LAIR-1 agonist antibody and method of use thereof

Abstract

A pharmaceutical composition for effectively eliminating cancer cells while avoiding normal, healthy cells. This pharmaceutical composition may contain a humanized monoclonal antibody that agonizes LAIR-1. The composition may also contain other therapeutic agents that act synergistically to treat cancers, including but not limited to acute myeloid leukemia (AML). The composition can be administered in various ways to subjects requiring it for treatment.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 485,848, filed on February 17, 2023, the entire content of which is incorporated herein by reference.

[0002] The present disclosure generally relates to the field of immunotherapy, and more specifically, to compositions and methods using LAIR - 1 antibodies.

Background Art

[0003] Acute leukemia is characterized by the uncontrolled production of malignant hematopoietic progenitor cells. Acute myeloid leukemia (AML) is the most common adult acute leukemia (ACS, Cancer Facts and Figures 2022). As a result of extensive research, new therapies for AML have recently been approved (Stanchina 2020), but the needs of patients who do not respond to standard of care (SoC) or who relapse after standard treatment remain significantly unmet. This may be mainly due to the persistence of leukemia stem cells (LSC) (van Gils et al., 2021).

[0004] LSC are leukemia - initiating cells at the apex of the hierarchy of myeloid leukemia cells with the ability of self - replication. LSC give rise to daughter leukemic blasts, develop leukemia when transplanted into immunodeficient animals, and propagate upon serial transplantation (Majeti et al 2017). LSC are an important target for next - generation therapeutics in AML because they cause recurrence and relapse in 50% of all patients who achieve remission after initial treatment due to their self - replication ability (Yilmaz et al., 2019).

[0005] Leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) is an immunoglobulin superfamily protein possessing two immunoreceptor tyrosine-dependent inhibitory motifs (ITIMs) that can sequester the Src homology domain 2-domain-containing phosphatase-1 (SHP-1) and C-terminal Src kinase (CSK) (Meyaard 2006, Meyaard 2008). LAIR-1 is limited to hematopoietic compartments, particularly myeloid cells, as well as T cells, B cells, and NK cells (Meyaard 2008). The IgV domain of LAIR-1 has a unique ability to specifically bind to collagen domain-containing ligands such as collagen, complement protein C1q, surfactant protein D (SP-D), mannose-binding lectin (MBL), and collectin-12 (Meyaard, 2008, Keerthivasan S, 2021). When a ligand binds to LAIR-1, receptor clustering leads to phosphorylation of the ITIM domain of LAIR-1, resulting in gradual phosphorylation of SHP-1 phosphatase and triggering downstream immunosuppressive signaling (Meyaard 2008). LAIR-1 has limited or redundant function in healthy cells and homeostatic environments, but weakens the immune response in non-homeostatic or diseased environments (Meyaard 2010, Son and Diamond 2014, Jin et al., 2018).

[0006] More recently, the function of LAIR-1 has been associated with the stem cell nature of cells and disease development in leukemia (Kang, Lu et al., 2015). Ligand-independent LAIR-1 constitutive phosphorylation and signaling, as well as the SHP-1 phosphorylation-independent pathway, have been described (Kang, Lu et al., 2015), highlighting the importance of the context of LAIR-1 function. Specifically, LAIR-1 on AML cells triggers downstream signaling via Ca++ / calmodulin-dependent protein kinase (CAMK1) and cAMP response element-binding protein (CREB) (Kang, Lu et al., 2015), which is involved in the persistence of AML stem cell activity (Kang, Lu et al., 2015; Kang, Kim et al., 2016). LAIR-1 is not necessarily required for normal hematopoiesis (Tang et al., 2012; Kang, Lu et al., 2015), but knockdown of LAIR-1 in human leukemia cells increases apoptosis in vitro and reduces the incidence of AML in mouse models (Kang, Lu et al., 2015).

[0007] In contrast, another study found that ligation of LAIR-1 on leukemia cells inhibits IκBα activation, preventing the translocation of nuclear factor kappa B (NF-κB) into the nucleus and leading to programmed cell death (Poggi et al., 2000). Indeed, follow-up studies have shown that receptor clustering blocks the proliferation of AML blasts and leads to subsequent cellular apoptosis, an effect dependent on SHP-1-mediated LAIR-1 ITIM signaling (Zocchi et al., 2001).

[0008] In this field, there is still a need to provide improved methods and compositions for selectively targeting LAIR-1 for the treatment of various cancers. [Overview of the project]

[0009] Please understand that this summary is not a comprehensive overview of the disclosure. This summary is illustrative and not limiting, and is not intended to identify or describe the scope of any key or essential elements of the disclosure. The sole purpose of this summary is to explain and illustrate certain concepts of the disclosure as an introduction to the full and extensive detailed description below.

[0010] This disclosure relates to a pharmaceutical composition containing an immunomodulator. The immunomodulator can bind to LAIR-1 as an agonist. In this manner, the composition effectively treats cancer by eliminating cancer cells while keeping normal, healthy cells at bay. In non-limiting examples, cancer cells include leukemia stem cells (LSCs) and leukemic blasts. In further non-limiting examples, healthy cells include normal hematopoietic stem cells. This agent may be used to treat all solid tumors and hematological malignancies. This agent may be an antibody, which may be a humanized monoclonal antibody. The humanized monoclonal antibody may be selected as a LAIR-1 mAb.

[0011] This disclosure relates to a humanized monoclonal antibody having a variable light chain having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a variable light chain having an amino acid sequence selected from the group of variable light chains consisting of SEQ ID NOs: 6, 7, 11, 13, 14, 15, and 16. According to one aspect of the present invention, a LAIR-1 antibody is provided that includes a variable light chain domain having 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 14 and 16. According to another aspect, a LAIR-1 antibody is provided that includes a light chain having 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 15 and 17.

[0012] This disclosure relates to a humanized monoclonal antibody having a variable heavy chain having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a variable heavy chain having an amino acid sequence selected from the group of variable heavy chains consisting of SEQ ID NOs: 8, 17, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, and 43. In one embodiment, a LAIR-1 antibody is provided that contains a variable heavy chain domain having 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 21, 24, 27, 30, 33, 36, 39, and 42. In another embodiment, LAIR-1 antibodies are provided that contain heavy chains having 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with respect to sequences selected from the group consisting of SEQ ID NOs: 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37, 38, 40, 41, 43, and 44.

[0013] This disclosure relates to a humanized monoclonal antibody having a variable light chain having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a variable light chain having an amino acid sequence selected from the variable light chain group consisting of SEQ ID NOs: 6, 7, 11, 13, 14, 15, and 16, and a variable heavy chain having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a variable heavy chain having an amino acid sequence selected from the variable heavy chain group consisting of SEQ ID NOs: 8, 17, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, and 43.

[0014] In one other embodiment, LAIR-1 antibodies are provided that include variable light chain domains and variable heavy chain domains having 95%, 96%, 98%, 99%, and 100% sequence identity with sequences selected from the group consisting of SEQ ID NOs: 14 and 21, 14 and 24, 14 and 27, 14 and 30, 14 and 33, 14 and 36, 14 and 39, 14 and 42.

[0015] In another embodiment, LAIR-1 antibodies are provided that include light and heavy chains having 95%, 96%, 98%, 99%, and 100% sequence identity with sequences selected from the group consisting of SEQ ID NOs: 15 and 22, 15 and 23, 15 and 25, 15 and 26, 15 and 28, 15 and 29, 15 and 31, 15 and 32, 15 and 34, 15 and 35, 15 and 37, 15 and 38, 15 and 40, 15 and 41, 15 and 43, 15 and 44, 17 and 22, 17 and 23, 17 and 25, 17 and 26, 17 and 28, 17 and 29, 17 and 31, 17 and 32, 17 and 34, 17 and 35, 17 and 37, 17 and 38, 17 and 40, 17 and 41, 17 and 43, and 17 and 44.

[0016] This disclosure relates to a humanized monoclonal antibody having a heavy chain having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to a heavy chain having an amino acid sequence selected from the group of heavy chains consisting of SEQ ID NOs: 9, 10, 18, and 19. In one embodiment, the present invention provides a LAIR-1 antibody comprising a variable heavy chain domain having 95%, 96%, 98%, 99%, and 100% sequence identity with respect to SEQ ID NO: 9. In another embodiment, the present invention provides a LAIR-1 antibody comprising an antigen-binding domain having 95%, 96%, 98%, 99%, and 100% sequence identity with respect to SEQ ID NO: 9.

[0017] This disclosure relates to a humanized monoclonal antibody having a light chain having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a light chain having an amino acid sequence selected from the light chain group consisting of SEQ ID NOs. 7 and 12.

[0018] This disclosure relates to a humanized monoclonal antibody comprising a light chain having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a light chain having an amino acid sequence selected from the light chain group consisting of SEQ ID NOs: 7 and 12, and a heavy chain having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a heavy chain having an amino acid sequence selected from the heavy chain group consisting of SEQ ID NOs: 9, 10, 18, and 19.

[0019] According to one other embodiment, a LAIR-1 antibody is provided comprising a variable light chain domain having 95%, 96%, 98%, 99%, and 100% sequence identity with respect to SEQ ID NO: 7. According to another embodiment, a LAIR-1 antibody is provided comprising an antigen-binding domain having 95%, 96%, 98%, 99%, and 100% sequence identity with respect to SEQ ID NO: 7.

[0020] This disclosure relates to a pharmaceutical composition comprising one or more therapeutic agents, in addition to an immunomodulatory agent disclosed herein. In non-limiting examples, the therapeutic agents include venetoclax, azacitidine, and a CD47 antibody. Combinations of immunomodulatory agents with one or more therapeutic agents have synergistic effects in the treatment of cancer, including but not limited to acute myeloid leukemia (AML). In one embodiment, the present invention provides a combination of therapeutic agents comprising venetoclax, azacitidine, and a CD47 antibody, in combination with any one of the LAIR-1 antibodies of any of the above sequences.

[0021] This disclosure relates to a method for treating a subject in need by administering a pharmaceutical composition comprising an effective amount of an immunomodulator for binding to LAIR-1 as disclosed herein. In one embodiment, a method is provided for treating a subject in need by administering a pharmaceutical composition comprising an antibody to LAIR-1 of any one of the sequences described above. In one embodiment, the subject to be treated is suffering from carcinoma, squamous cell carcinoma, leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt lymphoma, acute myeloid leukemia, chronic myeloid leukemia, promyelocytic leukemia, fibrosarcoma, rhabdomyosarcoma, malignant melanoma, seminomas, teratocarcinoma, neuroblastoma, glioma, astrocytoma, neuroblastoma, glioma, schwannoma, fibrosarcoma, rhabdomyosarcoma, osteosarcoma, xeroderma pigmentosum, keratosacral cell tumor, seminomas, follicular carcinoma of the thyroid gland, or teratocarcinoma. In another embodiment, the subject being treated is suffering from acute myeloid leukemia. Administration can be carried out by any method known in the art, including but not limited to parenteral administration, oral administration, and topical administration.

[0022] This disclosure relates to a kit of the compositions disclosed herein. The kit may comprise one or more agents, including but not limited to one LAIR-1 antibody of any of the sequences provided herein, venetoclax, azacitidine, and anti-CD47, or a combination thereof.

[0023] The features and components shown in the following figures are presented to highlight the general principles of this disclosure. Throughout the drawings, corresponding features and components may be designated by matching reference letters for consistency and clarity. [Brief explanation of the drawing]

