LILRB Polypeptides and Uses Thereof

Abstract

Provided is a LILRB polypeptide capable of binding to the HLA-G polypeptide set forth in SEQ ID NO:3 and having at least one mutation located within amino acids 40-60 of the DI domain of LILRB, the LILRB polypeptide having increased stability and / or increased affinity for HLA-G compared to a LILRB polypeptide of the same length and sequence not comprising the at least one mutation. Also provided are polynucleotides encoding the LILRB polypeptide, host cells expressing the LILRB polypeptide, and methods of producing and using the same.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 293,264, filed December 23, 2021, and U.S. Provisional Patent Application No. 63 / 407,763, filed September 19, 2022, the contents of which are incorporated by reference in their entireties herein.

[0002] Description of sequence listing The XML file entitled 94087.xml was created on Dec. 21, 2022, contains 206,301 bytes, was filed concurrently with the filing of this application, and is incorporated herein by reference.

[0003] The present invention, in some embodiments, relates to LILRB polypeptides and uses thereof. [Background technology]

[0004] One of the main immune escape mechanisms in cancer is the expression of inhibitory molecules on the surface of cancerous cells that impair immune activation signaling. Many of these inhibitory molecules are considered immune checkpoints (ICPs), which belong to a number of inhibitory pathways that were originally demonstrated to maintain self-tolerance and modulate the duration and range of action of physiological immune responses in peripheral tissues to avoid collateral tissue damage.

[0005] HLA-G is a non-classical MHC class I molecule that was initially known to confer protection to the fetus against destruction by its mother's immune system, thus contributing greatly to maternal-fetal immune tolerance [Carosella et al. Adv Immunol (2015) 127:33-144]. HLA-G is membrane-bound or soluble and binds strongly to its inhibitory receptors on immune cells, inhibiting their effector functions, thus functioning as an ICP molecule to induce immune inhibition. Reported receptors for HLA-G include leukocyte immunoglobulin-like receptor B1 (LILRB1, also known as ILT2 or CD85j), LILRB2 (also known as ILT4 or CD85d), and KIR2DL4 (killer cell Ig-like receptor 2DL4, also known as CD158d), which is expressed on natural killer cells and some T cells [e.g., Shiroishi M et al., Proc Natl Acad Sci USA. (2006) 103(44): 16412-16417, Attia JVD, et al. Int J Mol Sci. (2020) 21(22): 8678]. LILRB1 is expressed on various dendritic cell subsets, including human macrophages, some T cells, NK cells, B cells, monocytes, myeloid, plasmacytoid and tolerogenic DCs, whereas LILRB2 is expressed on human monocytes, B cells and at lower levels on the cell surface of myeloid and plasmacytoid dendritic cells (Katz HR. Adv Immunol. (2006) 91:251-272, Kang X, et al. Cell Cycle. (2016) 15(1):25-40). Both LILRB1 and LILRB2 have immune receptor tyrosine-based inhibitory motifs in their cytoplasmic tails to recruit protein tyrosine phosphatase SHP-1, resulting in inhibitory signaling. Because LILRB is expressed on a wide range of leukocytes and mediates inhibitory signals, HLA-G is thought to have a central role in a wide range of immune suppressive functions in the placenta.

[0006] While HLA-G is primarily expressed on placental trophoblasts and thymic epithelial cells (e.g., Shiroishi M et al., 2006), many tumors (including, e.g., pancreatic, breast, skin, colorectal, gastric, ovarian) have been shown to upregulate HLA-G expression (e.g., Lin, A. et al, Mol Med. 21 (2015) 782-791; Amiot, L., et al, Cell Mol Life Sci. 68(2011) 417-431). In addition, it has been shown that tumor cells escape host immune surveillance by inducing immune tolerance / suppression via HLA-G expression, and that expression of HLA-G is associated with poor prognosis. Furthermore, HLA-G can also be de novo expressed and / or upregulated in other pathological conditions, such as viral infections, autoimmune and inflammatory diseases, or after allogeneic transplantation. For example, viruses such as HCMV, HSV-1, RABV, HCV, IAV and HIV-1 are thought to upregulate the expression of HLA-G to prevent infected cells from being recognized and attacked by immune cells.

[0007] Due to the widespread demonstration of the relevance of HLA-G-LILRB1 / 2 signaling as an escape mechanism used by pathological cells, several approaches have been developed to target this pathway [e.g., Carosella ED et al., Trends Immunol (2008) 29:125-32, Carosella ED et al., Blood (2008) 111:4862-70, Yan WH, Endocr Metab Immune Disord Drug Targets (2011) 11:76-89, Blaschitz A et al., Hum Immunol 2000;61:1074-85, Menier C, et al., Hum Immunol 2003;64:315-26, Francois et al., J Immunother Cancer (2021) 9(3):e001998, U.S. Patent Application Publication No. 20210301020, and WO 2014 / 072534, WO 2017207775, and WO 2018091580.

[0008] Additional background art includes: Shiroishi M et al., Proc Natl Acad Sci USA. (2003) 100(15):8856-61, Shiroishi M et al., J Biol Chem. (2006) 281(15):10439-47, Clements CS et al., Proc Natl Acad Sci USA. (2005) 102(9):3360-5 Examples include: Summary of the Invention

[0009] According to one aspect of some embodiments of the present invention, there is provided a LILRB polypeptide capable of binding to the HLA-G polypeptide set forth in SEQ ID NO: 3 and having at least one mutation located within amino acids 40 to 60 of the D1 domain of LILRB, said LILRB polypeptide having increased stability and / or increased affinity for HLA-G compared to a LILRB polypeptide of the same length and sequence not containing the at least one mutation.

[0010] According to some embodiments of the invention, the LILRB is selected from the group consisting of LILRB1 and LILRB2.

[0011] According to some embodiments of the invention, the LILRB is LILRB2 and the at least one mutation is at a position corresponding to an amino acid selected from the group consisting of S45, I49, T50 and V57 of SEQ ID NO:1.

[0012] According to an aspect of some embodiments of the present invention, there is provided a LIL2B2 polypeptide capable of binding to an HLA-G polypeptide as set forth in SEQ ID NO: 3 and having at least one mutation at a position corresponding to an amino acid selected from the group consisting of S45, I49, T50 and V57 of SEQ ID NO: 1, wherein the LILRB2 polypeptide has increased stability and / or increased affinity for HLA-G compared to a LILRB2 polypeptide of the same length and a sequence not comprising the at least one mutation.

[0013] According to some embodiments of the invention, the mutation in S45 comprises an S45R, S45N, S45Q, S45H, S45L, S45K, S45M, S45F, S45W or S45Y mutation, the mutation in I49 comprises an I49R, I49K, I49F or I49Y mutation, the mutation in T50 comprises a T50R, T50N, T50L, T50K, T50F, T50W or T50Y mutation, and / or the mutation in V57 comprises a V57R, V57K, V57F or V57W mutation.

[0014] According to some embodiments of the invention, the mutation in S45 comprises an S45Q mutation, the mutation in I49 comprises an I49K mutation, the mutation in T50 comprises a T50F mutation, and / or the mutation in V57 comprises a V57R mutation.

[0015] According to some embodiments of the invention, the at least one mutation comprises at least two mutations.

[0016] According to some embodiments of the invention, the at least one mutation comprises a mutation at S45 and additional mutations at I49, T50 and / or V57.

[0017] According to some embodiments of the invention, the LILRB2 polypeptide comprises an S45N and a T50R mutation, an S45Y and a T50K mutation, an S45R and an I49F mutation, an S45Q and a V57R mutation, an S45Q and an I49K mutation, or an S45Y and a T50N mutation.

[0018] According to some embodiments of the present invention, the LILRB2 polypeptide has increased affinity for HLA-G compared to the LILRB2 polypeptide shown in SEQ ID NO:1.

[0019] According to some embodiments of the invention, the LILRB2 polypeptide has increased stability compared to the LILRB2 polypeptide shown in SEQ ID NO:1.

[0020] According to some embodiments of the invention, the amino acid sequence of the LILRB2 polypeptide is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23.

[0021] According to some embodiments of the invention, the amino acid sequence of the LILRB2 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23.

[0022] According to some embodiments of the invention, the amino acid sequence of the LILRB2 polypeptide is as set forth in SEQ ID NO:5, 7, 9, 11, 13, 15, 17, 19, 21 or 23.

[0023] According to some embodiments of the invention, the LILRB is LILRB1 and the at least one mutation is at a position corresponding to an amino acid selected from the group consisting of T43, I47, T48 and V55 of SEQ ID NO:102.

[0024] According to some embodiments of the invention, the mutation in T43 comprises a T43R, T43N, T43Q, T43H, T43L, T43K, T43M, T43F, T43W or T43Y mutation, the mutation in I47 comprises a I47R, I47K, I47F or I47Y mutation, the mutation in T48 comprises a T48R, T48N, T48L, T48K, T48F, T48W or T48Y mutation, and / or the mutation in V55 comprises a V55R, V55K, V55F or V55W mutation.

[0025] According to some embodiments of the invention, the mutation at V55 comprises a V55R mutation.

[0026] According to some embodiments of the present invention, the LILRB1 polypeptide has increased affinity for HLA-G compared to the LILRB1 polypeptide shown in SEQ ID NO:102.

[0027] According to some embodiments of the present invention, the LILRB1 polypeptide has increased stability compared to the LILRB1 polypeptide set forth in SEQ ID NO:102.

[0028] According to an aspect of some embodiments of the present invention there is provided a composition comprising a LILRB polypeptide and a non-proteinaceous moiety bound to the LILRB polypeptide.

[0029] According to an aspect of some embodiments of the present invention there is provided a composition comprising a LILRB2 polypeptide and a non-proteinaceous moiety bound to the LILRB2 polypeptide.

[0030] According to some embodiments of the invention, the non-proteinaceous moiety is selected from the group consisting of a drug, a chemical, a small molecule, a polynucleotide, a detectable moiety, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), poly(styrene co maleic anhydride) (SMA), and divinyl ether and maleic anhydride copolymer (DIVEMA).

[0031] According to some embodiments of the invention, the non-proteinaceous moiety is a dimerization moiety.

[0032] According to an aspect of some embodiments of the present invention there is provided a fusion polypeptide comprising a LILRB polypeptide linked to a heterologous proteinaceous moiety.

[0033] According to an aspect of some embodiments of the present invention there is provided a fusion polypeptide comprising a LILRB2 polypeptide linked to a heterologous proteinaceous moiety.

[0034] According to some embodiments of the invention, the heterologous proteinaceous moiety is a dimerization moiety.

[0035] According to some embodiments of the invention, the heterologous proteinaceous moiety comprises an Fc domain of an antibody or a fragment thereof.

[0036] According to some embodiments of the invention, the Fc domain is of IgG1 or IgG4.

[0037] According to some embodiments of the invention, the Fc domain is modified to alter its binding to Fc receptors, to reduce its immunostimulatory function, and / or to improve the half-life of the fusion.

[0038] According to an aspect of some embodiments of the present invention there is provided a dimer comprising a LILRB polypeptide, composition or fusion polypeptide.

[0039] According to an aspect of some embodiments of the present invention there is provided a dimer comprising a LILRB2 polypeptide, composition or fusion polypeptide.

[0040] According to some embodiments of the invention, the dimer is a heterodimer.

[0041] According to some embodiments of the invention, a first monomer of the heterodimer comprises a LILRB polypeptide and a second monomer comprises an amino acid sequence of a protein selected from the group consisting of SIRPα, PD1, TIGIT and SIGLEC10, which amino acid sequence is capable of binding to its natural binding pair.

[0042] According to some embodiments of the invention, a first monomer of the heterodimer comprises a LILRB2 polypeptide and a second monomer comprises an amino acid sequence of a protein selected from the group consisting of SIRPα, PD1, TIGIT and SIGLEC10, which amino acid sequence is capable of binding to its natural binding pair.

[0043] According to some embodiments of the invention, a first monomer of the heterodimer comprises a LILRB polypeptide and a second monomer comprises an amino acid sequence of SIRPα, which amino acid sequence is capable of binding to CD47.

[0044] According to some embodiments of the invention, a first monomer of the heterodimer comprises a LILRB2 polypeptide and a second monomer comprises an amino acid sequence of SIRPα, which amino acid sequence is capable of binding to CD47.

[0045] According to one aspect of some embodiments of the present invention, there is provided a heterodimer comprising a first monomer comprising a fusion polypeptide and a second monomer comprising an amino acid sequence of SIRPα bound to an Fc domain of an antibody or fragment thereof, wherein the amino acid sequence of SIRPα is capable of binding to CD47.

[0046] According to some embodiments of the invention, the SIRPα amino acid sequence is at least 90% identical to SEQ ID NO:27 or 88.

[0047] According to some embodiments of the invention, the SIRPα amino acid sequence comprises SEQ ID NO:27 or 88.

[0048] According to some embodiments of the invention, the SIRPα amino acid sequence is as set forth in SEQ ID NO:27 or 88.

[0049] According to an aspect of some embodiments of the present invention there is provided a composition comprising a dimer, wherein the dimer is a predominant form of LILRB in the composition.

[0050] According to an aspect of some embodiments of the present invention there is provided a composition comprising a dimer, wherein the dimer is a predominant form of LILRB2 in the composition.

[0051] According to an aspect of some embodiments of the present invention there is provided a polynucleotide encoding the LILRB polypeptide, fusion polypeptide or dimer.

[0052] According to an aspect of some embodiments of the present invention there is provided a polynucleotide encoding a LILRB2 polypeptide, fusion polypeptide or dimer.

[0053] According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct comprising a polynucleotide encoding a LILRB polypeptide, fusion polypeptide or dimer and regulatory elements for directing expression of the polynucleotide in a host cell.

[0054] According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct comprising a polynucleotide encoding a LILRB2 polypeptide, fusion polypeptide or dimer and regulatory elements for directing expression of the polynucleotide in a host cell.

[0055] According to an aspect of some embodiments of the present invention there is provided a host cell comprising the LILRB polypeptide, fusion polypeptide or dimer, polynucleotide or nucleic acid construct.

[0056] According to an aspect of some embodiments of the present invention there is provided a host cell comprising the LILRB2 polypeptide, fusion polypeptide or dimer, polynucleotide or nucleic acid construct.

[0057] According to an aspect of some embodiments of the present invention there is provided a method of producing a polypeptide comprising introducing a polynucleotide or nucleic acid construct into a host cell or culturing a cell.

[0058] According to some embodiments of the invention, the method comprises isolating the LILRB polypeptide, fusion polypeptide or dimer.

[0059] According to some embodiments of the invention, the method comprises isolating the LILRB2 polypeptide, fusion polypeptide or dimer.

[0060] According to an aspect of some embodiments of the present invention there is provided a method of treating a disease associated with pathological cells expressing HLA-G in a subject in need of such treatment comprising administering to the subject a therapeutically effective amount of a LILRB polypeptide, composition, fusion polypeptide or dimer, polynucleotide, nucleic acid construct or host cell, thereby treating the disease in the subject.

[0061] According to an aspect of some embodiments of the present invention there is provided a method of treating a disease associated with pathological cells expressing HLA-G in a subject in need of such treatment comprising administering to the subject a therapeutically effective amount of a LILRB2 polypeptide, composition, fusion polypeptide or dimer, polynucleotide, nucleic acid construct or host cell, thereby treating the disease in the subject.

[0062] According to an aspect of some embodiments of the present invention there is provided a LILRB polypeptide, composition, fusion polypeptide or dimer, polynucleotide, nucleic acid construct or host cell for use in treating a disease associated with pathological cells expressing HLA-G in a subject in need of such treatment.

[0063] According to an aspect of some embodiments of the present invention there is provided a LILRB2 polypeptide, composition, fusion polypeptide or dimer, polynucleotide, nucleic acid construct or host cell for use in treating a disease associated with pathological cells expressing HLA-G in a subject in need of such treatment.

[0064] According to some embodiments of the invention, the disease is cancer.

[0065] According to some embodiments of the invention, the cancer is selected from the group consisting of pancreatic cancer, breast cancer, skin cancer, colorectal cancer, gastric cancer and ovarian cancer.

[0066] According to an aspect of some embodiments of the present invention there is provided a method of activating an immune cell comprising activating the immune cell in vitro in the presence of a LILRB polypeptide, composition, fusion polypeptide or dimer, polynucleotide, nucleic acid construct or host cell.

