequence of the mouse antibody. Furthermore, it is possible to obtain a humanized antibody with higher affinity by replacing a part of the replaced framework sequence with another amino acid. Specifically, non-human animals, for example, a portion that overlaps the terminal portion of a DNA sequence designed to link the complementarity determining region (CDR) of a mouse antibody and the framework region of the variable region of a human antibody It is synthesized by PCR from several oligonucleotides prepared so as to have The obtained DNA is obtained by ligating with a DNA encoding a human antibody constant region, then incorporating it into an expression vector, introducing it into a host and producing it (EP239400, International Publication WO96 / 02576, etc.) ). The heavy chain constant region of the human CDR-grafted antibody may be any as long as it belongs to human immunoglobulin (hIg), but is preferably of the hIgG class, and further, any of the subclasses of hIgG1, hIgG2, hIgG3, hIgG4 are used. be able to. The light chain constant region of the human CDR-grafted antibody may be any as long as it belongs to hIg, and those of κ class or λ class can be used.

  The amino acid sequence including the complementarity determining region includes at least one region of the heavy chain or light chain complementarity determining region. Multiple complementarity determining regions can be joined directly or via a suitable amino acid sequence linker.

  The monoclonal antibody of the present invention is characterized by having an excellent neutralizing activity. Here, the neutralizing activity is an activity that can reduce or suppress at least one activity of a protein containing an epitope to which the antibody specifically binds, for example, LR11. Such neutralizing activity may be confirmed either in vitro and / or in vivo.

  The monoclonal antibody of the present invention can reduce or suppress at least one activity possessed by LR11 on the cell surface, such as cell adhesion. More specifically, cell adhesion enhanced by LR11 is 90% or less when the value enhanced by LR11 is 100% at an antibody concentration of 10 μg / mL, 5 μg / mL, or 2.5 μg / mL. Preferably, it has a neutralizing activity that can be suppressed to 80% or less, 60% or less, or 50% or less.

The monoclonal antibody of the present invention is an anti-LR11 monoclonal antibody produced using a protein comprising the amino acid sequence of the Vps10p domain of LR11 as an antigen,
(A) can recognize LR11,
(B) has a specific binding ability to a protein consisting of the amino acid sequence of the Vps10p domain of LR11, and
(C) significantly suppresses cell adhesion enhanced by a protein consisting of the amino acid sequence of the Vps10p domain of human LR11 in the adhesion assay,
It is an anti-LR11 monoclonal antibody characterized by this.

The protein comprising the amino acid sequence of the LR11 Vps10p domain is LR11, preferably a protein comprising the Vps10p domain of human LR11. Human LR11 is a protein composed of 2214 amino acids (see Moerwald S., et al., Arterioscler Thromb Vasc Biol., 17 (5): 996-1002, 1997., etc.), and the Vps10p domain is , A part composed of 753 amino acids from the 1st to the 753rd from the N-terminal.
The “protein consisting of the amino acid sequence of the Vps10p domain of LR11” in the present invention may have various tags such as Myc-tag and His-tag for ease of handling and availability.

  In addition, “LR11 can be recognized” in the present invention means that it has binding ability, and it has binding ability when the antibody concentration is 10000 ng / mL or less, preferably 1000 ng / mL or less.

  As preferred monoclonal antibodies of the present invention, the amino acid sequences of three heavy chain CDRs are the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, and the light chain CDR amino acid sequences are shown in SEQ ID NOs: 4, 5, and 6, respectively. Antibodies containing the described amino acid sequences are mentioned. This antibody is hereinafter referred to as 265-5 antibody. The 265-5 antibody is an antibody produced using the Vps10p domain of human LR11 as an antigen, and is an antibody screened by an adhesion assay.