[0024] [Figure 1A] This shows targeted acute myeloid leukemia (AML) therapy. It displays LAIR-1 transcription levels in AML patient samples clustered according to the France-America-UK (FAB) classification, measured by RNA-Seq and quantified with RSEM software. UD = undiagnosed (non-AML), N = 1–47 patient samples per group. [Figure 1B] This shows targeted acute myeloid leukemia (AML) therapy. It shows molecular mutations. UD = undiagnosed (non-AML), N = 1 to 47 patient samples per group. [Figure 1C] This illustrates targeted acute myeloid leukemia (AML) therapy. The diagram shows leukocytogenesis from leukemia stem cell (LSC) precursors to granulocyte-monocyte progenitor cells (GMP-like) LSCs, lymphoid pluripotent progenitor cells (LMP-like) LSCs, or pluripotent progenitor cells (MPP-like) LSCs. [Figure 1D] This shows targeted acute myeloid leukemia (AML) therapy. It also shows the average fluorescence intensity of LAIR-1 cell surface expression in the LSC subpopulation. [Figure 1E] This illustrates targeted acute myeloid leukemia (AML) therapy. It shows a diagram of normal hematopoiesis from healthy stem cells (HSCs) to pluripotent progenitor cells (MPPs), common lymphoid progenitor cells (CLPs), common myeloid progenitor cells (CMPs), or granulocyte-monocyte progenitor cells (GMPs). [Figure 1F] This shows targeted acute myeloid leukemia (AML) therapy. It also shows the average fluorescence intensity of LAIR-1 cell surface expression in the HSC subpopulation. [Figure 1G] This study illustrates targeted acute myeloid leukemia (AML) therapy. It compares LAIR-1 cell surface expression in cells from equivalent compartments derived from AML patients or healthy donors (H). Each dot represents a unique donor / patient. n=7 healthy donors or 25 AML patients. P-values ​​are determined by Student's t-test. Data are presented as mean ± SEM. [Figure 2A] This demonstrates that LAIR-1 binding effectively removes LSCs while suppressing normal hematopoiesis. Representative images of LSC colonization ex vivo under the indicated therapeutic doses are shown. [Figure 2B] This shows that LAIR-1 binding effectively removed LSCs while suppressing normal hematopoiesis. The figures show colony-forming units (CFUs) formed by ex vivo dissemination of LSCs from the indicated AML donor during dose-controlled treatment with an anti-LAIR-1 agonist mAb. N = 3 technical replicates per group. P-values ​​were determined by one-way ANOVA using multiple comparisons. [Figure 2C] This indicates that LAIR-1 binding effectively removed LSCs while suppressing normal hematopoiesis. It shows CFU formation derived from bone marrow of healthy donors or AML patients treated with 5 ug / mL LAIR-1 mAb. Values ​​are normalized to isotype controls. N = 5 healthy biological replicates or 14 AML biological replicates. P-values ​​are determined by Student's t-test. Error bars represent the standard error of the mean. [Figure 3A] This study demonstrates that LAIR-1 binding eradicated primary and secondary AML in patient-derived xenograft models. A schematic diagram of a xenograft (PDX) model derived from an AML patient and a representative scatter plot of circulating human (H)CD33+CD45+ leukemia cells at a specified time after engraftment are shown. [Figure 3B] This study demonstrates the eradication of primary and secondary AML in patient-derived xenograft models by LAIR-1 conjugation. It shows leukemic proliferation, measured by the percentage of circulating HCD33+ HCD45+ cells, in PDX mice engrafted with bone marrow from donors with normal karyotype AML, monocytic AML, acute myelomonocytic leukemia (AMML), Flt3 ITD+ AML, or uncharacterized AML. Engrafted mice were treated with 5 mg / kg of IgG isotype control (gray) or LAIR-1 mAb (red). N = 3-5 mice per group. [Figure 3C] This shows that LAIR-1 binding eradicated primary and secondary AML in patient-derived xenograft models. A schematic diagram of a PDX secondary transplant model is shown, in which bone marrow was harvested from PDX mice that had engrafted and been treated as described above, and secondary transplanted into tumor- and treatment-naive recipient mice. The graph shows leukemia proliferation in secondary recipient mice after bone marrow administration from AMML PDX animals (top) or normal karyotype AML PDX animals (bottom) treated with 5 mg / kg IgG isotype control (gray) or LAIR-1 mAb (red). N = 3 mice per group. P-values ​​are calculated by two-way ANOVA. Error bars represent the standard error of the mean. [Figure 4A]This study demonstrates that the collagen matrix is ​​essential for LAIR-1-induced AML cell death. Flow cytometry quantification of primary whole blood leukocytes (live, dead, or apoptotic cells) after ex vivo treatment with 10 ug / mL IgG isotype control (gray) or LAIR-1 mAb (red) in the presence of exogenous collagen is shown. N=3 technical replicates. [Figure 4B] This study demonstrates that the collagen matrix is ​​essential for LAIR-1-induced AML cell death. Flow cytometry quantification of primary whole blood leukocytes (live, dead, or apoptotic cells) after ex vivo treatment with 10 ug / mL IgG isotype control (gray) or LAIR-1 mAb (red) in the absence of exogenous collagen is shown. N=3 technical replicates. [Figure 4C] This study demonstrates that the collagen matrix is ​​essential for LAIR-1-induced AML cell death. Flow cytometry quantification of CD45LoSSCLo blast cells in the presence of isotype controls or LAIR-1 mAbs is shown. N=3 technical replicates. [Figure 4D] This shows that the collagen matrix is ​​essential for LAIR-1-induced AML cell death. The percentage of dead cells (normalized to isotype control) derived from whole blood leukocytes (left) or CD45LoSSCLo blasts (right) after treatment with 10 ug / mL LAIR-1 mAb in the presence of collagen is shown and graphed as a function of LAIR-1 surface expression. Each dot represents an individual donor. Red shading highlights surface expression of any number of units greater than 20,000. Blue shading highlights expression of less than 20,000 units. The line represents a simple linear regression. [Figure 4E] This study demonstrates that the collagen matrix is ​​essential for LAIR-1-induced AML cell death. The images show total viable cells (left) or CD45LoSSCLo blast cells (right) from healthy blood donors or AML patient donors after ex vivo treatment with 10 ug / mL LAIR-1 mAb (normalized to isotype control) in the presence of collagen. N=4–7 donors. [Figure 4F]This study demonstrates that the collagen matrix is ​​essential for LAIR-1-induced AML cell death. Phosphorylated SHP-1 is shown in PBMCs from AML patients cultured under the indicated conditions. Mean pixel density is obtained from Western blotting, normalized to isotype control conditions and graphed against total SHP-1. N=3 technical replicates. [Figure 4G] This study demonstrates that the collagen matrix is ​​essential for LAIR-1-induced AML cell death. For MAPK activity, the mean pixel density from phosphate arrays of PBMCs from AML patients ex vivo-treated with 10 ug / mL IgG isotype control (gray), 50 ug / mL coated collagen (white), or 10 ug / mL LAIR-1 mAb+ coated collagen (red) is shown. N = 2 technical replicates. [Figure 4H] This study demonstrates that the collagen matrix is ​​essential for LAIR-1-induced AML cell death. Mean pixel density from phosphate arrays of PBMCs from AML patients ex vivo-treated with 10 ug / mL IgG isotype control (gray), 50 ug / mL coated collagen (white), or 10 ug / mL LAIR-1 mAb+ coated collagen (red) is shown for mTOR and NF-κB activity. N = 2 technical replicates. [Figure 4I] This demonstrates that the collagen matrix is ​​essential for LAIR-1-induced AML cell death. A schematic diagram of LAIR-1 mAb crosslinking and LAIR-1 clustering is shown, using anti-IgG to crosslink the Fc domain of LAIR-1 mAbs upon binding to cell surface LAIR-1. [Figure 4J] This demonstrates that the collagen matrix is ​​essential for LAIR-1-induced AML cell death. It shows in vitro proliferation of MV4-11-LAIR-1 overexpressing cells treated with 10 ug / mL IgG isotype control (gray) or LAIR-1 mAb (red) in the absence of anti-IgG crosslinking. [Figure 4K]This demonstrates that the collagen matrix is ​​essential for LAIR-1-induced AML cell death. It shows in vitro proliferation of MV4-11-LAIR-1 overexpressing cells treated with 10 ug / mL IgG isotype control (gray) or LAIR-1 mAb (red) in the presence of anti-IgG crosslinking. [Figure 4L] This demonstrates that the collagen matrix is ​​essential for LAIR-1-induced AML cell death. As shown in Figures 4I-4K, annexin V staining of treated cells is shown, and a representative scatter plot of apoptotic annexin V+ Live-Dead Aqua- cells on day 3 of culture is presented. [Figure 4M] This demonstrates that the collagen matrix is ​​essential for LAIR-1-induced AML cell death. Figures 4I-4K show 4E-BP1 expression in treated cells as measured by the Lumit assay. [Figure 4N] This demonstrates that the collagen matrix is ​​essential for LAIR-1-induced AML cell death. Figures 4I–4K show the cleaved caspase-7 measured by Western blotting of treated cells. The average pixel density is normalized to histone 3 for the three samples. [Figure 4O] This study demonstrates that the collagen matrix is ​​essential for LAIR-1-induced AML cell death. It shows the percentage of TUNEL+MV4-11-LAIR-1 overexpressing cells on day 3 of treatment with DMSO vehicle, 50 μM 220509-74-0 (caspase-3 / 7 inhibitor), or 50 μM MHY1485 (mTOR activator). Values ​​are normalized to isotype for each condition. n=3-4 technical replicates. P-values ​​are calculated by Student's t-test. Error bars represent SEM. [Figure 5A] This shows that LAIR-1 binding by LAIR-1 mAbs systemically reduced AML proliferation that was dependent on LAIR-1 expression levels but did not require or affect immune cells. A schematic diagram of a xenotransplant (CDX) model derived from AML MV4-11-luciferase cells or THP-1-luciferase cells is shown. [Figure 5B] This shows that LAIR-1 binding by LAIR-1 mAb systemically reduced AML proliferation that was dependent on LAIR-1 expression levels but did not require or affect immune cells. The image shows in vivo leukemia proliferation measured by whole-body luminescence of MV4-11-luciferase cells (left) or THP-1-luciferase cells (right) in CDX mice treated with 10 mg / kg IgG isotype control (gray) or LAIR-1 mAb (red). N = 8 mice per group. P-values ​​were determined by two-way ANOVA. [Figure 5C] This shows that LAIR-1 binding by LAIR-1 mAbs systemically reduced AML proliferation, which is dependent on LAIR-1 expression levels but does not require or affect immune cells. The graphs show the number of MV4-11 cells in the blood, spleen, or bone marrow of CDX mice treated with vehicle control (gray) or 10 ug / mL LAIR-1 mAb (red). [Figure 5D] This shows that LAIR-1 binding by LAIR-1 mAbs systemically reduced AML proliferation, which is dependent on LAIR-1 expression levels but does not require or affect immune cells. The percentage of MV4-11 cells in the blood, spleen, or bone marrow of CDX mice treated with vehicle control (gray) or 10 ug / mL LAIR-1 mAb (red). [Figure 5E] This shows that LAIR-1 binding by LAIR-1 mAb systemically reduced AML proliferation, which is dependent on LAIR-1 expression levels but does not require or affect immune cells. Total cell count or mouse (M)CD45+ cell count in the bone marrow of CDX mice treated with vehicle control (gray) or 10 ug / mL LAIR-1 mAb (red). N = 9-10 mice per group. [Figure 5F] This shows that LAIR-1 binding by LAIR-1 mAbs systemically reduced AML proliferation that was dependent on LAIR-1 expression levels but did not require or affect immune cells. Representative histograms of LAIR-1 cell surface expression in the indicated cell lines are shown. [Figure 5G] This shows that LAIR-1 binding by LAIR-1 mAbs systemically reduced AML proliferation that was dependent on LAIR-1 expression levels but did not require or affect immune cells. A schematic diagram of the CDX model system for testing the inhibition of leukemia proliferation as a function of LAIR-1 expression is shown. [Figure 5H] This shows that LAIR-1 binding by LAIR-1 mAb systemically reduced AML proliferation that was dependent on LAIR-1 expression levels but did not require or affect immune cells. The left image shows the percentage of inhibition of in vivo MV4-11-LAIR-1 knockout (green), MV4-11-LAIR-1 wild-type (purple), or MV4-11-LAIR-1 overexpressing cell proliferation (normalized to each isotype control) after treatment with 10 mg / kg mL of LAIR-1 mAb, and the right image shows the plot against the geometric mean fluorescence intensity of LAIR-1. [Figure 5I] This shows that LAIR-1 binding by LAIR-1 mAb systemically reduced AML proliferation that was dependent on LAIR-1 expression levels but did not require or affect immune cells. The percentage of inhibition of MV4-11 proliferation is shown, fitted to a log regression curve of LAIR-1 expression. The P-value is determined by Student's t-test. Error bars represent the standard error of the mean. [Figure 6A] This study demonstrates that LAIR-1 signaling restricts the AML survival signaling pathway in vivo. A schematic diagram and line graph of tumor growth are shown for an in vivo MV4-11 subcutaneous model in mice treated with 10 mg / kg isotype control (gray) or the anti-LAIR-1 agonist mAb LAIR-1 mAb (red). N = 3 mice per group. [Figure 6B] This study demonstrates that LAIR-1 signaling restricts the AML survival signaling pathway in vivo. A schematic diagram and line graph of MV4-11-luciferase proliferation in CDX mice, used for digital spatial imaging and target protein quantification, are shown. N = 6 mice per group. [Figure 6C] This study demonstrates that LAIR-1 signaling restricts the AML survival signaling pathway in vivo. Representative images of CDX mouse bone stained with DAPI and anti-human CD45 are shown, used to quantify the number of MV4-11 cells in the indicated region of interest (ROI) (highlighted). ROI areas are equal across samples. [Figure 6D] This study demonstrates that LAIR-1 signaling restricts the AML survival signaling pathway in vivo. Protein reads of human CD45, BCL-XL, or uncleaved PARP are shown, derived from bone or spleen isolated from CDX mice 22 days post-engraftment. Read counts are normalized for histone H3 and ribosomal protein S6. N=3–11 quantified tissue regions across two isotype-treated mice or two LAIR-1 mAb-treated mice. Error bars represent the mean standard error. P-values ​​are determined by Student's t-test. [Figure 7A] This shows that NC525 has a synergistic effect with standard treatment for AML. Ex vivo cytotoxicity of leukemia cells from AML patients treated with VEN / AZA. Values ​​are normalized to vehicle control. The line represents the linear regression of NC525 concentration against normalized cytotoxicity. [Figure 7B] This shows that NC525 has a synergistic effect with standard treatment for AML. Ex vivo killing of T cells or NK cells from AML patients treated with VEN / AZA. Values ​​are normalized to vehicle control. The line represents the linear regression of NC525 concentration against normalized cell killing. [Figure 7C] This study demonstrates that NC525 has a synergistic effect with standard AML treatments. It shows LAIR-1 surface expression on leukemia cells, T cells, or NK cells from AML patients treated with VEN / AZA. [Figure 7D]This study demonstrates that NC525 synergistically interacts with standard AML treatments. It shows in vivo leukemic proliferation measured by whole-body luminescence of MV4-11-luciferase cells in CDX mice treated with 10 mg / kg isotype control (gray) or NC525 (red), or 100 mg / kg VEN (pink), or 0.5 mg / kg AZA (blue), or combination therapy (green or yellow, respectively). BLoD is below the detection limit. n = 9 mice per group. P-values ​​were determined by two-way ANOVA or log-rank (Mantel-Cox) test, respectively. [Figure 7E] This study demonstrates that NC525 synergistically interacts with standard AML treatments. It shows in vivo leukemia proliferation measured by whole-body luminescence in viable cells of CDX mice treated with 10 mg / kg isotype control (gray) or NC525 (red), or 100 mg / kg VEN (pink), or 0.5 mg / kg AZA (blue), or combination therapy (green or yellow, respectively). BLoD is below the detection limit. n = 9 mice per group. P-values ​​were determined by two-way ANOVA or log-rank (Mantel-Cox) test, respectively. [Figure 7F] This study demonstrates that NC525 synergistically interacts with standard AML treatments. Leukemia proliferation in the blood of AML PDX mice treated with vehicle (gray), VEN / AZA (blue), NC525 (red), or VEN / AZA + NC525 (green) at 8 weeks post-transplantation is shown. n = 5–10 mice per group. AML cells from the spleen and brain masses are compared from 4–5 mice per group. Data are presented as mean ± SEM. P-values ​​are determined by two-way ANOVA or one-way ANOVA with multiple comparisons. [Figure 7G]This study demonstrates that NC525 synergistically interacts with standard AML treatments. It shows leukemic proliferation in the spleen and BM of AML PDX mice treated with vehicle (gray), VEN / AZA (blue), NC525 (red), or VEN / AZA + NC525 (green) at 8 weeks post-transplantation. n = 5–10 mice per group. AML cells from the spleen and BM are compared from 4–5 mice per group. Data are presented as mean ± SEM. P-values ​​are determined by two-way ANOVA or one-way ANOVA with multiple comparisons. [Figure 8] This is a schematic diagram of LAIR-1-induced cell death in leukemia cells. Binding of LAIR-1 on AML cells by the agonist mAb LAIR-1 mAb induces an inhibitory signal that blocks abnormal mTOR activity, suppressing constitutively active MAPK signaling and the self-renewal mechanism promoted by AKT and NF-κB. This loss of proliferative signaling induces BCL-XL deactivation, leading to the release of an apoptosis cascade via caspase-7 and PARP, and ultimately terminating in programmed cell death. [Figure 9] This shows the analysis of LAIR-1 cell surface expression. It is a schematic gating diagram for quantifying LAIR-1 in a subpopulation of bone marrow cells from primary patients. [Figure 10A] This document describes the characterization of LAIR-1 agonist monoclonal antibodies. A schematic diagram of the human (H)LAIR-1 reporter cell line UT-140 is shown (left). Human LAIR-1 fused to the CD3 zeta chain was introduced into UT-140 cells expressing GFP under an NFAT promoter. When LAIR-1 binds, signal transduction activates GFP fluorescence. [Figure 10B] This section describes the characterization of LAIR-1 agonist monoclonal antibodies. It also shows the binding profile of LAIR-1 mAbs to LAIR-1+UT-140 cells. [Figure 10C] This section describes the characterization of LAIR-1 agonist monoclonal antibodies. It also shows the binding profile of LAIR-1 mAb to parental mAb on mouse (M) LAIR-1 expressed on the cell surface. [Figure 10D]This document presents the characterization of LAIR-1 agonist monoclonal antibodies. It shows the profile of LAIR-1 ligand collagen-1 blockade by LAIR-1 mAbs, measured by UT-140 reporter cell activation. Isotype treatments are indicated by gray circles. LAIR-1 mAb-parental mAb treatments are indicated by red squares. [Figure 10E] This document describes the characterization of LAIR-1 agonist monoclonal antibodies. It also shows the activation profiles of UT-140 LAIR-1 reporter cells induced by LAIR-1 mAb or collagen under the indicated conditions. [Figure 10F] This document describes the characterization of a LAIR-1 agonist monoclonal antibody. Western blot (left) and quantification (pixel density normalized to histone H3) (right) of phosphorylated SHP-1 in blood monocytes from healthy donors treated under the indicated conditions are shown. Each line represents an individual donor. The collagen matrix is ​​essential for LAIR-1-induced AML cell death. [Figure 11] Representative flow cytometry and scatter plots of primary whole blood leukocytes (live, dead, or apoptotic cells) after ex vivo treatment with 10 ug / mL IgG isotype control (gray) or LAIR-1 mAb (red) in the presence or absence of exogenous collagen (A) or in the absence of exogenous collagen (B), or of CD45LoSSCLo blast cells in the presence of isotype control or LAIR-1 mAb (C). [Figure 12A] This shows LAIR-1 mAb-induced and collagen-induced phosphorylation signaling. A human phosphokinase array dot blot is also shown. [Figure 12B] This shows LAIR-1 mAb-induced and collagen-induced phosphorylation signaling. A dot blot of a human phosphoimmunoreceptor array is shown, along with the respective keys for PBMCs in AML patients treated with 10 ug / mL isotype control, LAIR-1 agonist mAb, 50 ug / mL collagen-1, and isotype control or collagen-1 and LAIR-1 mAb. [Figure 13]This shows LAIR-1 expression in AML cell lines. The histograms show LAIR-1 cell surface expression (blue) in the indicated AML cell lines compared to isotype control staining (red). [Figure 14] A and B show that LAIR-1 monoclonal antibodies do not affect healthy leukocytes. A shows a schematic diagram of a model system for defining the effect of LAIR-1 agonist mAbs on human (H) immune cells in vivo. B shows the cell count of human CD45 cells or human CD3 cells in the spleen or bone marrow of engrafted mice treated with the vehicle (gray) or 10 mg / kg anti-LAIR-1 agonist mAb. N = 7 mice per group. P-values ​​are determined by Student's t-test. Error bars represent the standard error of the mean. [Figure 15] This study demonstrates that LAIR-1 promotes cell survival in acute myeloid leukemia (AML). U937-RF-luc LAIR1 wild-type or knockout cells and MV-4-11-RF-luc LAIR1 WT or KO cells were cultured at 1 e5 / well for 48 hours. 10 μL of XTT mix was added, and the cells were incubated for a further 4 hours. Absorbance was measured at 450 nm as a measure of cell proliferation. [Figure 16] A and B demonstrate LAIR-1 mAb and anti-AML mechanisms. A shows LAIR-1-mediated survival of AML LSCs and blasts, where LAIR-1 is expressed on AML cells and interacts with its native ligand (C1Q, collagen) to promote the survival of AML LSCs and blasts. B shows diverse LAIR-1 mAb anti-leukemic activity, where blocking of the native ligand LAIR-1 mAb that binds to AML cells inhibits survival signals, and LAIR-1 mAb induces AML killing through Fc receptor-dependent mechanisms such as ADCP and ADCC. [Figure 17]A-C show the forms of LAIR-1 highly expressed in AML blasts and leukemia stem cells (LSCs). B shows LAIR-1 mRNA expression that does not change between AML subsets with LAIR-1 mRNA expression in peripheral blood AML blasts, according to the disease subtype and mutation status defined by the FAB classification. UD means undecided. C shows that LAIR-1 is highly expressed in blasts / LSCs but not in HSPCs. C shows flow cytometry analysis of LAIR-1 protein expression on the cell surface of leukemia stem cells (LSCs, CD34+CD38-CD90-CD45RA+ / - or CD34-CD117+CD244+ / -) and hematopoietic stem cells and progenitor cells (HSPCs, CD34+CD38-CD90+CD99-) derived from bone marrow aspirates of AML patients, as well as leukemia blasts derived from peripheral blood. [Figure 18] Figures A and B show that LAIR-1 expression is lower in healthy donors than in AML. Figure A shows interaction with hematopoietic stem cells (HSCs). Figure B shows that LAIR-1 is expressed at the AML blast gate (SSCloCD45lo) in both AML and healthy whole blood samples, and that healthy donor progenitor cells are predominantly CD34- and have less LAIR-1 than AML (MFI). LAIR1 protein levels were measured by flow cytometry. [Figure 19] Figures A and B show that LAIR-1 is expressed in CD33+ / -CD34+ / - myeloid progenitor cells in AML bone marrow. In Figure A, frozen bone marrow cells from AML patients were thawed, stained with CD33, CD34, and LAIR-1 antibodies, and expression was measured using flow cytometry. All myeloid progenitor cells and leukemia stem cells derived from the CD33 and CD34 populations of AML bone marrow showed LAIR-1 expression. LAIR-1 was most highly expressed in the CD33+CD34- population. [Figure 20A]This describes a humanized 11B3 LAIR-1 mAb that exhibits strong binding and signaling blockade. The h11B3 LAIR-1 mAb contains the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27, as shown for anti-LAIR-1 variant 6 in Table 1 of this specification. Such a LAIR-1 mAb exhibits in vitro activity with KD=2.1nM, cross-reactivity with Cyno=1.2nM, cell binding EC50=0.15nM, and signaling blockade IC50=0.25nM. In vivo activity includes efficacy in therapeutic NSG mouse models challenged with AML cell lines MV-411 and THP-1, efficacy in therapeutic NSG-SGM3 mouse models challenged with human primary AML cells, and selectivity for leukemia cells in NSG-SGM3 mouse models without removal, inhibition, or proliferation of healthy human primary immune cells. It exhibits binding to human LAIR-1+ cells. [Figure 20B] This describes a humanized 11B3 LAIR-1 mAb that exhibits strong binding and signaling blockade. The h11B3 LAIR-1 mAb contains the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27, as shown for anti-LAIR-1 variant 6 in Table 1 of this specification. Such a LAIR-1 mAb exhibits in vitro activity with KD=2.1nM, cross-reactivity with Cyno=1.2nM, cell binding EC50=0.15nM, and signaling blockade IC50=0.25nM. In vivo activity includes efficacy in therapeutic NSG mouse models challenged with AML cell lines MV-411 and THP-1, efficacy in therapeutic NSG-SGM3 mouse models challenged with human primary AML cells, and selectivity for leukemia cells in NSG-SGM3 mouse models without removal, inhibition, or proliferation of healthy human primary immune cells. It also exhibits binding to cynomolgus monkey LAIR-1+ cells. [Figure 20C]This invention presents a humanized 11B3 LAIR-1 mAb exhibiting strong binding and signaling blockade. The h11B3 LAIR-1 mAb contains the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27, as shown for anti-LAIR-1 variant 6 in Table 1 of this specification. Such a LAIR-1 mAb exhibits in vitro activity with KD=2.1nM, cross-reactivity with Cyno=1.2nM, cell binding EC50=0.15nM, and signaling blockade IC50=0.25nM. In vivo activity includes efficacy in therapeutic NSG mouse models challenged with AML cell lines MV-411 and THP-1, efficacy in therapeutic NSG-SGM3 mouse models challenged with human primary AML cells, and selectivity for leukemia cells in NSG-SGM3 mouse models without removal, inhibition, or proliferation of healthy human primary immune cells. It exhibits ligand-mediated signaling blockade. [Figure 20D] This invention presents a humanized 11B3 LAIR-1 mAb exhibiting strong binding and signaling blockade. The h11B3 LAIR-1 mAb contains the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27, as shown for anti-LAIR-1 variant 6 in Table 1 of this specification. Such a LAIR-1 mAb exhibits in vitro activity with KD=2.1nM, cross-reactivity with Cyno=1.2nM, cell binding EC50=0.15nM, and signaling blockade IC50=0.25nM. In vivo activity includes efficacy in therapeutic NSG mouse models challenged with AML cell lines MV-411 and THP-1, efficacy in therapeutic NSG-SGM3 mouse models challenged with human primary AML cells, and selectivity for leukemia cells in NSG-SGM3 mouse models without removal, inhibition, or proliferation of healthy human primary immune cells. It also exhibits ligand-mediated collagen blockade. [Figure 21]When measured by flow cytometry, h11B3 LAIR-1 mAb exhibits LAIR-1 binding properties, including binding to cells expressing human LAIR-1 or cynomolgus monkey LAIR-1. The h11B3 LAIR-1 mAb contains the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 21, as shown for anti-LAIR-1 variant 6 in Table 1 of this specification. A shows the binding of h11B3 to a 293T cell line overexpressing human LAIR-1. Cells were incubated on ice for 30 minutes with titration-level h11B3. AF647-conjugated secondary antibody was used at a 1:2000 dilution. B shows the binding of h11B3 to a 293T cell line overexpressing cynomolgus monkey LAIR-1. Cells were incubated on ice for 30 minutes with titration-level h11B3. AF647-conjugated secondary antibody was used at a 1:2000 dilution. [Figure 22] This shows the binding analysis of h11B3 LAIR-1 mAb(A) compared to its parent mAb 11B3(B). The h11B3 LAIR-1 mAb contains the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27, as shown for anti-LAIR-1 variant 6 in Table 1 herein. C shows cell binding analysis against MV4-11 cells. LAIR-1 expressing MV4-11 cells were blocked on ice for 10 minutes with 2% human serum + 2% mouse serum + 2% goat serum in FACs buffer, stained on ice for 30 minutes with the indicated concentrations of AF647-conjugated mAb, washed three times with FACs buffer, and then analyzed by flow cytometry. FACs buffer = phosphate-buffered saline + 2% fetal bovine serum + 1 mM EDTA. FC-G1 BioXcell Ref #BE0096. [Figure 23]A and B show molecular modeling and docking of h11B3 LAIR-1 mAbs, including the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27, which include in silico structure and interaction analysis of LAIR1 and h11B3, and predict the amino acid residues of h11B3 that bind to LAIR1. LAIR-1 amino acid residues including R28, S30, T37, R50, A77, S80, E81, D114, and Y115 were predicted to interact with h11B3. Analysis was performed using MOE software version 2020.09. B shows LAIR1 with the predicted h11B3v6 epitope in yellow. [Figure 24] A-C show that h11B3 LAIR-1 mAbs containing the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27 bind to LAIR-1 and block its functional signaling triggered by collagen-I in UT-140 reporter cells. 1e4 UT140 reporter cells / 100 μl were seeded in a 96-well flat-bottom plate. LAIR1 signaling was induced by treatment with 10 μg / ml coated collagen. Blockade of LAIR1 signaling by such h11B3 LAIR-1 mAbs was measured by adding different concentrations of such h11B3 LAIR-1 mAbs at 100 μL per well. Cultures were maintained for 20 hours and GFP was read by flow cytometry. A (left) shows the mechanism of the reporter cell line UT140. B (middle) shows LAIR-1 binding in UT140. C (right) shows blockade of LAIR-1 signaling. [Figure 25]Figures A and B show that h11B3 LAIR-1 mAbs containing the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27 block collagen-1 signaling. Figure A is a schematic diagram of the LAIR-1-TCRζ reporter assay using the UT140 NFκB-GFP cell line. Figure B shows that such human LAIR-1 mAbs competed with collagen-1 in UT140-LAIR-1-NFAT-GFP reporter cells. The indicated concentrations of protein were diluted in phosphate-buffered saline and coated onto 96-well round-bottom tissue culture plates, which were left standing overnight (O / N) at 4°C. UT140 reporter cells were suspended in complete RPMI medium containing 10 μg / mL of the indicated soluble protein and then incubated overnight (O / N) in the coated wells at 37°C. Reporter activation was quantified by flow cytometry. FC-G1 BioXcell Cat# BE0096. Collagen-1 R&D Cat # 6220-CL. [Figure 26] A and B demonstrate that the h11B3 LAIR-1 mAb, containing the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27, induces AML cell death. Whole blood from AML-diagnosed donors was decontaminated using the StemCell RBC decontamination kit according to the manufacturer's instructions. Leukocytes were resuspended in complete RPMI medium containing 3 μg / mL of soluble mAb, then seeded in 96-well round-bottom plates and incubated at 37°C for 48 hours. Cell death was analyzed by Annexin V and Fixed Live-Dead staining according to the manufacturer's instructions and quantified by flow cytometry. RBC-decontaminated StemCell Ref# 18170. Isotype = FC-G1 BioXcell Ref# BE0096. Live-Dead Stain Invitrogen Cat# L34966. Annexin V-AF647 Biolegend Cat# 640943. A shows the isotype results. B shows the results for h11B3 LAIR-1 mAb. [Figure 27]A and B demonstrate that h11B3 LAIR-1 mAbs containing the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27 induce AML cell death via antibody-dependent cell-mediated cytotoxicity (ADCC). A shows the results using a lactate dehydrogenase (LDH) release assay. ADCC was measured using CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega) according to the manufacturer's instructions. Briefly, PBMCs from AML patients were mixed with healthy donor NK cells at a target:effector ratio of 3:1 in a complete RPMI containing soluble h11B3 or Fc-G1 isotype control at the indicated concentrations. The mixed cells were incubated at 37°C for 4 hours. Four test wells were used in a series. The AML PBMC-donor #200018616 was newly diagnosed / untreated. NK cells from healthy donor #83 were isolated from PBMCs via StemCell Kit #17955 according to manufacturer's instructions. CytoTox 96® non-radioactive cytotoxicity assay Promega Cat# G1780. % cytotoxicity was quantified by target cell LDH release. % cytotoxicity = ((Test well LDH release - Effector cell spontaneous release - Target cell spontaneous release) / (Maximum target cell release - Target cell spontaneous release)) * 100. Target cells = AML PBMC donor #200018616, Effector cells = NK cells isolated from PBMCs of healthy donor #83. B shows results using an adenylate kinase assay. ADCC was measured using Toxilight Bioluminescent Cytotoxicity Assay (Lonza) according to manufacturer's instructions. In short, PBMCs from AML patients were mixed with healthy donor NK cells in a 3:1 target:effector ratio in a complete RPMI containing the indicated concentrations of soluble h11B3 or Fc-G1 isotype control. The mixed cells were incubated at 37°C for 4 hours. Six test wells were used in a series. AML PBMC donor #200003038 was newly diagnosed / untreated. NK cells healthy donor #120 were isolated from PBMCs via StemCell Kit #17955 according to manufacturer's instructions.Toxilight Bioluminescent Cytotoxicity Assay kit Lonza Cat# LT17-217. % cytotoxicity was quantified by adenylate kinase release from target cells, and ADCC was calculated as shown above. Target cells = AML PBMC donor #200003038, effector cells = NK cells isolated from healthy donor #120 PBMCs. [Figure 28] This study demonstrates that h11B3 LAIR-1 mAbs, containing the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27, may promote antibody-dependent cell phagocytosis (ADCP) in AML cells. LAIR-1 mAbs induce ADCP activity in an in vitro phagocytic assay using cell-trace violet-labeled mouse bone marrow macrophages (BMMs) co-cultured with MV4-11-Luc cells. Mouse BMMs were generated using 100 ng / ml M-CSF (Cat#416-ML-500) with seed BMM cells at a rate of 1 e5 / well in a 24-well plate. Staining was performed with cell trace violet (Thermofisher). Target MV411 cells were labeled with PKH26 (Sigma # PKH26gl-1kt) according to manufacturer's instructions and co-cultured with BMMs in a 5:1 ratio for 16 hours in the presence of 20 μg / ml h11B3 or a control antibody. The phagocytic activity was analyzed by flow cytometry. [Figure 29]A–C demonstrate that h11B3 LAIR-1 mAb containing the variable light chain of SEQ ID NO. 16 and the variable heavy chain of SEQ ID NO. 27 restricts the proliferation of leukemia cells in a CDX model. 2e6 luciferase-expressing MV4-11-Luc cells or THP-1-Luc cells were suspended in sterile PBS and transplanted into NSG mice via tail vein injection. Starting 8 days post-challenge, mice were treated twice weekly by intraperitoneal (ip) injection of h11B3 LAIR-1 mAb or isotype containing the variable light chain of SEQ ID NO. 16 and the variable heavy chain of SEQ ID NO. 27 (A). AML cell proliferation was monitored by intraperitoneal injection of D-luciferin once weekly, followed immediately by IVIS bioluminescence imaging and quantification of luminescence signals. Mouse strain NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ(NSG) #005557 purchased from Jackson Labs. Substrate Xenolight RediJect D-Luciferin Perkin Elmore Cat# 770504. Imaging Perkin Elmore IVIS Lumina XRMS Series III. B shows the results for MV-4-11. C shows the results for THP-1. [Figure 30] A and B demonstrate that the h11B3 LAIR-1 mAb, containing the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27, may promote antibody-dependent cell phagocytosis (ADCP) of AML cells. Spleen tissue was collected 35 days after MV411 tumor inoculation (A). A portion of the spleen was fixed to an OCT block. The spleen tissue was placed on a slide and immunofluorescently stained with hCD45 and mCD45 antibodies, and the nuclear tissue was stained with DAPI dye. Images were acquired using a NanoString DSP instrument (B). The remaining portion of the spleen was subjected to flow cytometry analysis (B). The spleen was processed to produce a single-cell suspension, and the percentage of dead MV411 cells was counted by staining with Annexin V and human CD45 antibody. [Figure 31]A–H demonstrate that h11B3 LAIR-1 mAb, containing the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27, reduces tumor burden and restores normal immune cells in AML MV411 using a mouse model (flow cytometry analysis). NSG mice were treated with h11B3 LAIR-1 mAb at a dose of 10 mg / kg every two weeks. Single cells isolated from the spleen and bone marrow of the mouse NSG mouse model were stained using human CD45 antibody and mouse CD45 antibody. A–C show reductions in AML amount and activity in multiple compartments. D–F show induction of AML cell death and specificity for AML cells. G–H show protection of normal immune cells and benefits for chemotherapy. [Figure 32] A–C show the results of dose studies in an MV41-1 model of h11B3 LAIR-1 mAb containing the variable light chain of SEQ ID NO. 16 and the variable heavy chain of SEQ ID NO. 27. 2e6 luciferase-expressing MV4-11-Luc cells were suspended in sterile PBS and transplanted into NSG mice via tail vein injection (A). From day 8 after challenge, mice were treated twice weekly with intraperitoneal (ip) injection of h11B3v6 or sterile PBS vehicle at the indicated concentrations. AML cell proliferation was monitored by intraperitoneal injection of D-luciferin once weekly, followed immediately by IVIS bioluminescence imaging and quantification of luminescence signals (B–C). Mouse strain NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ(NSG) #005557 purchased from Jackson Labs. Substrate Xenolight RediJect D-Luciferin Perkin Elmore Cat# 770504. Imaging Perkin Elmore IVIS Lumina XRMS Series III. [Figure 33]A-D show the results of a treatment timing study in the MV4-11 model. 2e6 luciferase-expressing MV4-11-Luc cells were suspended in sterile PBS and transplanted into NSG mice via tail vein injection. From the indicated day after challenge, mice were treated twice weekly by intraperitoneal (ip) injection of 10 mg / kg of h11B3 or sterile PBS vehicle. AML cell proliferation was monitored by intraperitoneal injection of D-luciferin once weekly, followed immediately by IVIS bioluminescence imaging and quantification of luminescence signals. Mouse NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ(NSG) strain #005557 purchased from Jackson Labs. Substrate Xenolight RediJect D-Luciferin Perkin Elmore Cat# 770504. Imaging Perkin Elmore IVIS Lumina XRMS Series III. [Figure 34]This report presents the results of pharmacokinetic studies of h11B3 LAIR-1 mAb, containing the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27, in balb / c mice, with a Tmax of 8 hours. Balb / c mice were intraperitoneally injected with 10 mg / kg of h11B3. Subsequently, the mice were bled by facial vein puncture at the indicated time points. Serum was isolated by centrifugation at 2500 RCF in a serum collection tube. Circulating h11B3 in serum was tested by a universal PK assay. Briefly, h11B3 in mouse serum was captured using biotin-labeled anti-human IgG in a nanoliter column of a Gyrolab CD. Alexa Fluor-labeled anti-human IgG was used for detection. The quantitative dynamic range was (500–100,000) ng / mL (LLOQ–ULOQ). Standard curves and controls were prepared in pooled Balb / C serum (Biochemed). Standard curves, controls, and samples were diluted 1:10 (MRD) in buffer. Samples exceeding the upper limit of quantification (100,000 ng / mL) were brought within range by dilution with Rexiip buffer. The amount of analyte present in the samples was detected using laser-induced fluorescence detection. Unknown sample concentrations were interpolated using the standard curve signal. All samples were analyzed in pairs, and acceptance criteria (±20% CV and bias) were assigned. Unknown sample concentrations were interpolated using 4PL with Y-weighted nonlinear regression using Gyrolab Evaluator software. [Figure 35A]This study demonstrates that h11B3 LAIR-1 mAb, containing the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27, does not induce the removal, inhibition, or proliferation of healthy human primary immune cells in the NSG-SGM3 mouse model. NSG-SGM3 mice were purchased from Jackson Laboratory. CD34+ umbilical cord blood cell donor #2523 was engrafted into the mice. From 18 weeks post-engraftment, mice were treated weekly for 4 weeks with 100 μg / mouse (5 mg / kg) of human G1 or G4P morphology anti-human LAIR-1 mAb clone 11B3 or control. Bleeding was induced at week 2 (one day before the second dose) and week 4 (one day before the fourth dose) for circulating immune cells. Mice were sacrificed one week after the final dose for analysis of the spleen, LN, and bone marrow. This shows the total body weight and survival of NSG-SGM3 mice reconstituted with human CD34+ umbilical cord blood cells and treated 11B3 antibody. [Figure 35B] This study demonstrates that h11B3 LAIR-1 mAb, containing the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27, does not induce the removal, inhibition, or proliferation of healthy human primary immune cells in the NSG-SGM3 mouse model. NSG-SGM3 mice were purchased from Jackson Laboratory. CD34+ umbilical cord blood cell donor #2523 was engrafted into the mice. From 18 weeks post-engraftment, mice were treated weekly for 4 weeks with 100 μg / mouse (5 mg / kg) of human G1 or G4P morphology anti-human LAIR-1 mAb clone 11B3 or control. Bleeding was induced at week 2 (one day before the second dose) and week 4 (one day before the fourth dose) for circulating immune cells. Mice were sacrificed one week after the final dose for analysis of the spleen, LN, and bone marrow. This shows the total body weight and survival of NSG-SGM3 mice reconstituted with human CD34+ umbilical cord blood cells and treated 11B3 antibody. [Figure 35C]This study demonstrates that h11B3 LAIR-1 mAb, containing the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27, does not induce the removal, inhibition, or proliferation of healthy human primary immune cells in the NSG-SGM3 mouse model. NSG-SGM3 mice were purchased from Jackson Laboratory. CD34+ umbilical cord blood cell donor #2523 was engrafted into the mice. From 18 weeks post-engraftment, mice were treated weekly for 4 weeks with 100 μg / mouse (5 mg / kg) of human G1 or G4P morphology anti-human LAIR-1 mAb clone 11B3 or control. Bleeding was induced at week 2 (one day before the second dose) and week 4 (one day before the fourth dose) for circulating immune cells. Mice were sacrificed one week after the final dose for analysis of the spleen, LN, and bone marrow. This shows the time-course tracking of changes in the number of immune cells in blood collected from NSG-SGM3 reconstituted with human CD34+ umbilical cord blood cells. [Figure 35D] This study demonstrates that h11B3 LAIR-1 mAb, containing the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27, does not induce the removal, inhibition, or proliferation of healthy human primary immune cells in the NSG-SGM3 mouse model. NSG-SGM3 mice were purchased from Jackson Laboratory. CD34+ umbilical cord blood cell donor #2523 was engrafted into the mice. From 18 weeks post-engraftment, mice were treated weekly for 4 weeks with 100 μg / mouse (5 mg / kg) of human G1 or G4P morphology anti-human LAIR-1 mAb clone 11B3 or control. Bleeding was induced at week 2 (one day before the second dose) and week 4 (one day before the fourth dose) for circulating immune cells. Mice were sacrificed one week after the final dose for analysis of the spleen, LN, and bone marrow. This shows the time-course tracking of changes in the number of immune cells in blood collected from NSG-SGM3 reconstituted with human CD34+ umbilical cord blood cells. [Figure 35E]This study demonstrates that h11B3 LAIR-1 mAb, containing the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27, does not induce the removal, inhibition, or proliferation of healthy human primary immune cells in the NSG-SGM3 mouse model. NSG-SGM3 mice were purchased from Jackson Laboratory. CD34+ umbilical cord blood cell donor #2523 was engrafted into the mice. From 18 weeks post-engraftment, mice were treated weekly for 4 weeks with 100 μg / mouse (5 mg / kg) of human G1 or G4P morphology anti-human LAIR-1 mAb clone 11B3 or control. Bleeding was induced at week 2 (one day before the second dose) and week 4 (one day before the fourth dose) for circulating immune cells. Mice were sacrificed one week after the final dose for analysis of the spleen, LN, and bone marrow. This shows the time-course tracking of changes in the number of immune cells in blood collected from NSG-SGM3 reconstituted with human CD34+ umbilical cord blood cells. [Figure 35F] This study demonstrates that h11B3 LAIR-1 mAb, containing the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27, does not induce the removal, inhibition, or proliferation of healthy human primary immune cells in the NSG-SGM3 mouse model. NSG-SGM3 mice were purchased from Jackson Laboratory. CD34+ umbilical cord blood cell donor #2523 was engrafted into the mice. From 18 weeks post-engraftment, mice were treated weekly for 4 weeks with 100 μg / mouse (5 mg / kg) of human G1 or G4P morphology anti-human LAIR-1 mAb clone 11B3 or control. Bleeding was induced at week 2 (one day before the second dose) and week 4 (one day before the fourth dose) for circulating immune cells. Mice were sacrificed one week after the final dose for analysis of the spleen, LN, and bone marrow. This shows the time-course tracking of changes in the number of immune cells in blood collected from NSG-SGM3 reconstituted with human CD34+ umbilical cord blood cells. [Figure 35G]This study demonstrates that h11B3 LAIR-1 mAb, containing the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27, does not induce the removal, inhibition, or proliferation of healthy human primary immune cells in the NSG-SGM3 mouse model. NSG-SGM3 mice were purchased from Jackson Laboratory. CD34+ umbilical cord blood cell donor #2523 was engrafted into the mice. From 18 weeks post-engraftment, mice were treated weekly for 4 weeks with 100 μg / mouse (5 mg / kg) of human G1 or G4P morphology anti-human LAIR-1 mAb clone 11B3 or control. Bleeding was induced at week 2 (one day before the second dose) and week 4 (one day before the fourth dose) for circulating immune cells. Mice were sacrificed one week after the final dose for analysis of the spleen, LN, and bone marrow. This shows the time-course tracking of changes in the number of immune cells in blood collected from NSG-SGM3 reconstituted with human CD34+ umbilical cord blood cells. [Figure 35H] This study demonstrates that h11B3 LAIR-1 mAb, containing the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27, does not induce the removal, inhibition, or proliferation of healthy human primary immune cells in the NSG-SGM3 mouse model. NSG-SGM3 mice were purchased from Jackson Laboratory. CD34+ umbilical cord blood cell donor #2523 was engrafted into the mice. From 18 weeks post-engraftment, mice were treated weekly for 4 weeks with 100 μg / mouse (5 mg / kg) of human G1 or G4P morphology anti-human LAIR-1 mAb clone 11B3 or control. Bleeding was induced at week 2 (one day before the second dose) and week 4 (one day before the fourth dose) for circulating immune cells. Mice were sacrificed one week after the final dose for analysis of the spleen, LN, and bone marrow. This shows an analysis of immune cell counts in the spleen and bone marrow collected from NSG-SGM3 reconstituted with human CD34+ umbilical cord blood cells. [Figure 35I]This study demonstrates that h11B3 LAIR-1 mAb, containing the variable light chain of SEQ ID NO: 16 and the variable heavy chain of SEQ ID NO: 27, does not induce the removal, inhibition, or proliferation of healthy human primary immune cells in the NSG-SGM3 mouse model. NSG-SGM3 mice were purchased from Jackson Laboratory. CD34+ umbilical cord blood cell donor #2523 was engrafted into the mice. From 18 weeks post-engraftment, mice were treated weekly for 4 weeks with 100 μg / mouse (5 mg / kg) of human G1 or G4P morphology anti-human LAIR-1 mAb clone 11B3 or control. Bleeding was induced at week 2 (one day before the second dose) and week 4 (one day before the fourth dose) for circulating immune cells. Mice were sacrificed one week after the final dose for analysis of the spleen, LN, and bone marrow. This shows an analysis of immune cell counts in the spleen and bone marrow collected from NSG-SGM3 reconstituted with human CD34+ umbilical cord blood cells. [Modes for carrying out the invention]