[0067] According to an aspect of some embodiments of the invention there is provided a method of activating an immune cell comprising activating the immune cell in vitro in the presence of a LILRB2 polypeptide, composition, fusion polypeptide or dimer, polynucleotide, nucleic acid construct or host cell.

[0068] According to some embodiments of the invention, activation is performed in the presence of cells expressing HLA-G.

[0069] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the present patent specification, including definitions, will control. In addition, the materials, methods, and examples are only illustrative and are not intended to be necessarily limiting.

[0070] Some embodiments of the present invention are described herein, by way of example only, and with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and are intended as illustrative discussion of embodiments of the invention. In this regard, the description taken in conjunction with the drawings will make apparent to those skilled in the art how embodiments of the invention may be practiced. [Brief description of the drawings]

[0071] [Figure 1-1] 1A-1D demonstrate the structural analysis of the LILRB2-HLA-G binding interface. FIG. 1A shows the PDB ID-2DYP complex structure of LILRB2 (SEQ ID NO: 1, also referred to herein as "WT LILRB2") and HLA-G (SEQ ID NO: 3). HLA-G is shown in dark grey surface representation, beta-2-microglobulin (SEQ ID NO: 4) is shown in grey ribbon display, and LILRB2 is shown in white surface representation. FIG. 1B shows the mapping of interface residues between HLA-G and LILRB2. Both HLA-G and beta-2-microglobulin are shown in dark grey ribbons, and LILRB2 is shown in grey ribbons. The interacting residues are represented as balls and sticks. FIG. 1C shows a close-up view of the interaction interface between HLA-G and LILRB2 shown in FIG. 1B. At the interaction interface, there is only a single predominant difference between HLA-G and its homologs in PHE195. FIG. 1D shows four amino acids in LILRB2, namely Ser45, Ile49, Thr50 and Val57, which were found to have a significant effect on the binding energy. [Figure 1-2] Same as above [Figure 2A] Figures 2A and 2B show photographs of SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis of several heterodimeric proteins (see Table 3 below for a full description of each heterodimer) containing either WT LILRB2 (SEQ ID NO: 1) or mutant LILRB2 (herein referred to as "LILRB2 variants")-Fc fusions and SIRPα-Fc fusions, separated under reducing and / or non-reducing conditions. Samples presented in the figures are either crude (unpurified, Figure 2A) or Protein A purified (Figure 2B) (day 5 supernatants). Supernatants are from expi 293F cells transfected with plasmids encoding the indicated recombinant proteins. [Figure 2B] Same as above [Figure 3A] Figures 3A-F demonstrate the binding of several heterodimeric proteins, including LILRB2 variants, to HLA-G expressed on the cell surface compared to WT LILRB2. Figures 3A-B demonstrate the expression levels of CD47 and HLA-G on HT1080 and HT1080-HLA-G cells (Figure 3A) and THP1-EV and THP1-HLA-G cells (Figure 3B). The cell surface expression levels of CD47 and HLA-G were determined by immunostaining of cell lines with anti-human CD47 antibody and IgG1 as an isotype control or anti-human HLA-G antibody and IgG4 as an isotype control followed by flow cytometry analysis. [Figure 3B] Same as above [Figure 3C] Figure 3C demonstrates binding of the indicated heterodimers to HLA-G overexpressing cells HT1080-HLA-G. Binding was determined after incubation by immunostaining of the IgG backbone using an anti-human IgG1 antibody followed by flow cytometry analysis. GMFI values ​​were used to generate binding curve graphs using GraphPad Prism software. [Figure 3D]FIG. 3D demonstrates the percent blocking of binding of the indicated heterodimers to HT1080-HLA-G cells by anti-HLA-G blocking antibodies. [Figure 3E] Figures 3E-F demonstrate binding of the indicated heterodimers to HT1080 cells or to HLA-G overexpressing cells HT1080-HLA-G (Figure 3E), or to THP-1-EV cells or to HLA-G overexpressing cells THP-1-HLA-G (Figure 3F), with or without blocking antibodies as indicated. Binding was determined after incubation by immunostaining of the IgG backbone using an anti-human IgG1 antibody followed by flow cytometry analysis. GMFI values ​​were used to generate binding curve graphs using GraphPad Prism software. [Figure 3F] Same as above [Figure 4] Figure 4 demonstrates the binding of homodimeric proteins containing LILRB2 variant LILRB-V12 to HLA-G expressed on the cell surface compared to homodimeric proteins containing WT LILRB2 (LILRB2-V5). Binding of homodimers to HT1080 cells or to HLA-G overexpressing cells HT1080-HLA-G cells was determined by immunostaining of the IgG backbone using anti-human IgG1 antibody followed by flow cytometry analysis after incubation with or without blocking antibodies. GMFI values ​​are presented and used to generate binding curve graphs using GraphPad Prism software. [Diagram 5]Figures 5A-C demonstrate the expression levels of M2 markers, CD163 (Figure 5A) and CD206 (Figure 5B), and M1 marker HLA-DR (Figure 5C) in M-CSF-treated macrophages co-cultured with HT1080-HLA-G cells and treated with the indicated concentrations of LILRB2 variant-Fc fusion and SIRPα-Fc fusion heterodimers, referred to herein as "DSP216-V12" (labeled "DSP" in the figures) (see Table 3 below for a full description of each heterodimer) or 1.5 μg / ml of anti-HLA-G antibody as a positive control. Cell surface expression levels were determined by immunostaining of cells with anti-human-CD163, anti-human-CD206 or anti-human HLA-DR antibodies followed by flow cytometry analysis. Binding curve graphs were generated using GraphPad Prism software using the MFI values. [Figure 6] Figures 6A-B demonstrate the levels of TNF-α (Figure 6A) or IL-6 (Figure 6B) in the supernatants of co-cultures of MCS-F treated macrophages and T1080HLA-G cells 24 hours after initiation of co-culture and treatment with the indicated concentrations of DSP216-V12 (labeled "DSP2016" in the figure) or 1.5 μg / ml of anti-HLA-G antibody as a positive control. Cytokine levels (ng / mL) were determined by immunostaining of the supernatants using a cytometric bead array (CBA) followed by flow cytometry analysis. The MFI values ​​were used to generate binding curve graphs using GraphPad Prism software. [Figure 7] Figure 7 demonstrates the expression levels of CD47 on RBCs, PBMCs or HT1080-HLA-G cells. Cell surface expression levels were determined by immunostaining of cells with anti-human-CD47 antibody or IgG1 as an isotype control followed by flow cytometry analysis. MFI values ​​were used to generate binding curve graphs using GraphPad Prism software. [Figure 8]Figures 8A-B demonstrate binding of DSP216-V12 to RBCs, PBMCs or HT1080-HLA-G cells as determined by flow cytometry. Graphs represent the mean (±SEM) of MFI from four independent donor samples. Figure 8A is a graph showing the mean (±SEM) binding of DS216-V12 to RBCs, PBMCs or HT1080-HLA-G cells at concentrations between 0.4 and 12.5 μg / mL. Figure 8B is a bar graph showing significantly higher binding of DSP216-V12 (labeled "DSP2016" in the figure) to HT1080-HLA-G cells compared to PBMCs and RBCs at all concentrations (1.56, 3.125, 6.25 μg / mL). *P≦0.05, **P≦0.01. P values ​​for the 3.125 μg / mL concentration were 0.0044 for PBMCs and 0.0027 for RBCs (T-test). [Figure 9] Figure 9 shows a photograph of SDS polyacrylamide gel electrophoresis analysis (SDS-PAGE) of several heterodimeric proteins comprising SIRPα-Fc fusion subunits and WT LILRB2- or LILRB2 variant-Fc fusion subunits (see Table 3 below for a full description of each heterodimer shown in the figure), separated under reducing and non-reducing conditions. Samples presented in the figure are crude (non-purified-day 5 supernatants). Supernatants are from expi 293F cells transfected with plasmids encoding the indicated recombinant proteins. [Figure 10] Figure 10 is a photograph of a Western blot analysis of several heterodimeric proteins, including SIRPα-Fc fusion subunits and LILRB2 variant-Fc fusion subunits (see Table 3 below for a full description of each heterodimer shown in the figure). Samples presented in the figure are Protein A purified (day 5 supernatants). Supernatants are from Expi 293F cells transfected with plasmids encoding the indicated heterodimers. Proteins were separated on SDS-PAGE under reducing and non-reducing conditions, followed by immunoblotting with anti-LILRB2 or anti-SIRPα antibodies. [Figure 11]FIG. 11 demonstrates binding of several heterodimeric proteins (see Table 3 below for a full description of each heterodimer shown in the figure) comprising SIRPα-Fc fusion subunits and WT LILRB2- or LILRB2 variant-Fc fusion subunits to CD47 expressed on the cell surface of HT1080, or to CD47 and HLA-G expressed on the cell surface of HT1080-HLA-G cells overexpressing HLA-G. Binding with or without anti-HLA-G (HLA-G / LILRB2 interaction blocking antibody) demonstrates binding to CD47 only compared to binding to both HLA-G and CD47. Binding was determined after immunostaining of the IgG backbone using an anti-human IgG1 antibody followed by flow cytometry analysis. GMFI values ​​are presented and used to generate binding curve graphs using GraphPad Prism software. [Figure 12] Figures 12A-C demonstrate the cell surface expression levels of M2 markers, CD163 (Figure 12A) and CD206 (Figure 12B), and M1 marker HLA-DR (Figure 12C) in M-CSF-treated macrophages co-cultured with HT1080-HLA-G cells and treated with the indicated concentrations of LILRB2 variant-Fc fusion, referred to herein as "DSP216-V12 short chain", and short chain SIRPα-Fc fusion heterodimer (see Table 3 below for a full description of the heterodimer) or 1.5 μg / ml of anti-HLA-G antibody as a control. Cell surface expression levels were determined by immunostaining of cells with anti-human-CD163, anti-human-CD206 or anti-human HLA-DR antibodies followed by flow cytometry analysis. The MFI values ​​were used to generate binding curve graphs using GraphPad Prism software. [Figure 13]Figures 13A-B demonstrate phagocytosis of cancer cells treated with DSP216-V12 or anti-CD47 antibody by M2c macrophages as determined by flow cytometry. The graph shows the mean % (±SD) of M2c cells positive after uptake of cancer cells stained for cell tracing violet. The graph shows the mean uptake by M2c macrophages of CD47+HLA-G-721.221 cells transduced with empty vector (721.221EV) (Figure 13A) or CD47+HLA-G+721.221 cells transduced with HLA-G expression vector (721.221-HLA-G) (Figure 13B). Both 721.221 cell lines were incubated in medium containing different concentrations of DSP216-V12 or 6 μg / mL of CD47 blocking antibody before mixing with M2c macrophages. *P≦0.05, **P≦0.01. P value for % phagocytosis in medium for CD47 antibody treatment at 6 μg / mL presented in FIG. 13A=0.0028, and P value for % phagocytosis in medium for DSP216-V12 treatment at 10 μg / mL presented in FIG. 13B=0.0465 (paired T-test). [Figure 14] FIG. 14 shows the blast homology analysis between LILRB2 and LILRB1. [Figure 15-1]Figure 15A-D demonstrates the structural analysis of the LILRB1-HLA-G binding interface. Figure 15A shows the PDB ID-2DYP complex structure of LILRB1 (SEQ ID NO: 102, also referred to herein as "WT LILRB1") and HLA-G (SEQ ID NO: 3). HLA-G is shown in grey surface representation, beta-2-microglobulin (SEQ ID NO: 4) is shown in dark grey ribbon display, and LILRB1 is shown in white surface representation. Figure 15B shows the mapping of interface residues between HLA-G and LILRB1. Both HLA-G and beta-2-microglobulin are shown in dark grey ribbon, and LILRB1 is shown in grey ribbon. Interacting residues are represented as balls and sticks. Figure 15C shows a close-up of the interaction interface between HLA-G and LILRB1 shown in Figure 15B, specifically demonstrating Val55 of LILRB1 and Phe195 of HLA-G. FIG. 15D demonstrates the proximity of Phe195 of HLA-G to amino acid residue 55 of LILRB1 following substitution of WT valine with arginine (variant LILRB1 sequence shown in SEQ ID NO: 103). [Figure 15-2] Same as above DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] The present invention, in some embodiments, relates to LILRB polypeptides and uses thereof.

[0073] Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following detailed description or illustrated by way of examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0074] HLA-G is a non-classical MHC class I molecule that was initially known to confer protection to the fetus from destruction by its mother's immune system, thus contributing greatly to maternal-fetal immune tolerance. Reported receptors for HLA-G include LILRB1, LILRB2 and KIR2DL4. HLA-G is mainly expressed on placental trophoblasts and thymic epithelial cells, whereas cells associated with various pathological conditions, including cancer, viral infection, autoimmune and inflammatory diseases, or cells after allogeneic transplantation, have been shown to overexpress HLA-G, and indeed HLA-G-LILRB1 / 2 signaling has been suggested as an immune escape mechanism used by pathological cells.

[0075] In the course of realizing the practice of specific embodiments of the present invention, the inventors used structure-function tools to introduce mutations in LILRB2 and LILRB1 aimed at increasing stability and binding affinity to HLA-G (Examples 1 and 11 in the Examples section below). Using this method, the inventors were able to generate novel polypeptides with mutations in specific regions in the D1 domain of the proteins that have improved stability and binding affinity to HLA-G compared to wild-type LILRB2 (Examples 1-9 in the Examples section below).

[0076] Consequently, specific embodiments of the present teachings suggest LILRB (eg, LILRB2, LILRB1) polypeptides having these novel mutations and their use in therapeutic methods.

[0077] Therefore, according to one aspect of the present invention, there is provided a LILRB polypeptide capable of binding to the HLA-G polypeptide shown in SEQ ID NO: 3 and having at least one mutation located within amino acids 40 to 60 of the D1 domain of LILRB, said LILRB polypeptide having increased stability and / or increased affinity for HLA-G compared to a LILRB polypeptide of the same length and having a sequence not containing the at least one mutation.

[0078] According to further or alternative aspects of the present invention, there is provided a LIL2B2 polypeptide capable of binding to an HLA-G polypeptide as set forth in SEQ ID NO:3 and having at least one mutation at a position corresponding to an amino acid selected from the group consisting of S45, I49, T50 and V57 of SEQ ID NO:1, wherein the LILRB2 polypeptide has increased stability and / or increased affinity for HLA-G compared to a LILRB2 polypeptide of the same length and a sequence not comprising the at least one mutation.

[0079] "LILRB (leukocyte immunoglobulin-like receptor subfamily B)" refers to a family of receptors that contain two to four extracellular immunoglobulin-like domains (specifically, C-type Ig-like domains, InterPro database entry IPR008424 or Pfam database entry PF05790), including LILRB1, LILRB2, LILRB3, LILRB4, and LILRB5, and a cytoplasmic tail that contains an ITIM domain.

[0080] According to a specific embodiment, the LILRB is a human LILRB.

[0081] According to a specific embodiment, the LILRB is LILRB2.

[0082] "LILRB2 (leukocyte immunoglobulin-like receptor subfamily B member 2)" refers to a polypeptide encoded by the LILRB2 gene (corresponding to human gene ID 10288). According to specific embodiments, LILRB2 is human LILRB2. According to specific embodiments, LILRB2 refers to human LILRB2, e.g., as provided in the following GenBank numbers NP_001074447, NP_001265332, NP_001265333, NP_001265334, NP_001265335, or Uniprot number Q8N423.

[0083] According to a specific embodiment, the LILRB is LILRB1.

[0084] "LILRB1 (leukocyte immunoglobulin-like receptor subfamily B member 1)" refers to a polypeptide encoded by the LILRB1 gene (corresponding to human gene ID 10859). According to a specific embodiment, LILRB1 is human LILRB1. According to a specific embodiment, LILRB1 refers to human LILRB1, for example, as provided in the following GenBank numbers NP_001075106, NP_001075107, NP_001075108, NP_001265327, NP_001265328, or Uniprot number Q8NHL6.

[0085] One of the known binding pairs of LILRB (eg, LILRB2, LILRB1) is major histocompatibility molecules (MHC, eg, HLA-G).