The adhesion assay is not particularly limited as long as it can assay cell adhesion enhanced by a protein consisting of the amino acid sequence of the Vps10p domain of human LR11. Tumor cells are preferred as the cells used in the assay. For example, as described in Example 2 described later, an assay using a human monocyte lymphoma cell line U937 cell and a vessel coated with vitronectin is used. Is mentioned.
The monoclonal antibody of the present invention may be any antibody that can be suppressed when the value enhanced by a protein consisting of the amino acid sequence of the human LR11 Vps10p domain is 100% in such an adhesion assay, Preferred are those that are suppressed to 80% or less, 85% or less, 70% or less, 75% or less, 60% or less, 65% or less, or 50% or less. In the adhesion assay, the concentration of the protein comprising the amino acid sequence of the Vps10p domain of human LR11 is not particularly limited, but preferably 0.1 μg / mL to 500 μg / mL, 0.1 μg / mL to 100 μg / mL, 1 μg / mL. -500 μg / mL, 1 μg / mL-100 μg / mL, or 1 μg / mL-50 μg / mL. The concentration of the antibody used at that time can be appropriately adjusted according to the concentration of the protein described above, and preferably 0.01 μg / mL to 100 μg / mL, 0.01 μg / mL to 50 μg / mL, 0.01 μg / ML to 20 μg / mL, 0.1 μg / mL to 100 μg / mL, 0.1 μg / mL to 50 μg / mL, or 0.1 μg / mL to 20 μg / mL.

Preferred antibodies in the present invention include the above-mentioned 265-5 antibody. The 265-5 antibody has the amino acid sequence set forth in SEQ ID NOs: 1 to 6 as CDRs. The 265-5 antibody has the amino acid sequence of H chain described in SEQ ID NO: 7 and the amino acid sequence of L chain described in SEQ ID NO: 8 as variable regions.
Another preferable antibody in the present invention is a monoclonal antibody containing at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6. The sequence represented by SEQ ID NO: 1, 2 or 3 is the complementarity-determining region (CDR) amino acid sequence of the heavy chain hypervariable region, and the sequence represented by SEQ ID NO: 4, 5 or 6 is a sequence exceeding the light chain It is the complementarity determining region (CDR) amino acid sequence of the variable region. Preferred examples include an antibody comprising a heavy chain CDR amino acid sequence represented by SEQ ID NO: 1, 2 or 3 and a light chain CDR amino acid sequence represented by SEQ ID NO: 4, 5 or 6. More preferably, an antibody comprising a heavy chain CDR amino acid sequence represented by SEQ ID NOs: 1, 2, and 3 and a light chain CDR amino acid sequence represented by SEQ ID NOs: 4, 5, and 6 is exemplified.

  The monoclonal antibody of the present invention has excellent neutralizing activity, and is particularly useful as a therapeutic and / or prophylactic agent for various diseases caused by LR11 expression, preferably overexpression. Or it can be used as a preventive agent.

  Examples of the malignant tumor in the present invention include hematopoietic tumors and epithelial malignant tumors. Examples of hematopoietic tumors include leukemia and malignant lymphoma. Examples of leukemia include acute myeloid leukemia, acute promyelocytic leukemia, adult acute lymphoblastic leukemia, and chronic myelogenous leukemia. Malignant lymphoma includes non-Hodgkin lymphoma. On the other hand, epithelial malignant tumors include stomach cancer, liver cancer, pancreatic cancer, lung cancer, prostate cancer, bladder cancer, esophageal cancer, breast cancer, cervical cancer, ovarian cancer, colon cancer, colon cancer, kidney cancer, gallbladder cancer, nerve Tumor (glioma), malignant melanoma (melanoma), etc. are mentioned.

  As a specific treatment target according to the present invention, among hematopoietic tumors, acute leukemia is more preferable among leukemias, and non-Hodgkin lymphoma is more preferable among malignant lymphomas. In the epithelial malignant tumor, liver cancer, pancreatic cancer, colon cancer, colon cancer, and gallbladder cancer are preferable.

  While it is possible for the antibody of the present invention to be administered alone, the active ingredient itself is usually mixed with one or more pharmacologically acceptable carriers and administered as a pharmaceutical composition. Can do. The pharmaceutical composition of the present invention can be a pharmaceutical composition suitable for oral or parenteral administration, preferably a pharmaceutical composition suitable for parenteral administration. Pharmaceutical compositions suitable for parenteral administration include, for example, injections such as intravenous injections and intramuscular injections, drops, suppositories, inhalants, eye drops, nasal drops, transdermal absorption agents, transmucosal Although an absorbent etc. can be mentioned, it is not limited to these.

