Anti-Plexin A1 agonist antibody

Abstract

To provide a method for producing anti-plexin A1 agonist antibodies.SOLUTION: Provided is a method for producing an anti-plexin A1 agonist antibody using a peptide consisting of a specific residue of a specific sequence as an antigen, the method comprising: producing a recombinant antibody by cloning antibody-encoding DNA from a lymphocyte sensitized with the antigen; and obtaining an antibody that binds to the antigen from an antibody library, the anti-plexin A1 agonist antibody having type 3 semaphorin-like activity, and the type 3 semaphorin-like activity being an activity that promotes the regression of a dendritic cell or the regression of a glioma cell.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] The present invention relates to an anti-Plexin A1 agonist antibody and uses thereof. [Background technology]

[0002] Semaphorins were discovered in the early 1990s as repulsive molecules for neuronal growth cones (non-specific References 1 and 2). To date, more than 20 members of semaphorins have been identified. The family shares and conserves a region of about 500 amino acids called the Sema domain. The difference in the structure of the C-terminal side following the Sema domain allows for the division into eight subclusters. They are classified into semaphores (Sema 1-7, V).

[0003] The receptors that mediate semaphorin activity are mainly plexins (plexins A1 / A2 / A3 / A4, ...3 / A4, plexins A4 / A4, plexins A4 / A4, plexins A5 / A5, plexins A6 / A6, plexins A7 / A7, plexins A8 / A8, plex Rexin B1 / B2 / B3, Plexin C1, Plexin D1), neuropilin (Nrp-1, Nrp-2) Semaphorins also bind to integrins, CD72, and Tim-2. It is known that plexins, which are semaphorin receptors, are involved in VEGFR-2, c -Met, Trem2 / DAP12, and other co-receptors. Perhaps reflecting the diverse functions of the receptors, they form complex ligand-receptor relationships. The biological activities of folin include blood vessel and angiogenesis, cancer metastasis and invasion, bone metabolism regulation, and retinal homeostasis. and immunoregulation, and is effective in a wide range of diseases, including allergic diseases, autoimmune diseases, bone metabolic diseases, and neurodegenerative diseases. The role of semaphorins in various diseases such as retinitis pigmentosa, sudden cardiac death, and cancer metastasis and invasion has been investigated. In recent years, a number of reports have been published regarding the involvement of semaphorins in the pathogenesis of encephalopathy (Non-patent Document 3). Therefore, research aimed at developing diagnostic and therapeutic methods for human diseases is currently being conducted vigorously.

[0004] Plexin A1 is a receptor for class III and class VI semaphorins. During chicken cardiac development, 1 not only forms receptors with VEGF receptors and Off-Track receptors, but also , which acts as a receptor for class III semaphorin together with Nrp-1 and as a receptor for neurorepulsive factor Furthermore, Plexin A1 is reported to bind to Semaphorin, a class VI semaphorin. It has also been reported that it acts as a receptor for 6C and Sema6D and is involved in axon guidance and cardiac organogenesis. are.

[0005] By suppressing the expression of Plexin A1 in mouse dendritic cells using shRNA, It has been reported that T cell immunity against non-self is attenuated in vivo or in vitro. Furthermore, analysis of Plexin A1 signaling in dendritic cells and osteoclasts has revealed that In these cells, Plexin-A1 forms a heteroreceptor with Trem-2 and DAP-12. Furthermore, it was confirmed that stimulation with recombinant soluble Sema6D protein induced IL production from dendritic cells. The expression of inflammatory cytokines such as -12 and the differentiation of osteoclasts from precursor cells are promoted. While ema6D binds to wild-type dendritic cells, it hardly binds to dendritic cells from Plexin-A1-deficient mice. In Plexin A1-deficient mice, the T cell immune response was significantly reduced. This weakens the bone mineral density and causes abnormalities in osteoclast differentiation, leading to spontaneous onset of osteopetrosis-like symptoms. It has been reported (Non-Patent Document 5). Plexin A1 mediates the immune synapse between dendritic cells and T cells through activation of the signaling factor Rho. It has been shown that it regulates the localization of the actin cytoskeleton to the nucleus (Non-Patent Document 6).

[0006] Furthermore, Plexin A1 mediates dendritic cell migration to lymph nodes and antigen-specific T cell responses. Furthermore, expression of Sema3A, but not Sema6C or Sema6D, has been reported. is required for dendritic cell migration through lymphatic endothelial cells, and Sema3A mediates this process by inhibiting myoglobin transport. It has been reported that it stimulates actomyosin-II activity and induces actomyosin contraction (Patent Document 1).

[0007] Recently, another molecule in the Plexin A family, Plexin A2, was reported to be a "S A low-resolution (7.0 A) structure of the ternary complex of Sema3A-Plexin A2-Nrp-1 has been disclosed. Although the binding between A and plexin A2 is weak and undetectable, the presence of Nrp-1 inhibits the biosynthesis of Sema3A. Although the interaction between Sema3A and Plexin A2 was weak and far from expressing biological activity, However, this structure was not clearly understood at very low resolution (Non-Patent Document 7). This complex is difficult to elucidate because it uses partial length proteins rather than full-length proteins. Further detailed investigation is required. On the other hand, Non-Patent Document 8 cites Non-Patent Document 7 and states: The structure of the ternary complex of "Sema3A-Plexin A1-Nrp-1" is presented. Reference 7 does not disclose the structure of the ternary complex of "Sema3A-Plexin A1-Nrp-1".

[0008] Sema3A is involved in pruritus in psoriasis and atopic dermatitis, allergic rhinitis, osteoporosis, and rheumatoid arthritis. Autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematosus, inflammatory diseases, and tumors. The therapeutic effects of these genes on various diseases have been demonstrated mainly through expression analysis in patients and animal models. This is suggested by experiments using

[0009] For example, it has been reported that Sema3A expression is reduced in the skin of patients with psoriasis and atopic dermatitis. It has been reported that Sema3A inhibits neurite outgrowth of C-fibers (Non-patent Documents 9 and 10). However, in the skin of patients with psoriasis or atopic dermatitis, the expression of Sema3A is reduced, resulting in As a result, the C-fiber neurites are elongated, making the skin more susceptible to itching. In fact, Sema3A was administered intradermally or intradermally to the skin of atopic dermatitis model mice. It has been reported that applying it as an ointment improves scratching behavior caused by atopic dermatitis. (Non-patent documents 11 and 12).

[0010] Sema3A has also been reported to be involved in airway hyperresponsiveness reactions such as allergic rhinitis (non- Patent Document 13) Sema3A expression is reduced in nasal epithelial cells of allergic rhinitis model mice. As a result, the nerve distribution density of the nasal turbinate lamina propria is high, and As a result, it is one of the factors that exacerbate hypersensitivity reactions such as sneezing and itching. When Sema3A was administered intranasally to mice with allergic rhinitis, the nasal It has been reported that this treatment reduces the nerve distribution density of the lamina propria of the intestinal lining and improves sneezing and scratching behavior. are.

[0011] Furthermore, Sema3A is known to be involved in the regulation of bone mineral density. It activates osteoblasts in vitro and inhibits osteoclast differentiation. Mice lacking Sema3A in the body exhibit osteoporosis-like symptoms. It has been reported that administering Sema3A to mouse models with induced osteoporosis-like symptoms improves bone density. Such direct action of Sema3A on osteoblasts and osteoclasts has been reported (Non-Patent Document 14). In addition, it has been reported that Sema3A regulates bone density through the distribution of sensory neurons to bones. Neuronal-specific Sema3A-deficient mice exhibit osteoporosis similar to that observed in mice lacking Sema3A throughout their body. Similar bone mineral density loss has been reported (Non-Patent Document 15).

[0012] Sema3A is also involved in autoimmune and inflammatory diseases such as rheumatoid arthritis and systemic lupus erythematosus. For example, compared with normal human peripheral blood, In the peripheral blood of patients, peripheral blood mononuclear cells (PBMCs), CD4+ T cells, and CD8+ T cells were isolated in vitro. Sema3A mRNA and protein were produced upon activation with anti-CD3 and anti-CD28 antibodies. Similarly, it has been reported that the amount of arthritis lymphocytes is reduced compared to normal human synovial tissue. It has been reported that Sema3A mRNA expression is decreased in synovial tissue from uterine arthritis patients. In addition, we administered a plasmid encoding Sema3A protein to collagen-induced arthritis model mice. Intraperitoneal administration of acetaminophen improved arthritis scores and swelling of the hind paws. It has been reported that (Non-Patent Document 16).

[0013] In addition, the Sema3A concentration in the peripheral blood of patients with systemic lupus erythematosus was significantly higher than that in healthy individuals. The results showed significantly lower values ​​compared to those of the peripheral blood of patients with systemic lupus erythematosus. CD19+CD25high B cells have reduced Sema3A expression compared to the same cells from healthy individuals It has been reported that Sema3A expression levels in patients with systemic lupus erythematosus It has been suggested that changes in this pathway may affect B cell activation (Non-patent Document 17). ).

[0014] Furthermore, the expression of Sema3A in melanoma skin tissue from melanoma patients was significantly higher than that in normal skin tissue. Furthermore, it has been reported that the Sema3A gene is stably expressed in The mouse melanoma cell line transfected with the Sema3A gene showed a significant increase in in vitro activity compared with the parental line without the Sema3A gene. It has been reported that ro reduces the migration and invasion ability of tumor cells and increases their sensitivity to anticancer drugs. In a mouse tumor cell subcutaneous transplant model, tumors stably expressing the Sema3A gene were also successfully treated. Mouse malignant melanoma cell lines showed suppression of tumor metastasis compared to parental lines not transfected with the Sema3A gene. Inhibition and delayed proliferation have been reported (Non-patent Document 18).

[0015] These reports suggest that Sema3A is involved in the pruritus of psoriasis and atopic dermatitis, allergic rhinitis, and Osteoporosis, autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosus, inflammatory diseases, and The usefulness of this compound as a therapeutic agent for various diseases, including cancer and tumors, was suggested.

[0016] However, it binds to one of its receptors, Plexin-A1, and has activity similar to that of Sema3A. An antibody (agonist antibody) that binds to Plexin-A1 has not been reported so far. What region of the amino acid sequence of Plexin-A1 does an antibody with agonistic activity bind to? It was not clear whether it had any of the characteristics required for this purpose. [Prior art documents] [Non-patent literature]

[0017] [Non-Patent Document 1] Kolodkin, AL. et al., Cell, 75, 1389-1399(1993) [Non-patent document 2] Tessier-Lavigne, M. et al., Science, 274, 1123-1133(1996) [Non-patent document 3] Pasterkamp, ​​RJ. et al., Curr. Opin. Neurobiol., 13, 79-89(2003) [Non-patent document 4] Wong AW et al., Nat. Immunol., 4(9), 891-898(2003) [Non-Patent Document 5] Takegahara N. et al., Nat. Cell. Biol., 8, 615-622(2006) [Non-patent document 6] Eun SY et al., J. Immunol., 177(7), 4271-4275(2006) [Non-Patent Document 7] Janssen. BJ et al., Nat. Struct. Mol. Biol., 19, 1293-1299(2012) [Non-patent document 8] Sachiko Matsunaga et al. Experimental Medicine 31(4), 523-530(2013) [Non-Patent Document 9] Kouo, K. et al., Acta Derm. Venereol., 92, 521-528(2012) [Non-Patent Document 10] Tominaga, M. et al., Journal of Dermatological Science, 55, 40-46(2009) [Non-Patent Document 11] Yamaguchi, J. et al., Journal of Investigative Dermatology, 128, 2842-2849(2008) [Non-Patent Document 12] Negi, O. et al., Journal of Dermatological Science, 66, 37-43(2012) [Non-Patent Document 13] Sawaki, H. et al., J. Pharmacol. Sci., 117, 34-44(2011) [Non-Patent Document 14] Hayashi, M. et al., Nature, 485, 69-74(2012) [Non-Patent Document 15] Fukuda, T. et al., Nature, 497, 490-493(2013) [Non-Patent Document 16] Catalano, A., The Journal of Immunology, 185, 6373-83(2010) [Non-Patent Document 17] Vadasz, Z. et al., Arthritis Research & Therapy, 14, R146(2012) [Non-Patent Document 18] Chakraborty, G. et al., PLoS ONE, 7(3), e33633(2012) Summary of the Invention [Problem to be solved by the invention]

[0018] The present invention has been made in view of the above circumstances, and provides a novel anti-Plexin A1 agonist. an agonist antibody, a pharmaceutical composition containing the antibody, and a type 3 antibody containing the pharmaceutical composition; Preventive and / or therapeutic agent for diseases associated with quantitative or qualitative functional decline of semaphorin The present invention also aims to provide a kit for this purpose. [Means for solving the problem]

[0019] As a result of intensive research to solve the above problems, the present inventors have discovered a novel anti-Plexin A1 antagonist. Furthermore, the anti-Plexin A1 agonist antibody was successfully obtained. Antagonist antibodies recognize epitopes present in different regions of Plexin-A1. The present invention is based on this finding and relates to the following items [1] to

[0015] .

[0020] [1] Anti-Plexin A1 agonist antibody. [2] The antibody described in [1], which has semaphorin type 3-like activity. [3] The antibody according to [2], wherein the semaphorin 3-like activity is semaphorin 3A-like activity. . [4] Semaphorin-3-like activity promotes dendritic cell retraction or glioma cell retraction. The antibody according to [2] or [3], which has the activity of [5] Specific binding to an epitope within the Sema domain of Plexin A1. The antibody according to any one of the above. [6] The epitope in the Sema domain is an epitope in the C-terminal region of the Sema domain. The antibody described in [5]. [7] The C-terminal region is residues 461 to 514 of SEQ ID NO: 3 or residues 459 to 514 of SEQ ID NO: 52 The antibody according to [6], which binds to 512. [8] This antibody cross-reacts with human plexin A1 and mouse plexin A1. [1]~[7] The antibody according to any one of the preceding claims. [9] The antibody according to any one of [1] to [8], which is a monoclonal antibody.

[10] Any of claims [1] to [9], which is a chimeric antibody, a humanized antibody, or a human antibody. The antibody described.

[11] The antibody according to any one of [1] to

[0010] , which is a Fab, scFv, F(ab')2, single-chain antibody, or bispecific antibody.

[0021]

[12] A pharmaceutical composition comprising the antibody according to any one of [1] to

[11] and a pharmaceutically acceptable carrier. thing.

[13] A pharmaceutical composition comprising the antibody according to any one of [1] to

[11] and a pharmaceutically acceptable carrier. Methods for producing pharmaceutical compositions.

[14] Prevention and / or treatment of diseases associated with quantitative or qualitative impairment of type 3 semaphorin function The pharmaceutical composition according to

[12] , which is used for the treatment of

[15] The antibody according to any one of [1] to

[11] or the pharmaceutical composition according to

[12] or

[14] . and the prevention and / or treatment of diseases associated with quantitative or qualitative decrease in the function of type 3 semaphorin. and a kit containing instructions for administering said antibody or pharmaceutical composition to treat a disease or condition. . [Effects of the Invention]

[0022] According to the present invention, a novel anti-Plexin A1 agonist antibody and a composition containing the agonist antibody are provided. Pharmaceutical composition and a pharmaceutical composition for quantitatively or qualitatively inhibiting the function of type 3 semaphorin The present invention provides a preventive and / or therapeutic agent for diseases associated with decreased brain function, and a kit therefor. [Brief explanation of the drawings]

[0023] [Figure 1]FIG. 1 is a graph showing, as numerical values, the cell regression when mouse bone marrow-derived dendritic cells were stimulated with mouse semaphorin 3A for 5 hours. [Figure 2] This figure shows the binding activity of the anti-mouse Plexin A1 antibodies PXB361b, PXB693, and PXB727 to mouse Plexin A1. The X axis shows antibody concentration, and the Y axis shows absorbance at 405 nm. The solid line shows binding to the Ba / F3 cell line with high mouse Plexin A1 expression, and the dashed line shows binding to Ba / F3 cells. [Figure 3] FIG. 1 shows the dose-dependent antagonist activity of a control antibody or anti-Plexin A1 antibody (rabbit IgG) at concentrations of 0 to 100 μg / mL on semaphorin 3A-dependent mouse bone marrow-derived dendritic cell regression. [Figure 4] Each antibody was bound to BaF3 cells expressing human Plexin A1 or mouse Plexin A1 on the cell membrane surface, and the binding response was shown as an absorbance value. [Figure 5] This figure shows the binding response when mouse chimeric antibodies hPANL#240-mFc, 359B2-2-3-6-mFc, PXB693-mFc, mouse chimeric PXB727-mFc, and mouse chimeric PXB361b-mFc were immobilized on a Protein G Biosensor and human Plexin A1 and mouse Plexin A1 were applied. [Figure 6] PXB693 was immobilized on a Protein G Biosensor, and after application of the antigen, each antibody was then applied to evaluate whether or not the binding of each antibody competed with each other. The figure shows the competitive rate of each antibody for PXB693. [Figure 7] FIG. 1 shows changes in cell index values ​​when U87-MG cells were stimulated with human semaphorin 3A for 1 hour using the xCELLigence system. [Figure 8]1 shows the dose-dependent semaphorin 3A-like agonist activity of anti-Plexin A1 antibody on mouse bone marrow-derived dendritic cell retraction. Sema3A(+) indicates the value obtained when 0.8 μg / mL of mouse semaphorin 3A was added, and Sema3A(-) indicates the value obtained when mouse semaphorin 3A was not added. [Figure 9] 1 shows the semaphorin 3A-like agonist activity of anti-Plexin A1 antibodies on U87-MG cells. Sema3A(+) represents the value when 1.6 μg / mL of human semaphorin 3A was added. [Figure 10A] This figure shows the binding affinity of the antagonistic mouse chimeric antibody PXB361b to mouse Plexin A1, mouse Plexin A2, and the chimeric protein of mouse Plexins A1 and A2. 1: Mouse Plexin A1 sema domain, 2: Mouse Plexin A1 / A2 sema domain chimera, 3: Mouse Plexin A2 sema domain, Control Ab: control antibody, PXB361b: mouse chimeric antibody PXB361b-mFc. [Figure 10B] This figure shows the binding affinity of the agonist mouse chimeric antibodies PXB693 and PXB727 to mouse Plexin A1, mouse Plexin A2, and the chimeric protein of mouse Plexin A1 and A2. 1: Mouse Plexin A1 sema domain, 2: Mouse Plexin A1 / A2 sema domain chimera, 3: Mouse Plexin A2 sema domain, Control Ab: Control antibody, PXB693: Mouse chimeric antibody PXB693-mFc, PBX727-mFc: Mouse chimeric antibody PXB727. [Figure 11] Figure 1 shows a comparison of the amino acid sequences of the mouse Plexin A1 sema domain and the mouse Plexin A2 sema domain, with the sites used to construct the mouse Plexin A1 / A2 chimeric protein underlined. [Figure 12]1 shows a comparison of the amino acid sequences of mouse PlexinA1 sema domain and human PlexinA1 sema domain, with the sites used to construct the mouse PlexinA1 / A2 chimeric protein underlined. DETAILED DESCRIPTION OF THE INVENTION

[0024] [Plexin A1] Plexin A1 may be, for example, human or mouse Plexin A1, particularly The amino acid sequence and nucleotide sequence of mouse Plexin A1 are, but are not limited to, , for example, Kameyama T et al. "Biochemical and biophysical research communications." Biochem Biophys Res Commun. 1996, 226(2), 524-9, Genbank Accession No. D86948 , which is published in NCBI Reference Sequence NP_032907.1. The amino acid sequence and nucleotide sequence of phenotype A1 are described, for example, in Tamagnone L et al. Cell. 1999, 99(1), 71-80, Genbank Accession No. X87832, NCBI Reference Sequence NP_115618.3, NCBI The sequence is available in Reference Sequence NM_032242. Plexin A1 can be cloned into the appropriate promoter within the scope of the intended use. The amino acid sequence or nucleotide sequence of Rexin A1 can be modified before use. The amino acid sequence of A1 is well conserved between humans and mice. Plexin A1 is also referred to as Plexin-A1, PlexinA1, or PlxnA1. When mentioned, it refers to human and / or mouse Plexin A1 and may include other factors (e.g., semaphorins). Similarly, human and / or mouse are also included in the context of folin 3A).

[0025] [Anti-Plexin A1 agonist antibody] The anti-Plexin A1 agonist antibody is, for example, an antibody that specifically binds to the Plexin A1 protein. The term "antibody" refers to an antibody that has signal transduction activity mediated by Plexin A1, and is also used in combination with semaphorin type 3. Examples of antibodies having similar activities include antibodies having the same activity as the antibody.

[0026] The type 3 semaphorin-like activity is a type 3 semaphorin-like activity that can be exerted in vitro or in vivo. Preferably, it means at least one of the activities of semaphorin 3A, i.e., semaphorin 3A-like activity. This is at least one of the activities that Folin 3A can exhibit in vitro or in vivo. The activity mediated by the complex of maphorin 3A, neuropilin 1, and plexin A1 The activity referred to here includes stimulatory and inhibitory activities. The activity mediated by the complex of neuropilin A, neuropilin 1, and plexin A1 is e.g., sema Folin 3A forms a ternary complex with neuropilin 1 and plexin A1 A specific example of semaphorin 3A-like activity is the regression of cell morphology. Examples of the activity include promoting the regression of dendritic cells or glioma cells. Examples of glioma cells include human glioblastoma-derived cells and human glioblastoma-derived cells. Examples include tumor- and astrocytoma-derived cells, and more specifically, U-87 MG cells. can be.

[0027] In one embodiment of the anti-Plexin A1 agonist antibody of the present invention, the antibody promotes the regression of cell morphology. Examples of antibodies include those having cytotoxic activity, such as antibodies that inhibit the cell morphology of dendritic cells or glioma cells. Examples of antibodies that have the regression-promoting activity include antibodies that have the following activity: It may also mean to enhance.

[0028] [Anti-Plexin A1 antagonist antibody] The anti-Plexin A1 antagonist antibody is, for example, an antibody specific to the Plexin A1 protein. and inhibiting or attenuating Plexin A1-mediated signal transduction activity. Examples of antibodies include antibodies that inhibit or attenuate type 3 semaphorin-like activity, and preferably antibodies that inhibit or attenuate type 3 semaphorin-like activity. Anti-Plexin A1 agonists are antibodies that block or attenuate Plexin A1-like activity. The antibody may be, for example, an antibody that inhibits the regression of cell morphology, and may further inhibit the regression of dendritic cells or glioblastoma. It is an antibody that inhibits the regression of tumor cell morphology.

[0029] [Semaphorin type 3] To date, over 20 members of semaphorins have been identified, each of which has a Sema domain. It is notable that the family shares and conserves a region of about 500 amino acids called They are classified into eight subclasses based on the differences in the C-terminal structure following the Sema domain. The type 3 semaphorins are Sema3A, Sema3B, Sema3C, Sema3D, Sema3E, and Sema3F. It is known that type 3 semaphorins include Sema3A, Sema3B, Sema3C, and Sema3D. , Sema3E, and Sema3F, but preferably Sema3A (semaphorin 3A).

[0030] [Semaphorin 3A] For example, human or mouse semaphorin 3A can be used. The amino acid sequence of human semaphorin 3A is, for example, N The sequence is published in CBI Reference Sequence NP_006071.1. The mouse amino acid sequence is published, for example, in NP_033178.2. Based on this information, semaphorin 3A can be easily cloned. In this case, the amino acid sequence of semaphorin 3A can be appropriately modified before use. In this paper, semaphorin 3A is also referred to as Sema3A.

[0031] [SemaDomain] The sema domain is generally located at positions 51 to 487 of SEQ ID NO: 3 in human Plexin-A1, and at positions 51 to 487 of SEQ ID NO: 3 in mouse Plexin-A2. In SEQ ID NO: 52, positions 49 to 485 are located in the case of mouse Plexin A1, and in SEQ ID NO: 52, positions 49 to 485 are located in the case of mouse Plexin A2. It is from positions 50 to 483 of SEQ ID NO: 53. In this specification, the sema domain is Also include:

[0032] [Semaphorin 3A-like activity] Semaphorin 3A-like activity, which is a preferred example of type 3 semaphorin-like activity, is specifically Specifically, the following activities are included: and the activity of promoting the migration of dendritic cells to the lymph nodes (U.S. Patent Publication No. 2012 / 032 No. 2085), activity to inhibit osteoclast differentiation, activity to promote osteoblast differentiation (Hayashi M et al., Nature, 2012, 485, 69-74) and nerve outgrowth inhibitory activity (U.S. Patent No. 7642362). The agonist antibody of the present invention exhibits at least one of the semaphorin type 3-like activities. Any substance having such a function may be used, for example, a substance that promotes the retraction of cell morphology, and a substance that promotes the retraction of cell morphology, for example, a substance that promotes the retraction of dendritic cells. It promotes the regression of cell morphology of glioma cells or glioma cells. The antibody may be one that inhibits at least one of the semaphorin-3-like activities, For example, it inhibits the regression of cell morphology, and is also useful for inhibiting the regeneration of cells, such as dendritic cells or glioma cells. It inhibits the regression of cell morphology.

[0033] The term "having" semaphorin-3-like activity means that the anti-Plexin A1 agonist of the present invention This refers to an increase in type 3 semaphorin-like activity in the presence of the substance compared to its absence. Preferably, 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 50% or more, 7% or more 5% or more, 80% or more, 90% or more, 95% or more, 100% or more, 200% or more, 300 % or more, 400% or more, 500% or more, 600% or more, 700% or more, 800% or more, 9 This means an increase of more than 1000% or 1000%.

[0034] "Preventing or attenuating" semaphorin-3-like activity means preventing or attenuating the activity of an anti-Plexin A1 antagonist. In the presence of the antibody, semaphorin-3-like activity was reduced compared to its absence. Preferably, 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 50% or more This refers to a decrease of 75% or more, 80% or more, 90% or more, or 95% or more.

[0035] [Cell morphology retraction and nerve outgrowth inhibition] Sema3A inhibits axonal growth by retracting the growth cone of neurons and inhibits axonal growth by inhibiting the growth of dendritic cells. In the process of passing through the microlymphatic vessels, the dendritic cells are reduced in size by inducing the morphological retraction of the cells. By controlling cell migration and showing retraction activity on various cells, It controls a variety of biological reactions, including cell formation and immune responses.

[0036] The method for measuring the regression of cell morphology may be any method known to those skilled in the art. However, for example, methods for directly analyzing cell morphology using images, or for measuring changes in cell morphology and adhesion using xCEL One example is a method of measuring electrical impedance using an apparatus such as Ligence (registered trademark). However, it is not limited to these.

[0037] [Method for direct image analysis of cell morphology] Various tumor cells, endothelial cells including HUVEC, dorsal root ganglion (DRG) neurons, and dendritic cells These cells are seeded onto a 96-well cell culture plate and cultured for several hours to a day to produce cells. Next, Sema3A was added and the cells were incubated at 37°C and 5% CO2 for 30 minutes to several hours. Incubate the cells in a refrigerated incubator. As a control, a well without Sema3A is set up. This is followed by microscopic observation or high content screening using ArrayScan® or similar. The cell morphology was photographed using a cell image analyzer and analyzed using image analysis software (Cell Changes in cell morphology are quantified using techniques such as omics-vHCSTM:Scan.

[0038] An example of an index used to quantify changes in cell morphology is "%High Object C One method is to use an index called the "convex Hull Area ratio." The ratio of the area of ​​the convex hull to the area of ​​the convex hull is calculated and the percentage of cells whose ratio exceeds a certain threshold is calculated. By comparing these values ​​between when Sema3A was not added and when it was added, the effect of Sema3A was The cell morphology regression-inducing activity was measured. Cell morphology regression was measured by measuring the cell morphology regression activity when Sema3A was added. This means that the above value has decreased compared to when the substance was added, and preferably by 1% or more, 5% or more, or 10% or more. Above, 20% or more, 30% or more, 50% or more, 75% or more, 80% or more, 90% or more, 9 A decrease of 5% or more.

[0039] The agonist antibody of the present invention has the activity of promoting regression, for example, when the above-mentioned method is used. means that the value is reduced in the presence of the antibody compared to its absence, and preferably 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 50% or more, 75% or more, This refers to a reduction of 80% or more, 90% or more, or 95% or more.

[0040] Another example of an index used to quantify changes in cell morphology is image analysis software. Neurite outgrowth was measured using the neurite outgrowth application of software (e.g., Cellomics-vHCS™:Scan). Neurite outgrowth can be quantified as the average neurite outgrowth / neuron / well. The regression of cell morphology is a decrease in the above values ​​when Sema3A is added compared to when Sema3A is not added. This means that the concentration is preferably 1% or more, 5% or more, 10% or more, 20% or more, or 30% or more. , a decrease of 50% or more, 75% or more, 80% or more, 90% or more, or 95% or more.

[0041] The agonist antibody of the present invention has the activity of promoting regression, for example, when the above-mentioned method is used. means that the value is reduced in the presence of the antibody compared to its absence, and preferably 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 50% or more, 75% or more, This refers to a reduction of 80% or more, 90% or more, or 95% or more.

[0042] In addition, for example, the area of ​​a cell is calculated from the captured image, and if it is smaller than a certain threshold, A method for determining that cells that have undergone retraction can also be used.

[0043] [Method for measuring changes in cell morphology and adhesiveness as electrical impedance] Various tumor cells, endothelial cells including HUVEC, dorsal root ganglion (DRG) neurons, and dendritic cells These cells are seeded on tissue culture E-plates with electrodes integrated on the bottom of the wells and incubated for several hours to 1 The cells are then cultured for several days to allow them to adhere. Next, Sema3A is added and the cells are allowed to adhere for several minutes to several hours. The cells were cultured in an incubator at 37°C and 5% CO2. Sema3A was added as a control. Set up wells that do not contain the cells. Changes in cell morphology and adhesion are monitored by electrophoresis using CELLigence®. This electrical impedance is detected as xCELLigence (registered trademark). ) analysis software, RTCA software (registered trademark), and the cell It is created as a unitless parameter called the Index (CI) or the current The normalized cell index (CI) is a unit-free index derived from the relative change in CI between cell states. The normalized CI values ​​are also calculated when Sema3A is not added and when it is added. The cell morphological regression-inducing activity of Sema3A is measured by measuring the cell morphological regression of Sema3A. This refers to a decrease in the CI value when Sema3A is added compared to when Sema3A is not added, and is preferably 1%. or more, 5% or more, 10% or more, 20% or more, 30% or more, 50% or more, 75% or more, 80 This means a decrease of % or more, 90% or more, or 95% or more.

[0044] The agonist antibody of the present invention has the activity of promoting regression, for example, when the above-mentioned method is used. means that the value is reduced in the presence of the antibody compared to its absence, and preferably 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 50% or more, 75% or more, This refers to a reduction of 80% or more, 90% or more, or 95% or more.

