HK40005591B - Methods for identifying lilrb-blocking antibodies - Google Patents

Description

Methods for identifying LILRB blocking antibodies

The invention was made with government support under fund number 1R01 CA172268, provided by the national institutes of health. The government has certain rights in the invention.

Priority of U.S. provisional application serial No. 62/368,672, filed on 29/07/2016, the entire contents of which are incorporated herein by reference.

The sequence listing is contained in the file entitled "UTFHP0332WO _ st25.Txt", 176KB (measured in Microsoft Windows), created at 27.07.2017, which was filed electronically with the present application and is incorporated by reference.

Background

1. Field of the invention

The present disclosure relates generally to the field of molecular biology. More particularly, it relates to methods and compositions for identifying LILRB antibodies.

2. Description of the related Art

Acute Myeloid Leukemia (AML) is the most common adult acute leukemia and the common pediatric cancer. Current treatments for AML involve intensive cytotoxic chemotherapy, usually followed by myeloablative conditioning and stem cell transplantation. However, despite treatment, most patients will relapse or die from the disease within 5 years ¹ . New therapies for AML have not been approved for more than 30 years. In order to effectively treat AML, new molecular targets and therapeutic approaches must be identified. Recently, it has been discovered that the inhibitory leukocyte immunoglobulin-like receptor (LILRB) and the receptor LAIR1 containing the associated immunoreceptor tyrosine-based inhibitory motif (ITIM) have tumor-promoting functions in a variety of hematopoietic and solid cancer cells ^2,3,4-18,19 . ITIM-containing receptors are expressed in a variety of immune cells and signal by recruitment of phosphatases SHP-1, SHP-2, or SHIP, resulting in negative regulation of immune cell activation ^20,21,22 . Similar to CTLA4 and PD-1 ²³ LILRB is considered to be an immunodetection point factor ²² 。

LILRB can inhibit the activity of multiple immune cell types that promote tumor immune escape ²² . LILRB4 is expressed on monocytes, macrophages and dendritic cells, and can suppress innate immunity in a cell-autonomous manner and inhibit T cell activation by an indirect mechanism ^24,25 . LILRB4 is a specific marker for monocytic AML including refractory and relapsed diseases ²⁶ . LILRB1-5 is primate and human specific, but there are two orthologs of mice: paired immunoglobulin-like receptor B (PirB) ²⁷ And gp49B1 ²⁸ . The receptor LAIR1, which contains the relevant immunoreceptor tyrosine-based inhibitory motif (ITIM), has both human and mouse versions of the protein. Since the value of mouse models is limited and ligands for several LILRB including LILRB4 are still unknown, little is known about the biological function and clinical significance of these receptors.

Disclosure of Invention

Embodiments of the present disclosure provide methods and compositions relating to modulation of LILRB activation by its ligands. In a first embodiment, there is provided a method of identifying a modulator of LILRB activation, the method comprising: (a) Contacting a reporter cell with a ligand of a LILRB and a candidate substance; and (b) detecting a level of LILRB activation in the reporter cell, wherein a change in the level of LILRB activation compared to a reference level indicates that the candidate substance is a modulator of LILRB activation. In certain aspects, the reporter cell is a mouse T-cell hybridoma cell.

In some aspects, the reporter cell expresses a receptor comprising the extracellular domain of LILRB. In certain aspects, the extracellular domain of LILRB is further defined as the extracellular domain of LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, LAIR1 (human or mouse), pirB, or gp49B 1. In a particular aspect, the LILRB is further defined as LILRB4. In certain aspects, the ligand of LILRB4 is ApoE or LFA-1. In certain aspects, the ligand of LILRB is MHC I, UL18, S100A8, S100A9, angptls, β -amyloid, myelin inhibitors, CD1d, collagen, or integrin α v β 3. In other aspects, the receptor is a chimeric receptor comprising an intracellular domain that pairs with immunoglobulin-like receptor beta (PILR β).

In certain aspects, the chimeric receptor is expressed in a reporter cell by a viral expression vector. In some aspects, the viral expression vector is a retroviral expression vector. In particular aspects, the level of LILRB activation is measured based on the morphology or mobility of the cell. In certain aspects, the reporter cell further comprises a reporter gene encoding a detectable label operably linked to a promoter that is regulated by activation of the receptor. In a particular aspect, the promoter is a nuclear factor of activated T cell (NFAT) promoter. In particular aspects, the promoter is a CCL2 promoter, a CCL4 promoter, a CCL5 promoter, an IL-6R promoter, an IL-8 promoter, a gp130 promoter, an OSM promoter, a TIMP-1/2 promoter, a TNF-R1/II promoter, a uPAR promoter, or an arginase-1 promoter.

In some aspects, the detectable label is a colorimetric label, a fluorescent label, a bioluminescent label, or a chemiluminescent label. In certain aspects, the detectable label is GFP, YFP, RFP, or D-luciferin. In a particular aspect, the detectable label is GFP. In some aspects, the detecting step comprises flow cytometry analysis or luminescence quantification.

In certain aspects, the candidate compound is an antibody. In some aspects, the antibody is a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, fab ', F (ab') 2, fv, or scFv. In a particular aspect, the antibody is a monoclonal antibody.

In some aspects, the reference level is obtained from a reporter cell contacted with ApoE alone. In certain aspects, the ApoE is recombinant. In a particular aspect, the ApoE is human ApoE. In some aspects, human ApoE is isolated from serum. In certain aspects, apoE is further defined as ApoE2, apoE3 or ApoE4.

In certain aspects, an increase in the level of LILRB activation compared to a reference level indicates that the modulator is an agonist. In certain aspects, a decrease in the level of LILRB activation compared to a reference level indicates that the modulator is an antagonist.

In certain aspects, the candidate substance is linked to a substrate. In certain aspects, the candidate agent is linked to a cell that expresses FcR.

A further embodiment provides a composition for identifying a modulator of LILRB activation. In one aspect, the composition comprises a candidate LILRB modulator, a ligand for a LILRB, and a reporter cell expressing a receptor comprising an extracellular domain of the LILRB, wherein the reporter cell has a phenotype indicative of LILRB activation. In certain aspects, the reporter cell further comprises a reporter gene encoding a detectable label operably linked to a promoter that is regulated by activation of the receptor. In some aspects, the receptor further comprises the intracellular domain of PILR β. In certain aspects, the candidate LILRB inhibitor is an antibody. In some aspects, the detectable label is GFP. In certain aspects, the composition further comprises a cell that expresses FcR.

A further embodiment provides a composition for identifying a modulator of LILRB activation in the absence of its known ligand. In one aspect, the composition comprises a candidate LILRB modulator and a reporter cell expressing a receptor comprising an extracellular domain of LILRB, wherein the reporter cell has a phenotype suggestive of LILRB activation. In certain aspects, the reporter cell further comprises a reporter gene encoding a detectable label operably linked to a promoter that is regulated by activation of the receptor. In some aspects, the receptor further comprises the intracellular domain of PILR β. In certain aspects, the candidate LILRB inhibitor is an antibody. In some aspects, the detectable label is GFP. In certain aspects, the composition further comprises a cell that expresses FcR.

A still further embodiment provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an inhibitor of ApoE-induced LILRB activation (e.g., identified by embodiments disclosed herein). In some aspects, the inhibitor of ApoE-induced LILRB activation is an antibody. In a particular aspect, the cancer is AML.

A further embodiment provides a method of treating an autoimmune disease or inhibiting the appearance of transplant rejection or treating an inflammatory disorder in a subject, the method comprising administering to the subject an effective amount of an agonist of ApoE-induced LILRB activation.

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the disclosure, are given by way of illustration only, since various changes and modifications will become apparent to those skilled in the art from this detailed description without departing from the spirit and scope of the disclosure.

Drawings

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the description of specific embodiments presented herein.

Fig. 1-schematic diagram of the LILRB4 reporting system.

FIG. 2-a diagram of a LILRB and its ligands.

Figure 3-schematic representation of an assay used to identify antibodies that block ApoE-induced LILRB4 activation. The reporter cells on the left are GFP-negative, indicating that the antibody is able to block ApoE from binding to LILRB4, or competes with ApoE for binding to LILRB4 and blocks ApoE induction of GFP. On the right, the antibody binds to regions of LILRB4 but does not block induction of GFP by ApoE, resulting in GFP-positive reporter cells.

Figure 4-schematic representation of an antibody assay for identifying activated LILRB4. On the left, the antibody is capable of binding to FcR on K562 cells and capable of binding to LILRB4 on reporter cells. But it does not induce GFP. On the right, the antibody was able to bind FcR on K562 cells and LILRB4 on reporter cells in a manner that induces GFP. The results indicate that the antibody on the right side is an activated antibody.

FIG. 5a-5 b-analysis of the correlation between immunomodulatory molecules in the TCGA database and overall survival of AML patients. FIG. 5a: individual analysis of patient survival curves for each gene; FIG. 5b: p-values for all 50 genes were summarized.

Figure 6-analysis of mRNA expression data from the TCGA database showed that there was a higher concentration of LILRB4mRNA in M4 and M5AML cells than in other subtypes. * P <0.01, p <0.001.

FIGS. 7a-7 m-LILRB 4 expressed on leukemia cells directly inhibited T cell proliferation in vitro. FIG. 7a: surface expression of LILRB4 was quantified by flow cytometry analysis of samples from 105 patients of UT Southwestern. The "other" category includes cells and tumor-associated macrophages from patients with acute undifferentiated cell leukemia (AUL). FIGS. 7b-c: surface expression of LILRB4 on normal monocytes and tumor monocytes was compared from healthy donors (n = 25) and AML patients (n = 53) (fig. 7 b), or from the same AML patients (n = 6) (as shown in fig. 7 c), respectively. MFI: mean fluorescence intensity. FIG. 7d: t cells isolated from healthy donors were incubated with irradiated lilrb 4-regulated THP-1 cells at the indicated E: T ratios. Representative cells were photographed using an inverted microscope after 5 days of incubation with anti-CD 3/CD28/CD137 coated beads and rhIL-2. The E cells are effector cells; t, THP-1 cells are target cells. FIG. 7e: total T cells were stained with anti-CD 3 antibody and analyzed by flow cytometry. FIG. 7f: the percentage of CTL cells determined by flow cytometry using anti-CD 3, anti-CD 8 and anti-CD 28 antibody staining. FIGS. 7g-h: t cells isolated from healthy donors were incubated in the lower chamber of a 96-well transwell plate. The irradiated THP-1 cells were incubated in the upper chamber. The pore size of the Transwell membrane was 3 μm. T = 2. After 7 days of culture with anti-CD 3/CD28 coated beads and rhIL-2, representative cells were photographed using an inverted microscope (fig. 7 g), and T cells were stained using anti-CD 3, anti-CD 4 and anti-CD 8 antibodies and analyzed by flow cytometry (fig. 7 h). FIG. 7i: CD8 to be stimulated by anti-CD 3/CD28/CD137 coated beads ⁺ T cells were co-cultured with THP-1 cells stably expressing GFP and treated with anti-LILRB 4 antibody or control IgG. GFP (green fluorescent protein) ⁺ The cells are THP-1 leukemia cells, CD8 ⁺ CD28 ⁺ Are activated CTLs, and CD8 ⁺ CD28 ^- The cells are inactive T cells or suppressor T cells. FIGS. 7j-l: quantification of the indicated cells indicated that anti-LILRB 4 antibodies reversed LILRB4 mediated CD8 ⁺ CD28 ⁺ Inhibition of T cell activation by cellular upregulation and LILRB4 ⁺ Killing of AML cells. FIG. 7m: anti-LILRB 4 antibodies increase CTL cytokine production. Numerals 1 to 10 represent transwell plates to which GM-CSF, IFN γ, IL-13, IL-1 β, IL-5, MCP-3, MCP-4, MIP-3 α, RANTES and TNF β were added, respectively. Red boxes indicate the increase caused by anti-LILRB 4 antibody treatment and green boxes indicate the decrease caused by anti-LILRB 4 antibody treatment; the blue box represents the internal control in the cytokine array.

FIG. 8-LILRB4 on normal CD34 ⁺ Not expressed on HSCs. Shown is the LILRB4 and CD34 co-staining pattern of human cord blood mononuclear cells (hCB MNC). N/G, neutrophils and granulocytes; M/D, monocytes, macrophages and dendritic cells; L/P, lymphocytes, hematopoietic stem cells and progenitor cells.

Fig. 9a-9 f-primary AML cells expressing LILRB4 inhibited T cell proliferation. FIGS. 9a-9b: t cells isolated from individual AML (fig. 9 a) or B-ALL (fig. 9B) patients were co-incubated with radiation-treated lilrb 4-positive or negative primary leukemia cells from the same patient. Fig. 9 c-9 f: t cells isolated from healthy donors were incubated with radiation-treated lilrb 4-positive or negative primary leukemia cells from the indicated AML (fig. 9c, fig. 9 e) or B-ALL (fig. 9d, fig. 9 f) patients. T = 10. After 5 days of culture with anti-CD 3/CD28/CD137 coated beads and rhIL-2, T cells were stained with anti-CD 3, anti-CD 4 and anti-CD 8 antibodies and analyzed by flow cytometry

FIGS. 10a-10 c-anti-LILRB 4 antibody blocks human serum-induced activation of LILRB4. FIG. 10a: schematic representation of the LILRB4 receptor system. FIG. 10b: flow cytometry indicated that anti-LILRB 4 antibodies bound to human LILRB4 reporter cells. FIG. 10c: activation of LILRB4 induced by 10% Human Serum (HS) was inhibited by anti-LILRB 4 antibodies. IgG was used as a control. * P <0.0001.

FIGS.11a-11 b-anti-LILRB 4 antibody did not affect the proliferation of THP-1 cells or the cell activation or proliferation of T cells. FIG. 11a: the growth of THP-1 cells was not altered after 7 days of treatment with IgG or anti-LILRB 4 antibody. FIG. 11b: the activation status of human primary CD8+ cells was not affected after 5 days of in vitro treatment with IgG or anti-LILRB 4 antibodies. n.s., no significance.

FIGS. 12a-12 n-LILRB 4 expressed on leukemia cells inhibits T cell proliferation in vivo. FIG. 12a: in hPPBMC-transplanted humanized NSG mice, administration of anti-LILRB 4 antibody inhibited subcutaneous engraftment of THP-1 cells. Leukemia progression over time was monitored by luminescence imaging. FIG. 12b: the luminous flux was determined on day 26 after leukemia cell implantation. FIG. 12c: CD8 at day 26 ⁺ T cells were increased in anti-LILRB 4-treated hPGM-humanized NSG mice. FIG. 12d: the percentage of human CD45+ LILRB4+ cells was examined by flow cytometry from cells harvested from indicated organs from mice injected with primary monocyte AML cells obtained from different patients following injection with control mIgG or anti-LILRB 4 antibody (C84). As shown in table 1, AMLs #1 to 8 were derived from patients #1402903, #1403615, #1403605, #1403986, #1500237, #1500245, #1500401 and #1502990, respectively. FIG. 12e: transplantation of primary human AML cells in NSG mouse bone marrow was investigated by flow cytometry (engraftment). CD45+ LILRB4+ represents AML leukemia cells from human patients; CD8+ CD28+ indicates active tumor killer T cells from the same patient. FIG. 12f: mouse AML C1498 cells (3X 10) expressing human LILRB4 ⁶ Individual cells/mouse) were subcutaneously implanted into C57bl/6 mice. anti-LILRB 4-N297A antibody or control IgG was administered intravenously on days 6, 9, 12, 15, 18 and 21 after tumor cell inoculation. Tumor size was monitored every 3 days. Tumor size was calculated by (width x length). n.s., no significance; * P is p<0.05，**，p<0.01. FIG. 12g: tumor weight was measured 27 days after tumor cell inoculation. FIG. 12h: mice given anti-LILRB 4-N297A antibody showed prolonged mouse survival. FIG. 12i: determination of CD3 in host spleen by flow cytometry ⁺ CD8 ⁺ Percentage of T cells, which inversely correlates with tumor size. FIG. 12j: mouse AML C1498 cells (3X 10) expressing human LILRB4 ⁶ Individual cells/mouse) were subcutaneously implanted into C57bl/6 mice. In the inoculation of tumor cellsAll mouse anti-CD 8 antibodies were administered 3, 6, 9, and 12 days later. anti-LILRB 4-N297A antibody or control IgG was injected intravenously 6, 9, 12, 15 and 18 days after tumor cell inoculation. Tumor size was monitored every 3 days. Tumor size was calculated by (width x length). n.s., no significance. FIG. 12k: tumor weight was measured 18 days after tumor cell inoculation. FIG. 21: when CD8 is used ⁺ Treatment of mice with anti-LILRB 4-N297A antibody showed no effect on survival of mice when T cells were depleted. FIG. 12m: CD3 in host spleen as determined by flow cytometry ⁺ CD8 ⁺ The percentage of T cells did not correlate with tumor size. FIG. 12n: 2x10 ⁷ Individual splenocytes from C1498 vaccinated with overexpressed human LILRB4 were transferred to wild type C57bl/6 mice (N = 5) and then treated with anti-LILRB 4-N297A antibody. At the same time, 2x10 ⁷ Splenocytes from normal wild-type C57bl/6 mice were transferred to wild-type C57bl/6 mice (n = 5) as a negative control. One month after adoptive transplantation, 1x10 was given to each mouse by subcutaneous implantation ⁶ And C1498 cells. Tumor size was monitored and calculated by (width x length). Arrows indicate use of 3x10 in mice that have been leukemic after adoptive transplantation ⁶ One C1498 cell failed a subcutaneous rechallenge.

Figure 13-anti-LILRB 4 antibody reduces the percentage of GFP + leukemia cells present in host tissues. C57bl/6 mice were subcutaneously implanted with mouse AML C1498 cells (3X 10) expressing human LILRb4 and expressing GFP ⁶ Individual cells/mouse). anti-LILRB 4-N297A antibody or control IgG was injected intravenously 6, 9, 12, 15, 18 and 21 days after tumor cell inoculation. anti-LILRB 4 antibody, but not control IgG, reduced the percentage of GFP + leukemic cells present in the host bone marrow, liver, and brain as determined by flow cytometry. * P is p<0.05，**，p<0.01。

FIGS. 14a-14dd-LILRB4 promoted migration of AML cells and supported leukemia progression. FIG. 14a: lilrb4 knockdown reduces THP-1 cell migration through endothelial cells. FIG. 14b: 2x10 ⁶ Individual lilrb 4-Knock Out (KO) or scrambled control (WT) THP-1 cells were injected into NSG mice (n = 5) and then the mice were sacrificed 20 hours after transplantation. Will be measured by flow cytometryThe number of leukemia cells (GFP positive) in liver, spleen and bone marrow was normalized to the number of corresponding cells in peripheral blood. FIG. 14c: 1x10 ⁶ Individual lilrb 4-Knock Out (KO) or scrambled control (WT) THP-1 cells were injected into NSG mice (n = 5). Mice were sacrificed for analysis on day 21 post-transplantation. Anti-human CD45 was used to detect THP-1 cells by flow cytometry. FIG. 14d: the overall survival and body weight of these mice were also examined (fig. 14 e). FIG. 14f: forced expression of human LILRB4 promoted migration of mouse AML C1498 cells. FIG. 14g: will be 3x10 ⁶ Individual C1498 cells expressing human lilrb4 (GFP-hlibrrb 4) or control (GFP) were injected into NSG mice (n = 5) and then the mice were sacrificed 20 hours after transplantation. The number of leukemic cells in liver, spleen and bone marrow (GFP-positive) as determined by flow cytometry was normalized to the number of corresponding cells in peripheral blood. The number of leukemia cells (GFP positive) in liver, spleen and bone marrow as determined by flow cytometry was normalized to the number of corresponding cells in peripheral blood. FIG. 14h: mix 3x10 ⁶ Individual C1498 cells expressing human lilrb4 (GFP-hlibrrb 4) or control (GFP) were injected into NSG mice (n = 5). Mice were sacrificed for analysis on day 16 post-transplantation. Overall survival of these mice was determined figure 14i and figure 14j body weight. FIG. 14k: migration of anti-LILRB 4 antibody grafted THP-1 cells. IgG was used as a control. FIG. 14l: 1x10 ⁶ Individual THP-1 cells were injected into NSG mice, immediately followed by treatment with IgG or anti-LILRB 4 antibody, and then the mice were sacrificed 20 hours after transplantation (n = 5). The number of leukemia cells (GFP positive) in liver, spleen and bone marrow as determined by flow cytometry was normalized to the number of corresponding cells in peripheral blood. FIG. 14m: will be 1x10 ⁶ Individual THP-1 cells were injected into NSG mice, followed immediately by treatment with IgG or anti-LILRB 4 antibodies. Mice were sacrificed for analysis on day 21 post-transplantation. Anti-human CD45 was used to detect THP-1 cells by flow cytometry. The overall survival (fig. 14 n) and body weight (fig. 14 o) of these mice were also examined. FIG. 14p: anti-LILRB 4 antibodies inhibit migration of MV4-11 cells. IgG was used as a control. FIG. 14q: will be 5x10 ⁶ One CFSE-labeled MV4-11 cell was injected into NSG mice (n = 5), followed immediately by administration of IgG or anti-LILRB 4 antibody, and then 20 hours after transplantationMice were sacrificed at time. The number of leukemia cells in liver, spleen and bone marrow (CFSE positive) as determined by flow cytometry was normalized to the number of corresponding cells in peripheral blood. FIG. 14r: 1x10 ⁶ Individual MV4-11 cells were injected into NSG mice (n = 5) immediately followed by administration of IgG or anti-LILRB 4 antibodies. Mice were sacrificed for analysis on day 21 post-transplantation. Anti-human CD45 was used to detect MV4-11 cells by flow cytometry. The overall survival (fig. 14 s) and body weight (fig. 14 t) of these mice were also examined. FIG. 14u: THP-1 leukemia development is detected by whole animal bioluminescence imaging. Mice were given either control IgG or anti-LILRB 4 antibody. FIG. 14v: representative mice were sacrificed for ex vivo bioluminescence imaging of internal organs 21 days after transplantation of luciferase-expressing THP-1. 1: the GI tract; 2: a leg; 3: the lung; 4: spleen; 5: a liver; 6: a kidney; 7: a brain; 8: the heart. Fig. 14 w-fig. 14z: mixing 5x10 ⁶ Individual CFSE-labeled MV4-11 cells were injected into NSG mice (N = 5) that had been pretreated to deplete innate immune cells, then were given IgG or anti-LILRB 4-N297A antibody immediately, and then were sacrificed 20 hours post-transplantation. The number of leukemia cells in liver, spleen and bone marrow (CFSE positive) as determined by flow cytometry was normalized to the number of corresponding cells in peripheral blood. Mice in (x), (y) and (z) were pre-treated with anti-asialo GM1 antibody, clodronate liposome and anti-Ly 6G antibody, respectively. FIG. 14 aa-FIG. 14dd: 1x10 ⁶ Individual LILRB 4-modified THP-1 cells were injected into NSG mice, followed immediately by administration of IgG or anti-LILRB 4 antibody, and then the mice were sacrificed 20 hours after transplantation (n = 5). The number of leukemic cells in liver, spleen and bone marrow (CFSE positive) as determined by flow cytometry was normalized to the number of corresponding cells in peripheral blood. WT, wild-type THP-1 cells with inducible Cas9 and interfering gRNA expression; KO, lilrb 4-knock-out THP-1 cells, selected by inducible Cas9 expression and interference with lilrb 4-specific gRNA expression; KO-wt, wild-type lilrb4cDNA lilrb 4-knock-out overexpression in THP-1 cells; KO-int. DELTA., deletion of the inner domain in THP-1 cells, lilrb4cDNA lilrb4 knock-out over-expression.

FIG. 15-forced expression of human LILRB4 promoted migration of mouse AML WEHI-3 cells. * P <0.05.

FIGS. 16a-16b-LILRB4 expression regulation did not affect AML cell proliferation. (FIG. 16 a) the growth of THP-1 cells was not altered by knocking out lilrb4. WT, wild-type THP-1 cells with inducible Cas9 and interfering gRNA expression; KO, lilrb 4-knock-out THP-1 cells, selected by inducible Cas9 expression and interference with lilrb 4-specific gRNA expression. (FIG. 16 b) the growth of mouse AML C1498 cells was not altered by forced expression of human lilrb4. n.s., no significance.

Figure 17-anti-LILRB 4 antibody reduces leukemic cell invasion in the liver. Mouse AML C1498 cells (3X 10) expressing human LILRB4 and expressing GFP ⁶ Individual cells/mouse) were implanted subcutaneously in C57bl/6 mice. All mice were given anti-CD 8 antibodies 3, 6, 9 and 12 days after tumor cell inoculation. anti-LILRB 4-N297A antibody or control IgG was administered intravenously 6, 9, 12, 15 and 18 days after tumor cell inoculation. anti-LILRB 4 antibody, but not control IgG, reduced the percentage of GFP + leukocytes in the host liver as measured by flow cytometry. * P is p<0.05。

Figure 18-expression of LILRB4 on the immortalized human AML cells shown, as measured by flow cytometry. Isotype IgG was used as control.

Figure 19-anti-LILRB 4 antibodies did not act on LILRB4 negative cancer cells. NSG mice were injected with LILRB 4-human AML U937 cells, followed by administration of anti-LILRB 4 antibodies. IgG was used as a control antibody. At the 25 th day post-transplantation, the Liver (LV), bone Marrow (BM), spleen (SP) and Peripheral Blood (PB) were analyzed by flow cytometry. The presence of human AML cells was detected by staining with anti-human CD45 antibody. n.s., no significance.

FIGS. 20a-20 b-anti-LILRB 4 antibody inhibited human AML transplant. (FIG. 20 a) schematic representation of antibody administration in AML transplants. Antibodies (control IgG or anti-LILRB 4 antibody) were administered as indicated by the arrows. (fig. 20 b) the percentage of human leukemia (THP-1, cd45 +) cells in the Liver (LV), bone Marrow (BM) and Spleen (SP) of recipient NSG mice (n = 6) was determined by flow cytometry for antibodies administered every 3 days from the indicated days. (FIGS. 20c-20 d) administration of antibody at day 0, day 0+ day 3 + day 6, all similarly blocked AML development triggered by transplanted THP-1 cells (FIG. 20 c) and MV4-11 cells (FIG. 20 d).

Figure 21-anti-LILRB 4 antibody inhibits human AML transplants. Each antibody was administered at 20 μ g on day 0 as indicated. THP-1 leukemia progression was monitored by whole animal bioluminescence imaging.

Figure 22-anti-LILRB 4 antibody inhibits human AML transplants. Each antibody was administered at 200 μ g on day 0, day 3 or day 6 as indicated. THP-1 leukemia progression was monitored by whole animal bioluminescence imaging.

Figures 23 a-c-graphs of representative flow cytometry show successful reduction in the frequency of NK cells (CD 45+ CD49b +), (figure 23 b) macrophages (CD 11b + F4/80 +) and (figure 23 c) neutrophils (CD 11b + CD11 c-) in NSG mice depleted in each immune cell subtype compared to non-depleted (wild type) NSG mice.

Figure 24-human and mouse integrin heterodimer proteins fail to activate the LILRB4 receptor. Human and mouse sera were used as positive controls. n.s., no significance. * P <0.0001.

FIGS. 25a-25l-APOE bind LILRB4 and support AML migration. FIG. 25a: GFP, e.g. in the LILRB4 reporter System ⁺ Analysis of the percentage of cells showed that human and mouse sera specifically activated LILRB4. FIG. 25b: recombinant APOE activated human LILRB4 and mouse PIRB in a reporter system. FIG. 25c: sera from APOE-nude mice failed to activate LILRB4. FIG. 25d: lipid-reconstituted APOE (APOE-POPC) activated human LILRB4 as well as recombinant APOE in the reporter system. FIG. 25e: lilrb 4-knock-out THP-1 cells showed reduced APOE binding as measured by flow cytometry. Cells stained with anti-His tag-APC were used as negative controls. FIG. 25f: the binding kinetics of human APOE-3 to LILRB4-ECD-Fc was measured using Surface Plasmon Resonance (SPR). LILRB4-ECD-Fc was immobilized on the protein a biosensor tip and incubated with APOE-3 at a concentration ranging from 1.5625nM to 100 nM. FIG. 25g: activation of LILRB4 by APOE was reduced by N-terminal mutation in APOE. FIG. 25h: activation of LILRB4 by APOE was reduced by a single amino acid mutation shown for LILRB4. FIG. 25 i-FIG. 25l: APOE is essential for LILRB4 mediated homing (homing)In (1). Forced expression of human lilrb4 on mouse leukemia C1498 cells increased homing of leukemia cells in Wild Type (WT) recipient mice (n = 5) (as shown in figure 25 i). However, forced expression of lilrb4 did not increase homing in APOE-null (KO) recipient mice (n = 5) (as shown in fig. 25 j). C1498 cells (l) expressing human lilrb4 in APOE-nude (KO) mice (n = 5) but not control GFP-expressing C1498 cells (fig. 25 k) had lower homing ability compared to WT mice (n = 5); mice were sacrificed 20 hours after leukemia cell injection. GFP was used to detect leukemic cells by flow cytometry.

FIGS. 26a-26 c-identification of LILRB4 potential ligands in human serum. FIG. 26a: flow chart of ligand screening. FIG. 26b: LILRB4 stimulating activity was isolated from human serum by FPLC. 10% human serum was used as a positive control. FIG. 26c: list of proteins identified from LILRB4 stimulated fractions by Mass Spectrometry (MS). PSM: peptide matching profiles.

Figure 27-both human and mouse APOE proteins were able to activate the LILRB4 receptor. Human and mouse sera were used as positive controls. n.s., no significance. * P <0.0001.

FIGS. 28a-28 b-APOE proteins from different sources all activated LILRB4. FIG. 28a: APOE purified from human plasma (20. Mu.g/ml), his-tagged or untagged recombinant human APOE (rhAPOE) expressed from 293T mammalian cells (20. Mu.g/ml) or rhAPOE expressed from bacteria (20. Mu.g/ml) both activated the LILRB4 receptor. These APOEs all represent human APOE3. FIG. 28b: APOE2, APOE3 and APOE4 all activated the LILRB4 reporter protein. APOE was coated on plates at 40. Mu.g/ml or added directly to cell culture medium (soluble).

FIGS. 29a-29 g-three APOE isoforms bind to human LILRB4. FIGS. 29a-c: the binding kinetics of APOE2, 3 and 4 to LILRB4-Fc were measured using surface plasmon resonance (SRP). LILRB4-Fc was immobilized on protein a biosensor tips and incubated with APOE at concentrations ranging from 1.5625nM to 100 nM. APOE2, APOE3 and APOE4 bind to LILRB4 with Kd of 5.525nM, 2.485nM and 3.573nM, respectively. (FIG. 29 d-f) binding kinetics of APOE2, 3 and 4 to LILRB4-Fc were measured using biolayer interferometry (Octet). LILRB4-Fc was immobilized on protein a biosensor tips and incubated with APOE at concentrations ranging from 44nM to 1176 nM. APOE2, APOE3 and APOE4 bind to LILRB4 with Kd of 60.68nM, 61.67nM and 48.39nM, respectively. (FIG. 29 g) binding kinetics of APOE3 to His-LILRB4 were measured using microcalorimetric electrophoresis (MST). APOE3 binds LILRB4 with a Kd of 210nM.

FIGS. 30a-30 b-Effect of LILRB4 mutated residues on the possible APOE binding interface based on the known structure of LILRB4 and APOE. FIG. 30a: the PDB structure based on LILRB4 (PDBID: 3P 2T) and APOE3 (PDBID: 2L 7B), the residues used for mutagenesis studies in four possible ligand-binding interfaces, and the resulting series of mutant LILRB4 reporter cells. FIG. 30b: mutations at two residues W106 and Y121 located in the linker between the first Ig domain and the two Ig domains, respectively, significantly reduced LILRB4 activation by APOE.