  The above pharmaceutical composition can be produced by adding pharmacologically and pharmaceutically acceptable additives. Examples of pharmacologically and pharmaceutically acceptable additives include, for example, excipients, binders, extenders, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, Examples include, but are not limited to, flavoring agents, fragrances, coating agents, and diluents.

  The dosage of the pharmaceutical agent of the present invention is not particularly limited, and can be appropriately selected according to the type of disease, the purpose of prevention or treatment, the type of active ingredient, etc., and the patient's weight and age, symptoms, administration route, etc. It can be increased or decreased as appropriate according to various factors that should be normally considered. For example, in the case of intravenous administration, the weight of the active ingredient per day for an adult can be used in the range of about 0.1 mg to 10 g, but the dosage can be appropriately selected by those skilled in the art and is limited to the above range. It will never be done. Preferably, the weight of the active ingredient per adult can be intravenously administered at a dose of about 0.1 mg to 1000 mg, more preferably about 0.5 mg to 500 mg. Moreover, administration can be performed once a day or divided into several times.

For the production, isolation and purification of anti-LR11 monoclonal antibodies, known monoclonal antibody production methods, for example, Takao Hamakubo (2012) “New Functional Antibody Development Handbook (NTS)” or Shinobu Oumi (1994) It can be prepared, isolated and purified according to "New Edition Anti-Peptide Antibody Experiment Protocol (Shyujunsha)".
The anti-LR11 monoclonal antibody is obtained by culturing the prepared hybridoma according to a conventional method and separating it from the culture supernatant, or by administering the hybridoma to a mammal compatible with the hybridoma and producing the antibody in ascites. Can be manufactured. The antibody can be used after further purification as necessary. As a method for purifying and isolating antibodies, conventionally known methods, for example, salting out such as ammonium sulfate precipitation, gel filtration, ion exchange, columns such as protein A or G, and columns on which immune antigens are immobilized There is an affinity purification method.

(Monoclonal antibody production)
Specifically, a monoclonal antibody against LR11 can be prepared, for example, as follows. As an immunizing antigen used for the production of an antibody, LR11 protein or a partial fragment (peptide) thereof can be used, and the Vps10p domain portion on the N-terminal side of LR11 is preferable. You may obtain by refine | purifying from the sample derived from a biological tissue and a cell, and you may use a recombinant protein and a synthetic peptide. Examples of animals to be immunized include rabbits, mice, and rats, but mice can be preferably used for the production of monoclonal antibodies. The immunization method is performed according to a known method. For example, in order to increase the ability to produce an antibody by itself, or a carrier, diluent, or antibody at a site where antibody production is possible by administration methods such as intraperitoneal injection, intravenous injection, and subcutaneous injection, complete Freund's adjuvant or You may mix and emulsify with fluids such as incomplete Freund's adjuvant. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total.

When preparing monoclonal antibodies, select animals that have been immunized with LR11 antigen, such as mice that have been confirmed to produce monoclonal antibodies against LR11, collect spleen or lymph nodes, and use them to produce antibody-producing cells in bone marrow. An anti-LR11 monoclonal antibody-producing hybridoma can be prepared by fusing with a tumor cell (myeloma). The cell fusion operation is performed according to a known method. Polyethylene glycol (PEG) may be used as a fusion accelerator or may be fused by electric pulse, but PEG is preferably used. Examples of myeloma cells include NS-1, P3U1, SP2 / 0, AP-1, etc., and P3U1 is preferably used.
Various methods can be used for screening anti-LR11 antibody-producing hybridomas. For example, the hybridoma culture supernatant is added to a solid phase (eg, microplate) adsorbed with LR11 or a partial peptide thereof, and then an enzyme, etc. And a method of detecting an anti-LR11 monoclonal antibody bound to a solid phase by adding an anti-immunoglobulin antibody labeled with (in which the mouse used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used). Screening and culture of anti-LR11 monoclonal antibody-producing hybridomas are usually carried out in a cell culture medium (eg, RPMI1640) containing 10-20% fetal calf serum with addition of HAT (hypoxanthine, aminopterin, thymidine).
Separation and purification of anti-LR11 monoclonal antibody is the same as separation and purification of ordinary polyclonal antibodies, such as immunoglobulin separation and purification methods (eg, salting out method, adsorption / desorption method using ion exchanger (eg, DEAE), gel filtration method, antigen A binding solid phase or an affinity purification method such as protein A or protein G). As described above, the antibody of the present invention can be produced by purifying the antibody from the culture supernatant obtained by culturing the hybridoma cells or the body fluid after growing in the body of an animal compatible with the hybridoma.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, the scope of the present invention is not limited by these Examples.
In the specification of the present invention, amino acids and the like are represented by abbreviations based on abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field, and examples thereof are described below.
PBS: Phosphate buffered saline
FCS: Fetal calf serum