[0086] "HLA-G (human leukocyte antigen G)" refers to a polypeptide encoded by the HLA-G gene (gene ID 3135). According to a specific embodiment, the HLA-G amino acid sequence is that provided in SEQ ID NO:3.

[0087] According to specific embodiments, LILRB (e.g., LILRB2, LILRB1) binds to HLA-G in the context of beta2-microglobulin. A non-limiting example of a beta2-microglobulin sequence is provided in SEQ ID NO:4.

[0088] According to specific embodiments, LILRB (eg, LILRB2, LILRB1) binds to free HLA-G or HLA-G that is not in the context of non-beta2-microglobulin.

[0089] According to specific embodiments, LILRB (eg, LILRB2, LILRB1) binds to free HLA-G (i.e., not bound to a peptide).

[0090] According to specific embodiments, LILRB (eg, LILRB2, LILRB1) binds to peptide-presenting HLA-G.

[0091] According to specific embodiments, LILRB (eg, LILRB2, LILRB1) binds to HLA-G independent of the peptide sequence.

[0092] Assays for testing binding are well known in the art and include, but are not limited to, flow cytometry, Biacore, Biolayer Interference Blitz® assay, HPLC.

[0093] As used herein, the terms "LILRB polypeptide," "LILRB2 polypeptide," and "LILRB1 polypeptide" refer to full-length LILRB, LILRB2, and LILRB1, functional fragments thereof, or homologs thereof that maintain at least the ability to bind to HLA-G. For example, according to specific embodiments, the amino acid sequence of the polypeptide contains substitution, addition, and / or deletion mutations compared to the sequence of the wild-type protein, as further described herein.

[0094] According to specific embodiments, the amino acid sequence of the LILRB2 polypeptide comprises substitution, addition and deletion mutations (eg, compared to the LILRB2 polypeptide shown in SEQ ID NO:1), as described above and further below.

[0095] According to specific embodiments, the amino acid sequence of the LILRB1 polypeptide includes substitution, addition and deletion mutations (eg, compared to the LILRB1 polypeptide shown in SEQ ID NO: 102), as described above and further below.

[0096] According to specific embodiments, the LILRB (e.g., LILRB2, LILRB1) polypeptide comprises the extracellular domain of LILRB (e.g., LILRB2, LILRB1) or at least a functional fragment thereof capable of binding to HLA-G.

[0097] As described above, the extracellular domain of LILRB contains two to four immunoglobulin-like domains, known in the art as D domains, which are numbered 1 to 4 according to their position from distal to proximal to the membrane (and corresponding to their order from N-terminus to C-terminus of the amino acid sequence of the protein).

[0098] The extracellular domain of LILRB2 or LILRB1 contains four Ig-like domains known as D1 to D4. Non-limiting examples of LILRB2 and LILRB1 extracellular domains are provided by SEQ ID NOs: 1, 100, and 102.

[0099] Thus, according to specific embodiments, the amino acid sequence of a LILRB (eg, LILRB2, LILRB1) polypeptide comprises at least one Ig-like domain or a functional fragment thereof.

[0100] According to specific embodiments, the amino acid sequence of a LILRB (eg, LILRB2, LILRB1) polypeptide comprises at least one Ig-like domain.

[0101] According to specific embodiments, the LILRB (eg, LILRB2, LILRB1) polypeptide comprises at least two Ig-like domains, at least three Ig-like domains, or four Ig-like domains, or a functional fragment thereof.

[0102] According to specific embodiments, the LILRB (eg, LILRB2, LILRB1) polypeptide comprises at least two Ig-like domains, at least three Ig-like domains, or four Ig-like domains.

[0103] According to specific embodiments, the LILRB (eg, LILRB2, LILRB1) polypeptide comprises at least the D1 domain.

[0104] According to specific embodiments, the LILRB2 polypeptide comprises domains D1 and D2 of LILRB2, domains D1, D2 and D3 of LILRB2, domains D1, D2 and D4 of LILRB2, or domains D1, D2, D3 and D4 of LILRB2.

[0105] According to specific embodiments, the LILRB1 polypeptide comprises domains D1 and D2 of LILRB1, domains D1, D2 and D3 of LILRB1, domains D1, D2 and D4 of LILRB1, or domains D1, D2, D3 and D4 of LILRB1.

[0106] According to specific embodiments, the LILRB (eg, LILRB2, LILRB1) polypeptide comprises at least 70, at least 80, at least 90, at least 100 amino acids, each possibility representing a separate embodiment of the present invention.

[0107] According to specific embodiments, the LILRB (e.g., LILRB2, LILRB1) polypeptide comprises between 100 and 597 amino acids, between 100 and 500 amino acids, between 100 and 400 amino acids, between 150 and 400 amino acids, between 300 and 400 amino acids, between 350 and 400 amino acids, or between 150 and 250 amino acids, each possibility representing a separate embodiment of the present invention.

[0108] The terms "LILRB polypeptide," "LILRB2 polypeptide," and "LILRB1 polypeptide" also encompass functional homologs that exhibit a desired activity (i.e., binding to MHC, e.g., HLA-G). Such homologs include, for example, those that are at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequences of LILRB, LILRB2 and LILRB1 described herein. or homologous to or may be at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polynucleotide sequence encoding it (described further below).

[0109] As used herein, "identity" or "sequence identity" refers to global identity, i.e., identity over the entire amino acid or nucleic acid sequence disclosed herein, and not identity over a portion thereof.

[0110] Sequence identity or homology can be determined using any protein or nucleic acid sequence alignment algorithm, such as BLAST, Clustal W, and MUSCLE.

[0111] Homologs can also refer to orthologs, deletions, insertions, or substitution variants including amino acid substitutions, as further described below.

[0112] According to specific embodiments, LILRB (e.g., LILRB2, LILRB1) polypeptides may include conservative and / or non-conservative amino acid substitutions (also referred to herein as "mutations"), as further detailed below.

[0113] The LILRB polypeptides described herein comprise at least one mutation in the D1 domain of LILRB. Non-limiting examples of the D1 domain of LILRB2 and LILRB1 are provided in SEQ ID NOs: 104 and 105, respectively.

[0114] Mutations can be, for example, point mutations, substitutions (replacing one amino acid with another), addition and / or deletion mutations.

[0115] According to a specific embodiment, at least one mutation is a substitution mutation.

[0116] According to a specific embodiment, the at least one mutation is a single mutation.

[0117] According to other specific embodiments, the at least one mutation comprises at least two mutations.

[0118] According to other specific embodiments, the at least one mutation comprises at least three or at least four mutations.

[0119] According to a specific embodiment, the at least one mutation is localized within amino acids 40 to 60 of the D1 domain of LILRB.

[0120] According to specific embodiments, at least one mutation is located within amino acids 43-45, 47-50 and / or 55-77 of LILRB.

[0121] According to a specific embodiment, at least one mutation is in an amino acid selected from the group consisting of S, I, T and V.

[0122] According to a specific embodiment, at least one mutation is in an amino acid at the interface between LILRB and Phe195 of HLA-G, the numbering corresponding to SEQ ID NO:3.

[0123] According to specific embodiments, the LILRB2 polypeptide comprises at least one mutation at a position corresponding to an amino acid selected from the group consisting of S45, I49, T50 and V57 of SEQ ID NO:1.

[0124] As used herein, the phrase "corresponding to SEQ ID NO:1" is intended to include the corresponding amino acid residues in any other LILRB2 amino acid sequence.

[0125] According to a specific embodiment, the mutation comprises a conservative substitution.

[0126] According to other specific embodiments, the mutation comprises a non-conservative substitution.

[0127] According to a specific embodiment, the mutation is a non-naturally occurring mutation.

[0128] According to specific embodiments, the amino acid corresponding to S45 in SEQ ID NO:1 comprises an S45R, S45N, S45Q, S45H, S45L, S45K, S45M, S45F, S45W or S45Y mutation.

[0129] According to a specific embodiment, the amino acid corresponding to S45 in SEQ ID NO:1 comprises an S45Q mutation.

[0130] According to specific embodiments, the mutation corresponding to I49 in SEQ ID NO:1 comprises an I49R, I49K, I49F or I49Y mutation.

[0131] According to a specific embodiment, the mutation corresponding to I49 in SEQ ID NO:1 comprises an I49K mutation.

[0132] According to specific embodiments, the mutation corresponding to T50 in SEQ ID NO:1 comprises a T50R, T50N, T50L, T50K, T50F, T50W or T50Y mutation.

[0133] According to a specific embodiment, the mutation corresponding to T50 in SEQ ID NO:1 comprises a T50F mutation.

[0134] According to a specific embodiment, the mutation corresponding to V57 in SEQ ID NO:1 comprises a V57R, V57K, V57F or V57W mutation.

[0135] According to a specific embodiment, the mutation corresponding to V57 in SEQ ID NO:1 comprises a V57R mutation.

[0136] According to a specific embodiment, the LILRB2 polypeptide comprises one of the disclosed mutations.

[0137] According to a specific embodiment, the LILRB2 polypeptide comprises at least two of the disclosed mutations.

[0138] Thus, according to specific embodiments, the LILRB2 polypeptide comprises a mutation in amino acids corresponding to S45 and I49 of SEQ ID NO:1, a mutation in amino acids corresponding to S45 and T50 of SEQ ID NO:1, a mutation in amino acids corresponding to S45 and V57 of SEQ ID NO:1, a mutation in amino acids corresponding to I49 and T50 of SEQ ID NO:1, a mutation in amino acids corresponding to I49 and V57 of SEQ ID NO:1, or a mutation in amino acids corresponding to T50 and V57 of SEQ ID NO:1.

[0139] According to specific embodiments, the LILRB2 polypeptide comprises a mutation at the amino acid corresponding to S45 of SEQ ID NO:1 and additional mutations at the amino acids corresponding to I49, T50 and / or V57 of SEQ ID NO:1.

[0140] According to a specific embodiment, the LILRB2 polypeptide comprises the S45N and T50R mutations corresponding to SEQ ID NO:1.

[0141] According to a specific embodiment, the LILRB2 polypeptide comprises the S45Y and T50K mutations corresponding to SEQ ID NO:1.

[0142] According to a specific embodiment, the LILRB2 polypeptide comprises the S45R and I49F mutations corresponding to SEQ ID NO:1.

[0143] According to a specific embodiment, the LILRB2 polypeptide comprises the S45Q and V57R mutations corresponding to SEQ ID NO:1.

[0144] According to a specific embodiment, the LILRB2 polypeptide comprises the S45Q and I49K mutations corresponding to SEQ ID NO:1.

[0145] According to a specific embodiment, the LILRB2 polypeptide comprises the S45Y and T50N mutations corresponding to SEQ ID NO:1.

[0146] According to specific embodiments, the amino acid sequence of the LILRB2 polypeptide has a sequence similar to or similar to the amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, It is at least 99% or 100% identical or homologous to, or is at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polynucleotide sequence encoding it (described further below).

[0147] According to specific embodiments, the amino acid sequence of the LILRB2 polypeptide is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23, each possibility representing a separate embodiment of the present invention.

[0148] According to specific embodiments, the amino acid sequence of the LILRB2 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23, each possibility representing a separate embodiment of the present invention.

[0149] According to specific embodiments, the amino acid sequence of the LILRB2 polypeptide is set forth in SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23, each possibility representing a separate embodiment of the present invention.

[0150] According to specific embodiments, the LILRB1 polypeptide comprises at least one mutation at a position corresponding to an amino acid selected from the group consisting of T43, I47, T48 and V55 of SEQ ID NO:102.

[0151] As used herein, the phrase "corresponding to SEQ ID NO: 102" is intended to include the corresponding amino acid residue in any other LILRB1 amino acid sequence.

[0152] According to a specific embodiment, the mutation comprises a conservative substitution.

[0153] According to other specific embodiments, the mutation comprises a non-conservative substitution.

[0154] According to a specific embodiment, the mutation is a non-naturally occurring mutation.

[0155] According to specific embodiments, the amino acid corresponding to T43 in SEQ ID NO: 102 comprises a T43R, T43N, T43Q, T43H, T43L, T43K, T43M, T43F, T43W or T43Y mutation.

[0156] According to specific embodiments, the mutation corresponding to I47 in SEQ ID NO: 102 comprises an I47R, I47K, I47F or I47Y mutation.

[0157] According to specific embodiments, the mutation corresponding to T48 in SEQ ID NO: 102 comprises a T48R, T48N, T48L, T48K, T48F, T48W or T48Y mutation.

[0158] According to a specific embodiment, the mutation corresponding to V55 in SEQ ID NO: 102 comprises a V55R, V55K, V55F or V55W mutation.

[0159] According to a specific embodiment, the mutation corresponding to V55 in SEQ ID NO: 102 comprises a V55R mutation.

[0160] According to a specific embodiment, the LILRB1 polypeptide comprises one of the disclosed mutations.

[0161] According to specific embodiments, the LILRB1 polypeptide comprises at least two of the disclosed mutations.

[0162] According to specific embodiments, the amino acid sequence of the LILRB1 polypeptide is at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:103. is homologous to or is at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polynucleotide sequence encoding it (described further below).

[0163] According to a specific embodiment, the amino acid sequence of the LILRB1 polypeptide is at least 90% identical to SEQ ID NO:103.

[0164] According to a specific embodiment, the amino acid sequence of the LILRB1 polypeptide comprises SEQ ID NO:103.

[0165] According to a specific embodiment, the amino acid sequence of the LILRB1 polypeptide is as set forth in SEQ ID NO:103.

[0166] According to specific embodiments, the LILRB (e.g., LILRB2, LILRB1) polypeptide has increased stability compared to a LILRB (e.g., LILRB2, LILRB1) polypeptide of the same length and sequence that does not contain at least one mutation.

[0167] According to a specific embodiment, the LILRB2 polypeptide has increased stability compared to the LILRB2 polypeptide shown in SEQ ID NO:1.

[0168] According to specific embodiments, the LILRB1 polypeptide has increased stability compared to the LILRB2 polypeptide set forth in SEQ ID NO:102.

[0169] As used herein, the phrase "increased stability" refers to a statistically significant increase in the stability of a LILRB (e.g., LILRB2, LILRB1) polypeptide comprising at least one mutation disclosed herein, compared to a LILRB (e.g., LILRB2, LILRB1) polypeptide of the same length and sequence not comprising at least one mutation. According to specific embodiments, the increased stability is manifested by an increased stability of the LILRB-HLAG complex. According to specific embodiments, the increase is at least 2%, 5%, 10%, 30%, 40% or even higher, for example, at 50%, 60%, 70%, 80%, 90% or 100%. According to specific embodiments, the increase is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, or at least 20-fold.

[0170] Methods for determining the stability of a polypeptide are well known in the art and include, for example, size-exclusion high performance liquid chromatography (SEC-HPLC) to define the physical state and time dependence of aggregate formation, SDS-PAGE to define the physical state and time dependence (integrity) of the protein, and analysis of the melting temperature (Tm) using, for example, differential scanning calorimetry (DSC).

[0171] According to specific embodiments, the LILRB (e.g., LILRB2, LILRB1) polypeptide has increased affinity for HLA-G compared to a LILRB (e.g., LILRB2, LILRB1) polypeptide of the same length and sequence that does not contain at least one mutation.

[0172] According to a specific embodiment, the LILRB2 polypeptide has increased affinity for HLA-G compared to the LILRB2 polypeptide shown in SEQ ID NO:1.

[0173] According to a specific embodiment, the LILRB1 polypeptide has increased affinity for HLA-G compared to the LILRB2 polypeptide set forth in SEQ ID NO:102.

[0174] As used herein, the phrase "increased affinity for HLA-G" refers to a statistically significant increase in the binding affinity of a LILRB (e.g., LILRB2, LILRB1) polypeptide comprising at least one mutation disclosed herein to an HLA-G polypeptide (e.g., as shown in SEQ ID NO:3) compared to a LILRB (e.g., LILRB2, LILRB1) polypeptide of the same length and sequence not comprising at least one mutation, which can be determined directly or via inhibition of binding of a natural ligand. The increased binding affinity can be manifested by a higher affinity (e.g., Kd, ​​Ka) for HLA-G and / or a higher selective binding to HLA-G compared to other HLA (e.g., HLA-A, HLA-B, HLA-C). According to specific embodiments, the increase is at least 2%, 5%, 10%, 30%, 40% or even higher, e.g., 50%, 60%, 70%, 80%, 90% or 100%. According to specific embodiments, the increase is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, or at least 20-fold. According to specific embodiments, the increase is at least 5, 10, 100, 1000, or 10000-fold.