FIGS. 31a-31w-LILRB4 mediated intracellular signaling controls AML cell migration and T cell suppression. FIG. 31a: western blots showed that shp-1, shp-2 and ship were knocked out separately by CRISP/Cas9 in THP-1 cells. Fig. 31 b-fig. 31c: t cells isolated from healthy donors were incubated in the lower chamber of a 96-well transwell plate. The irradiated THP-1 cells were incubated in the upper chamber. The pore size of the Transwell membrane was 3 μm. T = 2. Subsequently, the anti-CD 3/CD 28-coated beads and rhIL-2 were cultured for 7 days. Representative cells were photographed using an inverted microscope (fig. 31 b), and T cells were stained using anti-CD 3 and anti-CD 8 antibodies and analyzed by flow cytometry (fig. 31 c). Fig. 31 d-fig. 31e: knockout of shp-2 reduces THP-1 cell migration and slows leukemia progression in transplanted tumor mice. FIG. 31d: 2x10 ⁶ Individual shp-1-knockout (shp-1-KO), shp-2-knockout (shp-2-KO), shp-knockout (shp-KO) or scrambled control (WT) THP-1 cells were injected into NSG mice (n = 5), and then the mice were sacrificed 20 hours after transplantation. The number of leukemia cells (GFP positive) in liver, spleen and bone marrow as determined by flow cytometry was normalized to the number of corresponding cells in peripheral blood. Fig. 31e: 1x10 ⁶ The indicated THP-1 cells were injected into NSG mice (n = 5). Mice were sacrificed for analysis on day 21 post-transplantation. Anti-human CD45 was used to detect THP-1 cells by flow cytometry. FIG. 31f: lilrb 4-knock-out (lilrb 4-KO) and scrambling control (WT) THP-1 by RNA-seqThe cells were analyzed. Loss of Lilrb4 induces changes in transcription factor activity (Lilrb 4-KO vs WT). The yellow dots highlight the transcription factors involved in the JAK/STAT and NFkB pathways. FIG. 31g: western blotting showed that the lilrb 4-knock-out (lilrb 4-KO) decreased phosphorylation of SHP-2 and STAT-3 compared to that in scrambled control (WT) THP-1 cells. FIG. 31h: western blotting showed that the lilrb 4-knock-out (lilrb 4-KO) reduced phosphorylation of IKK compared to that in scrambled control (WT) THP-1 cells. FIG. 31i: western blots showed a reduction in NFkB expression in the lilrb 4-Knock Out (KO) core fraction compared to that in scrambled control (WT) THP-1 cells. Fig. 31 j-fig. 31k: t cells isolated from healthy donors were incubated in the lower chamber of a 96-well transwell plate. Irradiated THP-1 cells were incubated in the upper chamber, which had been pretreated with 1-5. Mu.M fludarabine or stattic for 24 hours. The pore size of the Transwell membrane was 3 μm. T = 2. After 7 days of culture with anti-CD 3/CD 28-coated beads and rhIL-2, representative cells were photographed using an inverted microscope (fig. 31 j), and T cells were stained with anti-CD 3 antibody and analyzed by flow cytometry (as shown in fig. 31 k). FIG. 31l: THP-1 cell migration through endothelial cells was reduced by inhibition of STAT-3 but not STAT-1 (2.5 μ M STAT (STAT-3 inhibitor) or fludarabine, respectively) by specific inhibitors. FIG. 31m: NSG mice (n = 5) were given 1x10 intravenous injections ⁶ THP-1 cells, which were pre-treated with 2.5uM STAT inhibitor for 24 hours. DMSO was used as a control. Mice were sacrificed for analysis on day 21 post-transplantation. Anti-human CD45 was used to detect THP-1 by flow cytometry. FIG. 31n: loss of lilrb4 reduces cytokine and chemokine production in THP-1 cells. The numbers 1-12 in the red boxes indicate CCL2, CCL5, CCL4, OSM, IL-6R, IL-8, gp130, TNFRSF1B, TNFRSF1A, TIMP-1, TIMP-2, and uPAR, respectively. The basket represents the internal control in the cytokine array. FIG. 31o: quantification of blot intensity by ImageJ software (as shown in figure 31 n). FIG. 31p: the results of Western blotting showed that the lilrb 4-knock-out (lilrb 4-KO) reduced the expression of uPAR and arginase-1 as compared to that in scrambled control (WT) THP-1 cells. FIG. 31q: in lilr, compared to the situation in scrambled control (WT) THP-1 cellsb 4-Knock Out (KO) medium with reduced arginase activity. Fig. 31 r-fig. 31s: t cells isolated from healthy donors were incubated with irradiated lilrb4-KO or WT THP-1 cells. These cells were supplemented with uPAR protein at the indicated concentrations for 7 days. Representative cells were photographed using an inverted microscope (fig. 31 r), and T cells were stained using anti-CD 3 antibody and analyzed by flow cytometry (fig. 31 s). Fig. 31 t-fig. 31u: t cells isolated from healthy donors were incubated with irradiated lilrb4-KO or WT THP-1 cells. Cultures were supplemented with the indicated concentrations of recombinant arginase-1 protein for 7 days. Representative cells were photographed using an inverted microscope (fig. 31T), and T cells were stained using anti-CD 3 antibody and analyzed by flow cytometry (fig. 31 u). Fig. 31 v-fig. 31w: supplementation of the culture medium with recombinant uPAR or arginase-1 rescued the decreased ability of lilrb4-KO THP-1 (fig. 31 v) or lilrb4-KO MV4-11 cells (fig. 31 w) to cross the endothelium.

FIG. 32-qPCR shows that lilrb 4-Knock Out (KO) reduces gene expression of cytokines and chemokines compared to that in scrambled control (WT) THP-1 cells.

Figure 33-effect of cytokines on T cells. T cells isolated from healthy donors were co-cultured with anti-CD 3/CD 28-coated beads and rhIL-2 and supplemented with the indicated proteins for 3 days. Representative cells were photographed using an inverted microscope.

FIG. 34-rescue of T cell activation function of LILRB4KO THP-1 cells by cytokines. T cells isolated from healthy donors were incubated in the lower chamber of a 96-well transwell plate. Irradiated lilrb4-WT or KO THP-1 cells were incubated in the upper chamber. The upper chamber containing lilrb4-KO THP-1 cells was supplemented with the indicated protein. The pore size of the Transwell membrane was 3 μm. T = 2. Representative cells were photographed using an inverted microscope after 3 days of incubation with anti-CD 3/CD 28-coated beads and rhIL-2. In the upper chamber there are only T cells, and no THP-1 cells. CCL4, 20. Mu.g/ml, CCL5, 10. Mu.g/ml, TIMP-2, 10. Mu.g/ml, IL-8, 10. Mu.g/ml, IL10, 20. Mu.g/ml, IL-27, 10. Mu.g/ml.

FIG. 35a-35c-CCL effect on AML cell infiltration. FIG. 35a: blockade of CCL4 but not CCL2 or CCL3 by specific neutralizing antibodies is reducedMigration of THP-1 cells across endothelial cells was initiated. FIG. 35b: NSG mice (n = 5) were given 1x10 injections ⁶ THP-1 cells, which were immediately subsequently treated with 200 μ g/mouse IgG, anti-CCL 2, anti-CCL 3, or anti-CCL 4 neutralizing antibody. Mice were sacrificed for analysis on day 21 post-transplantation. Anti-human CD45 was used to detect THP-1 cells by flow cytometry. FIG. 35c: t cells isolated from healthy donors were incubated with irradiated lilrb4-KO or WT THP-1 cells. These cells were supplemented with 20. Mu.g/ml CCL4 protein or with 10. Mu.g/ml IgG or anti-CCL 4 neutralizing antibody for 7 days. T cell numbers were analyzed by flow cytometry.

FIG. 36a-36 d-correlation analysis of LILRB4 regulatory genes. FIG. 36a: comparison of different gene expression patterns in LILRB4-KO versus WT and in those treated with anti-LILRB 4 versus IgG, identified by RNA-seq. For antibody treatment, THP-1 cells were treated with 1% human serum in the presence of antibodies for 24 hours. Blue indicates lilrb 4-negative regulatory gene and red indicates lilrb 4-positive regulatory gene. FIG. 36b: correlation analysis of lilrb 4-regulatory gene mRNA expression data from the TCGA database showed that expression of lilrb4 was positively correlated with expression of lilrb 4-forward regulatory genes; in contrast, data from RNA-seq indicate that expression of lilrb4 is negatively correlated with expression of lilrb 4-negative regulatory genes. Fig. 36 c-fig. 36d: analysis of mRNA expression data from the TCGA database and patient survival revealed a negative correlation between lilrb 4-positive regulatory gene expression (red lines) and gene expression and overall patient survival (figure 36 c); however, the expression of lilrb 4-negatively regulated genes (red lines) showed a positive correlation with gene expression and overall survival of the patients (FIG. 36 d).

FIGS. 37a-37 c-anti-LILRB 4 antibody accelerated MV4-11 cell migration to peripheral blood. (FIG. 37 a) schematic antibody administration. (FIG. 37 b) the number of leukemia cells in Peripheral Blood (PB) as determined by flow cytometry was normalized to the number of corresponding cells in peripheral blood. (FIG. 37 c) leukemia cell numbers in Liver (LV), spleen (SP) and Bone Marrow (BM) as determined by flow cytometry were normalized to the number of corresponding cells in peripheral blood. Anti-human CD45 was used to detect MV4-11 cells.

FIG. 38a-38 e-synergism of anti-LILRB 4 and chemotherapeutic agents. Fig. 38a-38 b: anti-LILRB 4 antibodies accelerated the migration of MV4-11 cells from Bone Marrow (BM), liver (LV), and Spleen (SP) (fig. 38 b) to Peripheral Blood (PB) (fig. 38 a). Anti-human CD45 was used to detect MV4-11 cells by flow cytometry. Mice in each group, n =6. Fig. 38 c-38 e: the synergistic effect of anti-LILRB 4 antibody treatment in combination with chemotherapeutic drugs cytarabine (fig. 38 d) or daunorubicin (fig. 38 e) inhibited AML progression. Mice in each group, n =6. Administration of chemotherapeutic agents and anti-LILRB 4 antibodies is shown in the figure (figure 38 c). Anti-human CD45 was used to detect human leukemia cells by flow cytometry.

FIG. 39-anti-LILRB 4 antibody did not affect homing of normal HSC. Human umbilical cord blood mononuclear cells (1 x 10) ⁷ ) Injected into NSG mice, followed immediately by antibody administration, and then mice were sacrificed 20 hours after transplantation (n = 3). CD45 in liver, spleen and bone marrow as measured by flow cytometry ⁺ CD34 ⁺ HSC numbers were normalized to the number of corresponding cells in peripheral blood.

Figure 40a-40 c-anti-LILRB 4 antibody inhibits leukemia progression in hCB-humanized NSG mice. FIG. 40a: strategy to test whether anti-LILRB 4 antibody C84 inhibits leukemia progression in hCB-humanized NSG mice. FIG. 40b: the progression of leukemia was monitored by luminescence imaging as a function of time. FIG. 40c: leukemia transplanted in peripheral blood 24 days after leukemia transplantation, frequency of change with time of normal human cells (including human B cells, human myeloid cells and human T cells) and frequency in hematopoietic tissues of hCB humanized mouse. BM: bone marrow; LV: a liver; SP: spleen; PB: peripheral blood.

Detailed Description

Targeted therapy can induce rapid tumor regression, while immunotherapy can achieve long-term anti-tumor effects. Therefore, it would be desirable to identify molecular targets that can strongly combine targeted therapy with immunotherapy. The function of the leukocyte immunoglobulin-like receptor family (LILRB) expressed on immune and leukemia cells remains to be understood. The present disclosure found that LILRB4, a surface marker of monocytic Acute Myeloid Leukemia (AML), maintained the progression of leukemia. APOE was found to bind specifically to LILRB4, activating LILRB 4-mediated signaling and supporting homing of AML cells to visceral organs. Inhibition of LILRB4 signaling by LILRB4 blocking antibodies in transplantation tumor mice abolished AML progression by direct targeting of the tumor, disrupting leukemic cell retention in the microenvironment and immunodetection point inhibition. Thus, LILRB4 represents a new target for the treatment of monocytic AML, and anti-LILRB 4 antibodies are promising drug candidates.

Thus, LILRB4 is an ideal target for the treatment of AML and potentially other cancers. Since LILRB4 is a marker for monocytic AML and is expressed by both primary and mature monocytic AML cells, it may be appropriate to study the potential therapeutic effects of LILRB4 on monocytic AML. Most unexpectedly, the data suggest that the anti-LILRB 4 blocking antibody strategy combines targeted therapy and immunotherapy. anti-LILRB 4 blocks signaling and LILRB4 ⁺ The interaction between AML cells and their microenvironment also mediates direct tumor killing. Furthermore, anti-LILRB 4 stimulates the activation of T cells, which leads to immune system mediated anticancer effects. Since LILRB4 is expressed on tumor-associated macrophages and MDSCs support escape of cancer cells through immunosuppression ²⁹ Thus, anti-LILRB 4 antibodies may also alleviate immunosuppression mediated by these bone marrow cells. Moreover, the results in the examples described below indicate that anti-LILRB 4 can enhance the effectiveness of standard chemotherapy regimens because the antibodies cause leukemia cells to migrate from the niche (niche) into the bloodstream where they may be more sensitive to cytotoxic chemotherapy.

Furthermore, LILRB4 targeting may have the lowest toxicity. LILRB4 is expressed on monocytes and macrophages, dendritic cells, progenitor mast cells, endothelial cells and osteoclasts. However, it has a higher level of expression on human AML cells than normal counterpart cells. Notably, anti-LILRB 4 had no effect on HSCs, which did not express LILRB4. Although LILRB4 is expressed by osteoclasts, mice that do not express PirB (the orthologous gene of mouse LILRB) do not have altered osteoclast function ³⁰ . Thus, anti-LILRB 4 antibodies are directed against mononuclear cellsTreatment of patients with AML and other malignancies holds great promise.

Thus, embodiments of the present disclosure provide methods of identifying LILRB antagonists (e.g., anti-LILRB antibodies) that specifically target ApoE-induced LILRB activation. The assays provided herein include administering a LILRB ligand ApoE to a reporter cell or population of reporter cells and a candidate antagonist of LILRB activation. The level of LILRB activation is then measured, such as by detecting a marker (e.g., NFAT-GFP) under control of LILRB activation. Comparing the level of LILRB activation to activation by administration of ApoE alone, and identifying a decrease in LILRB activation as an inhibitor of ApoE-mediated LILRB activation. Thus, the methods and compositions of the present disclosure provide methods of identifying ApoE-induced LILRB activation, and uses thereof in the treatment of cancer, particularly AML.

The following description of the present disclosure is intended only to illustrate various embodiments of the present disclosure. Therefore, the revisions discussed should not be construed to limit the scope of the disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the disclosure, and it is to be understood that such equivalent embodiments are to be included within the scope of the present application. All references, including publications, patents, and patent applications, cited in this application are hereby incorporated by reference in their entirety.

I. Definition of

As used in this application, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

Throughout this application, the term "about" is used to mean a value that includes the inherent variation of the error of the method used to determine the value or variation that exists between study subjects.

Autoimmune diseases include, but are not limited to, rheumatoid arthritis, crohn's disease, multiple sclerosis, autoimmune diabetes, systemic lupus erythematosus, lupus vulgaris, thyroiditis, edison's disease, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, glomerulonephritis, goodpasture's syndrome, graves ' disease, myasthenia gravis, neuritis, ophthalmia, bullous pemphigoid, pemphigus, polyendocrinopathy, purpura, lauter's disease, stiff person syndrome, autoimmune pulmonary inflammation, guillain-Barre syndrome, and autoimmune inflammatory ocular disease. Preferably, in the subject method, the subject is a human. In one embodiment, the polypeptide is administered to the subject during the onset of an autoimmune challenge. The method may also include the administration of other immunosuppressive drugs such as cytotoxic agents, cyclosporine, methotrexate, azathioprine, and glucocorticoids.

As used herein, an "antagonist" or "inhibitor" of LILRB activation refers to any substance that is capable of blocking or reducing LILRB activation in the presence of a LILRB ligand (e.g., apoE). In certain embodiments, the antagonist or inhibitor may be a protein, such as an antibody. In certain embodiments, the antagonist or inhibitor may be a small molecule, such as a chemical compound. In certain embodiments, the antagonist or inhibitor reduces activation of LILRB by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% as compared to a reference level (e.g., the level of activation of LILRB in the presence of LILRB ligand but in the absence of the antagonist or inhibitor).

As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody that binds to a specific antigen (or multiple antigens). A natural, intact antibody comprises two heavy chains and two light chains. Each heavy chain is composed of a variable region (V) _H ) And first, second and third constant regions (each C) _H 1、C _H 2 and C _H 3) Each light chain is composed of a variable region (V) _L ) And a constant region (C) _L ) And (4) forming. The variable regions of the light and heavy chains determine the binding of the antigen. Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and mammalian light chains as λ or κ. The antibody has "Y" type, and the Y-type stem is connected via disulfide bondThe second and third constant regions of the two heavy chains together. Each arm of the Y-shape comprises the variable region and the first constant region of a single heavy chain joined together with the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding and are often referred to as Fv (for variable fragments) or Fv fragments. The variable regions of these two chains typically contain three hypervariable loops called Complementarity Determining Regions (CDRs) (light chain (L) CDRs include LCDR1, LCDR2 and LCDR3 and heavy chain (H) CDRs include HCDR1, HCDR2, HCDR 3). The CDR boundary CDRs of the antibodies and antigen-binding fragments disclosed herein can be defined or identified by the Chothia, kabat or Al-Lazikani convention (Chothia, C. Et Al, J Mol Biol 186 (3): 651-63 (1985); chothia, C. And Lesk, A.M., J Mol Biol,196 (1987); chothia, C. Et Al, nature 342 (52): 877-83 (1989); kabat E.A. Et Al, national Institutes of Health, bethesda, md. (1991); al-Lazikani, B., chothia, C., lesk, A.M., J Mol l 273 (4): 927 (1997)). Three CDRs are inserted between flanking segments called Framework Regions (FRs), which are more highly conserved than CDRs and form a scaffold that supports hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit multiple effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant region. The main classes or isotypes of five antibodies are IgA, igD, igE, igG and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma and mu heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ 1 heavy chain), igG2 (γ 2 heavy chain), igG3 (γ 3 heavy chain), igG4 (γ 4 heavy chain), igA1 (α 1 heavy chain), or IgA2 (α 2 heavy chain) in humans, as well as IgG1 (γ 1 heavy chain), igG2a (γ 2a heavy chain), igG2b (γ 2b heavy chain), and IgG3 (γ 3 heavy chain) in mice. As used herein, an antibody also includes an antigen-binding fragment, i.e., a portion of a protein that is capable of specifically binding to an antigen. In a certain embodiment, the antigen-binding fragment is from an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise the entire native antibody structure. Examples of antigen binding fragments include, but are not limited to, diabodies, fab ', F (ab') ₂ Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv) ₂ Bispecific dsFv (dsFv-dsFv'), disulfide stabilized dimer (ds dimer)Single chain antibody molecules (scFv), scFv dimers (diabodies), multispecific antibodies, single domain antibodies (sdabs), camelid or nanobodies, domain antibodies, and bivalent domain antibodies.

The term "cancer" refers to a condition or disorder in which cells grow and divide at an unregulated, accelerated rate. Examples of cancer include Acute Lymphocytic Leukemia (ALL), acute myelogenous leukemia, adrenocortical carcinoma, anal carcinoma, astrocytoma, childhood cerebellar or cerebral basal cell carcinoma, cholangiocarcinoma, bladder carcinoma, bone tumors, brain cancer, cerebellar astrocytoma, cerebral astrocytoma/glioblastomas, ependymomas, medulloblastomas, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic gliomas, breast cancer, burkitt's lymphoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, emphysema, endometrial cancer, ependymomas, esophageal cancer, ewing's sarcoma, retinoblastoma, gastric cancer (stomach cancer), gliomas, head and neck cancer, cardiac cancer, hodgkin's lymphoma, islet cell carcinoma (endocrine pancreas), kaposi's sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, leukemia, liver cancer, lung cancer, neuroblastoma, non-hodgkin's lymphoma, ovarian cancer, pancreatic cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cell carcinoma (renal cancer), retinoblastoma, ewing's tumor family, skin cancer, stomach cancer, testicular cancer, pharyngeal cancer, thyroid cancer, vaginal cancer.

As used herein, a "cell" may be a prokaryotic cell or a eukaryotic cell. Prokaryotic cells include, for example, bacteria. Eukaryotic cells include, for example, fungi, plant cells, and animal cells. Types of animal cells (e.g., mammalian cells or human cells) include, for example, cells from the circulatory/immune system or organ, such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, helper T cells), natural killer cells, granulocytes (e.g., basophils, eosinophils, neutrophils, and multilobal neutrophils), monocytes or macrophages, erythrocytes (reticulocytes), mast cells, platelets or megakaryocytes, and dendritic cells; cells from the endocrine system or organ, such as thyroid cells (e.g., thyroid epithelial cells, parafollicular cells), parathyroid cells (e.g., parathyroid chief cells, eosinophils), adrenal cells (e.g., pheochromocytes), and pineal cells (e.g., pineal cells); cells from the nervous system or organs, such as glioblasts (e.g., astrocytes and oligodendrocytes), microglia, large cell neurosecretory cells, astrocytes, burtescher cells, and pituitary cells (e.g., gonadotropin, corticoids, thyroxine, growth hormone, and prolactin cells); cells from the respiratory system or organs, such as lung cells (type I and type II), clara cells, goblet cells, and alveolar macrophages; cells from the circulatory system or organ (e.g., cardiomyocytes and pericytes); cells from the digestive system or organ, e.g., gastric chief cells, parietal cells, goblet cells, panne cells, G cells, D cells, ECL cells, I cells, K cells, S cells, enteroendocrine cells, enterochromaffin cells, APUD cells, and hepatocytes (e.g., hepatocytes and kupffer cells); cells from the integumentary system or organ, such as bone cells (e.g., osteoblasts, osteocytes, and osteoclasts), dental cells (e.g., cementoblasts and ameloblasts), chondrocytes (e.g., chondroblasts and chondrocytes), skin/hair cells (e.g., hair cells, keratinocytes, and melanocytes (nevi cells)), muscle cells (e.g., muscle cells), adipocytes, fibroblasts, and tenocytes; cells from the urinary system or organ (e.g., podocytes, periglomerular cells, intraglomerular mesangial cells, extraglomerular mesangial cells, renal proximal tubule brush border cells, and macular density cells); and cells from the reproductive system or organ (e.g., sperm, supporting cells, interstitial cells, ova, oocytes). The cell may be a normal healthy cell; or diseased or unhealthy cells (e.g., cancer cells). Cells also include mammalian zygotes or stem cells, including embryonic stem cells, fetal stem cells, induced pluripotent stem cells, and adult stem cells. Stem cells are cells that are capable of undergoing a cell division cycle while remaining undifferentiated and differentiating into specialized cell types. The stem cell may be a pluripotent stem cell, a multipotent stem cell, an oligodynamic stem cell, or a unipotent stem cell, and any of them may be induced from a somatic cell. The stem cells may also include cancer stem cells. The mammalian cell can be a rodent cell, e.g., a mouse, rat, hamster cell. The mammalian cell may be a cell of the order lagomorpha, e.g., a rabbit cell. The mammalian cell can also be a primate cell, such as a human cell.

As used herein, "substantially free" with respect to a particular component is used herein to indicate that no particular component is purposefully formulated into the composition and/or is present only as a contaminant or in trace amounts. Thus, any unintentional contamination of the composition results in a total amount of the specific components well below 0.05%, preferably below 0.01%. Most preferred is a composition wherein no amount of a particular component can be detected using standard analytical methods.

Inflammatory disorders include, but are not limited to, (i) inflammatory diseases, such as chronic inflammatory lesions (including chronic inflammatory lesions such as, but not limited to, sarcoidosis, chronic inflammatory bowel disease, ulcerative colitis, and crohn's disease); (ii) Vascular inflammatory disorders such as, but not limited to, disseminated intravascular coagulation, atherosclerosis, kawasaki disease, and vasculitis syndromes (such as, but not limited to, polyarteritis nodosa, wegener's granulomatosis, henoch-Schonlein purpura, giant cell arthritis, and renal microvascular inflammation); (iii) chronic active hepatitis; (iv) sjogren's syndrome; (v) Spondyloarthropathies such as ankylosing spondylitis, psoriatic arthritis and spondylitis, enteropathic arthritis and spondylitis, reactive arthritis and inflammatory bowel disease-related arthritis; and (vi) uveitis. Preferably, in the subject method, the subject is a human. The method may also be combined with the administration of other anti-inflammatory agents. Anti-inflammatory agents include, but are not limited to, any of the known non-steroidal anti-inflammatory agents, such as salicylic acid derivatives (aspirin), p-aminophenol derivatives (acetaminophen), indole and indene acetic acid (indomethacin), heteroaryl acetic acid (ketorolac), aryl propionic acid (ibuprofen), anthranilic acid (mefenamic acid), enolic acids (oxicams) and alkanones (nabumetone), as well as any of the known steroidal anti-inflammatory agents, including corticosteroids and biologically active synthetic analogs with their associated glucocorticoid (metabolic) and mineralocorticoid (electrolyte modulating) activities. In addition, other drugs used in the treatment of inflammation include, but are not limited to, endocrine antagonists such as histamine, bradykinin receptor antagonists, leukotriene and prostaglandin receptor antagonists, and platelet activating factor receptor antagonists.

As used herein, the term "linked" refers to association by intramolecular interactions, e.g., covalent, metallic and/or ionic bonds, or intermolecular interactions, e.g., hydrogen or non-covalent bonds.

The term "operably linked" refers to an arrangement of elements wherein the components so described are configured to perform their usual function. Thus, a given signal peptide operably linked to a polypeptide indicates that the polypeptide is secreted from the cell. In the case of a promoter, a promoter operably linked to the coding cell will indicate expression of the coding sequence. A promoter or other control element need not be adjacent to a coding sequence, so long as it functions to direct expression of the coding sequence. For example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and a coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence.

Although the present disclosure supports definitions that refer only to alternatives and "and/or," the use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated otherwise that only alternatives or alternatives are mutually exclusive. As used herein, "another" may refer to at least a second or more.

As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Humans include prenatal or postpartum forms. In many embodiments, the subject is a human. The subject may be a patient, which refers to a person who is at a medical provider for diagnosis or treatment of a disease. The term "subject" as used in this application is used interchangeably with "individual" or "patient". A subject may be suffering from or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

As used herein, a "treating" or "treatment" of a condition includes preventing or alleviating the condition, delaying the onset or rate of progression of the condition, reducing the risk of developing the condition, preventing or delaying the progression of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, producing a complete or partial regression of the condition, curing the condition, or some combination thereof.

As used herein, the term "therapeutically useful amount" or "effective dose" refers to a dose or concentration of a drug effective to treat a disease or condition. For example, for the treatment of cancer using the monoclonal antibodies or antigen binding fragments thereof disclosed herein, a therapeutically effective amount is a dose or concentration at which the monoclonal antibodies or antigen binding fragments thereof are capable of reducing tumor volume, eradicating all or a portion of the tumor, inhibiting or slowing tumor growth or cancer cell infiltration into other organs, inhibiting a growth or proliferation-mediated cancer condition of cells, inhibiting or slowing tumor cell metastasis, ameliorating any symptoms or markers associated with the tumor or cancer condition, preventing or delaying progression of the tumor or cancer condition, or some combination thereof. LILR

Leukocyte immunoglobulin-like receptors (LILRs) are a family of receptors with extracellular immunoglobulin domains. It is also known as CD85, ILT and LIR, which can exert an immunomodulatory effect on a variety of immune cells. Human genes encoding these receptors are present in the gene cluster in the chromosomal region 19q13.4. It comprises LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, LILRB6 or LILRA6, and LILRB7 or LILRA5. A subset of LILRs recognize MHC class I molecules (also known as HLA class I in humans). Among these, the inhibitory receptors LILRB1 and LILRB2 show broad specificity for classical and non-classical MHC alleles, which preferentially bind to β 2 m-associated complexes. In contrast, the activation receptors LILRA1 and LILRA3 is preferably independent of MHC class I free heavy chain of b2m, especially HLA-C alleles. For the following description of LILR and LILRB1-5 and LAIR1, see for review ²² 。

A.LILRB1

Leukocyte immunoglobulin-like receptor subfamily B member 1 is a protein encoded by the LILRB1 gene in humans. This gene is a member of the immunoglobulin-like receptor (LIR) family of leukocytes, and is present in the gene cluster in the chromosomal region 19q13.4. The encoded proteins belong to the LIR receptor subfamily B class, which contains two or four extracellular immunoglobulin domains, a transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). This receptor is expressed on immune cells, where it binds to MHC class I molecules on antigen presenting cells and transduces a negative signal that inhibits stimulation of the immune response. ILIRB1 is also reported to be expressed on human gastric cancer cells and can enhance tumor growth. It is thought to control inflammatory responses and cytotoxicity to aid in focusing immune responses and to limit self-reactions. Different isoforms encoded by multiple transcript variants of the gene have been discovered.

B.LILRB2

Leukocyte immunoglobulin-like receptor subfamily B member 2 is a protein encoded by the LILRB2 gene in humans. This gene is a member of the immunoglobulin-like receptor (LIR) family of leukocytes, and is present in the gene cluster in the chromosomal region 19q13.4. The encoded proteins belong to the LIR receptor subfamily B class, which contains two or four extracellular immunoglobulin domains, a transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). This receptor is expressed on immune cells, where it binds to MHC class I molecules on antigen presenting cells and transduces a negative signal that inhibits stimulation of the immune response. It is thought to control inflammatory responses and cytotoxicity to aid in focusing immune responses and to limit self-reactions. The receptor is also expressed on human non-small cell lung cancer cells. Different isoforms encoded by multiple transcript variants of this gene have been discovered. LILRB2 has been found to interact with PTPN 6.

C.LILRB3

Leukocyte immunoglobulin-like receptor subfamily B member 3 is a protein encoded by the LILRB3 gene in humans. This gene is a member of the immunoglobulin-like receptor (LIR) family of leukocytes, and is present in the gene cluster of the chromosome region 19q13.4. The encoded proteins belong to LIR receptor subfamily B, which contains two or four extracellular immunoglobulin domains, a transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). This receptor is expressed on immune cells, where it binds to MHC class I molecules on antigen-presenting cells and transduces a negative signal that inhibits stimulation of an immune response. It is thought to control inflammatory responses and cytotoxicity to aid in focusing immune responses and to limit self-reactions. Different isoforms encoded by multiple transcript variants of the gene have been discovered.

D.LILRB4

Leukocyte immunoglobulin-like receptor subfamily B member 4 is a protein encoded by the LILRB4 gene in humans. This gene is a member of the immunoglobulin-like receptor (LIR) family of leukocytes, and is present in the gene cluster of the chromosome region 19q13.4. The encoded proteins belong to the LIR receptor subfamily B class, which contains two or four extracellular immunoglobulin domains, a transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). This receptor is expressed on immune cells, where it binds to MHC class I molecules on antigen presenting cells and transduces a negative signal that inhibits stimulation of the immune response. The receptor also has the function of antigen capture and presentation. It is thought to control inflammatory responses and cytotoxicity to aid in focusing immune responses and to limit self-reactions. LILRB4 is also expressed on gastric cancer cells and can enhance tumor growth. Different isoforms encoded by multiple transcript variants of the gene have been discovered. LILRB4 has been found to interact with PTPN 6.

E.LAIR1

Leukocyte-associated immunoglobulin-like receptor 1 is a protein encoded by the LAIR1 gene in humans. LAIR1 is also referred to as CD305 (cluster of differentiation 305). LAIR1 is a type I transmembrane glycoprotein that contains one extracellular Ig-like domain and two intracellular ITIMs. Similar to the gene encoding LILRB, lair1 is located in the human chromosome 19q13.4 leukocyte receptor complexSubstance (LRC). LAIR1 binds to collagen, and its ITIM recruits SHP-1 and SHP-2.LAIR1 in T cells, B cells, natural Killer (NK) cells, macrophages and dendritic cells, and including human CD34 ⁺ Expressed in hematopoietic progenitor cells, including cells.

Examples

The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

Example 1

LILRB4 expressed on leukemia cells leads to T cell suppression

To identify new mechanisms and molecular targets for leukemia immune escape, the inventors analyzed the correlation between gene expression of 50 known conceptual costimulatory and cosuppression receptors and overall survival of 173 AML patients in the tcGAaml database. The inventors found that the expression of the immunosuppressive receptor lilrb4 was most significantly negatively correlated with survival of AML patients (fig. 5).

LILRB4 has a restricted expression pattern on normal monocytes ²² And the expression in monocytic AML (or acute monocytic leukemia, which develops from the monocytic lineage and belongs to the FAB M4 and M5AML subtypes) cells was higher than in cells from the other subtypes (fig. 6). The present inventors analyzed the surface expression of LILRB4 on leukemic blast cells of 118 AML patient samples from the UT southwest medical center (UTSW) and found that LILRB4 was present only on blast cells of M4 and M5 monocytic AML subtypes and not other AML subtypes (fig. 7a and table 1). These results have been reported heretofore with LILRB4 being a specific marker for monocytic AML ²⁶ And (5) the consistency is achieved. Importantly, LILRB4 in monocytic AML cellsThe levels were higher than in normal monocytes (FIGS. 7 b-c) and were not expressed on normal Hematopoietic Stem Cells (HSCs) (FIG. 8). These results indicate that the monocyte AML marker LILRB4 is an attractive target for the treatment of this type of leukemia.

To test whether LILRB expressed on AML cells has immunosuppressive function, the inventors co-cultured LILRB 4-positive or LILRB 4-negative leukemia cells or normal hematopoietic cells with autologous T cells or T cells from healthy donors. LILRB4 positive primary monocyte AML cells significantly inhibited T cell proliferation (fig. 9). The inventors then deleted LILRB4 in human monocytic AML THP-1 cells using an inducible CRISPR/Cas9 system with LILRB 4-specific guide RNA. After knocking out lilrb4 (KO), the T cell suppressive ability of THP-1 cells was lost (FIG. 7 d-f). In contrast, wild-type lilrb4 was forced to be expressed in lilrb4-KO THP-1 cells, whereas the non-endodomain deleted mutant lilrb4 rescued the inhibitory function of such T cells (FIG. 7 d-f). Thus, LILRB4 on tumor cells effectively inhibits human T cell activity, and this function of LILRB4 is dependent on its intracellular signaling domain. This previously reported extracellular domain of LILRB4 responsible for inhibiting T cell activity ²⁵ The opposite is true. Surprisingly, separation of wild-type THP-1 cells from human T cells in a transwell still inhibited T cells. In contrast, lilrb4-KO THP-1 cells lost this ability. Furthermore, full-length, but not endodomain deleted, LILRB4 was able to rescue this phenotype, suggesting that non-contact T cell suppression is LILRB4 intracellular signaling dependent (fig. 7 g-h).