Production of monoclonal antibody 265-5 (1) Construction of human soluble LR11 (hsLR11) expression vector
The hsLR11 expression vector was constructed by inserting the cloned hsLR11 into the multicloning site of pcDNA3.1A-MycHis (V800-20, Life Technologies) using restriction enzymes EcoRI and PmeI by a conventional method. This hsLR11 expression vector was designated as pcDNA3.1-hsLR11-MycHis A.
(2) Construction of human Vps10p (hVps10p) expression vector
The hVps10p expression vector was constructed using the expression plasmid pcDNA3.1-hsLR11-MycHis A prepared in (1) above as a template, and using the primers shown in SEQ ID NOs: 9 and 10, within the nucleotide sequence of the human LR11 Vps10p domain. From the NheI recognition site to the C-terminal part of Vps10p was amplified by PCR.

Forward primer: 5'- CCGTG GCTAGC AAGACAAACGTG-3 '(SEQ ID NO: 9)
Reverse primer: 5'- CGA TCTAGA GGGACAGGGGACCAGCTCT-3 '(SEQ ID NO: 10)
Forward primer underline is NheI recognition site, Reverse primer underline is XbaI recognition site

The obtained amplification product was further purified by treatment with restriction enzymes NheI and XbaI. The amplification product thus obtained is a fragment encoding the amino acid sequence from the restriction enzyme Nhel site in the Vps10p domain to the restriction enzyme Xbal site at the C-terminal of the Vps10p domain.
On the other hand, pcDNA3.1-hsLR11-MycHis A, which is an hsLR11 expression vector, has a restriction enzyme Xbal site on the 3 ′ end side of the extracellular portion of LR11 and is treated with restriction enzyme Nhel and restriction enzyme Xbal. The C-terminal portion can be excised from the restriction enzyme Nhel site in the Vps10p domain of human soluble LR11 (hsLR11).
The hsLR11 expression vector pcDNA3.1-hsLR11-MycHis A was treated with restriction enzymes NheI and XbaI, and then the amplified product obtained above was conjugated to pcDNA3.1-hsLR11-MycHis A treated with Alkaline phosphatase. Since the obtained expression vector has a Myc-tag and a His-tag, it is an expression vector of hVps10p with a Myc-tag and a His-tag added to the C-terminus, and pcDNA3.1 / myc-His ( +)-hsVps10p.

(3) Preparation of human Vps10p protein The human LR11 Vps10p protein was prepared by transfecting the expression vector pcDNA3.1 / myc-His (+)-hsVps10p obtained in (2) above into FreeStyle 293-F cells as follows. Was expressed.
Add 37.5 μg of vector DNA to 600 μL of serum-free medium OptiPro SFM (Life Technologies), and gently mix with 37.5 μL of the transfection reagent FreeStyle MAX Reagent (Life Technologies) and 562.5 μL of OptiPro SFM (total 600 μL). After 10 minutes incubation at room temperature, it was added to FreeStyle 293-F cells (1 × 10 ⁶ cells / mL, 30 mL) in a 125 mL Erlenmeyer flask and swirled for 4 days (8% CO ₂ , 125 rpm, 37 ° C.) . The collected culture supernatant was concentrated approximately 10 times by ultrafiltration (Amicon Ultra, molecular weight cut off 30,000, Merck Millipore), diluted with equilibration buffer for cobalt column (Clontech), and equilibrated with equilibration buffer. Added to a cobalt column (1 mL, Clontech). The column was washed with 10% Elution buffer (Clontech) and eluted with 60% Elution buffer. The eluted fraction was further concentrated by ultrafiltration, and the buffer was exchanged with PBS using a PD-10 column (GE Healthcare) to obtain purified hVps10p protein.

