阅读说明：本技术 人类抗cd47抗体及其用途 (Human anti-CD 47 antibodies and uses thereof ) 是由 陈皇慈 柳钧翔 徐崇渊 李政格 王韵婷 林俐岑 S－T·陈 C-W·江 于 2019-01-22 设计创作，主要内容包括：本发明提供以高亲和性与特异性结合至CD47的抗体,及其在治疗癌症上的用途。在本发明的一实例中,抗体或其抗原结合片段阻断CD47与信号调节蛋白α(SIRP-α)的交互作用。在另一方面,本发明提供IgG4经重新格式化的CD47抗体。在又一方面,本发明提供一用于治疗癌症的方法,其包含投予有需求的个体一治疗上有效量的抗CD47抗体。在更一方面,本发明提供一医药组合物,其包含一治疗上可接受量的抗CD47抗体及一或多个医药上可接受的载体。具体而言,医药组合物可有效治疗癌症。(The present invention provides antibodies that bind with high affinity and specificity to CD47, and their use in the treatment of cancer. In one example of the invention, an antibody or antigen-binding fragment thereof blocks the interaction of CD47 with signal-regulated protein alpha (SIRP-alpha). In another aspect, the invention provides CD47 antibodies that are reformatted to IgG 4. In yet another aspect, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-CD 47 antibody. In a further aspect, the invention provides a pharmaceutical composition comprising a therapeutically acceptable amount of an anti-CD 47 antibody and one or more pharmaceutically acceptable carriers. In particular, the pharmaceutical composition is effective in treating cancer.)

1. An isolated human anti-CD 47 antibody or antigen-binding fragment thereof, comprising: (a) a heavy chain variable (Vh) region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, and 13; (b) a light chain region having an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, and 14; or (c) a reformatted heavy chain region having an amino acid sequence selected from the group consisting of SEQ ID NOs:15, 16, 17, 18, 19, 20, and 21.

2. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment thereof blocks the interaction of CD47 with signal-regulating protein a (SIRP-a).

3. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment promotes or enhances at least one of the two or more epitopes selected from the group consisting of: enhancing macrophage-mediated phagocytosis, inducing no antibody-dependent cell-mediated cytotoxicity, inducing no apoptosis, and inducing no hemagglutination of human RBCs.

4. The antibody or antigen-binding fragment of claim 1, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO. 1.

5. The antibody or antigen binding fragment of claim 1, comprising a light chain region having the amino acid sequence of SEQ ID NO 2.

6. The antibody or antigen-binding fragment of claim 1, comprising a reformatted heavy chain region having an amino acid sequence selected from the group consisting of SEQ ID NOs:15, 16, 17, 18, 19, 20, and 21.

7. The antibody or antigen-binding fragment of claim 6, comprising a reformatted heavy chain region having an amino acid sequence selected from the group consisting of SEQ ID NOs:15, 19, and 21.

8. The antibody or antigen binding fragment of claim 1, comprising a heavy chain variable region and a light chain region having an amino acid sequence selected from the group consisting of amino acid sequences SEQ ID NO 1 and SEQ ID NO 2, amino acid sequences SEQ ID NO 3 and SEQ ID NO 4, amino acid sequences SEQ ID NO 5 and SEQ ID NO 6, amino acid sequences SEQ ID NO 7 and SEQ ID NO 8, amino acid sequences SEQ ID NO 9 and SEQ ID NO 10, amino acid sequences SEQ ID NO 11 and SEQ ID NO 12, and amino acid sequences SEQ ID NO 13 and SEQ ID NO 14.

9. The antibody or antigen binding fragment of claim 1, comprising a reformatted heavy chain region and a light chain region having an amino acid sequence selected from the group consisting of amino acid sequences SEQ ID NO 15 and SEQ ID NO 2, amino acid sequences SEQ ID NO 16 and SEQ ID NO 4, amino acid sequences SEQ ID NO 17 and SEQ ID NO 6, amino acid sequences SEQ ID NO 18 and SEQ ID NO 8, amino acid sequences SEQ ID NO 19 and SEQ ID NO 10, amino acid sequences SEQ ID NO 20 and SEQ ID NO 12, and amino acid sequences SEQ ID NO 21 and SEQ ID NO 14.

10. A fragment that binds to CD47 that is a Fab, Fab', f (ab) of the antibody of any preceding claim₂、F(ab’)₂Or scFv.

11. The antibody or antigen-binding fragment of any one of the preceding claims, which promotes macrophage-mediated phagocytosis of cells expressing CD 47.

12. The antibody or antigen-binding fragment of any one of the preceding claims, comprising an IgG isoform selected from the group consisting of an IgG1 isoform and an IgG2 isoform.

13. The antibody of any one of the preceding claims, which is an IgG4 reformatted CD47 antibody.

14. A method of treating a cancer that expresses CD47 in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of the antibody or antigen-binding fragment of any one of the preceding claims, wherein the cancer is selected from the group consisting of acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, non-hodgkin's lymphoma, multiple myeloma, bladder cancer, breast cancer, head and neck squamous cell carcinoma, ovarian cancer, and colon cancer.

15. A pharmaceutical composition comprising an antibody or antigen-binding fragment that binds to human CD47 of any preceding claim and a pharmaceutically acceptable carrier and/or excipient.

Technical Field

The present invention relates to anti-CD 47 antibodies.