[0175] Methods for determining affinity are well known in the art and are described above and below, and include, for example, Biacore, HPLC, surface plasmon resonance assay (SPR) and flow cytometry (FACS).

[0176] According to specific embodiments, LILRB (eg, LILRB2, LILRB1) polypeptides may, for example, induce or enhance phagocytosis of cancer cells.

[0177] Methods for determining phagocytosis are known in the art and are further described in the Examples section below.

[0178] According to specific embodiments, LILRB (eg, LILRB2, LILRB1) polypeptides may prevent or reduce the induction of tumor-promoting M2 macrophages and result in the induction of tumor-suppressing M1 macrophages.

[0179] According to specific embodiments, LILRB (eg, LILRB2, LILRB1) polypeptides can convert M0 (M2-like) macrophages into M1 macrophages.

[0180] Methods for determining macrophage phenotype are known in the art and are further described in the Examples section below.

[0181] The LILRB (eg, LILRB2, LILRB1) polypeptides of some embodiments of the present invention may be conjugated to a non-proteinaceous or proteinaceous moiety.

[0182] Thus, according to one aspect of the present invention, there is provided a composition comprising a LILRB (e.g., LILRB2, LILRB1) polypeptide disclosed herein and a non-proteinaceous moiety bound to a LILRB (e.g., LILRB2, LILRB1) polypeptide.

[0183] The phrase "non-proteinaceous moiety" as used herein refers to a molecule that does not contain peptide-bound amino acids that are bound to a peptide. According to a specific embodiment, the non-proteinaceous moiety is a non-toxic moiety. Exemplary non-proteinaceous moieties that can be used according to the present teachings include, but are not limited to, drugs, chemicals, small molecules, polynucleotides, detectable moieties, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), poly(styrene-co-maleic anhydride) (SMA), and divinyl ether and maleic anhydride copolymer (DIVEMA). According to a specific embodiment of the present invention, the non-proteinaceous moiety comprises polyethylene glycol (PEG).

[0184] Such molecules are highly stable (resisting in vivo proteolytic activity, presumably due to steric hindrance imparted by the nonproteinaceous portion) and can be produced using inexpensive, highly efficient, general solid-phase synthetic methods, as described further below. It will be appreciated, however, that recombinant techniques can still be used, whereby the recombinant peptide product is subjected to in vitro modification (e.g., PEGylation, as described further below).

[0185] Bioconjugation of PEG to the amino acid sequence of a polypeptide (i.e., PEGylation) can be accomplished using PEG derivatives, such as N-hydroxysuccinimide (NHS) esters of PEG carboxylic acids, monomethoxy PEG2-NHS, succinimidyl esters of carboxymethylated PEG (SCM-PEG), benzotriazole carbonate derivatives of PEG, glycidyl ethers of PEG, PEG p-nitrophenyl carbonate (PEG-NPC, e.g., methoxy PEG-NPC), PEG aldehydes, PEG-orthopyridyl-disulfides, carbonyldimidazol-activated PEG, PEG-thiols, and PEG-maleimides. Such PEG derivatives are commercially available in a variety of molecular weights (see, for example, catalog, Polyethylene Glycol and Derivatives, 2000 (Shearwater Polymers, Inc., Huntsville, Ala.)). If desired, many of the above derivatives are available in monofunctional monomethoxy PEG (mPEG) form. In general, PEGs attached to the polypeptides of the invention should range in molecular weight (MW) from a few hundred Daltons to about 100 kDa (e.g., 3-30 kDa). Larger MW PEGs can be used, but may result in some loss of yield of the PEGylated polypeptide. The purity of the larger PEG molecules should also be monitored, since it may be difficult to obtain PEGs of higher MW with the same high purity as can be obtained with lower MW PEGs. It is preferred to use PEGs that are at least 85% pure, more preferably at least 90% pure, 95% pure, or higher.PEGylation of molecules is discussed further in, for example, Chapter 15, Hermanson, Bioconjugate Techniques, Academic Press San Diego, Calif. (1996), and Zalipsky et al., "Succinimidyl Carbonates of Polyethylene Glycol," in Polymeric Drugs and Drug Delivery Systems, edited by Dunn and Ottenbrite, American Chemical Society, Washington, DC (1991).

[0186] Conveniently, PEG can be attached to a selected position in the polypeptide by site-directed mutagenesis, as long as the activity of the conjugate is retained. The target for PEGylation can be any cysteine ​​residue at the N-terminus or C-terminus of the peptide sequence. Additionally or alternatively, other cysteine ​​residues can be added to the amino acid sequence of the polypeptide (e.g., at the N-terminus or C-terminus), thereby serving as targets for PEGylation. Computer analysis can be performed to select favorable positions for mutagenesis that do not reduce activity.

[0187] Various conjugation chemistries of activated PEG can be used, for example, PEG-maleimide, PEG-vinylsulfone (VS), PEG-acrylate (AC), PEG-orthopyridyl disulfide. Methods for preparing activated PEG molecules are known in the art. For example, PEG-VS can be prepared by reacting a dichloromethane (DCM) solution of PEG-OH with NaH under argon, followed by di-vinylsulfone (molar ratio: OH 1:NaH 5:divinylsulfone 50, 0.2 grams of PEG / mL of DCM). PEG-AC is made by reacting a DCM solution of PEG-OH with acryloyl chloride and triethylamine under argon (molar ratio: OH 1:acryloyl chloride 1.5:triethylamine 2, 0.2 grams of PEG / mL of DCM). Such chemical groups can be attached to linearized, 2-arm, 4-arm, or 8-arm PEG molecules.

[0188] The resulting conjugated molecules (eg, PEGylated or PVP-conjugated polypeptides) are isolated, purified, and quantified using, for example, high performance liquid chromatography (HPLC) and biological assays.

[0189] According to a specific embodiment, the non-proteinaceous moiety is a dimerization moiety. Such dimerization molecules are well known to those skilled in the art and are further described below.

[0190] According to further or alternative aspects of the present invention, there is provided a fusion polypeptide comprising a LILRB (e.g., LILRB2, LILRB1) polypeptide disclosed herein linked to a heterologous proteinaceous moiety.

[0191] As used herein, the term "fusion polypeptide" refers to an amino acid sequence having two or more moieties that are not naturally found together in a single amino acid sequence.

[0192] As used herein, the term "heterologous" refers to an amino acid sequence that is non-native at least in location to the referenced amino acid sequence (e.g., a LILRB polypeptide, e.g., a LILRB2 polypeptide, a LILRB1 polypeptide) or is completely absent from the native sequence of the referenced amino acid sequence.

[0193] Non-limiting examples of heterologous proteinaceous moieties that may be fused to the LILRB (e.g., LILRB2, LILRB1) polypeptides of some embodiments of the present invention include dimerization moieties, detectable moieties, therapeutic moieties, cleavable moieties, etc., as further described below.

[0194] According to a specific embodiment, the heterologous proteinaceous moiety is a dimerization moiety. Such dimerization moieties are well known to those skilled in the art and are further described below.

[0195] As used herein, the term "dimerization moiety" refers to a moiety that can bind to two different monomers to form a dimer. Such dimerization moieties are known in the art and include chemical and proteinaceous moieties.

[0196] According to a specific embodiment, the dimerization moiety is directly attached to the polypeptide.

[0197] According to a specific embodiment, the dimerization moiety is indirectly attached to the polypeptide.

[0198] According to a specific embodiment, the dimerization moiety is covalently attached to the polypeptide.

[0199] According to a specific embodiment, the dimerization moiety is non-covalently attached to the polypeptide.

[0200] According to a specific embodiment, the dimerization moiety is a composition of at least two different molecules.

[0201] According to specific embodiments, the dimerization moiety is a non-proteinaceous moiety, such as a cross-linker, an organic polymer, a synthetic polymer, a small molecule, and the like.

[0202] Many such non-proteinaceous moieties are known in the art and are commercially available, for example from Santa Cruz, Sigma-Aldrich, Proteochem, etc. According to a specific embodiment, the non-proteinaceous moiety is a heterobifunctional crosslinker. Heterobifunctional crosslinkers have two different reactive ends. Typically, in a first step, a monomer is modified with one reactive group of the heterobifunctional reagent and the remaining free reagent is removed. In a second step, the modified monomer is mixed with a second monomer, which is then reacted with the modifying group at the other end of the reagent. The most widely used couple proteins via amine and sulfhydryl groups (at least stable amine-reactive NHS-esters are coupled first, and after removal of uncoupled reagent, coupling to sulfhydryl groups proceeds). Sulfhydryl-reactive groups are generally maleimides, pyridyl disulfides and alpha-haloacetyl. Other crosslinkers include carbodiimides that bond between a carboxyl group (-COOH) and a primary amine (-NH2). Another approach is to modify the lysine residue of one monomer to a thiol, and the second monomer by the addition of a maleimide group followed by the formation of a stable thioester bond between the monomers. If one of the monomers has a natural thiol, those groups can be reacted directly with the maleimide attached to the other monomer. There are also heterobifunctional crosslinkers with one photoreactive terminus, e.g., bis[2-(4-azidosalicylamido)ethyl)] disulfide, BASED. When no specific group is available to react, a photoreactive group is used because it reacts nonspecifically upon exposure to UV light.Non-limiting examples of such heterobifunctional crosslinkers include, but are not limited to, alkyne-PEG4-maleimide, alkyne-PEG5-N-hydroxysuccinimidyl ester, maleimide-PEG-succinimidyl ester, azido-PEG4-phenyloxadiazole methylsulfone, LC-SMCC (Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate)), MPBH (4-(4-N-maleimidophenyl)butyric acid hydrazide hydrochloride + 1 / 2 dioxane), PDPH (3-(2-pyridyldithio)propionyl hydrazide), SIAB (N-succinimidyl(4-iodoacetyl)aminobenzoate), SMPH (Succinimidyl-6-((b-maleimidopropionamido)hexahydrochloride ... Sulfo-KMUS (N-(κ-maleimido undecanoyloxy) sulfosuccinimide ester), sulfo-SIAB (sulfosuccinimidyl (4-iodoacetyl) aminobenzoate), 3-(maleimido)propionic acid N-hydroxysuccinimide ester, methoxycarbonylsulfenyl chloride, propargyl-PEG-acid, amino-PEG-t-butyl ester, BocNH-PEG5-acid, BMPH (N-(β-maleimidopropionic acid) hydrazide, trifluoroacetate), ANB-NOS, BMPS, EMCS, GMBS, LC-SPDP, MBS, SBA, SIA, sulfo-SIA, SMCC, SMPB, SMPH, SPDP, sulfo-LC-SPDP, sulfo-MBS, sulfo-SANPAH, sulfo-SMCC.

[0203] According to other specific embodiments, the dimerization moiety is a proteinaceous moiety.

[0204] According to specific embodiments, the LILRB (eg, LILRB2, LILRB1) polypeptide is attached to the N-terminus of the dimerized proteinaceous moiety.

[0205] According to specific embodiments, the LILRB (eg, LILRB2, LILRB1) polypeptide is attached to the C-terminus of the dimerization proteinaceous moiety.

[0206] According to a specific embodiment, the dimerization portion comprises a member of an affinity pair polypeptide having two separate affinity portions for two different affinity complementary tags.Such affinity pairs are well known in the art and include, but are not limited to, hemagglutinin (HA), anti-HA, Abitag™, V5, Myc, T7, FLAG, HSV, VSV-G, His, biotin, avidin, streptavidin, rhizavedin, metal affinity tag, lecithin affinity tag.Those skilled in the art will know which tag to choose.

[0207] According to specific embodiments, the dimerization moiety comprises a leucine zipper or a helix-loop-helix.

[0208] According to a specific embodiment, the dimerization moiety is an Fc domain of an antibody or fragment thereof.

[0209] According to specific embodiments, the Fc is IgG, IgA, IgD or IgE.

[0210] According to a specific embodiment, the Fc domain is that of an IgG.

[0211] According to specific embodiments, the Fc domain is of IgG1 or IgG4.

[0212] According to a specific embodiment, the Fc domain is that of human IgG4.

[0213] A non-limiting example of a human IgG4 Fc domain that can be used in specific embodiments of the invention is provided in SEQ ID NO:63.

[0214] According to a specific embodiment, the Fc domain is that of human IgG1.

[0215] A non-limiting example of a human IgG1 Fc domain that can be used in specific embodiments of the present invention is provided in SEQ ID NO:64.

[0216] According to specific embodiments, the Fc domain may comprise conservative and non-conservative amino acid substitutions (a detailed description of conservative and non-conservative substitutions is provided below). Such substitutions in the Fc domain are known in the art and are further described below.

[0217] For example, there are numerous mechanisms that can be used to generate heterodimers using the Fc domain of an antibody, including, but not limited to, knobs-into-holes or charge pairs (see, e.g., Gunasekaran et al., J. Biol. Chem. (2010) 285 (25):19637, incorporated herein by reference in its entirety).

[0218] A representative example that can be used in specific embodiments of the present invention is the "knob-into-hole" ("KIH") form. Such knob and hole mutations are well known in the art and are disclosed, for example, in U.S. Pat. No. 8,216,805, the contents of which are incorporated herein by reference in their entirety; Shane Atwell et al. J. Mol. Biol. (1997) 270, 26-35; Cater et al. (Protein Engineering vol.9 no.7 pp.617-621, 1996; and A. Margaret Merchant et.al. Nature Biotechnology (1998) 16 July. In addition, as described in Merchant et al., Nature Biotech. 16:677 (1998), these "knob and hole" mutations can be combined with disulfide bonds to bias the formation towards heterodimerization.

[0219] Thus, according to a specific embodiment, one of the monomers comprises an Fc domain that includes a knob mutation and the other monomer comprises an Fc domain that includes a hole mutation.

[0220] It is within the skill of the art to select specific immunoglobulin Fc domains from particular immunoglobulin classes and subclasses, as well as first Fc variants for knob mutations and others for hole mutations. Non-limiting examples of substitutions that can be used in specific embodiments include S228P, L235E, T366W, Y349C, T366S, L368A, Y407V and / or E356C (according to EU numbering corresponding to human IgG4 as part of a full length antibody (Kabat, EA, TT Wu, M. Reid-Miller, HM Perry and KS Gottesman. 1987. Sequences of proteins of Immunological Interest. US. Dept. of Health and Human Services, Bethesda)), or L235A, Y349C, T366W, T354C, D356C, T366S, L368A and / or Y407V (according to EU numbering corresponding to human IgG1 as part of a full length antibody (Kabat, EA, TT Wu, M. Reid-Miller, HM Perry and KS Gottesman. 1987. Sequences of [Following the instructions given in the US Food and Drug Administration (FDA) at 105–149 C.I. 1999), the National Institute of Standards and Technology (NIS), National Institute of Health and Human Services, Bethesda, USA].

[0221] Non-limiting examples of IgG4 Fc domains containing knob mutations that can be used in specific embodiments of the invention are provided in SEQ ID NOs: 65, 59 and 60.

[0222] Non-limiting examples of IgG4 Fc domains containing hole mutations that can be used in specific embodiments of the invention are provided in SEQ ID NOs: 66, 61 and 62.

[0223] Non-limiting examples of IgG1 Fc domains containing knob mutations that can be used in specific embodiments of the invention are provided in SEQ ID NOs: 31, 67 and 86.

[0224] Non-limiting examples of IgG1 Fc domains containing hole mutations that can be used in specific embodiments of the present invention are provided in SEQ ID NOs: 29, 68 and 85.

[0225] According to specific embodiments, the Fc domain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 63, 64, 65, 59, 60, 66, 61, 62, 31, 67, 86, 29, 68, and 85, or a functional fragment thereof exhibiting a desired activity as disclosed herein. %, at least 98%, at least 99% or 100% identity or homology to the polynucleotide sequence encoding it, or an amino acid sequence which has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the polynucleotide sequence encoding it.

[0226] According to specific embodiments, the Fc domain is modified to alter its binding to Fc receptors, to reduce its immunostimulatory function, and / or to improve the half-life of the fusion.