The inventors tried to determine whether antagonizing LILRB4 could prevent AML progression by reversing LILRB 4-mediated immunosuppression. As described by the inventors ^2,31 To identify potential agonistic and antagonistic effects of LILRB, the inventors were based on the extracellular domain (ECD) of a single LILRB and its mouse ortholog PirB ²⁷ And gp49B1 ²⁸ Fusion of (a) results in a single stable chimeric receptor reporter cell interacting with the intracellular domains of paired immunoglobulin-like receptors β, signaling through the aptamer DAP-12 to activate NFAT promoter-driven GFP expression. In the systemWith the help of systems, the inventors generated novel anti-LILRB 4 blocking antibodies to further evaluate LILRB 4-mediated signaling (fig. 10). Although anti-LILRB 4 had no effect on cell activation or proliferation of T cells or THP-1 cells themselves (fig. 11), treatment with anti-LILRB 4 antibody blocked LILRB 4-mediated T cell inhibition (fig. 7 g-h). Moreover, in the co-culture of THP-1 cells and CTLs, this treatment with blocking antibody significantly reduced the number of THP-1 cells and increased the number of CTLs and cytokines produced by the CTLs (FIGS. 7 i-m). Taken together, these in vitro results indicate that LILRB4 expressed by AML cells inhibits T cell activity, and that anti-LILRB 4 blocking antibodies reverse this immunodetection site function, sensitizing tumor cells to the cytotoxic killing effects of T cells.

The present inventors attempted to confirm the immune checkpoint blocking function of LILRB4 in vivo using a humanized mouse graft tumor model and an immunocompetent mouse model. To generate a humanized mouse model, immunocompromised NOD-SCID Il2rg knockout (NSG) mice were sublethally irradiated and human peripheral blood mononuclear cells (hPBMC) were transplanted, enabling analysis of the effect of human T cells on tumor biology ³² . In humanized NSG mice, blockade of LILRB4 by anti-LILRB 4 inhibited tumor development by subcutaneously implanted THP-1 cells (fig. 12 a-b) and increased cytotoxic T cells (fig. 12 c). Importantly, blockade of LILRB4 significantly reduced leukemia progression in 8 primary human monocytic AML-derived transplantable tumors (fig. 12 d), and simultaneously increased allogeneic human CTL cells (fig. 12 e). These results demonstrate that anti-LILRB 4 antibodies reverse the LILRB 4-mediated T cell immunosuppression mediated by tumor cell expression.

To further validate the conclusions, the inventors subcutaneously implanted mouse C1498AML cells expressing human LILRB4 (C1498-huLILRB 4) into immune competent C57BL/6 mice. To rule out the anti-tumor effects of antibody-dependent cell-mediated cytotoxicity/phagocytosis or complement-dependent cytotoxicity (ADCC/ADCP/CDC), the inventors administered anti-LILRB 4 antibodies (anti-LILRB 4-N297A) with mutations in the Fc glycosylation site of tumor-bearing mice, which antibodies lacked ADCC/ADCP/CDC ³³ . LILRB4 blockade was able to effectively reduce tumor burden (fig. 12 f-h) by increasing effective T cells (fig. 12 i) compared to administration of control IgG. Phase(s)Trans, CD8 ⁺ T cell depletion abrogated the anti-tumor effect of the anti-LILRB 4 antibody (fig. 12 j-l), indicating that LILRB4 tumor support was dependent on T cell suppression. To determine whether anti-LILRB 4 antibody treatment generated memory T cells to prevent AML relapse, the inventors adoptively transplanted splenocytes from anti-LILRB 4 treated mice into normal recipient mice. Four of the five transplanted mice rejected parent C1498 mouse leukemia cells, and these mice also prevented re-challenge with three times the number of leukemia cells (fig. 12 n). Taken together, these in vitro and in vivo results indicate that LILRB4 is a specific marker for monocytic AML and that LILRB4 signaling in tumor cells requires inhibition of T cell-mediated anti-tumor immunity.

LILRB4 supports leukemia cell infiltration

One of the characteristics of monocytic AML is enhanced extramedullary infiltration of tumor cells. The inventors observed that blockade with LILRB4 antibody could result in a significant reduction of leukemic infiltration into internal organs including bone marrow, liver and brain (fig. 13). The inventors hypothesized that, in addition to T cell suppression, LILRB4 is able to promote leukemic infiltration for immune escape. To validate this hypothesis, they performed transendothelial migration and homing assays and monitored leukemic infiltration relative to LILRB4 expression. LILRB4KO in human AML THP-1 cells reduced transendothelial migration in vitro (fig. 14 a), reduced short-term (20 hr) homing to liver and bone marrow (fig. 14 b), reduced long-term (21 days) engraftment to hematopoietic organs (fig. 14 c), prolonged survival in transplanted tumor mice (fig. 14 d) and delayed weight loss (fig. 14 e). In contrast, forced expression of human LILRB4 in mouse AML C1498 or WEHI-3 cells had opposite effects (FIGS. 14f-j and 15). Notably, KO or ectopic expression of LILRB4 did not significantly affect leukemia growth in vitro and in vivo (fig. 16). Since NSG mice are functional T cell deficient, these results, particularly from transplantation tumor experiments, reveal a unique role for LILRB4 in AML-promoting migration and leukemic infiltration. This is consistent with previous studies showing circulating LILRB4 ⁺ The frequency of AML blasts is significantly lower than that of LILRB4 ^- AML blast cell ²⁶ And LILRB4 ⁺ Frequency of chronic lymphocytic leukemia cells, the latter more commonly associated with lymphoid tissue involvement ¹² 。

Although administration of anti-LILRB 4 antibody did not reduce the size of the subcutaneous C1498 tumor when CD8T cells were depleted in C57bl/6 mice (fig. 12 h), administration of anti-LILRB 4 antibody resulted in a reduction in infiltration of leukemic cells in the liver (fig. 17). To further investigate whether LILRB4 modulates cell migration/infiltration, the inventors administered anti-LILRB 4 antibodies to LILRB 4-positive (THP-1 and MV 4-11) and LILRB 4-negative (U937) human AML cells in an in vitro transwell as in vivo homing assay and transplantation tumor models (fig. 18). The inventors found that antibody-mediated blockade of LILRB4 had the same effect on LILRB 4-expressing MV4-11 and THP-1AML cells as LILRB4KO (fig. 14 k-t), but it was not effective on U937 cells that did not express LILRB4 (fig. 19). Importantly, whole animal and ex vivo bioluminescence imaging showed that anti-LILRB 4 antibodies significantly prevented leukemic infiltration into the lung, liver, bone marrow, brain, kidney, spleen, and gastrointestinal tract (fig. 14u-v, fig. 20, fig. 21, and fig. 22). To rule out that the observed migration inhibitory effect was probably due to an antibody mediated effect by innate immune cells present in NSG mice, the inventors administered glycosylation-deficient anti-LILRB 4 antibodies (anti-LILRB 4-N297A) to NK cells, macrophages and neutrophil-depleted NSG receptors transplanted with MV4-11 cells (fig. 23 a-c). Administration of anti-LILRB 4-N297A antibody significantly inhibited AML infiltration compared to isotype control under each condition of innate immune cell depletion (fig. 14 w-z).

To determine whether intracellular signaling of LILRB4 is required for leukemic cell infiltration, the inventors investigated the homing of wild-type (WT) or knock-out (KO) LILRB4 gene THP-1 cells, as well as KO THP-1 cells with wild-type LILRB4 expression (KO-WT) or rescued by mutant LILRB4 expression with deletion of the intracellular domain (KO-int Δ). It further examined the effect of anti-LILRB 4 antibodies. The inventors found that anti-LILRB 4 antibody reduced the homing ability of wild-type THP-1 and LILRB4KO THP-1 (reintroduced WT LILRB4 (KO-WT)) cells to liver and bone marrow to the same level as LILRB4KO or KO THP-1 (reintroduced mutant LILRB4 (KO-int Δ) lacking the endodomain)) cells (fig. 14aa, fig. 14 cc). In contrast, the ability to home to LILRB4KO THP-1 cells or KO-int Δ cells was not affected by administration of the antibody (fig. 14bb, fig. 14 dd). Taken together, these results suggest that LILRB4 promotes migration of AML cells to internal organs with immune-privileged sites (AML-privileged sites) and supports AML progression.

APOE activation of LILRB4 to support AML infiltration

Blockade by anti-LILRB 4 antibodies was effective in inhibiting the immunosuppressive and migratory functions of acute monocytic leukemia cells, suggesting that LILRB4 function on the surface of leukemia cells may be ligand-dependent. The inventors sought to identify extracellular binding proteins of LILRB4. The integrin-alpha has been previously described _v β ₃ Identified as a ligand for gp49B1, integrin-alpha _v β ₃ Is an orthologue of mouse LILRB4 ³⁴ . However, multiple integrin- α β complexes failed to activate human LILRB4 reporter cells (fig. 24).

Surprisingly, the inventors found that human serum and mouse serum were able to specifically stimulate LILRB4 reporter protein but not other LILRB reporter proteins (fig. 25 a). It identified human and mouse APOE-specifically activated LILRB4 reporter proteins (fig. 25b and fig. 27) by Fast Protein Liquid Chromatography (FPLC) fractionation followed by reporter protein assay and mass spectrometry (fig. 26). APOE purified from different sources activated LILRB4 (FIG. 28 a). Under both solidified and soluble conditions, all three isoforms of human APOE activated LILRB4 (fig. 28 b). Interestingly, recombinant APOE specifically activated mouse PirB but not gp49B1 (fig. 25B), which was considered to be an orthologue of mouse LILRB4 ²⁸ . Sera from wild-type but not APOE-deficient mice activated the LILRB4 reporter protein (fig. 25 c). Furthermore, the liposomal reconstituted APOE protein (APOE-POPC) had the same ability to activate LILRB4 reporter cells as the non-liquid APOE protein (fig. 25 d). LILRB4KO significantly reduced APOE binding to THP-1 cells (fig. 25 e).

The inventors confirmed specific binding of recombinant APOE to LILRB4 using Surface Plasmon Resonance (SPR), biolayer interferometry (Octet), and Microcalorimetry (MST), with a dissociation constant of 2nM as determined by SPR (fig. 25f and fig. 29). APOEComprises two functional domains, an N-terminal domain containing its receptor LDLR binding site (residues 136-150) and a C-terminal domain (residues 222-299). To determine which domain of APOE is required for LILRB4 binding, the inventors generated one N-terminal mutant (Mut-N: R142A/K143A/R145A/K146A/R147A/R150A) and two C-terminal mutants (Mut-C1: deletion residues 245-299; and Mut-C2: deletion residues 279-299) of human APOE. The N-terminal mutant significantly reduced LILRB4 activation (fig. 25 g). The inventors also designed a series of site-specific mutations in amino acids that might be critical for ligand binding to LILRB4 based on molecular modeling of LILRB4 to APOE (fig. 30). It was found that P35 and W106 in the first Ig domain and Y121 in the linker region between the two Ig domains were critical for APOE activation of the LILRB4 reporter (fig. 25 h). Activation of the immunosuppressive receptor LILRB4 by APOE is consistent with well-documented immunosuppressive function of APOE ^35,36 。

To further determine whether ApoE modulates the function of LILRB4, the inventors compared homing of mouse C1498AML cells with and without ectopically expressed LILRB4 in wild-type and ApoE-knockout mice. Expression of LILRB4 significantly increased homing of C1498 to bone marrow and liver of wild type mice but not APOE-deficient mice (fig. 25 i-l). Taken together, APOE binds to and activates LILRB4 to support migration of human acute monocytic leukemia cells.

LILRB4 supports AML infiltration and T cell suppression by downregulation of effectors in AML cells

Loss of the intracellular domain of LILRB4 could disable its function (fig. 7e-g and fig. 14 dd) suggesting that the downstream signaling of LILRB4 is required for T cell suppression and leukocyte infiltration. The intracellular domain of LILRB4 contains three ITIMs that can recruit the phosphatases SHP-1, SHP-2 or SHIP for downstream signaling. To determine whether one or more phosphatases mediate the function of LILRB4, shp-1, shp-2 and shp were deleted separately in THP-1 cells for T cell co-culture and migration assays by CRISPR/Cas9 technology. The loss of SHP-2 rescued T cell suppression of THP-1 cells (FIGS. 31 a-c). However, loss of SHP-2 but not SHP-1 or SHIP reduced the migratory capacity and engraftment of THP-1 cells (FIG. 31d andfig. 31 e). These results indicate that SHP-2 is a mediator for LILRB4 signaling to support leukemia migration and T cell suppression. The inventors further investigated gene expression of wild type and lilrb4-KO THP-1 cells by RNA-seq analysis. They found that 585 genes were significantly down-regulated and 445 genes were up-regulated in lilrb4-KO THP-1 cells compared to the wild-type counterpart. Consistent with the phenotypes observed by the inventors, these lilrb 4-up-regulated genes are specifically involved in cell migration, cytokine production, and immunosuppressive IL10 signaling. Upstream regulatory factor Analysis in Ingenity Pathway Analysis (IPA) found JAK-STAT (STAT 1, STAT2, STAT3, and STAT 4) and NFkB (NFkB 1, REL, and RELA) pathways (known to be downstream signals of SHP-2) ³⁷ ) The activity of key transcription factors was significantly inhibited by the loss of lilrb4 (fig. 31 f). In parallel with the decrease in SHP-2 phosphorylation, loss of LILRB4 down-regulated JAK/STAT and NFkB pathway activation (fig. 31 g-i). Inhibition of STAT in leukemia cells increased T cell proliferation (FIG. 31 j-k) and decreased migration in vitro (FIG. 31 l) and leukemia progression in vivo (FIG. 31 m).

Previous results by the inventors have shown that isolation of wild-type THP-1 cells and human T cells in transwell still enables T cell suppression (fig. 7 g-h), suggesting that proteins secreted from leukemic cells are key effectors of immunosuppression. Consistent with this hypothesis, the inventors found that mRNA levels of secreted proteins involved in monocyte AML cells involved in monocyte migration and immune regulation were down-regulated by the loss of lilrb4 (fig. 32). Furthermore, CCL2, CCL4, CCL5, IL-6R, IL-8, gp130, OSM, TIMP-1/2, TNF-R1/II and uPAR were reduced in the culture medium of lilrb4-KO cells compared to wild-type control cells (FIG. 31 n-o). Among these proteins, monocytic AML cells particularly highly express uPAR (urokinase receptor) ³⁸ And are known to promote cancer invasion, metastasis, survival and angiogenesis ³⁹ . uPAR is a target of NFkB in human cancer cells ^40-43 . Consistent with the reduction in secreted uPAR, there was a reduction in the mRA and intracellular protein levels of uPAR in lilrb4-KO cells (fig. 32 and 31 p). Next, the inventors found that the expression of arginase-1 in lilrb4-KO cells was significantly reduced (FIG. 31 p); arginase-1 reportedly is signaled by uPARLeads up-regulation and inhibits T cell proliferation and CTL production by increasing superoxide production in the T cell microenvironment ^44-46 . Since arginase-1 is reported to be secreted in AML cells-inhibiting T cell activity ⁴⁷ Thus, the inventors measured the level of arginase-1 in the medium and found that the secretion of arginase-1 by the cells of indeed lilrb4-KO THP-1 was also reduced (FIG. 31 q). To determine whether uPAR contributes to LILRB 4-mediated T cell suppression, the inventors treated LILRB4-KO cells with additional uPAR protein when co-cultured with T cells. Supplementation with uPAR reduced T cell proliferation in a dose-dependent manner in co-culture (fig. 31 r-s); this effect of uPAR was probably achieved by co-culture, as uPAR was not effective in directly reducing T cell proliferation (fig. 33). Similarly, supplementation with arginase-1 also reduced T cell proliferation in co-culture (fig. 31T-u). Furthermore, supplementation with uPAR or arginase-1 increased the ability of THP-1 (FIG. 31 v) or MV4-11 cells (FIG. 31 w) to migrate across endothelial cells. These results indicate that LILRB4 can increase arginase-1 expression in AML cells by SHP-2/NFkB/uPAR/arginase-1 signaling, which inhibits T cell activity and increases leukemia migration.

In addition to uPAR and arginase-1, EBI3, which forms a heterodimer of IL-27 and IL27p28, was reduced by the loss of lilrb4 (FIG. 32). There was also a reduction in IL-10 (FIG. 32), and these results indicate that LILRB4 controls the expression of IL-27/IL-10 to inhibit T cell proliferation and activation ^48,49 (FIG. 34). TIMP-2 is a natural inhibitor of MMP. Supplementation with TIMP-2 reduced T cell proliferation in co-culture (FIG. 34) or in the absence of THP-1 cells (FIG. 33). Interestingly, it has a common receptor CCR controlling monocyte and T cell migration ⁵⁰ CCL2, CCL3, CCL4 and CCL5 of (a) were down-regulated due to the deletion of lilrb4. Treatment of THP-1 cells with neutralizing antibodies alone showed that CCL4, but not CCL2 and CCL3, promoted leukemia cell migration and AML progression in a transplantation tumor model (fig. 35 a-c). Furthermore, consistent with the reports of direct induction of T cell apoptosis by CCL4 ⁵¹ The inventors found that supplementation of CCL4 in T/THP-1 co-culture medium inhibited T cell proliferation; and administration of anti-CCL 4 neutralizing antibodies rescued LILRB 4-mediated T cell suppression (fig. 34 and fig. 35 a-c). These results indicate that CCL4 is in LILKey effectors in RB4 signaling that control leukemia progression and immune escape.

To attempt to verify the function of the LILRB4 downstream effectors, the inventors performed gene expression analysis on LILRB4-KO versus wild-type THP-1 cells, versus untreated THP-1 cells using human serum treatment, and versus THP-1 cells treated with anti-LILRB 4 antibody using control IgG pre-treated with human serum. They found that 44 genes, including 10 LILRB4 positive regulatory genes and 34 LILRB4 negative regulatory genes, showed opposite trends in serum-activated and anti-LILRB 4 treated samples (fig. 36 a-d). The mRNA levels of these genes also significantly correlated with those of LILRB4 in human AML patients (fig. 36 a-d). The expression of these LILRB 4-positive regulatory genes was negatively correlated with patient survival (fig. 36 a-d). In contrast, expression of these LILRB 4-negatively regulated genes was positively correlated with patient survival (fig. 36 a-d). Taken together, these results suggest that LILRB4 supports immune escape and cancer infiltration through downstream signaling in leukemia cells.

In this study, the inventors attempted to answer two major biological questions. First, given the general lack of effectiveness of existing immune checkpoint blockade therapies for leukemia, is leukemia adopting a unique and unknown mechanism of tumor development and immune escape? Second, receptors containing immunosuppressive ITIMs generally need to work with activating receptors for immunomodulation ⁵² . Is these receptors able to initiate immune-related primary signal transduction? Here, the inventors obtained a positive answer to both of these questions. It identifies a new mechanism for acute monocytic leukemia tumor development and immune escape, and also demonstrates that ITIM-containing receptors are able to initiate primary immune escape signaling in tumor cells. To escape immune attack, acute monocytic leukemia relies on the inhibition of T cells by LILRB 4; different from the previous findings ⁵³ These data indicate that intracellular signaling of LILRB4 in cancer cells is required for this immunosuppression. Consistently, LILRB4 directs tumor cell migration to internal organs/tissues including immune-privileged sites. Notably, this also explains the mononuclear detailsCharacteristic extracellular infiltration of cellular AML.

As demonstrated by the inventors, the tumor invasion mechanism of acute monocytic leukemia is unique. Unlike direct immunosuppression by cell-cell contact exemplified by PD-L1/PD-1 conjugation (engagment), these leukemic cells infiltrate the tissue and inhibit T cell activity using LILRB 4-mediated signal transduction-creating a new immunosuppressive microenvironment. These findings suggest that a different tumor-blocking strategy than the existing strategies is needed to treat acute monocytic leukemia.

LILRB4 may be a fatal weakness of acute monocytic leukemia and is therefore an ideal target for treating this disease. Targeting LILRB4 could reactivate multiple immune cell types (including T cells and possibly monocytes/macrophages), prevent tumor infiltration into tissues/organs, and kill tumor cells directly (through antibody-dependent cell-mediated cytotoxicity or phagocytosis), thus combining perfectly with immunotherapy and targeted therapy. Furthermore, anti-LILRB 4 can mobilize leukemia cells from bone marrow to peripheral blood (fig. 37 a-c) and targeting LILRB4 in combination with other therapies (e.g., chemotherapy) may be beneficial because anti-LILRB 4 treatment results in leukemia cells migrating from the niche into the bloodstream where they may be more sensitive to cytotoxic chemotherapy (fig. 38 a-e). Importantly, the functional dependence of LILRB4 in acute monocytic leukemia suggests that LILRB4 downregulation has a low probability of causing drug resistance in the LILRB4 blocking strategy. Further, because the expression of LILRB4 on normal monocytes is limited ²² However, it was expressed at high levels on human monocytic AML cells and anti-LILRB 4 blocking antibodies did not affect normal HSC homing (fig. 39) and normal hematopoiesis in human cord blood cell reconstituting mice (fig. 40), so targeting LILRB4 may have very low toxicity.

In addition to AML, LILRB4 may play a role in other hematopoietic malignancies and solid cancers. LILRB4 in chronic lymphocytic leukemia ¹² And upregulation in certain solid cancer cells ^2210,16,17 . LILRB4 is also expressed on tumor-associated macrophages, myeloid-derived suppressor cells, and tolerogenic dendritic cells ^2210,16,17 This may contribute to the immunosuppressive environment of many tumors. These results, inferred in AML, might indicate that LILRB4 might promote metastasis of LILRB 4-positive solid cancer cells. Furthermore, it has been reported that monocytes are the source of IL-6, a major cytokine responsible for life-threatening cytokine release syndrome in some immunotherapies ⁵⁴ . Thus, targeting these LILRB 4-positive monocytes may control cytokine release syndrome. Blocking LILRB4 signaling can prove to be a new strategy for treating different types of cancer with minimal side effects.

Example 2 materials and methods

C57BL/6J and NOD-scid IL2R γ Nude (NSG) mice were purchased from the Central animal House of the southwest medical center (UTSW) of the university of Texas and housed there. APOE-deficient mice are described previously ⁵⁵ . All animal experiments were approved by the animal protection committee.

Chimeric receptor reporter cells As described previously, the inventors constructed a stable chimeric receptor reporter cell system ²³¹ To test the ability of ligands to bind to the ECD of LILRB, pirB and gp49B1 alone and trigger activation or inhibition of the endodomain of paired immunoglobulin-like receptor β chemofusion, with activation of the NFAT promoter signaled by the aptamer DAP-12. An increase or decrease in GFP expression is observed if an agonist or antagonist binds to the ECD and activates or inhibits the chimeric signaling domain, respectively.

LILRB4 blocking antibodies were screened using an APOE competition assay. Briefly, APOE proteins were pre-coated on 96-well plates for 3 hours at 37 ℃. After 2 washes with PBS, 2 × 10 ⁴ Individual LILRB4 reporter cells were seeded in each well; at the same time, the indicated anti-LILRB 4 antibody was added to the medium. After 16 hours, GFP was analyzed by flow cytometry ⁺ The percentage of cells is reported.

anti-LILRB 4 antibodies that can enhance LILRB4 activity were screened using a K562 co-culture assay. Briefly, 2 × 10 ⁴ A LILRB4 reporter cell with 2x10 ⁴ Individual K562 cells were mixed and cultured in wells of a 96-well plate; at the same time, the indicated anti-LILRB 4 antibody was added to the medium. 16 smallTime later, mouse CD45 was analyzed by flow cytometry ⁺ GFP ⁺ Percentage of cells.

Flow cytometry for flow cytometry analysis of mouse AML cells, peripheral blood or bone marrow cells were stained with anti-Mac-1-APC (M1/70, BD Pharmingen), anti-Gr-1-PE (RB 6-8C5, BD Pharmingen), anti-CD 3-APC (145-2C 11, BD Pharmingen), anti-B220-PE (RA 3-6B2, BD Pharmingen) or anti-Kit-PE (B8, BD Pharmingen) monoclonal antibodies. To analyze human hematopoietic transplantation in NSG mice, a previously published protocol was used ^2,56,57 . The inventors used anti-human CD45-PE (HI 30, BD Pharmingen), anti-human CD34-FITC (555821, BD Pharmingen), anti-human CD19-PE (HIB 19, eBioscience), anti-human CD20-PE (555623, BD Pharmingen), anti-human CD11b-APC (ICRF 44, eBioscience), anti-human LILRB4-APC (ZM 4.1, eBioscience), anti-human CD14-APC (61D3, eBioscience), anti-human CD4-APC (RPA-T4, eBioscience), anti-human CD8-PE (555367, BD Pharmingen), anti-human CD28-APC (CD 28.2, eBioscience), and anti-human CD40L-APC (24-31, eAPC) antibodies to quantify different hematopoietic lineages of human hematopoietic cells.

For viral packaging, retroviral constructs MSCV-MLL-AF9-IRES-YFP, XZ201-IRES-GFP, XZ201-LILRB4-IRES-GFP were mixed with PCL-ECO (2. Viral-containing supernatants were collected 48-72 hours post transfection and used for infection as described previously ⁵⁸ . Infected mouse Lin by retrobulbar injection ^- Cell (3x 10) ⁵ ) Or mouse leukemia C1498 cell (1x 10) ⁶ ) Transplantation into C57BL/6J mice (6-8 weeks old) irradiated with either a lethal dose (1,000rad) or a sublethal dose (250 rad). C1498 cells were purchased from ATCC. For secondary transplantation, the inventors isolated YFP from primary recipient mice using FACS ⁺ BM cells and 3000 cells were transplanted into non-irradiated recipient mice including wild-type C57BL/6J and APOE-nude mice. It monitored survival, examined size and histological properties of bone marrow, spleen and liver and analyzed in peripheral blood, bone marrow, spleen and liverThe number of leukemia cells and the infiltration. It also determined populations of different leukemia cells using flow cytometry.

Human and mouse leukemia cells primary human AML samples were from UTSW. Informed consent was obtained according to the protocol reviewed and approved by the UTSW institutional review board. The UTSW case population included 105 AML patients, representing AML subtypes M1 (n = 9), M2 (n = 34), M3 (n = 10), M4 (n = 34), M5 (n = 25), M6 (n = 2), and M7 (n = 1), as well as undifferentiated leukemia (AUL; n = 1) and transient myeloproliferative disorders (TAM; n = 2). The expression of LILRB4 in the samples was analyzed by flow cytometry. At 37 ℃,5% CO ₂ And normal level O ₂ Human leukemia cells (THP-1, MV4-11 and U937) and mouse leukemia cells (WEHI-3) (purchased from ATCC) were cultured in RPMI-1640 supplemented with 10% FBS. At 37 ℃,5% CO ₂ And normal level O ₂ Under the conditions of (1), mouse leukemia cells (C1498) (purchased from ATCC) were cultured in DMEM supplemented with 10% FBS.

TCGA analysis. Data were from the TCGA acute myeloid leukemia database (version: 10 months 29 years 2015). Dividing the patients into AML subtypes M0 (n = 16), M1 (n = 42), M2 (n = 39), M3 (n = 16), M4 (n = 35), M5 (n = 18), M6 (n = 2), M7 (n = 3); two cases were not classified by subtype. The level of LILRB4mRNA was determined by RNAseq (IlluminaHiSeq). RESM normalized counts were reported and data were displayed using UCSC Xena (Xena. To analyze overall survival, 160 patients with available survival data were divided into three groups according to whether they had high (n = 55), medium (n = 48), or low (n = 57) LILRB4 expression.

Bio-layer interference assay binding interactions between LILRB4-Fc and APOE2, APOE3 and APOE4 were performed on Octet RED96 (ForteBio, pall Corporation). All interaction studies were performed using protein a immersion and reading biosensors (ForteBio). All binding experiments were performed at 30 ℃ using Octet Red and kinetic buffer. The LILRB4-Fc coated biosensor was washed in kinetic buffer (using a 25 μ g/ml LILRB4-Fc load of 420 s) before monitoring APOE binding (300 s) and dissociation (600 s). The background wavelength shift was measured using a reference sensor loaded only with LILRB 4-Fc.

Microcalorimetric Swimming (MST) MST experiments were carried out on a Monolith NT.115 system (NanoTemper Technologies) using 80% LEDs and 20% IR-laser power. The laser switching times were set to 30s and 5s, respectively. Recombinant LILRB4-ECD protein (SinoBio) was labeled with 4488-NHS (NanoTemper Technologies) and applied at a final concentration of 5.9 nM. Two-fold serial dilutions of unlabeled His-APOE ((# CI06, novoprotein) were prepared in PBS and each dilution spot was similarly transferred to a LILRB4-ECD solution, the final concentration of His-APOE ranged from 12 μ M to 0.36nM, samples were filled into standard treated capillaries (NanoTemper Technologies) for measurement.

Tumor cell/T cell co-culture assay T cells isolated from peripheral blood of healthy donors (PB 009-1-0, allcells) were co-cultured with irradiated (28 Gy) THP-1 cells in U-bottom 96-well plates for 3-7 days. anti-CD 3/CD 28-coated beads (# 11161D, thermo Fisher), 50U/ml recombinant human IL-2 and 5ng/ml recombinant human IL-7 were added to the medium. In some experiments, THP-1 cells were cultured in the upper chamber of a U-bottom 96-well plate transwell insert (pore size 3. Mu.M, #09-761-80, thermo Fisher). For primary AML or B-ALL samples, CD3 was collected from patients ⁺ T cells, sorting leukemia cells of patients as CD33 for AML and B-ALL, respectively ⁺ And CD19 ⁺ 。

Stimulation of CD8 isolated from hPMCs (Interstate Blood Bank) of healthy donors with anti-CD 3/CD28/CD137 coated beads (11163D, thermo Fisher) in 96-well plates ⁺ T cells (5X 10 per well) ⁴ ) Or cultured without stimulation for 2 days. Then, add 5x10 ³ Individual leukemic THP-1-Luc-GFP cells and 50 to 500 μ g/ml of anti-LILRB 4 antibody C84 or control antibody mIgG. Cell numbers were determined in triplicate wells on day 7. anti-CD 8 and anti-CD 28 were used to detect human CTL cells; THP-1 cells were positive for GFP. Cell supernatants of co-cultures of stimulated CTL cells and THP-1 cells treated with C84 or mIgG were used to examine cytokine production using human cytokine arrays (AAH-CYT-6, rayBiotech). This experiment was repeated three times with similar results.

Transwell assay to examine cell plasticity, CFSE (Invitrogen) labeling was usedNotation 1x10 ⁵ MV4-11 cells were given 100. Mu.g/ml anti-LILRB 4 antibody C84 or control antibody mIgG and cultured in the upper chamber of a well of a transwell plate (Corning). After 18h, the cells in the lower chamber were counted. To examine the ability of AML cells to migrate through endothelial cells, 3x10 was cultured on a transwell membrane ⁵ Individual umbilical vein endothelial cells (HUVEC). After 3 days, 1x10 ⁵ Each CFSE-labeled MV4-11 cells was seeded in the upper chamber containing 100. Mu.g/ml C84 or mIgG. After 18h, the cells in the lower chamber were counted.

Homing and mobilization of leukemia and HSC cells CFSE-labeled MV4-11 cells (5X 10 cells per mouse) ⁶ Individual cells) were injected intravenously into NSG mice. Immediately after injection of leukemia cells, animals were given 200 μ g of control antibody mIgG or anti-LILRB 4 antibody C84 or 10% serum. Mice were sacrificed after 8 or 20 h. Peripheral blood, bone marrow, liver and spleen were collected and single cell suspensions were detected by flow cytometry. CFSE or anti-human CD45 was used to detect human leukemia cells. The number of leukemia cells in recipient liver, spleen and bone marrow is reported in percent relative to the cells of peripheral blood. To detect HSC homing, 1x10 was assigned ⁷ Individual cord blood mononuclear cells were injected intravenously into NSG mice. Immediately after injection of monocytes, mice were given 200 μ g mIgG or C84, and mice were sacrificed 20h later. Anti-human CD45 and anti-human CD34 were used to detect human HSC by flow cytometry. To detect homing of murine leukemia cells, 5x10 was used ⁶ One C1498-GFP-hLILRB4 cell or C1498-GFP was injected intravenously into wild type C57BL/6J or APOE-nude mice. Mice were sacrificed after 20 h. GFP was used to detect leukemic cells by flow cytometry. Leukemia cell numbers in recipient liver, spleen and bone marrow were normalized to the corresponding cell numbers in peripheral blood and reported as a percentage. To detect mobilization of leukemic cells, 5x10 was used ⁶ Individual MV4-11 cells were injected intravenously into each NSG mouse. 3 days after transplantation, mice were given 200 μ g of C84 or mIgG by intravenous injection. The day of first administration was designated as day 0. Then, the mice were given another dose of 200. Mu.g of C84 or mIgG, respectively, the following day. Leukemia cells in peripheral blood were detected 4hr (on day 0) and on days 1 and 4 after the first administration of antibody. Mice were sacrificed on day 4.Anti-human CD45 was used to detect human leukemia cells by flow cytometry.