[0045] [Activity to promote migration of dendritic cells to regional lymph nodes] Sema3A acts on the hetero-receptor complex of Plexin-A1 and Neuropilin-1 on dendritic cells. It exhibits the activity of inducing actomyosin contraction and induces changes in cell morphology, thereby promoting the formation of microlymphatic vessels. It controls passage through the intercellular space.

[0046] As a method for measuring the activity, any method known to those skilled in the art can be used. For example, a method for measuring migration of cells using an in vitro Boyden chamber cell migration assay is used. These include, but are not limited to, the law.

[0047] Specifically, a transwell (Corning) was filled with RPMI16 containing chemokines such as CCL21 and CXCL12. Dendritic cells were placed in a 24-well plate containing 0.6 mL of 0.1% BSA in 40 ml of PBS. Add the mixture to the upper chamber and incubate at 37°C for 1 to 3 hours. For transendothelial cell migration assays, lymphatic endothelial cells and blood The endothelial cells were layered on the upper chamber. Briefly, SVEC4-10 or HMVEC-dLy cells were layered on the upper chamber. on the top or bottom of transwell inserts coated with μg / mL fibronectin After culturing for 1-2 days, transendothelial cells were seeded in the cells in the same manner as in the cell migration assay described above. Migration assays are performed. When performing these assays, the upper chamber of the transwell Sema3A was added to the bar along with dendritic cells, and the migration of dendritic cells to the lower chamber was counted. The cell migration promoting activity of Sema3A was evaluated by comparing the results with those of wells to which Sema3A was not added. Measure.

[0048] The activity of promoting migration of dendritic cells to the draining lymph nodes was confirmed by Se in wells containing Sema3A. Compared with wells without ma3A, the migration count of dendritic cells to the lower chamber increased. This means that the content is preferably 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, This refers to an increase of 50% or more, 75% or more, 80% or more, 90% or more, or 95% or more.

[0049] [Activity to inhibit osteoclast differentiation, activity to promote osteoblast differentiation] Sema3A inhibits osteoclast differentiation via receptors expressed in osteoclasts and osteoblasts At the same time, it has been reported that it has a bone-protecting effect by promoting osteoblast activation. It has been reported.

[0050] As a method for measuring the activity of inhibiting osteoclast differentiation and promoting osteoblast differentiation For this purpose, methods known to those skilled in the art can be used appropriately, and examples thereof include the following methods. However, it is not limited to these.

[0051] [Osteoclast differentiation inhibitory activity] There are various methods for measuring activity, one of which is tartrate-resistant acid activity. Tartrate-resistant acid phosphatase (TRAP) staining was used to assess Specifically, bone marrow cells were cultured in α-MEM medium containing M-CSF for 48 hours or more to generate bone marrow monocytes / myocytes. The medium is replaced with RANKL-supplemented medium and cultured for several days. To evaluate Sema3A, the medium was replaced with Sema3A-supplemented medium, and RANKL was added after 10 to 12 hours. The medium was changed every few days, and osteoclast formation was confirmed. Then, TRAP staining and nuclear staining were performed. Images of the stained cells with Sema3A were observed under a microscope or ArrayScan. (registered trademark) or other high-content screening cell image analyzers. By comparing the staining images of cells without Sema3A, it was confirmed that Sema3A inhibits osteoclast differentiation. Specifically, the number of TRAP-positive cells per well or the number of TRAP-positive cells per well was measured. The number of multinucleated cells is measured and compared.

[0052] The inhibitory activity against osteoclast differentiation was greater in the wells containing Sema3A than in the wells without Sema3A. The number of TRAP-positive cells or the number of TRAP-positive and multinucleated cells was decreased compared with the control group. Preferably, 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 50% or more This refers to a decrease of 75% or more, 80% or more, 90% or more, or 95% or more.

[0053] [Osteoblast activation activity] There are various methods for measuring activity, for example, alkaline phosphatase Evaluation was performed using alkaline phosphatase (ALP) staining, ALP activity measurement, and calcification detection. Specifically, calvarial cells and MC3T3-E1 cells were cultured on collagen-coated plates. The cells were seeded and cultured in α-MEM medium containing ascorbic acid and β-glycerophosphate. For titration, Sema3A is added to the medium and cultured. The medium is changed every few days, and after the culture is completed, The ALP activity was measured by staining the cells with ALP and then photographing them under a microscope, or by measuring the ALP activity using an absorbance method. In addition, Alizarin red staining is used to detect calcification. Staining of cells with Sema3A added Images were taken using a microscope and compared with stained images of cells without Sema3A. To evaluate the osteoblast activation effect of Sema3A, we photographed the stained cells under a microscope. The intensity of ALP staining and Alizarin red staining is visually compared to determine the presence or absence of activity.

[0054] Osteoblast activation activity was significantly higher in wells with Sema3A than in wells without Sema3A. This refers to an increase in the absorbance of ALP activity or the intensity of Alizarin Red staining compared to control. Preferably, 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 50% or more, 7% or more 5% or more, 80% or more, 90% or more, 95% or more, 100% or more, 200% or more, 300 % or more, 400% or more, 500% or more, 600% or more, 700% or more, 800% or more, 9 This means an increase of more than 1000% or 1000%.

[0055] [antibody] The term "antibody" is used in the broadest sense and refers to any molecule that exhibits the desired biological activity. Monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., The antibody may be a polyspecific antibody, an antibody derivative, or a modified antibody (Miller K et al., J Immun ol., 2003, 170(9), 4854-61). Antibodies may be murine, human, humanized, or chimeric. or from other species. The antibodies disclosed herein may be derived from immunoglobulin molecules. Any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, Ig2), Immunoglobulins can be of any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. It can be derived from any species (e.g., human, mouse, or rabbit). The terms "globulin" and "immunoglobulin" are used interchangeably and in a broad sense. .

[0056] An "antibody derivative" is a portion of an antibody, preferably the variable domain of an antibody, or at least Antibody derivatives include, for example, Fab, Fab', F(ab')2, Fv fragments, linear antibodies, and the like. antibody, single chain antibody (scFv), sc(Fv)2, Fab3, domain antibody (dAb) (International Publication No. WO 2004 / 0588 21, WO 2003 / 002609), diabodies, triabodies, tetrabodies, minibodies These include, but are not limited to, multispecific antibodies formed from antibodies and antibody derivatives. Here, "Fv" is the smallest antibody derivative, and it contains a complete antigen-recognition region and an antigen-binding domain. The antibody derivative may also be a fusion with, for example, the Fc of an IgG antibody. , U.S. Pat. No. 5,641,870, Example 2; Zapata G et al., Protein Eng., 1995, 8(10) , 1057-1062; Olafsen T et al., Protein Eng. Design & Sel., 2004, 17(4):315-323; Holliger P et al., Nat. Biotechnol., 2005, 23(9);1126-36; Fischer N et al., Pat hobiology, 2007, 74(1):3-14; Shen J et al., J. Immunol Methods., 2007, 318, 65-7 4; Wu et al., Nat. Biotechnol., 2007, 25(11), 1290-7.

[0057] "Bispecific" antibodies have variable regions that recognize different epitopes within the same antibody molecule. A bispecific antibody may be an antibody that recognizes two or more different antigens. Alternatively, the antibody may recognize two or more different epitopes on the same antigen.

[0058] IgG type bispecific antibodies are produced by fusing two types of hybridomas that produce IgG antibodies. It can be secreted by hybrid hybridomas (quadromas) that arise from this process (Milstein C e (Nature, 1983, 305: 537-540). The genes of the L and H chains that make up the two types of IgG of interest were also identified. By introducing four genes into cells and co-expressing them, secretion of It can be done.

[0059] In this case, by making appropriate amino acid substitutions in the CH3 region of the H chain, heterogeneous H chains can be obtained. It is also possible to preferentially secrete a combination of IgG and IgG (Ridgway JB et al., Protein Engineering g, 1996, 9: 617-621; Merchant AM et al., Nature Biotechnology, 1998, 16: 677-681 ; WO2006 / 106905; Davis JH et al., Protein Eng. Des. Sel., 2010, 4: 195-202).

[0060] In addition, the L chain variable region has less diversity than the H chain variable region, so A common L chain that can confer binding ability to both chains can be obtained. By introducing the antibody into the target cell, IgG expression can be achieved, enabling efficient expression of bispecific IgG. become.

[0061] Here, the term "common L chain" refers to a chain that associates with two or more different H chains and binds to each antigen. "Different H chains" are preferably H chains of antibodies against different antigens. It refers to, but is not limited to, heavy chains that differ from each other in amino acid sequence. The common light chain can be obtained, for example, according to the method described in WO 2006 / 109592.

[0062] Bispecific antibodies can also be prepared by chemically cross-linking Fab'. Alternatively, leucine zippers derived from Fos, Jun, etc. can be used, and this method can be used to The present invention is not limited to this, but can also be applied to scFv, Fv, etc.

[0063] In addition, sc( Fv)2(Drug Discov. Today, 2005, Sep 15;10(18):1237-44), DVD-Ig(Nat. Biotechnol ., 2007, Nov;25(11):1290-7. Epub 2007 Oct 14, MAbs, 2009, Jul;1(4):339-47. Epub 2009 Jul 10, IDrugs, 2010, 13:698-700), as well as two-in-one antibodies (Science, 2009, Mar 20;323(5921):1610-4, Immunotherapy, 2009, Sep;1(5):749-51), Tri-Fab and Tande Bispecific antibodies such as human scFv and diabodies are also known (MAbs, 2009, November; 1(6) Furthermore, even when molecular forms such as scFv-Fc and scaffold-Fc are used, heterogeneous F c) (Ridgway JB et al., Protein Engineering, 1996, 9: 61 7-621, Merchant AM et al., Nature Biotechnology, 1998, 16: 677-681, WO2006 / 10690 5, Davis JH et al., Protein Eng. Des. Sel., 2010, 4:195-202), bispecific antibodies It can be produced efficiently.

[0064] "Cross-reactive antibodies" are also called cross-antibodies, cross-active antibodies, or cross-reactive antibodies, and are antibodies that react with multiple antigens. Here, multiple antigens refer to antibodies that recognize the same or similar epitopes in a single antigen. For example, antigens of the same species may be used, or antigens of different species may be used.

[0065] Examples of antibody modifications include those bound to various molecules such as polyethylene glycol (PEG). In the modified antibody of the present invention, the substance to be bound is not limited. To obtain such an antibody modification, the obtained antibody is chemically modified. These methods are already established in this field.

[0066] The antigen-binding region is, for example, an antibody, a scaffold molecule (antibody-like molecule), a peptide, or the like that binds to an antigen. Examples of scaffold molecules include the site required for binding, or a fragment containing that site. , a molecule that exerts its function by binding to a target molecule, and Any conformationally stable polypeptide capable of binding to a target antigen is suitable. Polypeptides can also be used. Examples of such polypeptides include For example, antibody variable regions, fibronectin (WO 2002 / 032925), Protein A domains, (International Publication No. 1995 / 001937), LDL receptor A domain (International Publication No. 2004 / 044011, Publication No. 2005 / 040229), ankyrin (International Publication No. 2002 / 020565), etc., as well as Nygren et al. ( Current Opinion in Structural Biology, 7:463-469(1997), Journal of Immunol. Meth ods, 290:3-28(2004)), Binz et al. (Nature Biotech., 23:1257-1266(2005)), Hosse et al. (Protein in Science, 15:14-27(2006)). ol. Ther., 2010 Aug;12(4):487-95 and Drugs, 2008;68(7):901-12. Peptide molecules capable of binding to target antigens can also be used.

[0067] The antibody of the present invention is a recombinant antibody produced using gene recombination technology. Recombinant antibodies are preferably prepared by transfecting DNA encoding the antibodies into hybridomas or antibody-producing cells. The antibody is cloned from antibody-producing cells such as sensitized lymphocytes, and then inserted into a vector. It can be obtained by introducing it into a host (host cell) and producing it.

[0068] The antibody of the present invention is not limited in terms of origin, and may be a human antibody, a mouse antibody, a rat antibody, or the like. Genetically modified antibodies such as chimeric antibodies and humanized antibodies may also be used.

[0069] Genetically modified antibodies can be produced using known methods. Chimeric antibodies are composed of the variable regions of the H and L chains of an antibody from an immunized animal and the H and L chains of a human antibody. The antibody is composed of the constant region of the antibody. The resulting DNA is linked to DNA encoding the constant region of a human antibody, and then incorporated into an expression vector and introduced into the host. Chimeric antibodies can be obtained by introducing and producing the antibodies.

[0070] Methods for obtaining human antibodies are already known. For example, The desired human antibody is obtained by immunizing a transgenic animal with the target antigen. It is possible to do this (International Publication Nos. 93 / 12227, 92 / 03918, 94 / 02602 (See International Publication Nos. WO 94 / 25585, WO 96 / 34096, and WO 96 / 33735).

[0071] Additionally, human antibodies can be produced by hybridoma-based methods. Human myeloma and mouse-human heterologous cell lines for the production of human monoclonal antibodies are available. available (Kozbor J., Immunol., 133: 3001 (1984); Brodeur et al., Monoclona lAntibody Production Techniques and Applications, pp51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991). Human B Human antibodies produced through cell hybridoma technology are also known (Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Human hybridoma technology (e.g., oma technique) is also available (Vollmers and Brandlein, Histology and Histopathology). hology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0072] Human antibodies can be derived from Fv clones selected from a human-derived phage display library. Such variable domain sequences can also be generated by isolating the variable domain sequences. The desired human constant domain may be combined with the human antibody. The techniques for this purpose are described below, but are not limited to human antibodies.

[0073] Many antibody libraries are already known, and Since the method for preparing the library is also known, those skilled in the art can obtain an appropriate antibody library. For example, regarding phage libraries, see Clackson et al., Nature, 1991, 3 52: 624-8, Marks et al., J. Mol. Biol., 1991, 222: 581-97, Waterhouses et al., N ucleic Acids Res., 1993, 21: 2265-6, Griffiths et al., EMBO J., 1994, 13: 324.0- 60, Vaughan et al., Nature Biotechnology, 1996, 14: 309-14, and International Publication No. WO 96 / 077 54, etc. In addition, there are methods for using eukaryotic cells as libraries (International It is possible to use known methods such as the method disclosed in Publication No. 95 / 15393 and ribosome display.

[0074] The library may be a library known to those skilled in the art (Methods Mol. Biol., 2002; 178: 87-100 J. Immunol. Methods, 2004 Jun; 289(1-2): 65-80; and Expert Opin. Biol. Ther ., 2007 May; 7(5): 763-79), human naive libraries, non-human animals, and humans These include immune libraries derived from HIV-1, semi-synthetic libraries, and synthetic libraries. However, the method is not particularly limited to these examples.

[0075] Furthermore, a technique for obtaining human antibodies by panning using a human antibody library is also known. For example, the variable region of a human antibody is expressed as a single chain antibody (scFv) and then displayed on a phage. The antigen can be expressed on the surface of the phage using the Ray method, and phages that bind to the antigen can be selected. By analyzing the genes of the selected phages, it is possible to identify the variable regions of human antibodies that bind to antigens. The DNA sequence of the scFv that binds to the antigen can be determined. For example, a suitable expression vector can be constructed based on the sequence, and a human antibody can be obtained. These methods are already known and are described in International Publication Nos. 92 / 01047, 92 / 20791, and 92 / 20792. International Publication No. 93 / 06213, International Publication No. 93 / 11236, International Publication No. 93 / 19172, International Publication No. 95 / 01438, Reference may be made to WO 95 / 15388.

[0076] Antibodies or antibody fragments isolated from a human antibody library are referred to herein as human antibodies or It is considered a fragment of a human antibody.

[0077] Humanized antibodies are modified antibodies also called reshaped human antibodies. , constructed by grafting the CDRs of antibodies derived from immunized animals onto the complementarity-determining regions of human antibodies. The general genetic recombination techniques are also known (European Patent Application Publication No. 239400, International Publication No. 96 / 02576, Sato K et al., Cancer Research, 1993, 53:851-856, WO99 / 5174 See 3).

[0078] As a preferred embodiment of the agonist antibody of the present invention, specifically, the following antibodies are described in the Examples below. Examples include antibodies comprising the amino acid sequences described below. (a) H chain CDRs of SEQ ID NOs: 23, 24, and 25 (hPANL#240 H chain CDRs) The amino acid sequences of DR1, 2, and 3 and SEQ ID NOs: 26, 27, and 28 (hPANL#240) An antibody comprising the amino acid sequences of L-chain CDR1, 2, and 3 described in (L-chain CDR). (b) H chain CDR of SEQ ID NOs: 29, 30, and 31 (359B2-2-3-6) The amino acid sequences of CDR1, 2, and 3 of the 359B2-2- An antibody comprising the amino acid sequences of L-chain CDR1, 2, and 3 described in (L-chain CDRs 3-6).

[0079] Furthermore, as a preferred embodiment of the agonist antibody of the present invention, specifically, Examples of antibodies include antibodies comprising the amino acid sequences described below. (a) The heavy chain variable region of SEQ ID NO: 21 (hPANL#240 heavy chain variable region) The amino acid sequence and the L chain variable region of SEQ ID NO: 22 (hPANL#240 L chain variable region) An antibody comprising the amino acid sequence of the region. (b) the heavy chain variable region of SEQ ID NO: 35 (the heavy chain variable region of 359B2-2-3-6) The amino acid sequence and the L chain variable region of SEQ ID NO: 36 (359B2-2-3-6) An antibody comprising the amino acid sequence of the chain variable region.

[0080] Furthermore, as a preferred embodiment of the agonist antibody of the present invention, specifically, Examples of antibodies include antibodies comprising the amino acid sequences described below. (a) The amino acid sequence of the heavy chain set forth in SEQ ID NO: 9 (hPANL#240 heavy chain) and the sequence An antibody comprising the amino acid sequence of the L chain described in No. 10 (L chain of hPANL#240). (b) the amino acid sequence of the H chain set forth in SEQ ID NO: 11 (the H chain of 359B2-2-3-6); An antibody comprising the amino acid sequence of the L chain set forth in SEQ ID NO: 12 (L chain of 359B2-2-3-6). body.

[0081] One embodiment of the present invention is a method for producing a human immunoglobulin E1-containing antibody comprising administering to a mammalian subject the antibody sequence of hPANL#240 or 359B2-2-3-6. The nucleic acid encoding the CDRs, variable regions, or full-length heavy and light chains of the same sequence. Another embodiment is a vector incorporating the nucleic acid. Yet another embodiment is a host cell transformed with the vector. Yet another embodiment is a method for producing said antibody by culturing said cells. do. Yet another embodiment is an antibody produced by the above method.

[0082] One embodiment of the present invention is directed to the use of hPANL#240 or It is an anti-Plexin A1 antibody that competes with 359B2-2-3-6. For binding to the Sema domain of A1, hPANL#240 or 359B2-2-3-6 were used. More preferably, it is a competitive anti-Plexin A1 antibody. Binding to residues 461-514 of SEQ ID NO: 3 in mouse Plexin A1 or the sema domain of mouse Plexin A1 For binding to residues 459-512 of SEQ ID NO: 52 in hPANL#240 or 35 This is an anti-Plexin A1 antibody that competes with 9B2-2-3-6.

[0083] The present invention also relates to a method for identifying an epitope that overlaps with the epitope bound by the agonist antibody of the present invention. Also provided are antibodies that bind to the same epitope as another antibody. The competition between antibodies can be confirmed by the competition between the two epitopes. This can be assessed by a binding assay, such as enzyme-linked immunosorbent assay (ELISA). ELISA), Fluorescence Energy Transfer Assay (FRET), Fluorescence Micrometrology (FMAT®), Electrochemiluminescence (ECL) method, AlphaScreen (chemically amplified luminescence proximity homogenizer) Genius Assay, Radioimmunoassay (RIA), Scintillation prox imity assay (SPA), Surface Acoustic Wave (SAW), MicroScale Thermophoresis (Nan oTemper Technologies), Quartz Crystal Microbalance (QCM), Octet (ForteBIO), Analysis of biomolecular interactions using surface plasmon resonance and other methods. Examples include Kinetic Exclusion Assay (KinExA®). The amount of antibody bound to the target epitope is determined by the amount of candidate competing antibodies ( This indirectly correlates with the binding ability of the test antibody (i.e., the binding ability of the test antibody to overlapping epitopes). The greater the amount or affinity of the test antibody, the lower the amount of binding of the antibody to the antigen. The amount of antibody binding increases. Specifically, the antibody is appropriately labeled with the antigen, and the test antigen is The antibody and the antibody are added simultaneously, and the bound antibody is detected using the label. The amount of the antibody can be easily measured by labeling the antibody in advance. The labeling method is selected depending on the technique, but is not limited to this. For example, fluorescent labeling is used to immobilize Plexin A1 on beads. The labeled antibody and the unlabeled antibody or the test antibody are added simultaneously, and the labeled antibody The detection is performed using fluorescence micromeasurement technology. In addition, biomolecular interaction analysis and binding equilibrium exclusion method are also used. When using a labeled antibody, it is not necessary to use a labeled antibody, and competition can be evaluated. The epitopes that bind to each other are not only the same epitopes of the two antibodies, but also epitopes that are partially identical. In addition, in the present specification, antibodies that bind to overlapping epitopes may be referred to as antibodies that bind to overlapping epitopes. Thus, they may also be described as antibodies that bind to competing epitopes, or as competing antibodies.

[0084] Here, "antibodies that bind to overlapping epitopes," "competing antibodies," or "competitors" are used. "Antibody that binds to an epitope that binds to Plexin A1" means that the antibody and the test antibody bind to an epitope that binds to Plexin A1. Examples of the term "not binding substantially simultaneously" include cases where the two molecules do not bind qualitatively at the same time. The binding activity of the antibody to Plexin A1 is 60% or less, usually 50% or less, preferably 30% or less. It is particularly preferable that the binding activity is 15% or less. This can be performed by methods known to those skilled in the art, for example, Western blotting. This can be done by the following:

[0085] An epitope to which the above-mentioned agonist antibody binds or an epitope overlapping with the epitope The multispecific antigen-binding molecule having the antigen-binding site of an antibody that binds to type 3 semaphorin-like It is possible to promote the activity of the antibody, for example, the regression of cell morphology. The antigen-binding site of an antibody that binds to an epitope that overlaps with the epitope of the target antigen is a better type 3 antibody. One or more amino acids can be modified to obtain maphorin-like activity. .

[0086] The term "monoclonal antibody" as used herein refers to a population of substantially homogeneous antibodies, i.e. The individual antibodies that make up the population are identical except for the small amounts of variants that may occur in nature. refers to an antibody obtained from a homogeneous antibody population. Monoclonal antibodies are highly specific. and generally act against a single antigenic site. Compared to conventional polyclonal antibody preparations containing different antibodies against different determinants (epitopes) In addition to their specificity, monoclonal antibodies are directed against a single antigenic determinant on an antigen. Monoclonal antibodies are synthesized by hybridoma cultures that are free from other antibodies. The modifier "monoclonal" refers to a substantially homogeneous antibody. The characteristics of the antibodies obtained from the population are indicative of whether the antibodies were produced by a particular method. For example, the monoclonal antibody used in the present invention does not require that the antibody be The cells can be cultured, for example, by the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495 (1975) ), or by recombinant methods (e.g., U.S. Pat. No. 4,816,567). The monoclonal antibodies used in the present invention may also be isolated from phage antibody libraries. (e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biology ol., 222:581-597 (1991)).

[0087] The antibody of the present invention can be produced by a method known to those skilled in the art. The DNA encoding the antibody is inserted into an expression vector. It is incorporated into an expression vector so that it is expressed under the control of an enhancer and promoter. The host cells are transformed with this expression vector to express the antibody. Any combination of host and expression vector can be used.

[0088] Examples of vectors include M13 vectors, pUC vectors, pBR322, pBluescript, and pCR-S. crypt, etc. Also, for the purpose of subcloning or excising cDNA, In addition to the above vectors, for example, pGEM-T, pDIRECT, pT7, etc. can also be used.

[0089] When vectors are used for the purpose of producing antibodies, expression vectors are particularly useful. As the expression vector, for example, the host may be a strain such as JM109, DH5α, HB101, or XL1-Blue. In the case of E. coli, a promoter that can be efficiently expressed in E. coli, e.g. , lacZ promoter (Ward et al., Nature, (1989) 341, 544-546; FASEB J., (1992) 6, 24 22-2427), the araB promoter (Better et al., Science, (1988) 240, 1041-1043), or T It is essential that the vector has a promoter such as In addition to the above vectors, pGEX-5X-1 (Pharmacia) and the QIAexpress system (QIAGEN) were used. , pEGFP, or pET (in this case, the host is preferably BL21 expressing T7 RNA polymerase). Examples include:

[0090] The vector may also contain a signal sequence for secretion of the polypeptide. As a signal sequence for polypeptide secretion, when produced in the periplasm of E. coli, In this case, for example, the pelB signal sequence (Lei, SP et al., J. Bacteriol., (1987) 169, 4397) The vector can be introduced into the host cell by, for example, the calcium chloride method, electo method, or the like. This can be done using the poration method.

[0091] In addition to E. coli expression vectors, other vectors for producing the antibodies of the present invention include, for example, Mammalian expression vectors (e.g., pcDNA3 (Invitrogen) and pEGF-BOS (Nuclei c Acids. Res., 1990, 18(17), p5322), pEF, pCDM8), insect cell-derived expression vectors ( For example, the "Bac-to-BAC baculovirus expression system" (GIBCO BRL), pBacPAK8 , plant-derived expression vectors (e.g., pMH1, pMH2), animal virus-derived expression vectors (e.g., For example, pHSV, pMV, pAdexLcw), retrovirus-derived expression vectors (for example, pZIPneo) , yeast-derived expression vectors (e.g., "Pichia Expression Kit" (Invitrogen)), p NV11, SP-Q01), and expression vectors derived from Bacillus subtilis (e.g., pPL608, pKTH50).

[0092] For expression in animal cells such as CHO cells, COS cells, and NIH3T3 cells, A promoter necessary for expression, such as the SV40 promoter (Mulligan et al., Nature, (1979) 277, 108), MMTV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res., (1990) 18, 5322), CAG promoter (Gene, (1991) 108, 193), CMV promoter It is essential that the cells have motors and other functions, and the genes for selecting transformed cells are also included. It is more preferable that the gene be present. There are drug resistance genes that can be identified by drugs such as neomycin and G418. Examples of vectors having these properties include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP1. 3, etc.

[0093] Furthermore, we aim to stably express genes and amplify the copy number of genes in cells. In this case, CHO cells lacking a nucleic acid synthesis pathway are transfected with a DHFR gene that complements the defective nucleic acid synthesis pathway. A method in which a vector (e.g., pCHOI) is introduced and amplified with methotrexate (MTX). For transient gene expression, a method for expressing SV40 T antigen can be used. Using COS cells that have the gene on their chromosomes, a vector (such as pcD) with the SV40 replication origin was used. Origins of replication include those derived from polyoma viruses, adenoviruses, and the like. Those derived from the human ovarian cancer virus (HUVEC), bovine papillomavirus (BPV), etc. can also be used. Furthermore, the expression vector contains an amino acid sequence as a selectable marker for gene copy number amplification in the host cell line. aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Enterococcus coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, dihydro It may contain the folate reductase (dhfr) gene, etc.

[0094] The antibody of the present invention thus obtained can be isolated from inside or outside the host cell (such as from the medium). The antibody can be purified as a substantially pure and homogeneous antibody. Any separation and purification method used in antibody purification may be used, and there is no limitation. For example, chromatography columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis Antibodies can be separated and purified by appropriately selecting and combining methods such as precipitation, recrystallization, etc.

[0095] Examples of chromatography include affinity chromatography and ion exchange chromatography. Chromatography, hydrophobic chromatography, gel filtration, reversed phase chromatography, Adsorption chromatography and other methods are listed (Strategies for Protein Purification and Ch aracterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Sp (Harbor Laboratory Press, 1996). These chromatography methods are liquid-phase chromatography. The separation can be carried out using liquid phase chromatography such as HPLC or FPLC. The columns used for affinity chromatography include Protein A columns, Protein For example, columns using Protein A include Hyper D, POROS, and Seph arose FF (GE Amersham Biosciences), etc. The present invention relates to these purification methods. The present invention also includes highly purified antibodies.

[0096] The obtained antibodies can be purified to homogeneity. Separation and purification of antibodies are performed in the same way as conventional protein purification. For example, affinity chromatography can be used. Chromatography columns such as filters, ultrafiltration, salting out, dialysis, SDS polyacetal By appropriately selecting and combining acrylamide gel electrophoresis, isoelectric focusing, etc., antibodies can be separated and purified. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, C (Old Spring Harbor Laboratory, 1988), but is not limited to these. The columns used for chromatography are Protein A column and Protein G column. Examples include:

[0097] One embodiment of the present invention is a method for the production of a plexin-A1 gene comprising the sequence of SEQ ID NO: 3 in the sema domain of human plexin-A1. A peptide consisting of residues 461 to 514, or the sequence numbers in the Sema domain of mouse Plexin A1 Anti-Plexin A1 agonist antibody was prepared using a peptide consisting of residues 459-512 of Plexin A1 as an antigen. Another embodiment of the present invention is a method for producing a body produced by the above-mentioned production method. The anti-Plexin A1 agonist antibody is an anti-Plexin A1 agonist antibody. It can be manufactured using known methods.

[0098] [Uses of anti-Plexin A1 agonist antibodies] The anti-Plexin A1 agonist antibody of the present invention binds to Plexin A1 and thereby To exhibit type 3 semaphorin-like activity, quantitative or quantitative analysis of type 3 semaphorin, preferably Sema3A, is required. It is useful for treating and preventing diseases associated with qualitative functional impairment, especially psoriasis and Itching of atopic dermatitis, allergic rhinitis, osteoporosis, rheumatoid arthritis and systemic lupus erythematosus It is used to treat a variety of diseases, including autoimmune diseases, including rheumatoid arthritis, inflammatory diseases, and tumors. It is useful for treating and preventing

[0099] [Pharmaceutical composition] The present invention also provides a pharmaceutical composition containing the anti-Plexin A1 agonist antibody of the present invention. As described above, the anti-Plexin A1 agonist antibody of the present invention binds to type 3 semaphorin, preferably Preferably, diseases associated with quantitative or qualitative functional impairment of Sema3A (e.g., psoriasis and atopic dermatitis) are Itching from allergic dermatitis, allergic rhinitis, osteoporosis, rheumatoid arthritis and systemic lupus erythematosus It is useful as a therapeutic or preventive agent for autoimmune diseases, including rheumatoid arthritis, inflammatory diseases, and tumors. In addition, when the anti-Plexin A1 agonist antibody of the present invention is used as a pharmaceutical composition, In terms of antigenicity and the like, a human antibody or a humanized antibody is preferred.