[0025] This disclosure can be more readily understood by referring to the following detailed description, examples, drawings, and claims, as well as the preceding and following descriptions thereof. However, before disclosing and describing the compositions and / or methods, it should be understood that this disclosure is not limited to any specific compositions and / or methods disclosed, unless otherwise specified, and is therefore naturally subject to change. It should also be understood that the terms used herein are for the purpose of describing only specific aspects and are not intended to limit them.

[0026] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this disclosure pertains. Any compositions and methods similar or equivalent to those described herein may be used in the practice or testing of the present invention. All publications mentioned herein are incorporated herein in their entirety by reference.

[0027] Unless otherwise defined, all percentage values ​​of compositions used herein are given in weight percent.

[0028] In the context of describing the currently claimed invention (particularly in the context of the claims), the terms "a," "an," "the," and similar references are to be interpreted as encompassing both the singular and plural, unless otherwise indicated in the specification or unless it is clearly inconsistent with the context.

[0029] Unless otherwise stated herein, the descriptions of value ranges are intended solely as a concise way of referring to each distinct value within that range individually, and each distinct value is incorporated into the specification as if it were described individually herein.

[0030] The use of the term "approximately" is intended to describe a value that is either above or below the stated value, within a range of approximately + / - 10%. In other embodiments, the value may be within a range of approximately + / - 5% above or below the stated value. In other embodiments, the value may be within a range of approximately + / - 2% above or below the stated value. In other embodiments, the value may be within a range of approximately + / - 1% above or below the stated value. The above ranges are intended to be clear from the context and are not further limiting. All methods described herein may be performed in any suitable order unless otherwise indicated herein or unless it is clearly inconsistent from the context. Any and all examples provided herein, or the use of exemplary language (e.g., "etc."), are intended solely to better illustrate the invention and, unless otherwise claimed, do not impose any limitation on the scope of the invention. No expression herein should be construed as indicating that an unclaimed element is essential to the practice of the invention.

[0031] As used herein, “administration,” when applied to humans, primates, mammals, mammalian subjects, animals, veterinary subjects, placebo subjects, research subjects, experimental subjects, cells, tissues, organs, or biological fluids, means bringing an exogenous ligand, reagent, placebo, small molecule, drug, therapeutic agent, diagnostic agent, or composition into contact with a subject, cell, tissue, organ, or biological fluid, etc. “Administration” may refer to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. Treatment of cells includes contact of reagents with cells, as well as contact of reagents with fluids, where the fluid is in contact with the cells. “Administration” also includes, for example, in vitro and ex vivo treatment of cells with reagents, diagnostics, conjugated compositions, or other cells.

[0032] An "agonist" is related to both ligands and receptors, and therefore includes molecules, combinations of molecules, complexes, or combinations of reagents that stimulate a receptor. For example, agonists of granulocyte-macrophage colony-stimulating factor (GM-CSF) can include GM-CSF, mutant proteins or derivatives of GM-CSF, peptide mimes of GM-CSF, small molecules that mimic the biological function of GM-CSF, or antibodies that stimulate the GM-CSF receptor.

[0033] As used herein, “analog” or “derivative” of a peptide, polypeptide, or protein means another peptide, polypeptide, or protein that has similar or identical function to the original peptide, polypeptide, or protein, but does not necessarily contain a similar or identical amino acid sequence or structure to the original peptide, polypeptide, or protein. The analog preferably satisfies at least one of the following: (a) a protein agent having an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the original amino acid sequence; (b) a protein agent encoded by a nucleotide sequence that hybridizes under strict conditions to the nucleotide sequence encoding the original amino acid sequence; and (c) a protein agent encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence encoding the original amino acid sequence.

[0034] As used herein, the term “antigen-binding fragment” of an antibody refers to one or more portions of an antibody that exhibit the ability to bind immunospecifically to an antigen, comprising the antibody’s complementarity-determining region (“CDR”) and framework residues optionally containing the antibody’s “variable region” antigen-recognition site. Such fragments include Fab’, F(ab’)2, Fv, single-chain (ScFv) and their variants, naturally occurring variants, and fusion proteins containing the antibody’s “variable region” antigen-recognition site and heterologous proteins (e.g., toxins, antigen-recognition sites for different antigens, enzymes, receptors, or receptor ligands).

[0035] As used herein, "antibody" refers to a peptide or polypeptide derived from, modeled on, or substantially encoded by immunoglobulin gene(s) or fragments thereof that can specifically bind to an antigen or an epitope (Wilson, J. Immunol. Methods, 1994; Yarmush, J. Biochem. Biophys., 1992). The term antibody includes an antigen-binding portion that retains the ability to bind to an antigen, i.e., an "antigen-binding site" (e.g., a fragment, a subsequence, a complementarity-determining region (CDR)), and includes (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a divalent fragment containing two Fab fragments linked by a disulfide bond in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of a VH domain (Ward, et al., Nature, 1989); and (vi) an isolated complementarity-determining region (CDR). Single-chain antibodies are also included within the term "antibody" by reference.

[0036] As used herein, an "antigen-presenting cell" (APC) is a cell of the immune system that is used to present an antigen to a T cell. APCs include dendritic cells, monocytes, macrophages, marginal zone Kupffer cells, microglia, Langerhans cells, T cells, and B cells. Dendritic cells develop from at least two lineages. The first lineage includes pre-DC1, myeloid DC1, and mature DC1. The second lineage includes CD34 + CD45RA - early progenitor pluripotent cells, CD34 + CD45RA + cells, CD34 + CD45RA + CD4 + IL-3Rα + pro-DC2 cells, CD4 + CD11c - include plasmacytoid pre-DC2 cells, lymphoid human DC2 plasmacytoid-derived DC2, and mature DC2.

[0037] As used herein, the term “cancer” refers to a neoplasm or tumor resulting from the abnormal and uncontrolled proliferation of cells. As used herein, cancer expressly includes sarcomas, carcinomas, leukemias, and lymphomas. The term “cancer” refers to a disease involving cells that may metastasize distally and exhibit phenotypic traits distinct from non-cancerous cells, such as colonization in three-dimensional substrates like soft agar or formation of tubular networks or web-like matrices in three-dimensional basement membranes or extracellular matrix preparations. Non-cancerous cells do not colonize in soft agar and form distinct spherical structures in three-dimensional basement membranes or extracellular matrix preparations.

[0038] As used herein, the term “chimeric receptor” is defined as a cell surface receptor containing an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic costimulatory signaling domain in a combination not naturally present together on a single protein. This includes, in particular, receptors in which the extracellular and cytoplasmic domains are not naturally present together on a single receptor protein. Furthermore, chimeric receptors are distinct from TCRs expressed on native T-cell lymphocytes.

[0039] As used herein, the term “co-stimulus” signal includes both positive co-stimulus signals (e.g., signals that result in enhanced activity) and negative co-stimulus signals (e.g., signals that result in inhibited activity).

[0040] The term "derivative" refers to an antibody or its antigen-binding fragment that binds immunospecifically to the same target as the parent antibody or reference antibody, but whose amino acid sequence differs from that of the parent antibody or reference antibody or its antigen-binding fragment by including substitutions, additions, deletions, or modifications of one, two, three, four, or five or more amino acids. In some embodiments, such derivatives have substantially the same immunospecificity and / or properties, or the same immunospecificity and properties, as the parent antibody or reference antibody or its antigen-binding fragment. The amino acid substitutions or additions of such derivatives may include naturally occurring (i.e., DNA-encoded) amino acid residues or non-spontaneously occurring amino acid residues. The term "derivative" includes, for example, chimeric variants or humanized variants, as well as variants in which the CH1, hinge, CH2, CH3, or CH4 regions are altered to form antibodies, for example, those having variant Fc regions in which effector properties or binding properties are enhanced or weakened.

[0041] As used herein, “effective dose” includes, but is not limited to, an amount that can improve, restore, alleviate, prevent or diagnose the symptoms or signs of a medical condition or disorder. Unless otherwise expressly or contextually indicated, “effective dose” is not limited to the minimum amount sufficient to improve the medical condition.

[0042] As used herein, “epitope” refers to an antigenic determinant that can specifically bind to an antibody. Epitopes typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually possess specific three-dimensional structural properties and specific charge properties. Conformal epitopes and non-conformal epitopes are distinguished in that the former loses binding to a denaturing solvent, while the latter does not.

[0043] As used herein, “extracellular fluid” includes serum, plasma, blood, interstitial fluid, cerebrospinal fluid, secretions, lymph, bile, sweat, feces, and urine. “Extracellular fluid” may also include colloids or suspensions, such as whole blood or coagulated blood.

[0044] As used herein, in the context of polypeptides, “fragment” includes peptides or polypeptides comprising an amino acid sequence of at least five consecutive amino acid residues, at least ten consecutive amino acid residues, at least fifteen consecutive amino acid residues, at least twenty consecutive amino acid residues, at least twenty-five consecutive amino acid residues, at least forty consecutive amino acid residues, at least fifty consecutive amino acid residues, at least sixty consecutive amino acid residues, at least seventy consecutive amino acid residues, at least eighty consecutive amino acid residues, at least ninety consecutive amino acid residues, at least one hundred consecutive amino acid residues, at least one hundred and two

[0045] As used herein, the term “humanized antibody” refers to an immunoglobulin containing a human framework region and one or more CDRs derived from non-human (usually mouse or rat) immunoglobulins. The non-human immunoglobulin providing the CDRs is called the “donor,” and the human immunoglobulin providing the framework is called the “acceptor.” A constant region is not necessarily required, but if present, it should be substantially identical to the human immunoglobulin constant region, i.e., at least about 85–99% or about 95% or more identical. Thus, all parts of a humanized immunoglobulin (except possibly the CDRs) are substantially identical to the corresponding parts of the native human immunoglobulin sequence. A humanized antibody is an antibody containing humanized light chain and humanized heavy chain immunoglobulins. For example, a humanized antibody does not encompass a typical chimeric antibody, for example, because the entire variable region of a chimeric antibody is non-human.

[0046] As used herein, “immune cells” means any cell of hematopoietic origin, including, but not limited to, T cells, B cells, NK cells, monocytes, dendritic cells, and macrophages.

[0047] "Immunogenic drugs" or "immunogens," when administered to mammals in combination with adjuvants in an optional manner, can induce an immunological response to themselves.

[0048] As used herein, the terms “immunological,” “immunological,” or “immune” response refer to the occurrence of a beneficial humoral (antibody-mediated) and / or cellular (antigen-specific T cell-mediated or secreted product-mediated) response to a peptide in a recipient patient. Such a response may be an active response induced by the administration of an immunogen, or a passive response induced by the administration of an antibody or primed T cell. Cellular immune responses are triggered by the presentation of polypeptide epitopes associated with class I or class II MHC molecules and antigen-specific CD4 + T helper cells and / or CD8 +It activates cytotoxic T cells. This response may also include activation or escalation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, neutrophils, or other innate immune components. The presence of a cell-mediated immunological response is confirmed in proliferation assays (CD4 + This can be determined by T cell (CTL) or CTL (cytotoxic T lymphocyte) assays. The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating antibodies and T cells from immunized syngenes and measuring their protective or therapeutic effects in a second target.

[0049] As used herein, “inflammatory molecules” refers to molecules that cause an inflammatory response and includes, but is not limited to, IL-1β, TNF-α, TGF-beta, IFN-γ, IL-18, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs, as well as cytokines and metalloproteinases.

[0050] As used herein, “ligand” refers to a small molecule, peptide, polypeptide, or membrane-associated or membrane-bound molecule that is an agonist or antagonist of a receptor. “Ligand” also includes binders that are neither agonists nor antagonists and do not possess the properties of either agonist or antagonist. By convention, if a ligand is membrane-bound on a first cell, the receptor usually arises on a second cell. The second cell may have the same ID (same name) as the first cell, or it may have a different ID (different name). The ligand or receptor may be entirely intracellular; that is, it may reside in the cytosol, nucleus, or any other intracellular compartment. The ligand or receptor may change its location, for example, from an intracellular compartment to the outer surface of the plasma membrane. The ligand-receptor complex is called a “ligand-receptor complex.” When the ligand and receptor are involved in a signaling pathway, the ligand arises upstream of the signaling pathway, and the receptor arises downstream.