Human AML transplantable tumors substantially as described ^2,3,56,59 . Briefly, 6-8 week old NSG mice were used for transplantation. Human leukemia cells were resuspended in 200. Mu.l of PBS containing 1% FBS. Mice were given 1x10 by tail vein injection ⁶ Leukemia cells cultured individually or 5 to 10x 10 ⁶ Personal primary AML cells. Implantation of peripheral blood, bone marrow, spleen and liver was evaluated 1 to 4 months after transplantation.

For the hPPMC transplantable tumor model, 1x10 ⁷ Human PBMCs were injected intravenously into each NSG mouse. 3 weeks after inoculation, mice had 30 to 50% human T cell engraftment. 3 weeks after inoculation, 1X10 subcutaneous inoculation ⁶ Human AML THP-1 cells (THP-1-Luc-GFP cells) stably expressing luciferase. Mice were immediately given 200 μ g of C84 or mIgG intravenously every two weeks until sacrifice. Tumor growth was monitored over time by luminescence imaging.

For the human umbilical cord blood (hCB) HSC reconstitution of the transplantable tumor model, 3X10 was injected via the retrobulbar route ⁴ Individual cord blood CD34 ⁺ Cells were injected intravenously into 6-8 week old NSG mice irradiated with a sublethal dose (2.5 Gy). As hereinbefore described ^56,57,60 Multilineage artificial blood reconstitution was confirmed by flow cytometry at various time points between day 21 and day 41 post-transplantation. At day 42, intravenous inoculation 1x10 ⁶ Human THP-1-Luc-GFP cells. Mice were immediately given 200 μ g of C84 or mouse IgG by intravenous injection. Tumor growth was monitored over time by luminescence imaging. Multilineage human hematopoietic reconstitution was detected by flow cytometry at various time points between day 12 and day 24 post leukemia cell transplantation. CD19 and CD20 were used to identify human B cells; CD11b, CD14 and LILRB4 were used for human myeloid cells; CD4, CD8, CD28 and CD40L are used in human T cell populations.

For the survival curve experiments, mice were scored dead when dying animals.

CRISPR/Cas 9-based LILRB4 knockdown in AML cells THP1 cells were infected with a lentivirus expressing doxycycline-induced Cas9 (pCW-Cas 9, addgene 50661). After selection with 1 μ g/ml puromycin, surviving cells were infected with sgRNA-expressing lentiviruses generated from a modified pSLQ1651 (adddge 51024) plasmid that was sorted using GFP instead of puro-mcherry. A control sgRNA (control sgRNA 5-. After 1 week of treatment with 1 μ g/ml doxycycline, these cells were stained with anti-LILRB 4 antibody and LILRB4 negative cells were sorted as LILRB4 knockout cells.

SDS-PAGE and cytosolic/nucleoprotein separation for SDS-PAGE, samples were mixed with 4 XLoading buffer containing β -mercaptoethanol (BME) and loaded onto 10% SDS gels. The nuclear and cytoplasmic cell compartments were separated by NE-nuclear/cytoplasmic extraction kit (# 78833, thermo Fisher) and these protein extracts were mixed with 4 Xloading buffer containing beta-mercaptoethanol (BME) and loaded onto 10% SDS gels. anti-SHP-1 (# 3759), anti-SHP-2 (# 3397), anti-SHIP (# 2727), anti-phospho-SHP-2 (Tyr 580) (# 3703), anti-Nf-kB p65 (# 8242), anti-IKKA (# 11930), anti-IKKb (# 8943), anti-phospho-IKKA/B (Ser 176/180) (# 2697), anti-phospho-Stat 1 (Tyr 701) (# 7649), anti-phospho-Stat-3 (Ser 727) (# 9134), anti-Lamin-B2 12255), and anti-arginase-1 (# 9819) were purchased from Cell Signaling Technology Inc (# 9819). anti-uPAR antibodies (MON R-4-02, thermo Fisher) and anti-a-tubulin (# MABT205, sigma) were purchased from other companies.

RNA-seq analysis. RNA was purified from sorted cells using Qiagen RNeasy Mini kit according to the manufacturer's instructions, and then reverse transcribed using SuperScript III reverse transcriptase (Invitrogen). RNA-seq was performed in UTSW genomics and microarray-centric laboratories. The cDNA was sonicated using a Covaris S2 sonicator and the library was prepared using the KAPA high throughput library preparation kit. Samples were end-repaired, 3' ends adenylated and barcoded with multiplex aptamers. The PCR-amplified library was purified using AmpureXP beads and validated on an Agilent 2100 bioanalyzer. Prior to normalization and mixing, samples were quantified by Qubit (Invitrogen) and then run on an Illumina Hiseq 2500 instrument using PE100SBS v3 reagent to generate 51-bp single-ended readings. Before mapping, the readings were trimmed to remove terminal low mass regions. The trimmed reads were mapped onto the human genome (HM 19) using TopHat v2.0.1227 from Illumina with UCSC iGenomes GTF file.

The method of data normalization and analysis is based on the use of "internal standards" to characterize some aspects of system behavior, such as technical variability, as shown elsewhere. Log is to ₂ (fold change)>2，P<0.01 and RPKM>The gene of 0.1 was considered to have a significant difference in expression between the two cases and was used for pathway analysis and upstream transcription factor analysis. Pathway analysis was performed using DAVID (https:// DAVID. Ncifcrf. Gov/tools. Jsp). Upstream transcription factor analysis was performed using the Ingeneity tool from QIAGEN. Gene heatmaps were clustered by hierarchical clustering (Cluster and Java Treeview).

Quantitative RT-PCR according to the protocol provided, total RNA was extracted using RNAeasy kit (QIAGEN) and reverse transcribed to cDNA using SuperScript III reverse transcriptase (Invitrogen). Real-time PCR was performed using the primers listed in Table 2 using SYBR Green Master Mix (Bio-Rad). mRNA levels were normalized to the level of GAPDH or 18S rRNA transcripts present in the same sample.

Statistical analysis data are expressed as mean ± SEM. Data were analyzed using Student t-test and p <0.05 was considered statistically significant. The survival of both groups was analyzed using the log rank test and p <0.05 was considered statistically significant. In all figures, denotes p <0.05; * Denotes p <0.01; * Denotes p <0.001; * Represents p <0.0001; otherwise, the p-value is represented as an exact value.

Table 1: use of 8AML patient samples from a population of UTSW cases for mouse transplantation tumor model

All methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

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60.Huynh,H.et al.IGF binding protein 2 supports the survival and cycling of hematopoietic stem cells.Blood118,3236-3243,doi:10.1182/blood-2011-01-331876(2011).

Sequence listing

<110> board of university of Texas

C. (Sheet)

Z. (Ampere)

N. (Sheet)

M. (Deng)

J. (Gold)

X. (cinnamon)

<120> method for identifying an LILRB blocking antibody

<130> UTFH.P0332WO

<150> 62368672

<151> 2016-07-29

<160> 196

<170> PatentIn 3.5 edition

<210> 1

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> control sgRNA

<400> 1

gaacgactag ttaggcgtgt a 21

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> sgRNA1

<400> 2

tgttactatc gcagccctgt 20

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> sgRNA2

<400> 3

gtaggtcccc ccgtgcactg 20

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> sgRNA3

<400> 4

cctgtgacct cagtgcacgg 20

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 5

tggcaaaacg tcttcaggag g 21

<210> 6

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 6

agcttgactt agtggctttg g 21

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 7

aactcatttt gcggtcgcta t 21

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 8

tccctgaatt gctcgctcac 20

<210> 9

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 9

accctgacca tccaagtcaa a 21

<210> 10

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 10

ttggcctcgc atcttagaaa g 21

<210> 11

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 11

accgctttgc ggaatctca 19

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 12

aggtcaggga aacatcaggg a 21

<210> 13

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 13

actcacctct tcagaacgaa ttg 23

<210> 14

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 14

ccatctttgg aaggttcagg ttg 23

<210> 15

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 15

agagctgtga taagccagtt cc 22

<210> 16

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 16

aattcctcga aagtgaagtg tgt 23

<210> 17

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 17

agttctctgc atcacttgct g 21

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 18

cggcttcgct tggttaggaa 20

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 19

atcagagctg ggctgcgata 20

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 20

caggtcgaac gactctctcc 20

<210> 21

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 21

atgatggctt attacagtgg caa 23

<210> 22

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 22

gtcggagatt cgtagctgga 20

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 23

atgcccagac atctgtgtcc 20

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 24

ggggtctcta tgcccaacaa 20

<210> 25

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 25

cagccagatg caatcaatgc c 21

<210> 26

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 26

tggaatcctg aacccacttc t 21

<210> 27

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 27

cagccgcagg gttctctac 19

<210> 28

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 28

gatctggcag aagacgatgg t 21

<210> 29

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 29

cagcgagtgg accagatcc 19

<210> 30

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 30

ccaaagagga ggtagtggct atc 23

<210> 31

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 31

cagcttgact caaaattcct gga 23

<210> 32

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 32

tgaagattac gcttgctttt cct 23

<210> 33

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 33

ccaacgtgac ggacttccc 19

<210> 34

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 34

tacacgacta tgcggtacag c 21

<210> 35

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 35

ccaagcccgg aattgtcttc a 21

<210> 36

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 36

gggttggtac agacggaatg g 21

<210> 37

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 37

ccagctttac tcgcacagc 19

<210> 38

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 38

agccttggaa tcatcactcc c 21

<210> 39

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 39

ccgacagatt ccacagaa 18

<210> 40

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 40

ttggttagga agatgacact 20

<210> 41

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 41

ccggaggcgc tttactacc 19

<210> 42

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 42

taggggtgta ggcaggttca c 21

<210> 43

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 43

cctgcgcctc aagaccttc 19

<210> 44

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 44

gtcactgcgc tccagtagaa 20

<210> 45

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 45

cgcatctcct ccatactc 18

<210> 46

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 46

acctaataca ataatacagc acat 24

<210> 47

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 47

ctcagggaca acagtcagtt c 21

<210> 48

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 48

acagggctat cagggagca 19

<210> 49

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 49

ctcctgcaag agtttgatgt cc 22

<210> 50

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 50

tcatgccgat gtcatggtag g 21

<210> 51

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 51

ctgaaccgta agcccattga g 21

<210> 52

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 52

cgctccacac cacgatgac 19

<210> 53

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 53

ctgggccagc gtatagctc 19

<210> 54

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 54

agcaagctgc tttcgtccc 19

<210> 55

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 55

ctgtgctgat cccagtgaat c 21

<210> 56

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 56

tcagttcagt tccaggtcat aca 23

<210> 57

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 57

gctcctcact gttgttctac g 21

<210> 58

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 58

cgggccgctg aaagtcatt 19

<210> 59

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 59

ggaccccaac aacaagag 18

<210> 60

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 60

gtgaggaggc ttcaagac 18

<210> 61

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 61

ggagtcgaga catcttgact ga 22

<210> 62

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 62

atgaggaccg ttttatgggc t 21

<210> 63

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 63

gtggcattca aggagtacct c 21

<210> 64

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 64

tgatggcctt cgattctgga tt 22

<210> 65

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 65

gtggtgctgt cgctcttgat a 21

<210> 66

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 66

ccccagaaaa tggttcacgc t 21

<210> 67

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 67

tacaggctgg ctcaggacta t 21

<210> 68

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 68

cgcaacattt tgtagcactc tg 22

<210> 69

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 69

tcaggctgtt tgtctctgaa gg 22

<210> 70

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 70

catgctctca tactgacacc ac 22

<210> 71

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 71

tcattgccac gtacaggctc 20

<210> 72

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 72

gggtcgggct tgatgatgtg 20

<210> 73

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 73

tcccaggccc agttcataaa t 21

<210> 74

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 74

tgctttgtga gatgtaggag gt 22

<210> 75

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 75

tcctgtggaa gatcaccaat gt 22

<210> 76

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 76

gcaggcacaa cttgtagcc 19

<210> 77

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 77

tgaggtcacg gacgattaca t 21

<210> 78

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 78

gtaggcccac gaaacaaatg at 22

<210> 79

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 79

tgctgtacca agagtttgct c 21

<210> 80

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 80

cgcacacaga caactttttc ttt 23

<210> 81

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 81

tggagagcta cacaagaatc acc 23

<210> 82

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 82

tggtccagat gcttcatgga a 21

<210> 83

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 83

tggtagtagc aaccaacggg a 21

<210> 84

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 84

actttgattg agggcgtcat tc 22

<210> 85

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 85

tgtatatgtc atctcagt 18

<210> 86

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 86

taataacaat atgcttcca 19

<210> 87

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 87

tgttggccca atggattgaa a 21

<210> 88

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 88

ggaaacacga cctaactgtt cat 23

<210> 89

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 89

ttcagtatca caacctcagc aag 23

<210> 90

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 90

tggacctgca agttaaaatc cc 22

<210> 91

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 91

ttttgccaag gagtgctaaa ga 22

<210> 92

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 92

aaccctctgc acccagtttt c 21

<210> 93

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 93

ggagcgagat ccctccaaaa t 21

<210> 94

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 94

ggctgttgtc atacttctca tgg 23

<210> 95

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 95

catgtacgtt gctatccagg c 21

<210> 96

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 96

ctccttaatg tcacgcacga t 21

<210> 97

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 97

gtaacccgtt gaaccccatt 20

<210> 98

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 98

ccatccaatc ggtagtagcg 20

<210> 99

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 99

cagcagcttg acacacggta 20

<210> 100

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 100

aaacaccaaa gtggcatgtg a 21

<210> 101

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 101

ttgcctcgga gatcggagaa 20

<210> 102

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 102

ttcctcggaa tagccaccat c 21

<210> 103

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 103

ctcagcggta cttggaccc 19

<210> 104

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 104

cgagtggcaa agtcaatacc t 21

<210> 105

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 105

ctatcgacgc ccctaagact t 21

<210> 106

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 106

catcgctggg ccttactct 19

<210> 107

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 107

cggagcgcaa atacctgaag 20

<210> 108

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 108

ggttgatgat ggtgcgactg t 21

<210> 109

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 109

accacttgga atgaaattgg aca 23

<210> 110

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 110

gcggtgtgtt ataggagact ct 22

<210> 111

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 111

tccttggggc tagatggttt c 21

<210> 112

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 112

tccacgatca catggttctt tg 22

<210> 113

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 113

gactttaagg gttacctggg ttg 23

<210> 114

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 114

tcacatgcgc cttgatgtct g 21

<210> 115

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 115

aggttgggat tcgtgtctgg 20

<210> 116

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 116

agttgtttgt agttctcagg cag 23

<210> 117

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 117

accctcatct atatcctttg gca 23

<210> 118

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 118

caccaacgga accgaccag 19

<210> 119

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 119

gcaccatgca caactacctg t 21

<210> 120

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 120

attcgccacc gatggcatag 20

<210> 121

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 121

tcaccaagag ggtatctacc g 21

<210> 122

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 122

gtgcccttag gaaactcgtc t 21

<210> 123

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 123

ggactcccct tagcaacgtg 20

<210> 124

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 124

aggacatttg agagggtgtg a 21

<210> 125

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 125

cgaggggtga gcttatggaa c 21

<210> 126

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 126

gctttcccgt tgtagtcgaa ta 22

<210> 127

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 127

tgtaagacca acggggattg c 21

<210> 128

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 128

agccagtccg atagctcagg 20

<210> 129

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 129

accaccttat accagcgtta tga 23

<210> 130

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 130

ggtgtagacg aaccggatgt c 21

<210> 131

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 131

gctgcgagtg caagatcac 19

<210> 132

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 132

tggtgcccgt tgatgttctt c 21

<210> 133

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 133

tcaccgcttc agaaaaccac c 21

<210> 134

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 134

ggtccactgt gcaagaagag a 21

<210> 135

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 135

tgaaacatca gacgtggtgt g 21

<210> 136

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 136

tgcaaatatc cgtggatgaa gtc 23

<210> 137

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 137

ccagcagtcg tctttgtcac 20

<210> 138

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 138

ctctgggttg gcacacactt 20

<210> 139

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 139

gtggaaactt gcatggacaa c 21

<210> 140

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 140

aatcctggca catcgggaat c 21

<210> 141

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 141

cgcgagtgca ttccatcct 19

<210> 142

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 142

tccaaagtct tttaggtggc ag 22

<210> 143

<211> 1953

<212> DNA

<213> Intelligent people

<220>

<221> sources

<222> (1)..(1953)

<223> cDNA LILRB1

<400> 143

atgaccccca tcctcacggt cctgatctgt ctcgggctga gtctgggccc ccggacccac 60

gtgcaggcag ggcacctccc caagcccacc ctctgggctg aaccaggctc tgtgatcacc 120

caggggagtc ctgtgaccct caggtgtcag gggggccagg agacccagga gtaccgtcta 180

tatagagaaa agaaaacagc accctggatt acacggatcc cacaggagct tgtgaagaag 240

ggccagttcc ccatcccatc catcacctgg gaacacacag ggcggtatcg ctgttactat 300

ggtagcgaca ctgcaggccg ctcagagagc agtgaccccc tggagctggt ggtgacagga 360

gcctacatca aacccaccct ctcagcccag cccagccccg tggtgaactc aggagggaat 420

gtaaccctcc agtgtgactc acaggtggca tttgatggct tcattctgtg taaggaagga 480

gaagatgaac acccacaatg cctgaactcc cagccccatg cccgtgggtc gtcccgcgcc 540

atcttctccg tgggccccgt gagcccgagt cgcaggtggt ggtacaggtg ctatgcttat 600

gactcgaact ctccctatga gtggtctcta cccagtgatc tcctggagct cctggtccta 660

ggtgtttcta agaagccatc actctcagtg cagccaggtc ctatcgtggc ccctgaggag 720

accctgactc tgcagtgtgg ctctgatgct ggctacaaca gatttgttct gtataaggac 780

ggggaacgtg acttccttca gctcgctggc gcacagcccc aggctgggct ctcccaggcc 840

aacttcaccc tgggccctgt gagccgctcc tacgggggcc agtacagatg ctacggtgca 900

cacaacctct cctccgagtg gtcggccccc agcgaccccc tggacatcct gatcgcagga 960

cagttctatg acagagtctc cctctcggtg cagccgggcc ccacggtggc ctcaggagag 1020

aacgtgaccc tgctgtgtca gtcacaggga tggatgcaaa ctttccttct gaccaaggag 1080

ggggcagctg atgacccatg gcgtctaaga tcaacgtacc aatctcaaaa ataccaggct 1140

gaattcccca tgggtcctgt gacctcagcc catgcgggga cctacaggtg ctacggctca 1200

cagagctcca aaccctacct gctgactcac cccagtgacc ccctggagct cgtggtctca 1260

ggaccgtctg ggggccccag ctccccgaca acaggcccca cctccacatc tggccctgag 1320

gaccagcccc tcacccccac cgggtcggat ccccagagtg gtctgggaag gcacctgggg 1380

gttgtgatcg gcatcttggt ggccgtcatc ctactgctcc tcctcctcct cctcctcttc 1440

ctcatcctcc gacatcgacg tcagggcaaa cactggacat cgacccagag aaaggctgat 1500

ttccaacatc ctgcaggggc tgtggggcca gagcccacag acagaggcct gcagtggagg 1560

tccagcccag ctgccgatgc ccaggaagaa aacctctatg ctgccgtgaa gcacacacag 1620

cctgaggatg gggtggagat ggacactcgg agcccacacg atgaagaccc ccaggcagtg 1680

acgtatgccg aggtgaaaca ctccagacct aggagagaaa tggcctctcc tccttcccca 1740

ctgtctgggg aattcctgga cacaaaggac agacaggcgg aagaggacag gcagatggac 1800

actgaggctg ctgcatctga agccccccag gatgtgacct acgcccagct gcacagcttg 1860

accctcagac gggaggcaac tgagcctcct ccatcccagg aagggccctc tccagctgtg 1920

cccagcatct acgccactct ggccatccac tag 1953

<210> 144

<211> 650

<212> PRT

<213> Intelligent people

<220>

<221> sources

<222> (1)..(650)

<223> LILRB1

<400> 144

Met Thr Pro Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly

1 5 10 15

Pro Arg Thr His Val Gln Ala Gly His Leu Pro Lys Pro Thr Leu Trp

20 25 30

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

35 40 45

Cys Gln Gly Gly Gln Glu Thr Gln Glu Tyr Arg Leu Tyr Arg Glu Lys

50 55 60

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

65 70 75 80

Gly Gln Phe Pro Ile Pro Ser Ile Thr Trp Glu His Ala Gly Arg Tyr

85 90 95

Arg Cys Tyr Tyr Gly Ser Asp Thr Ala Gly Arg Ser Glu Ser Ser Asp

100 105 110

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

115 120 125

Ala Gln Pro Ser Pro Val Val Asn Ser Gly Gly Asn Val Ile Leu Gln

130 135 140

Cys Asp Ser Gln Val Ala Phe Asp Gly Phe Ser Leu Cys Lys Glu Gly

145 150 155 160

Glu Asp Glu His Pro Gln Cys Leu Asn Ser Gln Pro His Ala Arg Gly

165 170 175

Ser Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg

180 185 190

Trp Trp Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro Tyr Glu Trp

195 200 205

Ser Leu Pro Ser Asp Leu Leu Glu Leu Leu Val Leu Gly Val Ser Lys

210 215 220

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

225 230 235 240

Thr Leu Thr Leu Gln Cys Gly Ser Asp Ala Gly Tyr Asn Arg Phe Val

245 250 255

Leu Tyr Lys Asp Gly Glu Arg Asp Phe Leu Gln Leu Ala Gly Ala Gln

260 265 270

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

275 280 285

Arg Ser Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Leu Ser

290 295 300

Ser Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Ala Gly

305 310 315 320

Gln Phe Tyr Asp Arg Val Ser Leu Ser Val Gln Pro Gly Pro Thr Val

325 330 335

Ala Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Gln Gly Trp Met

340 345 350

Gln Thr Phe Leu Leu Thr Lys Glu Gly Ala Ala Asp Asp Pro Trp Arg

355 360 365

Leu Arg Ser Thr Tyr Gln Ser Gln Lys Tyr Gln Ala Glu Phe Pro Met

370 375 380

Gly Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser

385 390 395 400

Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser Asp Pro Leu Glu

405 410 415

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

420 425 430

Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr Gly

435 440 445

Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val Val Ile Gly

450 455 460

Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu Leu Leu Leu Phe

465 470 475 480

Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln

485 490 495

Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro

500 505 510

Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln

515 520 525

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

530 535 540

Val Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val

545 550 555 560

Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser

565 570 575

Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln

580 585 590

Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala

595 600 605

Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg

610 615 620

Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val

625 630 635 640

Pro Ser Ile Tyr Ala Thr Leu Ala Ile His

645 650

<210> 145

<211> 438

<212> PRT

<213> Intelligent people

<220>

<221> sources

<222> (1)..(438)

<223> ectodomain sequence of LILRB1

<400> 145

Gly His Leu Pro Lys Pro Thr Leu Trp Ala Glu Pro Gly Ser Val Ile

1 5 10 15

Thr Gln Gly Ser Pro Val Thr Leu Arg Cys Gln Gly Gly Gln Glu Thr

20 25 30

Gln Glu Tyr Arg Leu Tyr Arg Glu Lys Lys Thr Ala Leu Trp Ile Thr

35 40 45

Arg Ile Pro Gln Glu Leu Val Lys Lys Gly Gln Phe Pro Ile Pro Ser

50 55 60

Ile Thr Trp Glu His Ala Gly Arg Tyr Arg Cys Tyr Tyr Gly Ser Asp

65 70 75 80

Thr Ala Gly Arg Ser Glu Ser Ser Asp Pro Leu Glu Leu Val Val Thr

85 90 95

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

100 105 110

Asn Ser Gly Gly Asn Val Ile Leu Gln Cys Asp Ser Gln Val Ala Phe

115 120 125

Asp Gly Phe Ser Leu Cys Lys Glu Gly Glu Asp Glu His Pro Gln Cys

130 135 140

Leu Asn Ser Gln Pro His Ala Arg Gly Ser Ser Arg Ala Ile Phe Ser

145 150 155 160

Val Gly Pro Val Ser Pro Ser Arg Arg Trp Trp Tyr Arg Cys Tyr Ala

165 170 175

Tyr Asp Ser Asn Ser Pro Tyr Glu Trp Ser Leu Pro Ser Asp Leu Leu

180 185 190

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

195 200 205

Pro Gly Pro Ile Val Ala Pro Glu Glu Thr Leu Thr Leu Gln Cys Gly

210 215 220

Ser Asp Ala Gly Tyr Asn Arg Phe Val Leu Tyr Lys Asp Gly Glu Arg

225 230 235 240

Asp Phe Leu Gln Leu Ala Gly Ala Gln Pro Gln Ala Gly Leu Ser Gln

245 250 255

Ala Asn Phe Thr Leu Gly Pro Val Ser Arg Ser Tyr Gly Gly Gln Tyr

260 265 270

Arg Cys Tyr Gly Ala His Asn Leu Ser Ser Glu Trp Ser Ala Pro Ser

275 280 285

Asp Pro Leu Asp Ile Leu Ile Ala Gly Gln Phe Tyr Asp Arg Val Ser

290 295 300

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

305 310 315 320

Leu Leu Cys Gln Ser Gln Gly Trp Met Gln Thr Phe Leu Leu Thr Lys

325 330 335

Glu Gly Ala Ala Asp Asp Pro Trp Arg Leu Arg Ser Thr Tyr Gln Ser

340 345 350

Gln Lys Tyr Gln Ala Glu Phe Pro Met Gly Pro Val Thr Ser Ala His

355 360 365

Ala Gly Thr Tyr Arg Cys Tyr Gly Ser Gln Ser Ser Lys Pro Tyr Leu

370 375 380

Leu Thr His Pro Ser Asp Pro Leu Glu Leu Val Val Ser Gly Pro Ser

385 390 395 400

Gly Gly Pro Ser Ser Pro Thr Thr Gly Pro Thr Ser Thr Ser Gly Pro

405 410 415

Glu Asp Gln Pro Leu Thr Pro Thr Gly Ser Asp Pro Gln Ser Gly Leu

420 425 430

Gly Arg His Leu Gly Val

435

<210> 146

<211> 1797

<212> DNA

<213> Intelligent people

<220>

<221> sources

<222> (1)..(1797)

<223> LILRB2

<400> 146

atgaccccca tcgtcacagt cctgatctgt ctcgggctga gtctgggccc caggacccgc 60

gtgcagacag ggaccatccc caagcccacc ctgtgggctg agccagactc tgtgatcacc 120

caggggagtc ccgtcaccct cagttgtcag gggagccttg aagcccagga gtaccgtcta 180

tatagggaga aaaaatcagc atcttggatt acacggatac gaccagagct tgtgaagaac 240

ggccagttcc acatcccatc catcacctgg gaacacacag ggcgatatgg ctgtcagtat 300

tacagccgcg ctcggtggtc tgagctcagt gaccccctgg tgctggtgat gacaggagcc 360

tacccaaaac ccaccctctc agcccagccc agccctgtgg tgacctcagg aggaagggtg 420

accctccagt gtgagtcaca ggtggcattt ggcggcttca ttctgtgtaa ggaaggagaa 480

gatgaacacc cacaatgcct gaactcccag ccccatgccc gtgggtcgtc ccgcgccatc 540

ttctccgtgg gccccgtgag cccgaatcgc aggtggtcgc acaggtgcta tggttatgac 600

ttgaactctc cctatgtgtg gtcttcaccc agtgatctcc tggagctcct ggtcccaggt 660

gtttctaaga agccatcact ctcagtgcag ccgggtcctg tcatggcccc tggggaaagc 720

ctgaccctcc agtgtgtctc tgatgtcggc tatgacagat ttgttctgta caaggagggg 780

gaacgtgacc ttcgccagct ccctggccgg cagccccagg ctgggctctc ccaggccaac 840

ttcaccctgg gccctgtgag ccgctcctac gggggccagt acagatgcta cggtgcacac 900

aacctctcct ctgagtgctc ggcccccagc gaccccctgg acatcctgat cacaggacag 960

atccgtggca cacccttcat ctcagtgcag ccaggcccca cagtggcctc aggagagaac 1020

gtgaccctgc tgtgtcagtc atggcggcag ttccacactt tccttctgac caaggcggga 1080

gcagctgatg ccccactccg tctaagatca atacacgaat atcctaagta ccaggctgaa 1140

ttccccatga gtcctgtgac ctcagcccac gcggggacct acaggtgcta cggctcactc 1200

aactccgacc cctacctgct gtctcacccc agtgagcccc tggagctcgt ggtctcagga 1260

ccctccatgg gttccagccc cccacccacc ggtcccatct ccacacctgc aggccctgag 1320

gaccagcccc tcacccccac tgggtcggat ccccaaagtg gtctgggaag gcacctgggg 1380

gttgtgatcg gcatcttggt ggccgtcgtc ctactgctcc tcctcctcct cctcctcttc 1440

ctcatcctcc gacatcgacg tcagggcaaa cactggacat cgacccagag aaaggctgat 1500

ttccaacatc ctgcaggggc tgtggggcca gagcccacag acagaggcct gcagtggagg 1560

tccagcccag ctgccgacgc ccaggaagaa aacctctatg ctgccgtgaa ggacacacag 1620

cctgaagatg gggtggagat ggacactcgg gctgctgcat ctgaagcccc ccaggatgtg 1680

acctacgccc agctgcacag cttgaccctc agacggaagg caactgagcc tcctccatcc 1740

caggaaaggg aacctccagc tgagcccagc atctacgcca ccctggccat ccactag 1797

<210> 147

<211> 598

<212> PRT

<213> Intelligent people

<220>

<221> sources

<222> (1)..(598)

<223> LILRB2

<400> 147

Met Thr Pro Ile Val Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly

1 5 10 15

Pro Arg Thr His Val Gln Thr Gly Thr Ile Pro Lys Pro Thr Leu Trp

20 25 30

Ala Glu Pro Asp Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu Ser

35 40 45

Cys Gln Gly Ser Leu Glu Ala Gln Glu Tyr Arg Leu Tyr Arg Glu Lys

50 55 60

Lys Ser Ala Ser Trp Ile Thr Arg Ile Arg Pro Glu Leu Val Lys Asn

65 70 75 80

Gly Gln Phe His Ile Pro Ser Ile Thr Trp Glu His Thr Gly Arg Tyr

85 90 95

Gly Cys Gln Tyr Tyr Ser Arg Ala Arg Trp Ser Glu Leu Ser Asp Pro

100 105 110

Leu Val Leu Val Met Thr Gly Ala Tyr Pro Lys Pro Thr Leu Ser Ala

115 120 125

Gln Pro Ser Pro Val Val Thr Ser Gly Gly Arg Val Thr Leu Gln Cys

130 135 140

Glu Ser Gln Val Ala Phe Gly Gly Phe Ile Leu Cys Lys Glu Gly Glu

145 150 155 160

Glu Glu His Pro Gln Cys Leu Asn Ser Gln Pro His Ala Arg Gly Ser

165 170 175

Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Asn Arg Arg Trp

180 185 190

Ser His Arg Cys Tyr Gly Tyr Asp Leu Asn Ser Pro Tyr Val Trp Ser

195 200 205

Ser Pro Ser Asp Leu Leu Glu Leu Leu Val Pro Gly Val Ser Lys Lys

210 215 220

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

225 230 235 240

Leu Thr Leu Gln Cys Val Ser Asp Val Gly Tyr Asp Arg Phe Val Leu

245 250 255

Tyr Lys Glu Gly Glu Arg Asp Leu Arg Gln Leu Pro Gly Arg Gln Pro

260 265 270

Gln Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser Arg

275 280 285

Ser Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Leu Ser Ser

290 295 300

Glu Cys Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Thr Gly Gln

305 310 315 320

Ile Arg Gly Thr Pro Phe Ile Ser Val Gln Pro Gly Pro Thr Val Ala

325 330 335

Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Trp Arg Gln Phe His

340 345 350

Thr Phe Leu Leu Thr Lys Ala Gly Ala Ala Asp Ala Pro Leu Arg Leu

355 360 365

Arg Ser Ile His Glu Tyr Pro Lys Tyr Gln Ala Glu Phe Pro Met Ser

370 375 380

Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser Leu

385 390 395 400

Asn Ser Asp Pro Tyr Leu Leu Ser His Pro Ser Glu Pro Leu Glu Leu

405 410 415

Val Val Ser Gly Pro Ser Met Gly Ser Ser Pro Pro Pro Thr Gly Pro

420 425 430

Ile Ser Thr Pro Ala Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr Gly

435 440 445

Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val Val Ile Gly

450 455 460

Ile Leu Val Ala Val Val Leu Leu Leu Leu Leu Leu Leu Leu Leu Phe

465 470 475 480

Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln

485 490 495

Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro

500 505 510

Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln

515 520 525

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

530 535 540

Val Glu Met Asp Thr Arg Ala Ala Ala Ser Glu Ala Pro Gln Asp Val

545 550 555 560

Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Lys Ala Thr Glu

565 570 575

Pro Pro Pro Ser Gln Glu Arg Glu Pro Pro Ala Glu Pro Ser Ile Tyr

580 585 590

Ala Thr Leu Ala Ile His

595

<210> 148

<211> 440

<212> PRT

<213> Intelligent people

<220>

<221> Source

<222> (1)..(440)