[0227] According to other specific embodiments, the Fc domain has not been modified to alter its binding to Fc receptors, to reduce its immunostimulatory function, and / or to improve the half-life of the fusion.

[0228] According to specific embodiments, the Fc domain is modified to reduce or prevent binding to Fc receptors (e.g., Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII) in vivo. Such modifications have been described, for example, by Clark et al. (Armour et al., 1999; Armour et al., 2002), who designed and described a series of mutant IgG1, IgG2 and IgG4 Fc domains and their Fc.gamma.R binding properties. Additional or alternative modifications in the Fc of human IgG1 to reduce its binding to Fc receptors have been described by CHAPPEL et al. (Proc. Natl. Acad. Sci (1991) 88:9036-9040, the contents of which are incorporated herein by reference in their entirety), who identified amino acids L234 and L235 [according to EU numbering corresponding to full-length antibodies (Kabat et al.)] as essential for Fc receptor binding. An additional substitution of P329 to G further weakens binding, and this LALA-PG substitution combination has been described, for example, by Schlothauer, T., et al. (2016) Protein Eng. Des. Sel. 29, 457-466, and WO 2012 / 130831, the contents of which are incorporated herein by reference in their entirety. Additional or alternative modifications in the Fc of human IgG4 to disrupt Fab arm exchange and reduce its binding to Fc receptors have been described by John-Paul Silva et al. (THE JOURNAL OF BIOLOGICAL CHEMISTRY (2015), 290:9, 5462-5469, the contents of which are incorporated herein by reference in their entirety) and Newman et al. (Clinical Immunology (2001) 98:2, the contents of which are incorporated herein by reference in their entirety), who identified S228P and L235E [according to EU numbering corresponding to full-length antibodies (Kabat et al.)], respectively.

[0229] According to specific embodiments, the Fc domain is modified to maximize FcγRIIIa binding. Such modifications are described, for example, by Shields RL J Biol Chem. (2001) 276:6591, Smith P, Proc Natl Acad Sci USA. (2012) 109:6181, Stavenhagen et al., Cancer Res (2007) 67:8882, Lazar et al., Proc Natl Acad Sci USA (2006) 103:4005, Richards et al., 2008 Cancer Ther 7:2517, and Mimoto et al., (2013) MAbs 5:229, the contents of which are incorporated herein by reference in their entirety. Non-limiting examples of such modifications that can be used in specific embodiments include substitutions of one or more amino acid residues selected from S298, E333 and K334 [according to EU numbering (Kabat et al.) corresponding to a full length antibody] (e.g., S298A, E333A, K334A), G236A, S239A, A330L and I332E, F243L, R292P, Y300L, V305I and P396L, S239D, I332E and A330L, 236A, S239D and I332E, and asymmetric substitutions -L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in the heavy chain and D270E / K326D / A330M / K334E in the opposite heavy chain.

[0230] According to specific embodiments, the Fc domain is modified to alter effector function, e.g., to reduce complement binding and / or to reduce or abolish complement dependent cytotoxicity. Such modifications are described, for example, in U.S. Pat. Nos. 5,624,821 and 5,648,260, 6,194,551, WO 99 / 51642, Wines et al., 2000, Idusogie et al. (2000) J. Immunol. 164:4178, Tao et al. (1993) J. Exp. Med. 178:661, and Canfield & Morrison (1991) J. Exp. Med. 173:1483, the contents of which are incorporated herein by reference in their entirety. Non-limiting examples of such modifications that can be used in specific embodiments include substitutions of one or more amino acids at positions selected from 234, 235, 236, 237, 297, 318, 320 and 322, 329, 331 and 322, L234 and / or L235 (according to EU numbering (Kabat et al.) corresponding to a full length antibody) (e.g., L234A and / or L235A), D270, K322, P329 and P331 (e.g., D270A, K322A, P329A and P331A).

[0231] According to a specific embodiment, the Fc domain is modified to improve the half-life of the fusion protein. Such modifications are described, for example, in US Pat. Nos. 5,869,046 and 6,121,022, the contents of which are incorporated herein by reference in their entirety. For example, the substitution of one or more amino acids at positions (according to EU numbering (Kabat et al.) corresponding to full-length antibodies) selected from 252 (for example, to introduce Thr), 254 (for example, to introduce Ser) and 256 (for example, to introduce Phe). Another modification to improve half-life can be by alteration of the CH1 or CL region to introduce a salvage receptor motif, such as those found in the two loops of the CH2 domain of the Fc region of IgG.

[0232] Maximizing FcRn binding and extending half-life is also described, for example, in Stapleton NM, Nat Commun. (2011) 2:599, Shields RL. J Biol Chem. (2001) 276:6591, Dall'acqua WF J Immunol. (2002) 169:5171, Zalevsky J, Nat Biotechnol. (2010) 28:157, Ghetie V,. Nat. Biotechnol. (1997) 15:637, and Monnet C, MAbs. (2014) 6:422, the contents of which are incorporated herein by reference in their entireties. Non-limiting examples of such modifications that can be used in specific embodiments include substitution of one or more amino acid residues selected from Arg435His, Asn434Ala, Met252Tyr, Ser254Thr, and Thr256Glu, Met428Leu and Asn434Ser, Thr252Leu, Thr253Ser and Thr254Phe, Glu294delta, Thr307Pro and Asn434Tyr, Thr256Asn, Ala378Val, Ser383Asn and Asn434Tyr (according to EU numbering (Kabat et al.) corresponding to a full-length antibody).

[0233] Non-limiting examples of LILRB2-Fc fusion sequences that can be used in specific embodiments of the present invention are set forth in Table 3 below.

[0234] Non-limiting examples of LILRB2-Fc fusion sequences that can be used in specific embodiments of the present invention are provided in SEQ ID NOs: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 96, 98, and 101.

[0235] According to specific embodiments, the LILRB2-Fc fusion has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 41, 43, 45, 47, 49, 51, 53, 55, and 57. 00% identity or homology thereto, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a polynucleotide sequence encoding same (described further below).

[0236] According to specific embodiments, the LILRB2-Fc fusion amino acid sequence is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 41, 43, 45, 47, 49, 51, 53, 55, and 57, with each possibility representing a separate embodiment of the present invention.

[0237] According to specific embodiments, the LILRB2-Fc fusion amino acid sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 41, 43, 45, 47, 49, 51, 53, 55, and 57, each possibility representing a separate embodiment of the present invention.

[0238] According to specific embodiments, the LILRB2-Fc fusion amino acid sequences are as set forth in SEQ ID NOs: 39, 41, 43, 45, 47, 49, 51, 53, 55, and 57, each possibility representing a separate embodiment of the present invention.

[0239] As some embodiments of the LILRB (e.g., LILRB2, LILRB1) polypeptides comprise a dimerization moiety, further or alternative aspects of the invention provide dimers comprising the LILRB (e.g., LILRB2, LILRB1) polypeptides disclosed herein, compositions comprising same, or fusion polypeptides comprising same.

[0240] According to further or alternative aspects of the present invention, there is provided a composition comprising a dimer disclosed herein, wherein the dimer is the predominant form of LILRB (e.g., LILRB2, LILRB1) in the composition.

[0241] According to specific embodiments, 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%, at least 96%, at least 97%, at least 98%, at least 99% of the LILRB2 polypeptides in the composition are dimers as disclosed herein.

[0242] According to specific embodiments, at least 90% of the LILRB (eg, LILRB2, LILRB1) polypeptides in the composition are dimers as disclosed herein.

[0243] Methods for determining dimerization are well known in the art and include, but are not limited to, native-PAGE, SEC-HPLC 2D gel, gel filtration, SEC-MALS, analytical ultracentrifugation (AUC) mass spectrometry (MS), capillary gel electrophoresis (CGE).

[0244] According to a specific embodiment, the monomers of the dimer are not covalently linked.

[0245] According to other specific embodiments, the monomers of the dimer are covalently linked.

[0246] According to other specific embodiments, the monomers of the dimer are linked by disulfide bonds.

[0247] The dimer can be a homodimer or a heterodimer.

[0248] According to a specific embodiment, the dimer is a heterodimer.

[0249] As used herein, the term "heterodimer" refers to a non-naturally occurring dimeric protein formed by the artificial association of two different proteins (referred to herein as monomers).

[0250] Thus, according to further or alternative aspects of the present invention, there is provided a heterodimer comprising a first monomer comprising a LILRB (e.g., LILRB2, LILRB1) polypeptide disclosed herein, a composition comprising the same, or a fusion polypeptide comprising the same, and a second monomer comprising a polypeptide distinct from LILRB (e.g., LILRB2, LILRB1).

[0251] It is within the skill of the art to select suitable distinct polypeptides to be included in the second monomer. Non-limiting examples of such polypeptides include type I membrane proteins, type II membrane proteins, and immune checkpoint proteins, or functional fragments or homologs thereof capable of binding to at least their natural binding pairs.

[0252] As used herein, the phrase "type I membrane protein" refers to a transmembrane protein having an N-terminal extracellular domain.

[0253] Non-limiting examples of such type I membrane proteins that can be used in specific embodiments of the invention include PD1, SIRPα, LAG3, BTN3A1, CD27, CD80, CD86, ENG, NLGN4X, CD84, TIGIT, CD40, IL-8, IL-10, CD164, LY6G6F, CD28, CTLA4, BTLA, LILRB1, LILRB4, TYROBP, ICOS, VEGFA, CSF1, CSF1R, VEGFB, BMP2, BMP3, GDNF, PDGFC, PDGFD, RAET1E, CD155, CD166, MICA, NRG1, HVEM, DR3, TEK, TGFBR (e.g., TGFBR1), LY96, CD96, KIT, CD244 GFER, and SIGLEC (e.g., SIGLEC10).

[0254] According to specific embodiments, the type I membrane protein is selected from the group consisting of PD1, SIRPα, LAG3, TIGIT, LILRB1, CSF1, CSF1R and TGFBR.

[0255] According to a specific embodiment, the type I membrane protein is selected from the group consisting of PD1, SIRPα, TIGIT and SIGLEC.

[0256] According to a specific embodiment, the type I membrane protein is an immune modulator.

[0257] As used herein, the term "immunomodulator" refers to a protein that modulates an immune cell response (i.e., activation or function). An immune modulator may positively regulate immune cell activation or function, or may negatively regulate immune cell activation or function. Such immune modulators are known in the art and include immune checkpoint proteins, cytokines, and the like.

[0258] According to a specific embodiment, the immune modulator is an immune activator.

[0259] According to other specific embodiments, the immune modulator is an immune suppressor or inhibitor.

[0260] Non-limiting examples of type I membrane protein immunomodulators include, but are not limited to, PD1, SIRPα, CD28, CSF1R, IL-8, IL-10, CTLA4, ICOS, CD27, CD80, CD86, SIGLEC10, and TIGIT.

[0261] As used herein, the phrase "type II membrane protein" refers to a transmembrane protein having a C-terminal extracellular domain.

[0262] Non-limiting examples of such type II membrane proteins that can be used in specific embodiments of the invention include 4-1BBL, FasL, TRAIL, TNF-alpha, TNF-beta, OX40L, CD40L, CD27L, CD30L, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEGI, GITRL, EDAI / 2, lymphotoxin alpha and lymphotoxin beta.

[0263] According to a specific embodiment, the type II membrane protein is selected from the group consisting of 4-1BBL, OX40L, CD40L, LIGHT and GITRL.

[0264] According to a specific embodiment, the type II membrane protein is an immune modulator.

[0265] Such immune modulators include, but are not limited to, 4-1BBL, TNF-alpha, TNF-beta, OX40L, CD40L, CD27L and CD30L.

[0266] As used herein, the term "immune checkpoint protein" refers to a protein that regulates immune cell activation or function. Immune checkpoint proteins can be either costimulatory proteins (i.e., transducing stimulatory signals that lead to activation of immune cells) or inhibitory proteins (i.e., transducing inhibitory signals that lead to suppression of immune cell activity). According to some embodiments, immune checkpoint proteins regulate T cell activation or function. Numerous checkpoint proteins are known in the art, including, but not limited to, PD1, PDL-1, B7H2, B7H4, CTLA-4, CD80, CD86, LAG-3, TIM-3, KIR, IDO, CD19, OX40, 4-1BB (CD137), CD27, CD70, CD40, GITR, CD28, and ICOS (CD278).

[0267] According to specific embodiments, a first monomer of the heterodimer comprises a LILRB (e.g., LILRB2, LILRB1) polypeptide disclosed herein, and a second monomer comprises an amino acid sequence of a protein selected from the group consisting of SIRPα, PD1, TIGIT and SIGLEC10, which amino acid sequence is capable of binding to its natural binding pair.

[0268] According to specific embodiments, the first monomer of the heterodimer comprises a LILRB (e.g., LILRB2, LILRB1) polypeptide disclosed herein, and the second monomer comprises an amino acid sequence of SIRPα, which amino acid sequence can bind to CD47.

[0269] Non-limiting examples of LILRB2 and SIRPα heterodimer sequences that can be used in specific embodiments of the present invention are set out in Table 3 below.

[0270] "SIRPα (signal regulatory protein alpha, also known as CD172a)" refers to a polypeptide encoded by the SIRPA gene (Gene ID 140885). According to specific embodiments, SIRPα is human SIRPα. According to specific embodiments, SIRPα refers to human SIRPα, e.g., as provided in the following GenBank numbers NP_001035111, NP_001035112, NP_001317657, or NP_542970.

[0271] A known binding pair of SIRPα is CD47. According to specific embodiments, the CD47 protein refers to the human protein provided, for example, under the following GenBank numbers NP_001768 or NP_942088.

[0272] As used herein, the term "SIRPα polypeptide" refers to full-length SIRPα, functional fragments thereof that maintain at least the ability to bind to CD47, or homologs thereof. For example, according to specific embodiments, the amino acid sequence of SIRPα further comprises substitution, addition and deletion mutations as described above and below.

[0273] According to a specific embodiment, the SIRPα polypeptide comprises the extracellular domain of SIRPα or a functional fragment thereof capable of binding to CD47.

[0274] According to a specific embodiment, the SIRPα polypeptide comprises SEQ ID NO:27, or a functional fragment thereof capable of binding to CD47.

[0275] According to a specific embodiment, the SIRPα polypeptide comprises SEQ ID NO:27.

[0276] According to a specific embodiment, the SIRPα polypeptide consists of SEQ ID NO:27.

[0277] According to a specific embodiment, the SIRPα polypeptide comprises SEQ ID NO:88, or a functional fragment thereof capable of binding to CD47.

[0278] According to a specific embodiment, the SIRPα polypeptide comprises SEQ ID NO:88.

[0279] According to a specific embodiment, the SIRPα polypeptide consists of SEQ ID NO:88.

[0280] The term "SIRPα polypeptide" also encompasses functional homologs that exhibit a desired activity (i.e., binding to CD47). Such homologs may be, for example, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the SIRPα sequences described herein (e.g., SEQ ID NOs: 27, 88). or homologous to, or may be at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polynucleotide sequence encoding it (described further below).

[0281] According to specific embodiments, the SIRPα amino acid sequence is at least 90% identical to SEQ ID NO:27 or 88.

[0282] According to specific embodiments, SIRPα polypeptides may include conservative and non-conservative amino acid substitutions (detailed descriptions of conservative and non-conservative substitutions are provided below). Non-limiting examples of such substitutions are known in the art and are disclosed, for example, in Weiskopf K et al. Science. (2013), 341 (6141):88-91, the contents of which are incorporated herein by reference in their entirety.

[0283] According to specific embodiments, the SIRPα polypeptide comprises between 100 and 504, 100 and 500 amino acids, 150 and 450 amino acids, 200 and 400 amino acids, 250 and 400 amino acids, 300 and 400 amino acids, 320 and 420 amino acids, 340 and 350 amino acids, 300 and 400 amino acids, 340 and 450 amino acids, 100 and 200 amino acids, 100 and 150 amino acids, 100 and 125 amino acids, 100 and 120 amino acids, 100 and 119 amino acids, 105 and 119 amino acids, 110 and 119 amino acids, 115 and 119 amino acids, 105 and 118 amino acids, 110 and 118 amino acids, 115 and 118 amino acids, 105 and 117 amino acids, 110 and 117 amino acids, 115 and 117 amino acids, each possibility representing a separate embodiment of the present invention.