[0051] As used herein, the term “modulation” refers to the ability to alter an effect, outcome, or activity (e.g., signaling). Such modulation may be agonistic or antagonistic. Antagonistic modulation may be partial (i.e., attenuating but not inactivating) or may completely inactivate (e.g., neutralize) such activity. Modulation may include the internalization of a receptor after antibody binding or a reduction in the expression of the receptor on target cells. Agonistic modulation can enhance, increase, or augment activity (e.g., signaling). In further embodiments, such modulation may alter the nature of the interaction between a ligand and its homologous receptor in such a way as to alter the nature of the induced signaling. For example, a molecule can alter its ability to bind to other ligands or receptors by binding to a ligand or receptor, thereby altering their overall activity. In some embodiments, such modulation results in a change of at least 10% of measurable immune system activity, a change of at least 50% of such activity, or a change of at least 2-fold, 5-fold, 10-fold, or at least 100-fold in such activity.

[0052] As used herein, the terms “sequence identity percentage” and “sequence identity %” refer to the percentage of sequence similarity found by the comparison or alignment of two or more amino acid sequences or nucleic acid sequences. The identity percentage can be determined by directly comparing the sequence information of two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. An algorithm for calculating the identity percentage is the Smith-Waterman homology search algorithm (see, for example, Kann and Goldstein (2002) Proteins 48:367-376, Arslan, et al. (2001) Bioinformatics 17:327-337).

[0053] As used herein, “peptide” refers to a short sequence of amino acids linked together by peptide bonds. Peptides can occur in free form or be bound to another part, such as a polymer, lipid, oligosaccharide or polysaccharide, and / or polypeptide. Even when a peptide is incorporated into a polypeptide chain, the term “peptide” may still be used to specifically refer to a short sequence of amino acids. A “peptide” can be linked to another part via peptide bonds or any other type of bond. A peptide is the length of at least two amino acids, and the maximum length varies by convention or context.

[0054] As used herein, “pharmaceutically acceptable excipients” or “diagnostically acceptable excipients” include, but are not limited to, sterile distilled water, physiological saline, phosphate buffer, amino acid-based buffer, or bicarbonate buffer. The selected excipient and the amount of excipient used will depend on the method of administration. Administration includes injection, infusion, or a combination thereof.

[0055] As used herein, the term “polypeptide” refers to an amino acid chain of any length, regardless of modification (e.g., phosphorylation or glycosylation). The term polypeptide includes proteins and their fragments. Polypeptides can be “exogenous,” meaning “heterogeneous,” i.e., heterogeneous to the host cell from which they are utilized, such as human polypeptides produced by bacterial cells. Polypeptides are disclosed herein as amino acid residue sequences. Their sequences are described from left to right, from the amino terminus to the carboxyl terminus. According to standard nomenclature, amino acid residue sequences are named by either a three-letter or one-letter code, such as alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0056] As used herein, “protein” generally refers to a sequence of amino acids comprising a polypeptide chain. A protein can also refer to the three-dimensional structure of a polypeptide. A “denatured protein” refers to a partially denatured polypeptide that retains some degree of three-dimensional structure, or alternatively, to an essentially random three-dimensional structure, as in the case of a completely denatured protein. Polypeptide variants can be produced by glycosylation, phosphorylation, sulfation, disulfide bond formation, deamidation, isomerization, cleavage points in signal or leader sequence processing, covalent and non-covalent cofactors, oxidative variants, and the like.

[0057] As used herein, “recombination” refers to the introduction of exogenous non-natural nucleic acids, modification of natural nucleic acids, or modification by whole or partial induction from recombinant nucleic acids, cells, viruses, plasmids, or vectors, when used in relation to nucleic acids, cells, animals, viruses, plasmids, vectors, etc. Recombinant proteins refer to proteins produced or secreted from recombinant nucleic acids, viruses, plasmids, vectors, etc.

[0058] As used herein, “Sample” refers to a sample from a human, animal, placebo, or research sample, such as cells, tissues, organs, bodily fluids, gases, aerosols, slurries, colloids, or coagulating substances. “Sample” may be tested in vivo (i.e., without removal from a human or animal) or in vitro. A sample may be tested after processing, such as by histological methods. “Sample” also refers to cells containing bodily fluids or tissue samples, or cells isolated from bodily fluids or tissue samples. “Sample” may also refer to cells, tissues, organs, or bodily fluids freshly collected from a human or animal, or cells, tissues, organs, or bodily fluids that have been processed or stored.

[0059] "Specifically" or "selectively" binding refers to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biologics, when referring to ligand / receptor, nucleic acid / complementary nucleic acid, antibody / antigen, or other binding pairs (e.g., cytokines to cytokine receptors). Thus, under specified conditions, a particular ligand will bind to a particular receptor and not in significant amounts to other proteins present in the sample. Specific binding can also mean, for example, that a binding compound, nucleic acid ligand, antibody, or binding composition derived from the antigen-binding site of an antibody in the intended method binds to a target with an affinity that is often at least 25%, more often at least 50%, most often at least 100% (2x), usually at least 10x, more usually at least 20x, and most usually at least 100x higher than the affinity of other binding compounds.

[0060] As used herein, the term “subject” refers to a human or non-human organism. Therefore, the methods and compositions described herein are applicable to both human and veterinary diseases. In certain embodiments, the subject is a “patient,” such as a living human being receiving medical treatment for a disease or condition. The subject also includes individuals who are being investigated for signs of pathology, or whose disease is not clearly identified.

[0061] When used in the context of binding effects or demonstrated effects, the term “substantially” is intended to indicate that the observed effect is physiologically or therapeutically relevant. For example, a molecule can substantially inhibit the activity of a ligand or receptor if the degree of blockade is physiologically or therapeutically appropriate (e.g., if such a degree is greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 97%). Similarly, a molecule is said to have substantially the same immunospecificity and / or properties as another molecule if such immunospecificity and properties are 60% or more identical, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 97% identical.

[0062] As used herein, the term “therapeutic dose” is defined as the amount of reagent or pharmaceutical composition sufficient to induce a desired immune response specific to the encoded heterologous antigen, thereby providing a patient benefit (e.g., reducing, preventing, or improving the symptoms of the condition being treated). If the drug or pharmaceutical composition contains a diagnostic agent, “diagnostic dose” is defined as the amount sufficient to produce a signal, image, or other diagnostic parameter. The effective dose of a pharmaceutical formulation varies depending on factors such as the degree of susceptibility of the individual, the individual’s age, sex, and weight, and the individual’s specific response (US5,888,530).

[0063] As used herein, “treatment” and “to treat” (with respect to a medical condition or disease) refer to an approach to obtain a beneficial or desired outcome, preferably including a clinical outcome. For the purposes of this disclosure, a beneficial or desired outcome with respect to a disease includes, but is not limited to, one or more of the following: improvement of a medical condition, cure of the disease, reduction of the severity of the disease, delay of the progression of the disease, relief of one or more symptoms associated with the disease, improvement of the quality of life of a person suffering from the disease, and / or extension of survival. Similarly, for the purposes of this disclosure, a beneficial or desired outcome with respect to a medical condition includes, but is not limited to, one or more of the following: improvement of a medical condition, cure of the disease, reduction of the severity of the disease, delay of the progression of the disease, relief of one or more symptoms associated with the disease, improvement of the quality of life of a person suffering from the disease, and / or extension of survival.

[0064] As used herein, “tumor microenvironment” or “TME” refers to the normal cells, molecules, fibroblasts, immune cells, and blood vessels that surround and nourish tumor cells. The tumor microenvironment also includes proteins, including the extracellular matrix (ECM), that are produced by all cells present within the tumor and support the growth of cancer cells.

[0065] As used herein, the term “variant” refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide has an amino acid sequence that differs from another reference polypeptide. Generally, the differences are limited, so the sequences of the reference polypeptide and the variant are very similar overall and identical in many regions. Variants and reference polypeptides may have different amino acid sequences due to one or more modifications (e.g., substitutions, additions, and / or deletions). Substituted or inserted amino acid residues may or may not be encoded by the genetic code. Variants of polypeptides may be naturally occurring or not known to occur naturally, such as allele variants.

[0066] II. LAIR-1 Leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) is a highly expressed target in leukemia stem cells and blast cells, mediating their survival. This disclosure relates to a LAIR-1 antibody that specifically kills leukemia stem cells and blast cells while preserving healthy hematopoietic stem cells.

[0067] LAIR-1 is the only known collagen receptor with inhibitory signaling capability. This further suggests a crucial role for LAIR-1-mediated inhibitory signaling in AML cells (Ricard-Blum, The Collagen Family Review, 2011; Bo An et al., Collagen Interactions: Drug Design and Delivery, 2015). Collagen and ECM regulate the dynamics of a wide variety of cell membranes (as outlined in Hu et al., 2022), and ECM ligands may influence the dynamics of signaling and subsequent cell fate in leukemia cells, particularly in the bone marrow (Shin et al, 2016; Galan-Diez et al, 2018; Zanetti and Krause, 2020). These differing signaling dynamics offer a possible explanation for the discrepancy between reports showing LAIR-1 activation of cell survival mechanisms such as CAMK1 / CREB (Kang, Lu et al., 2015; Kang, Kim et al., 2016) and reports showing LAIR-1-mediated cessation of proliferation and cell death (Poggi et al., 2000; Zhocchi et al., 2001).

[0068] The sequence of LAIR-1 is provided. In some embodiments, the preceding methionine amino acid is cleaved in the posttranslational form of the protein.

[0069] A. Human LAIR-1 The sequence of human LAIR-1 is publicly known in the art. For example, the consensus sequence of LAIR-1a (isoform 1) is as follows: MSPHPTALLGLVLCLAQTIHT QEEDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTYNDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQSDYLELLVKETSGGPDSPDTEPGSSAGPTQRPSDNSHNEHAPASQGLKAEHLY ILIGVSVVFLFCLLLLVLFCLHRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDTSALAAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVARH

[0070] Amino acids 1-21 form the signal sequence, amino acids 22-165 (underlined) form the extracellular domain, amino acids 166-186 form the transmembrane domain, and amino acids 187-287 form the cytoplasmic domain (SEQ ID NO: 1, UniProtKB-Q6GTX8(LAIR1_HUMAN)). Amino acids 29-117 form the Ig-like C2 domain. Amino acids 249-254 and 279-284 form ITIM motifs 1 and 2, respectively. LAIR-1b (also known as isoform 2) lacks amino acids 122-138 compared to SEQ ID NO: 1. LAIR-1c (also known as isoform 3) lacks amino acids 23-23 and 122-138 compared to SEQ ID NO: 1. LAIR-1d (also known as isoform 4) lacks amino acids 210-287 compared to SEQ ID NO: 1.

[0071] As described above, the extracellular domain of human LAIR-1 may be as follows: QEEDLPR PSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTYNDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQSDYLE LLVKETSGGPDSPDTEPGSSAGPTQRPSDNSHNEHAPASQGLKAEHLY

[0072] (SEQ ID NO: 2) or a fragment thereof, for example, the Ig-like C2 domain (amino acids 8-96 in the underlined section of SEQ ID NO: 2) or the cysteine-enclosed region that forms a disulfide bond between amino acids 49-101 of SEQ ID NO: 1 (amino acids 28-80 in SEQ ID NO: 2, shown in italics).

[0073] Known variants and mutants of LAIR-1 include E63D, Y251F, and Y215F for SEQ ID NO: 1. Evidence has shown that Y215F causes decreased tyrosine phosphorylation, loss of binding to PTPN6 and CSK, complete loss of inhibitory activity, and loss of phosphorylation and calcium mobilization inhibition when bound to F-281 (Xu, et al., J. Biol. Chem. 275:17440-17446 (2000), Verbrugge, et al., Int. Immunol., 15:1349-1358 (2003), Verbrugge, et al., Eur.J. Immunol., 36:190-198 (2006)). Y281F shows decreased tyrosine phosphorylation and loss of binding to PTPN6, as well as partial inhibition of cytotoxic activity.

[0074] Meyaard, 2008, J. Leukoc. Biol. 83:799-803, demonstrates that LAIR-1 is widely expressed in human immune cells. Investigations of actual flow cytometry expression data in research papers show that LAIR-1 is expressed much more highly in myeloid cells such as monocytes, macrophages, and dendritic cells than in T cells and NK cells (Meyaard et al., 1997, Immunity 7:283-290). However, B cells differentially express high levels of LAIR-1 during differentiation (van der Vuurst de Vries et al., 1999, Eur. J. Immunol. 29:3160-3167). LAIR-1 has been found to be expressed in acute myeloid leukemia cells, acute lymphoblastic leukemia cells, and chronic lymphocytic leukemia cells (van der Vuurst de Vries et al., 1999, Eur. J.Immunol. 29:3160-3167, Poggi et al., 2000, Eur. J.Immunol. 30:2751-2758, Zocchi et al., 2001, Eur. J.Immunol. 31:3667-3675, Perbellini et al., 2014, Haematologica, 99:881-887, (Kang et al., 2015, Nat. Cell Biol. 17:665-677). Finally, LAIR-1 has been shown to be expressed in several human tumor cell lines (Meyaard et al. al., 1997, Immunity 7:283-290, Cao et al., 2015, Biochem.

[0075] In humans and mice, LAIR-1 binds to several types of collagen with high affinity (Meyaard, 2008, J. Leukoc. Biol. 83:799-803 and Meyaard, 2010, Immunol. Lett. 128:26-28). In humans, LAIR-1 has also been shown to bind to complement component C1q (Son et al., 2012, Proc. Natl. Acad. Sci. USA 109:E3160-3167) and surfactant protein-D (SP-D), a collagenous C-type lectin. SP-D is a collagenous carbohydrate-binding glycoprotein (collectin) that plays an important role in the innate immune response of the lung to microbial and antigen challenges (Olde Nordkamp et al., 2014, J. Leukoc. Biol. 96:105-111). The ability of mouse LAIR-1 to bind to C1q and SP-D has not been investigated.

[0076] B. Mouse LAIR-1 The sequence of mouse LAIR-1 (mLAIR-1) is known in the art. For example, the consensus sequence of mLAIR-1a (isoform 1) is as follows: MSLHPVILLVLVLCLGWKINT QEGSLPDITIFPNSSLMISQGTFVTVVCSYSDKHDLYNMVRLEKDGSTFMEKSTEPYKTEDEFEIGPVNETITGHYSCIYSKGITWSERSKTLELKVIKENVIQTPAPPGPTSDTSWLKTYSIY IFTVVSVIFLLCLSALLFCFLRHRQKKQGLPNNKRQQQRPEERLNLATNGLEMTPDIVADDRLPEDRWTETWTPVAGDLQEVTYIQLDHHSLTQRAVGAVTSQSTDMAESSTYAAIIRH

[0077] Amino acids 1-21 form the signal sequence, amino acids 22-144 (underlined) form the extracellular domain, amino acids 145-165 form the transmembrane domain, and amino acids 166-263 form the cytoplasmic domain (SEQ ID NO: 3, UniProtKB-Q8BG84(LAIR1_MOUSE)). Amino acids 27-115 form the Ig-like C2 domain. Amino acids 226-231 and 255-260 form ITIM motifs 1 and 2, respectively. mLAIR-1b (also known as isoform 2) is missing amino acids 124-133 compared to SEQ ID NO: 3. Isoform 3 has amino acids 25-56 replaced by ELCLWFLLYPWATLELIMCTWDAWKETLEYFL (SEQ ID NO: 5) [SLPDITIFPNSSLMISQGTFVTVVCSYSDKHD (SEQ ID NO: 4) of SEQ ID NO: 3], and is missing amino acids 57-263 compared to SEQ ID NO: 3. mLAIR-1d (also known as isoform 5) is missing amino acids 24-172 compared to SEQ ID NO: 3. mLAIR-1e (also known as isoform 6) is missing amino acids 134-172.

[0078] As described above, the extracellular domain of mouse LAIR-1 may be as follows: QEGSL PDITIFPNSSLMISQGTFVTVVCSYSDKHDLYNMVRLEKDGSTFMEKSTEPYKTEDEFEIGPVNETITGHYSCIYSKGITWSERSKTLE LKVIKENVIQTPAPGPTSDTSWLKTYSIY

[0079] (SEQ ID NO: 6) or a fragment thereof, for example, the Ig-like C2 domain (amino acids 6-94 in the underlined section of SEQ ID NO: 6) or the cysteine-enclosed region that forms a disulfide bond between amino acids 49-99 of SEQ ID NO: 3 (amino acids 28-78 of SEQ ID NO: 6, shown in italics). Exemplary alignments of the human extracellular domain and the mouse extracellular domain are shown below.

[0080] Known variants and mutants of LAIR-1 include IYI→MYM, V149G, L154P, and H263R at amino acid positions 143-145, as per SEQ ID NO: 3.

[0081] Meyaard (2008, J. Leukoc. Biol. 83:799-803) revealed that LAIR-1 is widely expressed in mouse immune cells, but one significant difference is that LAIR-1 is negative in B cells, while it is highly expressed in a subset of human B cells. Similar to the human expression pattern, actual flow cytometry data of LAIR-1 expression have been found to be highly expressed in monocytes, macrophages, and DCs, while being expressed at relatively low levels in T cells, NK cells, and Gr-1+ cells (Lebbink et al., 2007 Int. Immunol. 19:1011-1019, Tang et al., 2012, J. Immunol. 188:548-558).

[0082] Tang et al. (2012, J.Immunol.188:548-558) investigated the phenotype of LAIR-1-deficient mice. The knockout mice were healthy, fertile, and exhibited altered immune function, but without the prominent autoimmunity or inflammation observed in CTLA-4 knockout mice. LAIR-1 knockout mice showed increased numbers of dendritic cells, splenic B cells, and regulatory T cells, as well as higher frequencies of activated and memory T cells, indicating enhanced T cell reactivity. However, there were no differences between LAIR-1 WT and knockout mice in EAE and colitis disease models. These disease models may not have been optimal for investigating the LAIR-1 knockout phenotype, and in vitro functional testing of LAIR-1-deficient immune cell subsets was not performed. Furthermore, it is speculated that LAIR-1 knockout mice may not represent the role of LAIR-1 in humans due to differences in the expression and presence of soluble LAIR-2 in humans. Differences in the LAIR-1 genetic pathway between mouse and human viscera are discussed in Sun, et al., Gene, 552:14-145 (2014), and can be considered when designing and evaluating experiments using mouse models.

[0083] III. Immunomodulators This disclosure relates to immunomodulatory agents comprising LAIR-1 agonists. In non-limiting examples, such immunomodulatory agents include anti-LAIR-1 antibodies, as further described below. LAIR-1 agonists typically induce, enhance, or activate negative LAIR-1 signaling. The compositions and methods can be used, for example, to modulate LAIR-1 negative signaling in myeloid cells, T cells, natural killer (NK) cells, or combinations thereof, including antigen-presenting cells (e.g., monocytes, macrophages, or dendritic cells). In some embodiments, the compositions specifically target one or more cell types. Exemplary molecules that may be LAIR-1 agonists are discussed in more detail herein.

[0084] A.Antibodies Immunomodulators can be antibodies. Suitable antibodies are listed below. This sequence can be used by those skilled in the art to prepare antibodies or antigen-binding fragments specific to LAIR-1, as will be discussed in more detail below. Thus, antibodies or antigen-binding fragments can be agonists of LAIR-1.

[0085] The activity of antibodies or antigen-binding fragments specific to LAIR-1 is determined using functional assays known in the art, including those discussed below. Typically, the assay involves determining whether the antibody or antigen-binding fragment increases signaling via LAIR-1 (i.e., is an agonist). In some embodiments, the assay involves determining whether the antibody or antigen-binding fragment reduces the immune response negatively regulated by LAIR-1 (i.e., is an agonist).

[0086] In some embodiments, the disclosed antibody and its antigen-binding fragment bind immunospecifically to LAIR-1. In some embodiments, the antibody binds to the extracellular domain of LAIR-1.

[0087] For example, the following molecules are provided that can bind immunospecifically to LAIR-1: (I) Molecules arranged on the surface of cells (especially living cells), (II) Molecules arranged at endogenous concentrations on the surface of cells (especially living cells), (III) Molecules arranged on the surface of living cells that regulate the binding between LAIR-1 and its ligand, (IV) Molecules arranged on the surface of living cells that reduce or inhibit LAIR-1-mediated immunosuppression, (V) Molecules arranged on the surface of living cells that induce or enhance LAIR-1-mediated immunosuppression. (VI) Myeloid cells including antigen-presenting cells (e.g., monocytes, macrophages, or dendritic cells), wherein molecules are arranged on the surface of living cells that are T cells, natural killer (NK) cells, or a combination thereof. (VII) Molecules of combinations of I-IV and VI, (VIII) Molecules in combination of I-III and V-IV, (IX) Molecules that are arranged on the surface of living cancer cells (AML or ALL) derived from bone marrow or lymphoid cells, promoting apoptosis and differentiation and reducing the self-renewal of cancer stem cells.

[0088] In some embodiments, these molecules can induce apoptosis in LAIR-1 expressing cells via antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cell-mediated cytotoxicity (CDC), or other mechanisms.

[0089] To prepare antibodies or antigen-binding fragments that specifically bind to LAIR-1, purified proteins, polypeptides, fragments, fusions, or epitopes, or polypeptides expressed from their nucleic acid sequences, can be used against LAIR-1. The antibodies or antigen-binding fragments may be prepared using any preferred method known in the art, as will be discussed in more detail below.

[0090] i. Composition of humanized LAIR-1 antibody Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in the human body and, when administered to humans, can potentially elicit an undesirable immune response. Therefore, the use of human or humanized antibodies in this method helps reduce the likelihood that antibodies administered to humans will induce an undesirable immune response.

[0091] When inducing immunization, genetically modified animals (e.g., mice) that can produce a complete repertoire of human antibodies without producing endogenous immunoglobulins can be used. For example, it has been described that homozygous deletion of the antibody heavy chain binding region (J(H)) gene in chimeric mice or germline mutant mice completely inhibits endogenous antibody production. Transmission of a human germline immunoglobulin gene array in such germline mutant mice induces the production of human antibodies upon antigen challenge.

[0092] Selectively, antibodies are produced in other species and “humanized” for administration to humans. The humanized form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody) containing minimal sequences derived from non-human immunoglobulin. A humanized antibody contains human immunoglobulin (recipient antibody), in which residues in the complementarity-determining region (CDR) of the recipient antibody are replaced with residues from a CDR (donor antibody) of a non-human species such as mouse, rat, or rabbit, possessing the desired specificity, affinity, and capability. In some cases, Fv framework residues of the human immunoglobulin are replaced with corresponding non-human residues. The humanized antibody may also contain residues not found in the recipient antibody or in the transferred CDR or framework sequence. Generally, humanized antibodies substantially contain all of at least one, typically two, variable domains, with all or substantially all of the CDR regions in the variable domains corresponding to the CDR regions of non-human immunoglobulins, and all or substantially all of the FR regions being the FR regions of the human immunoglobulin consensus sequence. Humanized antibodies also optimally contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin constant region (Fc).

[0093] Methods for humanizing non-human antibodies are well known in the art. See, for example, Jones, PT, et al. (1986). Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature 321, 522-525. Generally, a humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often called "import" residues, usually obtained from the "import" variable domain. Antibody humanization techniques generally involve the use of recombinant DNA techniques to manipulate the DNA sequences encoding one or more polypeptide chains of the antibody molecule. Humanization can essentially be performed by substituting a rodent CDR or CDR sequence with the corresponding sequence of a human antibody. Thus, the humanized form of a non-human antibody (or fragment thereof) is a chimeric antibody or fragment in which a substantially smaller portion than the complete human variable domain is replaced by the corresponding sequence from a non-human species. In fact, humanized antibodies are typically human antibodies in which several CDR residues and possibly several FR residues are replaced with residues derived from similar sites in rodent antibodies.