<223> extracellular domain of LILRB2

<400> 148

Gln Thr Gly Thr Ile Pro Lys Pro Thr Leu Trp Ala Glu Pro Asp Ser

1 5 10 15

Val Ile Thr Gln Gly Ser Pro Val Thr Leu Ser Cys Gln Gly Ser Leu

20 25 30

Glu Ala Gln Glu Tyr Arg Leu Tyr Arg Glu Lys Lys Ser Ala Ser Trp

35 40 45

Ile Thr Arg Ile Arg Pro Glu Leu Val Lys Asn Gly Gln Phe His Ile

50 55 60

Pro Ser Ile Thr Trp Glu His Thr Gly Arg Tyr Gly Cys Gln Tyr Tyr

65 70 75 80

Ser Arg Ala Arg Trp Ser Glu Leu Ser Asp Pro Leu Val Leu Val Met

85 90 95

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

100 105 110

Val Thr Ser Gly Gly Arg Val Thr Leu Gln Cys Glu Ser Gln Val Ala

115 120 125

Phe Gly Gly Phe Ile Leu Cys Lys Glu Gly Glu Glu Glu His Pro Gln

130 135 140

Cys Leu Asn Ser Gln Pro His Ala Arg Gly Ser Ser Arg Ala Ile Phe

145 150 155 160

Ser Val Gly Pro Val Ser Pro Asn Arg Arg Trp Ser His Arg Cys Tyr

165 170 175

Gly Tyr Asp Leu Asn Ser Pro Tyr Val Trp Ser Ser Pro Ser Asp Leu

180 185 190

Leu Glu Leu Leu Val Pro Gly Val Ser Lys Lys Pro Ser Leu Ser Val

195 200 205

Gln Pro Gly Pro Val Val Ala Pro Gly Glu Ser Leu Thr Leu Gln Cys

210 215 220

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

225 230 235 240

Arg Asp Leu Arg Gln Leu Pro Gly Arg Gln Pro Gln Ala Gly Leu Ser

245 250 255

Gln Ala Asn Phe Thr Leu Gly Pro Val Ser Arg Ser Tyr Gly Gly Gln

260 265 270

Tyr Arg Cys Tyr Gly Ala His Asn Leu Ser Ser Glu Cys Ser Ala Pro

275 280 285

Ser Asp Pro Leu Asp Ile Leu Ile Thr Gly Gln Ile Arg Gly Thr Pro

290 295 300

Phe Ile Ser Val Gln Pro Gly Pro Thr Val Ala Ser Gly Glu Asn Val

305 310 315 320

Thr Leu Leu Cys Gln Ser Trp Arg Gln Phe His Thr Phe Leu Leu Thr

325 330 335

Lys Ala Gly Ala Ala Asp Ala Pro Leu Arg Leu Arg Ser Ile His Glu

340 345 350

Tyr Pro Lys Tyr Gln Ala Glu Phe Pro Met Ser Pro Val Thr Ser Ala

355 360 365

His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser Leu Asn Ser Asp Pro Tyr

370 375 380

Leu Leu Ser His Pro Ser Glu Pro Leu Glu Leu Val Val Ser Gly Pro

385 390 395 400

Ser Met Gly Ser Ser Pro Pro Pro Thr Gly Pro Ile Ser Thr Pro Ala

405 410 415

Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr Gly Ser Asp Pro Gln Ser

420 425 430

Gly Leu Gly Arg His Leu Gly Val

435 440

<210> 149

<211> 1896

<212> DNA

<213> Intelligent people

<220>

<221> Source

<222> (1)..(1896)

<223> cDNA of LILRB3

<400> 149

atgacgcccg ccctcacagc cctgctctgc cttgggctga gtctgggccc caggacccgc 60

atgcaggcag ggcccttccc caaacccacc ctctgggctg agccaggctc tgtgatcagc 120

tgggggagcc ccgtgaccat ctggtgtcag gggagcctgg aggcccagga gtaccaactg 180

gataaagagg gaagcccaga gccctgggac agaaataacc cactggaacc caagaacaag 240

gccagattct ccatcccatc catgacacag caccatgcag ggagataccg ctgccactat 300

tacagctctg caggctggtc agagcccagc gaccccctgg agctggtgat gacaggattc 360

tacaacaaac ccaccctctc agccctgccc agccctgtgg tggcctcagg ggggaatatg 420

accctccgat gtggctcaca gaagggatat caccattttg ttctgatgaa ggaaggagaa 480

caccagctcc cccggaccct ggactcacag cagctccaca gtggggggtt ccaggccctg 540

ttccctgtgg gccccgtgac ccccagccac aggtggaggt tcacatgcta ttactattat 600

acaaacaccc cctgggtgtg gtcccacccc agtgaccccc tggagattct gccctcaggc 660

gtgtctagga agccctccct cctgaccctg cagggccctg tcctggcccc tgggcagagc 720

ctgaccctcc agtgtggctc tgatgtcggc tacgacagat ttgttctgta taaggagggg 780

gaacgtgact tcctccagcg ccctggccag cagccccagg ctgggctctc ccaggccaac 840

ttcaccctgg gccctgtgag ccgctcctac gggggccagt acaggtgcta tggtgcacac 900

aacctctcct ccgagtggtc ggcccccagt gaccccctgg acatcctgat cacaggacag 960

atctatgaca ccgtctccct gtcagcacag ccgggcccca cagtggcctc aggagagaac 1020

atgaccctgc tgtgtcagtc acgggggtat tttgacactt tccttctgac caaagaaggg 1080

gcagcccatc ccccactgcg tctgagatca atgtacggag ctcataagta ccaggctgaa 1140

ttccccatga gtcctgtgac ctcagcccac gcggggacct acaggtgcta cggctcacgc 1200

agctccaacc cccacctgct gtctttcccc agtgagcccc tggaactcat ggtctcagga 1260

cactctggag gctccagcct cccacccaca gggccgccct ccacacctgg tctgggaaga 1320

tacctggagg ttttgattgg ggtctcggtg gccttcgtcc tgctgctctt cctcctcctc 1380

ttcctcctcc tcctccgtca gcgtcacagc aaacacagga catctgacca gagaaagact 1440

gatttccagc gtcctgcagg ggctgcggag acagagccca aggacagggg cctgctgagg 1500

aggtccagcc cagctgctga cgtccaggaa gaaaacctct atgctgctgt gaaggacaca 1560

cagtctgagg acagggtgga gctggacagt cagagcccac acgatgaaga cccccaggca 1620

gtgacgtatg ccccggtgaa acactccagt cctaggagag aaatggcctc tcctccctcc 1680

tcactgtctg gggaattcct ggacacaaag gacagacagg tggaagagga caggcagatg 1740

gacactgagg ctgctgcatc tgaagcctcc caggatgtga cctacgccca gctgcacagc 1800

ttgaccctta gacggaaggc aactgagcct cctccatccc aggaagggga acctccagct 1860

gagcccagca tctacgccac tctggccatc cactag 1896

<210> 150

<211> 631

<212> PRT

<213> Intelligent

<220>

<221> Source

<222> (1)..(631)

<223> LILRB3

<400> 150

Met Thr Pro Ala Leu Thr Ala Leu Leu Cys Leu Gly Leu Ser Leu Gly

1 5 10 15

Pro Arg Thr Arg Val Gln Ala Gly Pro Phe Pro Lys Pro Thr Leu Trp

20 25 30

Ala Glu Pro Gly Ser Val Ile Ser Trp Gly Ser Pro Val Thr Ile Trp

35 40 45

Cys Gln Gly Ser Gln Glu Ala Gln Glu Tyr Arg Leu His Lys Glu Gly

50 55 60

Ser Pro Glu Pro Leu Asp Arg Asn Asn Pro Leu Glu Pro Lys Asn Lys

65 70 75 80

Ala Arg Phe Ser Ile Pro Ser Met Thr Glu His His Ala Gly Arg Tyr

85 90 95

Arg Cys His Tyr Tyr Ser Ser Ala Gly Trp Ser Glu Pro Ser Asp Pro

100 105 110

Leu Glu Met Val Met Thr Gly Ala Tyr Ser Lys Pro Thr Leu Ser Ala

115 120 125

Leu Pro Ser Pro Val Val Ala Ser Gly Gly Asn Met Thr Leu Arg Cys

130 135 140

Gly Ser Gln Lys Gly Tyr His His Phe Val Leu Met Lys Glu Gly Glu

145 150 155 160

His Gln Leu Pro Arg Thr Leu Asp Ser Gln Gln Leu His Ser Arg Gly

165 170 175

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

180 185 190

Arg Phe Thr Cys Tyr Tyr Tyr Tyr Thr Asn Thr Pro Trp Val Trp Ser

195 200 205

His Pro Ser Asp Pro Leu Glu Ile Leu Pro Ser Gly Val Ser Arg Lys

210 215 220

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

225 230 235 240

Leu Thr Leu Gln Cys Gly Ser Asp Val Gly Tyr Asn Arg Phe Val Leu

245 250 255

Tyr Lys Glu Gly Glu Arg Asp Phe Leu Gln Arg Pro Gly Gln Gln Pro

260 265 270

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

275 280 285

Ser Asn Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Leu Ser Ser

290 295 300

Glu Trp Ser Ala Pro Ser Asp Pro Leu Asn Ile Leu Met Ala Gly Gln

305 310 315 320

Ile Tyr Asp Thr Val Ser Leu Ser Ala Gln Pro Gly Pro Thr Val Ala

325 330 335

Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Trp Trp Gln Phe Asp

340 345 350

Thr Phe Leu Leu Thr Lys Glu Gly Ala Ala His Pro Pro Leu Arg Leu

355 360 365

Arg Ser Met Tyr Gly Ala His Lys Tyr Gln Ala Glu Phe Pro Met Ser

370 375 380

Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser Tyr

385 390 395 400

Ser Ser Asn Pro His Leu Leu Ser His Pro Ser Glu Pro Leu Glu Leu

405 410 415

Val Val Ser Gly His Ser Gly Gly Ser Ser Leu Pro Pro Thr Gly Pro

420 425 430

Pro Ser Thr Pro Gly Leu Gly Arg Tyr Leu Glu Val Leu Ile Gly Val

435 440 445

Ser Val Ala Phe Val Leu Leu Leu Phe Leu Leu Leu Phe Leu Leu Leu

450 455 460

Arg Arg Gln Arg His Ser Lys His Arg Thr Ser Asp Gln Arg Lys Thr

465 470 475 480

Asp Phe Gln Arg Pro Ala Gly Ala Ala Glu Thr Glu Pro Lys Asp Arg

485 490 495

Gly Leu Leu Arg Arg Ser Ser Pro Ala Ala Asp Val Gln Glu Glu Asn

500 505 510

Leu Tyr Ala Ala Val Lys Asp Thr Gln Ser Glu Asp Arg Val Glu Leu

515 520 525

Asp Ser Gln Ser Pro His Asp Glu Asp Pro Gln Ala Val Thr Tyr Ala

530 535 540

Pro Val Lys His Ser Ser Pro Arg Arg Glu Met Ala Ser Pro Pro Ser

545 550 555 560

Ser Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln Val Glu Glu

565 570 575

Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala Ser Gln Asp

580 585 590

Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Lys Ala Thr

595 600 605

Glu Pro Pro Pro Ser Gln Glu Gly Glu Pro Pro Ala Glu Pro Ser Ile

610 615 620

Tyr Ala Thr Leu Ala Ile His

625 630

<210> 151

<211> 420

<212> PRT

<213> Intelligent people

<220>

<221> sources

<222> (1)..(420)

<223> extracellular domain of LILRB3

<400> 151

Gly Pro Phe Pro Lys Pro Thr Leu Trp Ala Glu Pro Gly Ser Val Ile

1 5 10 15

Ser Trp Gly Ser Pro Val Thr Ile Trp Cys Gln Gly Ser Gln Glu Ala

20 25 30

Gln Glu Tyr Arg Leu His Lys Glu Gly Ser Pro Glu Pro Leu Asp Arg

35 40 45

Asn Asn Pro Leu Glu Pro Lys Asn Lys Ala Arg Phe Ser Ile Pro Ser

50 55 60

Met Thr Glu His His Ala Gly Arg Tyr Arg Cys His Tyr Tyr Ser Ser

65 70 75 80

Ala Gly Trp Ser Glu Pro Ser Asp Pro Leu Glu Met Val Met Thr Gly

85 90 95

Ala Tyr Ser Lys Pro Thr Leu Ser Ala Leu Pro Ser Pro Val Val Ala

100 105 110

Ser Gly Gly Asn Met Thr Leu Arg Cys Gly Ser Gln Lys Gly Tyr His

115 120 125

His Phe Val Leu Met Lys Glu Gly Glu His Gln Leu Pro Arg Thr Leu

130 135 140

Asp Ser Gln Gln Leu His Ser Arg Gly Phe Gln Ala Leu Phe Pro Val

145 150 155 160

Gly Pro Val Thr Pro Ser His Arg Trp Arg Phe Thr Cys Tyr Tyr Tyr

165 170 175

Tyr Thr Asn Thr Pro Trp Val Trp Ser His Pro Ser Asp Pro Leu Glu

180 185 190

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

195 200 205

Gly Pro Val Leu Ala Pro Gly Gln Ser Leu Thr Leu Gln Cys Gly Ser

210 215 220

Asp Val Gly Tyr Asn Arg Phe Val Leu Tyr Lys Glu Gly Glu Arg Asp

225 230 235 240

Phe Leu Gln Arg Pro Gly Gln Gln Pro Gln Ala Gly Leu Ser Gln Ala

245 250 255

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

260 265 270

Cys Tyr Gly Ala His Asn Leu Ser Ser Glu Trp Ser Ala Pro Ser Asp

275 280 285

Pro Leu Asn Ile Leu Met Ala Gly Gln Ile Tyr Asp Thr Val Ser Leu

290 295 300

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

305 310 315 320

Leu Cys Gln Ser Trp Trp Gln Phe Asp Thr Phe Leu Leu Thr Lys Glu

325 330 335

Gly Ala Ala His Pro Pro Leu Arg Leu Arg Ser Met Tyr Gly Ala His

340 345 350

Lys Tyr Gln Ala Glu Phe Pro Met Ser Pro Val Thr Ser Ala His Ala

355 360 365

Gly Thr Tyr Arg Cys Tyr Gly Ser Tyr Ser Ser Asn Pro His Leu Leu

370 375 380

Ser His Pro Ser Glu Pro Leu Glu Leu Val Val Ser Gly His Ser Gly

385 390 395 400

Gly Ser Ser Leu Pro Pro Thr Gly Pro Pro Ser Thr Pro Gly Leu Gly

405 410 415

Arg Tyr Leu Glu

420

<210> 152

<211> 1347

<212> DNA

<213> Intelligent

<220>

<221> sources

<222> (1)..(1347)

<223> cDNA of LILRB4

<400> 152

atgatcccca ccttcacggc tctgctctgc ctcgggctga gtctgggccc caggacccac 60

atgcaggcag ggcccctccc caaacccacc ctctgggctg agccaggctc tgtgatcagc 120

tgggggaact ctgtgaccat ctggtgtcag gggaccctgg aggctcggga gtaccgtctg 180

gataaagagg aaagcccagc accctgggac agacagaacc cactggagcc caagaacaag 240

gccagattct ccatcccatc catgacagag gactatgcag ggagataccg ctgttactat 300

cgcagccctg taggctggtc acagcccagt gaccccctgg agctggtgat gacaggagcc 360

tacagtaaac ccaccctttc agccctgccg agtcctcttg tgacctcagg aaagagcgtg 420

accctgctgt gtcagtcacg gagcccaatg gacacttttc ttctgatcaa ggagcgggca 480

gcccatcccc tactgcatct gagatcagag cacggagctc agcagcacca ggctgaattc 540

cccatgagtc ctgtgacctc agtgcacggg gggacctaca ggtgcttcag ctcacacggc 600

ttctcccact acctgctgtc acaccccagt gaccccctgg agctcatagt ctcaggatcc 660

ttggagggtc ccaggccctc acccacaagg tccgtctcaa cagctgcagg ccctgaggac 720

cagcccctca tgcctacagg gtcagtcccc cacagtggtc tgagaaggca ctgggaggta 780

ctgatcgggg tcttggtggt ctccatcctg cttctctccc tcctcctctt cctcctcctc 840

caacactggc gtcagggaaa acacaggaca ttggcccaga gacaggctga tttccaacgt 900

cctccagggg ctgccgagcc agagcccaag gacgggggcc tacagaggag gtccagccca 960

gctgctgacg tccagggaga aaacttctgt gctgccgtga agaacacaca gcctgaggac 1020

ggggtggaaa tggacactcg gcagagccca cacgatgaag acccccaggc agtgacgtat 1080

gccaaggtga aacactccag acctaggaga gaaatggcct ctcctccctc cccactgtct 1140

ggggaattcc tggacacaaa ggacagacag gcagaagagg acagacagat ggacactgag 1200

gctgctgcat ctgaagcccc ccaggatgtg acctacgccc ggctgcacag ctttaccctc 1260

agacagaagg caactgagcc tcctccatcc caggaagggg cctctccagc tgagcccagt 1320

gtctatgcca ctctggccat ccactaa 1347

<210> 153

<211> 448

<212> PRT

<213> Intelligent people

<220>

<221> sources

<222> (1)..(448)

<223> LILRB4

<400> 153

Met Ile Pro Thr Phe Thr Ala Leu Leu Cys Leu Gly Leu Ser Leu Gly

1 5 10 15

Pro Arg Thr His Met Gln Ala Gly Pro Leu Pro Lys Pro Thr Leu Trp

20 25 30

Ala Glu Pro Gly Ser Val Ile Ser Trp Gly Asn Ser Val Thr Ile Trp

35 40 45

Cys Gln Gly Thr Leu Glu Ala Arg Glu Tyr Arg Leu Asp Lys Glu Glu

50 55 60

Ser Pro Ala Pro Trp Asp Arg Gln Asn Pro Leu Glu Pro Lys Asn Lys

65 70 75 80

Ala Arg Phe Ser Ile Pro Ser Met Thr Glu Asp Tyr Ala Gly Arg Tyr

85 90 95

Arg Cys Tyr Tyr Arg Ser Pro Val Gly Trp Ser Gln Pro Ser Asp Pro

100 105 110

Leu Glu Leu Val Met Thr Gly Ala Tyr Ser Lys Pro Thr Leu Ser Ala

115 120 125

Leu Pro Ser Pro Leu Val Thr Ser Gly Lys Ser Val Thr Leu Leu Cys

130 135 140

Gln Ser Arg Ser Pro Met Asp Thr Phe Leu Leu Ile Lys Glu Arg Ala

145 150 155 160

Ala His Pro Leu Leu His Leu Arg Ser Glu His Gly Ala Gln Gln His

165 170 175

Gln Ala Glu Phe Pro Met Ser Pro Val Thr Ser Val His Gly Gly Thr

180 185 190

Tyr Arg Cys Phe Ser Ser His Gly Phe Ser His Tyr Leu Leu Ser His

195 200 205

Pro Ser Asp Pro Leu Glu Leu Ile Val Ser Gly Ser Leu Glu Asp Pro

210 215 220

Arg Pro Ser Pro Thr Arg Ser Val Ser Thr Ala Ala Gly Pro Glu Asp

225 230 235 240

Gln Pro Leu Met Pro Thr Gly Ser Val Pro His Ser Gly Leu Arg Arg

245 250 255

His Trp Glu Val Leu Ile Gly Val Leu Val Val Ser Ile Leu Leu Leu

260 265 270

Ser Leu Leu Leu Phe Leu Leu Leu Gln His Trp Arg Gln Gly Lys His

275 280 285

Arg Thr Leu Ala Gln Arg Gln Ala Asp Phe Gln Arg Pro Pro Gly Ala

290 295 300

Ala Glu Pro Glu Pro Lys Asp Gly Gly Leu Gln Arg Arg Ser Ser Pro

305 310 315 320

Ala Ala Asp Val Gln Gly Glu Asn Phe Cys Ala Ala Val Lys Asn Thr

325 330 335

Gln Pro Glu Asp Gly Val Glu Met Asp Thr Arg Gln Ser Pro His Asp

340 345 350

Glu Asp Pro Gln Ala Val Thr Tyr Ala Lys Val Lys His Ser Arg Pro

355 360 365

Arg Arg Glu Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu

370 375 380

Asp Thr Lys Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu

385 390 395 400

Ala Ala Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His

405 410 415

Ser Phe Thr Leu Arg Gln Lys Ala Thr Glu Pro Pro Pro Ser Gln Glu

420 425 430

Gly Ala Ser Pro Ala Glu Pro Ser Val Tyr Ala Thr Leu Ala Ile His

435 440 445

<210> 154

<211> 238

<212> PRT

<213> Intelligent people

<220>

<221> Source

<222> (1)..(238)

<223> extracellular domain of LILRB4

<400> 154

Gln Ala Gly Pro Leu Pro Lys Pro Thr Leu Trp Ala Glu Pro Gly Ser

1 5 10 15

Val Ile Ser Trp Gly Asn Ser Val Thr Ile Trp Cys Gln Gly Thr Leu

20 25 30

Glu Ala Arg Glu Tyr Arg Leu Asp Lys Glu Glu Ser Pro Ala Pro Trp

35 40 45

Asp Arg Gln Asn Pro Leu Glu Pro Lys Asn Lys Ala Arg Phe Ser Ile

50 55 60

Pro Ser Met Thr Glu Asp Tyr Ala Gly Arg Tyr Arg Cys Tyr Tyr Arg

65 70 75 80

Ser Pro Val Gly Trp Ser Gln Pro Ser Asp Pro Leu Glu Leu Val Met

85 90 95

Thr Gly Ala Tyr Ser Lys Pro Thr Leu Ser Ala Leu Pro Ser Pro Leu

100 105 110

Val Thr Ser Gly Lys Ser Val Thr Leu Leu Cys Gln Ser Arg Ser Pro

115 120 125

Met Asp Thr Phe Leu Leu Ile Lys Glu Arg Ala Ala His Pro Leu Leu

130 135 140

His Leu Arg Ser Glu His Gly Ala Gln Gln His Gln Ala Glu Phe Pro

145 150 155 160

Met Ser Pro Val Thr Ser Val His Gly Gly Thr Tyr Arg Cys Phe Ser

165 170 175

Ser His Gly Phe Ser His Tyr Leu Leu Ser His Pro Ser Asp Pro Leu

180 185 190

Glu Leu Ile Val Ser Gly Ser Leu Glu Asp Pro Arg Pro Ser Pro Thr

195 200 205

Arg Ser Val Ser Thr Ala Ala Gly Pro Glu Asp Gln Pro Leu Met Pro

210 215 220

Thr Gly Ser Val Pro His Ser Gly Leu Arg Arg His Trp Glu

225 230 235

<210> 155

<211> 1773

<212> DNA

<213> Intelligent people

<220>

<221> sources

<222> (1)..(1773)

<223> cDNA of LILRB5

<400> 155

atgaccctca ccctctcagt cctgatttgc ctcgggctga gtgtgggccc caggacctgc 60

gtgcaggcag gcaccctccc caaacccacc ctctgggctg agccagcctc tgtgatagct 120

cgggggaagc ccgtgaccct ctggtgtcag gggcccctgg agactgagga gtaccgtctg 180

gataaggagg gactcccatg ggcccggaag agacagaacc cactggagcc tggagccaag 240

gccaagttcc acattccatc cacggtgtat gacagtgcag ggcgataccg ctgctactat 300

gagacccctg caggctggtc agagcccagt gaccccctgg agctggtggc gacaggattc 360

tatgcagaac ccactctttt agccctgccg agtcctgtgg tggcctcagg aggaaatgtg 420

accctccagt gtgatacact ggacggactt ctcacgtttg ttcttgttga ggaagaacag 480

aagctcccca ggaccctgta ctcacagaag ctccccaaag ggccatccca ggccctgttc 540

cctgtgggtc ccgtgacccc cagctgcagg tggaggttca gatgctatta ctattacagg 600

aaaaaccctc aggtgtggtc gaaccccagt gacctcctgg agattctggt cccaggcgtg 660

tctaggaagc cctccctcct gatcccgcag ggctctgtcg tggcccgcgg aggcagcctg 720

accctgcagt gtcgctctga tgtcggctat gacatattcg ttctgtacaa ggagggggaa 780

catgacctcg tccagggctc tggccagcag ccccaggctg ggctctccca ggccaacttc 840

accctgggcc ctgtgagccg ctcccacggg ggccagtaca gatgctacgg tgcacacaac 900

ctctccccta ggtggtcggc ccccagcgac cccctggaca tcctgatcgc aggactgatc 960

cctgacatac ccgccctctc ggtgcagccg ggccccaagg tggcctcagg agagaacgtg 1020

accctgctgt gtcagtcatg gcatcagata gacactttct ttttgaccaa ggagggggca 1080

gcccatcccc cgctgtgtct aaagtcaaag taccagtctt atagacacca ggctgaattc 1140

tccatgagtc ctgtgacctc agcccagggt ggaacctacc gatgctacag cgcaatcagg 1200

tcctacccct acctgctgtc cagccctagt tacccccagg agctcgtggt ctcaggaccc 1260

tctggggatc ccagcctctc acctacaggc tccaccccca cacctggccc tgaggaccag 1320

cccctcaccc ccacggggtt ggatccccag agtggtctgg gaaggcacct gggggttgtg 1380

actggggtct cagtggcctt cgtcctgctg ctgttcctcc tcctcttcct cctcctccga 1440

catcggcatc agagcaaaca caggacatcg gcccatttct accgtcctgc aggggctgcg 1500

gggccagagc ccaaggacca gggcctgcag aagagggcca gcccagttgc tgacatccag 1560

gaggaaattc tcaatgctgc cgtgaaggac acacagccca aggacggggt ggagatggat 1620

gctccggctg ctgcatctga agccccccag gatgtgacct acgcccagct gcacagcttg 1680

accctcagac gggaggcaac tgagcctcct ccatcccagg aaagggaacc tccagctgaa 1740

cccagcatct acgcccccct ggccatccac tag 1773

<210> 156

<211> 590

<212> PRT

<213> Intelligent people

<220>

<221> Source

<222> (1)..(590)

<223> LILRB5

<400> 156

Met Thr Leu Thr Leu Ser Val Leu Ile Cys Leu Gly Leu Ser Val Gly

1 5 10 15

Pro Arg Thr Cys Val Gln Ala Gly Thr Leu Pro Lys Pro Thr Leu Trp

20 25 30

Ala Glu Pro Ala Ser Val Ile Ala Arg Gly Lys Pro Val Thr Leu Trp

35 40 45

Cys Gln Gly Pro Leu Glu Thr Glu Glu Tyr Arg Leu Asp Lys Glu Gly

50 55 60

Leu Pro Trp Ala Arg Lys Arg Gln Asn Pro Leu Glu Pro Gly Ala Lys

65 70 75 80

Ala Lys Phe His Ile Pro Ser Thr Val Tyr Asp Ser Ala Gly Arg Tyr

85 90 95

Arg Cys Tyr Tyr Glu Thr Pro Ala Gly Trp Ser Glu Pro Ser Asp Pro

100 105 110

Leu Glu Leu Val Ala Thr Gly Phe Tyr Ala Glu Pro Thr Leu Leu Ala

115 120 125

Leu Pro Ser Pro Val Val Ala Ser Gly Gly Asn Val Thr Leu Gln Cys

130 135 140

Asp Thr Leu Asp Gly Leu Leu Thr Phe Val Leu Val Glu Glu Glu Gln

145 150 155 160

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

165 170 175

Gln Ala Leu Phe Pro Val Gly Pro Val Thr Pro Ser Cys Arg Trp Arg

180 185 190

Phe Arg Cys Tyr Tyr Tyr Tyr Arg Lys Asn Pro Gln Val Trp Ser Asn

195 200 205

Pro Ser Asp Leu Leu Glu Ile Leu Val Pro Gly Val Ser Arg Lys Pro

210 215 220

Ser Leu Leu Ile Pro Gln Gly Ser Val Val Ala Arg Gly Gly Ser Leu

225 230 235 240

Thr Leu Gln Cys Arg Ser Asp Val Gly Tyr Asp Ile Phe Val Leu Tyr

245 250 255

Lys Glu Gly Glu His Asp Leu Val Gln Gly Ser Gly Gln Gln Pro Gln

260 265 270

Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser Arg Ser

275 280 285

His Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Leu Ser Pro Arg

290 295 300

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

305 310 315 320

Pro Asp Ile Pro Ala Leu Ser Val Gln Pro Gly Pro Lys Val Ala Ser

325 330 335

Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Trp His Gln Ile Asp Thr

340 345 350

Phe Phe Leu Thr Lys Glu Gly Ala Ala His Pro Pro Leu Cys Leu Lys

355 360 365

Ser Lys Tyr Gln Ser Tyr Arg His Gln Ala Glu Phe Ser Met Ser Pro

370 375 380

Val Thr Ser Ala Gln Gly Gly Thr Tyr Arg Cys Tyr Ser Ala Ile Arg

385 390 395 400

Ser Tyr Pro Tyr Leu Leu Ser Ser Pro Ser Tyr Pro Gln Glu Leu Val

405 410 415

Val Ser Gly Pro Ser Gly Asp Pro Ser Leu Ser Pro Thr Gly Ser Thr

420 425 430

Pro Thr Pro Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr Gly Leu Asp

435 440 445

Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val Val Thr Gly Val Ser

450 455 460

Val Ala Phe Val Leu Leu Leu Phe Leu Leu Leu Phe Leu Leu Leu Arg

465 470 475 480

His Arg His Gln Ser Lys His Arg Thr Ser Ala His Phe Tyr Arg Pro

485 490 495

Ala Gly Ala Ala Gly Pro Glu Pro Lys Asp Gln Gly Leu Gln Lys Arg

500 505 510

Ala Ser Pro Val Ala Asp Ile Gln Glu Glu Ile Leu Asn Ala Ala Val

515 520 525

Lys Asp Thr Gln Pro Lys Asp Gly Val Glu Met Asp Ala Arg Ala Ala

530 535 540

Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu

545 550 555 560

Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Arg Glu

565 570 575

Pro Pro Ala Glu Pro Ser Ile Tyr Ala Pro Leu Ala Ile His

580 585 590

<210> 157

<211> 435

<212> PRT

<213> Intelligent

<220>

<221> sources

<222> (1)..(435)

<223> extracellular domain of LILRB5

<400> 157

Gly Thr Leu Pro Lys Pro Thr Leu Trp Ala Glu Pro Ala Ser Val Ile

1 5 10 15

Ala Arg Gly Lys Pro Val Thr Leu Trp Cys Gln Gly Pro Leu Glu Thr

20 25 30

Glu Glu Tyr Arg Leu Asp Lys Glu Gly Leu Pro Trp Ala Arg Lys Arg

35 40 45

Gln Asn Pro Leu Glu Pro Gly Ala Lys Ala Lys Phe His Ile Pro Ser

50 55 60

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

65 70 75 80

Ala Gly Trp Ser Glu Pro Ser Asp Pro Leu Glu Leu Val Ala Thr Gly

85 90 95

Phe Tyr Ala Glu Pro Thr Leu Leu Ala Leu Pro Ser Pro Val Val Ala

100 105 110

Ser Gly Gly Asn Val Thr Leu Gln Cys Asp Thr Leu Asp Gly Leu Leu

115 120 125

Thr Phe Val Leu Val Glu Glu Glu Gln Lys Leu Pro Arg Thr Leu Tyr

130 135 140

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

145 150 155 160

Pro Val Thr Pro Ser Cys Arg Trp Arg Phe Arg Cys Tyr Tyr Tyr Tyr

165 170 175

Arg Lys Asn Pro Gln Val Trp Ser Asn Pro Ser Asp Leu Leu Glu Ile

180 185 190

Leu Val Pro Gly Val Ser Arg Lys Pro Ser Leu Leu Ile Pro Gln Gly

195 200 205

Ser Val Val Ala Arg Gly Gly Ser Leu Thr Leu Gln Cys Arg Ser Asp

210 215 220

Val Gly Tyr Asp Ile Phe Val Leu Tyr Lys Glu Gly Glu His Asp Leu

225 230 235 240

Val Gln Gly Ser Gly Gln Gln Pro Gln Ala Gly Leu Ser Gln Ala Asn

245 250 255

Phe Thr Leu Gly Pro Val Ser Arg Ser His Gly Gly Gln Tyr Arg Cys

260 265 270

Tyr Gly Ala His Asn Leu Ser Pro Arg Trp Ser Ala Pro Ser Asp Pro

275 280 285

Leu Asp Ile Leu Ile Ala Gly Leu Ile Pro Asp Ile Pro Ala Leu Ser

290 295 300

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

305 310 315 320

Cys Gln Ser Trp His Gln Ile Asp Thr Phe Phe Leu Thr Lys Glu Gly

325 330 335

Ala Ala His Pro Pro Leu Cys Leu Lys Ser Lys Tyr Gln Ser Tyr Arg

340 345 350

His Gln Ala Glu Phe Ser Met Ser Pro Val Thr Ser Ala Gln Gly Gly

355 360 365

Thr Tyr Arg Cys Tyr Ser Ala Ile Arg Ser Tyr Pro Tyr Leu Leu Ser

370 375 380

Ser Pro Ser Tyr Pro Gln Glu Leu Val Val Ser Gly Pro Ser Gly Asp

385 390 395 400

Pro Ser Leu Ser Pro Thr Gly Ser Thr Pro Thr Pro Gly Pro Glu Asp

405 410 415

Gln Pro Leu Thr Pro Thr Gly Leu Asp Pro Gln Ser Gly Leu Gly Arg

420 425 430

His Leu Gly

435

<210> 158

<211> 2526

<212> DNA

<213> mice

<220>

<221> Source

<222> (1)..(2526)