(4) Preparation of hybridoma Using the protein (hVps10p) having a partial amino acid sequence [1-753] of human LR11 prepared in (3) above as an immunizing antigen, 0.1 mg of this immunizing antigen was completely Freund's adjuvant. The mixture was emulsified and administered twice subcutaneously in 6-week-old female BALB / C mice at 2-week intervals, and the spleen and lymph nodes were removed 3 days after the second immunization. Cells obtained from the removed spleen and lymph nodes were mixed with myeloma cells P3U1, and the cells were fused in the presence of polyethylene glycol. The fused cells were suspended in HAT medium and dispensed into 96-well culture plates by the limiting dilution method. This was cultured at 37 ° C. in a 5% CO ₂ incubator, and the culture supernatant of the well in which the hybridoma had grown was cultured according to the following ELISA (Enzyme-Linked Immuno Sorbent Assay) method. hVps10p) was screened, positive wells were grown to 24-well culture plates, culture supernatants were screened again by ELISA and cell adhesion assay, positive wells were cloned by limiting dilution, and hybridoma strains (265- 5) was established.

(5) Preparation of monoclonal antibody
The hybridoma obtained above was intraperitoneally administered in an amount of 0.5 × 10 ⁶ cells to 12-week-old female BALB / C mice that had been injected intraperitoneally with 0.5 mL of pristane two weeks ago. About 14 days later, ascites was collected and centrifuged to obtain a supernatant. The supernatant was mixed with an equal volume of PBS, and filtered through a 0.45 μm filter. The filtrate was passed through an Ab-Capcher ExTra column (manufactured by Protenova) equilibrated with PBS, and the antibody was adsorbed to the column. After thoroughly washing the column with PBS, 0.1 M citrate buffer (pH 3.0) The column was eluted from the column, and the anti-LR11 monoclonal antibody (265-5 antibody) was purified.

(6) Analysis of amino acid sequence The amino acid sequences of the variable region and the hypervariable region of the obtained anti-LR11 monoclonal antibody were analyzed according to a conventional method. The results are shown in SEQ ID NOs: 1-8.

Measurement of neutralizing activity on the biological activity of soluble LR11 (1) Inhibitory action on the enhancement of cell adhesion of soluble LR11 Step 1: Preparation of soluble LR11 (sLR11) Full-length cDNA of human LR11 was introduced into cell line COS7 derived from African green monkey kidney The encoded expression plasmid (RC214154, Origene) was transiently transfected by the lipofection method using Effectene (Qiagen) and then incubated at 37 ° C. After 16 hours, the serum-free medium was replaced, and further cultured at 37 ° C. for 24 hours to express LR11 on the surface of COS7 cells. Since the extracellular domain of LR11 is released into the culture supernatant as sLR11, the cell culture supernatant is collected and concentrated approximately 100 times by ultrafiltration (Amicon Ultra, molecular weight cut off 100,000, Merck Millipore) And obtained sLR11.

Step 2: Inhibitory action of soluble LR11 on cell adhesion enhancing action A solution of an extracellular matrix human vitronectin (Becton Dickinson and Company, hereinafter referred to as “BD”) prepared in PBS to a concentration of 25 μg / mL is 96. Dispense 50 μL / well into a well black plate (BD), leave it at 37 ° C for 1 hour to coat, wash with PBS, and dispense 1% bovine serum albumin (BSA) at 50 μL / well. Removed and air dried. Human monocytic cell line U937 cells are cultured overnight in serum-free RPMI1640 medium, then suspended in 0.1% BSA-Hanks balanced salt solution (HBSS) containing 1 μM Calcein-AM, and incubated at 37 ° C for 30 minutes Then, Calcein-AM was incorporated into the cells and the cells were fluorescently labeled. Then, suspend the cells in serum-free medium to 3 × 10 ⁶ cells / mL, and add normal mouse IgG antibody or mouse anti-LR11 monoclonal antibody (265-5 antibody) to a final concentration of 2.5 μg / mL Immediately after that, 1/100 volume of sLR11 (COS7 culture supernatant) was added and reacted at 37 ° C. for 3 hours. For comparison, a well to which each antibody and sLR11 were not added and a well to which only sLR11 was added were also set. Next, dispense U937 cell suspension containing sLR11 and antibody on a plate coated with human vitronectin (3 × 10 ⁵ cells / 100 μL / well), spin at low speed (1800 rpm, 2 minutes), then at 37 ° C. Let stand for 20 minutes. After washing the wells three times with PBS, the fluorescence intensity (RFU) of the cells bound to the plate was measured with a fluorescence plate reader (PerkinElmer). All experiments were performed at N = 3. The result is shown in FIG.