Background

CD47 is a cell membrane protein belonging to the immunoglobulin superfamily, which contains an extracellular N-terminal Ig variable (IgV) domain with 5 transmembrane domains, and a short C-terminal intracellular tail. Up to now, four alternative splice isoforms of CD47 have been identified which differ in cytoplasmic tail length [ Brown, E. (2001) J Clin Invest 107(12): 1499-500; reinhold, M.I., et al (1995) J Cell Sci 108(Part 11):3419-25 ]. Recent studies have demonstrated that CD47 is a SIRP-alpha ligand expressed on phagocytes (phagocytic cells) of the immune system, including macrophages, dendritic cells, and neutrophiles [ Tsai et al (2008) J Cell Biol 180(5): 989-. The CD 47-SIRP-alpha interaction axis has now been shown to be involved in several cellular processes, including apoptosis [ Wang et al (2016) J Dent Res95(6): 697-; kaur et al (2013) Sci Rep 3: 1673), adhesion, and migration [ Rebres et al (2005) J Cell Physiol 205(2): 182-93; mock et al, (2012) Br J Pharmacol 167(7):1415-30 ]. In addition, studies have also shown that CD 47-SIRP-alpha interaction also plays an important role in the angiogenic process [ Zhang et al (2015) Brain Res 1623: 74-80; chao et al (2012) Curr Opin Immunol 24(2):225-32 ].

CD47 is ubiquitously expressed in all human cells and has been shown to be over-expressed in multiple tumor cells. Indeed, among the majority of human cancers studied to date, Acute Myelogenous Leukemia (AML), chronic myelogenous leukemia [ Jaiswal et al (2009) Cell 138(2):271-85], Acute Lymphocytic Leukemia (ALL) [ Chao et al (2011) Cancer Res 71(4): 1374-84), non-hodgkin' S lymphoma (NHL) [ Chao et al (2010) Cell 142(5): 699-. Studies have shown that high concentrations of CD47 protect cancer cells from phagocytosis (phagocytosis) due to binding of SIRP-alpha on phagocytes to CD 47. The interaction between CD47 and SIRP- α, providing a signal for phagocytes to "eat me" and subsequently prevent phagocytic elimination of cancer cells and suppression of T cell immune responses [ Oldenborg et al (2000) Science 288(5473): 2051-4; blazar et al (2001) J Exp Med 194(4): 541-9). Since tumor cells overexpress CD47 to evade surveillance by the host immune system, CD47 targeted therapy is aimed at restoring the tumor cell clearance effect currently being actively tested clinically.

Many therapeutic agents that target the CD 47-SIRP-alpha axis are currently under development, including anti-CD 47 antibodies, genetically engineered decoy receptors (decoy receptors), anti-SIRP-alpha antibodies, and bispecific agents. Upon administration, the anti-CD 47 antibody or derivative selectively inhibits the interaction between CD47 and SIRP-a. Blockade of CD47-SIRP- α interaction, abrogating SIRP- α mediated inhibition signals to phagocytes, and leading to phagocytosis such as macrophage activation and tumor cells [ Chao et al (2011) Cancer Res 71(4): 1374-84; chao et al (2010) Cell 142(5) 699-; majeti et al (2009) Cell 138(2) 286-99; chao et al (2010) Sci Transl Med 2(63):63ra94 ]. In addition, blocking signaling of CD47 transmission with antibodies also activates anti-tumor T lymphocyte immune responses and T Cell-mediated killing of tumor cells expressing CD47 [ Matozaki et al (2009) Trends Cell Biol 19(2): 72-80; latour et al (2001) J Immunol 167(5):2547-54 ].

Disclosure of Invention

Accordingly, the present invention provides novel human CD47 blocking antibodies identified from the Fab library of antibodies for cancer patients, hereinafter referred to as anti-CD 47 antibodies.

In one aspect, the invention provides an isolated human anti-CD 47 antibody, or antigen-binding fragment thereof, comprising: (a) a heavy chain variable (Vh) region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, and 13; (b) an L chain region having an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, and 14; or (c) a reformatted H chain region having an amino acid sequence selected from the group consisting of SEQ ID NOs:15, 16, 17, 18, 19, 20, and 21.

In one example of the invention, an antibody or antigen-binding fragment thereof blocks the interaction of CD47 with signal-regulated protein alpha (SIRP-alpha).

In another aspect, the invention provides IgG4 reformatted CD47 antibodies prepared and obtained from anti-CD 47 antibodies.

In one example of the invention, reformatted CD47 blocking antibodies to IgG4 were confirmed to be effective in treating cancer, since the present inventors found that in vitro, reformatted CD47 blocking antibodies to IgG4 can treat cancer cells (e.g., Jurkat-1 or HL60 cells) and induce strong phagocytic activity demonstrated by polarized THP-1 macrophages and Peripheral Blood Mononuclear Cells (PBMCs), rather than antibody-dependent cell-mediated cytotoxicity (ADCC) and apoptosis of the cancer cells tested.

In yet another aspect, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-CD 47 antibody.

In one embodiment, the mammalian cancer to be treated is selected from the group consisting of acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, bladder cancer, breast cancer, squamous cell carcinoma of the head and neck, ovarian cancer, and colon cancer.

In a further aspect, the invention provides a pharmaceutical composition comprising a therapeutically acceptable amount of an anti-CD 47 antibody and one or more pharmaceutically acceptable carriers. In particular, the pharmaceutical composition is effective in treating cancer.

In one embodiment of the invention, the anti-CD 47 antibody comprises a heavy chain variable (Vh) region having an amino acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, and 13.

In one embodiment of the invention, the anti-CD 47 antibody comprises a light chain region having an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, and 14.

In one embodiment of the invention, the anti-CD 47 antibody comprises a heavy chain region having an amino acid sequence selected from the group consisting of SEQ ID NOs:15, 16, 17, 18, 19, 20, and 21.

In yet another aspect, the invention provides a fragment that binds to CD47, comprising a Fab, Fab', F (ab) of an antibody of the invention₂、F(ab’)₂Or scFv.