[0094] The selection of human variable domains, both light and heavy chain, used in the production of humanized antibodies is crucial for reducing antigenicity. Following a "best-fit" method, the sequences of variable domains from rodent antibodies are screened against the entire library of known human variable domain sequences. The human sequence most closely resembling the rodent sequence is then accepted as the human framework (FR) for the humanized antibody. Alternatively, a specific framework derived from the consensus sequences of all human antibodies for a particular subgroup of the light or heavy chain is used. The same framework may be used for several different humanized antibodies.

[0095] It is even more important to humanize antibodies to retain high affinity for antigens and other desirable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analyzing the parent sequence and various conceptual humanized products using a three-dimensional model of the parent sequence and the humanized sequence. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that exemplify and display possible three-dimensional conformations of selected candidate immunoglobulin sequences. By examining these displays, it becomes possible to analyze the likely roles of residues in the function of the candidate immunoglobulin sequence, i.e., residues that affect the ability of the candidate immunoglobulin to bind to the antigen. In this way, FR residues can be selected and combined from the consensus sequence and the transfer sequence so that desired antibody properties, such as increased affinity for the target antigen(s), are achieved. Generally, CDR residues are directly and most substantially involved in the effect on antigen binding.

[0096] Antibodies can bind to a substrate, label a detectable portion, or both. The detectable portions intended in the compositions of the present invention include fluorescent markers, enzyme markers, and radioactive markers.

[0097] This disclosure relates to a composition comprising a monoclonal antibody targeting LAIR-1. In leukemia, there remains a significant unmet need for therapeutic agents that selectively eradicate leukemic stem cells (LSCs) and leukemic blasts, but suppress normal hematopoietic stem cells. LAIR-1 is an immune receptor previously identified as a potential target for therapeutic interventions in AML.

[0098] This disclosure presents that enhancement of LAIR-1 signaling in the physiological environment of natural collagen is important for its anti-cancer function, and that LAIR-1 signaling promotes the induction of AML cell death. This function manifests particularly in the context of collagen. This specification discloses novel functions of LAIR-1 in AML and strategies for treating AML by targeting LAIR-1 with agonist antibodies.

[0099] This disclosure relates to an agonist LAIR-1 monoclonal antibody that effectively eliminates LSCs and blast cells both ex vivo and in vivo, while conserving healthy cells. LAIR-1 mAbs show increased acceleration of LSC and AML blast cell death both in vivo and ex vivo in the context of LAIR-1 clustering. LAIR-1 mAbs show minimal effect on healthy HSCs or immune cells.

[0100] ii. Exemplary humanized antibodies In one embodiment, a humanized monoclonal antibody (mAb), LAIR-1 mAb, is provided. LAIR-1 mAb is produced by a hybridoma selected from the group consisting of 10D6 and 11B3.

[0101] In another embodiment, a humanized monoclonal LAIR-1 antibody is provided, having at least one light chain or at least one heavy chain of an antibody produced by one or more hybridomas selected from the group consisting of 10D6 and 11B3.

[0102] In another embodiment, a humanized monoclonal LAIR-1 antibody is provided having a variable light chain having at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to a variable light chain having an amino acid sequence according to SEQ ID NOs. 7, 8, 12, 14, 15, 16, or 17.

[0103] In another embodiment, a humanized monoclonal LAIR-1 antibody is provided having a variable heavy chain having at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to a variable heavy chain having an amino acid sequence according to SEQ ID NOs. 9, 18, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44.

[0104] In another embodiment, a humanized monoclonal LAIR-1 antibody is provided, comprising a variable light chain having at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to a variable light chain having an amino acid sequence according to SEQ ID NOs: 7, 8, 12, 14, 15, 16, or 17, and a variable heavy chain having at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to an amino acid sequence according to SEQ ID NOs: 9, 18, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44.

[0105] In another embodiment, a humanized monoclonal LAIR-1 antibody is provided, having a heavy chain having an amino acid sequence having at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 10, 11, 19, or 20, and / or a light chain having an amino acid sequence having at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 8 or 13.

[0106] IV. How to use humanized LAIR-1 This disclosure relates to methods for modulating the ability to specifically remove cancer cells while excluding normal cells, as a key component of effective cancer therapy. This disclosure provides a novel biological understanding of LAIR-1 and novel methods for therapeutic intervention of AML through targeting of LAIR-1 with agonist mAbs, including LAIR-1 mAbs. The methods and compositions disclosed herein can be used to treat patients with AML, relapsed or refractory chronic myelomonocytic leukemia (CMML), and high-risk myelodysplastic syndromes (MDS).

[0107] This disclosure relates to the use of LAIR-1 mAb, an agonist LAIR-1 monoclonal antibody that targets LAIR-1+ leukemia cells and induces potent SHP-1 signaling. This is a novel and effective approach for inhibiting and eliminating AML blasts and LSC precursor cells. As disclosed herein, LAIR-1 binding by the agonist mAb induces cell death in leukemia blasts and LSCs, maintaining therapeutic efficacy and reducing relapse. This is demonstrated in a secondary transplant PDX model (Figure 3C).

[0108] Furthermore, LAIR-1 mAbs potently inhibit AML in two CDX models and in the more difficult-to-treat PDX disease model, as shown in Figures 3A and 3B. The CDX models exhibit systemic inhibition of AML proliferation that is dependent on LAIR-1 expression levels and independent of adaptive immunity. The PDX models are consistent with in vitro results showing LAIR-1 mAb-mediated inhibition of LSC colony proliferation.

[0109] The intrinsic activity of LAIR-1 mAbs is effective in venetoclax / azacitidine (VEN / AZA)-poorly responsive AML. Combination therapy with VEN / AZA induces additive or synergistic therapeutic effects, opening up several treatment options for LAIR-1 mAb treatment in patients with unmet needs.

[0110] This disclosure demonstrates significant downregulation of extra-large B-cell lymphoma (BCL-XL) during LAIR-1 mAb therapy. As a non-limiting example, patients develop resistance to VEN / AZA due to upregulation of BCL-XL, even though B-cell lymphoma 2 (BCL-2) and myeloid leukemia 1 (MCL-1) are effectively suppressed by VEN and AZA, respectively. The combination of VEN / AZA and LAIR-1 mAb effectively suppresses BCL-2 / MCL-1 via BCL-XL, inducing synergistic elimination of AML cells.

[0111] LAIR-1 mAb activity can be enhanced by antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP). However, while LAIR-1 mAb activity is thought to be exerted by crosslinking of LAIR-1 mAbs via interaction with Fc receptors, the lack of LAIR-1 mAb activity on healthy LAIR-1+ immune cells suggests that ADCC and ADCP are not the primary mechanisms of action for LAIR-1 mAbs. Beyond potential interactions in the trans state, intracellular signaling via Fc receptors has been shown to influence cell survival in various situations, including leukemia (Parting et al, 2020). The interaction between the LAIR-1 mAb IgG domain and FcγR may not only facilitate downstream signaling but also stabilize focal synapses with cis-state LAIR-1 and ECM components to promote crosslinking and potentially overcome the signaling threshold required to initiate apoptosis.

[0112] In summary, the research presented herein identifies LAIR-1 as a central inhibitory receptor that modulates signaling in response to collagen and the ECM. This concept will be a major focus of future research. A better understanding of LAIR-1 localization and its communication with other collagen and ECM-interacting proteins may enable the modulation of signaling patterns in both homeostatic and non-homeostatic cellular environments. Importantly, understanding the cell fate-determining networks will lead to novel target discovery, information on ideal interventions for AML and other diseases, and the generation of optimized therapeutic combination strategies.

[0113] This disclosure further relates to the use of LAIR-1 mAb in further leukemia models. Such models include, but are not limited to, VEN / AZA non-responder models derived from M5 AML patients and further combinations known in the art.

[0114] This disclosure relates to the detection of increased CAMK1 / CREB activity in AML cells under LAIR-1 agonist conditions (Figure 10B). This may indicate that CAMK1 / CREB is not required for AML cell survival in the context of the collagen matrix, in which case the LAIR-1 signaling network suppresses alternative downstream mediators such as NF-κB, MAPK, and Src kinase, and that the signaling threshold overcomes the survival-promoting mechanism, instead leading to programmed cell death.

[0115] V. Pharmaceutical Compositions A pharmaceutical composition comprising an immunomodulator is provided. The pharmaceutical composition comprising the immunomodulator can be administered by parenteral (intramuscular, intraperitoneal, intravenous (IV), or subcutaneous injection), transdermal (passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration, or by using biodegradable inserts, and can be formulated into dosage forms suitable for each route of administration.

[0116] In some in vivo approaches, the compositions disclosed herein are administered to a subject in a therapeutically effective dose. As used herein, the terms “effective dose” or “therapeutically effective dose” mean a dose sufficient to treat, inhibit or alleviate one or more symptoms of the disorder being treated, or otherwise sufficient to produce the desired pharmacological and / or physiological effect. The exact dose will vary depending on various factors such as subject-dependent variables (e.g., age, immune system health), disease, and the treatment being administered.

[0117] As further research is conducted on the disclosed immunomodulators, information regarding appropriate dosage levels for treating various medical conditions in various patients will become clear, and those skilled in the art will be able to determine the appropriate dosage considering the treatment situation, age, and the recipient's overall health status. The dosage to be selected will depend on the desired therapeutic effect, route of administration, and desired duration of treatment. The disclosed immunomodulators are generally administered to mammals at a dosage level of 0.001 to 20 mg / kg body weight per day. Generally, dosages may be lower when administered by intravenous injection or infusion.

[0118] In certain embodiments, the immunomodulator is administered locally, for example, by direct injection into the site of treatment. Typically, injection increases the local concentration of the immunomodulatory composition to a higher level than that achievable by systemic administration. The immunomodulatory composition can be combined with a matrix as described above to help increase the local concentration of the polypeptide composition by reducing the passive diffusion of polypeptides from the site of treatment.

[0119] A. Formulations for parenteral administration In some embodiments, the compositions disclosed herein, including those containing peptides and polypeptides, are administered by parenteral injection in aqueous solution. The formulations may also be in the form of suspensions or emulsions. Generally, pharmaceutical compositions are provided that contain an effective amount of peptide or polypeptide and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions optionally include one or more of the following additives: diluents, sterile water, buffered salines having various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength, and detergents and solubilizers (e.g., Tween 20 (Polysorbate-20), Tween 80 (Polysorbate-80)), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol), and bulk substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations can be freeze-dried and redissolved / resuspended immediately before use. The formulations can be sterilized, for example, by filtration through a bacterial capture filter, by incorporating a sterilizing agent into the composition, by irradiating the composition with radiation, or by heating the composition.

[0120] B. Formulations for oral administration In embodiments, the composition is formulated for oral delivery. Oral solid dosage forms are generally described in Chapter 89 of Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co., Easton, Pa. 18042). Solid dosage forms include tablets, capsules, pills, lozenges, or cachets, pellets, powders, or granules, or the incorporation of the material into particulate preparations of polymer compounds such as polylactic acid or polyglycolic acid, or into liposomes. Such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the disclosed substance. See, for example, pages 1435-1712 of Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042), incorporated herein by reference. The composition may be prepared in liquid form or in dry powder form (e.g., lyophilized). Compositions can be formulated using liposome or proteinoid encapsulation. Liposome encapsulation can be used, and liposomes can be derivatized with various polymers (e.g., U.S. Patent No. 5,013,556). See also Marshall, K. In: Modern Pharmaceutics Edited by GS Banker and CT Rhodes Chapter 10, 1979. Generally, formulations include a peptide (or a chemically modified form thereof) and an inert component that protects the peptide in the gastric environment and releases a biologically active substance in the intestines.

[0121] Drugs can be chemically modified to facilitate oral delivery of derivatives. Generally, the intended chemical modification involves attaching at least one moiety to the component molecule itself, which enables uptake from the stomach or intestines into the bloodstream, or direct uptake into the intestinal mucosa. It is also desirable to increase the overall stability of the component(s) and extend their circulation time in the body. PEGylation is an exemplary chemical modification for pharmaceutical applications. Other usable ingredients include propylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyproline, poly-1,3-dioxolane, and poly-1,3,6-thioxocan (see, for example, Abuchowski and Davis (1981) “Soluble Polymer-Enzyme Adducts,” in Enzymes as Drugs. Hocenberg and Roberts, eds. (Wiley-Interscience: New York, NY) pp. 367-383; and Newmark, et al. (1982) J. Appl. Biochem. 4:185-189).

[0122] Another embodiment provides a liquid dosage form for oral administration, comprising pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, which may include adjuvants such as inert diluents, wetting agents, emulsifiers, and suspending agents, as well as other components including sweeteners, flavorings, and fragrances.

[0123] A controlled-release oral formulation may be preferable in some cases. This drug can be incorporated into an inert matrix that allows for release via diffusion or exudation mechanisms, such as gum. A slowly denatured matrix may also be incorporated into the formulation. Another form of controlled release is based on the Oros therapeutic system (Alza Corp.), in which the drug is encapsulated in a semipermeable membrane, allowing water to enter through a single small opening via osmotic effect and push the drug out.

[0124] In the case of oral formulations, the release site may be the stomach, small intestine (duodenum, jejunum, or ileum), or large intestine. In some embodiments, release avoids the harmful effects of the gastric environment by protecting the drug (or derivative) or by releasing the drug (or derivative) into the intestines beyond the gastric environment. To ensure complete gastric tolerance, a coating that does not penetrate to at least pH 5.0 is essential. Examples of more common inert components used as enteric coatings include cellulose trimellitate acetate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, vinyl polyphthalate acetate (PVAP), Eudragit L30D™, Aquateric™, cellulose phthalate acetate (CAP), Eudragit L™, Eudragit S™, and Shellac™. These coatings may be used as mixed films.

[0125] C. Preparations for topical administration The disclosed immunomodulatory agents may be applied topically. Topical administration is not effective for most peptide formulations, but may be particularly effective when applied to the lungs, nasal cavity, oral cavity (sublingual, buccal), vaginal, or rectal mucosa.

[0126] When the composition is delivered as either an aerosol or spray-dried particles having an aerodynamic diameter of less than approximately 5 microns, it can be delivered to the lungs during inhalation, pass through the pulmonary epithelial layer, and reach the bloodstream.

[0127] A wide range of mechanical devices designed to deliver therapeutic drugs to the lungs are available, including, but not limited to, nebulizers, metered-dose inhalers, and powder inhalers, all of which are well known to those skilled in the art. Some specific examples of commercially available devices include the Ultravent nebulizer (Mallinckrodt Inc., St. Louis, Mo.), the Acorn II nebulizer (Marquest Medical Products, Englewood, Colo.), the Ventolin metered-dose inhaler (Glaxo Inc., Research Triangle Park, NC), and the Spinhaler powder inhaler (Fisons Corp., Bedford, Mass.). Nektar, Alkermes, and Mannkind have all approved inhalable insulin powder preparations, or their technologies may be applicable to the formulations described herein in clinical trials.

[0128] Formulations for administration to mucous membranes are typically spray-dried drug particles and can be incorporated into tablets, gels, capsules, suspensions, or emulsions. Standard pharmaceutical excipients are available from any prescriber.

[0129] Transdermal formulations may also be prepared. These transdermal formulations are typically ointments, lotions, sprays, or patches, all of which can be prepared using standard techniques. Transdermal formulations may need to contain penetration enhancers.

[0130] D. Delivery-controlled polymer matrix The immunomodulatory agents disclosed herein may also be administered in controlled-release formulations. Controlled-release polymer devices may be manufactured for long-term systemic release after implantation of the polymer device (rod, cylinder, film, disc) or injection (microparticles). The matrix may be in the form of microparticles such as microspheres, in which case the drug is dispersed within a solid polymer matrix or microcapsule, and the core consists of a different material from the polymer shell, with the peptide dispersed or suspended within the core, which is essentially liquid or solid. Unless otherwise defined herein, microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, the polymer may be cast as a thin slab or film ranging from nanometers to 4 centimeters, a powder produced by grinding or other standard techniques, or even as a gel such as a hydrogel.

[0131] For the delivery of fusion polypeptides or nucleic acids encoding fusion polypeptides, either non-biodegradable or biodegradable matrices may be used, and in some embodiments, biodegradable matrices are preferred. These matrices may be natural polymers or synthetic polymers, and in some embodiments, synthetic polymers are preferred because they are better characterized by their degradation and release profiles. The polymer is selected based on the period over which release is desired. In some cases, linear release may be most useful, but in other cases, pulsed release or "bulk release" may yield more effective results. The polymer may be in the form of a hydrogel (typically absorbing up to about 90% by weight of water) and may optionally be crosslinked with polyvalent ions or polymers.

[0132] The matrix may be formed by solvent evaporation, spray drying, solvent extraction, and other methods known to those skilled in the art. Biodegradable microspheres may be prepared using any method developed for producing microspheres for drug delivery, such as those described in Mathiowitz and Langer, J. Controlled Release, 5:13-22 (1987), Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987), and Mathiowitz, et al., J. Appl. Polymer Sci., 35:755-774 (1988).

[0133] These devices may be formulated for local release (typically administering doses much lower than those used for systemic treatment) or systemic delivery to treat the implantation or injection site. They may be implanted or injected subcutaneously, intramuscularly, or intrafatally, or swallowed.

[0134] VI. Therapeutic composition The following compositions should be understood as exemplary compositions relating to this disclosure. Such compositions are not intended to limit the scope of this disclosure.

[0135] The compositions described herein may be administered to the host either alone or in combination with pharmaceutically acceptable excipients in an amount sufficient to induce a suitable antitumor response. Responses include, but are not limited to, specific immune responses, nonspecific immune responses, both specific and nonspecific responses, innate immunity, primary immune responses, adaptive immunity, secondary immune responses, memory immune responses, immune cell activation, immune cell proliferation, immune cell differentiation, and cytokine expression. LAIR-1 cell therapy may be of any structural type: autologous, allogeneic, universal, or armed.

[0136] The effective amount of the compositions described herein may be administered in a single dose, but is not limited to a single dose. Therefore, administration may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more doses of the composition. In the method of the present invention, if there are multiple doses, the doses may be spaced apart by time intervals of 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes or more, or by intervals of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, etc. In the context of time, the term "about" means any time interval within ±30 minutes. The administration may also be spaced apart by time intervals of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, and combinations thereof. The present invention is not limited to administration intervals that are spaced evenly over time, and non-limiting examples include non-uniform intervals, such as a priming schedule consisting of administrations of 1 day, 4 days, 7 days, and 25 days. In such embodiments, various compositions may be administered using regimens of different doses and intervals. In such embodiments, the first composition may be administered in one or more doses spaced at a specific time interval, while the second composition may be administered in different numbers of doses spaced at different time intervals. In such embodiments, the first and second compositions may differ in composition.

[0137] The compositions of this disclosure may be administered in a single dose or in several doses, if administered in several doses, each dose may contain at least 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, 9.5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, and 50 mg / kg body weight. In certain embodiments, a single dose may preferably contain at least 2 mg / kg, 4.5 mg / kg, 10 mg / kg, 20 mg / kg, and 30 mg / kg body weight. In yet another embodiment, a single dose most preferably contains 10 mg / kg body weight.

[0138] In the present invention, for example, administration schedules such as once a week, twice a week, three times a week, four times a week, five times a week, six times a week, seven times a week, once every two weeks, once every three weeks, once every four weeks, and once every five weeks are available. The administration schedule includes administration over a total period of, for example, one week, two weeks, three weeks, four weeks, five weeks, six weeks, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, and twelve months.

[0139] The cycles of the above administration plan are provided. The cycles can be repeated, for example, every 7 days, every 14 days, every 21 days, every 28 days, every 35 days, every 42 days, every 49 days, every 56 days, every 63 days, every 70 days, etc. Non-administration intervals may occur between cycles, and these intervals may be, for example, about 7 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, about 56 days, about 63 days, about 70 days, etc. In this context, the term "about" means ±1 day, ±2 days, ±3 days, ±4 days, ±5 days, ±6 days, or ±7 days.

[0140] The method of co-administration with additional therapeutic agents is well known in the art (Hardman, et al. (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, NY; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila., PA; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., PA).

[0141] The effective dose of a therapeutic agent is the amount that reduces or improves the symptoms by at least 10%, more typically at least 20%, most typically at least 30%, typically at least 40%, more typically at least 50%, most typically at least 60%, often at least 70%, more often at least 80%, most often at least 90%, conventionally at least 95%, more conventionally at least 99%, and most conventionally at least 99.9%.

[0142] Therapeutic formulations may be prepared for storage, for example, in the form of lyophilized powder, slurry, aqueous solution, or suspension, by mixing with physiologically acceptable carriers, excipients, or stabilizers.

[0143] While several embodiments are disclosed in the aforementioned specification, it will be understood by those skilled in the art that, by benefiting from the teachings presented in the aforementioned description and the associated drawings, many modifications and other embodiments relating to this disclosure will come to mind. Therefore, it will be understood that this disclosure is not limited to the specific embodiments disclosed above, but is intended to encompass many modifications and other embodiments within the scope of any claims that can describe the disclosed subject matter.

[0144] It should be emphasized that the above embodiments are merely feasible examples presented to clearly illustrate the principles of this disclosure. Process descriptions and blocks within the flowcharts should be understood as representing modules, segments, or code portions containing one or more executable instructions for implementing a particular logical function or step within the process, and, as those skilled in the art will understand, include alternative implementations, depending on the function included, that may include no function at all or no function performed at all, or that the functions are performed in a reverse order of the illustrated or described order, substantially simultaneous or in a reverse order. Many variations and modifications can be made to the above embodiments (or embodiments) without substantially departing from the spirit and principles of this disclosure. Furthermore, the scope of this disclosure is intended to include all possible combinations and subcombinations of all elements, features, and embodiments discussed above. All such modifications and variations are intended to be incorporated herein within the scope of this disclosure, and all conceivable claims for individual embodiments or combinations of elements or steps are intended to be supported by this disclosure.

[0145] VII. Manufacturing method A. Method for producing antibodies Antibodies can be produced in cell cultures, phages, or in a variety of animals, including but not limited to cattle, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, and apes. Therefore, in one embodiment, the antibody is a mammalian antibody. Phage techniques can be used to isolate the initial antibody or to generate variants with altered specificity or binding properties. Such techniques are commonplace and well known in the art. In one embodiment, the antibody is produced by recombinant means known in the art. For example, recombinant antibodies can be produced by transfecting host cells with a vector containing a DNA sequence encoding the antibody. One or more vectors can be used to transfect host cells with DNA sequences expressing at least one VL region and one VH region. Examples of descriptions of recombinant methods for antibody generation and production include Delves, Antibody Production: Essential Techniques (Wiley, 1997); Shephard, et al., Monoclonal Antibodies (Oxford University Press, 2000); Goding, Monoclonal Antibodies: Principles And Practice (Academic Press, 1993); and Current Protocols In Immunology (John Wiley & Sons, most recent edition).

[0146] The disclosed antibodies can be modified by recombinant means to further enhance their efficacy in mediating desired functions. Therefore, the modification of antibodies by substitution using recombinant means falls within the scope of the present invention. Typically, substitutions are conservative substitutions. For example, at least one amino acid within the constant region of an antibody can be replaced with a different residue. See, for example, U.S. Patent No. 5,624,821, U.S. Patent No. 6,194,551, Patent Application No. WO9958572, and Angal, et al., Mol. Immunol. 30:105-08 (1993). Amino acid modifications include amino acid deletion, addition, and substitution. In some cases, such modifications are made to reduce undesirable activity, such as complement-dependent cytotoxicity. Often, antibodies are labeled by covalently or noncovalently binding a substance that emits a detectable signal. A wide variety of labeling and conjugation techniques are known and widely reported in both scientific and patent literature. These antibodies may be screened for binding to LAIR-1 or LAIR-2 proteins, polypeptides, or fusion proteins. See, for example, Antibody Engineering: A Practical Approach (Oxford University Press, 1996).