[0100] The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. Examples of carriers that can be used include sterilized water, physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, acetic acid, etc.), and the like. phosphate, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Twe Examples of suitable low-molecular-weight compounds include chelating agents (EDTA, etc.), binders, etc. proteins such as high molecular weight polypeptides, serum albumin, gelatin, or immunoglobulins; Amino acids such as glycine, glutamine, asparagine, arginine and lysine, polysaccharides and It contains sugars such as monosaccharides, carbohydrates, and sugar alcohols such as mannitol and sorbitol. When preparing an aqueous solution for injection, it is acceptable to use a solution containing, for example, physiological saline, glucose, or other adjuvants. isotonic solutions containing, for example, D-sorbitol, D-mannose, D-mannitol, sodium chloride, Suitable solubilizing agents include alcohols (ethanol, etc.), polyalcohols ( propylene glycol, PEG, etc.), non-ionic surfactants (polysorbate 80, HCO-50), etc. They may be used in combination.

[0101] Furthermore, the pharmaceutical composition of the present invention may be optionally formulated into microcapsules (hydroxymethylcellulose). It can be enclosed in microcapsules such as sugar, gelatin, poly(methyl methacrylate), etc. L-oid drug delivery systems (liposomes, albumin microspheres, microspheres) emulsions, nanoparticles and nanocapsules, etc. ("Remington's Pharm (See, for example, "Oceanic Science 16th edition", Oslo Ed., 1980). Methods for preparing such agents are also known and can be applied to the present invention (Langer et al., J. Biomed. Mater. Re s., 1981, 15:167-277; Langer, Chem. Tech., 1982, 12:98-105; US3773919; EP58481; Sidman et al., Biopolymers, 1983, 22:547-556; EP133988).

[0102] Administration to patients can be either oral or parenteral, but parenteral administration is preferred. The form (dosage form) of the pharmaceutical composition of the present invention is not particularly limited, and may be in the form of an injection, a nasal administration, or the like. Examples of dosage forms include oral administration, pulmonary administration, transdermal administration, freeze-dried administration, and solution administration.

[0103] Lyophilization can be carried out by methods well known to those skilled in the art (Pharm. Biotechnol., 2002, 13 , 109-33, Int. J. Pharm., 2000, 203(1-2), 1-60, Pharm. Res., 1997, 14(8), 969-97 5) For example, the solution is dispensed in appropriate amounts into containers such as vials to be used for freeze-drying, and then stored in a freezer or This can be done in a freeze-drying chamber or by immersion in a refrigerant such as acetone / dry ice or liquid nitrogen. Furthermore, when preparing a highly concentrated solution of an antibody formulation, this can be carried out by a method well known to those skilled in the art. For example, as described in J. Pharm. Sc., 2004, 93(6), 1390-1402, A membrane concentration method using an FF membrane is used.

[0104] Injectable dosage forms are administered systemically, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc. Alternatively, it can be administered locally. The administration method can be selected appropriately depending on the age and symptoms of the patient. The dosage can be selected from, for example, 0.0001 mg / kg body weight to Alternatively, for example, the dose may be selected from the range of 0.001 to 100,000 mg per patient. However, the present invention is not limited to these doses and The present invention is not limited to the dosage and administration method.

[0105] A therapeutically or prophylactically effective amount of an anti-Plexin A1 agonist antibody is defined as the amount of an anti-Plexin A1 agonist antibody that, when administered to a subject, to prevent, delay, reduce, or inhibit symptoms or activity associated with a disease or disorder, The term "agonist" refers to an amount of agonist that is effective. Administration may consist of a single dose or multiple doses. It may be administered in combination with other pharmaceutical compositions.

[0106] [kit] The present invention relates to a method for treating at least the agonistic Plexin A1 antibody or pharmaceutical composition of the present invention. Diseases associated with quantitative or qualitative functional impairment of type 3 semaphorin, preferably Sema3A, including The present invention provides a kit for preventing and / or treating a disease, particularly an anti-Plexin A1 antibody. Anti-pruritic and anti-allergic agents for psoriasis and atopic dermatitis, including anti-inflammatory agents and pharmaceutical compositions rhinitis, osteoporosis, autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosus, inflammatory diseases A kit for preventing and / or treating diseases and tumors is provided. , other, syringes, needles, pharmaceutically acceptable vehicles, alcohol wipes, adhesive bandages, or Instructions for use may also be included in the package.

[0107] The present invention also relates to a method for treating fibroblasts comprising administering to a subject the anti-Plexin A1 agonist antibody or pharmaceutical composition of the present invention in a manner similar to that described above. Prevention of diseases associated with quantitative or qualitative decrease in function of type 3 semaphorin, preferably Sema3A and / or use in the manufacture of therapeutic agents, particularly anti-Plexin A1 agonists. Antibody or pharmaceutical composition for treating pruritus in psoriasis and atopic dermatitis, allergic rhinitis, bone Porosis, autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosus, inflammatory diseases, and and its use in the manufacture of a preventive and / or therapeutic agent for tumors. The present invention also provides a method for treating a type 3 semaphorin, preferably a type 3A semaphorin, by quantitatively or qualitatively reducing its function. The present invention provides an anti-Plexin A1 agonist for the prevention and / or treatment of diseases associated with Plexin A1. In particular, the present invention relates to a method for treating itching caused by psoriasis and atopic dermatitis, and a pharmaceutical composition thereof. rhinitis, osteoporosis, autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosus, inflammation Anti-Plexin A1 Agonist Antibody for the Prevention and / or Treatment of Cancer Diseases and Tumors - Patent application The present invention relates to a pharmaceutical composition.

[0108] The anti-Plexin A1 agonist antibody or pharmaceutical composition of the present invention may be used in combination with other therapeutic agents. The anti-Plexin A1 agonist antibody or pharmaceutical composition of the present invention can be used in combination with other therapeutic agents. The anti-inflammatory drug of the present invention may be administered simultaneously with or at different times. As a kit combining a Rexin A1 agonist antibody or pharmaceutical composition with other therapeutic agents Furthermore, the anti-Plexin A1 agonist antibody or pharmaceutical composition of the present invention may be used. When other therapeutic agents are used in combination, the effectiveness of each agent may be improved as desired compared to when either agent is used alone. Lower doses may also be possible.

[0109] As used herein, the phrase "comprising" The aspect is defined by the expression "essentially consisting of..." and "consisting of" The present invention encompasses various embodiments.

[0110] All patent and literature references expressly cited herein are hereby incorporated by reference in their entirety. is incorporated by reference.

[0111] The present invention is further illustrated by, but not limited to, the following examples. isn't it. [Example]

[0112] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples. It is not something that can be done.

[0113] Example 1: Preparation of recombinant semaphorin 3A protein The mouse semaphorin 3A protein has the sequence of NCBI Reference Sequence NP_033178.2. Based on this, gene synthesis was performed to identify the arginine residues (552, 555, and 7) of the protease recognition site. The signal peptide (20 positions from the N-terminus) was converted to alanine. The remaining amino acid residues (up to the alanine) were replaced with the artificial signal peptide HMM+38 (SEQ ID NO: 7), and the artificial signal peptide Between the peptide and the 21st asparagine, there is a glutamic acid-aspartic acid-arginine A His tag sequence was inserted via a spacer. Furthermore, the C-terminus contains serine at position 771 and serine at position 772. The amino acid sequence of the recombinant mouse semaphorin 3A protein was deleted. The sequence is shown in SEQ ID NO: 1. The prepared gene was incorporated into an expression vector and Semaphorin 3A was expressed in eeStyle293 cells, and the HisTrap semaphorin 3A protein was isolated from the culture supernatant. Purification was performed by affinity purification using excel (GE Healthcare) and gel filtration chromatography. Made.

[0114] Human semaphorin 3A protein was identified based on the sequence of NCBI Reference Sequence NP_006071.1. The arginine residues of the protease recognition site (552, 555, and 757) were synthesized. The signal peptide (the 20th amino acid from the N-terminus) was converted to alanine. ranin) was replaced with the artificial signal peptide HMM+38 (SEQ ID NO: 7), and the artificial signal peptide Between the 21st asparagine and the 22nd asparagine, there is a glutamic acid-aspartic acid-arginine spacer. A His tag sequence was inserted via the nucleotide sequence. Furthermore, a FLAG tag sequence (SEQ ID NO: 5) was inserted at the C-terminus. The amino acid sequence of the prepared human semaphorin 3A recombinant protein is shown in SEQ ID NO: The gene was inserted into an expression vector and transformed into Invitrogen FreeStyle293 cells. The semaphorin 3A protein was extracted from the culture supernatant using HisTrap excel (GE Health The purified product was purified by affinity purification using a ELISA kit (Care) and gel filtration chromatography.

[0115] Example 2: Construction of an evaluation system for mouse semaphorin 3A activity using mouse dendritic cell retraction as an indicator To evaluate the effect of semaphorin 3A protein on dendritic cell morphology and contraction, A collapse assay was performed using mouse bone marrow-derived dendritic cells. The test was carried out by the following method.

[0116] Murine bone marrow-derived dendritic cells were prepared according to the method of Inaba et al. (J Exp Med., 1992, 176(6):1693-702). The medium was prepared according to the manufacturer's instructions. 10% FBS (Moregate) and mouse GM-CSF (40 ng / mL; R&D Systems) Murine bone marrow-derived dendritic cells were suspended in RPMI1640 medium (Nacalai Tesque) containing A suspension of 2 x 10 4 After seeding cells / well into a 96-well plate, culture them at 37°C for 12 to 24 hours. The mouse semaphorin 3A protein was cultured at an appropriate concentration in RPMI1640 medium containing 10% FBS. The solution was diluted to 100% and added to the cell culture medium, which was then incubated at 37°C for an additional 5 hours. The cells were fixed by treatment with PBS containing aldehyde and then incubated with PBS containing 0.1% Triton X-100. The cell membrane was permeabilized by treatment with PBS solution containing Alexa Fluor 488 (registered trademark) ) labeled phalloidin and DAPI in PBS, respectively. F-actin and nuclei were stained. Cell morphology images were taken with an ArrayScan VTI (Thermo Fisher Scientific Inc.). is the analysis software Cellomics-vHCS TM :Scan (Thermo Fisher Scientific The morphological changes of dendritic cells were analyzed using the Morphology v4 protocol of Fick Corporation. was quantified using the index %High Object Convex Hull Area Ratio.

[0117] After 12 to 24 hours of culture, dendritic cells showed a spindle-shaped cell morphology with extended pseudopods. Incubation at 37°C for an additional 5 hours in the presence of mouse semaphorin 3A resulted in cell morphology. The regression of cell morphology was quantified using the method described above and the results are shown in Figure 1. show.

[0118] Example 3: Preparation of biotin-labeled human Plexin A1 The extracellular domain of human Plexin A1 (hPlexin A1), a single-pass transmembrane protein, is shown below. The amino acid sequence of NCBI Reference Sequence NP_115618.3 (SEQ ID NO: 3) was From the originally synthesized hPlexinA1 gene, the residues after alanine 1245, which are predicted to be the transmembrane region, were The remaining sequence was removed and a FLAG tag sequence (SEQ ID NO: 5) was added instead. The signal peptide (SEQ ID NO: 6) was replaced with the artificial signal peptide HMM+38 (SEQ ID NO: 7). The gene encoding the prepared hPlexinA1 (SEQ ID NO: 4) was inserted into an expression vector for animal cells. The vector was transfected into FreeStyle293 cells (Invitorgen) using 293Fectin (Invitorgen). In order to improve the expression efficiency of the target gene, a gene expressing EBNA1 (SEQ ID NO: 8) was used. The transfected cells were cultured at 37°C and 8% CO2 for 6 days. The target protein was secreted into the culture supernatant.

[0119] The cell culture medium containing the target hPlexinA1 was filtered through a 0.22 μm bottle-top filter. The supernatant was obtained. Anti-FLAG antibody M2 agarose (Sigma-Aldrich) equilibrated with D-PBS(-) (Wako Pure Chemical Industries, Ltd.) After applying the culture supernatant to the cells, D-PBS containing FLAG peptide was added to obtain the desired h Plexin A1 was then eluted using Superdex 200 (GE Healthcare) equilibrated with D-PBS(-). The fraction containing hPlexin A1 was separated by gel filtration chromatography.

[0120] The soluble hPlexinA1 prepared as described above was transfected with EZ-Link NHS-PEG4-Biotin (Thermo S Biotin-labeled hPlexinA1 was prepared using a reagent called a chromatograph.

[0121] Example 4: Preparation of soluble mouse Plexin A1 protein The soluble mouse PlexinA1 protein was synthesized using the NCBI Reference Sequence NP_032907.1 (SEQ ID NO: The signal peptide (from the N-terminus to the extracellular domain) was designed based on the amino acid sequence of the nucleotide ... The C A FLAG tag sequence (SEQ ID NO: 5) was inserted at the end. The gene was inserted into an expression vector and grown in Invitrogen FreeStyle293 cells. The soluble mouse Plexin A1 protein was isolated from the culture supernatant by the anti-FLAG M2 antibody A. Affinity purification and gel filtration chromatography using Affinity Gel (Sigma-Aldrich) The product was purified by filtration.

[0122] Example 5: Preparation of anti-Plexin A1 antibody Rabbits were immunized by the following method to produce anti-mouse Plexin A1 antibodies. The rabbits were initially immunized with soluble mouse IgG in complete Freund's adjuvant (CFA). A total of 100 μg of human Plexin A1 protein or soluble human Plexin A1 protein was injected intradermally. Then, soluble mouse PlexinA1 protein or soluble human PlexinA1 protein was added. Each time, 50 μg of the vaccine was administered at intervals of at least one week. The animals were then given two or more booster immunizations.

[0123] Subsequently, tissues were collected from the immunized rabbits and single cell suspensions were prepared. To identify the cause of the disease, spleens and blood samples were collected from individuals with elevated serum antibody titers, and peripheral blood mononuclear cells (PBMCs) were isolated from the blood. Peripheral blood mononuclear cells were prepared using the Ficoll method. After harvesting the spleen, a cell culture was performed. Single cell suspensions were generated by processing through a liner (BD Falcon) containing 0.5% BSA. Biotinylated soluble Plexin A1 protein was added to the cells in PBS(-) containing 1000 kJ / mL. The cell suspension was incubated on ice for 30 minutes. After washing with PBS(-) containing 0.5% BSA, the antibody bound to the cells was removed. Unbiotinylated soluble PlexinA1 protein was removed using MACS Streptavidin. The cells were added to thrombin beads (Miltenyi Biotec), suspended, and incubated on ice. Positive cell fraction bound to MACS streptavidin beads using the oMACS Pro Separator Anti-rabbit IgG-PE was added to the cells, and the cells were incubated on ice. Cells with high PE fluorescence were collected using CSAria (BD).

[0124] The collected B cells were cultured as follows. The culture medium was conditioned with activated rabbit T cells and EL4 cells ( The cells were collected in a 96-well plate containing the European Collection of Cell Cultures (ECC). Activated rabbit T cell conditioned medium is a medium prepared by inoculating thymocytes collected from rabbits with Phy tohaemagglutinin (Roche), phorbol 12-myristate 13-acetate (Sigma-Aldrich), and The medium was prepared by culturing these cells in RPMI-1640 containing FBS at 37°C and 5% CO2. After culturing for 5 to 12 days, a portion of the supernatant containing the secreted antibody was collected. The binding activity of the antibody to mouse Plexin A1 was evaluated.

[0125] Total RNA was collected from B cells with confirmed binding activity, and H-chain and L-chain antibodies were detected by RT-PCR. The variable region genes were synthesized. The rabbit antibody H chain constant region sequence (SEQ ID NO: 17) and rabbit The antibody L chain constant region sequence (SEQ ID NO: 18) was ligated to prepare an antibody expression vector. The H-chain and L-chain expression vectors derived from the WT were mixed and transfected into FreeStyle293 (Invitrogen). The antibody was purified from the culture supernatant.

[0126] The purified antibody was used to evaluate the antigen binding activity by ELISA using a mouse PlexinA1 expressing cell line. The results are shown in Figure 2. The anti-mouse Plexin A1 antibodies PXB361b, PXB693, and PXB727 It was confirmed that Plexin A1 specifically binds to cell lines expressing mouse Plexin A1 in a concentration-dependent manner. The amino acid sequences of the variable regions of the H and L chains of PXB361b are shown in SEQ ID NOs: 19 and 20, and the H and L chains of PXB693 are shown in SEQ ID NOs: 19 and 20. The amino acid sequences of the variable regions of the H and L chains of PXB727 are shown in SEQ ID NOs: 41 and 42. The sequences are shown in SEQ ID NOs: 43 and 44. The antagonist activity of anti-mouse Plexin A1 antibodies was evaluated using a marrow-derived dendritic cell retraction assay system. The results are shown in Figure 3. The obtained anti-mouse Plexin A1 antibody, PXB361b, showed a concentration-dependent Inhibition of dendritic cell retraction was observed, and antagonist activity of semaphorin 3A signaling was observed. It was confirmed that it exhibits

[0127] Example 6: Antibody evaluation by cell ELISA The antibodies obtained above were subjected to cell ELISA in the following manner. First, a 384-well plate was used. The mouse PlexinA1-expressing Ba / F3 cells and the Ba / F3 cells (see Example 11) were prepared separately. After washing each well of the plate with PBS, the prepared antibody was 0 μL / well and left to stand at room temperature for 1 hour. After that, each well was washed with 0.05% Tween 20-PBS. 20uL / well of HRP-labeled anti-rabbit IgG antibody (Betyl, A120-101P) diluted 10,000 times with 2% FBS-PBS was added. Each well of the plate was diluted with 0.05% Tween 20-PBS and left to stand at room temperature for 1 hour. After washing, 20 μL of substrate solution (ABTS peroxidase substrate system) was dispensed into each well and incubated at room temperature for 1 After allowing the color to develop for 2 hours, measure the absorbance at 405 nm using a Molecular Devices SpectraMax. Binding to mouse PlexinA1 was confirmed by

[0128] [Example 7: Preparation of a human antibody naive library] Poly(A) RNA prepared from human PBMC or commercially available human poly(A) RNA was used as a template for PCR. Gene libraries for antibody heavy chain variable regions and antibody light chain variable regions were amplified using the R method.

[0129] The constructed antibody heavy chain variable region gene library and antibody light chain variable region gene library The resulting vectors are combined and inserted into a phagemid vector to display a Fab domain consisting of a human antibody sequence. A human antibody phage display library was constructed using the Methods Mol Biol., (2002) 178, 87-100. The linker portion connecting the Fab of the mid and the phage pIII protein, and the helper phage p A trypsin cleavage sequence was inserted between the N2 and CT domains of the III protein gene. Sequences from a phage display library were used.

[0130] Example 8: Binding to hPlexinA1 from a human antibody naive library by bead panning Obtaining antibody fragments that bind to the antibody Phages were produced from E. coli harboring the constructed phagemid for phage display. By adding 2.5M NaCl / 10% PEG to the culture medium of E. coli in which phage was produced, The precipitated phage population was diluted with TBS to obtain a phage library solution. Next, BSA and CaCl2 were added to the phage library solution to a final concentration of 4% BSA. The calcium ion concentration was adjusted to 1.2 mM. The panning method using antigens immobilized on magnetic beads was referred to (J. Immunol. Me thods., (2008) 332 (1-2), 2-9, J. Immunol. Methods., (2001) 247 (1-2), 191-203, B iotechnol. Prog., (2002) 18(2) 212-20, Mol. Cell Proteomics, (2003) 2 (2), 61-9 As magnetic beads, NeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin- coated) or streptavidin-coated beads (Dynabeads M-280 Streptavidin) Ta.

[0131] Specifically, the prepared phage library solution was added with 250 pmol of the biotin-labeled hPlexi By adding nA1, the phage library solution was contacted with the antigen at room temperature for 60 minutes. BSA-blocked magnetic beads were added, and the complex between the antigen and the phage was attached to the magnetic beads. The beads were then incubated at room temperature for 15 minutes. The beads were then soaked in 1 mL of 1.2 mM CaCl2 / TBST (TBST containing 1.2 mM CaCl2). After washing three times with TBS (1.2 mM CaCl2), the cells were further washed with 1 mL of 1.2 mM CaCl2 / TBS (TBS containing 1.2 mM CaCl2). After washing twice, 0.5 mL of 1 mg / mL trypsin was added to the beads and suspended at room temperature for 15 minutes. Immediately afterwards, the beads were separated using a magnetic stand, and the phage solution was recovered. The phage solution was added to 10 mL of E. coli strain ER2738 in the logarithmic growth phase (OD600 0.4-0.7). The phages were transferred to the E. coli by gently stirring the E. coli at 37°C for 1 hour. The infected E. coli was inoculated onto a 225 mm x 225 mm plate. The phage library solution was prepared by recovering phages from the culture medium of the inoculated E. coli. This was prepared and used for the second panning.

[0132] In the second panning, 40 pmol of biotin-labeled antigen was added to the prepared phage library solution. The phage library solution was prepared by adding 100 ml of PBS and performing the same procedure as the first panning. This preparation was used for the third panning.

[0133] In the third panning, 10 pmol of biotin-labeled antigen was added to the prepared phage library solution. The same procedure as in the first and second panning was carried out, and the inoculated E. coli was recovered.

[0134] [Example 9: Evaluation by phage ELISA] From the single colony of E. coli obtained by the above method, the bacteria were isolated by the conventional method (Methods Mol. Biol. The phage-containing culture supernatant was collected according to the method described by I., (2002) 178, 133-145).

[0135] The culture supernatant containing phage supplemented with skim milk and CaCl2 was subjected to ELISA as follows: StreptaWell 96 microtiter plates (Roche) were prepared containing biotin-labeled antigen. Each well of the plate was coated with 100 μL of PBS containing 100 μL of PBS overnight. After removing the antigen, the wells were blocked with 250 μL of 0.02% skim milk-TBS for at least 1 hour. The plate was then filled with the prepared culture supernatant in each well, excluding the 0.02% skim milk-TBS. The plate was left to stand at 37°C for 1 hour, and the antibodies displayed on the phage were then transferred to each well. After washing with 1.2 mM CaCl2 / TBST, the plate was diluted with 1.2 mM CaCl2 / TBS for 2 h. A 5,000-fold diluted HRP-conjugated anti-M13 antibody (GE Healthcare, 27-9421-01) was added to each well. The plate was incubated for 1 hour. After washing with 1.2 mM CaCl2 / TBST, the plate was resuspended in TMB solution (LifeTechno The color reaction of the solution in each well to which IgG was added was stopped by adding sulfuric acid. Thereafter, the color development was measured by measuring the absorbance at 450 nm.

[0136] The clones subjected to the above phage ELISA were amplified using specific primers. The base sequence of the gene was analyzed, and based on the results of the phage ELISA and sequence analysis, The panning pools were converted from antibody fragments to full-length human antibodies and subjected to further evaluation.

[0137] Example 10: Conversion of antibody fragments to full-length antibodies, expression, and purification From the E. coli collected in the third panning, NucleoBond Xtra Midi Plus (MACHEREY-N The phagemid was extracted using the AGEL (740412.50). The variable region of the antibody was excised and the EF1 promoter and OriP, the origin of EBNA1 replication, were retained. The vector is ligated to a cassette vector containing an antibody constant region. The ligation product was used to transform Escherichia coli DH5α (TOYOBO, DNA-903), and the resulting From the single colonies, full-length antibody plasmids for expression in animal cells were extracted.

[0138] The antibody was expressed using the following method. (Invitrogen) were suspended in FreeStyle 293 Expression Medium (Invitrogen) and diluted to 5.0 × 10 4 Thin The cells were seeded in a 96-well plate at a density of 190 μL per well. Each plasmid was introduced into the cells by lipofection. The cells were cultured at 37°C, 8% CO2 for 5 days to allow the antibody to be secreted into the culture supernatant. The antibody was purified using Multiscreen HTS GV (Millipore, MSGVN2250).

[0139] Example 11: Construction of human and mouse Plexin A1-expressing cells The antigen-expressing cell line was constructed using the following method: CAG promoter and neomycin resistance gene. The human nucleotide sequence of the present invention, in which the signal peptide was replaced with HMM+38 (SEQ ID NO: 7), was added to the pCXND3 vector carrying the above-mentioned nucleotide sequence. PlexinA1 (NCBI Reference Sequence NP_115618.3) or mouse PlexinA1 (NCBI Reference Sequence NP_115618.3) e Sequence NP_032907.1) up to the transmembrane region (up to base 1300 in humans, 12 in mice) 90) was expressed as a protein fused to a Myc tag (SEQ ID NO: 51) at the C-terminus. A plasmid containing a cDNA constructed to encode human Plex was prepared. The amino acid sequence of mouse PlexinA1 is shown in SEQ ID NO: 15, and the amino acid sequence of mouse PlexinA1 is shown in SEQ ID NO: 37. Each is shown below.

[0140] The constructed plasmid was linearized by cleavage with the restriction enzyme PvuI and then transformed into Ba / F3 cells using GenePulser The cells were transfected by electroporation using X cell (Bio-Rad). After transfection, the cells were transfected by limiting dilution. The cells were seeded onto a 96-well plate and selected with G418. After 1-2 weeks, the selected cells were Visually check whether single colonies have formed in the cells. Some of the cells were collected and analyzed by Western blot analysis using Myc tag antibody. In parallel, cells were captured in a 96-well plate and incubated with 4% paraformaldehyde. , acetone, and methanol, and cells with holes in the cell membrane were treated with Myc tag antibody. These Western blot analyses and ELISA were performed to examine whether the antibody reacted with the IgG. The results of the SA method confirmed that human and mouse Plexin A1 were expressed in Ba / F3 cells. .

[0141] Example 12: Evaluation by cell ELISA The antibody supernatant obtained in Example 10 was subjected to cell ELISA in the following manner. Human Plexin A1- and mouse Plexin A1-expressing Ba / F3 cells were plated separately. Each well of the plate was washed with PBS, and then the prepared antibody was added. The supernatant was added at 20 μL / well and allowed to stand at room temperature for 1 hour. Then, each well was filled with 1 M Hepes (pH 7.4) Then, 20 μL / wet HRP-labeled anti-human IgG antibody (Invitrogen, AHI0304) diluted 5000 times with TBS was added. Each well of the plate was washed with 1M Hepes (pH 7.4). Then, 20 μL of substrate solution (ABTS peroxidase substrate system) was dispensed into each well and incubated at room temperature for 1 hour. After color development, the absorbance at 405 nm was measured using a SpectraMax manufactured by Molecular Devices. Thus, binding to human PlexinA1 and binding to mouse PlexinA1 were confirmed.

[0142] Absorbance to human PlexinA1 / BaF3 is 0.2 or higher and absorbance to mouse PlexinA1 / BaF3 is 0.2 or higher The antibodies were selected and subjected to the in vitro activity evaluation assay described below. The antibodies that showed the absorbance values ​​shown in Table 1 were human-mouse cross-reactive Plexin A1 antibodies (hPANL#240, hPANL#359 The vertical axis in Figure 4 is the absorbance value (405 nm) in cell ELISA, and the numerical values ​​are shown in the table. The amino acid sequence of the H chain of hPANL#240 is shown in SEQ ID NO: 9, and the amino acid sequence of the L chain is shown in SEQ ID NO: No. 10 (see also Table 2).

[0143] [Table 1]

[0144] [Table 2]

[0145] Example 13: Affinity maturation of selected antibodies The hPANL#359 selected in Example 12 was subjected to affinity maturation using a method known to those skilled in the art. Specifically, the Biochemical and Biophysical Research Communication ions, (2000), 275, 2, 553-557), and the light chain portion of the antibody is synthesized using a human light chain library. After creating a new library with substituted nucleotides, the library was panned twice against biotin-labeled human Plexin A1. From the resulting single colony of E. coli, the phage-containing culture supernatant was extracted. The cells were collected and subjected to phage ELISA in the same manner as described above.

[0146] The majority of the clones in question showed color development in the wells where the antigen was immobilized in phage ELISA. The base sequence was analyzed from the E. coli. Phagemid was also extracted from the E. coli and used for animal experiments. The vector was then introduced into the FreeStyle 293-F cell line (I The cells were transfected into a culture medium containing the antibody, and the culture supernatant containing the antibody was obtained.

[0147] Example 14: Confirmation of binding ability of obtained antibodies Using the antibody culture supernatant prepared in Example 13, the binding ability to human PlexinA1 was confirmed using Octet The assay was performed using RED384 (ForteBIO). Specifically, Protein G Biosensor (ForteBIO) was used. Antibody culture supernatant diluted to 5 μg / mL was immobilized, and then human Plexin A1 was applied. The binding response between the antibody immobilized on the sensor and the antigen was measured.

[0148] As a result, several antibodies were obtained that were confirmed to bind to human Plexin A1. The L chain (SEQ ID NO: 11, L chain (SEQ ID NO: 12)) showed better binding than the parent antibody before affinity maturation. It is an antibody with improved activity.

[0149] [Table 3]

[0150] In addition, these antibodies obtained from the human antibody naive library were used to express mouse kyphosphatase inhibitors using the method described below. Monomerized antibody (hPANL#240-mFc: H chain SEQ ID NO: 13, L chain SEQ ID NO: 10, 359B2-2-3-6-mFc H chain SEQ ID NO: 14, L chain SEQ ID NO: 12), and a mouse chimeric antibody derived from an immunized rabbit (PXB693 -mFc: H chain SEQ ID NO: 45, L chain SEQ ID NO: 46, PXB727-mFc: H chain SEQ ID NO: 47, L chain SEQ ID NO: 48, PXB361b-mFc: H chain SEQ ID NO: 49, L chain SEQ ID NO: 50) Evaluation was performed using Octet RED384 (ForteBIO). For the antibody, the L chain was not chimerized.

[0151] [Table 4]

[0152] Specifically, Protein G Biosensor (ForteBIO) was loaded with 10-fold diluted HBS-EP+ (GE healthcare, The antibody was prepared at 10 μg / mL using BR-1006-69) and immobilized on a solid phase, and the concentration was adjusted to 260 nM with HBS-EP+. Human and mouse Plexin A1 were applied. Protein G Biosensor was loaded with 10 mmol / L glycine-H. The regeneration was performed using Cl, pH 1.5 (GE Healthcare, BR-1003-54). After the measurement, the amount of binding was calculated. The analysis software Data analysis 7.0 (ForteBIO) was used for this purpose (Figure 5). PXB361b, PXB693, and PXB727 showed binding only to mouse PlexinA1, whereas hPANL#240 and 359B2- 2-3-6 was shown to be a human-mouse cross-reactive antibody. The amount of binding to mouse Plexin A1.

[0153] Example 15: Preparation of mouse chimeric antibodies The heavy chain variable region of the antibody was amplified with specific primers, and the mouse heavy chain constant region fragment was then cloned. The fragment was then linearized with restriction enzymes to form an animal cell expression vector, and the In-Fusion HD clone was then inserted into the vector. Cloning was performed using the ng kit (Takara Bio). hPANL#240, 359B2-2-3-6, PXB693, and PX B727 uses a heavy chain constant region derived from mouse IgG2a, and PXB361b uses a heavy chain constant region derived from mouse IgG1. The heavy chain constant region was used.