Inhibitory action of soluble LR11 on cell migration enhancing step 1: Partially purified human sLR11 protein (partial protein with Myc-tag and His-tag added to C-terminal of amino acid sequence 1-2110, hereinafter referred to as “hsLR11”) Preparation The hsLR11 expression vector pcDNA3.1-hsLR11-MycHis A obtained in (1) of Example 1 was transfected into FreeStyle 293-F cells (Life Technologies) to express the hsLR11 protein. As a transfection reagent, Targefect-293FS reagent (Targetingsystems) was used, and after the transfection, swirling culture (8% CO ₂ , 125 rpm, 37 ° C.) was performed for 3 days. The sLR11-expressing cells collected by centrifugation were suspended in Lysis buffer (20 mM Tris-HCl, pH 7.5-1% NP-40-150 mM NaCl), solubilized, and then anti-His-tag antibody column equilibrated with PBS ( And then washed with PBS, eluted under acidic conditions (pH 3.0), and immediately neutralized with 0.1 M Tris-HCl, pH 7.5. The eluted fraction is then added to an anti-Myc-tag antibody column (Medical and Biological Laboratories), washed with PBS, eluted under acidic conditions (pH 3.0), and immediately neutralized, partially purified hsLR11 protein Got.

Step 2: Inhibitory effect of soluble LR11 on cell migration enhancing effect
Experiments on cell migration of U937 cells were performed using a 24-well transwell (Corning) with a membrane with an 8 μm pore hole at the bottom of the upper chamber. U937 cells are cultured overnight in serum-free medium, suspended in 0.1% BSA-Hanks balanced salt solution (HBSS) containing 1 μM Calcein-AM, and incubated at 37 ° C for 30 minutes to allow Calcein-AM to enter the cells. Upon incorporation, U937 cells were fluorescently labeled. Suspend U937 cells fluorescently labeled to 1 × 10 ⁶ cells / mL in serum-free medium, and mix normal mouse IgG antibody or mouse anti-LR11 monoclonal antibody (265-5 antibody) with partially purified hsLR11 (antibody end) Concentration 130 ng / mL, hsLR11 final concentration 500 ng / mL). For comparison, a well to which each antibody and sLR11 were not added and a well to which only sLR11 was added were also set. Next, RPMI1640 medium containing 10% fetal calf serum (FCS) is dispensed into the lower chamber of the transwell (500 μL / well), and the U937 cell suspension containing the above partially purified hsLR11 and antibody is added to the upper chamber. Aliquots (1.5 × 10 ⁶ cells / 150 μL / well) were allowed to react at 37 ° C. for 4 hours. The number of cells that migrated (moved) from the upper chamber to the lower chamber was indicated by the fluorescence intensity of the cells as seen in FIG. After removing the upper chamber, the medium in the lower chamber is collected in a 1.5 mL tube. After centrifugation (1500 rpm, 5 min), the precipitated cells are suspended in 100 μL of PBS and dispensed into a 96-well black plate (BD). The fluorescence intensity of the cells was measured with a fluorescence plate reader (PerkinElmer). All experiments were performed at N = 3.