[0147] For example, suitable antibodies with desired biological activity can be identified using in vitro assays, including but not limited to in vivo assays for inhibition of proliferation, migration, adhesion, soft agar growth, angiogenesis, intercellular communication, apoptosis, transport, signaling, and tumor growth. The antibodies provided herein may also be useful for diagnostic applications. As capture antibodies or non-neutralizing antibodies, they can be screened for their ability to bind to a specific antigen without inhibiting the antigen's receptor binding or biological activity. As neutralizing antibodies, antibodies may be useful in competitive binding assays.

[0148] Antibodies usable in the disclosed compositions and methods include any class of whole immunoglobulin (i.e., a complete antibody), fragments thereof, and synthetic proteins containing at least the antigen-binding variable domain of the antibody. The variable domain has a different sequence for each antibody and is used for the binding and specificity of each antibody to a particular antigen. However, variability is not usually evenly distributed throughout the variable domain of an antibody. It is usually concentrated in three segments called complementarity-determining regions (CDRs) or highly variable regions in both the light and heavy chain variable domains. The more highly conserved portion of the variable domain is called the framework (FR). The natural heavy and light chain variable domains each consist of four FR regions, primarily employing a beta-sheet configuration, connected by three CDRs, forming loops that connect to the beta-sheet structure, and in some cases forming part of the beta-sheet structure. The CDRs within each chain are held together in very close proximity by the FR region and, together with CDRs from other chains, contribute to the formation of the antibody's antigen-binding site.

[0149] Antibody fragments possessing biological activity are also disclosed. These fragments include insertions, deletions, substitutions, or other selected modifications to specific regions or specific amino acid residues, whether or not they are bound to other sequences, but the activity of the fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment.

[0150] The technique can also be adapted for the production of single-chain antibodies specific to antigenic peptides. Methods for producing single-chain antibodies are well known to those skilled in the art. Single-chain antibodies can be created by fusing the variable domains of the heavy and light chains together using a short peptide linker, thereby reconstituting an antigen-binding site on a single molecule. Variable fragments of single-chain antibodies (scFv) have been developed in which the C-terminus of one variable domain is linked to the N-terminus of another variable domain via 15-25 amino acid peptides or linkers without significantly impairing antigen binding or binding specificity. The linker is selected so that the heavy and light chains can bind together in the appropriate conformation.

[0151] Divalent single-chain variable fragments (di-scFv) can be manipulated by linking two scFv. This can be done by generating a single peptide chain with two VH regions and two VL regions, resulting in a serial scFv. ScFv can also be designed using a linker peptide (about 5 amino acids) that is too short for the two variable regions to fold, thereby forcing the scFv to dimerize. This type is known as a diabody. Diabodies have been shown to have a dissociation constant up to 40 times lower than the corresponding scFv, meaning they have a much higher affinity for their target. Even shorter linkers (1 or 2 amino acids) lead to the formation of trimers (tribodies or tribodies). Tetrabodies have also been produced. They exhibit even higher affinity for their target than diabodies.

[0152] Monoclonal antibodies are obtained from a substantially homogeneous antibody population; that is, individual antibodies within the population are identical except for spontaneous mutations that may exist in a small subset of antibody molecules. Monoclonal antibodies include "chimeric" antibodies, insofar as they exhibit the desired antagonistic activity, in which a portion of the heavy chain and / or light chain is identical or homogeneous to a corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homogeneous to a corresponding sequence in an antibody from a different species or belonging to a different antibody class or subclass, as well as fragments of such antibodies.

[0153] Monoclonal antibodies can be produced using any procedure for generating monoclonal antibodies. In the hybridoma method, mice or other suitable host animals are typically immunized with an immunizer and draw lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizer. Alternatively, lymphocytes may be immunized in vitro.

[0154] Antibodies can also be produced using recombinant DNA methods. The DNA encoding the disclosed antibodies can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of mouse antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques.

[0155] Methods for producing antibodies using protein chemistry are also known in this art. One method for producing antibodies-containing proteins is to link two or more peptides or polypeptides using protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment by utilizing the Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl) chemical reaction (Applied Biosystems, Inc., Foster City, CA). Those skilled in the art will readily understand that peptides or polypeptides corresponding to antibodies can be synthesized, for example, by standard chemical reactions. For example, while a peptide or polypeptide can be synthesized, it cannot be cleaved from its synthetic resin, whereas the other fragment of an antibody can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group on the other fragment that is functionally blocked. By a peptide condensation reaction, these two fragments can be covalently linked via peptide bonds at their carboxyl and amino terminals, respectively, to form an antibody or fragment thereof, or the peptides or polypeptides can be synthesized independently in vivo as described above. Once isolated, these independent peptides or polypeptides can be linked and form antibodies or their antigen-binding fragments via similar peptide condensation reactions.

[0156] For example, enzymatic ligation of cloned or synthetic peptide segments allows relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides, or entire protein domains. Alternatively, the innate chemical ligation of synthetic peptides can be utilized to synthetically construct larger peptides or polypeptides from shorter peptide fragments. This method consists of a two-step chemical reaction. The first step is a chemoselective reaction between an unprotected synthetic peptide-α-thioester and another unprotected peptide segment containing an amino-terminal Cys residue, with a thioester-bonded intermediate being the initial conjugation product. Without changing the reaction conditions, this intermediate undergoes a spontaneous and rapid intramolecular reaction to form a innate peptide bond at the ligation site.

[0157] VIII. Patients to be treated A. Cancer The disclosed compositions and methods can be used to treat cancer. Such compositions and methods can be used to treat all solid tumors and hematological malignancies. Cancer cells acquire a characteristic set of functional capabilities through various mechanisms during their development. Such capabilities include evasion of apoptosis, self-sufficiency of proliferation signals, insensitivity to anti-proliferative signals, tissue invasion / metastasis, unlimited expressivity, and persistent angiogenesis. The term “cancer cells” means to encompass both precancerous cancer cells and malignant cancer cells. In some embodiments, cancer refers to a locally confined benign tumor. In other embodiments, cancer refers to a malignant tumor that invades and destroys adjacent body structures and metastasizes to distant sites. In yet another embodiment, cancer is associated with a specific cancer antigen (e.g., pan-cancer antigen (KS1 / 4), ovarian cancer antigen (CA125), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), CD19, CD20, HER2 / neu, etc.).

[0158] The methods and compositions disclosed herein are for carcinomas (including bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, skin cancer, and squamous cell carcinoma), lymphoid hematopoietic malignancies (including leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, and Burkitt lymphoma), myeloid hematopoietic malignancies (including acute myeloid leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia (CMML), high-risk myelodysplastic syndrome (MDS), and promyelocytic leukemia), and mesenchymal It is useful in the treatment or prevention of various cancers or other abnormal proliferative disorders, including (but not limited to) tumors of mesenchymal origin (including fibrosarcoma and rhabdomyosarcoma), other tumors (including malignant melanoma, seminomas, teratocarcinomas, neuroblastomas, and gliomas), tumors of the central and peripheral nervous systems (including astrocytomas, neuroblastomas, gliomas, and schwannomas), tumors of mesenchymal origin (including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma), and other tumors (including malignant melanoma, xeroderma pigmentosum, keratosarcoma, seminomas, follicular carcinoma of the thyroid gland, and teratocarcinomas).

[0159] Cancers caused by abnormalities in apoptosis can also be treated by the disclosed methods and compositions. Such cancers include, but are not limited to, follicular lymphoma, carcinomas with p53 mutations, hormone-dependent tumors of the breast, prostate, and ovaries, and precancerous lesions such as familial adenomatous polyposis and myelodysplastic syndrome. In certain embodiments, the methods and compositions treat or prevent malignancies or abnormal proliferative changes (such as metaplasia and dysplasia) or hyperproliferative disorders of the ovaries, bladder, breast, colon, lungs, skin, pancreas, or uterus. In other specific embodiments, the methods and compositions treat or prevent sarcomas, melanomas, or leukemias.

[0160] The disclosed compositions and methods are particularly useful for treating cancers associated with cells expressing abnormally high levels of LAIR-1, high levels of LAIR-1 ligand, or combinations thereof.

[0161] Certain cancers and related disorders that can be treated or prevented by the methods and compositions disclosed herein include, but are not limited to, leukemia (acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia (myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, and myelodysplastic syndromes, etc.), chronic leukemia (chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia, etc., but not limited to these)), polycythemia vera, lymphoma (e.g., Hodgkin's lymphoma or non-Hodgkin's lymphoma (e.g., diffuse anaplastic lymphoma)), This includes, but is not limited to, ALK-negative large B-cell lymphoma (DLBCL), ALK-positive large B-cell lymphoma (DLBCL), ALK-positive ALK+ anaplastic large B-cell lymphoma (ALCL), and acute myeloid lymphoma (AML), as well as multiple myeloma (e.g., smoldering multiple myeloma, non-secretory myeloma, osteosclerosing myeloma, plasmacytoma, solitary plasmacytoma, and extramedullary plasmacytoma), primary macroglobulinemia, and monoclonal lesions of unknown significance. Sexual immunoglobulinemia, benign monoclonal immunoglobulinemia, heavy chain disease, bone and connective tissue sarcomas (e.g., bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor of bone, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, vascular sarcoma (angiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, synovial sarcoma, but not limited to these), brain tumors (glioma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, nonungrian tumor, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineal cell tumor, pineoblastoma, primary This includes, but is not limited to, brain lymphoma, breast cancer (including, but is not limited to, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary carcinoma, mucinous carcinoma, tubular carcinoma, papillary carcinoma, Paget's disease, and inflammatory breast cancer), adrenal carcinoma (including, but is not limited to, pheochromocytoma and adrenocortical carcinoma), thyroid cancer (including, but is not limited to, papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma, and anaplastic thyroid carcinoma), pancreatic cancer (including, but is not limited to, insulinoma, gastrinoma, glucagonoma, VIP-producing tumors, somatostatin-secreting tumors, carcinoid tumors, or islet cell tumors),(Not limited to these), pituitary cancer (including but not limited to Cushing's disease, prolactin-secreting tumors, acromegaly, diabetes insipidus, etc.), eye cancer (including but not limited to ocular malignant melanoma such as iris melanoma, choroidal melanoma, ciliary melanoma, retinoblastoma, etc.), vaginal cancer (including but not limited to squamous cell carcinoma, adenocarcinoma, malignant melanoma, etc.), vulvar cancer (including but not limited to squamous cell carcinoma, malignant melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, Paget's disease, etc.), cervical cancer (including but not limited to squamous cell carcinoma, adenocarcinoma, etc.), uterus Cancer (including but not limited to endometrial cancer and uterine sarcoma), ovarian cancer (including but not limited to ovarian epithelial carcinoma, borderline malignant tumor, germ cell tumor, stromal tumor, etc.), esophageal cancer (including but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, malignant melanoma, plasmacytoma, verrucous carcinoma, oat cell (small cell) carcinoma, etc.), gastric cancer (including but not limited to adenocarcinoma, mycoplasmic (polypoid), ulcerative, superficial spreading type, widespread spreading type, malignant lymphoma, liposarcoma, fibrosarcoma and carcinosarcoma, etc.), colon cancer, rectal cancer, liver cancer (including but not limited to hepatocellular carcinoma and hepatoblastoma, etc.) (and not limited to these), gallbladder cancer (including but not limited to adenocarcinoma), bile duct cancer (including but not limited to papillary carcinoma, nodular carcinoma, diffuse carcinoma), lung cancer (including but not limited to non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, small cell lung cancer), testicular cancer (including but not limited to germblastoma, seminomas, anaplastic carcinoma, typical carcinoma, spermatocyte carcinoma, nonseminomas, embryonic carcinoma, teratoma carcinoma, choriocarcinoma (yolk sac tumor), etc.), prostate cancer (including but not limited to adenocarcinoma, leiomyosarcoma, rhabdomyosarcoma), penile cancer, oral cancer (squamous cell carcinoma, basal carcinoma, etc.) (including, but not limited to,) salivary gland cancer (including, but not limited to, adenocarcinoma, mucoepidermoid carcinoma, adenoid cystic carcinoma, etc.), pharyngeal cancer (including, but not limited to, squamous cell carcinoma, verrucous pharyngeal carcinoma, etc.), skin cancer (including, but not limited to, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, superficial spreading malignant melanoma, nodular malignant melanoma, lentigo malignant melanoma, acral lentiginous malignant melanoma, etc.), renal cancer (including, but not limited to, renal cell carcinoma, adenocarcinoma, adrenal tumor, fibrosarcoma, transitional cell carcinoma (pelvis and / or ureter), etc.), Wilms' tumor, bladder cancer (transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma,This includes, but is not limited to, carcinosarcoma. Furthermore, cancers include myxosarcoma, osteosarcoma, endothelioma, lymphangioendothelioma, mesothelioma, synovial carcinoma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic lung cancer, sweat gland carcinoma, sebaceous carcinoma, papillary carcinoma, and papillary adenocarcinoma (for a review of these disorders, see Fishman et al., 1985, Medicine, 2d Ed., JBLippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books USA, Inc., United States of America).

[0162] B. Combination therapy The disclosed immunomodulatory agent may be administered alone or in combination with one or more additional therapeutic agents to a subject requiring it. In some embodiments, the immunomodulatory agent and the additional therapeutic agents are administered separately and simultaneously. The immunomodulatory agent and the additional therapeutic agents may also be administered as part of the same composition. In other embodiments, the immunomodulatory agent and the second therapeutic agent are administered separately at different times as part of the same treatment plan.

[0163] The subject may be administered the first therapeutic agent at least 1, 2, 3, 4, 5, or 6 hours or at least 1, 2, 3, 4, 5, 6, or 7 days before administering the second therapeutic agent. In some embodiments, the subject may be administered the first drug at least once every 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, or 48 days prior to the first administration of the second drug. The immunomodulator may be the first or second therapeutic agent.

[0164] Immunomodulators and additional therapeutic agents may be administered as part of a treatment regimen. For example, if the first therapeutic agent may be administered to the subject every four days, the second therapeutic agent may be administered on day 1, day 2, day 3, or day 4, or a combination thereof. The first or second therapeutic agent may be administered repeatedly throughout the entire treatment plan.

[0165] Exemplary molecules include, but are not limited to, cytokines, chemotherapeutic agents, radionuclides, other immunotherapeutic agents, enzymes, antibiotics, antiviral agents (in particular protease inhibitors alone or in combination with nucleosides for the treatment of HIV or hepatitis B or C), antiparasitic (parasitic helminths, parasitic protozoa) preparations, growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and their bioactive fragments (including humanized antibodies, single-chain antibodies and chimeric antibodies), antigens and vaccine preparations (including adjuvants), peptide drugs, anti-inflammatory agents, ligands that bind to Toll-like receptors (including, but not limited to, CpG oligonucleotides) to activate the innate immune system, molecules that mobilize and optimize the adaptive immune system, other molecules that activate or upregulate the action of cytotoxic T lymphocytes, natural killer cells and helper T cells, and other molecules that inactivate or downregulate suppressor or regulatory T cells.

[0166] Additional therapeutic agents are selected based on the medical condition, disorder, or disease being treated. For example, immunomodulators may be administered concurrently with one or more additional agents that function to enhance or promote the immune response, or to reduce or inhibit the immune response.

[0167] IX. Kit The disclosed LAIR-1 immunomodulatory agents may be packaged in sealed containers such as ampoules or sachets indicating the dose. The agents may be supplied as dry, sterile lyophilized powder or anhydrous concentrate in a sealed container and may be reconstituted, for example, with water or saline to a concentration appropriate for administration to the subject. For example, the agents may be supplied as dry, sterile lyophilized powder in a sealed container in unit doses of at least 5 mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg or at least 75 mg. The lyophilized agents may be stored in their original containers at a temperature between 2 and 8°C and are typically administered within 12 hours, 6 hours, 5 hours, 3 hours, or 1 hour after reconstitution.

[0168] In alternative embodiments, the drug is supplied in liquid form in a sealed container indicating the volume and concentration. In some embodiments, the liquid form of the drug is supplied in a sealed container containing at least 1 mg / ml, at least 2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 50 mg / ml, at least 100 mg / ml, at least 150 mg / ml, and at least 200 mg / ml of the drug.

[0169] Pharmaceutical packs and kits are also provided, each containing one or more containers filled with a pharmaceutical agent. Additionally, one or more other prophylactic or therapeutic agents useful for treating a disease may also be included in the pharmaceutical pack or kit. The pharmaceutical pack or kit may contain one or more containers filled with one or more components of the disclosed pharmaceutical composition. Optionally, such container(s) may be accompanied by a notice in the form prescribed by the government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, which reflects that the manufacture, use, or sale for human administration has been approved by the government agency.

[0170] Kits designed for the above methods are also provided. Embodiments typically include one or more LAIR-1 immunomodulators. In certain embodiments, the kit also includes one or more other prophylactic or therapeutic agents useful for the treatment of cancer in one or more containers. In other embodiments, the kit also includes one or more anti-inflammatory agents useful for the treatment of inflammatory and autoimmune diseases in one or more containers. [Examples]

[0171] Example 1. LAIR antibody, and its heavy chain sequence and light chain sequence Materials and methods Mice were immunized with soluble human LAIR-1 (soluble LAIR-1 refers to the extracellular domain of LAIR-1) fused to mouse G2a Fc (SEQ ID NO: 10). Two weeks later, the mice were challenged with the same immunogen. Two weeks after that, the mice received a third dose of the antigen. Three days after the final boost, mouse spleen cells were harvested, resuspended in RPMI supplemented with 10% FBS and glutamine, and then fused to form hybridomas.

[0172] Heavy and light chain RACE (rapid amplification of cDNA ends) identification was performed according to the following protocol: (1) mRNA denaturation, (2) cDNA synthesis, (3) 5' RACE reaction, (4) analysis of PCR results (amplified DNA fragments were visualized on an agarose gel, and the correct antibody variable region DNA fragment should be 500-700 base pairs in size), (5) TOPO cloning of PCR-positive bands, (6) PCR amplification of TOPO clones, subsequent gel electrophoresis and recovery from agarose gel, (7) sequencing of a total of 218 clones, and (8) CDR analysis using sequencing data (the CDR region was defined using VBASE2, available from vbase2.org).

[0173] result In the following amino acid sequences, the underlined portion of the sequence is the complementarity-determining region (CDR).

[0174] 10D6 array 10D6 VL DIQMTQSPASQSASLGESVTITC LASQTIGTWLA WFQQKPGKSPQLLIY AATSLAD GVPSRFSGSGSGTKFSFKISSLQAEDFVSYYC QQLYSAPYT FGGGTKLEIK*(sequence number 7)

[0175] c10D6 light chain (human kappa constant domain) DIQMTQSPASQSASLGESVTITC LASQTIGTWLA WFQQKPGKSPQLLIY AATSLAD GVPSRFSGSGSGTKFSFKISSLQAEDFVSYYC QQLYSAPYT FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (Sequence ID 8)

[0176] 10D6 VH EVHLVETGGGLVQPKGSLKLSCAAS GFNFNTNAMN WVRQAPGKGLEWVA RIRTKSNNYATYYADSVKD RFTISRDDSQSMLYLQMNNLKTEDTAMYYCVS TPYFTY WGQGTLVTVSA*(Sequence ID 9)

[0177] c10D6 heavy chain (human IgG1 constant domain) EVHLVETGGGLVQPKGSLKLSCAAS GFNFNTNAMN WVRQAPGKGLEWVA RIRTKSNNYATYYADSVKD RFTISRDDSQSMLYLQMNNLKTEDTAMYYCVS TPYFTYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*(Sequence ID 10)

[0178] c10D6 heavy chain (human IgG4 constant domain) EVHLVETGGGLVQPKGSLKLSCAAS GFNFNTNAMN WVRQAPGKGLEWVA RIRTKSNNYATYYADSVKD RFTISRDDSQSMLYLQMNNLKTEDTAMYYCVS TPYFTY WGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*(Sequence ID 11)

[0179] 11B3 array 11B3 VL DIQMAQSSSSFSVSLGDRVTITC KASEDIYIRLAWYQQKPGNAPRLLIS TATLET GVPSRFSGSGSGKDYTLSITSLQTEDVATYYC QQYWSTPYT FGGGTRLEIK (SEQ ID NO: 12)

[0180] c11B3 light chain (human kappa constant domain) DIQMAQSSSSFSVSLGDRVTITC KASEDIYIRLA WYQQKPGNAPRLLIS TATLET GVPSRFSGSGSGKDYTLSITSLQTEDVATYYC QQYWSTPYT FGGGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*(SEQ ID NO: 13)

[0181] h11B3 VL-1 DIQMTQSPSSLSASVGDRVTITC KASEDIYIRLA WYQQKPGKAPRLLIS TATLET GVPSRFSGSGSGTDYTFTISSLQPEDIATYYC QQYWSTPYT FGGGTRLEIK*(SEQ ID NO: 14)

[0182] h11B3 VL-1 light chain (kappa constant domain) DIQMTQSPSSLSASVGDRVTITC KASEDIYIRLA WYQQKPGKAPRLLIS TATLET GVPSRFSGSGSGTDYTFTISSLQPEDIATYYC QQYWSTPYT FGGGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*(SEQ ID NO: 15)

[0183] h11B3 VL-2 DIQMTQSPSSLSASLGDRVTITC KASEDIYIRLAWYQQKPGKAPRLLIS TATLET GVPSRFSGSGSGTDYTFTISSLQPEDIATYYC QQYWSTPYT FGGGTRLEIK*(Sequence ID 16)

[0184] h11B3 VL-2 light chain (kappa constant domain) DIQMTQSPSSLSASLGDRVTITC KASEDIYIRLA WYQQKPGKAPRLLIS TATLET GVPSRFSGSGSGTDYTFTISSLQPEDIATYYC QQYWSTPYT FGGGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (Sequence ID 17)

[0185] 11B3 VH EVQLVESGGGLVQPKGSLKLSCAAS GFTFNTNAMY WVRQAPGKGLEWVA RIRSKSSNYATYYADSVKD RFTISRDDSQSMLYLQMNNLKTEDTARYYCVR GGSGFFAY WGQGTLVTVSA*(Sequence ID 18)

[0186] c11B3 heavy chain (human IgG1 constant domain) EVQLVESGGGLVQPKGSLKLSCAAS GFTFNTNAMY WVRQAPGKGLEWVA RIRSKSSNYATYYADSVKD RFTISRDDSQSMLYLQMNNLKTEDTARYYCVR GGSGFFAYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*(Sequence ID 19)

[0187] c11B3 heavy chain (human IgG4 constant domain) EVQLVESGGGLVQPKGSLKLSCAAS GFTFNTNAMY WVRQAPGKGLEWVA RIRSKSSNYATYYADSVKD RFTISRDDSQSMLYLQMNNLKTEDTARYYCVR GGSGFFAY WGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*(Sequence ID 20)

[0188] h11B3 VH-1 EVQLVESGGGLVQPGGSLKLSCAAS GFTFNTNAMY WVRQAPGKGLEWVA RIRSKSSNYATYYAAASVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAY WGQGTLVTVSS*(SEQ ID NO: 21)

[0189] h11B3 VH-1 heavy chain (IgG1 constant domain) EVQLVESGGGLVQPGGSLKLSCAAS GFTFNTNAMY WVRQAPGKGLEWVA RIRSKSSNYATYYAAASVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*(SEQ ID NO: 22)