Drawings

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there is shown in the drawings an embodiment which is presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

FIG. 1 shows a gel analysis of purified IgG4 reformatted CD47 antibody; wherein the CD47 antibody transiently expressed by HEK293F was purified from the culture supernatant using protein-G chromatography, and the purified antibody was analyzed by PAGE-gel under reducing and non-reducing conditions (. about.2. mu.g/lane) and visualized using Coomassie blue staining (M: protein molecular weight marker; 1: CD47 antibody under non-reducing conditions; 2: CD47 antibody under reducing conditions).

FIG. 2 provides the results of ELISA assays for binding of purified antibodies to CD47-Fc protein (R & D Systems, USA). FIG. 2A shows the results of a direct ELISA binding assay of purified antibody to CD47-Fc protein; wherein the purified antibody was shown to specifically bind to the CD47-Fc protein, but not SIRP-alpha, with Hu5F 9-G4. FIG. 2B shows a titration ELISA analysis of CD47-Fc protein by purified antibody; wherein the purified antibody was shown to bind to recombinant CD47 in a dose-dependent manner with Hu5F9-G4, wherein the purified CD47 antibody has variable binding activity.

Figure 3 shows flow cytometry analysis of cell surface CD47 binding using anti-CD 47 antibody; wherein anti-CD 47 antibodies from the repertoire bind to cell surface CD47 in a dose-dependent manner using flow cytometry analysis; and Jurkat-1 cells (FIG. 3A) or HL-60 cells (FIG. 3B) were stained with anti-CD 47 antibody, Hu5F9-G4 antibody, purified human IgG4 isotype control and analyzed for surface binding by flow cytometry, and goat anti-human Fc antibody conjugated with FITC (Jackson ImmunoResearch, USA) was used for detection.

FIG. 4 shows an antibody binding competition assay between cell surface CD47 and recombinant CD47-Fc protein using flow cytometry; wherein Jurkat-1 cells were stained with anti-CD 47 antibody (1. mu.g/ml) in the absence (right peak) or presence (left peak) of 20. mu.g/ml CD47-Fc fusion protein and analyzed for surface binding using flow cytometry; and a goat anti-human Fc antibody conjugated with FITC (Jackson ImmunoResearch, USA) was used for detection. Hu5F9-G4 was included for comparison and purified human IgG4 was used as a negative control group (upper left panel). Representative flow assays are shown using selected CD47 antibody, Hu5F9-G4, or human IgG4 isotype controls.

FIG. 5 shows the blocking of CD 47-SIRP-alpha interaction using an anti-CD 47 antibody. Human CD47-Fc binding to SIRP- α pre-coated culture wells was detected by ELISA in the absence or presence of increased concentrations of anti-CD 47 antibody or Hu5F9-G4 as a control. The anti-CD 47 antibody has different degrees of activity with Hu5F9-G4, and can block the interaction of CD47 and SIRP-alpha.

FIG. 6 shows that anti-CD 47 antibody can enhance antibody-dependent phagocytosis activity of HL-60 or Jurkat-1 cells using polarized THP-1 macrophages or PBMC. anti-CD 47 antibodies, CwP1a1, CwP2E8, BrP1F3, BrP1F11, and Hu5F9-G4 significantly enhance the phagocytosis of antibody-treated cells mediated by polarized THP-1-or PBMC.

Figure 7 shows that anti-CD 47 antibody does not elicit ADCC. ADCC Reporter Bioassay Complete Kit (Promega, USA) targeting Raji cells was used. Effectors were cultured for 6 hours at a 6 to 1 ratio to target cells at different concentrations of Hu5F9-G4, CwP1a1-IgG4, or BrP1F11-IgG4 as shown in the figure (x-axis). CwP1a1-IgG1 and HuIgG4 with human IgG1 Fc fragment were also included as controls.

FIG. 8 shows an apoptosis assay of Jurkat-1 or HL-60 cells in the presence of anti-CD 47 antibody. Cells were incubated with 10ug/ml anti-CD 47 antibody, Hu5F9-G4, or an IgG4 isotype control. Apoptotic cells were identified using annexin-V staining and analyzed by flow cytometry. Treatment with anti-CD 47 antibody or Hu5F9-G4 did not induce apoptosis in Jurkat-1 or HL-60 cells.

FIG. 9 shows RBC surface antigen binding assay (A) and RBC agglutination assay (B). Human anti-CD 47 antibodies (CwP1a1, CwP2F12, and BrP1F11) bind to the surface CD47 of RBCs in a dose-dependent manner. Additionally, CwP1A1 and BrP1F11 did not induce hemagglutination of human RBCs.

Figure 10 shows the anti-tumor activity of human anti-CD 47 antibody, CwP1a1, and BrP1F11 in an HL-60 mouse xenograft model. Male SCID mice were injected subcutaneously in the right flank with 100. mu.l Matrigel plus 1.0X10⁷HL-60 cells were subsequently intraperitoneally injected three times a week with an isoform control antibody (400. mu.g/mouse) Hu5F9-G4, or a human anti-CD 47 antibody BrP1F11-G4, in combination with an IgG1 or IgG4 isoform with CwP1A1 (400. mu.g/mouse), for three weeks. CwP1A1-G1, CwP1A1-G4, and BrP1F11-G4 antibodies exhibited significant anti-tumor activity against HL60 xenografts.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "antibody" means an immunoglobulin molecule consisting of four polypeptide chains, including two heavy (H) chains and two light (L) chains interconnected by a disulfide bond. Antibodies may include whole immunoglobulins and variants and portions of antibodies known in the art. In the present invention, there is provided a fragment which binds to CD47, including Fab, Fab', F (ab) of the antibody of the invention₂、F(ab’)₂Or scFv.

As used herein, the term "Fc" refers to the tail region of an antibody that interacts with a cell surface receptor. This property allows the antibody to activate the immune system. In contrast to Fab, one class of Fc region of all antibodies is the same in each species; it is constant rather than variable.