<Inhibitory effect of mouse anti-LR11 monoclonal antibody on cell invasion of lymphoma cell line>
Experiments on cell infiltration of human lymphoma cell lines were performed using collagen coated on the membrane at the bottom of the upper chamber of the transwell. Collagen type 1C (Cellmatrix, 1C-20) prepared with PBS to a concentration of 300 μg / mL was dispensed into the upper chamber of a 24-well transwell (BD) with a membrane with a pore of 8 μm pore size. Poured (200 μL / well), incubated for 30 minutes at room temperature, washed with PBS to prepare a collagen-coated transwell. Human lymphoma cell line Raji cells or Daudi cells are cultured overnight in serum-free medium, suspended in 0.1% BSA-Hanks balanced salt solution (HBSS) containing 1 μM Calcein-AM, and incubated at 37 ° C for 30 minutes. Then, Calcein-AM was incorporated into the cells, and Raji cells or Daudi cells were fluorescently labeled. Suspended Raji cells or Daudi cells fluorescently labeled to 1 × 10 ⁶ cells / mL in serum-free Dulbecco's modified Eagle medium and mixed with normal mouse IgG antibody or mouse anti-LR11 monoclonal antibody (antibody final concentration 10 μg / mL). Next, the cell suspension containing the antibody is dispensed into the upper chamber of the transwell (1.5 × 10 ⁵ cells / 150 μL / well), and the medium containing 10% FCS is dispensed into the lower chamber. The cell suspension was dispensed into the upper chamber at 1.5 × 10 ⁵ cells / 150 μL / well, and reacted at 37 ° C. for 24 hours. The number of cells infiltrating (moving) from the collagen-coated membrane at the bottom of the upper chamber into the lower chamber was indicated by the fluorescence intensity of the cells as seen in FIG. After removing the upper chamber, the medium in the lower chamber is collected in a 1.5 mL tube, centrifuged (1500 rpm, 5 min), and the precipitated cells are suspended in 100 μL of PBS and dispensed into a 96-well black plate. Was used to measure the fluorescence intensity of the cells. All experiments were performed at N = 3. The result is shown in FIG.

<Examination of LR11 specificity of mouse anti-LR11 monoclonal antibody>
Step 1: Preparation of human LR11 Vps10p protein As described in (3) of Example 1 using the expression vector pcDNA3.1 / myc-His (+)-hsVps10p of human LR11 Vps10p prepared in (2) of Example 1. In this way, purified hVps10p protein was obtained.
Step 2: ELISA of LR11 and Sortilin with Vps10p protein
Since LR11 belongs to the Vps10p domain protein family, the specificity of the mouse anti-LR11 monoclonal antibody for human LR11 was confirmed by ELISA reactivity to human Sortinin Vps10p protein and human LR11 Vps10p protein belonging to the same Vps10p domain protein family. ELISA was performed as follows. 50 μL of human Sortilin Vps10p protein (Se78-Asn755, R & D, 3154ST) or purified hVps10p protein prepared at 2 μg / mL in 96-well ELISA plate (Nunc) with 0.05 M carbonate-bicarbonate buffer (pH 9.6) After aliquoting and coating each well (25 ° C, 2 hours), the plate was washed 3 times with PBS containing 0.05% Tween20 (PBS-T), and then Block Ace diluted to 25% (DS Pharma Biomedical) Were added to the wells for blocking (25 ° C., 1 hour). After washing once with PBS-T, a diluted series solution of mouse anti-LR11 monoclonal antibody prepared with PBS-T (diluted with a common ratio of 4 from 4000 ng / mL) was dispensed (50 μL / well) and reacted for 2 hours. (25 ° C). After washing 3 times with PBS-T, peroxidase-labeled donkey anti-mouse IgG antibody (Merck Millipore) prepared to 0.3 μg / mL with PBS-T was added to the well (50 μL / well) and allowed to react for 1 hour (25 ° C). Next, the peroxidase substrate TMB (3,3 ', 5,5'-tetramethylbenzidine) was added (50 μL / well) to perform a color reaction, and after stopping the reaction with 1 M sulfuric acid (10 μL / well), the absorbance at 450 nm was measured. It was measured. As a result, the mouse anti-LR11 monoclonal antibody did not react at all with the human Sortilin Vps10p protein of the Vps10p domain protein family. On the other hand, reactivity was observed for the hVps10p protein, confirming the specificity of the mouse anti-LR11 monoclonal antibody for human LR11. The result is shown in FIG.

  The present invention provides a novel anti-LR11 monoclonal antibody having excellent neutralizing activity, and the anti-LR11 monoclonal antibody of the present invention is used for the treatment of diseases involving LR11 or a fragment protein thereof, for example, malignant tumor treatment and / or Or it is useful as a medicine for prevention, provides a useful medicine, and has industrial applicability.