[0190] h11B3 VH-1 heavy chain (IgG4 constant domain) EVQLVESGGGLVQPGGSLKLSCAAS GFTFNTNAMY WVRQAPGKGLEWVA RIRSKSSNYATYYAAASVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*(Sequence ID 23)

[0191] h11B3 VH-2 EVQLVESGGGLVQPGGSLKLSCAAS GFTFSTQAMY WVRQAPGKGLEWVA RIRSKSSNYATYYAAASVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAY WGQGTLVTVSS*(Sequence ID 24)

[0192] h11B3 VH-double chain (IgG1 constant domain) EVQLVESGGGLVQPGGSLKLSCAAS GFTFSTQAMY WVRQAPGKGLEWVA RIRSKSSNYATYYAAASVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*(Sequence ID 25)

[0193] h11B3 VH-double chain (IgG4 constant domain) EVQLVESGGGLVQPGGSLKLSCAAS GFTFSTQAMY WVRQAPGKGLEWVA RIRSKSSNYATYYAAASVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*(Sequence ID 26)

[0194] h11B3 VH-3 EVQLVESGGGLVQPGGSLKLSCAAS GFTFNTNAMY WVRQAPGKGLEWVG RIRSKSSNYATYYAAASVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAY WGQGTLVTVSS*(Sequence ID 27)

[0195] h11B3 VH-3 heavy chain (IgG1 constant domain) EVQLVESGGGLVQPGGSLKLSCAAS GFTFNTNAMY WVRQAPGKGLEWVG RIRSKSSNYATYYAAASVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*(Sequence ID 28)

[0196] h11B3 VH-3 heavy chain (IgG4 constant domain) EVQLVESGGGLVQPGGSLKLSCAAS GFTFNTNAMY WVRQAPGKGLEWVG RIRSKSSNYATYYAAASVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*(Sequence ID 29)

[0197] h11B3 VH-4 EVQLVESGGGLVQPGGSLKLSCAAS GFTFNTNAMY WVRQAPGKGLEWVA RIRSKSSNYATYYADSVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAY WGQGTLVTVSS*(Sequence ID 30)

[0198] h11B3 VH-4 heavy chain (IgG1 constant domain) EVQLVESGGGLVQPGGSLKLSCAAS GFTFNTNAMY WVRQAPGKGLEWVA RIRSKSSNYATYYADSVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*(Sequence ID 31)

[0199] h11B3 VH-4 heavy chain (IgG4 constant domain) EVQLVESGGGLVQPGGSLKLSCAAS GFTFNTNAMY WVRQAPGKGLEWVA RIRSKSSNYATYYADSVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*(Sequence ID 32)

[0200] h11B3 VH-5 EVQLVESGGGLVQPGGSLKLSCAAS GFTFNTNAMY WVRQAPGKGLEWVG RIRSKSSNYATYYADSVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAY WGQGTLVTVSS*(Sequence ID 33)

[0201] h11B3 VH-5 heavy chain (IgG1 constant domain) EVQLVESGGGLVQPGGSLKLSCAAS GFTFNTNAMY WVRQAPGKGLEWVG RIRSKSSNYATYYADSVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*(Sequence ID 34)

[0202] h11B3 VH-5 heavy chain (IgG4 constant domain) EVQLVESGGGLVQPGGSLKLSCAAS GFTFNTNAMY WVRQAPGKGLEWVG RIRSKSSNYATYYADSVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*(Sequence ID 35)

[0203] h11B3 VH-6 EVQLVESGGGLVQPGGSLKLSCAAS GFTFSTNAMY WVRQAPGKGLEWVG RIRSKSSNYATYYADSVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAY WGQGTLVTVSS*(Sequence ID 36)

[0204] h11B3 VH-6 heavy chain (IgG1 constant domain) EVQLVESGGGLVQPGGSLKLSCAAS GFTFSTNAMY WVRQAPGKGLEWVG RIRSKSSNYATYYADSVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*(Sequence ID 37)

[0205] h11B3 VH-6 heavy chain (IgG4 constant domain) EVQLVESGGGLVQPGGSLKLSCAAS GFTFSTNAMY WVRQAPGKGLEWVG RIRSKSSNYATYYADSVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*(Sequence ID 38)

[0206] h11B3 VH-7 EVQLVESGGGLVQPGGSLKLSCAAS GFTFNTQAMY WVRQAPGKGLEWVG RIRSKSSNYATYYADSVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAY WGQGTLVTVSS*(Sequence ID 39)

[0207] h11B3 VH-7 heavy chain (IgG1 constant domain) EVQLVESGGGLVQPGGSLKLSCAAS GFTFNTQAMY WVRQAPGKGLEWVG RIRSKSSNYATYYADSVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*(Sequence ID 40)

[0208] h11B3 VH-7 heavy chain (IgG4 constant domain) EVQLVESGGGLVQPGGSLKLSCAAS GFTFNTQAMY WVRQAPGKGLEWVG RIRSKSSNYATYYADSVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*(Sequence ID 41)

[0209] h11B3 VH-8 EVQLVESGGGLVQPGGSLKLSCAAS GFTFSTQAMY WVRQAPGKGLEWVG RIRSKSSNYATYYADSVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAY WGQGTLVTVSS*(Sequence ID 42)

[0210] h11B3 VH-8 heavy chain (IgG1 constant domain) EVQLVESGGGLVQPGGSLKLSCAAS GFTFSTQAMY WVRQAPGKGLEWVG RIRSKSSNYATYYADSVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*(Sequence ID 43)

[0211] h11B3 VH-8 heavy chain (IgG4 constant domain) EVQLVESGGGLVQPGGSLKLSCAAS GFTFSTQAMY WVRQAPGKGLEWVG RIRSKSSNYATYYADSVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR GGSGFFAY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*(Sequence ID 44)

[0212] Several humanized anti-LAIR-1 immunomodulatory variants have been synthesized using the sequence combinations provided above. Non-exclusive examples of such synthesized variants, and the sequence identifiers associated with such exemplary variants, are shown in Table 1 below. Table 1. Anti-LAIR-1 variants [Table 1]

[0213] Example 2. Methods and materials

[0214] Patient sample For ex vivo studies, whole blood from AML patients or healthy donors was purchased through StemExpress (Folsom, CA). Whole blood was collected in EDTA vacuum tubes, kept refrigerated, and tested within 24 hours of collection.

[0215] animal I purchased NSG (NOD-scid-IL2Rgammanull) and NSG-SGM3 (NOD.Cg-PrkdcscidIl2rgtm1WjlTg(CMV-IL3,CSF2,KITLG)1Eav / MloySzJ) mice from Jackson Laboratory.

[0216] Flow cytometry All human cell preparations were viable at over 95% by trypan blue exclusion. 1e6 thawed or fresh bone marrow mononuclear cells were stained with Zombie Aqua viability testing dye, Annexin-5-AlexaFlour647, and mAbs conjugated with FITC, PE-Cy7, BV785, BV421, APC-Cy7, BV605, PerCP-Cy5.5, or Alexa Flour647 specific to the human lineage markers CD34 (BD Biosciences, San Diego, CA), CD38, CD90, CD45RA, CD123, CD117 (BioLegend, San Diego, CA), or human LAIR-1 (Clone DX26, BD Biosciences, San Diego, CA). After staining, the cells were washed, resuspended in phosphate-buffered saline (PBS) containing 1-4% paraformaldehyde, and analyzed using a Celesta flow cytometer (BD Biosciences, San Diego, CA) or an Attune NXT flow cytometer (Thermofisher, Waltham, MA), and analyzed using FlowJo software (Treestar, San Carlos, CA).

[0217] In most analyses, bone marrow mononuclear cells were sequentially gated to analyze a total of at least 3e5 events. CD34 + For AML cells, lineage-negative cells are CD34 + CD38 - CD45RA - CD90 - (MPP-like leukemia stem cells (LSCs)), CD34 + CD38 - CD45RA + CD90 - (LMPP LSC), CD34 + CD38 + CD45RA + CD123 + Classified as (GMP-compliant LSC) CD34 - For AML cells, lineage-negative cells are CD34 - CD117 +LSCs were classified as (GM precursor-like LSCs) (Thomas and Majeti et al. Blood 2017;129:1577). LAIR-1 expression was evaluated in each LSC subset compared to isotype controls. For bone marrow from healthy donors, the same mAb conjugates were used, except that PerCP-Cy5.5 CD127 (Biolegend, San Diego, CA) was used instead of PerCP-Cy5.5 CD117 in the AML panel. Bone marrow cells from healthy donors were gated according to the recommendations of Pang et al. [REF: PNAS 2011, 108:20012, PMID: 22123971]. HSPCs were gated to CD34 + CD38 - CD45RA - CD90 + (HSC), CD34 + CD38 - CD45RA - CD90 - (MPP), CD34 + CD38 + CD127 + (CLP), CD34 + CD38 + CD45RA - CD123 + (CMP), CD34 + CD38 + CD45RA - CD123 - (MEP) and CD34 + CD38 + CD45RA + CD123 + They were classified as (GMP). LAIR-1 expression was evaluated in each HSPC subset compared to isotype controls. CD45RA was used in the analysis of both AML and healthy donor bone marrow cells. + CD45RA - Separated from, CD90 + CD90 - Separated from CD123 + CD123 - Separated from CD127 + It was separated from the CD127-group.

[0218] MV4-11-LAIR-1 KO Cells and MV-4-11 WT The specificity of LAIR-1 mAb was validated using cells. AML cell lines MV4-11, THP-1, HL-60, and U937 were purchased from ATCC, and Kasumi1, NB4, HEL1 (provided by Manoj Pillai, Yale University), and MOLM14 (provided by Martin Carroll, University of Pennsylvania) were used as described.

[0219] TCGA analysis Expected value-maximized RNA-seq (RSEM) normalized mRNA expression data from 162 samples from AML patients included in the Cancer Genome Atlas (TCGA) project, along with corresponding clinical, mutational, and cytogenetic parameter information, were downloaded from www.cbioportal.org (hosted by Memorial Sloan Kettering Cancer Center).

[0220] Antibody-cell binding and reporter cell assays For the cell binding assay, 5e4 UT-140 cells or mouse LAIR-1 transfected 293T cells per well were seeded in 200 μL of PBS in a 96-well round-bottom plate, centrifuged at 500 × g for 4 minutes, washed once, and blocked on ice for 10 minutes in flow cytometry buffer (PBS + 2% FBS + 0.1 mM EDTA) containing a 1:50 dilution of TruStain® Fc block (Biolegend, San Diego, CA). Subsequently, the cells were stained on ice for 30 minutes with titration concentrations of soluble AF647-labeled LAIR-1 mAb or AF647-labeled isotype control. The cells were then washed three times as described above, and binding was measured by flow cytometry using an Attune NXT flow cytometer. For the reporter cell assay, flat-bottom 96-well TC plates were coated overnight at 4°C with 10 μg / mL of the indicated antibody in PBS or 2 μg / mL of the ligand in 0.01N HCl per well. The wells were washed once with 200 μL of sterile PBS. 5e4–1e5 cells per well were seeded in 100 μL of RPMIc containing titrated LAIR-1 mAb or isotype control, and then incubated overnight at 37°C. The following day, the cells were transferred to a 96-well round-bottom plate, centrifuged at 500 × g for 4 minutes, and resuspended in 200 μL of flow cytometry buffer. The cells were then centrifuged, resuspended, and washed a total of three times. GFP expression was measured by flow cytometry using an Attune NXT flow cytometer.

[0221] Colony-forming unit assay Cryopreserved AML bone marrow cells or healthy donor CD34 +Cells were thawed and seeded in 96-well plates. Cells were treated with the indicated concentrations of LAIR-1 mAb or isotype control at room temperature for 30 minutes. Subsequently, the cells were diluted in IMDM containing 2% FBS and mixed with a semi-solid methylcellulose-based medium (MethoCult H4435 Enriched, StemCell Technologies, Vancouver, Canada) containing human cytokines (stem cell factors, IL-3, IL-6, EPO, G-CSF, GM-CSF). Subsequently, 1e4–2.5e5 LSCs or 1e3–5e3 healthy donor CD34 cells per well were added. + Cells were seeded in 6-well SmartDishes (StemCell Technologies, Vancouver, Canada). The seeded plates were incubated at 37°C in air with 5% CO2 and over 95% humidity. On day 14, colony formation was counted using the automated and standardized colony counting system STEMvision (StemCell Technologies, Vancouver, Canada). The automated results were manually reviewed and edited using STEMvision colony marker software.

[0222] Patient-derived xenotransplant (PDX) model Newborn (3-5 days post-birth) NSG-SGM3 offspring were irradiated with a near-lethal dose (200 cGy). Twelve hours after irradiation, mice were given 0.2e6 AML myeloid cells or normal CD34 cells from a cryopreserved stock. +HSCs were transplanted intrahepatically. Six weeks after engraftment, blood was collected and the transplantation of progenitor cells was evaluated by flow cytometry to stain for human CD45, human CD33, and human CD3. Once human cell engraftment was confirmed, 5 mg / kg of anti-human LAIR-1 mAb or isotype control was administered intraperitoneally once a week for a total of four doses. Blood was collected at the indicated post-treatment time points and AML cell proliferation was evaluated as a measure of LAIR-1 mAb anti-leukemia activity compared to isotype control. In the standard treatment combination study, PDX mice were treated with 20 mg / kg venetoclax for 5 days over 5 weeks, followed by a 2-day rest period, and then treated with 1.5 mg / kf-tiw azacitidine only 1 week prior. LAIR-1 mAb was administered intraperitoneally once a week over 5 weeks. Blood was collected as described above to measure leukemia proliferation, and the spleen was harvested at the endpoint and weighed to evaluate splenomegaly. In the secondary transplantation study, mice were engrafted as described above. Once human cell engraftment was confirmed, 5 mg / kg of anti-human LAIR-1 mAb or isotype control was administered intraperitoneally once a week for a total of four doses. Bone marrow from the treated mice was transplanted into naive NSG-SGM3 recipient mice that had been irradiated to a near-lethal dose. The recipient mice were not treated. The progression of leukemia in the recipient mice was monitored by human CD33 in peripheral blood at 4, 6, and 10 weeks post-transplantation. + Human CD45 + The percentage of cells was quantified and measured by flow cytometry.

[0223] To evaluate the effects of mAbs on normal human immune cells, NSG-SGM3-CD34 + Fully engrafted humanized mice (over 25% human CD45+ leukocytes in circulation) were purchased from Jackson Laboratory (stock #2523, Bar Harbor, ME). Mice were received approximately 12 weeks after engraftment, and experiments were initiated after a one-week acclimatization period. Mice were treated intraperitoneally with 5 mg / kg mAb weekly for four weeks. One week after the final mAb administration, the mice were euthanized, and spleen cells, lymph node cells, and bone marrow cells were collected to determine the total cell count, followed by CD45 counting. + CD3+ CD4 + CD8 + CD14 + CD11b + CD20 + CD56 + The percentage of cells was analyzed. The absolute number of cell subpopulations was calculated, and the total CD45 + The percentages for each population subset were calculated as percentages for cells and total leukocyte gates.

[0224] Cell-derived xenotransplant (CDX) model NSG mice were injected via tail vein with either 2e6 THP-1 luciferase cells or 2e6 MV4-11-luciferase cells. Leukemia progression was quantified by intraperitoneal injection of 100 μL of bioluminescent substrate and IVIS imaging within 8 minutes post-injection. Initial progression was visually assessed weekly starting from day 7 after the challenge. Treatment with 10 mg / kg of mab was administered intraperitoneally from day 8 and continued twice weekly until the end of the study. In the subcutaneous engraftment model, 5e6 MV4-11-Luc-LAIR-1 cells were transplanted into the right flank of NSG mice. OE Cells were injected subcutaneously. Mice were treated with 10 mg / kg of mAb intraperitoneal injection once a week. Tumor growth was measured twice a week with calipers until the endpoint.

[0225] Ex vivo removal assay Fresh whole blood RBCs were removed using EasySep® RBC Depletion Reagent (StemCell, Vancouver, BC) according to the manufacturer's protocol. Alternatively, frozen PBMCs were thawed in pre-warmed cRPMI medium. Cell counts were determined by a VI-Cell XR cell counter. 2e5 cells per well were seeded in 200 μL of cRPMI containing 10 μg / mL of soluble LAIR-1 mAb or isotype control antibody (NP782 (NextCure, Beltsville, MD) or InVivoMAb recombinant human IgG1 Fc (BioXcell, Lebanon, NH)) in 96-well plates (with or without pre-coated human collagen I (StemCell, Vancouver, BC) 50 ug / mL). The plates were centrifuged at 100 × g for 2 minutes and then incubated in a TC incubator at 37°C for 20 hours. At the end of the incubation period, the plates were centrifuged at 500 × g for 4 minutes. The cells were resuspended and then transferred to 96-well round-bottom plates for cell staining and flow cytometry as described above.

[0226] Western blot and phosphorylation array To evaluate signaling in healthy cells, human monocytes were isolated from PBMCs (Primary Biomedical Cells) from two donors using the StemCell Monocyte Isolation Kit (StemCell, Vancouver, BC). Cells were seeded on cRPMI at a rate of 2e6 cells per well in a 12-well plate and stimulated with 20 ng / mL C1q (CompTech) with or without 10 ug / mL LAIR-1 mAb or isotype control antibody (NextCure). Plates were centrifuged at 350 × g for 2 minutes. After incubation at 37°C for 5 minutes in a TC incubator, cells were pelleted, lysed in the presence of a phosphatase inhibitor, and processed for Western blotting. Equal amounts of protein were separated from each sample on a gradient gel, transferred to PVDF, blocked with 5% BSA, and probed with pSHP-1 antibody or histone H3 antibody (D11G5 and D1H2, respectively, Cell Signaling Technology, MA). Data were quantified using FIJI (NIH, MD).

[0227] Digital spatial imaging and quantification Protocols and reagents derived from NanoString were used for GeoMx digital spatial imaging (DSP). Briefly, formalin-fixed / paraffin-embedded (FFPE) sections of tumors and spleens were deparaffinized and rehydrated, antigens were recovered in citrate buffer, blocked, and stained overnight at 37°C with Human Immune Profiling Core and Cell Death Panel. H MV4-11 cells were identified using CD45-Alexaflour 647 antibody (NBP2-34528AF647 from Novus Biologicals, Centennial, CO) at a morphological marker of 5 ug / mL. After washing, the slides were fixed with 4% paraformaldehyde and the nuclei were stained with Syto13. The slides were scanned, and the region of interest (human CD45) was identified. +Regions of interest (ROIs) were acquired using a GeoMx DSP instrument (Nanostring, Seattle, WA). Approximately five ROIs were collected from each tissue sample, and the samples were duplicated and processed using the nCounter Prep Station and Digital Analyzer as recommended. The data were analyzed using DSP analysis software. After quality control, the data were normalized for housekeeping genes, and statistics were performed using a linear mixed model with Benjamin-Hochberg correction.

[0228] result LAIR-1 agonists inhibit the proliferation of bone marrow LSCs. Studies have reported abnormally elevated LAIR-1 expression in leukemia cells (Kang, Lu et al., 2015; Ramos et al., 2021). To extend these findings, we evaluated LAIR-1 expression in human AML. We analyzed LAIR1 mRNA levels from AML patients representing each disease subtype as described by the France-America-UK (FAB) classification system (M0-M7) in the cancer genome atlas, and similarly analyzed LAIR1 expression in patients with AML-related mutations. No differences in LAIR1 were observed among the various subtypes, and each subtype except M7 had a higher mean expression than undiagnosed donors (Figure 1A). Similarly, LAIR1 mRNA levels did not correlate with any specific mutation (Figure 1B). Since leukemic blasts in peripheral blood arise from a pool of self-replicating LSCs in the bone marrow, we used flow cytometry (Figure 7C) to identify LAIR-1 cell surface expression in different lineage subsets of human AML cells (Thomas and Majeti, 2017; Seita and Weissman, 2010) (Figure 1C). LAIR-1 levels varied among AML patients and were observed in GMP-like subsets and CD34 + CD38 + The subset expressed the highest total levels of LAIR-1 receptor (Figure 1D). Conversely, the healthy donor HSC subset (Figure 1E) showed little variability (Figure 1F). Consistent with mRNA data, CD34 derived from AML donors.+ CD38 + The subset showed higher LAIR-1 expression than healthy donors (Figure 1G).

[0229] LAIR-1 mAbs are humanized mAbs with a functional IgG1 scaffold that specifically bind to human LAIR-1 but not to mouse LAIR-1 (Figures 10A-10C), and block collagen binding to LAIR-1 (Figure 10D). LAIR-1 mAbs can induce human LAIR-1 signaling (agonist) during binding and crosslinking (Figures 10E-10F).

[0230] LAIR-1 agonists inhibit the proliferation of bone marrow LSCs but do not inhibit healthy HSCs. To test the binding effect of LAIR-1 to LSCs, ex vivo colony-forming unit (CFU) assays were performed on AML bone marrow collected from multiple patients, as shown in Table 2 below.

[0231] Table 2. Samples from AML patients [Table 2] TIFF2026513405000003.tif115159TIFF2026513405000004.tif110159TIFF2026513405000005.tif112159TIFF2026513405000006.tif115159 TIFF2026513405000007.tif18159

[0232] Colony formation in AML myeloid cells identified leukemic cells broadly defined as precursor cells of leukemic blasts, and therefore colony formation can be used to quantify LSCs (Sutherland et al. 2001). Myeloid cells from AML patients were cultured with titrated concentrations of LAIR-1 mAbs to promote increased levels of LAIR-1 binding. Compared to isotypes or vehicle controls, LAIR-1 binding via LAIR-1 mAbs significantly reduced CFU formation in myeloid cells from AML patients in a dose-response manner (Figure 2A, 2B). However, LAIR-1 binding via LAIR-1 mAbs on myeloid cells from healthy control donors did not induce any changes in CFU formation (Figure 2C), suggesting that LAIR-1 independently regulates atypical self-renewal in LSCs.

[0233] LAIR-1 binding eradicates primary and secondary AML in patient-derived xenograft models. To test the in vivo effects of LAIR-1 on LSCs, xenograft (PDX) modeling was performed using LSCs derived from AML patients. LSCs from AML patients were engrafted into non-lethally irradiated neonatal mice, and human cell proliferation was measured by quantifying the percentage of circulating leukemia cells (Figure 3A). PDX mice treated with the LAIR-1 agonist mAb did not develop disease and maintained human CD45 in circulation at all time points. + CD33 + While the cell count was less than 10%, control mice had up to 70% circulating leukemia cells by 12 weeks after engraftment (Figure 3B). LAIR-1-mediated AML suppression was observed in normal karyotype AML, monocytic AML, acute myelomonocytic leukemia (AMML), and FLT3 +This was observed in multiple donors and AML subtypes, including ITD AML and uncharacterized AML (Figure 3B). To clarify whether the in vivo suppressive effect was due to the removal of circulating blasts or the eradication of LSCs in the bone marrow, secondary transplantation experiments were performed from PDX donor mice that had engrafted either AMML patient LSCs or normal karyotype LSCs. Mouse bone marrow harvested from PDX animals treated for 4 weeks with either a LAIR-1 mAb or an isotype control was transplanted into tumor-naive mice (secondary transplantation) (Figure 3C). No further treatment was performed after secondary transplantation. Mice that received bone marrow from donors treated with an isotype control developed AML. LAIR-1 mAb did not cause disease development, indicating that LAIR-1 binding by the LAIR-1 mAb agonist mAb eradicated LSCs in the bone marrow of PDX donor animals (Figure 3C).