As used herein, the terms "antigen-binding Fragment" or "Fab" and the like refer to an antigen-binding Fragment on an antibody that binds to an antigen. A Fab fragment is an antibody structure that remains bound to antigen but is monovalent and free of an Fc portion. Fab consists of a constant domain and a variable domain for each of the heavy and light chains, where the variable domain contains the antigen binding site, which comprises a set of complementarity determining regions at the amino terminus of the monomer. Antibodies digested with papain produced two Fab fragments of about 50kDa each and an Fc fragment.

“F(ab')₂The term "as used herein means a fragment of an antibody produced by pepsin digestion of an entire IgG antibody to remove most of the Fc region while retaining some of the integrity of the hinge region. F (ab')₂Fragments have two antigen-binding F (ab) moieties linked together by a disulfide bond and are therefore bivalent, with a molecular weight of about 110 kDa.

As used herein, the term "single chain variable fragment" or "scFv" or the like means an antibody or a fusion protein of the heavy and light chain variable regions of an immunoglobulin, linked by a short linker peptide (e.g., a linker having 10 to 25 amino acids). This single chain variable fragment retains the specificity of the original antibody despite the removal of the constant region and the introduction of the linker.

As used herein, the term "antibody-dependent cell-mediated cytotoxicity (ADCC)" means an immune mechanism by which Fc receptor-bearing effector cells can recognize and kill antibody-coated target cells, expressing tumor-or pathogen-derived antigens on their surface.

The invention provides anti-CD 47 antibodies that specifically bind to human CD47 and block the interaction of CD47 with SIRP- α. According to the assays shown in examples 8 and 9, the anti-CD 47 antibodies of the present invention enhance macrophage-mediated phagocytosis without inducing apoptosis.

According to the present invention, anti-CD 47 antibodies specifically bind to human and mouse CD 47.

According to the present invention, the anti-CD 47 antibody comprises a heavy chain variable (Vh) region having an amino acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, and 13.

According to the present invention, the anti-CD 47 antibody comprises an L chain region having an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, and 14.

According to the invention, the anti-CD 47 antibody is a polypeptide comprising a sequence selected from the group consisting of:

a Vh region having an amino acid sequence shown in SEQ ID NO. 1, and an L chain region having an amino acid sequence shown in SEQ ID NO. 2;

a Vh region having an amino acid sequence shown in SEQ ID NO. 3, and an L chain region having an amino acid sequence shown in SEQ ID NO. 4;

a Vh region having an amino acid sequence shown in SEQ ID NO. 5, and an L chain region having an amino acid sequence shown in SEQ ID NO. 6;

a Vh region having an amino acid sequence shown in SEQ ID NO. 7, and an L chain region having an amino acid sequence shown in SEQ ID NO. 8;

a Vh region having an amino acid sequence shown by SEQ ID NO. 9, and an L chain region having an amino acid sequence shown by SEQ ID NO. 10;

a Vh region having an amino acid sequence shown as SEQ ID NO. 11, and an L chain region having an amino acid sequence shown as SEQ ID NO. 12; and

a Vh region having an amino acid sequence shown as SEQ ID NO. 13, and an L chain region having an amino acid sequence shown as SEQ ID NO. 14,

amino acids of the group.

In addition, the anti-CD 47 antibodies of the invention comprise a reformatted H chain having an amino acid sequence selected from the group consisting of SEQ ID NOs:15, 16, 17, 18, 19, 20, and 21.

The following table lists the amino acid sequences:

TABLE 1 amino acid sequences of anti-CD 47 Fabs binders screened from the library.

Table 2 amino acid sequence of heavy chain of IgG4 reformatted CD47 antibody.

According to the invention, the anti-CD 47 antibody is a polypeptide comprising a sequence selected from the group consisting of:

an H chain region having an amino acid sequence selected from the group consisting of SEQ ID NO. 15, and an L chain region having an amino acid sequence selected from the group consisting of SEQ ID NO. 2;

an H chain region having an amino acid sequence selected from the group consisting of SEQ ID NO 16, and an L chain region having an amino acid sequence selected from the group consisting of SEQ ID NO 4;

an H chain region having an amino acid sequence selected from the group consisting of SEQ ID NO 17, and an L chain region having an amino acid sequence selected from the group consisting of SEQ ID NO 6;

an H chain region having an amino acid sequence selected from the group consisting of SEQ ID NO. 18, and an L chain region having an amino acid sequence selected from the group consisting of SEQ ID NO. 8;

an H chain region having an amino acid sequence selected from the group consisting of SEQ ID NO 19, and an L chain region having an amino acid sequence selected from the group consisting of SEQ ID NO 10;

an H chain region having an amino acid sequence selected from the group consisting of SEQ ID NO 20, and an L chain region having an amino acid sequence selected from the group consisting of SEQ ID NO 12; and

an H chain region having an amino acid sequence selected from the group consisting of SEQ ID NO 21, and an L chain region having an amino acid sequence selected from the group consisting of SEQ ID NO 14,

amino acids of the group.

In this document, reference is made to the following representative anti-CD 47 antibodies of the invention. CwP1A1 represents an antibody having an H chain region corresponding to SEQ ID NO. 15 and an L chain region corresponding to SEQ ID NO. 2. CwP1C4 represents an antibody having an H chain region corresponding to SEQ ID NO. 16 and an L chain region corresponding to SEQ ID NO. 4. CwP2E8 represents an antibody having an H chain region corresponding to SEQ ID NO. 17 and an L chain region corresponding to SEQ ID NO. 6. CwP2F8 represents an antibody having an H chain region corresponding to SEQ ID NO. 18 and an L chain region corresponding to SEQ ID NO. 8. CwP2F12 represents an antibody having an H chain region corresponding to SEQ ID NO. 19 and an L chain region corresponding to SEQ ID NO. 10. BrP1F3 represents an antibody having an H chain region corresponding to SEQ ID NO. 20 and an L chain region corresponding to SEQ ID NO. 12. BrP1F11 represents an antibody having an H chain region corresponding to SEQ ID NO. 21 and an L chain region corresponding to SEQ ID NO. 14.