SEQ ID NO: 1: CDR1 of H chain of antibody 265-5
SEQ ID NO: 2: CDR2 of H chain of antibody 265-5
SEQ ID NO: 3: CDR3 of H chain of antibody 265-5
SEQ ID NO: 4: CDR1 of L chain of antibody 265-5
SEQ ID NO: 5: CDR2 of L chain of antibody 265-5
SEQ ID NO: 6: CDR3 of L chain of antibody 265-5
SEQ ID NO: 7: Full length of variable region of H chain of antibody 265-5 SEQ ID NO: 8: Full length of variable region of L chain of antibody 265-5 SEQ ID NO: 9: Vsp10p forward primer SEQ ID NO: 10: Vsp10p reverse primer sequence

Claims (15)

An anti-LR11 monoclonal antibody having, as a complementarity determining region (CDR), at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5 and 6;
(A) can recognize LR11,
(B) has a specific binding ability to a protein consisting of the amino acid sequence of the Vps10p domain of LR11, and
(C) significantly suppresses cell adhesion enhanced by a protein consisting of the amino acid sequence of the Vps10p domain of human LR11 in the adhesion assay,
An anti-LR11 monoclonal antibody characterized by that.   The anti-LR11 monoclonal antibody of Claim 1 which has an amino acid sequence of sequence number 1, 2, 3, 4, 5 and 6 as a complementarity determining region (CDR).   The SEQ ID NOs: 1, 2, and 3 are H chain complementarity determining regions (CDR), and the SEQ ID NOs: 4, 5, and 6 are L chain complementarity determining regions (CDR). Anti-LR11 monoclonal antibody.   The sequence of the variable region of the heavy chain is the amino acid sequence shown by SEQ ID NO: 7, and the sequence of the variable region of the light chain is the amino acid sequence shown by SEQ ID NO: 8, Anti-LR11 monoclonal antibody.   The anti-LR11 monoclonal antibody according to any one of claims 1 to 4, wherein the adhesion assay is an assay using a human monocyte lymphoma cell line U937 cell and a container coated with vitronectin.   In the adhesion assay, when the value enhanced by a protein comprising the amino acid sequence of the Vps10p domain of human LR11 is defined as 100%, the value can be suppressed to 80% or less. The anti-LR11 monoclonal antibody according to Item.   In the adhesion assay, the concentration of the protein consisting of the amino acid sequence of the Vps10p domain of human LR11 is 1 μg / mL to 100 μg / mL, and the concentration of the antibody is 0.1 μg / mL to 100 μg / mL. The anti-LR11 monoclonal antibody according to any one of the above.   The ability of the human LR11 Vps10p domain to bind specifically to a protein consisting of the amino acid sequence of the human LR11 Vps10p domain is higher than that of the protein consisting of the amino acid sequence of the human sortilin Vps10p domain at a concentration of antibody of at least 1 μg / mL. The anti-LR11 monoclonal antibody according to any one of claims 1 to 7, which has an ability to bind strongly to a protein comprising an amino acid sequence.   Furthermore, the anti-LR11 monoclonal antibody according to any one of claims 1 to 8, wherein the antibody has an inhibitory action on a cell migration enhancing action.   Furthermore, the anti-LR11 monoclonal antibody according to any one of claims 1 to 9, wherein the antibody has an ability to suppress a cell invasion effect of malignant tumor cells.   A preventive and / or therapeutic agent for malignant tumor, comprising the anti-LR11 monoclonal antibody according to any one of claims 1 to 10.   The preventive and / or therapeutic agent for a malignant tumor according to claim 11, wherein the malignant tumor is a hematopoietic tumor or an epithelial malignant tumor.   The preventive and / or therapeutic agent for malignant tumor according to claim 12, wherein the hematopoietic tumor is leukemia or malignant lymphoma.   A tumor cell adhesion suppression, migration suppression, or infiltration suppression agent comprising the anti-LR11 monoclonal antibody according to any one of claims 1 to 10.   The tumor cell adhesion suppressant, migration inhibitory, or infiltration suppressor according to claim 14, wherein the tumor cell is a hematopoietic tumor or an epithelial malignant tumor.