[0234] The collagen matrix is ​​essential for LAIR-1-induced AML cell death. To clarify the mechanisms of proliferation arrest and cell death in LAIR-1-mediated leukemia, blood samples from AML patients were tested ex vivo. First, whole blood from erythrocyte (RBC)-depleted AML patients was cultured ex vivo in the presence of LAIR-1 mAbs, and the degree of cell death induced by LAIR-1 ligation was quantified by measuring the viable and dead cell populations by flow cytometry (Figure 11A). To support the in vivo data, LAIR-1 binding was observed in whole AML patient cells (Figure 11B) or CD45 cells. 低 Side scattered light (SSC) 低Significant cell death was induced in the blast cell population (Figure 11C) as measured by gating. Surprisingly, LAIR-1-mediated cell death depended on the presence of plate-coated collagen mimicking the extracellular matrix (ECM) (Figures 4A-C). This is surprising because LAIR-1 agonist mAbs block the binding of collagen to LAIR-1 (Figure 10A). This intriguing finding suggests that the fate of leukemia cells is determined by LAIR-1 signaling coordination from the collagen matrix. Indeed, several studies have suggested that AML cells can undergo collagen-dependent reprogramming within the bone marrow niche (Galan-Diez et al, 2018). Ex vivo leukemia cell death in the presence of collagen was observed to depend on a threshold of LAIR-1 expression, as patient samples showing an average fluorescence intensity of LAIR-1 exceeding 20,000 arbitrary fluorescence units were more receptive to LAIR-1 mAb-induced cell death (Figure 4D). Importantly, LAIR-1 binding by LAIR-1 mAbs did not remove blood leukocytes from healthy (non-AML) donors, even in the presence of collagen ECM, indicating that LAIR-1 regulation of programmed cell death is specific to AML cell type (Figure 4E).

[0235] These findings led to an attempt to explain the phosphorylation network responsible for LAIR-1 mAb-induced cell death in the presence and absence of collagen. Since SHP-1 has been reported as a key adapter molecule involved in LAIR-1 signaling (Zocchi et al., 2001), we first treated primary patient AML blasts ex vivo with LAIR-1 agonist mAbs in or without collagen, and then measured the phosphorylation status of SHP-1. Compared to controls, collagen alone induced a limited increase in pSHP-1, while LAIR-1 mAbs increased phosphorylation by 25%, and co-treatment of cells with LAIR-1 mAbs and collagen induced an additive effect, increasing phosphorylation levels by 50% (Figure 4F). To further extrapolate the signaling axis regulated by LAIR-1 binding, we probed phosphoimmune receptors and downstream kinase molecules on PBMCs from another AML patient using dot blot arrays. LAIR-1 mAbs combined with natural collagen increased the phosphorylation of multiple ITIM-containing receptors and adapter molecules, including SHP-1 and LAIR-1 itself (Figures 12A-12B). Quantification of downstream intracellular phosphoactivity during LAIR-1 crosslinking and collagen treatment revealed a pattern of reduced phosphorylation in protein species crucial for cell proliferation and survival, including ERK1 / 2, GSK-3β, and JNK (Figures 4G and 12B). Furthermore, strong LAIR-1 agonism in the context of collagen reduced the activation of key leukemia cell survival factors AKT, mTORC, and NF-κB (Figures 4H and 12B). These data demonstrate a differentiated pattern of signal transduction resulting from the enhancement of LAIR-1 signaling in the context of collagen, where such enhancement would not exist without the collagen matrix.

[0236] Based on data suggesting differential LAIR-1 signaling kinetics in AML cells and prior studies (Poggi et al., 2000, Zocchi et al., 2001), a hypothesis was proposed that the major role of collagen in LAIR-1-mediated cell death is to promote the accumulation of local LAIR-1 receptors that can overcome the signaling threshold determining the fate of leukemia cells. Thus, strong LAIR-1 clustering results in AML inhibition by overcoming this threshold even in the absence of a collagen matrix. To test this, the MV4-11-LAIR-1 過剰発現 cell line was utilized. Crosslinking and LAIR-1 clustering on the cell surface were induced by culturing the cells with LAIR-1 mAb and an anti-human IgG antibody that binds to the Fc domain of the agonist LAIR-1 mAb (Figure 4I). LAIR-1 cultured with LAIR-1 mAb alone 過剰発現Cell proliferation was only slightly suppressed compared to isotype controls (Figure 4J). However, proliferation of cells cultured under crosslinking conditions was completely inhibited after 3 days of culture (Figure 4K), and annexin V+ cells increased accordingly (Figure 4L). Since proliferation completely stopped by day 5 under crosslinking conditions, it was hypothesized that potent LAIR-1 agonist signaling induces cell death programming. To test this, a caspase-3 / 7 assay was performed on anti-IgG crosslinked cells on day 5 after treatment, and a significant increase in activated caspase-7 activity was observed in the LAIR-1 mAb treated group (Figure 4M). This suggests that LAIR-1 clustering and signaling induced by LAIR-1 mAbs can replicate collagen-mediated LAIR-1 clustering in vitro, as can occur in vivo via enhanced Fc receptor clustering (Gogesch et al., 2021), thereby inducing disruptive signals to the leukemia proliferation process. The inventors observed significant suppression of mTORC1 target protein 4E-BP1 (Figure 4M) and substantial increase in activated caspase-7 (Figure 4N) in each NC525 treatment group. Furthermore, NC525-induced apoptosis in AML cells could be partially but significantly reversed by adding small molecule activators of mTOR or small molecule inhibitors of caspase-3 / 7 (Figure 4O). On the other hand, NC525 clustering of LAIR-1 in healthy CD34+ cells induced only slight changes in signaling activity. These data further support the idea that NC525 clustering of LAIR-1 induces a leukemia cell-specific signaling pathway that inhibits the proliferation process and promotes cell death.

[0237] In summary, these results detail a novel regulatory mechanism for AML proliferation in which the LAIR-1 receptor acts as a central collagen response element determining the fate of AML between proliferation tolerance and inhibition.

[0238] LAIR-1 binding by LAIR-1 mAbs systemically reduced AML proliferation that was dependent on LAIR-1 expression levels but did not require or affect immune cells. To evaluate the in vivo dynamics of LAIR-1-mediated suppression of leukemia cell proliferation, we used xenografts (CDX) derived from more manageable AML cells, which allow for mechanistic studies not readily available in the PDX model. As shown in Table 3 below, we first identified LAIR-1 expression in several AML cell lines. Table 3. Leukemia cell lines tested for LAIR-1 expression [Table 3]

[0239] Most AML cell lines were shown to be rich in LAIR-1 (Figure 13). MV4-11 and THP-1 cells were selected for CDX modeling based on established protocols (Etchin et al., 2013, Cantilena et al., 2022) and transduced to constitutively express the RedFluc luciferase reporter human LAIR1. + MV4-11 cells or LAIR-1 + THP-1 cells were engrafted into naive NSG mice lacking T cells or B cells (Figure 5A). These mice were then treated with a LAIR-1 mAb that binds to human LAIR-1 on the engrafted leukemia cells but does not bind to mouse LAIR-1 that is intrinsically expressed on mouse cells (Figures 10A-10B). Similar to the PDX study, LAIR-1 binding by the LAIR-1 mAb inhibited leukemia cell proliferation in vivo in both the MV4-11 CDX model (left in Figure 5B) and the THP-1 model (right in Figure 5B).

[0240] Focusing on the MV4-11 model, we found that mice treated with LAIR-1 mAb had virtually no AML cells in the blood, spleen, and bone marrow (Figure 5C). Simultaneously, the percentage of dead MV4-11 cells increased in the blood, spleen, and bone marrow (Figure 5D), supporting the hypothesis that LAIR-1 binding actively induces cell death of circulating and tissue-resident AML cells in vivo. Importantly, inhibition of AML growth in the bone marrow allowed for the retention of healthy mouse immune cells (Figure 5E). While the MV4-11 CDX model provides a powerful tool for explaining the cell-specific effects of LAIR-1 signaling on leukemia cells, this model does not extend to potential immunoinfluencing factors because NSG mice do not possess an intact human immune compartment.

[0241] To evaluate the effects of LAIR-1 agonists in vivo in the presence of a healthy immune cell population, healthy human CD34 + LAIR-1 mAbs were tested in NSG mice reconstituted with stem cells for 11–16 weeks (Figure 14A). Supporting the ex vivo data, this experiment showed that LAIR-1 binding by LAIR-1 mAbs had minimal effect on healthy immune cells in the spleen or bone marrow (Figure 13B).

[0242] Since the ex vivo data indicated that a threshold level of LAIR-1 expression is required for LAIR-1-induced cell death, we evaluated whether in vivo proliferation inhibition also depends on the LAIR-1 surface expression level. LAIR-1 knockout (KO) cells were generated in an MV4-11 background (LAIR-1 is not expressed on the cell surface), and LAIR-1 overexpression (OE) cell lines (constitutively overexpressing LAIR-1 compared to wild-type (WT) cells) were used (Figure 5F). CDX modeling as described above (Figure 5G) revealed that the in vivo inhibitory effect was dependent on LAIR-1 surface expression, and that LAIR-1 mAbs more strongly inhibited LAIR-1 OE AML proliferation compared to LAIR-1 WT cells (Figure 5H). Proliferation inhibition, measured by mean fluorescence intensity, fit a logarithmic curve of LAIR-1 expression y = 18 - 18ln(x) - 77.824 (Figure 5I). These data suggest that the arrest of AML cell proliferation via LAIR-1 signaling is a process regulated through the dynamics of additive signaling available to agonist mAbs such as LAIR-1 mAbs.

[0243] LAIR-1 signaling restricts AML survival pathways in vivo. LAIR-1 mAb ligation of LAIR-1 cells was evaluated in vivo. To capture the signaling changes at the onset of leukemia proliferation suppression, a subcutaneous MV4-11 CDX model was used, which allowed for the recovery of a sufficient quantity of AML cells at the time of the first observation of proliferation branching (Figure 6A). Phosphate compound analysis of MV4-11 cells grown in vivo showed that LAIR-1 mAbs significantly suppressed the MAPK pathway in vivo and inhibited the activation of survival and proliferation molecules mTORC, AKT, and NF-κB. These data supported results from AML patient samples.

[0244] Quantifying the phosphorylation status of cell survival molecules is a powerful tool for gaining insights into downstream signaling dynamics; however, this method is limited to measuring single post-translational modifications and fails to capture non-phosphorylated signaling events that may be important in the leukemia cell fate axis centered on LAIR-1 binding. To address this, we evaluated the effects of LAIR-1 on the in vivo regulation of leukemia cell homing and apoptosis by performing digital spatial imaging on mouse bone marrow and spleen tissue from MV4-11 CDX mice treated with LAIR-1 mAbs. To capture time-dependent cellular changes, mouse tissues were collected at the onset of MV4-11 proliferation branching between treatment groups (Figure 6A-6B). At this point, LAIR-1 mAbs did not induce any differences in MV4-11 tissue localization (Figure 6C). However, as supported by in vivo CDX disease profiling (Figure 5B), LAIR-1 binding was associated with AML cells (human CD45) in the bone of CDX mice. + This resulted in a decrease in (represented by cells) (Figure 6D). Furthermore, AML cells showed decreased levels of anti-apoptotic BCL-XL (Figure 6D) and uncleaved anti-apoptotic PARP (Figure 6D) in the bone of mice treated with LAIR-1 agonist mAbs, but did not show a decrease in caspase-9 or BCL6 (data not shown), and no such decrease was observed in the spleen. These results suggest that LAIR-1 mAb regulation of leukemia cell proliferation in vivo may be involved in the regulation of apoptosis regulators in collagen-rich bone marrow.

[0245] LAIR-1 mAb works synergistically with standard treatment for AML. The VEN / AZA combination regimen, consisting of venetoclax (VEN), which blocks the anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein, and azacitidine (AZA), which inhibits DNA methyltransferase, has become the standard of care (SoC) for the treatment of AML in elderly patients. Two of the AML whole blood samples that responded to ex vivo NC525 treatment came from patients receiving VEN therapy, and three of these samples came from patients previously treated with hypomethylating agents (Figure 4D and Table 4), supporting the hypothesis that NC525 induces apoptosis in AML cells in the patient population receiving SoC treatment. One reason why AML patients develop resistance to VEN / AZA is the upregulation of BCL-XL. Since we observed a significant decrease in BCL-XL during NC525 treatment, we tested the activity of NC525 with VEN / AZA using ex vivo assays as well as CDX and PDX in vivo models.

[0246] The inventors observed dose-dependent elimination of SoC-resistant AML cells by NC525 in fresh patient-derived BM leukemia cells treated with VEN / AZA, with up to 70% elimination observed with 5 μg / mL antibody (Figure 7A). Notably, no effect on healthy T cells or NK cells was observed (Figure 7B), but BM AML cells had 4-fold higher LAIR-1 expression compared to patient-adapted T cells or NK cells (Figure 7C). Next, the activity of NC525 was evaluated using the MV4-11 model, compared to AZA or VEN treatment, and in combination with them. NC525 monotherapy showed significantly superior activity compared to AZA monotherapy at physiologically relevant doses, and AZA combination therapy with NC525 did not inhibit NC525 activity (Figure 7D). Physiologically relevant regimens of VEN monotherapy suppressed in vivo proliferation of MV4-11 below the detection limit within the growth curve timeframe, but animals treated with the NC525 and VEN combination showed significantly increased survival after challenge compared to animals treated with either monotherapy alone (Figure 7E). To further clarify the potential of NC525 for SoC-resistant AML, PDX modeling was performed using engrafted BM from additional VEN / AZA-resistant patients. NC525 treatment significantly reduced AML disease, as measured by circulating blast cell volume (Figure 7F) and AML cells in the spleen and BM (Figure 7G). Combining NC525 with VEN / AZA further reduced AML disease and demonstrated synergistic activity (Figures 7F and 7G). These results indicate not only that LAIR-1 is a viable target for therapeutic intervention for AML, but also that NC525 agonist antibodies can work in conjunction with current clinical therapies to eradicate the disease and improve patient outcomes.

[0247] Overall, these collective results indicate that potent LAIR-1 signaling induction inhibits the proliferation of leukemic blasts and LSCs, but not the proliferation of immune cells or HSCs, promoting a programmed cell death phenotype without adversely affecting healthy cells. Importantly, LAIR-1 signaling enhanced in the context of collagen can regulate AML not only through programmed cell death of blasts but also through LSC elimination. These findings provide crucial insights into a novel biological mechanism by which the collagen matrix may provide survival and maintenance signals to leukemic cells, particularly LSCs in the collagen-rich bone marrow niche. However, LAIR-1 signaling enhanced via agonist mAbs disrupts a receptor pathway crucial for survival and instead triggers a repressive pathway that downregulates anti-apoptotic quiescence, ultimately leading to leukemic cell death (Figure 8). Therefore, agonist targeting of LAIR-1 is a unique and promising strategy for therapeutic intervention of AML.

[0248] Table 4. LAIR-1 expression between AML PDX models [Table 4] JPEG2026513405000010.jpg103159JPEG2026513405000011.jpg104159 JPEG2026513405000012.jpg91159

Claims

1. LAIR-1 antibody containing variable light chain domains having 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with sequences selected from the group consisting of SEQ ID NOs: 14 and 16.

2. The antibody according to claim 1, wherein the variable light chain domain is Sequence ID No.

14.

3. The antibody according to claim 1, wherein the variable light chain domain is Sequence ID No.

16.

4. LAIR-1 antibody containing a variable heavy chain domain having 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 21, 24, 27, 30, 33, 36, 39, and 42.

5. The antibody according to claim 4, wherein the variable heavy chain domain is Sequence ID No.

21.

6. The antibody according to claim 4, wherein the variable heavy chain domain is Sequence ID No.

24.

7. The antibody according to claim 4, wherein the variable heavy chain domain is Sequence ID No.

27.

8. The antibody according to claim 4, wherein the variable heavy chain domain is Sequence ID No.

30.

9. The antibody according to claim 4, wherein the variable heavy chain domain is Sequence ID No.

33.

10. The antibody according to claim 4, wherein the variable heavy chain domain is Sequence ID No.

36.

11. The antibody according to claim 4, wherein the variable heavy chain domain is Sequence ID No.

39.

12. The antibody according to claim 4, wherein the variable heavy chain domain is Sequence ID No.

42.

13. LAIR-1 antibody containing light chains having 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with sequences selected from the group consisting of SEQ ID NOs. 15 and 17.

14. The antibody according to claim 13, wherein the light chain is Sequence ID No.

15.

15. The antibody according to claim 13, wherein the light chain is Sequence ID No.

17.

16. LAIR-1 antibody containing heavy chains having 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with sequences selected from the group consisting of SEQ ID NOs: 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37, 38, 40, 41, 43, and 44.

17. The antibody according to claim 16, wherein the heavy chain is Sequence ID No.

22.

18. The antibody according to claim 16, wherein the heavy chain is Sequence ID No.

23.

19. The antibody according to claim 16, wherein the heavy chain is Sequence ID No.

25.

20. The antibody according to claim 16, wherein the heavy chain is Sequence ID No.

26.

21. The antibody according to claim 16, wherein the heavy chain is Sequence ID No.

28.

22. The antibody according to claim 16, wherein the heavy chain is Sequence ID No.

29.

23. The antibody according to claim 16, wherein the heavy chain is Sequence ID No.

31.

24. The antibody according to claim 16, wherein the heavy chain is Sequence ID No.

32.

25. The antibody according to claim 16, wherein the heavy chain is sequence number 34.

26. The antibody according to claim 16, wherein the heavy chain is Sequence ID No.

35.

27. The antibody according to claim 16, wherein the heavy chain is Sequence ID No.

37.

28. The antibody according to claim 16, wherein the heavy chain is sequence number 38.

29. The antibody according to claim 16, wherein the heavy chain is sequence number 40.

30. The antibody according to claim 16, wherein the heavy chain is sequence number 41.

31. The antibody according to claim 16, wherein the heavy chain is sequence number 43.

32. The antibody according to claim 16, wherein the heavy chain is sequence number 44.

33. LAIR-1 antibody containing variable light chain domains and variable heavy chain domains having 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with sequences selected from the group consisting of SEQ ID NOs: 14 and 21, 14 and 24, 14 and 27, 14 and 30, 14 and 33, 14 and 36, 14 and 39, 14 and 42, 16 and 21, 16 and 24, 16 and 27, 16 and 30, 16 and 33, 16 and 36, 16 and 39, and 16 and 42.

34. The antibody according to claim 33, wherein the variable light chain domain and the variable heavy chain domain are Sequence IDs 14 and 21.

35. The antibody according to claim 33, wherein the variable light chain domain and the variable heavy chain domain are Sequence IDs 14 and 24.

36. The antibody according to claim 33, wherein the variable light chain domain and the variable heavy chain domain are Sequence ID Nos. 14 and 27.

37. The antibody according to claim 33, wherein the variable light chain domain and the variable heavy chain domain are Sequence ID Nos. 14 and 30.

38. The antibody according to claim 33, wherein the variable light chain domain and the variable heavy chain domain are Sequence ID Nos. 14 and 33.

39. The antibody according to claim 33, wherein the variable light chain domain and the variable heavy chain domain are Sequence ID Nos. 14 and 36.

40. The antibody according to claim 33, wherein the variable light chain domain and the variable heavy chain domain are Sequence ID Nos. 14 and 39.

41. The antibody according to claim 33, wherein the variable light chain domain and the variable heavy chain domain are Sequence ID Nos. 14 and 42.

42. The antibody according to claim 33, wherein the variable light chain domain and the variable heavy chain domain are Sequence IDs 16 and 21.

43. The antibody according to claim 33, wherein the variable light chain domain and the variable heavy chain domain are Sequence IDs 16 and 24.

44. The antibody according to claim 33, wherein the variable light chain domain and the variable heavy chain domain are Sequence IDs 16 and 27.

45. The antibody according to claim 33, wherein the variable light chain domain and the variable heavy chain domain are Sequence ID Nos. 16 and 30.

46. The antibody according to claim 33, wherein the variable light chain domain and the variable heavy chain domain are Sequence IDs 16 and 33.

47. The antibody according to claim 33, wherein the variable light chain domain and the variable heavy chain domain are Sequence IDs 16 and 36.

48. The antibody according to claim 33, wherein the variable light chain domain and the variable heavy chain domain are Sequence ID Nos. 16 and 39.

49. The antibody according to claim 33, wherein the variable light chain domain and the variable heavy chain domain are Sequence IDs 16 and 42.

50. LAIR-1 antibody containing light and heavy chains having 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with sequences selected from the group consisting of SEQ ID NOs: 15 and 22, 15 and 23, 15 and 25, 15 and 26, 15 and 28, 15 and 29, 15 and 31, 15 and 32, 15 and 34, 15 and 35, 15 and 37, 15 and 38, 15 and 40, 15 and 41, 15 and 43, 15 and 44, 17 and 22, 17 and 23, 17 and 25, 17 and 26, 17 and 28, 17 and 29, 17 and 31, 17 and 32, 17 and 34, 17 and 35, 17 and 37, 17 and 38, 17 and 40, 17 and 41, 17 and 43, and 17 and 44.

51. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 15 and 22.

52. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 15 and 23.

53. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 15 and 25.

54. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 15 and 26.

55. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 15 and 28.

56. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 15 and 29.

57. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 15 and 31.

58. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 15 and 32.

59. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 15 and 34.

60. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 15 and 35.

61. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 15 and 37.

62. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 15 and 38.

63. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 15 and 40.

64. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 15 and 41.

65. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 15 and 43.

66. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 15 and 44.

67. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 17 and 22.

68. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 17 and 23.

69. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 17 and 25.

70. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 17 and 26.

71. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 17 and 28.

72. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 17 and 29.

73. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 17 and 31.

74. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 17 and 32.

75. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 17 and 34.

76. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 17 and 35.

77. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 17 and 37.

78. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 17 and 38.

79. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 17 and 40.

80. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 17 and 41.

81. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 17 and 43.

82. The antibody according to claim 50, wherein the light chain and the heavy chain are Sequence ID Nos. 17 and 44.

83. LAIR-1 antibody containing variable light chain domains having 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with SEQ ID NO:

7.

84. LAIR-1 antibodies containing antigen-binding domains having 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with SEQ ID NO:

7.

85. LAIR-1 antibody containing variable heavy chain domains having 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with SEQ ID NO:

9.

86. LAIR-1 antibodies containing antigen-binding domains having 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with SEQ ID NO:

9.

87. A combination of therapeutic agents comprising any one of the LAIR-1 antibodies described in any one of claims 1 to 86, and a combination of venetoclax and azacitidine.

88. A method for treating a subject in need by administering a pharmaceutical composition containing the LAIR-1 antibody described in any one of claims 1 to 86.

89. The method according to claim 89, wherein the subject to be treated is suffering from carcinoma, squamous cell carcinoma, leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt lymphoma, acute myeloid leukemia, chronic myeloid leukemia, promyelocytic leukemia, fibrosarcoma, rhabdomyosarcoma, malignant melanoma, seminomas, teratocarcinoma, neuroblastoma, glioma, astrocytoma, neuroblastoma, glioma, schwannoma, fibrosarcoma, rhabdomyosarcoma, osteosarcoma, xeroderma pigmentosum, keratosacral cell tumor, seminomas, follicular carcinoma of the thyroid gland, or teratocarcinoma.

90. The method according to claim 90, wherein the subject to be treated is suffering from acute myeloid leukemia.

91. The method according to claim 89, wherein the pharmaceutical composition is administered parenterally, orally, or topically.

92. A kit for administering a pharmaceutical composition to a subject that requires it, a. A first agent comprising the LAIR-1 antibody described in any one of claims 1 to 86, b. A second drug including venetoclax, c. The kit comprising a third drug containing azacitidine.

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