[0154] In addition, the light chain variable region of PXB693, PXB727, and PXB361b was amplified with specific primers, The mouse light chain constant region was linearized with a restriction enzyme and inserted into an animal cell expression vector. Cloning was performed using the -Fusion HD cloning kit (Takara Bio).

[0155] A heavy chain expression vector with a mouse constant region and a human light chain sequence expression vector or a mouse The light chain expression vector containing the constant region was transfected into FreeStyle 293-F by lipofection. The cells were then transfected into a strain (Invitrogen) and incubated in a CO2 incubator (37°C, 8% CO2, 90 rpm) for 5 days. After the incubation, the cells were purified by rProtein A Sepharose™ Fast Flow (Amersham Biosciences) according to the method known to those skilled in the art. The antibody was purified from the culture supernatant obtained above by a known method. Purification was performed using a spectrophotometer. The absorbance of the antibody solution at 280 nm is measured, and the extinction coefficient calculated by the PACE method is used. The antibody concentration was calculated from the measured values ​​(Protein Science, (1995) 4, 2 411-2423).

[0156] Example 16: Epitope binning Five types of mouse chimeric antibodies (PXB361b-mFc, PXB693-mFc, PXB727-mFc) prepared in Example 15 c, hPANL#240-mFc, 359B2-2-3-6-mFc), any combination of the two antibodies We investigated whether the antibody could bind to the original mouse Plexin A1 without competing with it by using Octet RED 384 (ForteBIO). By examining the competitive patterns, five types of mouse chimeric antibodies were identified as having the same epitope. See general epitope binning methods. This was carried out (mAbs (2012) 5:2, 270-278).

[0157] Specifically, five types of Protein G biosensors were loaded with 10 μg / mL of “Ab1.” Antibodies (tentatively referred to as antibodies A to E) were immobilized on the solid phase, and mouse Plexin A1 was prepared at 260 nM. Then, antibody A was applied as "Ab2" to five cells on which five types of "Ab1" were immobilized. The Protein G Biosensor was bound to a 10 mmol / L Glycine-HCl, pH 1.5 (GE Healthcare After regeneration using the antibody, five types of Ab1 were immobilized on the sensor again. After applying the antigen, antibody B was then bound as "Ab2." This procedure was repeated up to antibody E while regenerating.

[0158] The parameters were calculated using the analysis software Data Analysis 7.0 (ForteBIO). When antibody A and antibody B compete, the antigen bound to immobilized antibody A is stimulated by the binding response of antibody B. Furthermore, when antibody A and antibody B are non-competitive, no signal is observed binding to immobilized antibody A. An increase in the binding response of antibody B to the antigen can be confirmed.

[0159] {"Ab2; antibody B" binds to the antigen and the binding response after 200 seconds} to {"Ab1; antibody A" The binding response of the bound antigen was subtracted from the value of {"Ab1; Antibody B" Protein G Bios The value obtained by dividing the antibody B by the immobilization response to the sensor was used as the competitive ratio of antibody B to antibody A (Figure 6). PXB693 was used as Ab2.

[0160] As a result, PXB361b, which was obtained as an antagonist antibody, was obtained as an agonist antibody. It was revealed that the antibody did not compete with the other four antibodies obtained. It was shown that the two (PXB693, PXB727, hPANL#240, 359B2-2-3-6) compete with each other.

[0161] Example 17: Evaluation of semaphorin 3A activity on U87-MG cells using the xCELLigence system Valence System Construction To evaluate the effects of Semaphorin 3A protein in human cells, we used the xCELLigence sy The assay was performed using stem (ACEA). The xCELLigence system uses a dedicated plate bottom. The electrical resistance generated between the macroelectrodes placed on the cell surface and the cell adhesion surface can be used to evaluate morphological changes and migration. The assay was carried out as follows.

[0162] U87-MG cells, a human glioblastoma cell line, cultured in EMEM medium (ATCC) containing 5% FBS U87-MG cells were plated on an E-plate 96 (ACEA), a dedicated plate for the xCELLigence system, at 1 x10 4 The cells were plated at a density of 12 cells / well and cultured at 37°C for 12-24 hours. The solution was diluted to an appropriate concentration with 100% FBS-containing EMEM medium, added to the cell culture medium, and then injected into the xCELLigence system (AC The cell index value of each well was measured using a chromatograph (EA). Cell index 1 hour after addition of semaphorin 3A The values ​​are shown in Figure 7. A concentration-dependent decrease in the cell index value due to semaphorin 3A was confirmed. The cell index value was used as an index of the size of the cell morphology or the adhesive strength of the cells. .

[0163] Example 18: Evaluation of activity of anti-mouse Plexin A1 antibody Mouse semaphorin 3A-dependent mouse bone marrow-derived dendritic cell retraction assay system described in Example 2 The activity of the prepared anti-mouse Plexin A1 antibody and anti-human / mouse Plexin A1 antibody was evaluated using Specifically, mouse bone marrow derived cells were cultured in RPMI1640 medium containing FBS and mouse GM-CSF. 2 × 10 dendritic cells 4 Cells / well were seeded onto a 96-well plate and cultured at 37°C for 12 to 24 hours. Anti-mouse Plexin A1 antibody and anti-human / mouse Plexin A1 antibody were used, as well as the same antibody as a control. Isotype anti-KLH antibody and mouse semaphorin 3A were incubated in RPMI 1640 medium containing 10% FBS. The mixture was diluted to a suitable concentration, added to the cell culture medium, and cultured at 37°C for 5 hours. The regression of dendritic cell morphology was evaluated by quantifying it using the method. The results are shown in Figure 8. Anti-Plexin A1 antibodies (PXB693-mFc, PXB727-mFc) and anti-human / mouse Plexin A1 antibodies (359B2- 2-3-6-mFc) promotes dendritic cell retraction in a concentration-dependent manner, and is responsive to semaphorin 3A signaling. It was confirmed that the compound exhibited similar activity.

[0164] Example 19: Evaluation of activity of anti-human / mouse Plexin A1 antibodies Using the xCELLigence system for measuring human semaphorin 3A activity described in Example 17, The activity of the generated anti-human / mouse Plexin A1 antibodies was evaluated. U87-MG cells were cultured in xCELLigence 1 × 10 4 The cells were plated at a density of 12 cells / well and incubated at 37°C for 12 h. The cells were cultured for 24 hours. Anti-human / mouse Plexin A1 antibody and the same isotype as a control were used. Anti-KLH antibody was diluted to an appropriate concentration in EMEM medium containing 5% FBS and added to the wells. One hour later, The cell index value of each well was measured. The results are shown in Figure 9. Anti-human / mouse Plexin A1 antibody (hPANL#240, 359B2-2-3-6) reduced the cell index in a concentration-dependent manner and suppressed the expression of semaphorin 3A. It was confirmed that the activity was similar to that of the signal.

[0165] Example 20: Preparation of recombinant mouse Plexin A1 sema domain protein The mouse PlexinA1 sema domain protein gene is available from NCBI Reference Sequence NP_03290 Based on the sequence of 7.1, a signal peptide (from the N-terminus to the 24th isoleucine) was created as an artificial signal peptide. The peptide HMM+38 (SEQ ID NO: 7) was substituted, and a FLAG tag (SEQ ID NO: 5) was inserted after the serine at position 512. ) and a stop codon were added, and the sequence was synthesized. The amino acid sequence is shown in SEQ ID NO: 38. The prepared gene was inserted into an expression vector and then purified by Invitrogen. The mouse PlexinA1 sema domain protein was isolated from the culture supernatant of FreeStyle293 cells. The protein was purified by affinity purification using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich). It was purified by gel filtration chromatography.

[0166] Example 21: Preparation of recombinant mouse Plexin A2 sema domain protein The mouse PlexinA2 sema domain protein gene is available from NCBI Reference Sequence NP_03290. Based on the sequence of 8.2 (SEQ ID NO: 53), the signal peptide (from the N-terminus to the 31st glycine) The artificial signal peptide HMM+38 (SEQ ID NO: 7) was substituted with a FLAG tag after the 510th serine. The sequence was designed to encode the sequence (SEQ ID NO: 5) with a stop codon added, and gene synthesis was carried out. The amino acid sequence is shown in SEQ ID NO: 39. The gene thus prepared was incorporated into an expression vector. The mouse Plexin A2 was expressed in Invitrogen FreeStyle293 cells and purified from the culture supernatant. The ema domain protein was purified using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich). It was purified by affinity purification and gel filtration chromatography.

[0167] Example 22: Preparation of mouse Plexin A1 / A2 sema domain chimeric protein The mouse PlexinA1 / A2 sema domain chimeric protein gene is based on the NCBI Reference Sequence Based on the sequence of NP_032907.1, a signal peptide (from the N-terminus to the 24th isoleucine) was artificially created. The signal peptide was replaced with HMM+38 (SEQ ID NO: 7), and the mouse PlexinA1 sema domain protein The sequence from isoleucine at position 458 of the protein is 459 of the mouse PlexinA2 sema domain protein. The sequence from arginine at position 510 to serine at position 510, a FLAG tag (SEQ ID NO: 5), and a stop code The amino acid sequence was designed to encode a sequence with an added amino acid sequence, and was produced by gene synthesis. The gene was inserted into an expression vector and then transferred to Invitrogen FreeStyle2. The mouse Plexin A1 / A2 sema domain protein was extracted from the culture supernatant of 93 cells. Affinity purification and gel filtration using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich) Purification was carried out by chromatography.

[0168] Example 23: Identification of binding site region of anti-Plexin A1 antibody The mouse chimeric antibodies PXB693-mFc (mouse IgG2a) and PXB727-mFc (mouse IgG2a) prepared in Example 15 were PXB361b-mFc (mouse IgG2a), PXB361b-mFc (mouse IgG1), and KLH antibodies of each isotype were used. After binding to protein G-immobilized magnetic beads, the protein solution prepared in Examples 20 to 22 was After the reaction, the magnetic beads were collected and diluted 4 times with a sample buffer (3-mercapto-1 Three types of mouse Plex were added and heated. The inA1 sema domain protein and the protein released from the beads were separated by SDS-PAGE, and PV After transferring to a DF membrane, the samples were analyzed using the iBind Western System (Life Technologies) using alkaline phosphatase. After reacting with phospholipase-labeled anti-FLAG antibody (Sigma-Aldrich), the BCIP-NBT solution kit (Nacalai) was used. The target protein was detected using a Western blot assay (TESQ). The results of the Western blot are shown in Figure 10. As a result, the agonist antibodies (PXB693-mFc, PXB727-mFc) were found to have the same amino acid sequence as PlexinA1. It was found that the area underlined in Figure 11 was recognized. The underlined region of the human PlexinA1 sema domain corresponds to the region shown in The homology between the PlexinA1 sema domain and the mouse PlexinA1 sema domain is quite high. From the data, it can be seen that the anti-human Plexin A1 agonist antibody of the present invention binds to the underlined region of human Plexin A1. It is thought that it exerts its agonistic action and effect by binding to the epitope present in the It should be noted that, with reference to the description in Examples 20 to 22, human PlexinA1 sema domain recombinant Recombinant protein, human PlexinA2 sema domain recombinant protein and human Plexin A1 / A2 sema domain chimeric protein was prepared and cloned into hPANL#240 and 35 by the method described in Example 23. The region where 9B2-2-3-6 binds can be confirmed. [Sequence List Free Text]