In the present invention, anti-CD 47 antibodies promote macrophage-mediated phagocytosis of CD47 expressing cells.

In the present invention, the anti-CD 47 antibody does not induce antibody-dependent cell-mediated cytotoxicity or apoptotic activity.

In the present invention, the antibody may comprise an IgG isoform selected from the group consisting of the IgG1 isoform and the IgG2 isoform.

In the present invention, the antibody may be prepared as a reformatted CD47 antibody that is IgG 4.

In one embodiment, the invention provides a method for treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of an anti-CD 47 antibody.

In one embodiment, the anti-CD 47 mammalian cancer to be treated is selected from the group consisting of acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, non-hodgkin's lymphoma, multiple myeloma, bladder cancer, breast cancer, head and neck squamous cell carcinoma, ovarian cancer, and colon cancer.

In a further embodiment, the invention provides a pharmaceutical composition comprising a therapeutically acceptable amount of an anti-CD 47 antibody and one or more pharmaceutically acceptable carriers. In particular, the pharmaceutical composition is effective in treating cancer.

In one embodiment of the invention, the recombinant CD47 protein (R) is commercially available as recombinant&D Systems, USA) screened a library of antibodies for study Fab, TRB800-01, to isolate fully human antibodies to CD 47. To be administered orally from a patient containing 16x, 22x esophagus, and15x prostate cancer (IRB No. VGHKS17-CT11-13) and a capacity of-1 x10¹⁰Construct TRB800-01 from a blood sample collected from a clinically diagnosed patient.

In another embodiment of the invention, the titer of the eluted phage is significantly increased via panning, representing enrichment of the CD 47-specific binding partner. 384 clones randomly selected from round 3 panning were further confirmed using a direct ELISA assay for CD47 protein, and OD was selected₄₅₀Positive binders greater than 0.5 were subjected to sequencing analysis. 7 unique clusters were identified and selected for reformatting into full-length human IgG4 for analysis in FreeStyle^TMExpression using HEK293 cells in the 293-F system (Invitrogen, USA) was performed and further confirmed.

The invention is further illustrated by the following examples, which are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent.

Example 1 human antibody Fab library screening for recombinant CD47 protein

From the blood of selected cancer patients (diversity-10 thereof)¹⁰) Constructing a human antibody Fab library. Briefly, the identification of CD47 antibodies for the antibody library was performed as follows. ELISA wells at 500 ng/well (in PBS) of human CD47-Fc protein (R) at 4 ℃&D Systems, USA) for 16 hours. After blocking,. about.10¹⁰Phage were added and incubated on a shaker at room temperature for 1 hour. Unbound phage were removed and the culture wells were washed 10 times with PBS containing 0.05% Tween-20 (PBS-T). After washing, bound phage were eluted by adding 100. mu.l of 0.1M TEA solution (Sigma, USA) and neutralized with 50. mu.l of 1M Tris-HCl, pH 7.4. Subsequently, the eluted phages were used to infect log-phase TG-1 cells, plated on 1.5% agar plates containing antibiotics and glucose, and incubated overnight at 30 ℃. In the morning of the morning, with disintegrated (scarped) bacteria (30 OD)₆₀₀) For phage rescue, M13Hyperphages (PROGEN, Germany) was used. After centrifugation, rescued phagesPrecipitated with PEG and reconstituted in PBS, titrated, and used for the next round of panning. The panning process was repeated two additional times. After the last round of screening, individual clones were selected, cultured and induced with 1mM IPTG to produce Fab. Supernatants containing expressed Fabs from each individual clone were analyzed using an ELISA for CD47-Fc protein to identify binders.

Example 2 sequencing analysis and reformatting of combinations of CD47

Selection of OD₄₅₀>0.5 ELISA positive binding for sequencing analysis. The heavy chain of the identified CD47 binding body was genetically engineered to be a human IgG4 framework to minimize the recruitment of Fc-dependent effector functions (such as ADCC and CDC). The amino acid sequences of the light and heavy chain variable (Vh) regions, and the reformatted full-length heavy chain sequence (in the form of an IgG4 isoform) of the identified CD47 binder are listed in tables 1 and 2. Subsequently, individual reformatted CD47 antibody heavy chains and corresponding light chains were sub-cloned into individual pCI-neo vectors (Promega, USA) for paired HEK293 transfection, expression, purification, and validation.

Example 3 antibody expression and purification

HEK293 cells and FreeStyle^TM293 Expression Medium (Invitrogen, USA) was used to generate recombinant antibodies. Transient transfections were performed according to the manufacturer's instructions (Invitrogen, USA). To purify the antibody, the culture supernatant was applied to Pierce protein G agarose resin (ThermoFisher, USA) and dialyzed against PBS. The purified antibodies were analyzed on a 4-12% Bolt Bis-Tris plus gel (Invitrogen, USA) by SDS-PAGE under reducing or non-reducing conditions and the results were visualized by Coomassie blue staining (Invitrogen, USA).

The results in fig. 1 show that all of the reformatted anti-CD 47 antibodies to IgG4 were properly expressed and produced. At the same time, each individual antibody was obtained with a purity of greater than 90% using protein-G chromatography.