[0284] According to specific embodiments, the first monomer of the heterodimer has at least at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23. and a second monomer comprises an amino acid sequence of SIRPα, the amino acid sequence having at least at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 27 or 88.

[0285] According to specific embodiments, the first monomer of the heterodimer comprises a LILRB2 polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23, and the second monomer comprises an amino acid sequence of SIRPα, wherein the amino acid sequence comprises SEQ ID NO: 27 or 88.

[0286] According to a specific embodiment, the first monomer of the heterodimer has an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 41, 43, 45, 47, 49, 51, 53, 55 and 57, which is at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. or 100% identity to SEQ ID NOs: 35, 90, 94, and 98, and the second monomer of the heterodimer comprises an amino acid sequence having at least at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NOs: 35, 90, 94, and 98.

[0287] According to specific embodiments, the first monomer of the heterodimer comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55 and 57, and the second monomer of the heterodimer comprises SEQ ID NO: 35.

[0288] According to specific embodiments, the first monomer of the heterodimer is as set forth in SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55 or 57, and the second monomer of the heterodimer is as set forth in SEQ ID NO: 35, 90, 94 or 98.

[0289] According to specific embodiments, heterodimers comprising a LILRB (e.g., LILRB2, LILRB1) polypeptide disclosed herein and an amino acid sequence of SIRPα may, for example, induce or enhance phagocytosis of cancer cells.

[0290] According to specific embodiments, heterodimers comprising a LILRB (e.g., LILRB2, LILRB1) polypeptide disclosed herein and an amino acid sequence of SIRPα can prevent or reduce the induction of tumor-promoting M2 macrophages and result in the induction of tumor-suppressing M1 macrophages.

[0291] According to specific embodiments, a heterodimer comprising a LILRB (e.g., LILRB2, LILRB1) polypeptide disclosed herein and an amino acid sequence of SIRPα can convert M0 (M2-like) macrophages into M1 macrophages.

[0292] According to specific embodiments, heterodimers comprising a LILRB (e.g., LILRB2, LILRB1) polypeptide disclosed herein and an amino acid sequence of SIRPα bind to cells expressing both CD47 and HLA-G, and do not significantly bind to cells expressing only one of CD47 and HLA-G, as determined, for example, by flow cytometry analysis.

[0293] According to specific embodiments, heterodimers comprising a LILRB (e.g., LILRB2, LILRB1) polypeptide disclosed herein and an amino acid sequence of SIRPα do not significantly bind to red blood cells (RBCs) as determined by flow cytometry analysis.

[0294] According to specific embodiments, the LILRB (e.g., LILRB2, LILRB1) polypeptide or a composition, fusion or dimer comprising same is linked to or comprises a heterologous therapeutic moiety. The therapeutic moiety can be any molecule, including small molecule compounds and polypeptides.

[0295] Non-limiting examples of therapeutic moieties that can be used in specific embodiments of the invention include cytotoxic moieties, toxic moieties, cytokine moieties, immunomodulatory moieties (e.g., immune checkpoint moieties, cytokines, as further described above), polypeptides, antibodies, drugs, chemicals and / or radioisotopes.

[0296] According to some embodiments of the invention, the therapeutic moiety is conjugated by translationally fusing a polynucleotide encoding a polypeptide of some embodiments of the invention with a nucleic acid sequence encoding the therapeutic moiety.

[0297] Additionally or alternatively, a therapeutic moiety may be chemically conjugated (coupled) to the LILRB (e.g., LILRB2, LILRB1) polypeptide of some embodiments of the present invention or a composition, fusion or dimer comprising same using any conjugation method known to one of skill in the art. For example, peptides may be conjugated to peptides using 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (also called N-succinimidyl 3-(2-pyridyldithio)propionate) ("SDPD") (Sigma, Catalog No. P-3415; see, e.g., Cumber et al. 1985, Methods of Enzymology 112:207-224), glutaraldehyde conjugation procedures (see, e.g., G.T. Hermanson 1996, "Antibody Modification and Conjugation" in Bioconjugate Techniques, Academic Press, San Diego) or carbodiimide conjugation procedures [see, e.g., J. March, Advanced Organic Chemistry: Reaction's, Mechanism, and Structure, pp.349-50 & 372-74 (3d ed.), 1985; B. Neises et al. 1978, Angew. Chem., Int. Ed. Engl. 17:522; A. Hassner et al. 1978, Tetrahedron Lett. 4475; EP Boden et al. 1986, J. Org. Chem. 50:2394 and LJ Mathias 1979, Synthesis 561] can be used to conjugate to the drug of interest.

[0298] The therapeutic moiety can be attached to the LILRB (e.g., LILRB2, LILRB1) polypeptide of some embodiments of the present invention or compositions, fusions or dimers comprising same using any suitable chemical linkage, for example, directly or indirectly, via a peptide bond (when the functional moiety is a polypeptide) or via a covalent bond to an intervening linker element, for example, a linker peptide or other chemical moiety, for example, an organic polymer, using standard chemical synthesis techniques commonly practiced in the art (see, for example, hypertexttransferprotocol: / / worldwideweb(dot)chemistry(dot)org / portal / Chemistry). Chimeric peptides can be attached via a bond at the carboxy (C) or amino (N) terminus of the peptide, or via an internal chemical group, for example, a linear, branched or cyclic side chain, a bond to an internal carbon or nitrogen atom, etc.

[0299] According to specific embodiments, the LILRB (e.g., LILRB2, LILRB1) polypeptide or a composition, fusion or dimer comprising same comprises a detectable tag. As used herein, in one embodiment, the term "detectable tag" refers to any moiety that can be detected by a practitioner using techniques known in the art. The detectable tag may be a peptide sequence. Optionally, the detectable tag may be removable by chemicals or by enzymatic means, e.g., proteolysis. The detectable tags of some embodiments of the present invention may be used for purification of the polypeptide, composition, fusion or dimer. For example, the term "detectable tag" includes chitin-binding protein (CBP)-tags, maltose-binding protein (MBP)-tags, glutathione-S-transferase (GST)-tags, poly(His)-tags, FLAG tags, epitope tags, such as V5-tags, c-myc-tags, and HA-tags, and fluorescent tags, such as green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), and cyan fluorescent protein (CFP), as well as derivatives of these tags, or any tag known in the art. The term "detectable tag" also includes the term "detectable marker."

[0300] According to specific embodiments, the LILRB (e.g., LILRB2, LILRB1) polypeptide or a composition, fusion or dimer comprising the same comprises a cleavable moiety. Thus, for example, to facilitate recovery, the expressed coding sequence can be engineered to encode the polypeptide of some embodiments of the present invention and a cleavable moiety fused thereto. In one embodiment, the polypeptide is designed such that it is easily isolated by affinity chromatography, for example, by immobilization on a column specific for the cleavable moiety. In one embodiment, a cleavage site is engineered between the polypeptide and the cleavable moiety, and the peptide can be released from the chromatography column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site [see, for example, Booth et al., Immunol. Lett. 19:65-70 (1988), and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)].

[0301] According to specific embodiments, each of the moieties in the compositions, fusions or dimers disclosed herein may include a linker between the moieties, e.g., separating the polypeptide (e.g., LILRB, LILRB2, LILRB1, SIRPα) and the dimerization moiety.

[0302] According to other specific embodiments, the compositions, fusions or dimers disclosed herein do not comprise a linker between the polypeptide (e.g., LILRB, LILRB2, LILRB1, SIRPα) and the dimerization moiety.

[0303] Any linker known in the art can be used in specific embodiments of the present invention.

[0304] According to specific embodiments, the linker may be derived from a naturally occurring multidomain protein or is an experimental linker, for example, as described in Chichili et al., (2013), Protein Sci. 22(2):153-167; Chen et al., (2013), Adv Drug Deliv Rev. 65 (10):1357-1369, the entire contents of which are incorporated herein by reference. In some embodiments, the linker can be designed using linker design databases and computer programs, such as those described in Chen et al., (2013), Adv Drug Deliv Rev. 65 (10):1357-1369 and Crasto et al., (2000), Protein Eng. 13 (5):309-312, the entire contents of which are incorporated herein by reference.

[0305] According to a specific embodiment, the linker is a synthetic linker, for example PEG.

[0306] According to specific embodiments, the linker may be functional. For example, but not limited to, the linker may function to improve the folding and / or stability, improve expression, improve pharmacokinetics, and / or improve biological activity of the composition. In another example, the linker may function to target the composition to a specific cell type or location.

[0307] According to a particular embodiment, the linker is a polypeptide.

[0308] Non-limiting examples of polypeptide linkers include the sequences LE, GGGGS (SEQ ID NO:69), (GGGGS) n (n=1 to 4) (SEQ ID NO: 70), GGGGSGGGG (SEQ ID NO: 71), (GGGGS) × 2 (SEQ ID NO: 33), (GGGGS) × 2 + GGGG (SEQ ID NO: 72), (GGGGS) × 3 (SEQ ID NO: 73), (GGGGS) × 4 (SEQ ID NO: 74), (Gly) 8 (SEQ ID NO: 75), (Gly) 6 (SEQ ID NO: 76), (EAAAK) n(n = 1 to 3) (SEQ ID NO: 77), A (EAAAK) n A(n=2-5) (SEQ ID NO: 78), AEAAAKEAAAKA (SEQ ID NO: 79), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 80), PAPAP (SEQ ID NO: 81), KESGSVSSEQLAQFRSLD (SEQ ID NO: 82), EGKSSGSGSESKST (SEQ ID NO: 83), GSAGSAAGSGEF (SEQ ID NO: 84), and (XP) n (wherein X designates any amino acid, for example, Ala, Lys, or Glu).

[0309] According to a specific embodiment, the linker is (GGGGS)x2 (SEQ ID NO:33).

[0310] According to a specific embodiment, the linker is from 1 to 6 amino acids in length.

[0311] According to a specific embodiment, the linker is (GGGGS)x3 (SEQ ID NO:73).

[0312] According to a specific embodiment, the linker is from 1 to 6 amino acids in length.

[0313] According to specific embodiments, the linker is substantially composed of glycine and / or serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% or 100% glycine and serine).

[0314] According to a specific embodiment, the linker is a single amino acid linker.

[0315] In some embodiments of the invention, one amino acid is glycine.

[0316] According to specific embodiments, the compositions disclosed herein (e.g., LILRB (e.g., LILRB2, LILRB1) polypeptides, compositions comprising same, fusions and dimers) are soluble (i.e., not immobilized on a synthetic or naturally occurring surface).

[0317] According to specific embodiments, the compositions disclosed herein (e.g., LILRB (e.g., LILRB2, LILRB1) polypeptides, compositions comprising same, fusions and dimers) are immobilized on a synthetic or naturally occurring surface.

[0318] Because the compositions disclosed herein (e.g., LILRB (e.g., LILRB2, LILRB1) polypeptides, compositions comprising same, fusions and dimers, polynucleotides encoding same, or host cells expressing same) comprise LILRB (e.g., LILRB2, LILRB1) polypeptides, they can be used in methods of activating immune cells in vitro, ex vivo, and / or in vivo.

[0319] Thus, according to one aspect of the present invention, there is provided a method of activating an immune cell, the method comprising activating an immune cell in vitro in the presence of a LILRB (e.g., LILRB2, LILRB1) polypeptide, a composition comprising same, a fusion polypeptide or dimer, a polynucleotide encoding same, or a host cell expressing same.

[0320] As used herein, the term "peripheral blood mononuclear cells (PBMCs)" refers to blood cells with a single nucleus, and includes lymphocytes, monocytes and dendritic cells (DCs).

[0321] According to specific embodiments, the PBMCs are selected from the group consisting of dendritic cells (DCs), macrophages, polymorphonuclear cells, T cells, B cells, NK cells and NKT cells.

[0322] Methods for obtaining PBMCs are well known in the art, for example, whole blood drawing from a subject and collection in a container containing an anticoagulant (e.g., heparin or citrate), and apheresis. Then, according to a specific embodiment, at least one type of PBMCs is purified from the peripheral blood. There are several methods and reagents known to those skilled in the art for purifying PBMCs from whole blood, for example, leukapheresis, sedimentation, density gradient centrifugation (e.g., Ficoll), centrifugal elutriation, fractionation, for example, chemical lysis of red blood cells (e.g., by ACK), selection of specific cell types using cell surface markers (e.g., using a FACS sorter or magnetic cell separation technology, such as those commercially available from Invitrogen, Stemcell Technologies, Cellpro, Advanced Magnetics, or Miltenyi Biotec), and depletion of specific cell types by methods such as eradication (e.g., killing) with specific antibodies, or by affinity-based purification based on negative selection (e.g., using magnetic cell separation technology, FACS sorter, and / or capture ELISA label). Such methods are described, for example, in THE HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, volumes 1 to 4 (ed. DN Weir) and FLOW CYTOMETRY AND CELL SORTING (ed. A. Radbruch, Springer Verlag, 2000).

[0323] According to a specific embodiment, the immune cells comprise tumor infiltrating lymphocytes.

[0324] As used herein, the term "tumor infiltrating lymphocytes (TILs)" refers to mononuclear white blood cells that have left the bloodstream and migrated into a tumor.

[0325] According to a specific embodiment, the TILs are selected from the group consisting of T cells, B cells, NK cells and monocytes.

[0326] Methods for obtaining TILs are well known in the art, for example, by obtaining a tumor sample from a subject, for example, by biopsy or autopsy, and preparing a single cell suspension thereof. The single cell suspension can be obtained in any suitable manner, for example, mechanically (for example, by disaggregating the tumor using a GentleMACS™ dissociator, Miltenyi Biotec, Auburn, Calif.) or enzymatically (for example, collagenase or DNase). At least one type of TILs can then be purified from the cell suspension. There are several methods and reagents known to those skilled in the art for purifying the desired type of TIL, such as selection of specific cell types using cell surface markers (e.g., using FACS sorters or magnetic cell separation techniques, such as those commercially available from Invitrogen, Stemcell Technologies, Cellpro, Advanced Magnetics, or Miltenyi Biotec), and depletion of specific cell types by methods such as eradication (e.g., killing) with specific antibodies, or by affinity-based purification based on negative selection (e.g., using magnetic cell separation techniques, FACS sorters, and / or capture ELISA labels). Such methods are described, for example, in THE HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, volumes 1-4 (ed. DN Weir) and FLOW CYTOMETRY AND CELL SORTING (ed. A. Radbruch, Springer Verlag, 200).

[0327] According to a specific embodiment, the immune cells comprise phagocytic cells.

[0328] As used herein, the term "phagocytic cells" refers to cells capable of phagocytosis, including both professional and non-professional phagocytic cells. Methods for analyzing phagocytosis are well known in the art and include, for example, killing assays, flow cytometry and / or microscopic evaluation (live cell imaging, fluorescence microscopy, confocal microscopy, electron microscopy). According to specific embodiments, the phagocytic cells are selected from the group consisting of monocytes, dendritic cells (DCs) and granulocytes.

[0329] According to a specific embodiment, the phagocytes include granulocytes.

[0330] According to a specific embodiment, the phagocytes include monocytes.

[0331] According to a specific embodiment, the immune cells comprise monocytes.

[0332] According to specific embodiments, the term "monocytes" refers to both circulating monocytes and macrophages (also called mononuclear phagocytes) present in tissues.

[0333] According to specific embodiments, the monocytes include macrophages, whose cell surface phenotype typically includes CD14, CD40, CD11b, CD64, F4 / 80 (mouse) / EMR1 (human), lysozyme M, MAC-1 / MAC-3, and CD68.

[0334] According to specific embodiments, the monocytes include circulating monocytes. Typically, the cell surface phenotype of circulating monocytes includes CD14 and CD16 (e.g., CD14++CD16-, CD14+CD16++, CD14++CD16+).

[0335] According to a specific embodiment, the immune cells comprise DCs.

[0336] As used herein, the term "dendritic cell (DC)" refers to any member of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. DCs are a class of professional antigen-presenting cells with a high capacity to sensitize HLA-restricted T cells. DCs include, for example, plasmacytoid dendritic cells, myeloid dendritic cells (including immature and mature dendritic cells), Langerhans cells, interdigitating cells, and follicular dendritic cells. Dendritic cells can be recognized functionally or phenotypically, particularly by cell surface phenotype. These cells are characterized by their distinct morphology with veil-like projections on the cell surface, moderate to high levels of surface HLA class II expression, and the ability to present antigens to T cells, particularly naive T cells (see Steinman R, et al., Ann. Rev. Immunol. 1991, 9:271-196). Typically, the cell surface phenotype of DC includes CD1a+, CD4+, CD86+, or HLA-DR. The term DC encompasses both immature and mature DC.