[0169] SEQ ID NO: 1: Amino acid sequence of mouse semaphorin 3A recombinant protein SEQ ID NO: 2: Amino acid sequence of human semaphorin 3A recombinant protein SEQ ID NO: 3: Amino acid sequence of human Plexin A1 (NCBI Reference Sequence NP_115618.3 ) SEQ ID NO: 4: Amino acid sequence of human Plexin A1 recombinant protein SEQ ID NO: 5: FLAG tag sequence SEQ ID NO: 6: Amino acid sequence of the signal peptide of human Plexin A1 SEQ ID NO: 7: Amino acid sequence of artificial signal peptide HMM+38 SEQ ID NO: 8: Amino acid sequence of EBNA1 SEQ ID NO: 9: Amino acid sequence of the H chain of human-mouse cross-reactive PlexinA1 antibody hPANL#240 SEQ ID NO: 10: Amino acid sequence of the L chain of human-mouse cross-reactive PlexinA1 antibody hPANL#240 SEQ ID NO: 11: Amino acid sequence of the H chain of human-mouse cross-reactive PlexinA1 antibody 359B2-2-3-6 SEQ ID NO: 12: Amino acid sequence of the L chain of human-mouse cross-reactive PlexinA1 antibody 359B2-2-3-6 SEQ ID NO: 13: Amino acid sequence of the H chain of mouse chimeric human-mouse cross-reactive PlexinA1 antibody hPANL#240-mFc Acid sequence SEQ ID NO: 14: H chain sequence of mouse chimeric human-mouse cross-reactive PlexinA1 antibody 359B2-2-3-6-mFc amino acid sequence SEQ ID NO: 15: Amino acid sequence of human Plexin A1 recombinant protein SEQ ID NO: 16: Amino acid sequence of soluble mouse PlexinA1 protein SEQ ID NO: 17: Rabbit antibody H chain constant region sequence SEQ ID NO: 18: Rabbit antibody L chain constant region sequence SEQ ID NO: 19: Amino acid sequence of the H chain variable region of anti-mouse PlexinA1 antibody PXB361b SEQ ID NO: 20: Amino acid sequence of the L chain variable region of anti-mouse PlexinA1 antibody PXB361b SEQ ID NO: 21: Amino acid sequence of the heavy chain variable region of hPANL#240 SEQ ID NO: 22: Amino acid sequence of the L chain variable region of hPANL#240 SEQ ID NO: 23: Amino acid sequence of H chain CDR1 of hPANL#240 SEQ ID NO: 24: Amino acid sequence of H chain CDR2 of hPANL#240 SEQ ID NO: 25: Amino acid sequence of the H chain CDR3 of hPANL#240 SEQ ID NO: 26: Amino acid sequence of L chain CDR1 of hPANL#240 SEQ ID NO: 27: Amino acid sequence of L chain CDR2 of hPANL#240 SEQ ID NO: 28: Amino acid sequence of the L chain CDR3 of hPANL#240 SEQ ID NO: 29: Amino acid sequence of H chain CDR1 of 359B2-2-3-6 SEQ ID NO: 30: Amino acid sequence of the heavy chain CDR2 of 359B2-2-3-6 SEQ ID NO: 31: Amino acid sequence of the heavy chain CDR3 of 359B2-2-3-6 SEQ ID NO: 32: Amino acid sequence of L chain CDR1 of 359B2-2-3-6 SEQ ID NO: 33: Amino acid sequence of L chain CDR2 of 359B2-2-3-6 SEQ ID NO: 34: Amino acid sequence of L chain CDR3 of 359B2-2-3-6 SEQ ID NO: 35: Amino acid sequence of the heavy chain variable region of 359B2-2-3-6 SEQ ID NO: 36: Amino acid sequence of the light chain variable region of 359B2-2-3-6 SEQ ID NO: 37: Amino acid sequence of mouse Plexin A1 recombinant protein SEQ ID NO: 38: Amino acid sequence of mouse PlexinA1 sema domain recombinant protein SEQ ID NO: 39: Amino acid sequence of mouse PlexinA2 sema domain recombinant protein SEQ ID NO: 40: Amino acid sequence of mouse PlexinA1 / A2 sema domain chimeric protein SEQ ID NO: 41: Amino acid sequence of the H chain variable region of anti-mouse PlexinA1 antibody PXB693 SEQ ID NO: 42: Amino acid sequence of the L chain variable region of anti-mouse PlexinA1 antibody PXB693 SEQ ID NO: 43: Amino acid sequence of the H chain variable region of anti-mouse PlexinA1 antibody PXB727 SEQ ID NO: 44: Amino acid sequence of the L chain variable region of anti-mouse PlexinA1 antibody PXB727 SEQ ID NO: 45: Amino acid sequence of the heavy chain of mouse chimeric anti-mouse PlexinA1 antibody PXB693 SEQ ID NO: 46: Amino acid sequence of the L chain of mouse chimeric anti-mouse PlexinA1 antibody PXB693 SEQ ID NO: 47: Amino acid sequence of the heavy chain of mouse chimeric anti-mouse PlexinA1 antibody PXB727 SEQ ID NO: 48: Amino acid sequence of the L chain of mouse chimeric anti-mouse PlexinA1 antibody PXB727 SEQ ID NO: 49: Amino acid sequence of the heavy chain of mouse chimeric anti-mouse PlexinA1 antibody PXB361b SEQ ID NO: 50: Amino acid sequence of the L chain of mouse chimeric anti-mouse PlexinA1 antibody PXB361b SEQ ID NO: 51: Myc tag sequence SEQ ID NO: 52: Amino acid sequence of mouse PlexinA1 (NCBI Reference Sequence NP_032907.1 ) SEQ ID NO: 53: Amino acid sequence of mouse PlexinA2 (NCBI Reference Sequence NP_032908.2 ) SEQUENCE LISTING <110> Osaka University Cyugai Seiyaku Kabushiki Kaisha <120> ANTI-PLEXIN A1 AGONIST ANTIBODY <130> PCG-9044WO <150> JP2015-132067 <151> 2015-06-30 <160> 53 <170> PatentIn version 3.5 <210> 1 <211> 780 <212> PRT <213> Artificial Sequence <220> <223> Mouse semaphorin 3A recombinant protein <400> 1 Met Trp Trp Arg Leu Trp Trp Leu Leu Leu Leu Leu Leu Leu Leu Trp 1 5 10 15 Pro Met Val Trp Ala Glu Asp Arg His His His His His Asn Tyr 20 25 30 Ala Asn Gly Lys Asn Asn Val Pro Arg Leu Lys Leu Ser Tyr Lys Glu 35 40 45 Met Leu Glu Ser Asn Asn Val Ile Thr Phe Asn Gly Leu Ala Asn Ser 50 55 60 Ser Ser Tyr His Thr Phe Leu Leu Asp Glu Glu Arg Ser Arg Leu Tyr 65 70 75 80 Val Gly Ala Lys Asp His Ile Phe Ser Phe Asn Leu Val Asn Ile Lys 85 90 95 Asp Phe Gln Lys Ile Val Trp Pro Val Ser Tyr Thr Arg Arg Asp Glu 100 105 110 Cys Lys Trp Ala Gly Lys Asp Ile Leu Lys Glu Cys Ala Asn Phe Ile 115 120 125 Lys Val Leu Glu Ala Tyr Asn Gln Thr His Leu Tyr Ala Cys Gly Thr 130 135 140 Gly Ala Phe His Pro Ile Cys Thr Tyr Ile Glu Val Gly His His Pro 145 150 155 160 Glu Asp Asn Ile Phe Lys Leu Gln Asp Ser His Phe Glu Asn Gly Arg 165 170 175 Gly Lys Ser Pro Tyr Asp Pro Lys Leu Leu Thr Ala Ser Leu Leu Ile 180 185 190 Asp Gly Glu Leu Tyr Ser Gly Thr Ala Ala Asp Phe Met Gly Arg Asp 195 200 205 Phe Ala Ile Phe Arg Thr Leu Gly His His His Pro Ile Arg Thr Glu 210 215 220 Gln His Asp Ser Arg Trp Leu Asn Asp Pro Arg Phe Ile Ser Ala His 225 230 235 240 Leu Ile Pro Glu Ser Asp Asn Pro Glu Asp Asp Lys Val Tyr Phe Phe 245 250 255 Phe Arg Glu Asn Ala Ile Asp Gly Glu His Ser Gly Lys Ala Thr His 260 265 270 Ala Arg Ile Gly Gln Ile Cys Lys Asn Asp Phe Gly Gly His Arg Ser 275 280 285 Leu Val Asn Lys Trp Thr Thr Phe Leu Lys Ala Arg Leu Ile Cys Ser 290 295 300 Val Pro Gly Pro Asn Gly Ile Asp Thr His Phe Asp Glu Leu Gln Asp 305 310 315 320 Val Phe Leu Met Asn Ser Lys Asp Pro Lys Asn Pro Ile Val Tyr Gly 325 330 335 Val Phe Thr Thr Ser Ser Asn Ile Phe Lys Gly Ser Ala Val Cys Met 340 345 350 Tyr Ser Met Ser Asp Val Arg Arg Val Phe Leu Gly Pro Tyr Ala His 355 360 365 Arg Asp Gly Pro Asn Tyr Gln Trp Val Pro Tyr Gln Gly Arg Val Pro 370 375 380 Tyr Pro Arg Pro Gly Thr Cys Pro Ser Lys Thr Phe Gly Gly Phe Asp 385 390 395 400 Ser Thr Lys Asp Leu Pro Asp Asp Val Ile Thr Phe Ala Arg Ser His 405 410 415 Pro Ala Met Tyr Asn Pro Val Phe Pro Ile Asn Asn Arg Pro Ile Met 420 425 430 Ile Lys Thr Asp Val Asn Tyr Gln Phe Thr Gln Ile Val Val Asp Arg 435 440 445 Val Asp Ala Glu Asp Gly Gln Tyr Asp Val Met Phe Ile Gly Thr Asp 450 455 460 Val Gly Thr Val Leu Lys Val Val Ser Val Pro Lys Glu Thr Trp His 465 470 475 480 Asp Leu Glu Glu Val Leu Leu Glu Glu Met Thr Val Phe Arg Glu Pro 485 490 495 Thr Thr Ile Ser Ala Met Glu Leu Ser Thr Lys Gln Gln Gln Leu Tyr 500 505 510 Ile Gly Ser Thr Ala Gly Val Ala Gln Leu Pro Leu His Arg Cys Asp 515 520 525 Ile Tyr Gly Lys Ala Cys Ala Glu Cys Cys Leu Ala Arg Asp Pro Tyr 530 535 540 Cys Ala Trp Asp Gly Ser Ser Cys Ser Arg Tyr Phe Pro Thr Ala Lys 545 550 555 560 Arg Ala Thr Arg Ala Gln Asp Ile Arg Asn Gly Asp Pro Leu Thr His 565 570 575 Cys Ser Asp Leu Gln His His Asp Asn His His Gly Pro Ser Leu Glu 580 585 590 Glu Arg Ile Ile Tyr Gly Val Glu Asn Ser Ser Thr Phe Leu Glu Cys 595 600 605 Ser Pro Lys Ser Gln Arg Ala Leu Val Tyr Trp Gln Phe Gln Arg Arg 610 615 620 Asn Glu Asp Arg Lys Glu Glu Ile Arg Met Gly Asp His Ile Ile Arg 625 630 635 640 Thr Glu Gln Gly Leu Leu Leu Arg Ser Leu Gln Lys Lys Asp Ser Gly 645 650 655 Asn Tyr Leu Cys His Ala Val Glu His Gly Phe Met Gln Thr Leu Leu 660 665 670 Lys Val Thr Leu Glu Val Ile Asp Thr Glu His Leu Glu Glu Leu Leu 675 680 685 His Lys Asp Asp Asp Gly Asp Gly Ser Lys Ile Lys Glu Met Ser Ser 690 695 700 Ser Met Thr Pro Ser Gln Lys Val Trp Tyr Arg Asp Phe Met Gln Leu 705 710 715 720 Ile Asn His Pro Asn Leu Asn Thr Met Asp Glu Phe Cys Glu Gln Val 725 730 735 Trp Lys Arg Asp Arg Lys Gln Arg Arg Gln Arg Pro Gly His Ser Gln 740 745 750 Gly Ser Ser Asn Lys Trp Lys His Met Gln Glu Ser Lys Lys Gly Ala 755 760 765 Asn Ala Ala Thr His Glu Phe Glu Arg Ala Pro Arg 770 775 780 <210> 2 <211> 789 <212> PRT <213> Artificial Sequence <220> <223> Human semaphorin 3A recombinant protein <400> 2 Met Trp Trp Arg Leu Trp Trp Leu Leu Leu Leu Leu Leu Leu Leu Trp 1 5 10 15 Pro Met Val Trp Ala Glu Asp Arg His His His His His His Asn Tyr 20 25 30 Gln Asn Gly Lys Asn Asn Val Pro Arg Leu Lys Leu Ser Tyr Lys Glu 35 40 45 Met Leu Glu Ser Asn Asn Val Ile Thr Phe Asn Gly Leu Ala Asn Ser 50 55 60 Ser Ser Tyr His Thr Phe Leu Leu Asp Glu Glu Arg Ser Arg Leu Tyr 65 70 75 80 Val Gly Ala Lys Asp His Ile Phe Ser Phe Asp Leu Val Asn Ile Lys 85 90 95 Asp Phe Gln Lys Ile Val Trp Pro Val Ser Tyr Thr Arg Arg Asp Glu 100 105 110 Cys Lys Trp Ala Gly Lys Asp Ile Leu Lys Glu Cys Ala Asn Phe Ile 115 120 125 Lys Val Leu Lys Ala Tyr Asn Gln Thr His Leu Tyr Ala Cys Gly Thr 130 135 140 Gly Ala Phe His Pro Ile Cys Thr Tyr Ile Glu Ile Gly His His Pro 145 150 155 160 Glu Asp Asn Ile Phe Lys Leu Glu Asn Ser His Phe Glu Asn Gly Arg 165 170 175 Gly Lys Ser Pro Tyr Asp Pro Lys Leu Leu Thr Ala Ser Leu Leu Ile 180 185 190 Asp Gly Glu Leu Tyr Ser Gly Thr Ala Ala Asp Phe Met Gly Arg Asp 195 200 205 Phe Ala Ile Phe Arg Thr Leu Gly His His His Pro Ile Arg Thr Glu 210 215 220 Gln His Asp Ser Arg Trp Leu Asn Asp Pro Lys Phe Ile Ser Ala His 225 230 235 240 Leu Ile Ser Glu Ser Asp Asn Pro Glu Asp Asp Lys Val Tyr Phe Phe 245 250 255 Phe Arg Glu Asn Ala Ile Asp Gly Glu His Ser Gly Lys Ala Thr His 260 265 270 Ala Arg Ile Gly Gln Ile Cys Lys Asn Asp Phe Gly Gly His Arg Ser 275 280 285 Leu Val Asn Lys Trp Thr Thr Phe Leu Lys Ala Arg Leu Ile Cys Ser 290 295 300 Val Pro Gly Pro Asn Gly Ile Asp Thr His Phe Asp Glu Leu Gln Asp 305 310 315 320 Val Phe Leu Met Asn Phe Lys Asp Pro Lys Asn Pro Val Val Tyr Gly 325 330 335 Val Phe Thr Thr Ser Ser Asn Ile Phe Lys Gly Ser Ala Val Cys Met 340 345 350 Tyr Ser Met Ser Asp Val Arg Arg Val Phe Leu Gly Pro Tyr Ala His 355 360 365 Arg Asp Gly Pro Asn Tyr Gln Trp Val Pro Tyr Gln Gly Arg Val Pro 370 375 380 Tyr Pro Arg Pro Gly Thr Cys Pro Ser Lys Thr Phe Gly Gly Phe Asp 385 390 395 400 Ser Thr Lys Asp Leu Pro Asp Asp Val Ile Thr Phe Ala Arg Ser His 405 410 415 Pro Ala Met Tyr Asn Pro Val Phe Pro Met Asn Asn Arg Pro Ile Val 420 425 430 Ile Lys Thr Asp Val Asn Tyr Gln Phe Thr Gln Ile Val Val Asp Arg 435 440 445 Val Asp Ala Glu Asp Gly Gln Tyr Asp Val Met Phe Ile Gly Thr Asp 450 455 460 Val Gly Thr Val Leu Lys Val Val Ser Ile Pro Lys Glu Thr Trp Tyr 465 470 475 480 Asp Leu Glu Glu Val Leu Leu Glu Glu Met Thr Val Phe Arg Glu Pro 485 490 495 Thr Ala Ile Ser Ala Met Glu Leu Ser Thr Lys Gln Gln Gln Leu Tyr 500 505 510 Ile Gly Ser Thr Ala Gly Val Ala Gln Leu Pro Leu His Arg Cys Asp 515 520 525 Ile Tyr Gly Lys Ala Cys Ala Glu Cys Cys Leu Ala Arg Asp Pro Tyr 530 535 540 Cys Ala Trp Asp Gly Ser Ala Cys Ser Arg Tyr Phe Pro Thr Ala Lys 545 550 555 560 Arg Ala Thr Arg Ala Gln Asp Ile Arg Asn Gly Asp Pro Leu Thr His 565 570 575 Cys Ser Asp Leu His His Asp Asn His His Gly His Ser Pro Glu Glu 580 585 590 Arg Ile Ile Tyr Gly Val Glu Asn Ser Ser Thr Phe Leu Glu Cys Ser 595 600 605 Pro Lys Ser Gln Arg Ala Leu Val Tyr Trp Gln Phe Gln Arg Arg Asn 610 615 620 Glu Glu Arg Lys Glu Glu Ile Arg Val Asp Asp His Ile Ile Arg Thr 625 630 635 640 Asp Gln Gly Leu Leu Leu Arg Ser Leu Gln Gln Lys Asp Ser Gly Asn 645 650 655 Tyr Leu Cys His Ala Val Glu His Gly Phe Ile Gln Thr Leu Leu Lys 660 665 670 Val Thr Leu Glu Val Ile Asp Thr Glu His Leu Glu Glu Leu Leu His 675 680 685 Lys Asp Asp Asp Gly Asp Gly Ser Lys Thr Lys Glu Met Ser Asn Ser 690 695 700 Met Thr Pro Ser Gln Lys Val Trp Tyr Arg Asp Phe Met Gln Leu Ile 705 710 715 720 Asn His Pro Asn Leu Asn Thr Met Asp Glu Phe Cys Glu Gln Val Trp 725 730 735 Lys Arg Asp Arg Lys Gln Arg Arg Gln Arg Pro Gly His Thr Pro Gly 740 745 750 Asn Ser Asn Lys Trp Lys His Leu Gln Glu Asn Lys Lys Gly Ala Asn 755 760 765 Ala Ala Thr His Glu Phe Glu Arg Ala Pro Arg Ser Val Asp Tyr Lys 770 775 780 Asp Asp Asp Asp Lys 785 <210> 3 <211> 1896 <212> PRT <213> Homo sapiens <400> 3 Met Pro Leu Pro Pro Arg Ser Leu Gln Val Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Pro Gly Met Trp Ala Glu Ala Gly Leu Pro Arg 20 25 30 Ala Gly Gly Gly Ser Gln Pro Pro Phe Arg Thr Phe Ser Ala Ser Asp 35 40 45 Trp Gly Leu Thr His Leu Val Val His Glu Gln Thr Gly Glu Val Tyr 50 55 60 Val Gly Ala Val Asn Arg Ile Tyr Lys Leu Ser Gly Asn Leu Thr Leu 65 70 75 80 Leu Arg Ala His Val Thr Gly Pro Val Glu Asp Asn Glu Lys Cys Tyr 85 90 95 Pro Pro Pro Ser Val Gln Ser Cys Pro His Gly Leu Gly Ser Thr Asp 100 105 110 Asn Val Asn Lys Leu Leu Leu Leu Asp Tyr Ala Ala Asn Arg Leu Leu 115 120 125 Ala Cys Gly Ser Ala Ser Gln Gly Ile Cys Gln Phe Leu Arg Leu Asp 130 135 140 Asp Leu Phe Lys Leu Gly Glu Pro His His Arg Lys Glu His Tyr Leu 145 150 155 160 Ser Ser Val Gln Glu Ala Gly Ser Met Ala Gly Val Leu Ile Ala Gly 165 170 175 Pro Pro Gly Gln Gly Gln Ala Lys Leu Phe Val Gly Thr Pro Ile Asp 180 185 190 Gly Lys Ser Glu Tyr Phe Pro Thr Leu Ser Ser Arg Arg Leu Met Ala 195 200 205 Asn Glu Glu Asp Ala Asp Met Phe Gly Phe Val Tyr Gln Asp Glu Phe 210 215 220 Val Ser Ser Gln Leu Lys Ile Pro Ser Asp Thr Leu Ser Lys Phe Pro 225 230 235 240 Ala Phe Asp Ile Tyr Tyr Val Tyr Ser Phe Arg Ser Glu Gln Phe Val 245 250 255 Tyr Tyr Leu Thr Leu Gln Leu Asp Thr Gln Leu Thr Ser Pro Asp Ala 260 265 270 Ala Gly Glu His Phe Phe Thr Ser Lys Ile Val Arg Leu Cys Val Asp 275 280 285 Asp Pro Lys Phe Tyr Ser Tyr Val Glu Phe Pro Ile Gly Cys Glu Gln 290 295 300 Ala Gly Val Glu Tyr Arg Leu Val Gln Asp Ala Tyr Leu Ser Arg Pro 305 310 315 320 Gly Arg Ala Leu Ala His Gln Leu Gly Leu Ala Glu Asp Glu Asp Val 325 330 335 Leu Phe Thr Val Phe Ala Gln Gly Gln Lys Asn Arg Val Lys Pro Pro 340 345 350 Lys Glu Ser Ala Leu Cys Leu Phe Thr Leu Arg Ala Ile Lys Glu Lys 355 360 365 Ile Lys Glu Arg Ile Gln Ser Cys Tyr Arg Gly Glu Gly Lys Leu Ser 370 375 380 Leu Pro Trp Leu Leu Asn Lys Glu Leu Gly Cys Ile Asn Ser Pro Leu 385 390 395 400 Gln Ile Asp Asp Asp Phe Cys Gly Gln Asp Phe Asn Gln Pro Leu Gly 405 410 415 Gly Thr Val Thr Ile Glu Gly Thr Pro Leu Phe Val Asp Lys Asp Asp 420 425 430 Gly Leu Thr Ala Val Ala Ala Tyr Asp Tyr Arg Gly Arg Thr Val Val 435 440 445 Phe Ala Gly Thr Arg Ser Gly Arg Ile Arg Lys Ile Leu Val Asp Leu 450 455 460 Ser Asn Pro Gly Gly Arg Pro Ala Leu Ala Tyr Glu Ser Val Val Ala 465 470 475 480 Gln Glu Gly Ser Pro Ile Leu Arg Asp Leu Val Leu Ser Pro Asn His 485 490 495 Gln Tyr Leu Tyr Ala Met Thr Glu Lys Gln Val Thr Arg Val Pro Val 500 505 510 Glu Ser Cys Val Gln Tyr Thr Ser Cys Glu Leu Cys Leu Gly Ser Arg 515 520 525 Asp Pro His Cys Gly Trp Cys Val Leu His Ser Ile Cys Ser Arg Arg 530 535 540 Asp Ala Cys Glu Arg Ala Asp Glu Pro Gln Arg Phe Ala Ala Asp Leu 545 550 555 560 Leu Gln Cys Val Gln Leu Thr Val Gln Pro Arg Asn Val Ser Val Thr 565 570 575 Met Ser Gln Val Pro Leu Val Leu Gln Ala Trp Asn Val Pro Asp Leu 580 585 590 Ser Ala Gly Val Asn Cys Ser Phe Glu Asp Phe Thr Glu Ser Glu Ser 595 600 605 Val Leu Glu Asp Gly Arg Ile His Cys Arg Ser Pro Ser Ala Arg Glu 610 615 620 Val Ala Pro Ile Thr Arg Gly Gln Gly Asp Gln Arg Val Val Lys Leu 625 630 635 640 Tyr Leu Lys Ser Lys Glu Thr Gly Lys Lys Phe Ala Ser Val Asp Phe 645 650 655 Val Phe Tyr Asn Cys Ser Val His Gln Ser Cys Leu Ser Cys Val Asn 660 665 670 Gly Ser Phe Pro Cys His Trp Cys Lys Tyr Arg His Val Cys Thr His 675 680 685 Asn Val Ala Asp Cys Ala Phe Leu Glu Gly Arg Val Asn Val Ser Glu 690 695 700 Asp Cys Pro Gln Ile Leu Pro Ser Thr Gln Ile Tyr Val Pro Val Gly 705 710 715 720 Val Val Lys Pro Ile Thr Leu Ala Ala Arg Asn Leu Pro Gln Pro Gln 725 730 735 Ser Gly Gln Arg Gly Tyr Glu Cys Leu Phe His Ile Pro Gly Ser Pro 740 745 750 Ala Arg Val Thr Ala Leu Arg Phe Asn Ser Ser Ser Leu Gln Cys Gln 755 760 765 Asn Ser Ser Tyr Ser Tyr Glu Gly Asn Asp Val Ser Asp Leu Pro Val 770 775 780 Asn Leu Ser Val Val Trp Asn Gly Asn Phe Val Ile Asp Asn Pro Gln 785 790 795 800 Asn Ile Gln Ala His Leu Tyr Lys Cys Pro Ala Leu Arg Glu Ser Cys 805 810 815 Gly Leu Cys Leu Lys Ala Asp Pro Arg Phe Glu Cys Gly Trp Cys Val 820 825 830 Ala Glu Arg Arg Cys Ser Leu Arg His His Cys Ala Ala Asp Thr Pro 835 840 845 Ala Ser Trp Met His Ala Arg His Gly Ser Ser Arg Cys Thr Asp Pro 850 855 860 Lys Ile Leu Lys Leu Ser Pro Glu Thr Gly Pro Arg Gln Gly Gly Thr 865 870 875 880 Arg Leu Thr Ile Thr Gly Glu Asn Leu Gly Leu Arg Phe Glu Asp Val 885 890 895 Arg Leu Gly Val Arg Val Gly Lys Val Leu Cys Ser Pro Val Glu Ser 900 905 910 Glu Tyr Ile Ser Ala Glu Gln Ile Val Cys Glu Ile Gly Asp Ala Ser 915 920 925 Ser Val Arg Ala His Asp Ala Leu Val Glu Val Cys Val Arg Asp Cys 930 935 940 Ser Pro His Tyr Arg Ala Leu Ser Pro Lys Arg Phe Thr Phe Val Thr 945 950 955 960 Pro Thr Phe Tyr Arg Val Ser Pro Ser Arg Gly Pro Leu Ser Gly Gly 965 970 975 Thr Trp Ile Gly Ile Glu Gly Ser His Leu Asn Ala Gly Ser Asp Val 980 985 990 Ala Val Ser Val Gly Gly Arg Pro Cys Ser Phe Ser Trp Arg Asn Ser 995 1000 1005 Arg Glu Ile Arg Cys Leu Thr Pro Pro Gly Gln Ser Pro Gly Ser 1010 1015 1020 Ala Pro Ile Ile Ile Asn Ile Asn Arg Ala Gln Leu Thr Asn Pro 1025 1030 1035 Glu Val Lys Tyr Asn Tyr Thr Glu Asp Pro Thr Ile Leu Arg Ile 1040 1045 1050 Asp Pro Glu Trp Ser Ile Asn Ser Gly Gly Thr Leu Leu Thr Val 1055 1060 1065 Thr Gly Thr Asn Leu Ala Thr Val Arg Glu Pro Arg Ile Arg Ala 1070 1075 1080 Lys Tyr Gly Gly Ile Glu Arg Glu Asn Gly Cys Leu Val Tyr Asn 1085 1090 1095 Asp Thr Thr Met Val Cys Arg Ala Pro Ser Val Ala Asn Pro Val 1100 1105 1110 Arg Ser Pro Pro Glu Leu Gly Glu Arg Pro Asp Glu Leu Gly Phe 1115 1120 1125 Val Met Asp Asn Val Arg Ser Leu Leu Val Leu Asn Ser Thr Ser 1130 1135 1140 Phe Leu Tyr Tyr Pro Asp Pro Val Leu Glu Pro Leu Ser Pro Thr 1145 1150 1155 Gly Leu Leu Glu Leu Lys Pro Ser Ser Pro Leu Ile Leu Lys Gly 1160 1165 1170 Arg Asn Leu Leu Pro Pro Ala Pro Gly Asn Ser Arg Leu Asn Tyr 1175 1180 1185 Thr Val Leu Ile Gly Ser Thr Pro Cys Thr Leu Thr Val Ser Glu 1190 1195 1200 Thr Gln Leu Leu Cys Glu Ala Pro Asn Leu Thr Gly Gln His Lys 1205 1210 1215 Val Thr Val Arg Ala Gly Gly Phe Glu Phe Ser Pro Gly Thr Leu 1220 1225 1230 Gln Val Tyr Ser Asp Ser Leu Leu Thr Leu Pro Ala Ile Val Gly 1235 1240 1245 Ile Gly Gly Gly Gly Gly Leo Leo Leo Leo Val Ile Val Ala Val 1250 1255 1260 Leu Ile Ala Tyr Lys Arg Lys Ser Arg Asp Ala Asp Arg Thr Leu 1265 1270 1275 Lys Arg Leu Gln Leu Gln Met Asp Asn Leu Glu Ser Arg Val Ala 1280 1285 1290 Leu Glu Cys Lys Glu Ala Phe Ala Glu Leu Gln Thr Asp Ile His 1295 1300 1305 Glu Leu Thr Asn Asp Leu Asp Gly Ala Gly Ile Pro Phe Leu Asp 1310 1315 1320 Tyr Arg Thr Tyr Ala Met Arg Val Leu Phe Pro Gly Ile Glu Asp 1325 1330 1335 His Pro Val Leu Lys Glu Met Glu Val Gln Ala Asn Val Glu Lys 1340 1345 1350 Ser Leu Thr Leu Phe Gly Gln Leu Leu Thr Lys Lys His Phe Leu 1355 1360 1365 Leu Thr Phe Ile Arg Thr Leu Glu Ala Gln Arg Ser Phe Ser Met 1370 1375 1380 Arg Asp Arg Gly Asn Val Ala Ser Leu Ile Met Thr Ala Leu Gln 1385 1390 1395 Gly Glu Met Glu Tyr Ala Thr Gly Val Leu Lys Gln Leu Leu Ser 1400 1405 1410 Asp Leu Ile Glu Lys Asn Leu Glu Ser Lys Asn His Pro Lys Leu 1415 1420 1425 Leu Leu Arg Arg Thr Glu Ser Val Ala Glu Lys Met Leu Thr Asn 1430 1435 1440 Trp Phe Thr Phe Leu Leu Tyr Lys Phe Leu Lys Glu Cys Ala Gly 1445 1450 1455 Glu Pro Leu Phe Met Leu Tyr Cys Ala Ile Lys Gln Gln Met Glu 1460 1465 1470 Lys Gly Pro Ile Asp Ala Ile Thr Gly Glu Ala Arg Tyr Ser Leu 1475 1480 1485 Ser Glu Asp Lys Leu Ile Arg Gln Gln Ile Asp Tyr Lys Thr Leu 1490 1495 1500 Thr Leu Asn Cys Val Asn Pro Glu Asn Glu Asn Ala Pro Glu Val 1505 1510 1515 Pro Val Lys Gly Leu Asp Cys Asp Thr Val Thr Gln Ala Lys Glu 1520 1525 1530 Lys Leu Leu Asp Ala Ala Tyr Lys Gly Val Pro Tyr Ser Gln Arg 1535 1540 1545 Pro Lys Ala Ala Asp Met Asp Leu Glu Trp Arg Gln Gly Arg Met 1550 1555 1560 Ala Arg Ile Ile Leu Gln Asp Glu Asp Val Thr Thr Lys Ile Asp 1565 1570 1575 Asn Asp Trp Lys Arg Leu Asn Thr Leu Ala His Tyr Gln Val Thr 1580 1585 1590 Asp Gly Ser Ser Val Ala Leu Val Pro Lys Gln Thr Ser Ala Tyr 1595 1600 1605 Asn Ile Ser Asn Ser Ser Thr Phe Thr Lys Ser Leu Ser Arg Tyr 1610 1615 1620 Glu Ser Met Leu Arg Thr Ala Ser Ser Pro Asp Ser Leu Arg Ser 1625 1630 1635 Arg Thr Pro Met Ile Thr Pro Asp Leu Glu Ser Gly Thr Lys Leu 1640 1645 1650 Trp His Leu Val Lys Asn His Asp His Leu Asp Gln Arg Glu Gly 1655 1660 1665 Asp Arg Gly Ser Lys Met Val Ser Glu Ile Tyr Leu Thr Arg Leu 1670 1675 1680 Leu Ala Thr Lys Gly Thr Leu Gln Lys Phe Val Asp Asp Leu Phe 1685 1690 1695 Glu Thr Ile Phe Ser Thr Ala His Arg Gly Ser Ala Leu Pro Leu 1700 1705 1710 Ala Ile Lys Tyr Met Phe Asp Phe Leu Asp Glu Gln Ala Asp Lys 1715 1720 1725 His Gln Ile His Asp Ala Asp Val Arg His Thr Trp Lys Ser Asn 1730 1735 1740 Cys Leu Pro Leu Arg Phe Trp Val Asn Val Ile Lys Asn Pro Gln 1745 1750 1755 Phe Val Phe Asp Ile His Lys Asn Ser Ile Thr Asp Ala Cys Leu 1760 1765 1770 Ser Val Val Ala Gln Thr Phe Met Asp Ser Cys Ser Thr Ser Glu 1775 1780 1785 His Lys Leu Gly Lys Asp Ser Pro Ser Asn Lys Leu Leu Tyr Ala 1790 1795 1800 Lys Asp Ile Pro Asn Tyr Lys Ser Trp Val Glu Arg Tyr Tyr Ala 1805 1810 1815 Asp Ile Ala Lys Met Pro Ala Ile Ser Asp Gln Asp Met Ser Ala 1820 1825 1830 Tyr Leu Ala Glu Gln Ser Arg Leu His Leu Ser Gln Phe Asn Ser 1835 1840 1845 Met Ser Ala Leu His Glue Ile Tyr Ser Tyr Ile Thr Lys Tyr Lys 1850 1855 1860 Asp Glu Ile Leu Ala Ala Leu Glu Lys Asp Glu Gln Ala Arg Arg 1865 1870 1875 Gln Arg Leu Arg Ser Lys Leu Glu Gln Val Val Asp Thr Met Ala 1880 1885 1890 Leo Ser Ser 1895 <210> 4 <211> 1247 <212> PRT <213> Artificial Sequence <220> <223> Human plexin A1 recombinant protein <400> 4 Met Trp Trp Arg Leu Trp Trp Leu Leu Leu Leu Leu Leu Leu Leu Trp 1 5 10 15 Pro Met Val Trp Ala Glu Ala Gly Leu Pro Arg Ala Gly Gly Gly Ser 20 25 30 Gln Pro Pro Phe Arg Thr Phe Ser Ala Ser Asp Trp Gly Leu Thr His 35 40 45 Leu Val Val His Glu Gln Thr Gly Glu Val Tyr Val Gly Ala Val Asn 50 55 60 Arg Ile Tyr Lys Leu Ser Gly Asn Leu Thr Leu Leu Arg Ala His Val 65 70 75 80 Thr Gly Pro Val Glu Asp Asn Glu Lys Cys Tyr Pro Pro Pro Ser Val 85 90 95 Gln Ser Cys Pro His Gly Leu Gly Ser Thr Asp Asn Val Asn Lys Leu 100 105 110 Leu Leu Leu Asp Tyr Ala Ala Asn Arg Leu Leu Ala Cys Gly Ser Ala 115 120 125 Ser Gln Gly Ile Cys Gln Phe Leu Arg Leu Asp Asp Leu Phe Lys Leu 130 135 140 Gly Glu Pro His His Arg Lys Glu His Tyr Leu Ser Ser Val Gln Glu 145 150 155 160 Ala Gly Ser Met Ala Gly Val Leu Ile Ala Gly Pro Pro Gly Gln Gly 165 170 175 Gln Ala Lys Leu Phe Val Gly Thr Pro Ile Asp Gly Lys Ser Glu Tyr 180 185 190 Phe Pro Thr Leu Ser Ser Arg Arg Leu Met Ala Asn Glu Glu Asp Ala 195 200 205 Asp Met Phe Gly Phe Val Tyr Gln Asp Glu Phe Val Ser Ser Gln Leu 210 215 220 Lys Ile Pro Ser Asp Thr Leu Ser Lys Phe Pro Ala Phe Asp Ile Tyr 225 230 235 240 Tyr Val Tyr Ser Phe Arg Ser Glu Gln Phe Val Tyr Tyr Leu Thr Leu 245 250 255 Gln Leu Asp Thr Gln Leu Thr Ser Pro Asp Ala Ala Gly Glu His Phe 260 265 270 Phe Thr Ser Lys Ile Val Arg Leu Cys Val Asp Asp Pro Lys Phe Tyr 275 280 285 Ser Tyr Val Glu Phe Pro Ile Gly Cys Glu Gln Ala Gly Val Glu Tyr 290,295,300 Arg Leu Val Gln Asp Ala Tyr Leu Ser Arg Pro Gly Arg Ala Leu Ala 305 310 315 320 His Gln Leu Gly Leu Ala Glu Asp Glu Asp Val Leu Phe Thr Val Phe 325 330 335 Ala Gln Gly Gln Lys Asn Arg Val Lys Pro Pro Lys Glu Ser Ala Leu 340 345 350 Cys Leu Phe Thr Leu Arg Ala Ile Lys Glu Lys Ile Lys Glu Arg Ile 355 360 365 Gln Ser Cys Tyr Arg Gly Glu Gly Lys Leu Ser Leu Pro Trp Leu Leu 370 375 380 Asn Lys Glu Leu Gly Cys Ile Asn Ser Pro Leu Gln Ile Asp Asp Asp 385 390 395 400 Phe Cys Gly Gln Asp Phe Asn Gln Pro Leu Gly Gly Thr Val Thr Ile 405 410 415 Glu Gly Thr Pro Leu Phe Val Asp Lys Asp Asp Gly Leu Thr Ala Val 420 425 430 Ala Ala Tyr Asp Tyr Arg Gly Arg Thr Val Val Phe Ala Gly Thr Arg 435 440 445 Ser Gly Arg Ile Arg Lys Ile Leu Val Asp Leu Ser Asn Pro Gly Gly 450 455 460 Arg Pro Ala Leu Ala Tyr Glu Ser Val Val Ala Gln Glu Gly Ser Pro 465 470 475 480 Ile Leu Arg Asp Leu Val Leu Ser Pro Asn His Gln Tyr Leu Tyr Ala 485 490 495 Met Thr Glu Lys Gln Val Thr Arg Val Pro Val Glu Ser Cys Val Gln 500 505 510 Tyr Thr Ser Cys Glu Leu Cys Leu Gly Ser Arg Asp Pro His Cys Gly 515 520 525 Trp Cys Val Leu His Ser Ile Cys Ser Arg Arg Asp Ala Cys Glu Arg 530 535 540 Ala Asp Glu Pro Gln Arg Phe Ala Ala Asp Leu Leu Gln Cys Val Gln 545 550 555 560 Leu Thr Val Gln Pro Arg Asn Val Ser Val Thr Met Ser Gln Val Pro 565 570 575 Leu Val Leu Gln Ala Trp Asn Val Pro Asp Leu Ser Ala Gly Val Asn 580 585 590 Cys Ser Phe Glu Asp Phe Thr Glu Ser Glu Ser Val Leu Glu Asp Gly 595 600 605 Arg Ile His Cys Arg Ser Pro Ser Ala Arg Glu Val Ala Pro Ile Thr 610 615 620 Arg Gly Gln Gly Asp Gln Arg Val Val Lys Leu Tyr Leu Lys Ser Lys 625 630 635 640 Glu Thr Gly Lys Lys Phe Ala Ser Val Asp Phe Val Phe Tyr Asn Cys 645 650 655 Ser Val His Gln Ser Cys Leu Ser Cys Val Asn Gly Ser Phe Pro Cys 660 665 670 His Trp Cys Lys Tyr Arg His Val Cys Thr His Asn Val Ala Asp Cys 675 680 685 Ala Phe Leu Glu Gly Arg Val Asn Val Ser Glu Asp Cys Pro Gln Ile 690 695 700 Leu Pro Ser Thr Gln Ile Tyr Val Pro Val Gly Val Val Lys Pro Ile 705 710 715 720 Thr Leu Ala Ala Arg Asn Leu Pro Gln Pro Gln Ser Gly Gln Arg Gly 725 730 735 Tyr Glu Cys Leu Phe His Ile Pro Gly Ser Pro Ala Arg Val Thr Ala 740 745 750 Leu Arg Phe Asn Ser Ser Ser Leu Gln Cys Gln Asn Ser Ser Tyr Ser 755 760 765 Tyr Glu Gly Asn Asp Val Ser Asp Leu Pro Val Asn Leu Ser Val Val 770 775 780 Trp Asn Gly Asn Phe Val Ile Asp Asn Pro Gln Asn Ile Gln Ala His 785 790 795 800 Leu Tyr Lys Cys Pro Ala Leu Arg Glu Ser Cys Gly Leu Cys Leu Lys 805 810 815 Ala Asp Pro Arg Phe Glu Cys Gly Trp Cys Val Ala Glu Arg Arg Cys 820 825 830 Ser Leu Arg His His Cys Ala Ala Asp Thr Pro Ala Ser Trp Met His 835 840 845 Ala Arg His Gly Ser Ser Arg Cys Thr Asp Pro Lys Ile Leu Lys Leu 850 855 860 Ser Pro Glu Thr Gly Pro Arg Gln Gly Gly Thr Arg Leu Thr Ile Thr 865 870 875 880 Gly Glu Asn Leu Gly Leu Arg Phe Glu Asp Val Arg Leu Gly Val Arg 885 890 895 Val Gly Lys Val Leu Cys Ser Pro Val Glu Ser Glu Tyr Ile Ser Ala 900 905 910 Glu Gln Ile Val Cys Glu Ile Gly Asp Ala Ser Ser Val Arg Ala His 915 920 925 Asp Ala Leu Val Glu Val Cys Val Arg Asp Cys Ser Pro His Tyr Arg 930 935 940 Ala Leu Ser Pro Lys Arg Phe Thr Phe Val Thr Pro Thr Phe Tyr Arg 945 950 955 960 Val Ser Pro Ser Arg Gly Pro Leu Ser Gly Gly Thr Trp Ile Gly Ile 965 970 975 Glu Gly Ser His Leu Asn Ala Gly Ser Asp Val Ala Val Ser Val Gly 980 985 990 Gly Arg Pro Cys Ser Phe Ser Trp Arg Asn Ser Arg Glu Ile Arg Cys 995 1000 1005 Leu Thr Pro Pro Gly Gln Ser Pro Gly Ser Ala Pro Ile Ile Ile 1010 1015 1020 Asn Ile Asn Arg Ala Gln Leu Thr Asn Pro Glu Val Lys Tyr Asn 1025 1030 1035 Tyr Thr Glu Asp Pro Thr Ile Leu Arg Ile Asp Pro Glu Trp Ser 1040 1045 1050 Ile Asn Ser Gly Gly Thr Leu Leu Thr Val Thr Gly Thr Asn Leu 1055 1060 1065 Ala Thr Val Arg Glu Pro Arg Ile Arg Ala Lys Tyr Gly Gly Ile 1070 1075 1080 Glu Arg Glu Asn Gly Cys Leu Val Tyr Asn Asp Thr Thr Met Val 1085 1090 1095 Cys Arg Ala Pro Ser Val Ala Asn Pro Val Arg Ser Pro Pro Glu 1100 1105 1110 Leu Gly Glu Arg Pro Asp Glu Leu Gly Phe Val Met Asp Asn Val 1115 1120 1125 Arg Ser Leu Leu Val Leu Asn Ser Thr Ser Phe Leu Tyr Tyr Pro 1130 1135 1140 Asp Pro Val Leu Glu Pro Leu Ser Pro Thr Gly Leu Leu Glu Leu 1145 1150 1155 Lys Pro Ser Ser Pro Leu Ile Leu Lys Gly Arg Asn Leu Leu Pro 1160 1165 1170 Pro Ala Pro Gly Asn Ser Arg Leu Asn Tyr Thr Val Leu Ile Gly 1175 1180 1185 Ser Thr Pro Cys Thr Leu Thr Val Ser Glu Thr Gln Leu Leu Cys 1190 1195 1200 Glu Ala Pro Asn Leu Thr Gly Gln His Lys Val Thr Val Arg Ala 1205 1210 1215 Gly Gly Phe Glu Phe Ser Pro Gly Thr Leu Gln Val Tyr Ser Asp 1220 1225 1230 Ser Leu Leu Thr Leu Pro Asp Tyr Lys Asp Asp Asp Asp Lys 1235 1240 1245 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> FLAG tag sequence <400> 5 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 6 <211> 26 <212> PRT <213> Homo sapiens <400> 6 Met Pro Leu Pro Pro Arg Ser Leu Gln Val Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Pro Gly Met Trp Ala 20 25 <210> 7 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Artificial signal peptide HMM+38 <400> 7 Met Trp Trp Arg Leu Trp Trp Leu Leu Leu Leu Leu Leu Leu Leu Trp 1 5 10 15 Pro Met Val Trp Ala 20 <210> 8 <211> 641 <212> PRT <213> Homo sapiens <400> 8 Met Ser Asp Glu Gly Pro Gly Thr Gly Pro Gly Asn Gly Leu Gly Glu 1 5 10 15 Lys Gly Asp Thr Ser Gly Pro Glu Gly Ser Gly Gly Ser Gly Pro Gln 20 25 30 Arg Arg Gly Gly Asp Asn His Gly Arg Gly Arg Gly Arg Gly Arg Gly 35 40 45 Arg Gly Gly Gly Arg Pro Gly Ala Pro Gly Gly Ser Gly Ser Gly Pro 50 55 60 Arg His Arg Asp Gly Val Arg Arg Pro Gln Lys Arg Pro Ser Cys Ile 65 70 75 80 Gly Cys Lys Gly Thr His Gly Gly Thr Gly Ala Gly Ala Gly Ala Gly 85 90 95 Gly Ala Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala Gly 100 105 110 Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly 115 120 125 Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala 130 135 140 Gly Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly Ala Gly 145 150 155 160 Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly 165 170 175 Ala Gly Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly 180 185 190 Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala Gly Gly Ala Gly 195 200 205 Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala 210 215 220 Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala 225 230 235 240 Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly 245 250 255 Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly 260 265 270 Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly 275 280 285 Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Gly Ala Gly 290 295 300 Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala Gly 305 310 315 320 Gly Ala Gly Ala Gly Gly Gly Gly Arg Gly Arg Gly Gly Ser Gly Gly 325 330 335 Arg Gly Arg Gly Gly Ser Gly Gly Arg Gly Arg Gly Gly Ser Gly Gly 340 345 350 Arg Arg Gly Arg Gly Arg Glu Arg Ala Arg Gly Gly Ser Arg Glu Arg 355 360 365 Ala Arg Gly Arg Gly Arg Gly Arg Gly Glu Lys Arg Pro Arg Ser Pro 370 375 380 Ser Ser Gln Ser Ser Ser Ser Gly Ser Pro Pro Arg Arg Pro Pro Pro 385 390 395 400 Gly Arg Arg Pro Phe Phe His Pro Val Gly Glu Ala Asp Tyr Phe Glu 405 410 415 Tyr His Gln Glu Gly Gly Pro Asp Gly Glu Pro Asp Val Pro Pro Gly 420 425 430 Ala Ile Glu Gln Gly Pro Ala Asp Asp Pro Gly Glu Gly Pro Ser Thr 435 440 445 Gly Pro Arg Gly Gln Gly Asp Gly Gly Arg Arg Lys Lys Gly Gly Trp 450 455 460 Phe Gly Lys His Arg Gly Gln Gly Gly Ser Asn Pro Lys Phe Glu Asn 465 470 475 480 Ile Ala Glu Gly Leu Arg Ala Leu Leu Ala Arg Ser His Val Glu Arg 485 490 495 Thr Thr Asp Glu Gly Thr Trp Val Ala Gly Val Phe Val Tyr Gly Gly 500 505 510 Ser Lys Thr Ser Leu Tyr Asn Leu Arg Arg Gly Thr Ala Leu Ala Ile 515 520 525 Pro Gln Cys Arg Leu Thr Pro Leu Ser Arg Leu Pro Phe Gly Met Ala 530 535 540 Pro Gly Pro Gly Pro Gln Pro Gly Pro Leu Arg Glu Ser Ile Val Cys 545 550 555 560 Tyr Phe Met Val Phe Leu Gln Thr His Ile Phe Ala Glu Val Leu Lys 565 570 575 Asp Ala Ile Lys Asp Leu Val Met Thr Lys Pro Ala Pro Thr Cys Asn 580 585 590 Ile Arg Val Thr Val Cys Ser Phe Asp Asp Gly Val Asp Leu Pro Pro 595 600 605 Trp Phe Pro Pro Met Val Glu Gly Ala Ala Ala Glu Gly Asp Asp Gly 610 615 620 Asp Asp Gly Asp Glu Gly Gly Asp Gly Asp Glu Gly Glu Glu Gly Gln 625 630 635 640 Glu <210> 9 <211> 450 <212> PRT <213> Homo sapiens <400> 9 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Leu Ser Ser Thr 20 25 30 Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Gly Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ser Val 85 90 95 Tyr Tyr Cys Ala Arg Asp Arg Gly Tyr Tyr Asn Gly Val Asp Val Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro 450 <210> 10 <211> 212 <212> PRT <213> Homo sapiens <400> 10 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Leu Leu Val Ile Tyr 35 40 45 Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Gln Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met 65 70 75 80 Asp Glu Ala Asp Tyr Phe Cys Gln Ala Trp Asp Ser Gly Thr Phe Val 85 90 95 Phe Gly Thr Gly Thr Lys Val Thr Ile Leu Arg Gln Pro Lys Ala Asn 100 105 110 Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn 115 120 125 Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val 130 135 140 Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Ala Gly Val Glu 145 150 155 160 Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser 165 170 175 Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser 180 