Example 4 CD47 binding Studies

To check and confirm the binding specificity, the purified anti-CD 47 antibody was first validated in a direct ELISA assay. Briefly, at 4 ℃, 96-well ELISA culture dish (Nunc, Denmark) with 1. mu.g/ml recombinant CD47-Fc or SIRP-alpha (R)&D Systems, USA) for 16 hours. At room temperature, CD47 or SIRP-alpha pre-coated culture wells were blocked with 5% skim milk in PBS for 1 hour, followed by addition of 100ul of 0.5. mu.g/ml human anti-CD 47 antibody and shake culture at room temperature for 1 hour. After incubation and washing, 100. mu.l of a 1:2500 diluted HRP conjugated goat anti-human Fc antibody (Jackson ImmunoResearch, USA) was added to each culture well and incubated at room temperature for 1 hour. After the final wash, bound antibodies were detected using TMB solution (Invitrogen, USA). The reaction was stopped by adding 50. mu.l of 1M HCl and stopped at OD₄₅₀The absorbance of each well was read at nm.

The results in FIG. 2A indicate that, although the binding activity varied from clone to clone, all candidate antibodies showed specificity for recombinant CD47 with Hu5F9-G4 and no cross-reactivity to SIRP-alpha protein (R & D Systems, USA). Hu5F9-G4 is a humanized antibody targeting CD47, has remarkable biological activity in vitro and in vivo, and can be used as a positive control group for comparison.

Titration ELISA assays were used to examine the binding activity of those candidate antibodies. In the titration ELISA assay, culture wells were coated with 1. mu.g/ml recombinant CD47-Fc for 16 hours at 4 ℃. CD47 culture wells were blocked with 5% skim milk in PBS for 1 hour at room temperature, followed by addition of 100ul of a 1:3 serial dilution of anti-CD 47 antibody starting at 30nM and shake culture at room temperature for 1 hour. After incubation and washing, 100ul of a 1:2500 dilution of HRP conjugated goat anti-human Fc antibody (Jackson ImmunoResearch, USA) was added to each well and incubated at room temperature for 1 hour. After the final wash, bound antibodies were detected using TMB solution (Invitrogen, USA). The reaction was stopped with 1M HCl and at OD₄₅₀The absorbance of each well was read at nm. Subsequently, the EC of each tested CD47 antibody was calculated using GraphPad Software₅₀(antibody concentration required for half-maximal absorbance).

As shown in fig. 2B, all tested anti-CD 47 antibodies bound to recombinant CD47 in a dose-dependent manner, and Hu5F9-G4, CwP1a1, CwP1C4 were determinedEC of CwP2E8, CwP2F8, CwP2F12, BrP1F3, and BrP1F11₅₀0.1442nM, 0.1835nM, 0.2271nM, 0.4694nM, 0.3264nM, 0.4413nM, 10.1nM, and 1.21nM, respectively.

Example 5 cell surface antigen binding assay

In one embodiment of the invention, differences between recombinant and native CD47 proteins were examined by flow cytometry for cell surface CD47 binding. The binding activity of anti-CD 47 antibodies with increasing concentration was against cell surface CD47 on Jurkat-1 or HL-60 cells (BCRC, taiwan, china). In the cell surface antigen binding assay, 1X10 cells were incubated at 4 deg.C⁵Jurkat-1 cells (BCRC, Taiwan, China) were cultured with 0.001, 0.01, 0.1, 1, 10, or 100. mu.l/ml of anti-CD 47 antibody for 15min in 100. mu.L. After incubation, cells were stained with ice-cold staining buffer (1 XPBS + 2% FBS + 0.05% NaN)₃) Washed three times and then incubated with a goat anti-human Fc antibody conjugated with FITC (Jackson ImmunoResearch, USA). Finally, the cells were treated with BD Accuri^TMC6 Plus flow cytometer (BD Biosciences, USA) washing and analysis. The Mean Fluorescence Intensity (MFI) values are plotted.

The results collected from figure 3 show that in the two cell lines tested, the anti-CD 47 antibody was dose-dependent on cell surface binding of Hu5F9-G4, whereas the human IgG4 isoform control group (BioLegend, USA) was not.

Example 6 competitive assay for CD47 binding Using flow cytometry

In another specific embodiment of the invention, the binding specificity of the anti-CD 47 antibody to native CD47 is performed by antibody binding competition assays using flow cytometry, with or without excess recombinant CD47-Fc protein. 1X10 at 4 ℃ in the absence or presence of 10. mu.g/ml of recombinant CD47-Fc protein⁵Each Jurkat-1 cell was incubated with 1. mu.g/ml human anti-CD 47 antibody for 15 minutes. Thereafter, in BD Accuri^TMPrior to flow cytometry analysis by C6 Plus (BD Biosciences, USA), cells were washed and stained with FITC conjugated goat anti-human Fc antibody (Jackson ImmunoResearch, USA).

FIG. 4 shows that the surface CD47 binding activity of anti-CD 47 antibodies to Hu5F9-G4 was shown to be blocked by the presence of 10. mu.g/ml of CD47-Fc fusion protein, representing the specific binding effect of all antibodies tested on CD 47.

Example 7 CD 47-SIRP-alpha interaction Block assay

Recombinant anti-CD 47 antibodies were tested for CD 47-sirpa interaction blocking activity using an ELISA assay. ELISA culture wells (Nunc, Denmark) were incubated with 1. mu.g/ml of recombinant human His-tagged SIRP-alpha protein (R) at 4 ℃&D Systems, USA) for 16 hours. After blocking, 1. mu.g/ml of recombinant CD47-Fc protein was added with 3-fold serial dilutions of anti-CD 47 antibody, starting at 30nM, and incubated for 1 hour at room temperature. After incubation, the culture wells were washed and incubated with HRP conjugated goat anti-human Fc antibody (1:2500 dilution, Jackson ImmunoResearch, USA) for 1 hour. After the final wash, bound CD47-Fc protein was detected with TMB substrate. The reaction was stopped by addition of 1M HCl and an OD450 reading was obtained. IC was calculated for each tested CD47 antibody using GraphPad Software₅₀(antibody concentration required to suppress half-maximal absorbance).