[0337] According to a specific embodiment, the immune cells comprise granulocytes.

[0338] As used herein, the term "granulocyte" refers to a polymorphonuclear leukocyte characterized by the presence of granules in the cytoplasm.

[0339] According to a specific embodiment, the granulocytes comprise neutrophils.

[0340] According to a specific embodiment, the granulocytes comprise mast cells.

[0341] According to a specific embodiment, the immune cells comprise T cells.

[0342] As used herein, the term "T cell" refers to a T cell receptor (TCR)+-bearing differentiated lymphocyte with either a CD3+, CD4+, or CD8+ phenotype. T cells can be either effector or regulatory T cells.

[0343] As used herein, the term "effector T cell" refers to a T cell that activates or directs other immune cells, e.g., by production of cytokines, or has cytotoxic activity, e.g., CD4+, Th1 / Th2, CD8+ cytotoxic T lymphocytes.

[0344] As used herein, the term "regulatory T cells" or "Treg" refers to T cells that negatively regulate the activation of other T cells, including effector T cells, and cells of the innate immune system. Treg cells are characterized by sustained suppression of effector T cell responses. According to a specific embodiment, Treg are CD4+CD25+Foxp3+ T cells.

[0345] According to a specific embodiment, the T cells are CD4+ T cells.

[0346] According to other specific embodiments, the T cells are CD8+ T cells.

[0347] According to specific embodiments, the T cells are memory T cells. Non-limiting examples of memory T cells include effector memory CD4+ T cells with a CD3+ / CD4+ / CD45RA- / CCR7- phenotype, central memory CD4+ T cells with a CD3+ / CD4+ / CD45RA- / CCR7+ phenotype, effector memory CD8+ T cells with a CD3+ / CD8+CD45RA- / CCR7- phenotype, and central memory CD8+ T cells with a CD3+ / CD8+CD45RA- / CCR7+ phenotype.

[0348] According to specific embodiments, the T cells include engineered T cells transduced with a nucleic acid sequence encoding an expression product of interest.

[0349] According to specific embodiments, the expression product of interest is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0350] As used herein, the phrase "transduced with a nucleic acid sequence encoding a TCR" or "transducing with a nucleic acid sequence encoding a TCR" refers to the cloning of variable α and β chains from a T cell with specificity for a desired antigen presented in the context of an MHC. Methods of transducing with a TCR are known in the art and are disclosed, for example, in Nicholson et al. Adv Hematol.2012, 2012:404081, Wang and Riviere Cancer Gene Ther. 2015 Mar, 22(2):85-94), and Lamers et al, Cancer Gene Therapy (2002) 9,613-623.

[0351] As used herein, the phrase "transduced with a nucleic acid sequence encoding a CAR" or "transducing with a nucleic acid sequence encoding a CAR" refers to the cloning of a nucleic acid sequence encoding a chimeric antigen receptor (CAR), which comprises an antigen recognition portion and a T cell activation portion. A chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide that contains the antigen binding domain of an antibody (e.g., a single chain variable fragment (scFv)) linked to a T cell signaling or T cell activation domain. Methods of transducing with CARs are known in the art and have been disclosed, for example, in Davila et al. Oncoimmunology. 2012 Dec 1, 1(9): 1577-1583; Wang and Riviere Cancer Gene Ther. 2015 Mar, 22 (2): 85-94); Maus et al. Blood. 2014 Apr 24, 123 (17): 2625-35; Porter DL The New England journal of medicine. 2011, 365 (8): 725-733; Jackson HJ, Nat Rev Clin Oncol. 2016, 13 (6): 370-383; and Globerson-Levin et al. Mol Ther. 2014, 22 (5): 1029-1038.

[0352] According to a specific embodiment, the immune cells comprise B cells.

[0353] As used herein, the term "B cells" refers to lymphocytes with a B cell receptor (BCR)+, CD19+ and / or B220+ phenotype. B cells are characterized by their ability to bind specific antigens and elicit a humoral response.

[0354] According to a specific embodiment, the immune cells comprise NK cells.

[0355] As used herein, the term "NK cells" refers to differentiated lymphocytes with a CD16+CD56+ and / or CD57+TCR- phenotype. NKs are characterized by their ability to bind to and kill cells that may not express "self" MHC / HLA antigens by activating specific cytolytic enzymes, to kill tumor cells or other diseased cells that express ligands for NK activating receptors, and to release protein molecules called cytokines that stimulate or inhibit the immune response.

[0356] According to a specific embodiment, the immune cells comprise NKT cells.

[0357] As used herein, the term "NKT cells" refers to a specialized population of T cells that express the semi-invariant αβ T cell receptor but also express various molecular markers typically associated with NK cells, e.g., NK1.1. NKT cells include NK1.1+ and NK1.1-, as well as CD4+, CD4-, CD8+ and CD8- cells. The TCR on NKT cells is unique in that it recognizes glycolipid antigens presented by the MHCI-like molecule CD1d. NKT cells can have either protective or deleterious effects due to their ability to produce cytokines that promote either inflammation or immune tolerance.

[0358] According to a specific embodiment, the immune cells are obtained from a healthy subject.

[0359] According to a specific embodiment, the immune cells are obtained from a subject suffering from a pathology (eg, cancer).

[0360] According to a specific embodiment, activation is performed in the presence of cells expressing HLA-G.

[0361] According to a specific embodiment, the cells expressing HLA-G include diseased cells, such as cancer cells.

[0362] According to specific embodiments, activation is performed in the presence of a stimulatory agent capable of transmitting at least a primary activation signal (e.g., ligation of the T cell receptor (TCR) with a major histocompatibility complex (MHC) / peptide complex on an antigen-presenting cell (APC)) that results in cell proliferation, maturation, cytokine production, phagocytosis, and / or induction of immune cell regulation or effector functions. According to specific embodiments, the stimulatory agent may also transmit a secondary co-stimulatory signal.

[0363] Stimulatory agents can activate immune cells in an antigen-dependent or -independent (ie, polyclonal) manner.

[0364] Methods for determining the amount of stimulatory agent and the ratio of stimulatory agent to immune cells are well within the skill of one of ordinary skill in the art and therefore are not prescribed herein.

[0365] According to a specific embodiment, the immune cells are purified after activation.

[0366] Thus, the present invention also contemplates isolated immune cells obtained according to the methods of the present invention.

[0367] According to specific embodiments, immune cells used and / or obtained according to the present invention can be freshly isolated, stored, and cryopreserved (i.e., frozen) at any stage, e.g., at liquid nitrogen temperatures, for extended periods (e.g., months, years) for future use, and can be cell lines.

[0368] Methods of cryopreservation are generally known by those of skill in the art and are disclosed, for example, in WO2007054160 and WO2001039594, and US20120149108.

[0369] According to a specific embodiment, the cells obtained according to the present invention may be stored in a cell bank or depository or repository.

[0370] Consequently, the present teachings further suggest uses and methods of the isolated immune cells of the present invention, including, but not limited to, as a resource for adoptive immune cell therapy.

[0371] Thus, according to a specific embodiment, the methods of the invention comprise adoptive transfer of immune cells following activation into a subject in need thereof.

[0372] According to a specific embodiment, there is provided immune cells obtained according to the methods of the invention for use in adoptive cell therapy.

[0373] The cells used in accordance with specific embodiments of the present invention may be autologous or non-autologous, it may be syngeneic or non-syngeneic: allogeneic or xenogeneic to the subject, each possibility representing a separate embodiment of the present invention.

[0374] The present teachings also contemplate the use of compositions of the invention (e.g., LILRB (e.g., LILRB2, LILRB1) polypeptides, compositions, fusions or dimers comprising same, polynucleotides encoding same, or host cells expressing same) in methods of treating diseases associated with pathological cells expressing HLA-G.

[0375] Thus, according to one aspect of the present invention, there is provided a method of treating a disease associated with pathological cells expressing HLA-G in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of a LILRB (e.g., LILRB2, LILRB1) polypeptide, composition, fusion polypeptide or dimer disclosed herein, a polynucleotide encoding same, or a host cell expressing same, thereby treating the disease in the subject.

[0376] According to further or alternative aspects of the invention, there is provided a LILRB (e.g., LILRB2, LILRB1) polypeptide, composition, fusion polypeptide or dimer disclosed herein, a polynucleotide encoding same, or a host cell expressing same, for use in treating a disease associated with pathological cells expressing HLA-G in a subject in need of such treatment.

[0377] As used herein, the term "subject" refers to a human or non-human individual having an MHC system, e.g., an HLA system in humans. The subject may be of any sex and any age.

[0378] According to a specific embodiment, the subject is a human subject.

[0379] According to specific embodiments, the subject has been diagnosed with or is at risk of developing a disease (eg, cancer).

[0380] According to a specific embodiment, the diseased cells of the subject exhibit a level of HLA-G above a predefined threshold, as further described below.

[0381] Thus, according to specific embodiments, the methods disclosed herein further comprise determining the level of HLA-G in a biological sample from the subject, e.g., prior to administration of a LILRB (e.g., LILRB2, LILRB1) polypeptide, a composition, fusion or dimer comprising same, a polynucleotide encoding same, or a host cell expressing same, and treating the subject accordingly.

[0382] As used herein, the term "treatment" refers to inhibiting, preventing, or arresting the development of a pathology (disease, disorder, or condition, e.g., cancer) and / or causing the reduction, remission, or regression of a pathology. Those skilled in the art will appreciate that a variety of methods and assays can be used to assess the development of a pathology, and similarly, a variety of methods and assays can be used to assess the reduction, remission, or regression of a pathology.

[0383] According to specific embodiments, treatment can be assessed by a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with the cancerous condition.

[0384] As used herein, the phrase "disease associated with pathological cells expressing HLA-G" refers to a disease in which pathological cells presenting HLA-G drive the development and / or progression of the disease.

[0385] According to a specific embodiment, the diseased cells exhibit a level of HLA-G above a predefined threshold.

[0386] Such a predetermined threshold can be experimentally determined by comparing the expression level in a biological sample from a subject diagnosed with a disease (e.g., cancer) with a biological sample obtained from a healthy subject (e.g., without a disease, e.g., cancer). Alternatively or additionally, such a predetermined threshold can be experimentally determined by comparing the expression level in a diseased cell (e.g., cancer cell) with the expression level in a healthy cell obtained from the same subject. Alternatively, such levels can be obtained from the scientific literature and from databases.

[0387] According to specific embodiments, a level above a predetermined threshold is statistically significant.

[0388] According to specific embodiments, the increase from the pre-determined threshold is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100% or more, greater than about 2-fold, greater than about 3-fold, greater than about 4-fold, greater than about 5-fold, greater than about 6-fold, greater than about 7-fold, greater than about 8-fold, greater than about 9-fold, greater than about 20-fold, greater than about 50-fold, greater than about 100-fold, greater than about 200-fold, greater than about 350, greater than about 500-fold, greater than about 1000-fold, or more, as compared to a control sample measured using the same assay.

[0389] Methods for determining HLA-G expression are known in the art and include, for example, flow cytometry, immunohistochemistry, ELISA, and the like.

[0390] Alternatively or additionally, such predetermined threshold is the detectable level of HLA-G as determined by ELISA, immunohistochemistry or flow cytometry.

[0391] Non-limiting examples of diseases that can be treated with specific embodiments of the present invention include cancer, viral infections (e.g., HCMV, HSV-1, RABV, HCV, IAV and HIV-1), autoimmune and inflammatory diseases, post-allogeneic transplantation, GVHD.

[0392] According to a particular embodiment, the disease is cancer.

[0393] Cancers that may be treated by some embodiments of the present invention may be any solid or non-solid tumor, cancer metastasis, and / or pre-cancer.

[0394] According to a specific embodiment, the cancer is a malignant cancer.

[0395] Examples of cancer include, but are not limited to, carcinoma, blastoma, sarcoma, and lymphoma. More specific examples of such cancers include, but are not limited to, tumors of the gastrointestinal tract (colon carcinoma, rectal carcinoma, colorectal carcinoma, colorectal cancer, colorectal adenoma, hereditary nonpolyposis 1, hereditary nonpolyposis 2, hereditary nonpolyposis 3, hereditary nonpolyposis 6, colorectal cancer, hereditary nonpolyposis 7, small intestine and / or large intestine carcinoma, esophageal cancer, calluses with esophageal cancer, gastric carcinoma, pancreatic carcinoma, pancreatic endocrine tumors. tumor), endometrial carcinoma, dermatofibrosarcoma protuberans, gallbladder carcinoma, biliary tract tumor, prostate cancer, prostate adenocarcinoma, kidney cancer (e.g., Wilms' tumor type 2 or type 1), liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma, hepatocellular carcinoma), bladder cancer, embryonal rhabdomyosarcoma, germ cell tumor, trophoblastic tumor, testicular germ cell tumor, ovary, uterus, immature teratoma of epithelial ovary, sacrococcygeal tumor, choriocarcinoma, placental trophoblastic tumor, epithelial adult tumor (epithelial adult tumor, ovarian carcinoma, serous ovarian carcinoma, ovarian sex cord tumor, cervical carcinoma, uterine cervix carcinoma, small cell and non-small cell lung carcinoma, nasopharyngeal carcinoma, breast carcinoma (e.g., ductal carcinoma, invasive intraductal carcinoma, sporadic, breast cancer, susceptibility to breast cancer, type 4 breast cancer, stage 1 breast cancer, stage 3 breast cancer, ovarian cancer), squamous cell carcinoma (e.g., in the head and neck), neurogenic tumor, astrocytoma, ganglioblastoma, neuroblastoma, lymphoma (e.g., Hodgkin's disease, non-Hodgkin's lymphoma, B cell, Burkitt's, cutaneous T cell, histiocytic, lymphoblastic, T cell, thymic), glioma, adenocarcinoma, adrenal tumor, hereditary adrenal cortical carcinoma, brain malignant tumor (tumor), various other carcinomas (e.g., bronchogenic large cell cell), ductal, Ehrlich-Lettl ascites, epidermoid, large cell, Lewis lung, medullary, mucoepidermoid, oat cell, small cell, spindle cell, spinous cell, transitional cell, undifferentiated, carcinosarcoma, choriocarcinoma, cystadenocarcinoma), ependymoblastoma, epithelioma, erythroleukemia (e.g., Friend, lymphoblastic), fibrosarcoma, giant cell tumor, glial tumor, glioblastoma (e.g., pleomorphic, astrocytoma), gliomaHepatoma, heterohybridoma, heteromyeloma, histiocytoma, hybridoma (e.g., B-cell), adrenal tumor, insulinoma, pancreatic islet tumor, keratosis, leiomyoblastoma, leiomyosarcoma, leukemia (e.g., acute lymphocytic leukemia, acute lymphoblastic leukemia, precursor B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute megakaryoblastic leukemia, monocytic leukemia, acute myelogenous ... myeloid leukemia, acute myeloid leukemia with eosinophilia, B-cell leukemia, basophilic leukemia, chronic myeloid leukemia, chronic leukemia, B-cell leukemia, eosinophilic leukemia, Friend leukemia, granulocytic or myeloid leukemia, hairy cell leukemia, lymphocytic leukemia, megakaryoblastic leukemia, monocytic leukemia, monocytic-macrophagic leukemia, myeloblastic leukemia , myeloid leukemia, myelomonocytic leukemia, plasma cell leukemia, precursor B-cell leukemia, promyelocytic leukemia, subacute leukemia, T-cell leukemia, lymphocytic neoplasms, predisposition to myeloid malignancies, acute nonlymphocytic leukemia), lymphosarcoma, melanoma, breast tumor, mast cell tumor, medulloblastoma, mesothelioma, metastatic tumor, monocytic tumor, multiple myeloma, myelodysplastic syndrome, myeloma, Nephroblastoma, neuroglial tumors, neuroneuronal tumors, neurofibromatosis, neuroblastoma, oligodendroglioma, osteochondroma, myeloma, osteosarcoma (e.g., Ewing's sarcoma), papilloma, transitional cell, pheochromocytoma, pituitary tumor (invasive), plasmacytoma, retinoblastoma, rhabdomyosarcoma, sarcoma (e.g., Ewing's sarcoma, histiocytic cell sarcoma, Jensen's sarcoma, osteosarcoma, reticulum cell sarcoma), schwannoma, subcutaneous tumor, teratocarcinoma (e.g., multipotent), teratomas, testicular tumors, thymoma and trichoepithelioma, gastric cancer, fibrosarcoma, glioblastoma multiforme, multiple glomus tumors, Li-Fraumeni syndrome, liposarcoma, Lynch familial cancer syndrome type II, male germ cell tumors, mast cell leukemia, medullary thyroid, multiple meningiomas, endocrine tumors These include myxosarcoma, paraganglioma, familial achromoblastoma, pilomatrixoma, papillary, familial and sporadic, rhabdoid predisposition syndrome, familial, rhabdoid tumor, soft tissue sarcoma, and Turcot's syndrome with glioblastoma.