<223> cDNA of PirB

<400> 158

atgtcctgca ccttcacagc cctgctccgt cttggactga ctctgagcct ctggatccca 60

gtgctgacag ggtccctccc taagcctatc ctcagagtac agccagactc tgtggtctcc 120

aggaggacta aggtgacctt cttgtgtgaa gagacaattg gagccaatga gtaccgcctc 180

tataaagatg gaaagctata taaaaccgta acaaagaaca aacagaagcc agaaaacaag 240

gctgaattct cattctcaaa tgtagacctg agtaatgcag gtcaatatcg atgttcctac 300

agcacccagt ataaatcatc aggctacagt gacctcctgg agctggtggt gacaggacac 360

tactggacac ccagcctttt agcccaagcc agccctgtgg taacttcagg agggtatgtc 420

accctccagt gtgagtcctg gcacaacgat cacaagttca ttctgactgt agaaggacca 480

cagaagctct cgtggacaca agactcacag tataattact ctacaaggaa gtaccacgcc 540

ctgttctctg tgggccctgt gacccccaac cagagatgga tatgcagatg ttacagttat 600

gacaggaaca gaccatatgt gtggtcacct ccaagtgaat ccgtggagct cctggtctca 660

ggtaatctcc aaaaaccaac catcaaggct gaaccaggat ctgtgatcac ctccaaaaga 720

gcaatgacca tctggtgtca ggggaacctg gatgcagaag tatattttct gcataatgag 780

aaaagccaaa aaacacagag cacacagacc ctacaggagc ctgggaacaa gggcaagttc 840

ttcatccctt ctgtgacact acaacatgca gggcaatatc gctgttattg ttacggctca 900

gctggttggt cacagcccag tgacaccctg gagctggtgg tgacaggaat ctatgaatac 960

tatgaaccca ggctgtcagt actgcccagc cctgtggtga cagctggagg gaacatgaca 1020

ctccactgtg cctcagactt tccctacgat aaattcattc tcaccaagga agataagaaa 1080

ttcggcaact cactggacac agagcatata tcttctagtg gacagtaccg agccctgttt 1140

attataggac ccacaacccc aacccataca ggggcattca gatgttacgg ttactacaag 1200

aatgccccac agctgtggtc agtacctagt gctctccaac aaatactcat ctcagggctg 1260

tccaagaagc cctctctgct gactcaccaa ggccatatcc tggaccctgg aatgaccctc 1320

accctgcagt gtttctctga catcaactat gacagatttg ctctgcacaa ggtgggggga 1380

gctgacatca tgcagcactc tagccagcag actgacactg gcttctctgt ggccaacttc 1440

acactgggct atgtgagtag ctccactgga ggccaataca gatgctatgg tgcacacaac 1500

ctctcctctg agtggtcagc ctccagtgag cccctggaca tcctgatcac aggacagctc 1560

cctctcactc cttccctctc agtgcagcct aaccacacag tgcactcagg agagaccgtg 1620

agcctgctgt gttggtcaat ggactctgtg gatactttca ttctgtccaa ggagggatca 1680

gcccagcaac ccctgcgact aaaatcaaag tcccatgatc agcagtccca ggcagaattc 1740

tccatgagtg ctgtgacctc ccatctctca ggcacctaca ggtgctatgg agctcaagac 1800

tcatctttct acctcttgtc atctgccagt gcccctgtgg agctcacagt ctcaggaccc 1860

atcgaaacct ctaccccgcc acccacaatg tccatgccac taggtggact gcatatgtac 1920

ctgaaggctc tcattggagt gtctgtggcc ttcatcctgt tcctcttcat cttcatcttc 1980

attcttcttc gacgaagaca tcggggaaaa ttcaggaaag atgtccagaa agagaaagac 2040

ttgcaacttt cttcaggagc tgaagagccc ataaccagga aaggagaact ccagaagagg 2100

cccaacccag ctgctgccac ccaggaagaa agcctatatg cttcagtgga ggacatgcaa 2160

actgaggatg gagtggagct gaacagctgg acaccacctg aggaagatcc ccagggagag 2220

acttatgccc aggtgaaacc ctccaggctc aggaaggcag gacatgtctc accttctgtc 2280

atgtcaaggg aacaactgaa cacagaatat gaacaagcag aagagggcca aggagcaaac 2340

aatcaggctg ccgaatctgg ggagtcccag gatgtgacct atgcccagct gtgcagcagg 2400

acactcagac agggggcagc tgcatctcct ctctcccagg caggggaagc cccagaggag 2460

cccagtgtat atgctactct ggcggctgct cgtccagagg ctgttcccaa ggacatggag 2520

caatga 2526

<210> 159

<211> 841

<212> PRT

<213> mouse

<220>

<221> sources

<222> (1)..(841)

<223> PirB

<400> 159

Met Ser Cys Thr Phe Thr Ala Leu Leu Arg Leu Gly Leu Thr Leu Ser

1 5 10 15

Leu Trp Ile Pro Val Leu Thr Gly Ser Leu Pro Lys Pro Ile Leu Arg

20 25 30

Val Gln Pro Asp Ser Val Val Ser Arg Arg Thr Lys Val Thr Phe Leu

35 40 45

Cys Glu Glu Thr Ile Gly Ala Asn Glu Tyr Arg Leu Tyr Lys Asp Gly

50 55 60

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

65 70 75 80

Ala Glu Phe Ser Phe Ser Asn Val Asp Leu Ser Asn Ala Gly Gln Tyr

85 90 95

Arg Cys Ser Tyr Ser Thr Gln Tyr Lys Ser Ser Gly Tyr Ser Asp Leu

100 105 110

Leu Glu Leu Val Val Thr Gly His Tyr Trp Thr Pro Ser Leu Leu Ala

115 120 125

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

130 135 140

Glu Ser Trp His Asn Asp His Lys Phe Ile Leu Thr Val Glu Gly Pro

145 150 155 160

Gln Lys Leu Ser Trp Thr Gln Asp Ser Gln Tyr Asn Tyr Ser Thr Arg

165 170 175

Lys Tyr His Ala Leu Phe Ser Val Gly Pro Val Thr Pro Asn Gln Arg

180 185 190

Trp Ile Cys Arg Cys Tyr Ser Tyr Asp Arg Asn Arg Pro Tyr Val Trp

195 200 205

Ser Pro Pro Ser Glu Ser Val Glu Leu Leu Val Ser Gly Asn Leu Gln

210 215 220

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

225 230 235 240

Ala Met Thr Ile Trp Cys Gln Gly Asn Leu Asp Ala Glu Val Tyr Phe

245 250 255

Leu His Asn Glu Lys Ser Gln Lys Thr Gln Ser Thr Gln Thr Leu Gln

260 265 270

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

275 280 285

His Ala Gly Gln Tyr Arg Cys Tyr Cys Tyr Gly Ser Ala Gly Trp Ser

290 295 300

Gln Pro Ser Asp Thr Leu Glu Leu Val Val Thr Gly Ile Tyr Glu Tyr

305 310 315 320

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

325 330 335

Gly Asn Met Thr Leu His Cys Ala Ser Asp Phe Pro Tyr Asp Lys Phe

340 345 350

Ile Leu Thr Lys Glu Asp Lys Lys Phe Gly Asn Ser Leu Asp Thr Glu

355 360 365

His Ile Ser Ser Ser Gly Gln Tyr Arg Ala Leu Phe Ile Ile Gly Pro

370 375 380

Thr Thr Pro Thr His Thr Gly Ala Phe Arg Cys Tyr Gly Tyr Tyr Lys

385 390 395 400

Asn Ala Pro Gln Leu Trp Ser Val Pro Ser Ala Leu Gln Gln Ile Leu

405 410 415

Ile Ser Gly Leu Ser Lys Lys Pro Ser Leu Leu Thr His Gln Gly His

420 425 430

Ile Leu Asp Pro Gly Met Thr Leu Thr Leu Gln Cys Phe Ser Asp Ile

435 440 445

Asn Tyr Asp Arg Phe Ala Leu His Lys Val Gly Gly Ala Asp Ile Met

450 455 460

Gln His Ser Ser Gln Gln Thr Asp Thr Gly Phe Ser Val Ala Asn Phe

465 470 475 480

Thr Leu Gly Tyr Val Ser Ser Ser Thr Gly Gly Gln Tyr Arg Cys Tyr

485 490 495

Gly Ala His Asn Leu Ser Ser Glu Trp Ser Ala Ser Ser Glu Pro Leu

500 505 510

Asp Ile Leu Ile Thr Gly Gln Leu Pro Leu Thr Pro Ser Leu Ser Val

515 520 525

Gln Pro Asn His Thr Val His Ser Gly Glu Thr Val Ser Leu Leu Cys

530 535 540

Trp Ser Met Asp Ser Val Asp Thr Phe Ile Leu Ser Lys Glu Gly Ser

545 550 555 560

Ala Gln Gln Pro Leu Arg Leu Lys Ser Lys Ser His Asp Gln Gln Ser

565 570 575

Gln Ala Glu Phe Ser Met Ser Ala Val Thr Ser His Leu Ser Gly Thr

580 585 590

Tyr Arg Cys Tyr Gly Ala Gln Asp Ser Ser Phe Tyr Leu Leu Ser Ser

595 600 605

Ala Ser Ala Pro Val Glu Leu Thr Val Ser Gly Pro Ile Glu Thr Ser

610 615 620

Thr Pro Pro Pro Thr Met Ser Met Pro Leu Gly Gly Leu His Met Tyr

625 630 635 640

Leu Lys Ala Leu Ile Gly Val Ser Val Ala Phe Ile Leu Phe Leu Phe

645 650 655

Ile Phe Ile Phe Ile Leu Leu Arg Arg Arg His Arg Gly Lys Phe Arg

660 665 670

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

675 680 685

Glu Pro Ile Thr Arg Lys Gly Glu Leu Gln Lys Arg Pro Asn Pro Ala

690 695 700

Ala Ala Thr Gln Glu Glu Ser Leu Tyr Ala Ser Val Glu Asp Met Gln

705 710 715 720

Thr Glu Asp Gly Val Glu Leu Asn Ser Trp Thr Pro Pro Glu Glu Asp

725 730 735

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

740 745 750

Ala Gly His Val Ser Pro Ser Val Met Ser Arg Glu Gln Leu Asn Thr

755 760 765

Glu Tyr Glu Gln Ala Glu Glu Gly Gln Gly Ala Asn Asn Gln Ala Ala

770 775 780

Glu Ser Gly Glu Ser Gln Asp Val Thr Tyr Ala Gln Leu Cys Ser Arg

785 790 795 800

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

805 810 815

Ala Pro Glu Glu Pro Ser Val Tyr Ala Thr Leu Ala Ala Ala Arg Pro

820 825 830

Glu Ala Val Pro Lys Asp Met Glu Gln

835 840

<210> 160

<211> 618

<212> PRT

<213> mouse

<220>

<221> Source

<222> (1)..(618)

<223> extracellular domain of PirB

<400> 160

Ser Leu Pro Lys Pro Ile Leu Arg Val Gln Pro Asp Ser Val Val Ser

1 5 10 15

Arg Arg Thr Lys Val Thr Phe Leu Cys Glu Glu Thr Ile Gly Ala Asn

20 25 30

Glu Tyr Arg Leu Tyr Lys Asp Gly Lys Leu Tyr Lys Thr Val Thr Lys

35 40 45

Asn Lys Gln Lys Pro Glu Asn Lys Ala Glu Phe Ser Phe Ser Asn Val

50 55 60

Asp Leu Ser Asn Ala Gly Gln Tyr Arg Cys Ser Tyr Ser Thr Gln Tyr

65 70 75 80

Lys Ser Ser Gly Tyr Ser Asp Leu Leu Glu Leu Val Val Thr Gly His

85 90 95

Tyr Trp Thr Pro Ser Leu Leu Ala Gln Ala Ser Pro Val Val Thr Ser

100 105 110

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

115 120 125

Phe Ile Leu Thr Val Glu Gly Pro Gln Lys Leu Ser Trp Thr Gln Asp

130 135 140

Ser Gln Tyr Asn Tyr Ser Thr Arg Lys Tyr His Ala Leu Phe Ser Val

145 150 155 160

Gly Pro Val Thr Pro Asn Gln Arg Trp Ile Cys Arg Cys Tyr Ser Tyr

165 170 175

Asp Arg Asn Arg Pro Tyr Val Trp Ser Pro Pro Ser Glu Ser Val Glu

180 185 190

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

195 200 205

Gly Ser Val Ile Thr Ser Lys Arg Ala Met Thr Ile Trp Cys Gln Gly

210 215 220

Asn Leu Asp Ala Glu Val Tyr Phe Leu His Asn Glu Lys Ser Gln Lys

225 230 235 240

Thr Gln Ser Thr Gln Thr Leu Gln Glu Pro Gly Asn Lys Gly Lys Phe

245 250 255

Phe Ile Pro Ser Val Thr Leu Gln His Ala Gly Gln Tyr Arg Cys Tyr

260 265 270

Cys Tyr Gly Ser Ala Gly Trp Ser Gln Pro Ser Asp Thr Leu Glu Leu

275 280 285

Val Val Thr Gly Ile Tyr Glu Tyr Tyr Glu Pro Arg Leu Ser Val Leu

290 295 300

Pro Ser Pro Val Val Thr Ala Gly Gly Asn Met Thr Leu His Cys Ala

305 310 315 320

Ser Asp Phe Pro Tyr Asp Lys Phe Ile Leu Thr Lys Glu Asp Lys Lys

325 330 335

Phe Gly Asn Ser Leu Asp Thr Glu His Ile Ser Ser Ser Gly Gln Tyr

340 345 350

Arg Ala Leu Phe Ile Ile Gly Pro Thr Thr Pro Thr His Thr Gly Ala

355 360 365

Phe Arg Cys Tyr Gly Tyr Tyr Lys Asn Ala Pro Gln Leu Trp Ser Val

370 375 380

Pro Ser Ala Leu Gln Gln Ile Leu Ile Ser Gly Leu Ser Lys Lys Pro

385 390 395 400

Ser Leu Leu Thr His Gln Gly His Ile Leu Asp Pro Gly Met Thr Leu

405 410 415

Thr Leu Gln Cys Phe Ser Asp Ile Asn Tyr Asp Arg Phe Ala Leu His

420 425 430

Lys Val Gly Gly Ala Asp Ile Met Gln His Ser Ser Gln Gln Thr Asp

435 440 445

Thr Gly Phe Ser Val Ala Asn Phe Thr Leu Gly Tyr Val Ser Ser Ser

450 455 460

Thr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Leu Ser Ser Glu

465 470 475 480

Trp Ser Ala Ser Ser Glu Pro Leu Asp Ile Leu Ile Thr Gly Gln Leu

485 490 495

Pro Leu Thr Pro Ser Leu Ser Val Gln Pro Asn His Thr Val His Ser

500 505 510

Gly Glu Thr Val Ser Leu Leu Cys Trp Ser Met Asp Ser Val Asp Thr

515 520 525

Phe Ile Leu Ser Lys Glu Gly Ser Ala Gln Gln Pro Leu Arg Leu Lys

530 535 540

Ser Lys Ser His Asp Gln Gln Ser Gln Ala Glu Phe Ser Met Ser Ala

545 550 555 560

Val Thr Ser His Leu Ser Gly Thr Tyr Arg Cys Tyr Gly Ala Gln Asp

565 570 575

Ser Ser Phe Tyr Leu Leu Ser Ser Ala Ser Ala Pro Val Glu Leu Thr

580 585 590

Val Ser Gly Pro Ile Glu Thr Ser Thr Pro Pro Pro Thr Met Ser Met

595 600 605

Pro Leu Gly Gly Leu His Met Tyr Leu Lys

610 615

<210> 161

<211> 1008

<212> DNA

<213> mice

<220>

<221> sources

<222> (1)..(1008)

<223> cDNA for gp49B1

<400> 161

atgatcgcca tgctcacagt gctgctatac cttggtctta ttctggaacc caggactgca 60

gtacaggcag gacacctccc aaagcccatc atctgggctg agccaggctc tgtgatcgct 120

gcgtatacat ctgtgattac ctggtgtcag ggttcctggg aggcccagta ttatcatctg 180

tataaagaga aaagtgtaaa tccttgggac actcaagtcc ctctggaaac caggaataag 240

gccaagttca acattccaag catgacaacc tcatatgcag gcatatataa gtgttactat 300

gagagtgctg ctggcttctc agagcacagt gatgccatgg agctggtgat gacaggagca 360

tatgaaaatc ccagcctgtc agtctatccc agctctaatg tgacctctgg agtttccata 420

tcctttagtt gcagctcatc catagtattt ggcagattca ttctgatcca ggaaggaaag 480

catggcctct cttggaccct ggactcacag catcaggcca atcagccatc ctatgctact 540

tttgttctgg atgctgttac tcccaaccac aatggaacat tcagatgcta tggctacttt 600

agaaatgaac cacaggtgtg gtcaaaacca agtaactccc tagacctcat gatctcagaa 660

accaaggacc agtcctctac acccactgaa gatggactgg aaacatacca gaagattttg 720

attggagtcc tggtgtcatt cctcctgctt ttcttcctcc tgctttttct catcctcatc 780

ggataccagt atgggcacaa aaagaaggct aatgcttctg tgaagaacac acaatctgag 840

aacaatgcag agctgaacag ttggaaccca caaaatgaag acccccaggg aattgtctac 900

gcccaggtaa aaccctccag gcttcagaag gacactgcat gcaaagagac ccaggatgta 960

acctatgccc agttgtgcat caggacacag gaacagaaca acagctga 1008

<210> 162

<211> 335

<212> PRT

<213> mice

<220>

<221> sources

<222> (1)..(335)

<223> gp49B1

<400> 162

Met Ile Ala Met Leu Thr Val Leu Leu Tyr Leu Gly Leu Ile Leu Glu

1 5 10 15

Pro Arg Thr Ala Val Gln Ala Gly His Leu Pro Lys Pro Ile Ile Trp

20 25 30

Ala Glu Pro Gly Ser Val Ile Ala Ala Tyr Thr Ser Val Ile Thr Trp

35 40 45

Cys Gln Gly Ser Trp Glu Ala Gln Tyr Tyr His Leu Tyr Lys Glu Lys

50 55 60

Ser Val Asn Pro Trp Asp Thr Gln Val Pro Leu Glu Thr Arg Asn Lys

65 70 75 80

Ala Lys Phe Asn Ile Pro Ser Met Thr Thr Ser Tyr Ala Gly Ile Tyr

85 90 95

Lys Cys Tyr Tyr Glu Ser Ala Ala Gly Phe Ser Glu His Ser Asp Ala

100 105 110

Met Glu Leu Val Met Thr Gly Ala Tyr Glu Asn Pro Ser Leu Ser Val

115 120 125

Tyr Pro Ser Ser Asn Val Thr Ser Gly Val Ser Ile Ser Phe Ser Cys

130 135 140

Ser Ser Ser Ile Val Phe Gly Arg Phe Ile Leu Ile Gln Glu Gly Lys

145 150 155 160

His Gly Leu Ser Trp Thr Leu Asp Ser Gln His Gln Ala Asn Gln Pro

165 170 175

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

180 185 190

Thr Phe Arg Cys Tyr Gly Tyr Phe Arg Asn Glu Pro Gln Val Trp Ser

195 200 205

Lys Pro Ser Asn Ser Leu Asp Leu Met Ile Ser Glu Thr Lys Asp Gln

210 215 220

Ser Ser Thr Pro Thr Glu Asp Gly Leu Glu Thr Tyr Gln Lys Ile Leu

225 230 235 240

Ile Gly Val Leu Val Ser Phe Leu Leu Leu Phe Phe Leu Leu Leu Phe

245 250 255

Leu Ile Leu Ile Gly Tyr Gln Tyr Gly His Lys Lys Lys Ala Asn Ala

260 265 270

Ser Val Lys Asn Thr Gln Ser Glu Asn Asn Ala Glu Leu Asn Ser Trp

275 280 285

Asn Pro Gln Asn Glu Asp Pro Gln Gly Ile Val Tyr Ala Gln Val Lys

290 295 300

Pro Ser Arg Leu Gln Lys Asp Thr Ala Cys Lys Glu Thr Gln Asp Val

305 310 315 320

Thr Tyr Ala Gln Leu Cys Ile Arg Thr Gln Glu Gln Asn Asn Ser

325 330 335

<210> 163

<211> 215

<212> PRT

<213> mouse

<220>

<221> sources

<222> (1)..(215)

<223> extracellular domain of gp49B1

<400> 163

Gly His Leu Pro Lys Pro Ile Ile Trp Ala Glu Pro Gly Ser Val Ile

1 5 10 15

Ala Ala Tyr Thr Ser Val Ile Thr Trp Cys Gln Gly Ser Trp Glu Ala

20 25 30

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

35 40 45

Gln Val Pro Leu Glu Thr Arg Asn Lys Ala Lys Phe Asn Ile Pro Ser

50 55 60

Met Thr Thr Ser Tyr Ala Gly Ile Tyr Lys Cys Tyr Tyr Glu Ser Ala

65 70 75 80

Ala Gly Phe Ser Glu His Ser Asp Ala Met Glu Leu Val Met Thr Gly

85 90 95

Ala Tyr Glu Asn Pro Ser Leu Ser Val Tyr Pro Ser Ser Asn Val Thr

100 105 110

Ser Gly Val Ser Ile Ser Phe Ser Cys Ser Ser Ser Ile Val Phe Gly

115 120 125

Arg Phe Ile Leu Ile Gln Glu Gly Lys His Gly Leu Ser Trp Thr Leu

130 135 140

Asp Ser Gln His Gln Ala Asn Gln Pro Ser Tyr Ala Thr Phe Val Leu

145 150 155 160

Asp Ala Val Thr Pro Asn His Asn Gly Thr Phe Arg Cys Tyr Gly Tyr

165 170 175

Phe Arg Asn Glu Pro Gln Val Trp Ser Lys Pro Ser Asn Ser Leu Asp

180 185 190

Leu Met Ile Ser Glu Thr Lys Asp Gln Ser Ser Thr Pro Thr Glu Asp

195 200 205

Gly Leu Glu Thr Tyr Gln Lys

210 215

<210> 164

<211> 864

<212> DNA

<213> Intelligent people

<220>

<221> Source

<222> (1)..(864)

<223> cDNA of Lair1

<400> 164

atgtctcccc accccaccgc cctcctgggc ctagtgctct gcctggccca gaccatccac 60

acgcaggagg aagatctgcc cagaccctcc atctcggctg agccaggcac cgtgatcccc 120

ctggggagcc atgtgacttt cgtgtgccgg ggcccggttg gggttcaaac attccgcctg 180

gagagggaca gtagatccac atacaatgat actgaagatg tgtctcaagc tagtccatct 240

gagtcagagg ccagattccg cattgactca gtaagagaag gaaatgccgg gctttatcgc 300

tgcatctatt ataagccccc taaatggtct gagcagagtg actacctgga gctgctggtg 360

aaagaaagct ctggaggccc ggactccccg gacacagagc ccggctcctc agctggaccc 420

acgcagaggc cgtcggacaa cagtcacaat gagcatgcac ctgcttccca aggcctgaaa 480

gctgagcatc tgtatattct catcggggtc tcagtggtct tcctcttctg tctcctcctc 540

ctggtcctct tctgcctcca tcgccagaat cagataaagc aggggccccc cagaagcaag 600

gacgaggagc agaagccaca gcagaggcct gacctggctg ttgatgttct agagaggaca 660

gcagacaagg ccacagtcaa tggacttcct gagaaggaca gagagacgga cacctcggcc 720

ctggctgcag ggagttccca ggaggtgacg tatgctcagc tggaccactg ggccctcaca 780

cagaggacag cccgggctgt gtccccacag tccacaaagc ccatggccga gtccatcacg 840

tatgcagccg ttgccagaca ctga 864

<210> 165

<211> 287

<212> PRT

<213> Intelligent people

<220>

<221> Source

<222> (1)..(287)

<223> Lair1

<400> 165

Met Ser Pro His Pro Thr Ala Leu Leu Gly Leu Val Leu Cys Leu Ala

1 5 10 15

Gln Thr Ile His Thr Gln Glu Glu Asp Leu Pro Arg Pro Ser Ile Ser

20 25 30

Ala Glu Pro Gly Thr Val Ile Pro Leu Gly Ser His Val Thr Phe Val

35 40 45

Cys Arg Gly Pro Val Gly Val Gln Thr Phe Arg Leu Glu Arg Glu Ser

50 55 60

Arg Ser Thr Tyr Asn Asp Thr Glu Asp Val Ser Gln Ala Ser Pro Ser

65 70 75 80

Glu Ser Glu Ala Arg Phe Arg Ile Asp Ser Val Ser Glu Gly Asn Ala

85 90 95

Gly Pro Tyr Arg Cys Ile Tyr Tyr Lys Pro Pro Lys Trp Ser Glu Gln

100 105 110

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

115 120 125

Ser Pro Asp Thr Glu Pro Gly Ser Ser Ala Gly Pro Thr Gln Arg Pro

130 135 140

Ser Asp Asn Ser His Asn Glu His Ala Pro Ala Ser Gln Gly Leu Lys

145 150 155 160

Ala Glu His Leu Tyr Ile Leu Ile Gly Val Ser Val Val Phe Leu Phe

165 170 175

Cys Leu Leu Leu Leu Val Leu Phe Cys Leu His Arg Gln Asn Gln Ile

180 185 190

Lys Gln Gly Pro Pro Arg Ser Lys Asp Glu Glu Gln Lys Pro Gln Gln

195 200 205

Arg Pro Asp Leu Ala Val Asp Val Leu Glu Arg Thr Ala Asp Lys Ala

210 215 220

Thr Val Asn Gly Leu Pro Glu Lys Asp Arg Glu Thr Asp Thr Ser Ala

225 230 235 240

Leu Ala Ala Gly Ser Ser Gln Glu Val Thr Tyr Ala Gln Leu Asp His

245 250 255

Trp Ala Leu Thr Gln Arg Thr Ala Arg Ala Val Ser Pro Gln Ser Thr

260 265 270

Lys Pro Met Ala Glu Ser Ile Thr Tyr Ala Ala Val Ala Arg His

275 280 285

<210> 166

<211> 144

<212> PRT

<213> Intelligent

<220>

<221> Source

<222> (1)..(144)

<223> extracellular domain of Lair1

<400> 166

Gln Glu Glu Asp Leu Pro Arg Pro Ser Ile Ser Ala Glu Pro Gly Thr

1 5 10 15

Val Ile Pro Leu Gly Ser His Val Thr Phe Val Cys Arg Gly Pro Val

20 25 30

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

35 40 45

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

50 55 60

Phe Arg Ile Asp Ser Val Ser Glu Gly Asn Ala Gly Pro Tyr Arg Cys

65 70 75 80

Ile Tyr Tyr Lys Pro Pro Lys Trp Ser Glu Gln Ser Asp Tyr Leu Glu

85 90 95

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

100 105 110

Pro Gly Ser Ser Ala Gly Pro Thr Gln Arg Pro Ser Asp Asn Ser His

115 120 125

Asn Glu His Ala Pro Ala Ser Gln Gly Leu Lys Ala Glu His Leu Tyr

130 135 140

<210> 167

<211> 792

<212> DNA

<213> mice

<220>

<221> sources

<222> (1)..(792)

<223> cDNA of Lair1

<400> 167

atgtcacttc atccagttat cctgctggtg cttgtgctgt gcctgggatg gaaaattaac 60

acacaggagg gttctctgcc tgatattacc atcttcccta attcaagtct tatgatctcc 120

caagggactt ttgtaactgt tgtgtgctca tactctgata aacacgactt gtataacatg 180

gtccgcctgg agaaggacgg cagcaccttt atggaaaaga gcactgagcc ttataaaaca 240

gaggatgaat ttgagattgg gccagtgaat gaaaccatta ctggacatta tagctgtatc 300

tattcgaagg ggattacctg gtccgaacgt agtaagacgc tggagctaaa ggtgatcaaa 360

gaaaatgtca tccagactcc tgccccaggt ccaacctcag atacatcttg gctaaagaca 420

tacagcattt acatttttac tgtggtctct gtgattttcc tcctttgtct ttccgccctt 480

ctgttctgct tcctcaggca ccgtcagaaa aagcagggac tcccaaacaa caaaagacag 540

cagcagaggc cagaagagag gctaaatcta gctactaatg gcctggagat gactccagac 600

atagttgcag atgacaggct tcctgaggac agatggacag aaacctggac cccagttgca 660

ggagaccttc aagaggtgac gtatatccag ctggaccatc actccctcac acagagggca 720

gtcggagctg tgacctcaca gagcacagat atggctgagt ccagcacata tgcagccatc 780

atcagacact ga 792

<210> 168

<211> 263

<212> PRT

<213> mice

<220>

<221> sources

<222> (1)..(263)

<223> Lair

<400> 168

Met Ser Leu His Pro Val Ile Leu Leu Val Leu Val Leu Cys Leu Gly

1 5 10 15

Trp Lys Ile Asn Thr Gln Glu Gly Ser Leu Pro Asp Ile Thr Ile Phe

20 25 30

Pro Asn Ser Ser Leu Met Ile Ser Gln Gly Thr Phe Val Thr Val Val

35 40 45

Cys Ser Tyr Ser Asp Lys His Asp Leu Tyr Asn Met Val Arg Leu Glu

50 55 60

Lys Asp Gly Ser Thr Phe Met Glu Lys Ser Thr Glu Pro Tyr Lys Thr

65 70 75 80

Glu Asp Glu Phe Glu Ile Gly Pro Val Asn Glu Thr Ile Thr Gly His

85 90 95

Tyr Ser Cys Ile Tyr Ser Lys Gly Ile Thr Trp Ser Glu Arg Ser Lys

100 105 110

Thr Leu Glu Leu Lys Val Ile Lys Glu Asn Val Ile Gln Thr Pro Ala

115 120 125

Pro Gly Pro Thr Ser Asp Thr Ser Trp Leu Lys Thr Tyr Ser Ile Tyr

130 135 140

Ile Phe Thr Val Val Ser Val Ile Phe Leu Leu Cys Leu Ser Ala Leu

145 150 155 160

Leu Phe Cys Phe Leu Arg His Arg Gln Lys Lys Gln Gly Leu Pro Asn

165 170 175

Asn Lys Arg Gln Gln Gln Arg Pro Glu Glu Arg Leu Asn Leu Ala Thr

180 185 190

Asn Gly Leu Glu Met Thr Pro Asp Ile Val Ala Asp Asp Arg Leu Pro

195 200 205

Glu Asp Arg Trp Thr Glu Thr Trp Thr Pro Val Ala Gly Asp Leu Gln

210 215 220

Glu Val Thr Tyr Ile Gln Leu Asp His His Ser Leu Thr Gln Arg Ala

225 230 235 240

Val Gly Ala Val Thr Ser Gln Ser Thr Asp Met Ala Glu Ser Ser Thr

245 250 255

Tyr Ala Ala Ile Ile Arg His

260

<210> 169

<211> 123

<212> PRT

<213> mouse

<220>

<221> sources

<222> (1)..(123)

<223> extracellular domain of Lair1

<400> 169

Gln Glu Gly Ser Leu Pro Asp Ile Thr Ile Phe Pro Asn Ser Ser Leu

1 5 10 15

Met Ile Ser Gln Gly Thr Phe Val Thr Val Val Cys Ser Tyr Ser Asp

20 25 30

Lys His Asp Leu Tyr Asn Met Val Arg Leu Glu Lys Asp Gly Ser Thr

35 40 45

Phe Met Glu Lys Ser Thr Glu Pro Tyr Lys Thr Glu Asp Glu Phe Glu

50 55 60

Ile Gly Pro Val Asn Glu Thr Ile Thr Gly His Tyr Ser Cys Ile Tyr

65 70 75 80

Ser Lys Gly Ile Thr Trp Ser Glu Arg Ser Lys Thr Leu Glu Leu Lys

85 90 95

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

100 105 110

Asp Thr Ser Trp Leu Lys Thr Tyr Ser Ile Tyr

115 120

<210> 170

<211> 1470

<212> DNA

<213> Intelligent

<220>

<221> sources

<222> (1)..(1470)