185 190 Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro 195 200 205 Thr Glu Cys Ser 210 <210> 11 <211> 448 <212> PRT <213> Homo sapiens <400> 11 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Pro Gly His Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 12 <211> 214 <212> PRT <213> Homo sapiens <400> 12 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Glu Asp Lys Tyr Ala 20 25 30 Ser Trp Tyr Gln Leu Lys Pro Gly His Ser Pro Val Leu Val Ile Tyr 35 40 45 Gln Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Arg Ala Trp Asp Ser Asn Thr Gly Asp 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 <210> 13 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> H chain of human / mouse chimeric and human / mouse corss-reactive anti-plexin A1 antibody hPANL#240-mFc <400> 13 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Leu Ser Ser Thr 20 25 30 Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Gly Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ser Val 85 90 95 Tyr Tyr Cys Ala Arg Asp Arg Gly Tyr Tyr Asn Gly Val Asp Val Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro 115 120 125 Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser 130 135 140 Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val 180 185 190 Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His 195 200 205 Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro 210 215 220 Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Arg 225 230 235 240 Arg Gly Pro Lys Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu 245 250 255 Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser 260 265 270 Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu 275 280 285 Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr 290 295 300 Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser 305 310 315 320 Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro 325 330 335 Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln 340 345 350 Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val 355 360 365 Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val 370 375 380 Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg 405 410 415 Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val 420 425 430 Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg 435 440 445 Thr Pro Gly Lys 450 <210> 14 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> H chain of human / mouse chimeric and human / mouse corss-reactive anti-plexin A1 antibody 359B2-2-3-6-mFc <400> 14 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Pro Gly His Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val 115 120 125 Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr 130 135 140 Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr 145 150 155 160 Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser 180 185 190 Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala 195 200 205 Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile 210 215 220 Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Arg Arg Gly 225 230 235 240 Pro Lys Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile 245 250 255 Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp 260 265 270 Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His 275 280 285 Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg 290 295 300 Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys 305 310 315 320 Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu 325 330 335 Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr 340 345 350 Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu 355 360 365 Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp 370 375 380 Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu 405 410 415 Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His 420 425 430 Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro 435 440 445 Gly Lys 450 <210> 15 <211> 1305 <212> PRT <213> Artificial Sequence <220> <223> Human plexin A1 recombinant protein <400> 15 Met Trp Trp Arg Leu Trp Trp Leu Leu Leu Leu Leu Leu Leu Leu Trp 1 5 10 15 Pro Met Val Trp Ala Glu Ala Gly Leu Pro Arg Ala Gly Gly Gly Ser 20 25 30 Gln Pro Pro Phe Arg Thr Phe Ser Ala Ser Asp Trp Gly Leu Thr His 35 40 45 Leu Val Val His Glu Gln Thr Gly Glu Val Tyr Val Gly Ala Val Asn 50 55 60 Arg Ile Tyr Lys Leu Ser Gly Asn Leu Thr Leu Leu Arg Ala His Val 65 70 75 80 Thr Gly Pro Val Glu Asp Asn Glu Lys Cys Tyr Pro Pro Pro Ser Val 85 90 95 Gln Ser Cys Pro His Gly Leu Gly Ser Thr Asp Asn Val Asn Lys Leu 100 105 110 Leu Leu Leu Asp Tyr Ala Ala Asn Arg Leu Leu Ala Cys Gly Ser Ala 115 120 125 Ser Gln Gly Ile Cys Gln Phe Leu Arg Leu Asp Asp Leu Phe Lys Leu 130 135 140 Gly Glu Pro His His Arg Lys Glu His Tyr Leu Ser Ser Val Gln Glu 145 150 155 160 Ala Gly Ser Met Ala Gly Val Leu Ile Ala Gly Pro Pro Gly Gln Gly 165 170 175 Gln Ala Lys Leu Phe Val Gly Thr Pro Ile Asp Gly Lys Ser Glu Tyr 180 185 190 Phe Pro Thr Leu Ser Ser Arg Arg Leu Met Ala Asn Glu Glu Asp Ala 195 200 205 Asp Met Phe Gly Phe Val Tyr Gln Asp Glu Phe Val Ser Ser Gln Leu 210 215 220 Lys Ile Pro Ser Asp Thr Leu Ser Lys Phe Pro Ala Phe Asp Ile Tyr 225 230 235 240 Tyr Val Tyr Ser Phe Arg Ser Glu Gln Phe Val Tyr Tyr Leu Thr Leu 245 250 255 Gln Leu Asp Thr Gln Leu Thr Ser Pro Asp Ala Ala Gly Glu His Phe 260 265 270 Phe Thr Ser Lys Ile Val Arg Leu Cys Val Asp Asp Pro Lys Phe Tyr 275 280 285 Ser Tyr Val Glu Phe Pro Ile Gly Cys Glu Gln Ala Gly Val Glu Tyr 290,295,300 Arg Leu Val Gln Asp Ala Tyr Leu Ser Arg Pro Gly Arg Ala Leu Ala 305 310 315 320 His Gln Leu Gly Leu Ala Glu Asp Glu Asp Val Leu Phe Thr Val Phe 325 330 335 Ala Gln Gly Gln Lys Asn Arg Val Lys Pro Pro Lys Glu Ser Ala Leu 340 345 350 Cys Leu Phe Thr Leu Arg Ala Ile Lys Glu Lys Ile Lys Glu Arg Ile 355 360 365 Gln Ser Cys Tyr Arg Gly Glu Gly Lys Leu Ser Leu Pro Trp Leu Leu 370 375 380 Asn Lys Glu Leu Gly Cys Ile Asn Ser Pro Leu Gln Ile Asp Asp Asp 385 390 395 400 Phe Cys Gly Gln Asp Phe Asn Gln Pro Leu Gly Gly Thr Val Thr Ile 405 410 415 Glu Gly Thr Pro Leu Phe Val Asp Lys Asp Asp Gly Leu Thr Ala Val 420 425 430 Ala Ala Tyr Asp Tyr Arg Gly Arg Thr Val Val Phe Ala Gly Thr Arg 435 440 445 Ser Gly Arg Ile Arg Lys Ile Leu Val Asp Leu Ser Asn Pro Gly Gly 450 455 460 Arg Pro Ala Leu Ala Tyr Glu Ser Val Val Ala Gln Glu Gly Ser Pro 465 470 475 480 Ile Leu Arg Asp Leu Val Leu Ser Pro Asn His Gln Tyr Leu Tyr Ala 485 490 495 Met Thr Glu Lys Gln Val Thr Arg Val Pro Val Glu Ser Cys Val Gln 500 505 510 Tyr Thr Ser Cys Glu Leu Cys Leu Gly Ser Arg Asp Pro His Cys Gly 515 520 525 Trp Cys Val Leu His Ser Ile Cys Ser Arg Arg Asp Ala Cys Glu Arg 530 535 540 Ala Asp Glu Pro Gln Arg Phe Ala Ala Asp Leu Leu Gln Cys Val Gln 545 550 555 560 Leu Thr Val Gln Pro Arg Asn Val Ser Val Thr Met Ser Gln Val Pro 565 570 575 Leu Val Leu Gln Ala Trp Asn Val Pro Asp Leu Ser Ala Gly Val Asn 580 585 590 Cys Ser Phe Glu Asp Phe Thr Glu Ser Glu Ser Val Leu Glu Asp Gly 595 600 605 Arg Ile His Cys Arg Ser Pro Ser Ala Arg Glu Val Ala Pro Ile Thr 610 615 620 Arg Gly Gln Gly Asp Gln Arg Val Val Lys Leu Tyr Leu Lys Ser Lys 625 630 635 640 Glu Thr Gly Lys Lys Phe Ala Ser Val Asp Phe Val Phe Tyr Asn Cys 645 650 655 Ser Val His Gln Ser Cys Leu Ser Cys Val Asn Gly Ser Phe Pro Cys 660 665 670 His Trp Cys Lys Tyr Arg His Val Cys Thr His Asn Val Ala Asp Cys 675 680 685 Ala Phe Leu Glu Gly Arg Val Asn Val Ser Glu Asp Cys Pro Gln Ile 690 695 700 Leu Pro Ser Thr Gln Ile Tyr Val Pro Val Gly Val Val Lys Pro Ile 705 710 715 720 Thr Leu Ala Ala Arg Asn Leu Pro Gln Pro Gln Ser Gly Gln Arg Gly 725 730 735 Tyr Glu Cys Leu Phe His Ile Pro Gly Ser Pro Ala Arg Val Thr Ala 740 745 750 Leu Arg Phe Asn Ser Ser Ser Leu Gln Cys Gln Asn Ser Ser Tyr Ser 755 760 765 Tyr Glu Gly Asn Asp Val Ser Asp Leu Pro Val Asn Leu Ser Val Val 770 775 780 Trp Asn Gly Asn Phe Val Ile Asp Asn Pro Gln Asn Ile Gln Ala His 785 790 795 800 Leu Tyr Lys Cys Pro Ala Leu Arg Glu Ser Cys Gly Leu Cys Leu Lys 805 810 815 Ala Asp Pro Arg Phe Glu Cys Gly Trp Cys Val Ala Glu Arg Arg Cys 820 825 830 Ser Leu Arg His His Cys Ala Ala Asp Thr Pro Ala Ser Trp Met His 835 840 845 Ala Arg His Gly Ser Ser Arg Cys Thr Asp Pro Lys Ile Leu Lys Leu 850 855 860 Ser Pro Glu Thr Gly Pro Arg Gln Gly Gly Thr Arg Leu Thr Ile Thr 865 870 875 880 Gly Glu Asn Leu Gly Leu Arg Phe Glu Asp Val Arg Leu Gly Val Arg 885 890 895 Val Gly Lys Val Leu Cys Ser Pro Val Glu Ser Glu Tyr Ile Ser Ala 900 905 910 Glu Gln Ile Val Cys Glu Ile Gly Asp Ala Ser Ser Val Arg Ala His 915 920 925 Asp Ala Leu Val Glu Val Cys Val Arg Asp Cys Ser Pro His Tyr Arg 930 935 940 Ala Leu Ser Pro Lys Arg Phe Thr Phe Val Thr Pro Thr Phe Tyr Arg 945 950 955 960 Val Ser Pro Ser Arg Gly Pro Leu Ser Gly Gly Thr Trp Ile Gly Ile 965 970 975 Glu Gly Ser His Leu Asn Ala Gly Ser Asp Val Ala Val Ser Val Gly 980 985 990 Gly Arg Pro Cys Ser Phe Ser Trp Arg Asn Ser Arg Glu Ile Arg Cys 995 1000 1005 Leu Thr Pro Pro Gly Gln Ser Pro Gly Ser Ala Pro Ile Ile Ile 1010 1015 1020 Asn Ile Asn Arg Ala Gln Leu Thr Asn Pro Glu Val Lys Tyr Asn 1025 1030 1035 Tyr Thr Glu Asp Pro Thr Ile Leu Arg Ile Asp Pro Glu Trp Ser 1040 1045 1050 Ile Asn Ser Gly Gly Thr Leu Leu Thr Val Thr Gly Thr Asn Leu 1055 1060 1065 Ala Thr Val Arg Glu Pro Arg Ile Arg Ala Lys Tyr Gly Gly Ile 1070 1075 1080 Glu Arg Glu Asn Gly Cys Leu Val Tyr Asn Asp Thr Thr Met Val 1085 1090 1095 Cys Arg Ala Pro Ser Val Ala Asn Pro Val Arg Ser Pro Pro Glu 1100 1105 1110 Leu Gly Glu Arg Pro Asp Glu Leu Gly Phe Val Met Asp Asn Val 1115 1120 1125 Arg Ser Leu Leu Val Leu Asn Ser Thr Ser Phe Leu Tyr Tyr Pro 1130 1135 1140 Asp Pro Val Leu Glu Pro Leu Ser Pro Thr Gly Leu Leu Glu Leu 1145 1150 1155 Lys Pro Ser Ser Pro Leu Ile Leu Lys Gly Arg Asn Leu Leu Pro 1160 1165 1170 Pro Ala Pro Gly Asn Ser Arg Leu Asn Tyr Thr Val Leu Ile Gly 1175 1180 1185 Ser Thr Pro Cys Thr Leu Thr Val Ser Glu Thr Gln Leu Leu Cys 1190 1195 1200 Glu Ala Pro Asn Leu Thr Gly Gln His Lys Val Thr Val Arg Ala 1205 1210 1215 Gly Gly Phe Glu Phe Ser Pro Gly Thr Leu Gln Val Tyr Ser Asp 1220 1225 1230 Ser Leu Leu Thr Leu Pro Ala Ile Val Gly Ile Gly Gly Gly Gly 1235 1240 1245 Gly Leu Leu Leu Leu Val Ile Val Ala Val Leu Ile Ala Tyr Lys 1250 1255 1260 Arg Lys Ser Arg Asp Ala Asp Arg Thr Leu Lys Arg Leu Gln Leu 1265 1270 1275 Gln Met Asp Asn Leu Glu Ser Arg Val Ala Leu Glu Cys Lys Glu 1280 1285 1290 Ala Phe Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1295 1300 1305 <210> 16 <211> 1247 <212> PRT <213> Artificial Sequence <220> <223> Soluble mouse plexin A1 protein <400> 16 Met Trp Trp Arg Leu Trp Trp Leu Leu Leu Leu Leu Leu Leu Leu Trp 1 5 10 15 Pro Met Val Trp Ala Ala Ile Ser Ser Pro Pro Ala Gly Leu Gly Pro 20 25 30 Gln Pro Ala Phe Arg Thr Phe Val Ala Ser Asp Trp Gly Leu Thr His 35 40 45 Leu Val Val His Glu Gln Thr Gly Glu Val Tyr Val Gly Ala Val Asn 50 55 60 Arg Ile Tyr Lys Leu Ser Gly Asn Leu Thr Leu Leu Arg Ala His Val 65 70 75 80 Thr Gly Pro Val Glu Asp Asn Glu Lys Cys Tyr Pro Pro Pro Ser Val 85 90 95 Gln Ser Cys Pro His Gly Leu Gly Ser Thr Asp Asn Val Asn Lys Leu 100 105 110 Leu Leu Leu Asp Tyr Ala Ala Asn Arg Leu Leu Ala Cys Gly Ser Ala 115 120 125 Ser Gln Gly Ile Cys Gln Phe Leu Arg Leu Asp Asp Leu Phe Lys Leu 130 135 140 Gly Glu Pro His His Arg Lys Glu His Tyr Leu Ser Ser Val Arg Glu 145 150 155 160 Ala Gly Ser Met Ala Gly Val Leu Ile Ala Gly Pro Pro Gly Gln Gly 165 170 175 Gln Ala Lys Leu Phe Val Gly Thr Pro Ile Asp Gly Lys Ser Glu Tyr 180 185 190 Phe Pro Thr Leu Ser Ser Arg Arg Leu Met Ala Asn Glu Glu Asp Ala 195 200 205 Asp Met Phe Gly Phe Val Tyr Gln Asp Glu Phe Val Ser Ser Gln Leu 210 215 220 Lys Ile Pro Ser Asp Thr Leu Ser Lys Phe Pro Ala Phe Asp Ile Tyr 225 230 235 240 Tyr Val Tyr Ser Phe Arg Ser Glu Gln Phe Val Tyr Tyr Leu Thr Leu 245 250 255 Gln Leu Asp Thr Gln Leu Thr Ser Pro Asp Ala Ala Gly Glu His Phe 260 265 270 Phe Thr Ser Lys Ile Val Arg Leu Cys Val Asn Asp Pro Lys Phe Tyr 275 280 285 Ser Tyr Val Glu Phe Pro Ile Gly Cys Glu Gln Ala Gly Val Glu Tyr 290 295 300 Arg Leu Val Gln Asp Ala Tyr Leu Ser Arg Pro Gly Gln Ala Leu Ala 305 310 315 320 Lys Gln Leu Gly Leu Ala Glu Asp Glu Glu Val Leu Phe Thr Val Phe 325 330 335 Ala Gln Gly Gln Lys Asn Arg Val Lys Pro Pro Lys Glu Ser Ala Leu 340 345 350 Cys Leu Phe Thr Leu Arg Ala Ile Lys Glu Lys Ile Lys Glu Arg Ile 355 360 365 Gln Ser Cys Tyr Arg Gly Glu Gly Lys Leu Ser Leu Pro Trp Leu Leu 370 375 380 Asn Lys Glu Leu Gly Cys Ile Asn Ser Pro Leu Gln Ile Asp Asp Asp 385 390 395 400 Phe Cys Gly Gln Asp Phe Asn Gln Pro Leu Gly Gly Thr Val Thr Ile 405 410 415 Glu Gly Thr Pro Leu Phe Val Asp Lys Glu Asp Gly Leu Thr Ala Val 420 425 430 Ala Ala Tyr Asp Tyr Gln Gly Arg Thr Val Val Phe Ala Gly Thr Arg 435 440 445 Ser Gly Arg Ile Arg Lys Ile Leu Val Asp Leu Ala Asn Pro Ser Gly 450 455 460 Arg Pro Ala Leu Ala Tyr Glu Ser Val Val Ala Gln Glu Gly Asn Pro 465 470 475 480 Ile Leu Arg Asp Leu Val Leu Ser Pro Asn Arg Gln Tyr Leu Tyr Ala 485 490 495 Met Thr Glu Lys Gln Val Thr Gln Val Pro Val Glu Ser Cys Val Gln 500 505 510 Tyr Thr Ser Cys Glu Leu Cys Leu Gly Ser Arg Asp Pro His Cys Gly 515 520 525 Trp Cys Val Leu His Ser Ile Cys Ser Arg Gln Asp Ala Cys Glu Arg 530 535 540 Ala Glu Glu Pro Gln Arg Phe Ala Ser Asp Leu Leu Gln Cys Val Gln 545 550 555 560 Leu Thr Val Gln Pro Arg Asn Val Ser Val Thr Met Ser Gln Val Pro 565 570 575 Leu Val Leu Gln Ala Trp Asn Val Pro Asp Leu Ser Ala Gly Val Asn 580 585 590 Cys Ser Phe Glu Asp Phe Thr Glu Thr Glu Ser Ile Leu Glu Asp Gly 595 600 605 Arg Ile His Cys His Ser Pro Ser Ala Arg Glu Val Ala Pro Ile Thr 610 615 620 Gln Gly Gln Gly Asp Gln Arg Val Val Lys Leu Tyr Leu Lys Ser Lys 625 630 635 640 Glu Thr Gly Lys Lys Phe Ala Ser Val Asp Phe Val Phe Tyr Asn Cys 645 650 655 Ser Val His Gln Ser Cys Leu Ala Cys Val Asn Gly Ser Phe Pro Cys 660 665 670 His Trp Cys Lys Tyr Arg His Val Cys Thr Asn Asn Ala Ala Asp Cys 675 680 685 Ala Phe Leu Glu Gly Arg Val Asn Met Ser Glu Asp Cys Pro Gln Ile 690 695 700 Leu Pro Ser Thr His Ile Tyr Val Pro Val Gly Val Val Lys Pro Ile 705 710 715 720 Thr Leu Ala Ala Arg Asn Leu Pro Gln Pro Gln Ser Gly Gln Arg Gly 725 730 735 Tyr Glu Cys Leu Phe His Ile Pro Gly Ser Pro Ala Arg Val Thr Ala 740 745 750 Leu Arg Phe Asn Ser Ser Ser Leu Gln Cys Gln Asn Ser Ser Tyr Ser 755 760 765 Tyr Glu Gly Asn Asp Val Ser Asp Leu Pro Val Asn Leu Ser Val Val 770 775 780 Trp Asn Gly Asn Phe Val Ile Asp Asn Pro Gln Asn Ile Gln Ala His 785 790 795 800 Leu Tyr Lys Cys Pro Ala Leu Arg Gln Ser Cys Gly Leu Cys Leu Lys 805 810 815 Ala Asp Pro Arg Phe Glu Cys Gly Trp Cys Val Ala Glu Arg Arg Cys 820 825 830 Ser Leu Arg His His Cys Pro Ala Asp Ser Pro Ala Ser Trp Met His 835 840 845 Ala His His Gly Ser Ser Arg Cys Thr Asp Pro Lys Ile Leu Lys Leu 850 855 860 Ser Pro Glu Thr Gly Pro Arg Gln Gly Gly Thr Arg Leu Thr Ile Thr 865 870 875 880 Gly Glu Asn Leu Gly Leu Arg Phe Glu Asp Val Arg Leu Gly Val His 885 890 895 Val Gly Lys Val Leu Cys Ser Pro Val Glu Ser Glu Tyr Ile Ser Ala 900 905 910 Glu Gln Ile Val Cys Glu Ile Gly Asp Ala Ser Thr Leu Arg Ala His 915 920 925 Asp Ala Leu Val Glu Val Cys Val Arg Asp Cys Ser Leu His Tyr Arg 930 935 940 Ala Leu Ser Pro Lys Arg Phe Thr Phe Val Thr Pro Thr Phe Tyr Arg 945 950 955 960 Val Ser Pro Ser Arg Gly Pro Leu Ser Gly Gly Thr Trp Ile Gly Ile 965 970 975 Glu Gly Ser His Leu Asn Ala Gly Ser Asp Val Ala Val Ser Ile Gly 980 985 990 Gly Arg Pro Cys Ser Phe Ser Trp Arg Asn Ser Arg Glu Ile Arg Cys 995 1000 1005 Leu Thr Pro Pro Gly His Thr Pro Gly Ser Ala Pro Ile Val Ile 1010 1015 1020 Asn Ile Asn Arg Ala Gln Leu Ser Asn Pro Glu Val Lys Tyr Asn 1025 1030 1035 Tyr Thr Glu Asp Pro Thr Ile Leu Arg Ile Asp Pro Glu Trp Ser 1040 1045 1050 Ile Asn Ser Gly Gly Thr Leu Leu Thr Val Thr Gly Thr Asn Leu 1055 1060 1065 Ala Thr Val Arg Glu Pro Arg Ile Arg Ala Lys Tyr Gly Gly Ile 1070 1075 1080 Glu Arg Glu Asn Ser Cys Met Val Tyr Asn Asp Thr Thr Met Val 1085 1090 1095 Cys Arg Ala Pro Ser Ile Asp Asn Pro Lys Arg Ser Pro Pro Glu 1100 1105 1110 Leu Gly Glu Arg Pro Asp Glu Ile Gly Phe Ile Met Asp Asn Val 1115 1120 1125 Arg Thr Leu Leu Val Leu Asn Ser Ser Ser Phe Leu Tyr Tyr Pro 1130 1135 1140 Asp Pro Val Leu Glu Pro Leu Ser Pro Thr Gly Leu Leu Glu Leu 1145 1150 1155 Lys Pro Ser Ser Pro Leu Ile Leu Lys Gly Arg Asn Leu Leu Pro 1160 1165 1170 Pro Ala Pro Gly Asn Ser Arg Leu Asn Tyr Thr Val Leu Ile Gly 1175 1180 1185 Ser Thr Pro Cys Ile Leu Thr Val Ser Glu Thr Gln Leu Leu Cys 1190 1195 1200 Glu Ala Pro Asn Leu Thr Gly Gln His Lys Val Thr Val Arg Ala 1205 1210 1215 Gly Gly Phe Glu Phe Ser Pro Gly Met Leu Gln Val Tyr Ser Asp 1220 1225 1230 Serum Leu Leu Thr Leu Pro Asp Tyr Lys Asp Asp Asp Asp Lys 1235 1240 1245 <210> 17 <211> 323 <212> PRT <213> Oryctolagus cuniculus <400> 17 Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly 1 5 10 15 Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr Asn 35 40 45 Gly Val Arg Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr Cys 65 70 75 80 Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val Ala 85 90 95 Pro Ser Thr Cys Ser Lys Pro Met Cys Pro Pro Pro Glu Leu Leu Gly 100 105 110 Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 115 120 125 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 130 135 140 Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln Val 145 150 155 160 Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile 165 170 175 Arg Val Val Ser Thr Leu Pro Ile Ala His Gln Asp Trp Leu Arg Gly 180 185 190 Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro Ile 195 200 205 Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys Val 210 215 220 Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val Ser 225 230 235 240 Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu 245 250 255 Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro Thr 260 265 270 Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val 275 280 285 Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val Met 290 295 300 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg Ser 305 310 315 320 Pro Gly Lys <210> 18 <211> 104 <212> PRT <213> Oryctolagus cuniculus <400> 18 Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp 1 5 10 15 Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr 20 25 30 Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr 35 40 45 Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr 50 55 60 Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser 65 70 75 80 His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val 85 90 95 Gln Ser Phe Asn Arg Gly Asp Cys 100 <210> 19 <211> 124 <212> PRT <213> Oryctolagus cuniculus <400> 19 Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro Glu Gly Ser 1 5 10 15 Leu Thr Leu Ser Cys Thr Ala Ser Gly Phe Thr Ile Ser Ser Ser Tyr 20 25 30 Trp Ile Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ala Cys Ile Tyr Val Gly Ser Gly Asp Gly Tyr Thr Tyr Tyr Ala Ser 50 55 60 Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val 65 70 75 80 Thr Leu Arg Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe 85 90 95 Cys Ala Arg Gly Gly Asp Gly Gly Val Gly Gly Phe Asp Phe Phe Asp 100 105 110 Pro Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 20 <211> 109 <212> PRT <213> Oryctolagus cuniculus <400> 20 Asp Val Val Leu Thr Gln Thr Pro Ala Ser Val Ser Glu Pro Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Thr Glu Ser Ile Asn Arg Asn 20 25 30 Cys Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Cys Thr Asp Tyr Gly Thr Tyr 85 90 95 Tyr Val Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 <210> 21 <211> 122 <212> PRT <213> Homo sapiens <400> 21 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Leu Ser Ser Thr 20 25 30 Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Gly Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ser Val 85 90 95 Tyr Tyr Cys Ala Arg Asp Arg Gly Tyr Tyr Asn Gly Val Asp Val Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 22 <211> 106 <212> PRT <213> Homo sapiens <400> 22 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Leu Leu Val Ile Tyr 35 40 45 Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Gln Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met 65 70 75 80 Asp Glu Ala Asp Tyr Phe Cys Gln Ala Trp Asp Ser Gly Thr Phe Val 85 90 95 Phe Gly Thr Gly Thr Lys Val Thr Ile Leu 100 105 <210> 23 <211> 7 <212> PRT <213> Homo sapiens <400> 23 Ser Thr Ser Ala Ala Trp Asn 1 5 <210> 24 <211> 18 <212> PRT <213> Homo sapiens <400> 24 Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val 1 5 10 15 Light Sees <210> 25 <211> 10 <212> PRT <213> Homo sapiens <400> 25 Asp Arg Gly Tyr Tyr Asn Gly Val Asp Val 1 5 10 <210> 26 <211> 11 <212> PRT <213> Homo sapiens <400> 26 Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala Ser 1 5 10 <210> 27 <211> 7 <212> PRT <213> Homo sapiens <400> 27 Gln Asp Asn Lys Arg Pro Ser 1 5 <210> 28 <211> 9 <212> PRT <213> Homo sapiens <400> 28 Gln Ala Trp Asp Ser Gly Thr Phe Val 1 5 <210> 29 <211> 5 <212> PRT <213> Homo sapiens <400> 29 Ser Tyr Tyr Met His 1 5 <210> 30 <211> 17 <212> PRT <213> Homo sapiens <400> 30 Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 31 <211> 11 <212> PRT <213> Homo sapiens <400> 31 Ala Pro Gly His Tyr Tyr Tyr Gly Met Asp Val 1 5 10 <210> 32 <211> 11 <212> PRT <213> Homo sapiens <400> 32 Ser Gly Asp Lys Leu Glu Asp Lys Tyr Ala Ser 1 5 10 <210> 33 <211> 7 <212> PRT <213> Homo sapiens <400> 33 Gln Asp Ser Lys Arg Pro Ser 1 5 <210> 34 <211> 11 <212> PRT <213> Homo sapiens <400> 34 Arg Ala Trp Asp Ser Asn Thr Gly Asp Val Val 1 5 10 <210> 35 <211> 120 <212> PRT <213> Homo sapiens <400> 35 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Pro Gly His Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 36 <211> 108 <212> PRT <213> Homo sapiens <400> 36 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Glu Asp Lys Tyr Ala 20 25 30 Ser Trp Tyr Gln Leu Lys Pro Gly His Ser Pro Val Leu Val Ile Tyr 35 40 45 Gln Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Arg Ala Trp Asp Ser Asn Thr Gly Asp 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 37 <211> 1297 <212> PRT <213> Artificial Sequence <220> <223> Mouse plexin A1 recombinant protein <400> 37 Met Trp Trp Arg Leu Trp Trp Leu Leu Leu Leu Leu Leu Leu Leu Trp 1 5 10 15 Pro Met Val Trp Ala Ala Ile Ser Ser Pro Pro Ala Gly Leu Gly Pro 20 25 30 Gln Pro Ala Phe Arg Thr Phe Val Ala Ser Asp Trp Gly Leu Thr His 35 40 45 Leu Val Val His Glu Gln Thr Gly Glu Val Tyr Val Gly Ala Val Asn 50 55 60 Arg Ile Tyr Lys Leu Ser Gly Asn Leu Thr Leu Leu Arg Ala His Val 65 70 75 80 Thr Gly Pro Val Glu Asp Asn Glu Lys Cys Tyr Pro Pro Pro Ser Val 85 90 95 Gln Ser Cys Pro His Gly Leu Gly Ser Thr Asp Asn Val Asn Lys Leu 100 105 110 Leu Leu Leu Asp Tyr Ala Ala Asn Arg Leu Leu Ala Cys Gly Ser Ala 115 120 125 Ser Gln Gly Ile Cys Gln Phe Leu Arg Leu Asp Asp Leu Phe Lys Leu 130 135 140 Gly Glu Pro His His Arg Lys Glu His Tyr Leu Ser Ser Val Arg Glu 145 150 155 160 Ala Gly Ser Met Ala Gly Val Leu Ile Ala Gly Pro Pro Gly Gln Gly 165 170 175 Gln Ala Lys Leu Phe Val Gly Thr Pro Ile Asp Gly Lys Ser Glu Tyr 180 185 190 Phe Pro Thr Leu Ser Ser Arg Arg Leu Met Ala Asn Glu Glu Asp Ala 195 200 205 Asp Met Phe Gly Phe Val Tyr Gln Asp Glu Phe Val Ser Ser Gln Leu 210 215 220 Lys Ile Pro Ser Asp Thr Leu Ser Lys Phe Pro Ala Phe Asp Ile Tyr 225 230 235 240 Tyr Val Tyr Ser Phe Arg Ser Glu Gln Phe Val Tyr Tyr Leu Thr Leu 245 250 255 Gln Leu Asp Thr Gln Leu Thr Ser Pro Asp Ala Ala Gly Glu His Phe 260 265 270 Phe Thr Ser Lys Ile Val Arg Leu Cys Val Asn Asp Pro Lys Phe Tyr 275 280 285 Ser Tyr Val Glu Phe Pro Ile Gly Cys Glu Gln Ala Gly Val Glu Tyr 290,295,300 Arg Leu Val Gln Asp Ala Tyr Leu Ser Arg Pro Gly Gln Ala Leu Ala 305 310 315 320 Lys Gln Leu Gly Leu Ala Glu Asp Glu Glu Val Leu Phe Thr Val Phe 325 330 335 Ala Gln Gly Gln Lys Asn Arg Val Lys Pro Pro Lys Glu Ser Ala Leu 340 345 350 Cys Leu Phe Thr Leu Arg Ala Ile Lys Glu Lys Ile Lys Glu Arg Ile 355 360 365 Gln Ser Cys Tyr Arg Gly Glu Gly Lys Leu Ser Leu Pro Trp Leu Leu 370 375 380 Asn Lys Glu Leu Gly Cys Ile Asn Ser Pro Leu Gln Ile Asp Asp Asp 385 390 395 400 Phe Cys Gly Gln Asp Phe Asn Gln Pro Leu Gly Gly Thr Val Thr Ile 405 410 415 Glu Gly Thr Pro Leu Phe Val Asp Lys Glu Asp Gly Leu Thr Ala Val 420 425 430 Ala Ala Tyr Asp Tyr Gln Gly Arg Thr Val Val Phe Ala Gly Thr Arg 435 440 445 Ser Gly Arg Ile Arg Lys Ile Leu Val Asp Leu Ala Asn Pro Ser Gly 450 455 460 Arg Pro Ala Leu Ala Tyr Glu Ser Val Val Ala Gln Glu Gly Asn Pro 465 470 475 480 Ile Leu Arg Asp Leu Val Leu Ser Pro Asn Arg Gln Tyr Leu Tyr Ala 485 490 495 Met Thr Glu Lys Gln Val Thr Gln Val Pro Val Glu Ser Cys Val Gln 500 505 510 Tyr Thr Ser Cys Glu Leu Cys Leu Gly Ser Arg Asp Pro His Cys Gly 515 520 525 Trp Cys Val Leu His Ser Ile Cys Ser Arg Gln Asp Ala Cys Glu Arg 530 535 540 Ala Glu Glu Pro Gln Arg Phe Ala Ser Asp Leu Leu Gln Cys Val Gln 545 550 555 560 Leu Thr Val Gln Pro Arg Asn Val Ser Val Thr Met Ser Gln Val Pro 565 570 575 Leu Val Leu Gln Ala Trp Asn Val Pro Asp Leu Ser Ala Gly Val Asn 580 585 590 Cys Ser Phe Glu Asp Phe Thr Glu Thr Glu Ser Ile Leu Glu Asp Gly 595 600 605 Arg Ile His Cys His Ser Pro Ser Ala Arg Glu Val Ala Pro Ile Thr 610 615 620 Gln Gly Gln Gly Asp Gln Arg Val Val Lys Leu Tyr Leu Lys Ser Lys 625 630 635 640 Glu Thr Gly Lys Lys Phe Ala Ser Val Asp Phe Val Phe Tyr Asn Cys 645 650 655 Ser Val His Gln Ser Cys Leu Ala Cys Val Asn Gly Ser Phe Pro Cys 660 665 670 His Trp Cys Lys Tyr Arg His Val Cys Thr Asn Asn Ala Ala Asp Cys 675 680 685 Ala Phe Leu Glu Gly Arg Val Asn Met Ser Glu Asp Cys Pro Gln Ile 690 695 700 Leu Pro Ser Thr His Ile Tyr Val Pro Val Gly Val Val Lys Pro Ile 705 710 715 720 Thr Leu Ala Ala Arg Asn Leu Pro Gln Pro Gln Ser Gly Gln Arg Gly 725 730 735 Tyr Glu Cys Leu Phe His Ile Pro Gly Ser Pro Ala Arg Val Thr Ala 740 745 750 Leu Arg Phe Asn Ser Ser Ser Leu Gln Cys Gln Asn Ser Ser Tyr Ser 755 760 765 Tyr Glu Gly Asn Asp Val Ser Asp Leu Pro Val Asn Leu Ser Val Val 770 775 780 Trp Asn Gly Asn Phe Val Ile Asp Asn Pro Gln Asn Ile Gln Ala His 785 790 795 800 Leu Tyr Lys Cys Pro Ala Leu Arg Gln Ser Cys Gly Leu Cys Leu Lys 805 810 815 Ala Asp Pro Arg Phe Glu Cys Gly Trp Cys Val Ala Glu Arg Arg Cys 820 825 830 Ser Leu Arg His His Cys Pro Ala Asp Ser Pro Ala Ser Trp Met His 835 840 845 Ala His His Gly Ser Ser Arg Cys Thr Asp Pro Lys Ile Leu Lys Leu 850 855 860 Ser Pro Glu Thr Gly Pro Arg Gln Gly Gly Thr Arg Leu Thr Ile Thr 865 870 875 880 Gly Glu Asn Leu Gly Leu Arg Phe Glu Asp Val Arg Leu Gly Val His 885 890 895 Val Gly Lys Val Leu Cys Ser Pro Val Glu Ser Glu Tyr Ile Ser Ala 900 905 910 Glu Gln Ile Val Cys Glu Ile Gly Asp Ala Ser Thr Leu Arg Ala His 915 920 925 Asp Ala Leu Val Glu Val Cys Val Arg Asp Cys Ser Leu His Tyr Arg 930 935 940 Ala Leu Ser Pro Lys Arg Phe Thr Phe Val Thr Pro Thr Phe Tyr Arg 945 950 955 960 Val Ser Pro Ser Arg Gly Pro Leu Ser Gly Gly Thr Trp Ile Gly Ile 965 970 975 Glu Gly Ser His Leu Asn Ala Gly Ser Asp Val Ala Val Ser Ile Gly 980 985 990 Gly Arg Pro Cys Ser Phe Ser Trp Arg Asn Ser Arg Glu Ile Arg Cys 995 1000 1005 Leu Thr Pro Pro Gly His Thr Pro Gly Ser Ala Pro Ile Val Ile 1010 1015 1020 Asn Ile Asn Arg Ala Gln Leu Ser Asn Pro Glu Val Lys Tyr Asn 1025 1030 1035 Tyr Thr Glu Asp Pro Thr Ile Leu Arg Ile Asp Pro Glu Trp Ser 1040 1045 1050 Ile Asn Ser Gly Gly Thr Leu Leu Thr Val Thr Gly Thr Asn Leu 1055 1060 1065 Ala Thr Val Arg Glu Pro Arg Ile Arg Ala Lys Tyr Gly Gly Ile 1070 1075 1080 Glu Arg Glu Asn Ser Cys Met Val Tyr Asn Asp Thr Thr Met Val 1085 1090 1095 Cys Arg Ala Pro Ser Ile Asp Asn Pro Lys Arg Ser Pro Pro Glu 1100 1105 1110 Leu Gly Glu Arg Pro Asp Glu Ile Gly Phe Ile Met Asp Asn Val 1115 1120 1125 Arg Thr Leu Leu Val Leu Asn Ser Ser Ser Phe Leu Tyr Tyr Pro 1130 1135 1140 Asp Pro Val Leu Glu Pro Leu Ser Pro Thr Gly Leu Leu Glu Leu 1145 1150 1155 Lys Pro Ser Ser Pro Leu Ile Leu Lys Gly Arg Asn Leu Leu Pro 1160 1165 1170 Pro Ala Pro Gly Asn Ser Arg Leu Asn Tyr Thr Val Leu Ile Gly 1175 1180 1185 Ser Thr Pro Cys Ile Leu Thr Val Ser Glu Thr Gln Leu Leu Cys 1190 1195 1200 Glu Ala Pro Asn Leu Thr Gly Gln His Lys Val Thr Val Arg Ala 1205 1210 1215 Gly Gly Phe Glu Phe Ser Pro Gly Met Leu Gln Val Tyr Ser Asp 1220 1225 1230 Ser Leu Leu Thr Leu Pro Ala Ile Val Gly Ile Gly Gly Gly Gly 1235 1240 1245 Gly Leu Leu Leu Leu Val Ile Val Ala Val Leu Ile Ala Tyr Lys 1250 1255 1260 Arg Lys Ser Arg Asp Ala Asp Arg Thr Leu Lys Arg Leu Gln Leu 1265 1270 1275 Gln Met Asp Asn Leu Glu Ser Arg Val Glu Gln Lys Leu Ile Ser 1280 1285 1290 Glu Glu Asp Leu 1295 <210> 38 <211> 515 <212> PRT <213> Artificial Sequence <220> <223> Mouse plexin A1 sema domain recombinant protein <400> 38 Met Trp Trp Arg Leu Trp Trp Leu Leu Leu Leu Leu Leu Leu Leu Trp 1 5 10 15 Pro Met Val Trp Ala Ala Ile Ser Ser Pro Pro Ala Gly Leu Gly Pro 20 25 30 Gln Pro Ala Phe Arg Thr Phe Val Ala Ser Asp Trp Gly Leu Thr His 35 40 45 Leu Val Val His Glu Gln Thr Gly Glu Val Tyr Val Gly Ala Val Asn 50 55 60 Arg Ile Tyr Lys Leu Ser Gly Asn Leu Thr Leu Leu Arg Ala His Val 65 70 75 80 Thr Gly Pro Val Glu Asp Asn Glu Lys Cys Tyr Pro Pro Pro Ser Val 85 90 95 Gln Ser Cys Pro His Gly Leu Gly Ser Thr Asp Asn Val Asn Lys Leu 100 105 110 Leu Leu Leu Asp Tyr Ala Ala Asn Arg Leu Leu Ala Cys Gly Ser Ala 115 120 125 Ser Gln Gly Ile Cys Gln Phe Leu Arg Leu Asp Asp Leu Phe Lys Leu 130 135 140 Gly Glu Pro His His Arg Lys Glu His Tyr Leu Ser Ser Val Arg Glu 145 150 155 160 Ala Gly Ser Met Ala Gly Val Leu Ile Ala Gly Pro Pro Gly Gln Gly 165 170 175 Gln Ala Lys Leu Phe Val Gly Thr Pro Ile Asp Gly Lys Ser Glu Tyr 180 185 190 Phe Pro Thr Leu Ser Ser Arg Arg Leu Met Ala Asn Glu Glu Asp Ala 195 200 205 Asp Met Phe Gly Phe Val Tyr Gln Asp Glu Phe Val Ser Ser Gln Leu 210 215 220 Lys Ile Pro Ser Asp Thr Leu Ser Lys Phe Pro Ala Phe Asp Ile Tyr 225 230 235 240 Tyr Val Tyr Ser Phe Arg Ser Glu Gln Phe Val Tyr Tyr Leu Thr Leu 245 250 255 Gln Leu Asp Thr Gln Leu Thr Ser Pro Asp Ala Ala Gly Glu His Phe 260 265 270 Phe Thr Ser Lys Ile Val Arg Leu Cys Val Asn Asp Pro Lys Phe Tyr 275 280 285 Ser Tyr Val Glu Phe Pro Ile Gly Cys Glu Gln Ala Gly Val Glu Tyr 290 295 300 Arg Leu Val Gln Asp Ala Tyr Leu Ser Arg Pro Gly Gln Ala Leu Ala 305 310 315 320 Lys Gln Leu Gly Leu Ala Glu Asp Glu Glu Val Leu Phe Thr Val Phe 325 330 335 Ala Gln Gly Gln Lys Asn Arg Val Lys Pro Pro Lys Glu Ser Ala Leu 340 345 350 Cys Leu Phe Thr Leu Arg Ala Ile Lys Glu Lys Ile Lys Glu Arg Ile 355 360 365 Gln Ser Cys Tyr Arg Gly Glu Gly Lys Leu Ser Leu Pro Trp Leu Leu 370 375 380 Asn Lys Glu Leu Gly Cys Ile Asn Ser Pro Leu Gln Ile Asp Asp Asp 385 390 395 400 Phe Cys Gly Gln Asp Phe Asn Gln Pro Leu Gly Gly Thr Val Thr Ile 405 410 415 Glu Gly Thr Pro Leu Phe Val Asp Lys Glu Asp Gly Leu Thr Ala Val 420 425 430 Ala Ala Tyr Asp Tyr Gln Gly Arg Thr Val Val Phe Ala Gly Thr Arg 435 440 445 Ser Gly Arg Ile Arg Lys Ile Arg Ala Asp Gly Pro Pro His Gly Gly 450 455 460 Val Gln Tyr Glu Met Val Ser Val Phe Lys Asp Gly Ser Pro Ile Leu 465 470 475 480 Arg Asp Met Ala Phe Ser Ile Asn Gln Leu Tyr Leu Tyr Val Met Ser 485 490 495 Glu Arg Gln Val Thr Arg Val Pro Val Glu Ser Asp Tyr Lys Asp Asp 500 505 510 Asp Asp Lys 515 <210> 39 <211> 508 <212> PRT <213> Artificial Sequence <220> <223> Mouse plexin A2 sema domain recombinant protein <400> 39 Met Trp Trp Arg Leu Trp Trp Leu Leu Leu Leu Leu Leu Leu Leu Trp 1 5 10 15 Pro Met Val Trp Ala Thr Thr Gly Met Pro Gln Tyr Ser Thr Phe His 20 25 30 Ser Glu Asn Arg Asp Trp Thr Phe Asn His Leu Thr Val His Arg Arg 35 40 45 Thr Gly Ala Val Tyr Val Gly Ala Ile Asn Arg Val Tyr Lys Leu Thr 50 55 60 Gly Asn Leu Thr Ile Gln Val Ala His Lys Thr Gly Pro Glu Glu Asp 65 70 75 80 Asn Lys Ala Cys Tyr Pro Pro Leu Ile Val Gln Pro Cys Ser Glu Val 85 90 95 Leu Thr Leu Thr Asn Asn Val Asn Lys Leu Leu Ile Ile Asp Tyr Ser 100 105 110 Glu Asn Arg Leu Leu Ala Cys Gly Ser Leu Tyr Gln Gly Val Cys Lys 115 120 125 Leu Leu Arg Leu Asp Asp Leu Phe Ile Leu Val Glu Pro Ser His Lys 130 135 140 Lys Glu His Tyr Leu Ser Ser Val Asn Lys Thr Gly Thr Met Tyr Gly 145 150 155 160 Val Ile Val Arg Ser Glu Gly Glu Asp Gly Lys Leu Phe Ile Gly Thr 165 170 175 Ala Val Asp Gly Lys Gln Asp Tyr Phe Pro Thr Leu Ser Ser Arg Lys 180 185 190 Leu Pro Arg Asp Pro Glu Ser Ser Ala Met Leu Asp Tyr Glu Leu His 195 200 205 To be Asp Phe Val To be Leu Ile Lys Ile Pro To be Asp Thr Leu Ala 210 215 220 Leu Val Ser His Phe Asp Ile Phe Tyr Ile Tyr Gly Phe Ala Ser Gly 225 230 235 240 Gly Phe Val Tyr Phe Leu Thr Val Gln Pro Glu Thr Pro Asp Gly Met 245 250 255 Only Gly Asp Leu Phe Tyr Thr Ser Arg And Val Arg 260 265 270 Leu Cys Lys Asp Asp Pro Lys Phe His Ser Tyr Val Ser Leu Pro Phe 275 280 285 Gly Cys Thr Arg Ala Gly Val Glu Tyr Arg Leu Leu Gln Ala Ala Tyr 290,295,300 Leu Ala Lys Pro Gly Glu Ala Leu Ala Gln Ala Phe Asn Ile Ser Ser 305 310 315 320 Asp Glu Asp Val Leu Phe Ala Ile Phenotype Lys Gly Gln Lys Gln Tyr 325 330 335 His His Pro Pro Asp Asp Ser Ala Leu Cys Ala Phe Pro Ile Arg