The anti-CD 47 antibody was shown in figure 5 to block the interaction between recombinant human CD47 and recombinant human SIRP- α. Calculated ICs for Hu5F9-G4, CwP1a1, CwP1C4, CwP2E8, CwP2F8, and CwP2F12₅₀0.063nM, 0.053nM, 0.155nM, 0.556nM, 0.068nM, and 0.063nM, respectively. The results of this study showed CwP1A1, CwP2F8, and CwP2F12 to have blocking activity similar to or better than Hu5F 9-G4.

Example 8 in vitro antibody-mediated phagocytosis assay

Subsequently, it was examined whether the CD 47-SIRP-alpha interaction-blocking antibody could induce CD47⁺Macrophage-mediated phagocytosis of cancer cells. The in vitro phagocytosis assay is performed briefly as follows. Using PMA, LPS (Sigma, USA), and IFN-gamma (R)&D Systems, USA), inducing differentiation of THP-1(BCRC, taiwan) monocytes into macrophages. Subsequently, the polarized THP-1 cells were stained with CellTracker Red (CTR, Life Technology, USA) prior to use. At 5. mu.g/mlCFSE-labeled human cells Jurkat-1 or HL-60 cells (BCRC, Taiwan) were cultured with polarized THP-1 macrophages at 37 ℃ for 3 hours in the presence of a configuration control antibody, Hu5F9-G4, or human anti-CD 47 antibody (IgG 4). Cells were washed prior to flow cytometry analysis to determine the phagocytic index (number of cells ingested per 100 macrophages). PBMC-derived macrophages were induced by treatment with 50ng/ml rhM-CSF (Peprotech, USA) for 7 days and confirmed by staining with anti-CD 14 antibody (Abcam, USA). CFSE-labeled HL-60 cells were cultured with macrophages in the presence of 10. mu.g/ml of an isotype control antibody, Hu5F9-G4, or human anti-CD 47 antibody for 2 hours at 37 ℃. Subsequently, the cells were analyzed by flow cytometry to determine the phagocytic index.

HL-60AML and Jurkat-1 ALL cells were used as target cells. Phagocytic activity was examined using polarized THP-1 cells and human peripheral blood-derived macrophages. Phagocytic activity was determined by flow cytometry. As shown in FIGS. 6A-C, human anti-CD 47 antibody and Hu5F9-G4 significantly promoted phagocytosis of tumor cells by polarized THP-1 and PBMC-derived macrophages.

Example 9 ADCC assay

To assess whether human CD47 antibody induces ADCC in addition to phagocytosis, ADCC activities of CwP1a1-IgG4, BrP1F11-IgG4, and Hu5F9-G4 having an IgG4 framework were examined using an ADCC reporter bioassay. In this study, CwP1a1-IgG1 and HuIgG4 with human IgG1 Fc fragment were also included as controls, respectively. ADCC Reporter Bioassay Complete Kit (Promega, USA) targeting Raji cells was used according to the manufacturer's instructions. Effectors were cultured for 6 hours at a 6 to 1 ratio to target cells at different concentrations of HuIgG4, Hu5F9-G4, CwP1a1-IgG1, CwP1a1-IgG4, or BrP1F11-IgG4 as shown in the figure (x-axis). After incubation, Bio-Glo luciferase assay reagent was added and the luminescence read. The samples were run in duplicate and the mean of the duplicate values was plotted, with error bars representing the Standard Error (SEM) of the mean.

The results shown in FIG. 7 indicate that CwP1A1-IgG1 can induce ADCC activity in a dose-dependent manner. On the other hand, CwP1A1-IgG4, BrP1F11-IgG4, Hu5F9-G4, and HuIgG4 did not induce ADCC. In general, the mechanism of action of TRB human CD47 antibody, CwP1a1-IgG4, and BrP1F11-IgG4 did not induce ADCC or apoptosis, but rather activated antibody-dependent phagocytosis.

Example 10 apoptosis assay

An apoptosis assay was performed to examine whether binding of anti-CD 47 antibody to CD47 directly induces apoptosis in cancer cells. Human cells Jurkat-1, HL-60, or Raji cells at 1x10⁶Each cell/ml was resuspended in RPMI-1640 medium containing 10% FBS. Mu.g/ml of the isotype control, 2D3, Hu5F9-G4, or human anti-CD 47 antibody (IgG4) were added and the cells were cultured at 37 ℃ for 3 hours. In the case of the HL-60 cell line, staurosporine (staurosporine) was used as a positive control group. Apoptotic cells were identified by annexin-V and PI staining according to the manufacturer's instructions (BD Biosciences, USA) and analyzed by flow cytometry.

As shown in FIG. 8A, it was found that the anti-CD 47 antibody tested did not induce apoptosis of Jurkat-1 cells similar to the isoform control group, as compared to Hu5F 9-G4. FIG. 8B shows that human anti-CD 47 antibody did not induce apoptosis of HL-60 cells, unlike treatment with staurosporine. Similar results were obtained with Raji cells (data not shown). Overall, the inventors' results indicate that anti-human CD47 antibodies induce phagocytosis rather than apoptotic activity.

In the present invention, anti-CD 47 antibodies with therapeutic potential are currently tested for their efficacy against acute myeloid leukemia and solid tumors in xenograft animal models.

Example 11 RBC surface antigen binding assay and RBC agglutination assay

In the context of the RBC surface antigen binding assay, cell surface CD47 binding activity was determined using flow cytometry. Human erythrocytes were isolated by Ficoll-Paque Plus (Sigma, Sweden) using density gradient centrifugation. Purified human RBCs were incubated with 1, 10, and 100. mu.g/ml anti-CD 47 antibody (IgG4) in 100. mu.l for 15min at 4 ℃. After incubation, cells were stained with ice-cold staining buffer (1 XPBS + 2% FBS + 0.05% NaN)₃) Cleaning threeNext, the cells were then incubated with a goat anti-human Fc antibody conjugated with FITC (Jackson ImmunoResearch, USA). The cells are treated with BD Accuri^TMC6 Plus flow cytometer (BD Biosciences, USA) washing and analysis. The Mean Fluorescence Intensity (MFI) values are plotted. In the context of RBC agglutination assays, human RBCs are first diluted in PBS and then added to 96-well round bottom culture plates (Thermo, Denmark) at multiple antibody concentrations. The plates were incubated at 37 ℃ for 4 hours. Non-menstruating RBCs were defined as breakpoint (breakthrough dot) and HA index was calculated using Image J software.