<223> cDNA of LILRA1

<400> 170

atgaccccca tcgtcacagt cctgatctgt ctcaggctga gtctgggccc ccggacccac 60

gtgcaggcag ggaccctccc caagcccaca ctctgggctg agccaggctc tgtgatcacc 120

caggggagtc ccgtgaccct ctggtgtcag gggatcctgg agacccagga gtaccgtctg 180

tatagagaaa agaaaacagc accctggatt acacggatcc cacaggagat tgtgaagaag 240

ggccagttcc ccatcccatc catcacctgg gaacacacag ggcggtatcg ctgtttctac 300

ggtagccaca ctgcaggctg gtcagagccc agtgaccccc tggagctggt ggtgacagga 360

gcctacatca aacccaccct ctcagctcta cccagccctg tggtgacctc aggagggaac 420

gtgaccctcc attgtgtctc acaggtggca tttggcagct tcattctgtg taaggaagga 480

gaagatgaac acccacaatg cctgaactca cagccccgta cccatgggtg gtcccgggcc 540

atcttctctg tgggccccgt gagcccgagt cgcaggtggt cgtacaggtg ctatgcttat 600

gactcgaact ctccccatgt gtggtctcta cccagtgatc tcctggagct cctggtccta 660

ggtgtttcta agaagccatc actctcagtg cagccaggtc ctatagtggc ccctggggag 720

agcctgaccc tccagtgtgt ttctgatgtc agctacgaca gatttgttct gtataaggag 780

ggagaacgtg acttcctcca gctccctggc ccacagcccc aggctgggct ctcccaggcc 840

aacttcaccc tgggccctgt gagccgctcc tacgggggcc agtacagatg ctccggtgca 900

tacaacctct cctccgagtg gtcggccccc agcgaccccc tggacatcct gatcgcagga 960

cagttccgtg gcagaccctt catctcggtg catccgggcc ccacggtggc ctcaggagag 1020

aacgtgaccc tgctgtgtca gtcatggggg ccgttccaca ctttccttct gaccaaggcg 1080

ggagcagctg atgcccccct ccgtctcaga tcaatacacg aatatcctaa gtaccaggct 1140

gaattcccta tgagtcctgt gacctcagcc cactcgggga cctacaggtg ctacggctca 1200

ctcagctcca acccctacct gctgtctcac cccagtgact ccctggagct catggtctca 1260

ggagcagctg agaccctcag cccaccacaa aacaagtccg attccaaggc tggagcagct 1320

aacaccctca gcccatcaca aaacaagact gcctcacacc cccaggatta cacagtggag 1380

aatctcatcc gcatgggcat agctggcttg gtcctggtgg tcctcgggat tctgctattt 1440

gaggctcagc acagccagag aagcctctga 1470

<210> 171

<211> 489

<212> PRT

<213> Intelligent people

<220>

<221> sources

<222> (1)..(489)

<223> LILRA1

<400> 171

Met Thr Pro Ile Val Thr Val Leu Ile Cys Leu Arg Leu Ser Leu Gly

1 5 10 15

Pro Arg Thr His Val Gln Ala Gly Thr Leu Pro Lys Pro Thr Leu Trp

20 25 30

Ala Glu Pro Gly Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu Trp

35 40 45

Cys Gln Gly Ile Leu Glu Thr Gln Glu Tyr Arg Leu Tyr Arg Glu Lys

50 55 60

Lys Thr Ala Pro Trp Ile Thr Arg Ile Pro Gln Glu Ile Val Lys Lys

65 70 75 80

Gly Gln Phe Pro Ile Pro Ser Ile Thr Trp Glu His Thr Gly Arg Tyr

85 90 95

Arg Cys Phe Tyr Gly Ser His Thr Ala Gly Trp Ser Glu Pro Ser Asp

100 105 110

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

115 120 125

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

130 135 140

Cys Val Ser Gln Val Ala Phe Gly Ser Phe Ile Leu Cys Lys Glu Gly

145 150 155 160

Glu Asp Glu His Pro Gln Cys Leu Asn Ser Gln Pro Arg Thr His Gly

165 170 175

Trp Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg

180 185 190

Trp Ser Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro His Val Trp

195 200 205

Ser Leu Pro Ser Asp Leu Leu Glu Leu Leu Val Leu Gly Val Ser Lys

210 215 220

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

225 230 235 240

Ser Leu Thr Leu Gln Cys Val Ser Asp Val Ser Tyr Asp Arg Phe Val

245 250 255

Leu Tyr Lys Glu Gly Glu Arg Asp Phe Leu Gln Leu Pro Gly Pro Gln

260 265 270

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

275 280 285

Arg Ser Tyr Gly Gly Gln Tyr Arg Cys Ser Gly Ala Tyr Asn Leu Ser

290 295 300

Ser Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Ala Gly

305 310 315 320

Gln Phe Arg Gly Arg Pro Phe Ile Ser Val His Pro Gly Pro Thr Val

325 330 335

Ala Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Trp Gly Pro Phe

340 345 350

His Thr Phe Leu Leu Thr Lys Ala Gly Ala Ala Asp Ala Pro Leu Arg

355 360 365

Leu Arg Ser Ile His Glu Tyr Pro Lys Tyr Gln Ala Glu Phe Pro Met

370 375 380

Ser Pro Val Thr Ser Ala His Ser Gly Thr Tyr Arg Cys Tyr Gly Ser

385 390 395 400

Leu Ser Ser Asn Pro Tyr Leu Leu Ser His Pro Ser Asp Ser Leu Glu

405 410 415

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

420 425 430

Ser Asp Ser Lys Ala Gly Ala Ala Asn Thr Leu Ser Pro Ser Gln Asn

435 440 445

Lys Thr Ala Ser His Pro Gln Asp Tyr Thr Val Glu Asn Leu Ile Arg

450 455 460

Met Gly Ile Ala Gly Leu Val Leu Val Val Leu Gly Ile Leu Leu Phe

465 470 475 480

Glu Ala Gln His Ser Gln Arg Ser Leu

485

<210> 172

<211> 445

<212> PRT

<213> Intelligent people

<220>

<221> Source

<222> (1)..(445)

<223> extracellular domain of LILRA1

<400> 172

Pro Arg Thr His Val Gln Ala Gly Thr Leu Pro Lys Pro Thr Leu Trp

1 5 10 15

Ala Glu Pro Gly Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu Trp

20 25 30

Cys Gln Gly Ile Leu Glu Thr Gln Glu Tyr Arg Leu Tyr Arg Glu Lys

35 40 45

Lys Thr Ala Pro Trp Ile Thr Arg Ile Pro Gln Glu Ile Val Lys Lys

50 55 60

Gly Gln Phe Pro Ile Pro Ser Ile Thr Trp Glu His Thr Gly Arg Tyr

65 70 75 80

Arg Cys Phe Tyr Gly Ser His Thr Ala Gly Trp Ser Glu Pro Ser Asp

85 90 95

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

100 105 110

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

115 120 125

Cys Val Ser Gln Val Ala Phe Gly Ser Phe Ile Leu Cys Lys Glu Gly

130 135 140

Glu Asp Glu His Pro Gln Cys Leu Asn Ser Gln Pro Arg Thr His Gly

145 150 155 160

Trp Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg

165 170 175

Trp Ser Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro His Val Trp

180 185 190

Ser Leu Pro Ser Asp Leu Leu Glu Leu Leu Val Leu Gly Val Ser Lys

195 200 205

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

210 215 220

Ser Leu Thr Leu Gln Cys Val Ser Asp Val Ser Tyr Asp Arg Phe Val

225 230 235 240

Leu Tyr Lys Glu Gly Glu Arg Asp Phe Leu Gln Leu Pro Gly Pro Gln

245 250 255

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

260 265 270

Arg Ser Tyr Gly Gly Gln Tyr Arg Cys Ser Gly Ala Tyr Asn Leu Ser

275 280 285

Ser Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Ala Gly

290 295 300

Gln Phe Arg Gly Arg Pro Phe Ile Ser Val His Pro Gly Pro Thr Val

305 310 315 320

Ala Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Trp Gly Pro Phe

325 330 335

His Thr Phe Leu Leu Thr Lys Ala Gly Ala Ala Asp Ala Pro Leu Arg

340 345 350

Leu Arg Ser Ile His Glu Tyr Pro Lys Tyr Gln Ala Glu Phe Pro Met

355 360 365

Ser Pro Val Thr Ser Ala His Ser Gly Thr Tyr Arg Cys Tyr Gly Ser

370 375 380

Leu Ser Ser Asn Pro Tyr Leu Leu Ser His Pro Ser Asp Ser Leu Glu

385 390 395 400

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

405 410 415

Ser Asp Ser Lys Ala Gly Ala Ala Asn Thr Leu Ser Pro Ser Gln Asn

420 425 430

Lys Thr Ala Ser His Pro Gln Asp Tyr Thr Val Glu Asn

435 440 445

<210> 173

<211> 1452

<212> DNA

<213> Intelligent people

<220>

<221> sources

<222> (1)..(1452)

<223> cDNA of LILRA2

<400> 173

atgaccccca tcctcacggt cctgatctgt ctcgggctga gtctgggccc caggacccac 60

gtgcaggcag ggcacctccc caagcccacc ctctgggctg agccaggctc tgtgatcatc 120

cagggaagtc ctgtgaccct caggtgtcag gggagccttc aggctgagga gtaccatcta 180

tatagggaaa acaaatcagc atcctgggtt agacggatac aagagcctgg gaagaatggc 240

cagttcccca tcccatccat cacctgggaa cacgcagggc ggtatcactg tcagtactac 300

agccacaatc actcatcaga gtacagtgac cccctggagc tggtggtgac aggagcctac 360

agcaaaccca ccctctcagc tctgcccagc cctgtggtga cctcaggagg gaacgtgacc 420

ctccagtgtg tctcacaggt ggcatttgac ggcttcattc tgtgtaagga aggagaagat 480

gaacacccac aacgcctgaa ctcccattcc catgcccgtg ggtggtcctg ggccatcttc 540

tccgtgggcc ccgtgagccc gagtcgcagg tggtcgtaca ggtgctatgc ttatgactcg 600

aactctccct atgtgtggtc tctacccagt gatctcctgg agctcctggt cccaggtgtt 660

tctaagaagc catcactctc agtgcagcca ggtcctatgg tggcccctgg ggagagcctg 720

accctccagt gtgtctctga tgtcggctac gacagatttg ttctgtataa ggagggagaa 780

cgtgacttcc tccagcgccc tggttggcag ccccaggctg ggctctccca ggccaacttc 840

accctgggcc ctgtgagccc ctcccacggg ggccagtaca gatgctacag tgcacacaac 900

ctctcctccg agtggtcggc ccccagtgac cccctggaca tcctgatcac aggacagttc 960

tatgacagac cctctctctc ggtgcagccg gtccccacag tagccccagg aaagaacgtg 1020

accctgctgt gtcagtcacg ggggcagttc cacactttcc ttctgaccaa ggagggggca 1080

ggccatcccc cactgcatct gagatcagag caccaagctc agcagaacca ggctgaattc 1140

cgcatgggtc ctgtgacctc agcccacgtg gggacctaca gatgctacag ctcactcagc 1200

tccaacccct acctgctgtc tctccccagt gaccccctgg agctcgtggt ctcagaagca 1260

gctgagaccc tcagcccatc acaaaacaag acagactcca cgactacatc cctaggccaa 1320

cacccccagg attacacagt ggagaatctc atccgcatgg gtgtggctgg cttggtcctg 1380

gtggtcctcg ggattctgct atttgaggct cagcacagcc agagaagcct acaagatgca 1440

gccgggaggt ga 1452

<210> 174

<211> 483

<212> PRT

<213> Intelligent

<220>

<221> sources

<222> (1)..(483)

<223> LILRA2

<400> 174

Met Thr Pro Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly

1 5 10 15

Pro Arg Thr His Val Gln Ala Gly His Leu Pro Lys Pro Thr Leu Trp

20 25 30

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

35 40 45

Cys Gln Gly Ser Leu Gln Ala Glu Glu Tyr His Leu Tyr Arg Glu Asn

50 55 60

Lys Ser Ala Ser Trp Val Arg Arg Ile Gln Glu Pro Gly Lys Asn Gly

65 70 75 80

Gln Phe Pro Ile Pro Ser Ile Thr Trp Glu His Ala Gly Arg Tyr His

85 90 95

Cys Gln Tyr Tyr Ser His Asn His Ser Ser Glu Tyr Ser Asp Pro Leu

100 105 110

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

115 120 125

Pro Ser Pro Val Val Thr Leu Gly Gly Asn Val Thr Leu Gln Cys Val

130 135 140

Ser Gln Val Ala Phe Asp Gly Phe Ile Leu Cys Lys Glu Gly Glu Asp

145 150 155 160

Glu His Pro Gln Arg Leu Asn Ser His Ser His Ala Arg Gly Trp Ser

165 170 175

Trp Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg Trp Ser

180 185 190

Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro Tyr Val Trp Ser Leu

195 200 205

Pro Ser Asp Leu Leu Glu Leu Leu Val Pro Gly Val Ser Lys Lys Pro

210 215 220

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

225 230 235 240

Thr Leu Gln Cys Val Ser Asp Val Gly Tyr Asp Arg Phe Val Leu Tyr

245 250 255

Lys Glu Gly Glu Arg Asp Phe Leu Gln Arg Pro Gly Trp Gln Pro Gln

260 265 270

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

275 280 285

His Gly Gly Gln Tyr Arg Cys Tyr Ser Ala His Asn Leu Ser Ser Glu

290 295 300

Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Thr Gly Gln Phe

305 310 315 320

Tyr Asp Arg Pro Ser Leu Ser Val Gln Pro Val Pro Thr Val Ala Pro

325 330 335

Gly Lys Asn Val Thr Leu Leu Cys Gln Ser Arg Gly Gln Phe His Thr

340 345 350

Phe Leu Leu Thr Lys Glu Gly Ala Gly His Pro Pro Leu His Leu Arg

355 360 365

Ser Glu His Gln Ala Gln Gln Asn Gln Ala Glu Phe Arg Met Gly Pro

370 375 380

Val Thr Ser Ala His Val Gly Thr Tyr Arg Cys Tyr Ser Ser Leu Ser

385 390 395 400

Ser Asn Pro Tyr Leu Leu Ser Leu Pro Ser Asp Pro Leu Glu Leu Val

405 410 415

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

420 425 430

Ser Thr Thr Thr Ser Leu Gly Gln His Pro Gln Asp Tyr Thr Val Glu

435 440 445

Asn Leu Ile Arg Met Gly Val Ala Gly Leu Val Leu Val Val Leu Gly

450 455 460

Ile Leu Leu Phe Glu Ala Gln His Ser Gln Arg Ser Leu Gln Asp Ala

465 470 475 480

Ala Gly Arg

<210> 175

<211> 426

<212> PRT

<213> Intelligent people

<220>

<221> sources

<222> (1)..(426)

<223> extracellular domain of LILRA2

<400> 175

Gly His Leu Pro Lys Pro Thr Leu Trp Ala Glu Pro Gly Ser Val Ile

1 5 10 15

Ile Gln Gly Ser Pro Val Thr Leu Arg Cys Gln Gly Ser Leu Gln Ala

20 25 30

Glu Glu Tyr His Leu Tyr Arg Glu Asn Lys Ser Ala Ser Trp Val Arg

35 40 45

Arg Ile Gln Glu Pro Gly Lys Asn Gly Gln Phe Pro Ile Pro Ser Ile

50 55 60

Thr Trp Glu His Ala Gly Arg Tyr His Cys Gln Tyr Tyr Ser His Asn

65 70 75 80

His Ser Ser Glu Tyr Ser Asp Pro Leu Glu Leu Val Val Thr Gly Ala

85 90 95

Tyr Ser Lys Pro Thr Leu Ser Ala Leu Pro Ser Pro Val Val Thr Leu

100 105 110

Gly Gly Asn Val Thr Leu Gln Cys Val Ser Gln Val Ala Phe Asp Gly

115 120 125

Phe Ile Leu Cys Lys Glu Gly Glu Asp Glu His Pro Gln Arg Leu Asn

130 135 140

Ser His Ser His Ala Arg Gly Trp Ser Trp Ala Ile Phe Ser Val Gly

145 150 155 160

Pro Val Ser Pro Ser Arg Arg Trp Ser Tyr Arg Cys Tyr Ala Tyr Asp

165 170 175

Ser Asn Ser Pro Tyr Val Trp Ser Leu Pro Ser Asp Leu Leu Glu Leu

180 185 190

Leu Val Pro Gly Val Ser Lys Lys Pro Ser Leu Ser Val Gln Pro Gly

195 200 205

Pro Met Val Ala Pro Gly Glu Ser Leu Thr Leu Gln Cys Val Ser Asp

210 215 220

Val Gly Tyr Asp Arg Phe Val Leu Tyr Lys Glu Gly Glu Arg Asp Phe

225 230 235 240

Leu Gln Arg Pro Gly Trp Gln Pro Gln Ala Gly Leu Ser Gln Ala Asn

245 250 255

Phe Thr Leu Gly Pro Val Ser Pro Ser His Gly Gly Gln Tyr Arg Cys

260 265 270

Tyr Ser Ala His Asn Leu Ser Ser Glu Trp Ser Ala Pro Ser Asp Pro

275 280 285

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

290 295 300

Val Gln Pro Val Pro Thr Val Ala Pro Gly Lys Asn Val Thr Leu Leu

305 310 315 320

Cys Gln Ser Arg Gly Gln Phe His Thr Phe Leu Leu Thr Lys Glu Gly

325 330 335

Ala Gly His Pro Pro Leu His Leu Arg Ser Glu His Gln Ala Gln Gln

340 345 350

Asn Gln Ala Glu Phe Arg Met Gly Pro Val Thr Ser Ala His Val Gly

355 360 365

Thr Tyr Arg Cys Tyr Ser Ser Leu Ser Ser Asn Pro Tyr Leu Leu Ser

370 375 380

Leu Pro Ser Asp Pro Leu Glu Leu Val Val Ser Glu Ala Ala Glu Thr

385 390 395 400

Leu Ser Pro Ser Gln Asn Lys Thr Asp Ser Thr Thr Thr Ser Leu Gly

405 410 415

Gln His Pro Gln Asp Tyr Thr Val Glu Asn

420 425

<210> 176

<211> 1320

<212> DNA

<213> Intelligent people

<220>

<221> sources

<222> (1)..(1320)

<223> cDNA of LILRA3

<400> 176

atgaccccca tcctcacggt cctgatctgt ctcgggctga gcctggaccc caggacccac 60

gtgcaggcag ggcccctccc caagcccacc ctctgggctg agccaggctc tgtgatcacc 120

caagggagtc ctgtgaccct caggtgtcag gggagcctgg agacgcagga gtaccatcta 180

tatagagaaa agaaaacagc actctggatt acacggatcc cacaggagct tgtgaagaag 240

ggccagttcc ccatcctatc catcacctgg gaacatgcag ggcggtattg ctgtatctat 300

ggcagccaca ctgcaggcct ctcagagagc agtgaccccc tggagctggt ggtgacagga 360

gcctacagca aacccaccct ctcagctctg cccagccctg tggtgacctc aggagggaat 420

gtgaccatcc agtgtgactc acaggtggca tttgatggct tcattctgtg taaggaagga 480

gaagatgaac acccacaatg cctgaactcc cattcccatg cccgtgggtc atcccgggcc 540

atcttctccg tgggccccgt gagcccaagt cgcaggtggt cgtacaggtg ctatggttat 600

gactcgcgcg ctccctatgt gtggtctcta cccagtgatc tcctggggct cctggtccca 660

ggtgtttcta agaagccatc actctcagtg cagccgggtc ctgtcgtggc ccctggggag 720

aagctgacct tccagtgtgg ctctgatgcc ggctacgaca gatttgttct gtacaaggag 780

tggggacgtg acttcctcca gcgccctggc cggcagcccc aggctgggct ctcccaggcc 840

aacttcaccc tgggccctgt gagccgctcc tacgggggcc agtacacatg ctccggtgca 900

tacaacctct cctccgagtg gtcggccccc agcgaccccc tggacatcct gatcacagga 960

cagatccgtg ccagaccctt cctctccgtg cggccgggcc ccacagtggc ctcaggagag 1020

aacgtgaccc tgctgtgtca gtcacaggga gggatgcaca ctttcctttt gaccaaggag 1080

ggggcagctg attccccgct gcgtctaaaa tcaaagcgcc aatctcataa gtaccaggct 1140

gaattcccca tgagtcctgt gacctcggcc cacgcgggga cctacaggtg ctacggctca 1200

ctcagctcca acccctacct gctgactcac cccagtgacc ccctggagct cgtggtctca 1260

ggagcagctg agaccctcag cccaccacaa aacaagtccg actccaaggc tggtgagtga 1320

<210> 177

<211> 439

<212> PRT

<213> Intelligent people

<220>

<221> sources

<222> (1)..(439)

<223> LILRA3

<400> 177

Met Thr Pro Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Asp

1 5 10 15

Pro Arg Thr His Val Gln Ala Gly Pro Leu Pro Lys Pro Thr Leu Trp

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Gly Gln Phe Pro Ile Leu Ser Ile Thr Trp Glu His Ala Gly Arg Tyr

85 90 95

Cys Cys Ile Tyr Gly Ser His Thr Ala Gly Leu Ser Glu Ser Ser Asp

100 105 110

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

115 120 125

Ala Leu Pro Ser Pro Val Val Thr Ser Gly Gly Asn Val Thr Ile Gln

130 135 140

Cys Asp Ser Gln Val Ala Phe Asp Gly Phe Ile Leu Cys Lys Glu Gly

145 150 155 160

Glu Asp Glu His Pro Gln Cys Leu Asn Ser His Ser His Ala Arg Gly

165 170 175

Ser Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg

180 185 190

Trp Ser Tyr Arg Cys Tyr Gly Tyr Asp Ser Arg Ala Pro Tyr Val Trp

195 200 205

Ser Leu Pro Ser Asp Leu Leu Gly Leu Leu Val Pro Gly Val Ser Lys

210 215 220

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

225 230 235 240

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

245 250 255

Leu Tyr Lys Glu Trp Gly Arg Asp Phe Leu Gln Arg Pro Gly Arg Gln

260 265 270

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

275 280 285

Arg Ser Tyr Gly Gly Gln Tyr Thr Cys Ser Gly Ala Tyr Asn Leu Ser

290 295 300

Ser Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Thr Gly

305 310 315 320

Gln Ile Arg Ala Arg Pro Phe Leu Ser Val Arg Pro Gly Pro Thr Val

325 330 335

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

340 345 350

His Thr Phe Leu Leu Thr Lys Glu Gly Ala Ala Asp Ser Pro Leu Arg

355 360 365

Leu Lys Ser Lys Arg Gln Ser His Lys Tyr Gln Ala Glu Phe Pro Met

370 375 380

Ser Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser

385 390 395 400

Leu Ser Ser Asn Pro Tyr Leu Leu Thr His Pro Ser Asp Pro Leu Glu

405 410 415

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

420 425 430

Ser Asp Ser Lys Ala Gly Glu

435

<210> 178

<211> 416

<212> PRT

<213> Intelligent

<220>

<221> Source

<222> (1)..(416)

<223> extracellular domain of LILRA3

<400> 178

Gly Pro Leu Pro Lys Pro Thr Leu Trp Ala Glu Pro Gly Ser Val Ile

1 5 10 15

Thr Gln Gly Ser Pro Val Thr Leu Arg Cys Gln Gly Ser Leu Glu Thr

20 25 30

Gln Glu Tyr His Leu Tyr Arg Glu Lys Lys Thr Ala Leu Trp Ile Thr

35 40 45

Arg Ile Pro Gln Glu Leu Val Lys Lys Gly Gln Phe Pro Ile Leu Ser

50 55 60

Ile Thr Trp Glu His Ala Gly Arg Tyr Cys Cys Ile Tyr Gly Ser His

65 70 75 80

Thr Ala Gly Leu Ser Glu Ser Ser Asp Pro Leu Glu Leu Val Val Thr

85 90 95

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

100 105 110

Thr Ser Gly Gly Asn Val Thr Ile Gln Cys Asp Ser Gln Val Ala Phe

115 120 125

Asp Gly Phe Ile Leu Cys Lys Glu Gly Glu Asp Glu His Pro Gln Cys

130 135 140

Leu Asn Ser His Ser His Ala Arg Gly Ser Ser Arg Ala Ile Phe Ser

145 150 155 160

Val Gly Pro Val Ser Pro Ser Arg Arg Trp Ser Tyr Arg Cys Tyr Gly

165 170 175

Tyr Asp Ser Arg Ala Pro Tyr Val Trp Ser Leu Pro Ser Asp Leu Leu

180 185 190

Gly Leu Leu Val Pro Gly Val Ser Lys Lys Pro Ser Leu Ser Val Gln

195 200 205

Pro Gly Pro Val Val Ala Pro Gly Glu Lys Leu Thr Phe Gln Cys Gly

210 215 220

Ser Asp Ala Gly Tyr Asp Arg Phe Val Leu Tyr Lys Glu Trp Gly Arg

225 230 235 240

Asp Phe Leu Gln Arg Pro Gly Arg Gln Pro Gln Ala Gly Leu Ser Gln

245 250 255

Ala Asn Phe Thr Leu Gly Pro Val Ser Arg Ser Tyr Gly Gly Gln Tyr

260 265 270

Thr Cys Ser Gly Ala Tyr Asn Leu Ser Ser Glu Trp Ser Ala Pro Ser

275 280 285

Asp Pro Leu Asp Ile Leu Ile Thr Gly Gln Ile Arg Ala Arg Pro Phe

290 295 300

Leu Ser Val Arg Pro Gly Pro Thr Val Ala Ser Gly Glu Asn Val Thr

305 310 315 320

Leu Leu Cys Gln Ser Gln Gly Gly Met His Thr Phe Leu Leu Thr Lys

325 330 335

Glu Gly Ala Ala Asp Ser Pro Leu Arg Leu Lys Ser Lys Arg Gln Ser

340 345 350

His Lys Tyr Gln Ala Glu Phe Pro Met Ser Pro Val Thr Ser Ala His

355 360 365

Ala Gly Thr Tyr Arg Cys Tyr Gly Ser Leu Ser Ser Asn Pro Tyr Leu

370 375 380

Leu Thr His Pro Ser Asp Pro Leu Glu Leu Val Val Ser Gly Ala Ala

385 390 395 400

Glu Thr Leu Ser Pro Pro Gln Asn Lys Ser Asp Ser Lys Ala Gly Glu

405 410 415

<210> 179

<211> 1500

<212> DNA

<213> Intelligent people

<220>

<221> sources

<222> (1)..(1500)

<223> cDNA of LILRA4

<400> 179

atgaccctca ttctcacaag cctgctcttc tttgggctga gcctgggccc caggacccgg 60

gtgcaggcag aaaacctact caaacccatc ctgtgggccg agccaggtcc cgtgatcacc 120

tggcataacc ccgtgaccat ctggtgtcag ggcaccctgg aggcccaggg gtaccgtctg 180

gataaagagg gaaactcaat gtcgaggcac atattaaaaa cactggagtc tgaaaacaag 240

gtcaaactct ccatcccatc catgatgtgg gaacatgcag ggcgatatca ctgttactat 300

cagagccctg caggctggtc agagcccagc gaccccctgg agctggtggt gacagcctac 360

agcagaccca ccctgtccgc actgccaagc cctgtggtga cctcaggagt gaacgtgacc 420

ctccggtgtg cctcacggct gggactgggc aggttcactc tgattgagga aggagaccac 480

aggctctcct ggaccctgaa ctcacaccaa cacaaccatg gaaagttcca ggccctgttc 540

cccatgggcc ccctgacctt cagcaacagg ggtacattca gatgctacgg ctatgaaaac 600

aacaccccat acgtgtggtc ggaacccagt gaccccctgc agctactggt gtcaggcgtg 660

tctaggaagc cctccctcct gaccctgcag ggccctgtcg tgacccccgg agagaatctg 720

accctccagt gtggctctga tgtcggctac atcagataca ctctgtacaa ggagggggcc 780

gatggcctcc cccagcgccc tggccggcag ccccaggctg ggctctccca ggccaacttc 840

accctgagcc ctgtgagccg ctcctacggg ggccagtaca gatgctacgg cgcacacaac 900

gtctcctccg agtggtcggc ccccagtgac cccctggata tcctgatcgc aggacagatc 960

tctgacagac cctccctctc agtgcagccg ggccccacgg tgacctcagg agagaaggtg 1020

accctgctgt gtcagtcatg ggacccgatg ttcactttcc ttctgaccaa ggagggggca 1080

gcccatcccc cgttgcgtct gagatcaatg tacggagctc ataagtacca ggctgaattc 1140

cccatgagtc ctgtgacctc agcccacgcg gggacctaca ggtgctacgg ctcacgcagc 1200

tccaacccct acctgctgtc tcaccccagt gagcccctgg agctcgtggt ctcaggagca 1260

actgagaccc tcaatccagc acaaaagaag tcagattcca agactgcccc acacctccag 1320

gattacacag tggagaatct catccgcatg ggtgtggctg gcttggtcct gctgttcctc 1380

gggattctgt tatttgaggc tcagcacagc cagagaagcc ccccaaggtg cagccaggag 1440

gcaaacagca gaaaggacaa tgcacccttc agagtggtgg agccttggga acagatctga 1500

<210> 180

<211> 499

<212> PRT

<213> Intelligent people

<220>

<221> Source

<222> (1)..(499)

<223> LILRA4

<400> 180

Met Thr Leu Ile Leu Thr Ser Leu Leu Phe Phe Gly Leu Ser Leu Gly

1 5 10 15

Pro Arg Thr Arg Val Gln Ala Glu Asn Leu Pro Lys Pro Ile Leu Trp

20 25 30

Ala Glu Pro Gly Pro Val Ile Thr Trp His Asn Pro Val Thr Ile Trp

35 40 45

Cys Gln Gly Thr Leu Glu Ala Gln Gly Tyr Arg Leu Asp Lys Glu Gly

50 55 60

Asn Ser Met Ser Arg His Ile Leu Lys Thr Leu Glu Ser Glu Asn Lys

65 70 75 80

Val Lys Leu Ser Ile Pro Ser Met Met Trp Glu His Ala Gly Arg Tyr

85 90 95

His Cys Tyr Tyr Gln Ser Pro Ala Gly Trp Ser Glu Pro Ser Asp Pro

100 105 110

Leu Glu Leu Val Val Thr Ala Tyr Ser Arg Pro Thr Leu Ser Ala Leu

115 120 125

Pro Ser Pro Val Val Thr Ser Gly Val Asn Val Thr Leu Arg Cys Ala

130 135 140

Ser Arg Leu Gly Leu Gly Arg Phe Thr Leu Ile Glu Glu Gly Asp His

145 150 155 160

Arg Leu Ser Trp Thr Leu Asn Ser His Gln His Asn His Gly Lys Phe

165 170 175

Gln Ala Leu Phe Pro Met Gly Pro Leu Thr Phe Ser Asn Arg Gly Thr

180 185 190

Phe Arg Cys Tyr Gly Tyr Glu Asn Asn Thr Pro Tyr Val Trp Ser Glu

195 200 205

Pro Ser Asp Pro Leu Gln Leu Leu Val Ser Gly Val Ser Arg Lys Pro

210 215 220

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

225 230 235 240

Thr Leu Gln Cys Gly Ser Asp Val Gly Tyr Ile Arg Tyr Thr Leu Tyr

245 250 255

Lys Glu Gly Ala Asp Gly Leu Pro Gln Arg Pro Gly Arg Gln Pro Gln

260 265 270

Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Ser Pro Val Ser Arg Ser

275 280 285

Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Val Ser Ser Glu

290 295 300

Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Ala Gly Gln Ile

305 310 315 320

Ser Asp Arg Pro Ser Leu Ser Val Gln Pro Gly Pro Thr Val Thr Ser

325 330 335

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

340 345 350

Phe Leu Leu Thr Lys Glu Gly Ala Ala His Pro Pro Leu Arg Leu Arg

355 360 365

Ser Met Tyr Gly Ala His Lys Tyr Gln Ala Glu Phe Pro Met Ser Pro

370 375 380

Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser Arg Ser

385 390 395 400

Ser Asn Pro Tyr Leu Leu Ser His Pro Ser Glu Pro Leu Glu Leu Val

405 410 415

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

420 425 430

Ser Lys Thr Ala Pro His Leu Gln Asp Tyr Thr Val Glu Asn Leu Ile

435 440 445

Arg Met Gly Val Ala Gly Leu Val Leu Leu Phe Leu Gly Ile Leu Leu

450 455 460

Phe Glu Ala Gln His Ser Gln Arg Ser Pro Pro Arg Cys Ser Gln Glu

465 470 475 480

Ala Asn Ser Arg Lys Asp Asn Ala Pro Phe Arg Val Val Glu Pro Trp

485 490 495

Glu Gln Ile

<210> 181

<211> 423

<212> PRT

<213> Intelligent people

<220>

<221> sources

<222> (1)..(423)