Ala 340 345 350 Ile Asn Leu Gln Ile Lys Glu Arg Leu Gln Ser Cys Tyr His Gly Glu 355 360 365 Gly Asn Leu Glu Leu Asn Trp Leu Leu Gly Lys Asp Val Gln Cys Thr 370 375 380 Lys Ala Pro Val Pro Ile Asp Asp Asn Phe Cys Gly Leu Asp Ile Asn 385 390 395 400 Gln Pro Leu Gly Gly Ser Thr Pro Val Glu Gly Leu Thr Leu Tyr Thr 405 410 415 Thr Ser Arg Asp Arg Leu Thr Ser Val Ala Ser Tyr Val Tyr Asn Gly 420 425 430 Tyr Ser Val Val Phe Val Gly Thr Lys Ser Gly Lys Leu Lys Lys Ile 435 440 445 Arg Ala Asp Gly Pro Pro His Gly Gly Val Gln Tyr Glu Met Val Ser 450 455 460 Val Phe Lys Asp Gly Ser Pro Ile Leu Arg Asp Met Ala Phe Ser Ile 465 470 475 480 Asn Gln Leu Tyr Leu Tyr Val Met Ser Glu Arg Gln Val Thr Arg Val 485 490 495 Pro Val Glu Ser Asp Tyr Lys Asp Asp Asp Asp Lys 500 505 <210> 40 <211> 515 <212> PRT <213> Artificial Sequence <220> <223> Mouse plexin A1 / A2 sema domain chimeric protein <400> 40 Met Trp Trp Arg Leu Trp Trp Leu Leu Leu Leu Leu Leu Leu Leu Trp 1 5 10 15 Pro Met Val Trp Ala Ala Ile Ser Ser Pro Pro Ala Gly Leu Gly Pro 20 25 30 Gln Pro Ala Phe Arg Thr Phe Val Ala Ser Asp Trp Gly Leu Thr His 35 40 45 Leu Val Val His Glu Gln Thr Gly Glu Val Tyr Val Gly Ala Val Asn 50 55 60 Arg Ile Tyr Lys Leu Ser Gly Asn Leu Thr Leu Leu Arg Ala His Val 65 70 75 80 Thr Gly Pro Val Glu Asp Asn Glu Lys Cys Tyr Pro Pro Pro Ser Val 85 90 95 Gln Ser Cys Pro His Gly Leu Gly Ser Thr Asp Asn Val Asn Lys Leu 100 105 110 Leu Leu Leu Asp Tyr Ala Ala Asn Arg Leu Leu Ala Cys Gly Ser Ala 115 120 125 Ser Gln Gly Ile Cys Gln Phe Leu Arg Leu Asp Asp Leu Phe Lys Leu 130 135 140 Gly Glu Pro His His Arg Lys Glu His Tyr Leu Ser Ser Val Arg Glu 145 150 155 160 Ala Gly Ser Met Ala Gly Val Leu Ile Ala Gly Pro Pro Gly Gln Gly 165 170 175 Gln Ala Lys Leu Phe Val Gly Thr Pro Ile Asp Gly Lys Ser Glu Tyr 180 185 190 Phe Pro Thr Leu Ser Ser Arg Arg Leu Met Ala Asn Glu Glu Asp Ala 195 200 205 Asp Met Phe Gly Phe Val Tyr Gln Asp Glu Phe Val Ser Ser Gln Leu 210 215 220 Lys Ile Pro Ser Asp Thr Leu Ser Lys Phe Pro Ala Phe Asp Ile Tyr 225 230 235 240 Tyr Val Tyr Ser Phe Arg Ser Glu Gln Phe Val Tyr Tyr Leu Thr Leu 245 250 255 Gln Leu Asp Thr Gln Leu Thr Ser Pro Asp Ala Ala Gly Glu His Phe 260 265 270 Phe Thr Ser Lys Ile Val Arg Leu Cys Val Asn Asp Pro Lys Phe Tyr 275 280 285 Ser Tyr Val Glu Phe Pro Ile Gly Cys Glu Gln Ala Gly Val Glu Tyr 290,295,300 Arg Leu Val Gln Asp Ala Tyr Leu Ser Arg Pro Gly Gln Ala Leu Ala 305 310 315 320 Lys Gln Leu Gly Leu Ala Glu Asp Glu Glu Val Leu Phe Thr Val Phe 325 330 335 Ala Gln Gly Gln Lys Asn Arg Val Lys Pro Pro Lys Glu Ser Ala Leu 340 345 350 Cys Leu Phe Thr Leu Arg Ala Ile Lys Glu Lys Ile Lys Glu Arg Ile 355 360 365 Gln Ser Cys Tyr Arg Gly Glu Gly Lys Leu Ser Leu Pro Trp Leu Leu 370 375 380 Asn Lys Glu Leu Gly Cys Ile Asn Ser Pro Leu Gln Ile Asp Asp Asp 385 390 395 400 Phe Cys Gly Gln Asp Phe Asn Gln Pro Leu Gly Gly Thr Val Thr Ile 405 410 415 Glu Gly Thr Pro Leu Phe Val Asp Lys Glu Asp Gly Leu Thr Ala Val 420 425 430 Ala Ala Tyr Asp Tyr Gln Gly Arg Thr Val Val Phe Ala Gly Thr Arg 435 440 445 Ser Gly Arg Ile Arg Lys Ile Arg Ala Asp Gly Pro Pro His Gly Gly 450 455 460 Val Gln Tyr Glu Met Val Ser Val Phe Lys Asp Gly Ser Pro Ile Leu 465 470 475 480 Arg Asp Met Ala Phe Ser Ile Asn Gln Leu Tyr Leu Tyr Val Met Ser 485 490 495 Glu Arg Gln Val Thr Arg Val Pro Val Glu Ser Asp Tyr Lys Asp Asp 500 505 510 Asp Asp Lys 515 <210> 41 <211> 116 <212> PRT <213> Oryctolagus cuniculus <400> 41 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Ser Ala 20 25 30 Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Ile Asn Asn Ser Asn Ile Ala Ala Tyr Ala Ser Trp Thr Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr 65 70 75 80 Ser Pro Ile Ile Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Asp 85 90 95 Val Gly Thr Thr His Tyr Phe Asn Ile Trp Gly Pro Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 42 <211> 111 <212> PRT <213> Oryctolagus cuniculus <400> 42 Ala Ala Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Val Val Gly 1 5 10 15 Gly Thr Val Ser Ile Ser Cys Gln Ser Ser Lys Ser Val Tyr Asn Asn 20 25 30 Asn Trp Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Ala Ile Ser Asp Val 65 70 75 80 Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ile Asn 85 90 95 Ser Asp Asn Phe Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 43 <211> 121 <212> PRT <213> Oryctolagus cuniculus <400> 43 Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Ala Ser 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser Gly Gly Phe 20 25 30 Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ala Cys Ile Gly Thr Gly Ser Ser Gly Asn Thr Asp Tyr Ala Ser Trp 50 55 60 Ala Lys Gly Arg Phe Thr Ile Thr Gly Ser Ala Ser Leu Asn Thr Val 65 70 75 80 Thr Leu Gln Leu Asn Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe 85 90 95 Cys Ala Arg Gly Pro Phe Gly Ala Gly Ser Tyr Tyr Asn Leu Trp Gly 100 105 110 Pro Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 44 <211> 108 <212> PRT <213> Oryctolagus cuniculus <400> 44 Ala Phe Glu Leu Thr Gln Thr Pro Ala Ser Val Glu Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Phe Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Glu Val Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Wing Asp Wing Wing Thr Tyr Cys Gln Ser Tyr Tyr Gly Val Thr Thr 85 90 95 Asn Ile Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 <210> 45 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> H chain of human / mouse chimeric anti-plexin A1 antibody PXB693 <400> 45 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Ser Ala 20 25 30 Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Ile Asn Asn Ser Asn Ile Ala Ala Tyr Ala Ser Trp Thr Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr 65 70 75 80 Ser Pro Ile Ile Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Asp 85 90 95 Val Gly Thr Thr His Tyr Phe Asn Ile Trp Gly Pro Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala 115 120 125 Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu 130 135 140 Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly 145 150 155 160 Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp 165 170 175 Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro 180 185 190 Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys 195 200 205 Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro 210 215 220 Pro Cys Lys Cys Pro Ala Pro Asn Leu Arg Arg Gly Pro Lys Val Phe 225 230 235 240 Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro 245 250 255 Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val 260 265 270 Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr 275 280 285 Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala 290 295 300 Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys 305 310 315 320 Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser 325 330 335 Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro 340 345 350 Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val 355 360 365 Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly 370 375 380 Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp 405 410 415 Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His 420 425 430 Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 435 440 445 <210> 46 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> L chain of human / mouse chimeric anti-plexin A1 antibody PXB693 <400> 46 Ala Ala Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Val Val Gly 1 5 10 15 Gly Thr Val Ser Ile Ser Cys Gln Ser Ser Lys Ser Val Tyr Asn Asn 20 25 30 Asn Trp Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Ala Ile Ser Asp Val 65 70 75 80 Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ile Asn 85 90 95 Ser Asp Asn Phe Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys Arg 100 105 110 Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln 115 120 125 Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr 130 135 140 Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln 145 150 155 160 Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg 180 185 190 His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro 195 200 205 Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 210 215 <210> 47 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> H chain of human / mouse chimeric anti-plexin A1 antibody PXB727 <400> 47 Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Ala Ser 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser Gly Gly Phe 20 25 30 Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ala Cys Ile Gly Thr Gly Ser Ser Gly Asn Thr Asp Tyr Ala Ser Trp 50 55 60 Ala Lys Gly Arg Phe Thr Ile Thr Gly Ser Ala Ser Leu Asn Thr Val 65 70 75 80 Thr Leu Gln Leu Asn Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe 85 90 95 Cys Ala Arg Gly Pro Phe Gly Ala Gly Ser Tyr Tyr Asn Leu Trp Gly 100 105 110 Pro Gly Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser 115 120 125 Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val 130 135 140 Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu 145 150 155 160 Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr 180 185 190 Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro 195 200 205 Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr 210 215 220 Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Arg Arg 225 230 235 240 Gly Pro Lys Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met 245 250 255 Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu 260 265 270 Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val 275 280 285 His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu 290 295 300 Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly 305 310 315 320 Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile 325 330 335 Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val 340 345 350 Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr 355 360 365 Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu 370 375 380 Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val 405 410 415 Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val 420 425 430 His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr 435 440 445 Pro Gly Lys 450 <210> 48 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> L chain of human / mouse chimeric anti-plexin A1 antibody PXB727 <400> 48 Ala Phe Glu Leu Thr Gln Thr Pro Ala Ser Val Glu Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Phe Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Glu Val Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Tyr Tyr Gly Val Thr Thr 85 90 95 Asn Ile Phe Gly Gly Gly Thr Glu Val Val Val Lys Arg Ala Asp Ala 100 105 110 Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser 115 120 125 Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp 130 135 140 Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val 145 150 155 160 Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met 165 170 175 Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser 180 185 190 Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys 195 200 205 Ser Phe Asn Arg Asn Glu Cys 210 215 <210> 49 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> H chain of human / mouse chimeric anti-plexin A1 antibody PXB361b <400> 49 Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro Glu Gly Ser 1 5 10 15 Leu Thr Leu Ser Cys Thr Ala Ser Gly Phe Thr Ile Ser Ser Ser Tyr 20 25 30 Trp Ile Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ala Cys Ile Tyr Val Gly Ser Gly Asp Gly Tyr Thr Tyr Tyr Ala Ser 50 55 60 Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val 65 70 75 80 Thr Leu Arg Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe 85 90 95 Cys Ala Arg Gly Gly Asp Gly Gly Val Gly Gly Phe Asp Phe Phe Asp 100 105 110 Pro Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr 115 120 125 Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn 130 135 140 Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val 180 185 190 Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val 195 200 205 Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg 210 215 220 Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Lys Glu Val Ser Lys 225 230 235 240 Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu 245 250 255 Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro 260 265 270 Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala 275 280 285 Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val 290 295 300 Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe 305 310 315 320 Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile 340 345 350 Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys 355 360 365 Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp 370 375 380 Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp 385 390 395 400 Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser 405 410 415 Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly 420 425 430 Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 445 <210> 50 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> L chain of human / mouse chimeric anti-plexin A1 antibody PXB361b <400> 50 Asp Val Val Leu Thr Gln Thr Pro Ala Ser Val Ser Glu Pro Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Thr Glu Ser Ile Asn Arg Asn 20 25 30 Cys Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Cys Thr Asp Tyr Gly Thr Tyr 85 90 95 Tyr Val Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys Arg Ala Asp 100 105 110 Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr 115 120 125 Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys 130 135 140 Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly 145 150 155 160 Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn 180 185 190 Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val 195 200 205 Lys Ser Phe Asn Arg Asn Glu Cys 210 215 <210> 51 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Myc tag sequence <400> 51 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 <210> 52 <211> 1894 <212> PRT <213> Mus musculus <400> 52 Met Pro Leu Pro Pro Leu Ser Ser Arg Thr Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Leu Arg Gly Val Trp Ile Ala Ile Ser Ser Pro Pro Ala Gly 20 25 30 Leu Gly Pro Gln Pro Ala Phe Arg Thr Phe Val Ala Ser Asp Trp Gly 35 40 45 Leu Thr His Leu Val Val His Glu Gln Thr Gly Glu Val Tyr Val Gly 50 55 60 Ala Val Asn Arg Ile Tyr Lys Leu Ser Gly Asn Leu Thr Leu Leu Arg 65 70 75 80 Ala His Val Thr Gly Pro Val Glu Asp Asn Glu Lys Cys Tyr Pro Pro 85 90 95 Pro Ser Val Gln Ser Cys Pro His Gly Leu Gly Ser Thr Asp Asn Val 100 105 110 Asn Lys Leu Leu Leu Leu Asp Tyr Ala Ala Asn Arg Leu Leu Ala Cys 115 120 125 Gly Ser Ala Ser Gln Gly Ile Cys Gln Phe Leu Arg Leu Asp Asp Leu 130 135 140 Phe Lys Leu Gly Glu Pro His His Arg Lys Glu His Tyr Leu Ser Ser 145 150 155 160 Val Arg Glu Ala Gly Ser Met Ala Gly Val Leu Ile Ala Gly Pro Pro 165 170 175 Gly Gln Gly Gln Ala Lys Leu Phe Val Gly Thr Pro Ile Asp Gly Lys 180 185 190 Ser Glu Tyr Phe Pro Thr Leu Ser Ser Arg Arg Leu Met Ala Asn Glu 195 200 205 Glu Asp Ala Asp Met Phe Gly Phe Val Tyr Gln Asp Glu Phe Val Ser 210 215 220 Ser Gln Leu Lys Ile Pro Ser Asp Thr Leu Ser Lys Phe Pro Ala Phe 225 230 235 240 Asp Ile Tyr Tyr Val Tyr Ser Phe Arg Ser Glu Gln Phe Val Tyr Tyr 245 250 255 Leu Thr Leu Gln Leu Asp Thr Gln Leu Thr Ser Pro Asp Ala Ala Gly 260 265 270 Glu His Phe Phe Thr Ser Lys Ile Val Arg Leu Cys Val Asn Asp Pro 275 280 285 Lys Phe Tyr Ser Tyr Val Glu Phe Pro Ile Gly Cys Glu Gln Ala Gly 290 295 300 Val Glu Tyr Arg Leu Val Gln Asp Ala Tyr Leu Ser Arg Pro Gly Gln 305 310 315 320 Ala Leu Ala Lys Gln Leu Gly Leu Ala Glu Asp Glu Glu Val Leu Phe 325 330 335 Thr Val Phe Ala Gln Gly Gln Lys Asn Arg Val Lys Pro Pro Lys Glu 340 345 350 Ser Ala Leu Cys Leu Phe Thr Leu Arg Ala Ile Lys Glu Lys Ile Lys 355 360 365 Glu Arg Ile Gln Ser Cys Tyr Arg Gly Glu Gly Lys Leu Ser Leu Pro 370 375 380 Trp Leu Leu Asn Lys Glu Leu Gly Cys Ile Asn Ser Pro Leu Gln Ile 385 390 395 400 Asp Asp Asp Phe Cys Gly Gln Asp Phe Asn Gln Pro Leu Gly Gly Thr 405 410 415 Val Thr Ile Glu Gly Thr Pro Leu Phe Val Asp Lys Glu Asp Gly Leu 420 425 430 Thr Ala Val Ala Ala Tyr Asp Tyr Gln Gly Arg Thr Val Val Phe Ala 435 440 445 Gly Thr Arg Ser Gly Arg Ile Arg Lys Ile Leu Val Asp Leu Ala Asn 450 455 460 Pro Ser Gly Arg Pro Ala Leu Ala Tyr Glu Ser Val Val Ala Gln Glu 465 470 475 480 Gly Asn Pro Ile Leu Arg Asp Leu Val Leu Ser Pro Asn Arg Gln Tyr 485 490 495 Leu Tyr Ala Met Thr Glu Lys Gln Val Thr Gln Val Pro Val Glu Ser 500 505 510 Cys Val Gln Tyr Thr Ser Cys Glu Leu Cys Leu Gly Ser Arg Asp Pro 515 520 525 His Cys Gly Trp Cys Val Leu His Ser Ile Cys Ser Arg Gln Asp Ala 530 535 540 Cys Glu Arg Ala Glu Glu Pro Gln Arg Phe Ala Ser Asp Leu Leu Gln 545 550 555 560 Cys Val Gln Leu Thr Val Gln Pro Arg Asn Val Ser Val Thr Met Ser 565 570 575 Gln Val Pro Leu Val Leu Gln Ala Trp Asn Val Pro Asp Leu Ser Ala 580 585 590 Gly Val Asn Cys Ser Phe Glu Asp Phe Thr Glu Thr Glu Ser Ile Leu 595 600 605 Glu Asp Gly Arg Ile His Cys His Ser Pro Ser Ala Arg Glu Val Ala 610 615 620 Pro Ile Thr Gln Gly Gln Gly Asp Gln Arg Val Val Lys Leu Tyr Leu 625 630 635 640 Lys Ser Lys Glu Thr Gly Lys Lys Phe Ala Ser Val Asp Phe Val Phe 645 650 655 Tyr Asn Cys Ser Val His Gln Ser Cys Leu Ala Cys Val Asn Gly Ser 660 665 670 Phe Pro Cys His Trp Cys Lys Tyr Arg His Val Cys Thr Asn Asn Ala 675 680 685 Ala Asp Cys Ala Phe Leu Glu Gly Arg Val Asn Met Ser Glu Asp Cys 690 695 700 Pro Gln Ile Leu Pro Ser Thr His Ile Tyr Val Pro Val Gly Val Val 705 710 715 720 Lys Pro Ile Thr Leu Ala Ala Arg Asn Leu Pro Gln Pro Gln Ser Gly 725 730 735 Gln Arg Gly Tyr Glu Cys Leu Phe His Ile Pro Gly Ser Pro Ala Arg 740 745 750 Val Thr Ala Leu Arg Phe Asn Ser Ser Ser Leu Gln Cys Gln Asn Ser 755 760 765 Ser Tyr Ser Tyr Glu Gly Asn Asp Val Ser Asp Leu Pro Val Asn Leu 770 775 780 Ser Val Val Trp Asn Gly Asn Phe Val Ile Asp Asn Pro Gln Asn Ile 785 790 795 800 Gln Ala His Leu Tyr Lys Cys Pro Ala Leu Arg Gln Ser Cys Gly Leu 805 810 815 Cys Leu Lys Ala Asp Pro Arg Phe Glu Cys Gly Trp Cys Val Ala Glu 820 825 830 Arg Arg Cys Ser Leu Arg His His Cys Pro Ala Asp Ser Pro Ala Ser 835 840 845 Trp Met His Ala His His Gly Ser Ser Arg Cys Thr Asp Pro Lys Ile 850 855 860 Leu Lys Leu Ser Pro Glu Thr Gly Pro Arg Gln Gly Gly Thr Arg Leu 865 870 875 880 Thr Ile Thr Gly Glu Asn Leu Gly Leu Arg Phe Glu Asp Val Arg Leu 885 890 895 Gly Val His Val Gly Lys Val Leu Cys Ser Pro Val Glu Ser Glu Tyr 900 905 910 Ile Ser Ala Glu Gln Ile Val Cys Glu Ile Gly Asp Ala Ser Thr Leu 915 920 925 Arg Ala His Asp Ala Leu Val Glu Val Cys Val Arg Asp Cys Ser Leu 930 935 940 His Tyr Arg Ala Leu Ser Pro Lys Arg Phe Thr Phe Val Thr Pro Thr 945 950 955 960 Phe Tyr Arg Val Ser Pro Ser Arg Gly Pro Leu Ser Gly Gly Thr Trp 965 970 975 Ile Gly Ile Glu Gly Ser His Leu Asn Ala Gly Ser Asp Val Ala Val 980 985 990 Ser Ile Gly Gly Arg Pro Cys Ser Phe Ser Trp Arg Asn Ser Arg Glu 995 1000 1005 Ile Arg Cys Leu Thr Pro Pro Gly His Thr Pro Gly Ser Ala Pro 1010 1015 1020 Ile Val Ile Asn Ile Asn Arg Ala Gln Leu Ser Asn Pro Glu Val 1025 1030 1035 Lys Tyr Asn Tyr Thr Glu Asp Pro Thr Ile Leu Arg Ile Asp Pro 1040 1045 1050 Glu Trp Ser Ile Asn Ser Gly Gly Thr Leu Leu Thr Val Thr Gly 1055 1060 1065 Thr Asn Leu Ala Thr Val Arg Glu Pro Arg Ile Arg Ala Lys Tyr 1070 1075 1080 Gly Gly Ile Glu Arg Glu Asn Ser Cys Met Val Tyr Asn Asp Thr 1085 1090 1095 Thr Met Val Cys Arg Ala Pro Ser Ile Asp Asn Pro Lys Arg Ser 1100 1105 1110 Pro Pro Glu Leu Gly Glu Arg Pro Asp Glu Ile Gly Phe Ile Met 1115 1120 1125 Asp Asn Val Arg Thr Leu Leu Val Leu Asn Ser Ser Ser Phe Leu 1130 1135 1140 Tyr Tyr Pro Asp Pro Val Leu Glu Pro Leu Ser Pro Thr Gly Leu 1145 1150 1155 Leu Glu Leu Lys Pro Ser Ser Pro Leu Ile Leu Lys Gly Arg Asn 1160 1165 1170 Leu Leu Pro Pro Ala Pro Gly Asn Ser Arg Leu Asn Tyr Thr Val 1175 1180 1185 Leu Ile Gly Ser Thr Pro Cys Ile Leu Thr Val Ser Glu Thr Gln 1190 1195 1200 Leu Leu Cys Glu Ala Pro Asn Leu Thr Gly Gln His Lys Val Thr 1205 1210 1215 Val Arg Ala Gly Gly Phe Glu Phe Ser Pro Gly Met Leu Gln Val 1220 1225 1230 Tyr Ser Asp Ser Leu Leu Thr Leu Pro Ala Ile Val Gly Ile Gly 1235 1240 1245 Gly Gly Gly Gly Leu Leu Leu Val Ile Val Ala Val Leu Ile 1250 1255 1260 Ala Tyr Lys Arg Lys Ser Arg Asp Ala Asp Arg Thr Leu Lys Arg 1265 1270 1275 Leu Gln Leu Gln Met Asp Asn Leu Glu Ser Arg Val Ala Leu Glu 1280 1285 1290 Cys Lys Glu Ala Phe Ala Glu Leu Gln Thr Asp Ile His Glu Leu 1295 1300 1305 Thr Ser Asp Leu Asp Gly Ala Gly Ile Pro Phe Leu Asp Tyr Arg 1310 1315 1320 Thr Tyr Ala Met Arg Val Leu Phe Pro Gly Ile Glu Asp His Pro 1325 1330 1335 Val Leu Lys Glu Met Glu Val Gln Ala Asn Val Glu Lys Ser Leu 1340 1345 1350 Thr Leu Phe Gly Gln Leu Leu Thr Lys Lys His Phe Leu Leu Thr 1355 1360 1365 Phe Ile Arg Thr Leu Glu Ala Gln Arg Ser Phe Ser Met Arg Asp 1370 1375 1380 Arg Gly Asn Val Ala Ser Leu Ile Met Thr Ala Leu Gln Gly Glu 1385 1390 1395 Met Glu Tyr Ala Thr Gly Val Leu Lys Gln Leu Leu Ser Asp Leu 1400 1405 1410 Ile Glu Lys Asn Leu Glu Ser Lys Asn His Pro Lys Leu Leu Leu 1415 1420 1425 Arg Arg Thr Glu Ser Val Ala Glu Lys Met Leu Thr Asn Trp Phe 1430 1435 1440 Thr Phe Leu Leu Tyr Lys Phe Leu Lys Glu Cys Ala Gly Glu Pro 1445 1450 1455 Leu Phe Met Leu Tyr Cys Ala Ile Lys Gln Gln Met Glu Lys Gly 1460 1465 1470 Pro Ile Asp Ala Ile Thr Gly Glu Ala Arg Tyr Ser Leu Ser Glu 1475 1480 1485 Asp Lys Leu Ile Arg Gln Gln Ile Asp Tyr Lys Thr Leu Thr Leu 1490 1495 1500 Asn Cys Val Asn Pro Glu His Glu Asn Ala Pro Glu Val Pro Val 1505 1510 1515 Lys Gly Leu Asn Cys Asp Thr Val Thr Gln Val Lys Glu Lys Leu 1520 1525 1530 Leu Asp Ala Val Tyr Lys Gly Val Pro Tyr Ser Gln Arg Pro Lys 1535 1540 1545 Ala Gly Asp Met Asp Leu Glu Trp Arg Gln Gly Arg Met Ala Arg 1550 1555 1560 Ile Ile Leu Gln Asp Glu Asp Val Thr Thr Lys Ile Asp Asn Asp 1565 1570 1575 Trp Lys Arg Leu Asn Thr Leu Ala His Tyr Gln Val Thr Asp Gly 1580 1585 1590 Ser Ser Val Ala Leu Val Pro Lys Gln Thr Ser Ala Tyr Asn Ile 1595 1600 1605 Ser Asn Ser Ser Thr Phe Thr Lys Ser Leu Ser Arg Tyr Glu Ser 1610 1615 1620 Met Leu Arg Thr Ala Ser Ser Pro Asp Ser Leu Arg Ser Arg Thr 1625 1630 1635 Pro Met Ile Thr Pro Asp Leu Glu Ser Gly Thr Lys Leu Trp His 1640 1645 1650 Leu Val Lys Asn His Asp His Leu Asp Gln Arg Glu Gly Asp Arg 1655 1660 1665 Gly Ser Lys Met Val Ser Glu Ile Tyr Leu Thr Arg Leu Leu Ala 1670 1675 1680 Thr Lys Gly Thr Leu Gln Lys Phe Val Asp Asp Leu Phe Glu Thr 1685 1690 1695 Ile Phe Ser Thr Ala His Arg Gly Ser Ala Leu Pro Leu Ala Ile 1700 1705 1710 Lys Tyr Met Phe Asp Phe Leu Asp Glu Gln Ala Asp Lys His Gln 1715 1720 1725 Ile His Asp Ser Asp Val Arg His Thr Trp Lys Ser Asn Cys Leu 1730 1735 1740 Pro Leu Arg Phe Trp Val Asn Val Ile Lys Asn Pro Gln Phe Val 1745 1750 1755 Phe Asp Ile His Lys Asn Ser Ile Thr Asp Ala Cys Leu Ser Val 1760 1765 1770 Val Ala Gln Thr Phe Met Asp Ser Cys Ser Thr Ser Glu His Lys 1775 1780 1785 Leu Gly Lys Asp Ser Pro Ser Asn Lys Leu Leu Tyr Ala Lys Asp 1790 1795 1800 Ile Pro Asn Tyr Lys Ser Trp Val Glu Arg Tyr Tyr Ala Asp Ile 1805 1810 1815 Ala Lys Met Pro Ala Ile Ser Asp Gln Asp Met Ser Ala Tyr Leu 1820 1825 1830 Ala Glu Gln Ser Arg Leu His Leu Ser Gln Phe Asn Ser Met Ser 1835 1840 1845 Ala Leu His Glu Ile Tyr Ser Tyr Ile Ala Lys Tyr Lys Asp Glu 1850 1855 1860 Ile Leu Val Ala Leu Glu Lys Asp Glu Gln Ala Arg Arg Gln Arg 1865 1870 1875 Leu Arg Ser Lys Leu Glu Gln Val Val Asp Thr Met Ala Leu Ser 1880 1885 1890 Serum <210> 53 <211> 1894 <212> PRT <213> Mus musculus <400> 53 Met Glu Gln Arg Arg Phe Tyr Leu Arg Ala Met Gln Ala Asp Asn Leu 1 5 10 15 Ser Val Val Leu Leu Ser Val Ala Trp Leu Leu Leu Ala Arg Gly Thr 20 25 30 Thr Gly Met Pro Gln Tyr Ser Thr Phe His Ser Glu Asn Arg Asp Trp 35 40 45 Thr Phe Asn His Leu Thr Val His Arg Arg Thr Gly Ala Val Tyr Val 50 55 60 Gly Ala Ile Asn Arg Val Tyr Lys Leu Thr Gly Asn Leu Thr Ile Gln 65 70 75 80 Val Ala His Lys Thr Gly Pro Glu Glu Asp Asn Lys Ala Cys Tyr Pro 85 90 95 Pro Leu Ile Val Gln Pro Cys Ser Glu Val Leu Thr Leu Thr Asn Asn 100 105 110 Val Asn Lys Leu Leu Ile Ile Asp Tyr Ser Glu Asn Arg Leu Leu Ala 115 120 125 Cys Gly Ser Leu Tyr Gln Gly Val Cys Lys Leu Leu Arg Leu Asp Asp 130 135 140 Leu Phe Ile Leu Val Glu Pro Ser His Lys Lys Glu His Tyr Leu Ser 145 150 155 160 Ser Val Asn Lys Thr Gly Thr Met Tyr Gly Val Ile Val Arg Ser Glu 165 170 175 Gly Glu Asp Gly Lys Leu Phe Ile Gly Thr Ala Val Asp Gly Lys Gln 180 185 190 Asp Tyr Phe Pro Thr Leu Ser Ser Arg Lys Pro Arg Asp Pro Glu 195 200 205 Ser Ser Ala Met Leu Asp Tyr Glu Leu His Ser Asp Phe Val Ser Ser 210 215 220 Leu Ile Lys Ile Pro Ser Asp Thr Leu Ala Leu Val Ser His Phe Asp 225 230 235 240 Ile Phe Tyr Ile Tyr Gly Phe Ala Ser Gly Gly Phe Val Tyr Phe Leu 245 250 255 Thr Val Gln Pro Glu Thr Pro Asp Gly Met Ala Ile Asn Ser Ala Gly 260 265 270 Asp Leu Phe Tyr Thr Ser Arg Ile Val Arg Leu Cys Lys Asp Asp Pro 275 280 285 Lys Phe His Ser Tyr Val Ser Leu Pro Phe Gly Cys Thr Arg Ala Gly 290,295,300 Val Glu Tyr Arg Leu Leu Gln Ala Ala Tyr Leu Ala Lys Pro Gly Glu 305 310 315 320 Ala Leu Ala Gln Ala Phe Asn Ile Ser Ser Asp Glu Asp Val Leu Phe 325 330 335 Ala Ile Phe Ser Lys Gly Gln Lys Gln Tyr His His Pro Pro Asp Asp 340 345 350 Ser Ala Leu Cys Ala Phe Pro Ile Arg Ala Ile Asn Leu Gln Ile Lys 355 360 365 Glu Arg Leu Gln Ser Cys Tyr His Gly Glu Gly Asn Leu Glu Leu Asn 370 375 380 Trp Leu Leu Gly Lys Asp Val Gln Cys Thr Lys Ala Pro Val Pro Ile 385 390 395 400 Asp Asp Asn Phe Cys Gly Leu Asp Ile Asn Gln Pro Leu Gly Gly Ser 405 410 415 Thr Pro Val Glu Gly Leu Thr Leu Tyr Thr Thr Ser Arg Asp Arg Leu 420 425 430 Thr Ser Val Ala Ser Tyr Val Tyr Asn Gly Tyr Ser Val Val Phe Val 435 440 445 Gly Thr Lys Ser Gly Lys Leu Lys Lys Ile Arg Ala Asp Gly Pro Pro 450 455 460 His Gly Gly Val Gln Tyr Glu Met Val Ser Val Phe Lys Asp Gly Ser 465 470 475 480 Pro Ile Leu Arg Asp Met Ala Phe Ser Ile Asn Gln Leu Tyr Leu Tyr 485 490 495 Val Met Ser Glu Arg Gln Val Thr Arg Val Pro Val Glu Ser Cys Glu 500 505 510 Gln Tyr Thr Thr Cys Gly Glu Cys Leu Ser Ser Gly Asp Pro His Cys 515 520 525 Gly Trp Cys Ala Leu His Asn Met Cys Ser Arg Arg Asp Lys Cys Gln 530 535 540 Arg Ala Trp Glu Ala Asn Arg Phe Ala Ala Ser Ile Ser Gln Cys Met 545 550 555 560 Ser Leu Glu Val His Pro Asn Ser Ile Ser Val Ser Asp His Ser Arg 565 570 575 Leu Leu Ser Leu Val Val Asn Asp Ala Pro Asn Leu Ser Glu Gly Ile 580 585 590 Ala Cys Ala Phe Gly Asn Leu Thr Glu Val Glu Gly Gln Val Ser Gly 595 600 605 Ser Gln Val Ile Cys Ile Ser Pro Gly Pro Lys Asp Val Pro Val Ile 610 615 620 Pro Leu Asp Gln Asp Trp Phe Gly Leu Glu Leu Gln Leu Arg Ser Lys 625 630 635 640 Glu Thr Gly Lys Ile Phe Val Ser Thr Glu Phe Lys Phe Tyr Asn Cys 645 650 655 Ser Ala His Gln Leu Cys Leu Ser Cys Val Asn Ser Ala Phe Arg Cys 660 665 670 His Trp Cys Lys Tyr Arg Asn Leu Cys Thr His Asp Pro Thr Thr Cys 675 680 685 Ser Phe Gln Glu Gly Arg Ile Asn Val Ser Glu Asp Cys Pro Gln Leu 690 695 700 Val Pro Thr Glu Glu Ile Leu Ile Pro Val Gly Glu Val Lys Pro Ile 705 710 715 720 Thr Leu Lys Ala Arg Asn Leu Pro Gln Pro Gln Ser Gly Gln Arg Gly 725 730 735 Tyr Glu Cys Val Leu Ser Ile Gln Gly Ala Val His Arg Val Pro Ala 740 745 750 Leu Arg Phe Asn Ser Ser Ser Val Gln Cys Gln Asn Ser Ser Tyr Gln 755 760 765 Tyr Asp Gly Met Asp Ile Ser Asn Leu Ala Val Asp Phe Ala Val Val 770 775 780 Trp Asn Gly Asn Phe Ile Ile Asp Asn Pro Gln Asp Leu Lys Val His 785 790 795 800 Leu Tyr Lys Cys Ala Ala Gln Arg Glu Ser Cys Gly Leu Cys Leu Lys 805 810 815 Ala Asp His Lys Phe Glu Cys Gly Trp Cys Ser Gly Glu Arg Arg Cys 820 825 830 Thr Leu His Gln His Cys Pro Ser Thr Ser Ser Pro Trp Leu Asp Trp 835 840 845 Ser Ser His Asn Val Lys Cys Ser Asn Pro Gln Ile Thr Glu Ile Leu 850 855 860 Thr Val Ser Gly Pro Pro Glu Gly Gly Thr Arg Val Thr Ile His Gly 865 870 875 880 Val Asn Leu Gly Leu Asp Phe Ser Glu Ile Ala His His Val Gln Val 885 890 895 Ala Gly Val Pro Cys Thr Pro Ile Pro Gly Glu Tyr Ile Ile Ala Glu 900 905 910 Gln Ile Val Cys Glu Met Gly His Ala Val Ile Gly Thr Thr Ser Gly 915 920 925 Pro Val Arg Leu Cys Ile Gly Glu Cys Lys Pro Glu Phe Met Thr Lys 930 935 940 Ser His Gln Gln Tyr Thr Phe Val Asn Pro Ser Val Leu Ser Leu Ser 945 950 955 960 Pro Ile Arg Gly Pro Glu Ser Gly Gly Thr Met Val Thr Ile Thr Gly 965 970 975 His Tyr Leu Gly Ala Gly Ser Ser Val Ala Val Tyr Leu Gly Asn Gln 980 985 990 Thr Cys Glu Phe Tyr Gly Arg Ser Met Asn Glu Ile Val Cys Val Ser 995 1000 1005 Pro Pro Ser Ser Asn Gly Leu Gly Pro Val Pro Val Ser Val Ser 1010 1015 1020 Val Asp Arg Ala Arg Val Asp Ser Ser Leu Gln Phe Glu Tyr Ile 1025 1030 1035 Asp Asp Pro Arg Val Gln Arg Ile Glu Pro Glu Trp Ser Ile Thr 1040 1045 1050 Ser Gly His Thr Pro Leu Thr Ile Thr Gly Phe Asn Leu Asp Val 1055 1060 1065 Ile Gln Glu Pro Arg Val Arg Val Lys Phe Asn Gly Lys Glu Ser 1070 1075 1080 Val Asn Val Cys Thr Val Val Asn Thr Thr Thr Leu Thr Cys Leu 1085 1090 1095 Ala Pro Ser Leu Thr Ser Asp Tyr Arg Pro Gly Leu Asp Thr Val 1100 1105 1110 Glu Arg Pro Asp Glu Phe Gly Phe Leu Phe Asn Asn Val Gln Ser 1115 1120 1125 Leu Leu Ile Tyr Asn Asp Thr Lys Phe Ile Tyr Tyr Pro Asn Pro 1130 1135 1140 Thr Phe Glu Leu Leu Ser Pro Thr Gly Ile Leu Asp Gln Lys Pro 1145 1150 1155 Gly Ser Pro Ile Ile Leu Lys Gly Lys Asn Leu Cys Pro Pro Ala 1160 1165 1170 Ser Gly Gly Ala Lys Leu Asn Tyr Thr Val Met Ile Gly Glu Thr 1175 1180 1185 Pro Cys Thr Val Thr Val Ser Glu Thr Gln Leu Leu Cys Glu Pro 1190 1195 1200 Pro Asn Leu Thr Gly Gln His Lys Val Met Val His Val Gly Gly 1205 1210 1215 Met Val Phe Ser Pro Gly Ser Val Ser Val Ile Ser Asp Ser Leu 1220 1225 1230 Leu Thr Leu Pro Ala Ile Ile Ser Ile Ala Ala Gly Gly Ser Leu 1235 1240 1245 Leu Leu Ile Ile Val Ile Ile Val Leu Ile Ala Tyr Lys Arg Lys 1250 1255 1260 Ser Arg Glu Asn Asp Leu Thr Leu Lys Arg Leu Gln Met Gln Met 1265 1270 1275 Asp Asn Leu Glu Ser Arg Val Ala Leu Glu Cys Lys Glu Ala Phe 1280 1285 1290 Ala Glu Leu Gln Thr Asp Ile Asn Glu Leu Thr Ser Asp Leu Asp 1295 1300 1305 Arg Ser Gly Ile Pro Tyr Leu Asp Tyr Arg Thr Tyr Ala Met Arg 1310 1315 1320 Val Leu Phe Pro Gly Ile Glu Asp His Pro Val Leu Arg Glu Leu 1325 1330 1335 Glu Val Gln Gly Asn Gly Gln Gln His Val Glu Lys Ala Leu Lys 1340 1345 1350 Leu Phe Ala Gln Leu Ile Asn Asn Lys Val Phe Leu Leu Thr Phe 1355 1360 1365 Ile Arg Thr Leu Glu Leu Gln Arg Ser Phe Ser Met Arg Asp Arg 1370 1375 1380 Gly Asn Val Ala Ser Leu Ile Met Thr Gly Leu Gln Gly Arg Leu 1385 1390 1395 Glu Tyr Ala Thr Asp Val Leu Lys Gln Leu Leu Ser Asp Leu Ile 1400 1405 1410 Asp Lys Asn Leu Glu Asn Lys Asn His Pro Lys Leu Leu Leu Arg 1415 1420 1425 Arg Thr Glu Ser Val Ala Glu Lys Met Leu Thr Asn Trp Phe Ala 1430 1435 1440 Phe Leu Leu His Lys Phe Leu Lys Glu Cys Ala Gly Glu Pro Leu 1445 1450 1455 Phe Met Leu Tyr Cys Ala Ile Lys Gln Gln Met Glu Lys Gly Pro 1460 1465 1470 Ile Asp Ala Ile Thr Gly Glu Ala Arg Tyr Ser Leu Ser Glu Asp 1475 1480 1485 Lys Leu Ile Arg Gln Gln Ile Glu Tyr Lys Thr Leu Ile Leu Asn 1490 1495 1500 Cys Val Asn Pro Asp Asn Glu Asn Ser Pro Glu Ile Pro Val Lys 1505 1510 1515 Val Leu Asn Cys Asp Thr Ile Thr Gln Val Lys Glu Lys Ile Leu 1520 1525 1530 Asp Ala Val Tyr Lys Asn Val Pro Tyr Ser Gln Arg Pro Arg Ala 1535 1540 1545 Val Asp Met Asp Leu Glu Trp Arg Gln Gly Arg Ile Ala Arg Val 1550 1555 1560 Val Leu Gln Asp Glu Asp Ile Thr Thr Lys Ile Glu Gly Asp Trp 1565 1570 1575 Lys Arg Leu Asn Thr Leu Met His Tyr Gln Val Ser Asp Arg Ser 1580 1585 1590 Val Val Ala Leu Val Pro Lys Gln Thr Ser Ser Tyr Asn Ile Pro 1595 1600 1605 Ala Ser Ala Ser Ile Ser Arg Thr Ser Ile Ser Arg Tyr Asp Ser 1610 1615 1620 Ser Phe Arg Tyr Thr Gly Ser Pro Asp Ser Leu Arg Ser Arg Val 1625 1630 1635 Pro Met Ile Thr Pro Asp Leu Glu Ser Gly Val Lys Val Trp His 1640 1645 1650 Leu Val Lys Asn His Asp His Gly Asp Gln Lys Glu Gly Asp Arg 1655 1660 1665 Gly Ser Lys Met Val Ser Glu Ile Tyr Leu Thr Arg Leu Leu Ala 1670 1675 1680 Thr Lys Gly Thr Leu Gln Lys Phe Val Asp Asp Leu Phe Glu Thr 1685 1690 1695 Leu Phe Ser Thr Val His Arg Gly Ser Ala Leu Pro Leu Ala Ile 1700 1705 1710 Lys Tyr Met Phe Asp Phe Leu Asp Glu Gln Ala Asp Arg His Ser 1715 1720 1725 Ile His Asp Thr Asp Val Arg His Thr Trp Lys Ser Asn Cys Leu 1730 1735 1740 Pro Leu Arg Phe Trp Val Asn Val Ile Lys Asn Pro Gln Phe Val 1745 1750 1755 Phe Asp Ile His Lys Gly Ser Ile Thr Asp Ala Cys Leu Ser Val 1760 1765 1770 Val Ala Gln Thr Phe Met Asp Ser Cys Ser Thr Ser Glu His Arg 1775 1780 1785 Leu Gly Lys Asp Ser Pro Ser Asn Lys Leu Leu Tyr Ala Lys Asp 1790 1795 1800 Ile Pro Ser Tyr Lys Asn Trp Val Glu Arg Tyr Tyr Ala Asp Ile 1805 1810 1815 Ala Lys Leu Pro Ala Ile Ser Asp Gln Asp Met Asn Ala Tyr Leu 1820 1825 1830 Ala Glu Gln Ser Arg Leu His Ala Thr Glu Phe Asn Met Leu Ser 1835 1840 1845 Ala Leu Asn Glu Ile Tyr Ser Tyr Val Ser Lys Tyr Ser Glu Glu 1850 1855 1860 Leu Ile Gly Ala Leu Glu Gln Asp Glu Gln Ala Arg Arg Gln Arg 1865 1870 1875 Leu Ala Tyr Lys Val Glu His Leu Ile Asn Ala Met Ser Ile Glu 1880 1885 1890 Looking