As shown in fig. 9A, human anti-CD 47 antibodies (CwP1a1, CwP2F12, and BrP1F11) bound to the surface CD47 of RBCs in a dose-dependent manner. Fig. 9B shows that CwP1a1 and BrP1F11 did not induce hemagglutination of human RBCs. In contrast, Hu5F9-G4 (an anti-CD 47 antibody at the time of clinical examination) induced severe RBC hemagglutination. The results indicate that anti-CD 47 antibodies, CwP1a1, and BrP1F11 may have a better safety profile than Hu5F9-G4 with respect to hemagglutination of RBCs.

Example 12 in vivo evaluation of anti-CD 47 antibody efficacy Using a mouse xenograft model

The right flank of a male SCID mouse (BioLASCO, Taiwan, China) was injected subcutaneously with 100. mu.l of Matrigel (Corning, USA) plus 1.0X10⁷HL-60 cells (0.1mL of cell suspension). Mice were intraperitoneally injected three times per week with isotype control antibody (400 ug/mouse), Hu5F9-G4, or human anti-CD 47 antibody BrP1F11-G4 with IgG1 or IgG4 isotypes and CwP1a1(400 ug/mouse) for three weeks. Tumor volumes were measured twice per week. Using the equation V ═ LW²The tumor volume was calculated 2. After sacrifice of mice, tumor tissue was excised and fixed in formalin.

To evaluate the antitumor activity, anti-CD 47 antibodies BrP1F11-G4, CwP1a1-G1, and CwP1a1-G4 were tested in an HL-60 xenograft model and compared to Hu5F9-G4 antibody. As shown in FIG. 10A, 400 μ G of CwP1A1-G1, CwP1A1-G4, and BrP1F11-G4 antibodies administered intraperitoneally to each mouse demonstrated significant anti-tumor activity against human acute promyelocytic leukemia (HL 60 xenograft) and comparable to Hu5F 9-G4. Representative tumors for each treatment are shown in fig. 10B.

Example 13 pharmacokinetic evaluation

PK of anti-CD 47 antibody was assessed in rats (n ═ 5). The rat was administered 10mg/kg of anti-CD 47 antibody by tail vein injection. Blood samples were collected via the tail vein of each rat at 0 min, 1 hr, 4 hr, 8 hr, 12 hr, 1 day, 2 days, 3 days, 4 days, 6 days, 8 days, 10 days, 12 days, and 14 days after injection. The concentration of antibody in serum was determined by enzyme linked immunosorbent assay (ELISA). Serum concentrations of anti-CD 47 antibody were interpolated from a 4-parameter logistic regression of the standard curve on the same culture dish.

In view of the above, anti-CD 47 antibodies were genetically engineered on the human IgG4 framework to minimize recruitment of Fc-dependent effector functions (ADCC and CDC) and not involving apoptosis, therefore, the present invention suggests that anti-CD 47 antibodies (as well as Hu5F9-G4) can trigger activated macrophage-mediated phagocytosis by blocking SIRP- α interaction with CD 47.

Sequence listing

<110> SanYu Biotechnology Ltd

Chen and Huang Ci

<120> human anti-CD 47 antibody and use thereof

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Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln

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Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly

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Arg Asn Tyr Val Phe Gly Thr Gly Thr Lys Val Ser Val Leu Ser Gln

100 105 110

Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 15

<211> 449

<212> PRT

<213> Intelligent people

<400> 15

Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Arg

20 25 30

Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala Leu Ile Tyr Trp Asn Asp Asp Lys Arg Tyr Ser Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala His Leu Ile Thr Phe Gly Gly Arg Arg Ala Phe Asp Ile 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 Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr

210 215 220

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp

260 265 270

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445

Lys

<210> 16

<211> 449

<212> PRT

<213> Intelligent people

<400> 16

Gln Val Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Leu Ser Leu Ser Thr Ser

20 25 30

Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala Leu Ile Tyr Trp Asn Asp Asp Lys Arg Tyr Ser Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Val Thr Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala His Leu Ile Thr Phe Gly Gly Arg Arg Ala Phe Asp Ile 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 Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr

210 215 220

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp

260 265 270

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445

Lys

<210> 17

<211> 448

<212> PRT

<213> Intelligent people

<400> 17

Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Leu Phe Gly Pro Ser Arg Ser Ser Ala Phe Asp Ile Trp Gly

100 105 110

Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly

210 215 220

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

260 265 270

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 18

<211> 447

<212> PRT

<213> Intelligent people

<400> 18

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala 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 Ser Leu Arg Trp Leu His Arg Val Phe Asp Tyr 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 Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 19

<211> 444

<212> PRT

<213> Intelligent people

<400> 19

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Val Trp Met

35 40 45

Gly Thr Ser Ile Pro Thr Ala Ala Ser Gly Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Thr 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 Gly Gly Arg Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Pro

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 20

<211> 444

<212> PRT

<213> Intelligent people

<400> 20

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 Gly Gly Thr Leu Gly Met Asp Val Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 21

<211> 446

<212> PRT

<213> Intelligent people

<400> 21

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Ser Asn Ala Gly Asn Thr Gly Tyr Ala Gln Asn Phe

50 55 60

Gln Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Lys Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Pro Gly Met Gly Trp Tyr Met His His Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445