<223> extracellular domain of LILRA4

<400> 181

Glu Asn Leu Pro Lys Pro Ile Leu Trp Ala Glu Pro Gly Pro Val Ile

1 5 10 15

Thr Trp His Asn Pro Val Thr Ile Trp Cys Gln Gly Thr Leu Glu Ala

20 25 30

Gln Gly Tyr Arg Leu Asp Lys Glu Gly Asn Ser Met Ser Arg His Ile

35 40 45

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

50 55 60

Met Met Trp Glu His Ala Gly Arg Tyr His Cys Tyr Tyr Gln Ser Pro

65 70 75 80

Ala Gly Trp Ser Glu Pro Ser Asp Pro Leu Glu Leu Val Val Thr Ala

85 90 95

Tyr Ser Arg Pro Thr Leu Ser Ala Leu Pro Ser Pro Val Val Thr Ser

100 105 110

Gly Val Asn Val Thr Leu Arg Cys Ala Ser Arg Leu Gly Leu Gly Arg

115 120 125

Phe Thr Leu Ile Glu Glu Gly Asp His Arg Leu Ser Trp Thr Leu Asn

130 135 140

Ser His Gln His Asn His Gly Lys Phe Gln Ala Leu Phe Pro Met Gly

145 150 155 160

Pro Leu Thr Phe Ser Asn Arg Gly Thr Phe Arg Cys Tyr Gly Tyr Glu

165 170 175

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

180 185 190

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

195 200 205

Pro Val Val Thr Pro Gly Glu Asn Leu Thr Leu Gln Cys Gly Ser Asp

210 215 220

Val Gly Tyr Ile Arg Tyr Thr Leu Tyr Lys Glu Gly Ala Asp Gly Leu

225 230 235 240

Pro Gln Arg Pro Gly Arg Gln Pro Gln Ala Gly Leu Ser Gln Ala Asn

245 250 255

Phe Thr Leu Ser Pro Val Ser Arg Ser Tyr Gly Gly Gln Tyr Arg Cys

260 265 270

Tyr Gly Ala His Asn Val Ser Ser Glu Trp Ser Ala Pro Ser Asp Pro

275 280 285

Leu Asp Ile Leu Ile Ala Gly Gln Ile Ser Asp Arg Pro Ser Leu Ser

290 295 300

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

305 310 315 320

Cys Gln Ser Trp Asp Pro Met Phe Thr Phe Leu Leu Thr Lys Glu Gly

325 330 335

Ala Ala His Pro Pro Leu Arg Leu Arg Ser Met Tyr Gly Ala His Lys

340 345 350

Tyr Gln Ala Glu Phe Pro Met Ser Pro Val Thr Ser Ala His Ala Gly

355 360 365

Thr Tyr Arg Cys Tyr Gly Ser Arg Ser Ser Asn Pro Tyr Leu Leu Ser

370 375 380

His Pro Ser Glu Pro Leu Glu Leu Val Val Ser Gly Ala Thr Glu Thr

385 390 395 400

Leu Asn Pro Ala Gln Lys Lys Ser Asp Ser Lys Thr Ala Pro His Leu

405 410 415

Gln Asp Tyr Thr Val Glu Asn

420

<210> 182

<211> 900

<212> DNA

<213> Intelligent people

<220>

<221> sources

<222> (1)..(900)

<223> cDNA of LILRA5

<400> 182

atggcaccat ggtctcatcc atctgcacag ctgcagccag tgggaggaga cgccgtgagc 60

cctgccctca tggttctgct ctgcctcggg ctgagtctgg gccccaggac ccacgtgcag 120

gcagggaacc tctccaaagc caccctctgg gctgagccag gctctgtgat cagccggggg 180

aactctgtga ccatccggtg tcaggggacc ctggaggccc aggaataccg tctggttaaa 240

gagggaagcc cagaaccctg ggacacacag aacccactgg agcccaagaa caaggccaga 300

ttctccatcc catccatgac agagcaccat gcagggagat accgctgtta ctactacagc 360

cctgcaggct ggtcagagcc cagcgacccc ctggagctgg tggtgacagg attctacaac 420

aaacccaccc tctcagccct gcccagtcct gtggtgacct caggagagaa cgtgaccctc 480

cagtgtggct cacggctgag attcgacagg ttcattctga ctgaggaagg agaccacaag 540

ctctcctgga ccttggactc acagctgacc cccagtgggc agttccaggc cctgttccct 600

gtgggccctg tgacccccag ccacaggtgg atgctcagat gctatggctc tcgcaggcat 660

atcctgcagg tatggtcaga acccagtgac ctcctggaga ttccggtctc aggagcagct 720

gataacctca gtccgtcaca aaacaagtct gactctggga ctgcctcaca ccttcaggat 780

tacgcagtag agaatctcat ccgcatgggc atggccggct tgatcctggt ggtccttggg 840

attctgatat ttcaggattg gcacagccag agaagccccc aagctgcagc tggaaggtga 900

<210> 183

<211> 299

<212> PRT

<213> Intelligent people

<220>

<221> Source

<222> (1)..(299)

<223> LILRA5

<400> 183

Met Ala Pro Trp Ser His Pro Ser Ala Gln Leu Gln Pro Val Gly Gly

1 5 10 15

Asp Ala Val Ser Pro Ala Leu Met Val Leu Leu Cys Leu Gly Leu Ser

20 25 30

Leu Gly Pro Arg Thr His Val Gln Ala Gly Asn Leu Ser Lys Ala Thr

35 40 45

Leu Trp Ala Glu Pro Gly Ser Val Ile Ser Arg Gly Asn Ser Val Thr

50 55 60

Ile Arg Cys Gln Gly Thr Leu Glu Ala Gln Glu Tyr Arg Leu Val Lys

65 70 75 80

Glu Gly Ser Pro Glu Pro Trp Asp Thr Gln Asn Pro Leu Glu Pro Lys

85 90 95

Asn Lys Ala Arg Phe Ser Ile Pro Ser Met Thr Glu His His Ala Gly

100 105 110

Arg Tyr Arg Cys Tyr Tyr Tyr Ser Pro Ala Gly Trp Ser Glu Pro Ser

115 120 125

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

130 135 140

Ser Ala Leu Pro Ser Pro Val Val Thr Ser Gly Glu Asn Val Thr Leu

145 150 155 160

Gln Cys Gly Ser Arg Leu Arg Phe Asp Arg Phe Ile Leu Thr Glu Glu

165 170 175

Gly Asp His Lys Leu Ser Trp Thr Leu Asp Ser Gln Leu Thr Pro Ser

180 185 190

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

195 200 205

Arg Trp Met Leu Arg Cys Tyr Gly Ser Arg Arg His Ile Leu Gln Val

210 215 220

Trp Ser Glu Pro Ser Asp Leu Leu Glu Ile Pro Val Ser Gly Ala Ala

225 230 235 240

Asp Asn Leu Ser Pro Ser Gln Asn Lys Ser Asp Ser Gly Thr Ala Ser

245 250 255

His Leu Gln Asp Tyr Ala Val Glu Asn Leu Ile Arg Met Gly Met Ala

260 265 270

Gly Leu Ile Leu Val Val Leu Gly Ile Leu Ile Phe Gln Asp Trp His

275 280 285

Ser Gln Arg Ser Pro Gln Ala Ala Ala Gly Arg

290 295

<210> 184

<211> 227

<212> PRT

<213> Intelligent

<220>

<221> Source

<222> (1)..(227)

<223> extracellular domain of LILRA5

<400> 184

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

1 5 10 15

Ser Arg Gly Asn Ser Val Thr Ile Arg Cys Gln Gly Thr Leu Glu Ala

20 25 30

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

35 40 45

Gln Asn Pro Leu Glu Pro Lys Asn Lys Ala Arg Phe Ser Ile Pro Ser

50 55 60

Met Thr Glu His His Ala Gly Arg Tyr Arg Cys Tyr Tyr Tyr Ser Pro

65 70 75 80

Ala Gly Trp Ser Glu Pro Ser Asp Pro Leu Glu Leu Val Val Thr Gly

85 90 95

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

100 105 110

Ser Gly Glu Asn Val Thr Leu Gln Cys Gly Ser Arg Leu Arg Phe Asp

115 120 125

Arg Phe Ile Leu Thr Glu Glu Gly Asp His Lys Leu Ser Trp Thr Leu

130 135 140

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

145 150 155 160

Gly Pro Val Thr Pro Ser His Arg Trp Met Leu Arg Cys Tyr Gly Ser

165 170 175

Arg Arg His Ile Leu Gln Val Trp Ser Glu Pro Ser Asp Leu Leu Glu

180 185 190

Ile Pro Val Ser Gly Ala Ala Asp Asn Leu Ser Pro Ser Gln Asn Lys

195 200 205

Ser Asp Ser Gly Thr Ala Ser His Leu Gln Asp Tyr Ala Val Glu Asn

210 215 220

Leu Ile Arg

225

<210> 185

<211> 1446

<212> DNA

<213> Intelligent

<220>

<221> Source

<222> (1)..(1446)

<223> cDNA of LILRA6

<400> 185

atgaccccca ccctcgcagc cctgctctgc ctagggctga gtctgggccc caggacccac 60

gtgcaggcag ggcccttccc caaacccacc ctctgggctg agccaggctc tgtgatcagc 120

tgggggagcc ccgtgaccat ctggtgtcag gggagcctgg aggcccagga gtaccgactg 180

gataaagagg gaagcccaga gccctgggac agaaataacc cactggaacc caagaacaag 240

gccagattct ccatcccatc cataacagag caccatgcgg ggagataccg ctgccactat 300

tacagctctg caggctggtc agagcccagc gaccccctgg agctggtgat gacaggagcc 360

tatagcaaac ccaccctctc agccctgccc agccctgtgg tggcctcagg ggggaatatg 420

accctccaat gtggctcaca gaagggatat caccattttg ttctgatgaa ggaaggagaa 480

caccagctcc cccggaccct ggactcacag cagctccaca gtggggggtt ccaggccctg 540

ttccctgtgg gccccgtgaa ccccagccac aggtggaggt tcacatgcta ttactattat 600

atgaacaccc cccgggtgtg gtcccacccc agtgaccccc tggagattct gccctcaggc 660

gtgtctagga agccctccct cctgaccctg cagggccctg tcctggcccc tgggcagagc 720

ctgaccctcc agtgtggctc tgatgtcggc tacgacagat ttgttctgta taaggagggg 780

gaacgtgact tcctccagcg ccctggccag cagccccagg ctgggctctc ccaggccaac 840

ttcaccctgg gccctgtgag cccctcccac gggggccagt acaggtgcta tggtgcacac 900

aacctctcct ccgagtggtc ggcccccagc gaccccctga acatcctgat ggcaggacag 960

atctatgaca ccgtctccct gtcagcacag ccgggcccca cagtggcctc aggagagaac 1020

gtgaccctgc tgtgtcagtc atggtggcag tttgacactt tccttctgac caaagaaggg 1080

gcagcccatc ccccactgcg tctgagatca atgtacggag ctcataagta ccaggctgaa 1140

ttccccatga gtcctgtgac ctcagcccac gcggggacct acaggtgcta cggctcatac 1200

agctccaacc cccacctgct gtctttcccc agtgagcccc tggaactcat ggtctcagga 1260

cactctggag gctccagcct cccacccaca gggccgccct ccacacctgc ctcacacgcc 1320

aaggattaca cagtggagaa tctcatccgc atgggcatgg caggcttggt cctggtgttc 1380

ctcgggattc tgttatttga ggctcagcac agccagagaa acccccaaga tgcagccggg 1440

aggtga 1446

<210> 186

<211> 481

<212> PRT

<213> Intelligent people

<220>

<221> sources

<222> (1)..(481)

<223> LILRA6

<400> 186

Met Thr Pro Ala Leu Thr Ala Leu Leu Cys Leu Gly Leu Ser Leu Gly

1 5 10 15

Pro Arg Thr Arg Val Gln Ala Gly Pro Phe Pro Lys Pro Thr Leu Trp

20 25 30

Ala Glu Pro Gly Ser Val Ile Ser Trp Gly Ser Pro Val Thr Ile Trp

35 40 45

Cys Gln Gly Ser Leu Glu Ala Gln Glu Tyr Gln Leu Asp Lys Glu Gly

50 55 60

Ser Pro Glu Pro Leu Asp Arg Asn Asn Pro Leu Glu Pro Lys Asn Lys

65 70 75 80

Ala Arg Phe Ser Ile Pro Ser Met Thr Gln His His Ala Gly Arg Tyr

85 90 95

Arg Cys His Tyr Tyr Ser Ser Ala Gly Trp Ser Glu Pro Ser Asp Pro

100 105 110

Leu Glu Leu Val Met Thr Gly Phe Tyr Asn Lys Pro Thr Leu Ser Ala

115 120 125

Leu Pro Ser Pro Val Val Ala Ser Gly Gly Asn Met Thr Leu Arg Cys

130 135 140

Gly Ser Gln Lys Gly Tyr His His Phe Val Leu Met Lys Glu Gly Glu

145 150 155 160

His Gln Leu Pro Arg Thr Leu Asp Ser Gln Gln Leu His Ser Gly Gly

165 170 175

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

180 185 190

Arg Phe Thr Cys Tyr Tyr Tyr Tyr Thr Asn Thr Pro Arg Val Trp Ser

195 200 205

His Pro Ser Asp Pro Leu Glu Ile Leu Pro Ser Gly Val Ser Arg Lys

210 215 220

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

225 230 235 240

Leu Thr Leu Gln Cys Gly Ser Asp Val Gly Tyr Asp Arg Phe Val Leu

245 250 255

Tyr Lys Glu Gly Glu Arg Asp Phe Leu Gln Arg Pro Gly Gln Gln Pro

260 265 270

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

275 280 285

Ser His Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Leu Ser Ser

290 295 300

Glu Trp Ser Ala Pro Ser Asp Pro Leu Asn Ile Leu Met Ala Gly Gln

305 310 315 320

Ile Tyr Asp Thr Val Ser Leu Ser Ala Gln Pro Gly Pro Thr Val Ala

325 330 335

Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Arg Gly Tyr Phe Asp

340 345 350

Thr Phe Leu Leu Thr Lys Glu Gly Ala Ala His Pro Pro Leu Arg Leu

355 360 365

Arg Ser Met Tyr Gly Ala His Lys Tyr Gln Ala Glu Phe Pro Met Ser

370 375 380

Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser Tyr

385 390 395 400

Ser Ser Asn Pro His Leu Leu Ser Phe Pro Ser Glu Pro Leu Glu Leu

405 410 415

Met Val Ser Gly His Ser Gly Gly Ser Ser Leu Pro Pro Thr Gly Pro

420 425 430

Pro Ser Thr Pro Ala Ser His Ala Lys Asp Tyr Thr Val Glu Asn Leu

435 440 445

Ile Arg Met Gly Met Ala Gly Leu Val Leu Val Phe Leu Gly Ile Leu

450 455 460

Leu Phe Glu Ala Gln His Ser Gln Arg Asn Pro Gln Asp Ala Ala Gly

465 470 475 480

Arg

<210> 187

<211> 424

<212> PRT

<213> Intelligent people

<220>

<221> sources

<222> (1)..(424)

<223> LILRA6

<400> 187

Gly Pro Phe Pro Lys Pro Thr Leu Trp Ala Glu Pro Gly Ser Val Ile

1 5 10 15

Ser Trp Gly Ser Pro Val Thr Ile Trp Cys Gln Gly Ser Leu Glu Ala

20 25 30

Gln Glu Tyr Gln Leu Asp Lys Glu Gly Ser Pro Glu Pro Leu Asp Arg

35 40 45

Asn Asn Pro Leu Glu Pro Lys Asn Lys Ala Arg Phe Ser Ile Pro Ser

50 55 60

Met Thr Gln His His Ala Gly Arg Tyr Arg Cys His Tyr Tyr Ser Ser

65 70 75 80

Ala Gly Trp Ser Glu Pro Ser Asp Pro Leu Glu Leu Val Met Thr Gly

85 90 95

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

100 105 110

Ser Gly Gly Asn Met Thr Leu Arg Cys Gly Ser Gln Lys Gly Tyr His

115 120 125

His Phe Val Leu Met Lys Glu Gly Glu His Gln Leu Pro Arg Thr Leu

130 135 140

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

145 150 155 160

Gly Pro Val Thr Pro Ser His Arg Trp Arg Phe Thr Cys Tyr Tyr Tyr

165 170 175

Tyr Thr Asn Thr Pro Arg Val Trp Ser His Pro Ser Asp Pro Leu Glu

180 185 190

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

195 200 205

Gly Pro Val Leu Ala Pro Gly Gln Ser Leu Thr Leu Gln Cys Gly Ser

210 215 220

Asp Val Gly Tyr Asp Arg Phe Val Leu Tyr Lys Glu Gly Glu Arg Asp

225 230 235 240

Phe Leu Gln Arg Pro Gly Gln Gln Pro Gln Ala Gly Leu Ser Gln Ala

245 250 255

Asn Phe Thr Leu Gly Pro Val Ser Pro Ser His Gly Gly Gln Tyr Arg

260 265 270

Cys Tyr Gly Ala His Asn Leu Ser Ser Glu Trp Ser Ala Pro Ser Asp

275 280 285

Pro Leu Asn Ile Leu Met Ala Gly Gln Ile Tyr Asp Thr Val Ser Leu

290 295 300

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

305 310 315 320

Leu Cys Gln Ser Arg Gly Tyr Phe Asp Thr Phe Leu Leu Thr Lys Glu

325 330 335

Gly Ala Ala His Pro Pro Leu Arg Leu Arg Ser Met Tyr Gly Ala His

340 345 350

Lys Tyr Gln Ala Glu Phe Pro Met Ser Pro Val Thr Ser Ala His Ala

355 360 365

Gly Thr Tyr Arg Cys Tyr Gly Ser Tyr Ser Ser Asn Pro His Leu Leu

370 375 380

Ser Phe Pro Ser Glu Pro Leu Glu Leu Met Val Ser Gly His Ser Gly

385 390 395 400

Gly Ser Ser Leu Pro Pro Thr Gly Pro Pro Ser Thr Pro Ala Ser His

405 410 415

Ala Lys Asp Tyr Thr Val Glu Asn

420

<210> 188

<211> 675

<212> DNA

<213> mice

<220>

<221> sources

<222> (1)..(675)

<223> PILRbeta

<400> 188

atggctttgc tgatctcgct tcctggaggg actccagcca tggctcaggt cctgcttctg 60

ctctcatcag gctgtctgca tgctggaaat tcagaaagat acaacagaaa aaatggcttt 120

ggggtcaacc aacctgaacg ctgctctgga gtccagggtg gctccatcga catccccttc 180

tccttctatt tcccctggaa gttggccaag gatccacaga tgagcatagc ctggaaatgg 240

aaggatttcc atggggaagt catctacaac tcctccctgc ctttcataca tgagcacttc 300

aagggccggc tcatcctgaa ctggacacag ggtcagacat ctggagtcct cagaatcctg 360

aacttgaagg agtctgacca agcccagtac tttagtcgag ttaatctgca gtcgacagaa 420

ggcatgaagt tgtggcagtc aattcctgga acccaactca acgtgaccca agcactcaac 480

accaccatga ggagcccctt catcgtcacc tctgaattca ccacagctgg cctggagcac 540

acaagcgacc agaggaatcc ttcactgatg aacctgggag ccatggtcac gatgctcctg 600

gctaaagttt tggtcatagt cctagtctat ggatggatga tcttcctgag gtggaagcaa 660

aggccagcac actaa 675

<210> 189

<211> 224

<212> PRT

<213> mouse

<220>

<221> sources

<222> (1)..(224)

<223> PILRbeta

<400> 189

Met Ala Leu Leu Ile Ser Leu Pro Gly Gly Thr Pro Ala Met Ala Gln

1 5 10 15

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

20 25 30

Arg Tyr Asn Arg Lys Asn Gly Phe Gly Val Asn Gln Pro Glu Arg Cys

35 40 45

Ser Gly Val Gln Gly Gly Ser Ile Asp Ile Pro Phe Ser Phe Tyr Phe

50 55 60

Pro Trp Lys Leu Ala Lys Asp Pro Gln Met Ser Ile Ala Trp Lys Trp

65 70 75 80

Lys Asp Phe His Gly Glu Val Ile Tyr Asn Ser Ser Leu Pro Phe Ile

85 90 95

His Glu His Phe Lys Gly Arg Leu Ile Leu Asn Trp Thr Gln Gly Gln

100 105 110

Thr Ser Gly Val Leu Arg Ile Leu Asn Leu Lys Glu Ser Asp Gln Ala

115 120 125

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

130 135 140

Trp Gln Ser Ile Pro Gly Thr Gln Leu Asn Val Thr Gln Ala Leu Asn

145 150 155 160

Thr Thr Met Arg Ser Pro Phe Ile Val Thr Ser Glu Phe Thr Thr Ala

165 170 175

Gly Leu Glu His Thr Ser Asp Gln Arg Asn Pro Ser Leu Met Asn Leu

180 185 190

Gly Ala Met Val Thr Met Leu Leu Ala Lys Val Leu Val Ile Val Leu

195 200 205

Val Tyr Gly Trp Met Ile Phe Leu Arg Trp Lys Gln Arg Pro Ala His

210 215 220

<210> 190

<211> 24

<212> DNA

<213> mouse

<220>

<221> sources

<222> (1)..(24)

<223> intracellular domain of PILRbeta

<400> 190

aggtggaagc aaaggccagc acac 24

<210> 191

<211> 8

<212> PRT

<213> mouse

<220>

<221> sources

<222> (1)..(8)

<223> intracellular domain of PILRbeta

<400> 191

Arg Trp Lys Gln Arg Pro Ala His

1 5

<210> 192

<211> 900

<212> DNA

<213> Artificial sequence

<220>

<223> chimeric LILRB4-PLILRbeta receptor

<400> 192

atggatccca aaggatccct ttcctggaga atacttctgt ttctctccct ggcttttgag 60

ttgagctacg gactcgagca ggcagggccc ctccccaaac ccaccctctg ggctgagcca 120

ggctctgtga tcagctgggg gaactctgtg accatctggt gtcaggggac cctggaggct 180

cgggagtacc gtctggataa agaggaaagc ccagcaccct gggacagaca gaacccactg 240

gagcccaaga acaaggccag attctccatc ccatccatga cagaggacta tgcagggaga 300

taccgctgtt actatcgcag ccctgtaggc tggtcacagc ccagtgaccc cctggagctg 360

gtgatgacag gagcctacag taaacccacc ctttcagccc tgccgagtcc tcttgtgacc 420

tcaggaaaga gcgtgaccct gctgtgtcag tcacggagcc caatggacac ttttcttctg 480

atcaaggagc gggcagccca tcccctactg catctgagat cagagcacgg agctcagcag 540

caccaggctg aattccccat gagtcctgtg acctcagtgc acggggggac ctacaggtgc 600

ttcagctcac acggcttctc ccactacctg ctgtcacacc ccagtgaccc cctggagctc 660

atagtctcag gatccttgga gggtcccagg ccctcaccca caaggtccgt ctcaacagct 720

gcaggccctg aggaccagcc cctcatgcct acagggtcag tcccccacag tggtctgaga 780

aggcactggg agctcgagct gggagccatg gtcacgatgc tcctggctaa agttttggtc 840

atagtcctag tctatggatg gatgatcttc ctgaggtgga agcaaaggcc agcacactaa 900

<210> 193

<211> 299

<212> PRT

<213> Artificial sequence

<220>

<223> chimeric LILRB4-PLILRbeta receptor

<400> 193

Met Asp Pro Lys Gly Ser Leu Ser Trp Arg Ile Leu Leu Phe Leu Ser

1 5 10 15

Leu Ala Phe Glu Leu Ser Tyr Gly Leu Glu Gln Ala Gly Pro Leu Pro

20 25 30

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

35 40 45

Ser Val Thr Ile Trp Cys Gln Gly Thr Leu Glu Ala Arg Glu Tyr Arg

50 55 60

Leu Asp Lys Glu Glu Ser Pro Ala Pro Trp Asp Arg Gln Asn Pro Leu

65 70 75 80

Glu Pro Lys Asn Lys Ala Arg Phe Ser Ile Pro Ser Met Thr Glu Asp

85 90 95

Tyr Ala Gly Arg Tyr Arg Cys Tyr Tyr Arg Ser Pro Val Gly Trp Ser

100 105 110

Gln Pro Ser Asp Pro Leu Glu Leu Val Met Thr Gly Ala Tyr Ser Lys

115 120 125

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

130 135 140

Val Thr Leu Leu Cys Gln Ser Arg Ser Pro Met Asp Thr Phe Leu Leu

145 150 155 160

Ile Lys Glu Arg Ala Ala His Pro Leu Leu His Leu Arg Ser Glu His

165 170 175

Gly Ala Gln Gln His Gln Ala Glu Phe Pro Met Ser Pro Val Thr Ser

180 185 190

Val His Gly Gly Thr Tyr Arg Cys Phe Ser Ser His Gly Phe Ser His

195 200 205

Tyr Leu Leu Ser His Pro Ser Asp Pro Leu Glu Leu Ile Val Ser Gly

210 215 220

Ser Leu Glu Asp Pro Arg Pro Ser Pro Thr Arg Ser Val Ser Thr Ala

225 230 235 240

Ala Gly Pro Glu Asp Gln Pro Leu Met Pro Thr Gly Ser Val Pro His

245 250 255

Ser Gly Leu Arg Arg His Trp Glu Leu Glu Leu Gly Ala Met Val Thr

260 265 270

Met Leu Leu Ala Lys Val Leu Val Ile Val Leu Val Tyr Gly Trp Met

275 280 285

Ile Phe Leu Arg Trp Lys Gln Arg Pro Ala His

290 295

<210> 194

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> NFAT promoter

<400> 194

tgaaaatgaa aatgaaaa 18

<210> 195

<211> 954

<212> DNA

<213> Intelligent

<400> 195

atgaaggttc tgtgggctgc gttgctggtc acattcctgg caggatgcca ggccaaggtg 60

gagcaagcgg tggagacaga gccggagccc gagctgcgcc agcagaccga gtggcagagc 120

ggccagcgct gggaactggc actgggtcgc ttttgggatt acctgcgctg ggtgcagaca 180

ctgtctgagc aggtgcagga ggagctgctc agctcccagg tcacccagga actgagggcg 240

ctgatggacg agaccatgaa ggagttgaag gcctacaaat cggaactgga ggaacaactg 300

accccggtgg cggaggagac gcgggcacgg ctgtccaagg agctgcaggc ggcgcaggcc 360

cggctgggcg cggacatgga ggacgtgtgc ggccgcctgg tgcagtaccg cggcgaggtg 420

caggccatgc tcggccagag caccgaggag ctgcgggtgc gcctcgcctc ccacctgcgc 480

aagctgcgta agcggctcct ccgcgatgcc gatgacctgc agaagcgcct ggcagtgtac 540

caggccgggg cccgcgaggg cgccgagcgc ggcctcagcg ccatccgcga gcgcctgggg 600

cccctggtgg aacagggccg cgtgcgggcc gccactgtgg gctccctggc cggccagccg 660

ctacaggagc gggcccaggc ctggggcgag cggctgcgcg cgcggatgga ggagatgggc 720

agccggaccc gcgaccgcct ggacgaggtg aaggagcagg tggcggaggt gcgcgccaag 780

ctggaggagc aggcccagca gatacgcctg caggccgagg ccttccaggc ccgcctcaag 840

agctggttcg agcccctggt ggaagacatg cagcgccagt gggccgggct ggtggagaag 900

gtgcaggctg ccgtgggcac cagcgccgcc cctgtgccca gcgacaatca ctga 954

<210> 196

<211> 317

<212> PRT

<213> Intelligent people

<400> 196

Met Lys Val Leu Trp Ala Ala Leu Leu Val Thr Phe Leu Ala Gly Cys

1 5 10 15

Gln Ala Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu

20 25 30

Arg Gln Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu

35 40 45

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

50 55 60

Val Gln Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala

65 70 75 80

Leu Met Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu

85 90 95

Glu Glu Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser

100 105 110

Lys Glu Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp

115 120 125

Val Cys Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu

130 135 140

Gly Gln Ser Thr Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg

145 150 155 160

Lys Leu Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg

165 170 175

Leu Ala Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu

180 185 190

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

195 200 205

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

210 215 220

Ala Gln Ala Trp Gly Glu Arg Leu Arg Ala Arg Met Glu Glu Met Gly

225 230 235 240

Ser Arg Thr Arg Asp Arg Leu Asp Glu Val Lys Glu Gln Val Ala Glu

245 250 255

Val Arg Ala Lys Leu Glu Glu Gln Ala Gln Gln Ile Arg Leu Gln Ala

260 265 270

Glu Ala Phe Gln Ala Arg Leu Lys Ser Trp Phe Glu Pro Leu Val Glu

275 280 285

Asp Met Gln Arg Gln Trp Ala Gly Leu Val Glu Lys Val Gln Ala Ala

290 295 300

Val Gly Thr Ser Ala Ala Pro Val Pro Ser Asp Asn His

305 310 315

Claims (33)

1. A method of identifying a modulator of LILRB4 activation, the method comprising:

(a) Contacting a reporter cell with a ligand for LILRB4, which is ApoE, and a candidate substance, wherein the reporter cell expresses:

(i) LILRB4 or a receptor comprising the extracellular domain of LILRB4, and

(ii) A reporter gene encoding a detectable label operably linked to a promoter regulated by activation of LILRB4 or a receptor comprising the extracellular domain of LILRB 4; and

(b) Detecting a level of LILRB4 activation in the reporter cell, wherein a change in the level of LILRB4 activation compared to a reference level is indicative that the candidate substance is a modulator of LILRB4 activation, wherein the reference level is obtained from a reporter cell contacted with the ligand and not contacted with the candidate substance.

2. The method of claim 1, wherein the reporter cell expresses a receptor comprising the extracellular domain of LILRB4.

3. The method of claim 1, wherein the reporter cell is a mouse T-cell hybridoma cell.

4. The method of claim 2, wherein the receptor further comprises the intracellular domain of paired immunoglobulin-like receptor beta (PILR β).

5. The method of claim 1, wherein the receptor is expressed in the cell by a viral expression vector.

6. The method of claim 5, wherein the viral expression vector is a retroviral expression vector.

7. The method of claim 1, wherein the level of LILRB4 activation is detected based on morphology or mobility of the cells.

8. The method of claim 1, wherein the promoter is a nuclear factor of activated T cell (NFAT) promoter.

9. The method of claim 1, wherein the promoter is a CCL2 promoter, a CCL4 promoter, a CCL5 promoter, an IL-6R promoter, an IL-8 promoter, a gp130 promoter, an OSM promoter, a TIMP-1/2 promoter, a TNF-R1/II promoter, a uPAR promoter, or an arginase-1 promoter.

10. The method of claim 1, wherein the detectable label is luciferase in the presence of GFP, YFP, RFP, or D-luciferin.

11. The method of claim 1, wherein the detecting step comprises flow cytometry analysis or luminescence quantification.

12. The method of claim 1, wherein the candidate substance is an antibody.

13. The method of claim 12, wherein the antibody is a chimeric antibody, a CDR-grafted antibody, fab ', F (ab') 2, fv, or scFv.

14. The method of claim 13, wherein the antibody is a humanized antibody.

15. The method of claim 13, wherein the antibody is a monoclonal antibody.

16. The method of claim 1, wherein the reference level is obtained in the reporter cell when contacted with only the ligand of LILRB4.

17. The method of claim 1, wherein said ApoE is recombinant.

18. The method of claim 1, wherein the ApoE is human ApoE or mouse ApoE.

19. The method of claim 18 in which the human ApoE or mouse ApoE is isolated from human serum or mouse serum, respectively.

20. The method of claim 1, wherein said ApoE is ApoE2, apoE3 or ApoE4.

21. The method of claim 1, wherein an increase in the level of activation of LILRB4 as compared to the reference level indicates that the modulator is an agonist.

22. The method of claim 1, wherein a decrease in the level of LILRB4 activation as compared to the reference level indicates that the modulator is an antagonist.

23. The method of claim 1, wherein the candidate agent is linked to an FcR expressing cell.

24. A composition for identifying a modulator of LILRB4 activation, the composition comprising:

a candidate LILRB4 modulator;

ApoE; and

a reporter cell that expresses a receptor comprising the extracellular domain of LILRB4, wherein the reporter cell has a phenotype that suggests LILRB4 activation.

25. The composition of claim 24, wherein the candidate LILRB4 modulator is an antibody.

26. The composition of claim 24, wherein the receptor further comprises the intracellular domain of PILR β.

27. The composition of claim 24, wherein the reporter cell further comprises a reporter gene encoding a detectable marker and operably linked to a promoter regulated by activation of the receptor.

28. The composition of claim 27, wherein the detectable label is GFP.

29. The composition of claim 24, further comprising a cell that expresses FcR.

30. A composition for identifying an antagonist antibody for LILRB4 activation, the composition comprising:

candidate LILRB4 antibodies

A cell expressing FcR; and

a reporter cell that expresses a receptor comprising the extracellular domain of LILRB4, wherein the reporter cell has a phenotype that suggests LILRB4 activation.

31. The composition of claim 30, wherein the receptor further comprises the intracellular domain of PILR β.

32. The composition of claim 30, wherein the reporter cell further comprises a reporter gene, the reporter gene encoding a detectable marker and the reporter gene operably linked to a promoter regulated by activation of the receptor.

33. The composition of claim 32, wherein the detectable label is GFP.