Claims

1. A method for producing an anti-Plexin A1 agonist antibody, comprising: using a peptide consisting of residues 461 to 514 of SEQ ID NO: 3 or a peptide consisting of residues 459 to 512 of SEQ ID NO: 52 as an antigen to obtain an antibody that binds to the antigen; selecting an anti-Plexin A1 agonist antibody based on the type 3 semaphorin-like activity of the obtained antibody; A manufacturing method comprising:

2. The method of claim 1, comprising cloning DNA encoding the antibody from lymphocytes sensitized with the antigen to produce a recombinant antibody.

3. The method of claim 1 , further comprising obtaining an antibody that binds to the antigen from an antibody library.

4. The method according to claim 1, wherein the semaphorin 3-like activity is semaphorin 3A-like activity.

5. The method according to claim 4, wherein the type 3 semaphorin-like activity is an activity that promotes the regression of dendritic cells or the regression of glioma cells.

6. The method according to any one of claims 1 to 5, wherein the anti-Plexin A1 agonist antibody cross-reacts with human Plexin A1 and mouse Plexin A1.

7. The method according to any one of claims 1 to 6, wherein the anti-Plexin A1 agonist antibody is a monoclonal antibody.

8. The method of any one of claims 1 to 7, wherein the anti-Plexin A1 agonist antibody is a chimeric antibody, a humanized antibody, or a human antibody.

9. The method according to any one of claims 1 to 8, wherein the anti-Plexin A1 agonist antibody is Fab, scFv, F(ab')2, a single-chain antibody, or a bispecific antibody.

Images