[0396] According to a specific embodiment, the cancer is a pre-malignant cancer.

[0397] Precancers are well characterized and known in the art (see, e.g., Berman JJ. and Henson DE., 2003. Classifying the pre-cancers: a metadata approach. BMC Med Inform Decis Mak. 3:8). Examples of precancers include, but are not limited to, acquired small precancers, acquired large lesions with nuclear atypia, precursor lesions associated with hereditary hyperplastic syndromes that progress to cancer, and acquired diffuse hyperplasia and diffuse metaplasia. Non-limiting examples of small precancers include HGSIL (high grade squamous intraepithelial lesions of the cervix), AIN (anal intraepithelial neoplasia), vocal cord dysplasia, aberrant crypts (of the colon), and PIN (prostatic intraepithelial neoplasia).

[0398] Non-limiting examples of acquired large lesions with nuclear atypia include tubular adenoma, AILD (angioimmunoblastic lymphadenopathy with dysproteinemia), atypical meningioma, gastric polyp, large plaque psoriasis, spinal dysplasia, intraepithelial carcinoma of papillary transitional cells, refractory anemia with hyperblastic cells, and Schneider's papilloma. Non-limiting examples of precursor lesions associated with hereditary hyperplastic syndromes that progress to cancer include atypical nevus syndrome, C-cell adenomatosis, and MEA. Non-limiting examples of acquired diffuse hyperplasia and diffuse metaplasia include Paget's disease of bone and ulcerative colitis.

[0399] According to specific embodiments, the cancer is selected from the group consisting of pancreatic cancer, breast cancer, skin cancer, colorectal cancer, gastric cancer and ovarian cancer.

[0400] According to specific embodiments, the compositions disclosed herein (e.g., LILRB (e.g., LILRB2, LILRB2) polypeptides, compositions comprising same, fusions or dimers, polynucleotides encoding same and / or host cells expressing same) can be administered to a subject in combination with other established or experimental therapeutic regimens for treating disease, including, but not limited to, anesthetics, chemotherapeutic agents, radiation therapy agents, cytotoxic therapy (conditioning), hormonal therapy, antibodies and other therapeutic regimens known in the art (e.g., surgery).

[0401] According to specific embodiments, the therapeutic agent administered in combination with the compositions of some embodiments of the present invention comprises an antibody.

[0402] According to specific embodiments, the compositions disclosed herein (e.g., LILRB (e.g., LILRB2, LILRB1) polypeptides, compositions comprising same, fusions or dimers, polynucleotides encoding same and / or host cells expressing same) can be administered to a subject in combination with adoptive cell transfer, including but not limited to, transplantation of bone marrow cells, hematopoietic stem cells, PBMCs, umbilical cord blood stem cells and / or induced pluripotent stem cells.

[0403] According to specific embodiments, the therapeutic agent administered in combination with the compositions of some embodiments of the present invention comprises an anti-cancer agent.

[0404] According to specific embodiments, therapeutic agents administered in combination with the compositions of some embodiments of the present invention include anti-infective agents (eg, antibiotics and antiviral agents).

[0405] According to specific embodiments, the combination therapy has an additive effect.

[0406] According to specific embodiments, the combination therapy has a synergistic effect.

[0407] According to another aspect of the invention, there are provided packaging materials for packaging therapeutic agents for treating a disease, as well as articles of manufacture comprising a LILRB (e.g., LILRB2, LILRB1) polypeptide, a composition comprising same, a fusion or dimer, a polynucleotide encoding same, and / or a host cell expressing same.

[0408] According to a specific embodiment, the article of manufacture is identified for the treatment of a disease associated with pathological cells expressing HLA-G, such as cancer.

[0409] According to specific embodiments, a therapeutic agent for treating a disease and a LILRB (e.g., LILRB2, LILRB1) polypeptide, a composition comprising same, a fusion or dimer, a polynucleotide encoding same and / or a host cell expressing same are packaged in separate containers.

[0410] According to specific embodiments, a therapeutic agent for treating a disease and a LILRB (e.g., LILRB2, LILRB1) polypeptide, composition comprising same, fusion or dimer, polynucleotide encoding same and / or host cell expressing same are packaged in a co-formulation.

[0411] As used herein, the terms "amino acid sequence", "protein", "peptide", "polypeptide" and "proteinaceous moiety" are used interchangeably herein and include natural peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically synthetically synthesized peptides), as well as peptoids and semipeptoids, which are peptide analogs that may have modifications that make the peptide more stable while in the body or more permeable into cells. Such modifications include, but are not limited to, N-terminal modifications, C-terminal modifications, peptide bond modifications, backbone modifications, and residue modifications. Methods for preparing peptidomimetic compounds are well known in the art and are set forth, for example, in Quantitative Drug Design, CA Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this regard are provided below.

[0412] The peptide bond (-CO-NH-) in a peptide can be, for example, an N-methylated amide bond -N(CH3)-CO-), an ester bond (-C(=O)-O-), a ketomethylene bond (-CO-CH2-), a sulfinylmethylene bond (-S(=O)-CH2-), an α-aza bond (-NH-N(R)-CO-) (where R is any alkyl (e.g., methyl)), an amine bond (-CH2-NH-), a sulfide bond (-CH2-S- ), ethylene bond (-CH2-CH2-), hydroxyethylene bond (-CH(OH)-CH2-), thioamide bond (-CS-NH-), olefinic double bond (-CH=CH-), fluorinated olefinic double bond (-CF=CH-), retroamide bond (-NH-CO-), peptide derivatives (-N(R)-CH2-CO-), where R is a "normal" side chain naturally occurring on a carbon atom.

[0413] These modifications can occur at any of the bonds along the peptide chain or even at several (2-3) bonds simultaneously.

[0414] The natural aromatic amino acids, Trp, Tyr and Phe, can be replaced by unnatural aromatic amino acids, such as 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), naphthylalanine, ring-methylated derivatives of Phe, halogenated derivatives of Phe or O-methyl-Tyr.

[0415] Peptides of some embodiments of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (eg, fatty acids, complex carbohydrates, etc.).

[0416] The term "amino acid" or "amino acids" is understood to include the twenty naturally occurring amino acids, those that are frequently post-translationally modified in vivo, including, for example, hydroxyproline, phosphoserine, and phosphothreonine, as well as other unusual amino acids, including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine, and ornithine. Additionally, the term "amino acid" includes both D- and L-amino acids.

[0417] Tables 4 and 5 below list naturally occurring amino acids (Table 4), and unconventional or modified amino acids (eg, synthetic, Table 5) that can be used in some embodiments of the invention.

[0418] [Table 1]

[0419] [Table 2] TIFF2025500456000003.tif196124

[0420] The polypeptides of some embodiments of the present invention are preferably utilized in linear form, although it will be recognized that cyclic forms of the peptides can also be utilized where cyclization does not significantly interfere with the peptide characteristics.

[0421] Because the present polypeptides are preferably utilized in therapeutic applications which require the polypeptide to be in a soluble form, the polypeptides of some embodiments of the invention contain one or more unnatural or natural polar amino acids, including, but not limited to, serine and threonine, which may increase peptide solubility due to hydroxyl-containing side chains.

[0422] The amino acids of the polypeptides of the present invention may be substituted in a conservative or non-conservative manner.

[0423] The term "conservative substitution" as used herein refers to the replacement of an amino acid present in a natural sequence in a peptide with a naturally occurring or non-naturally occurring amino acid or peptidomimetic having similar steric properties. If the side chain of the natural amino acid to be replaced is either polar or hydrophobic, the conservative substitution should use a naturally occurring amino acid, a non-naturally occurring amino acid, or a peptidomimetic moiety that is also polar or hydrophobic (in addition to having the same steric properties as the side chain of the amino acid to be replaced).

[0424] Since naturally occurring amino acids are typically classified according to their properties, conservative substitutions with naturally occurring amino acids can be readily determined in view of the fact that, according to the present invention, replacement of a charged amino acid with a sterically similar uncharged amino acid is considered a conservative substitution.

[0425] It is also possible to use amino acid analogues (synthetic amino acids) well known in the art to provide conservative substitutions with non-naturally occurring amino acids. Peptide mimetics of naturally occurring amino acids are well documented in the literature known to practitioners.

[0426] When making a conservative substitution, the substituting amino acid should have the same or a similar functional group in its side chain as the original amino acid.

[0427] Conservative substitution tables providing functionally similar amino acids are well known in the art. Guidance on which amino acid charges are likely to be phenotypically silent can also be found in Bowie et al., 1990, Science 247:1306 1310. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles. Exemplary conservative substitutions include, but are not limited to, 1) alanine (A), glycine (G), 2) aspartic acid (D), glutamic acid (E), 3) asparagine (N), glutamine (Q), 4) arginine (R), lysine (K), 5) isoleucine (I), leucine (L), methionine (M), valine (V), 6) phenylalanine (F), tyrosine (Y), tryptophan (W), 7) serine (S), threonine (T), and 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, Proteins (1984)). Amino acids can be substituted based on the properties associated with their side chains, e.g., amino acids with polar side chains, e.g., serine (S) and threonine (T), amino acids based on the charge of the side chain, e.g., arginine (R) and histidine (H), and amino acids with hydrophobic side chains, e.g., valine (V) and leucine (L). As indicated, the changes are typically minor, e.g., conservative amino acid substitutions that do not significantly affect the folding or activity of the protein.

[0428] The phrase "non-conservative substitution" as used herein refers to the replacement of an amino acid present in a parent sequence with another naturally occurring or non-naturally occurring amino acid having different electrochemical and / or steric properties. Thus, the side chain of the substituted amino acid may be significantly larger (or smaller) than the side chain of the natural amino acid being substituted and / or may have a functional group with significantly different electronic properties than the amino acid being substituted. Examples of this type of non-conservative substitution include the substitution of phenylalanine or cyclohexylmethylglycine for alanine, the substitution of isoleucine for glycine, or the substitution of -NH-CH[(-CH2)5-COOH]-CO- for aspartic acid. Those non-conservative substitutions that fall within the scope of the present invention still constitute peptides with antimicrobial properties.

[0429] The N- and C-termini of the peptides and compositions of the invention can be protected by functional groups. Suitable functional groups are described in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, Chapters 5 and 7, 1991, the teachings of which are incorporated herein by reference. Preferred protecting groups are those that facilitate the transport of the compound attached thereto into cells, for example by reducing the hydrophilicity and increasing the lipophilicity of the compound.

[0430] According to specific embodiments, one or more of the amino acids can be modified (conceptually considered as "chemically modified"), for example by the addition of a functional group. For example, the side chain amino acid residues present in the native sequence can be optionally modified, but alternatively, other parts of the protein can be optionally modified in addition to or instead of the side chain amino acid residues, as described below. The modification can be optionally performed during the synthesis of the molecule, for example by adding a chemically modified amino acid, if a chemical synthesis process is followed. However, chemical modification of an amino acid is also possible if it is already present in the molecule ("in situ" modification). Modifications to peptides or proteins can be introduced, for example, by gene synthesis, site-directed (e.g., PCR-based) or random mutagenesis by exonuclease deletion (e.g., EMS), by chemical modification, or by fusion of polynucleotide sequences encoding heterologous domains or binding proteins.

[0431] As used herein, the term "chemical modification" refers to a peptide, in which at least one of its amino acid residues has been modified by natural processes, such as processing or other post-translational modifications, or by any of the chemical modification techniques known in the art.Non-limiting exemplary types of modification include carboxymethylation, acetylation, acylation, phosphorylation, glycosylation, amidation, ADP-ribosylation, fatty acid acylation, farnesyl group, isofarnesyl group, carbohydrate group, fatty acid group, addition of linker for conjugation, functionalization, GPI anchor formation, covalent attachment of lipid or lipid derivative, methylation, myristylation, pegylation, prenylation, phosphorylation, ubiquitination, or any similar process and known protecting / blocking group.An ether bond can be optionally used to link the hydroxyl of serine or threonine to the hydroxyl of sugar. Amide bonds can optionally be used to link the carboxyl group of glutamate or aspartate to the amino group of the sugar (Garg and Jeanloz, Advances in Carbohydrate Chemistry and Biochemistry, Vol.43, Academic Press(1985), Kunz, Ang. Chem. Int. Ed. English 26:294-308 (1987)). Acetal and ketal bonds can also be optionally formed between amino acids and carbohydrates. For example, fatty acid acyl derivatives can be optionally generated by acylation of free amino groups (e.g., lysine) (Toth et al., Peptides: Chemistry, Structure and Biology, Rivier and Marshal, eds., ESCOM Publ., Leiden, 1078-1079 (1990)).

[0432] According to specific embodiments, the modifications include the addition of cycloalkane moieties to the peptides, as described in PCT application WO 2006 / 050262, which is incorporated by reference as if fully set forth herein. These moieties are designed for use with biomolecules and can optionally be used to confer various properties to proteins.

[0433] In addition, any point on the peptide can be optionally modified. For example, pegylation of glycosylated moieties on the protein can be optionally performed as described in PCT application WO 2006 / 050247, which is incorporated by reference as if fully set forth herein, and as further described above. One or more polyethylene glycol (PEG) groups can be optionally added to O-linked and / or N-linked glycosylation. The PEG groups can be optionally branched or linear. Optionally, any type of water-soluble polymer can be attached to the glycosylation site on the protein via a glycosyl linker.

[0434] According to specific embodiments, the peptide is modified to have an altered glycosylation pattern (i.e., altered from the original or native glycosylation pattern). As used herein, "altered" means having one or more carbohydrate moieties deleted and / or having at least one glycosylation site added to the original protein.

[0435] Glycosylation of proteins is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars, N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0436] Addition of glycosylation sites to a peptide is conveniently accomplished by altering the amino acid sequence of the peptide to contain one or more of the above tripeptide sequences (for N-linked glycosylation sites). Alterations can also be made by the addition of, or substitution by, one or more serine or threonine residues in the sequence of the original peptide (for O-linked glycosylation sites). The amino acid sequence of the peptide can also be altered by the introduction of changes at the DNA level.

[0437] Another means of increasing the number of carbohydrate moieties on a peptide is by chemical or enzymatic coupling of glycosides to the amino acid residues of the peptide. Depending on the coupling method used, sugars can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as those of cysteine, (d) free hydroxyl groups, such as those of serine, threonine, or hydroxyproline, (e) aromatic residues, such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described, for example, in WO 87 / 05330 and in Aplin and Wriston, CRC Crit. Rev. Biochem., 22:259-306 (1981).

[0438] Removal of any carbohydrate moieties present on a peptide can be accomplished chemically, enzymatically, or by the introduction of changes at the DNA level. Chemical deglycosylation requires exposure of the peptide to trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine) while leaving the amino acid sequence intact.

[0439] Chemical deglycosylation is described by Hakimuddin et al., Arch. Biochem. Biophys., 259:52 (1987), and Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on peptides can be accomplished by the use of a variety of endo- and exoglycosidases, as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).

[0440] Compositions of some embodiments of the present invention (e.g., LILRB (e.g., LILRB2, LILRB1) polypeptides, compositions comprising same, fusions or dimers) can be synthesized and purified by any technique known to those skilled in the art of peptide synthesis, including, but not limited to, solid phase and recombinant techniques.

[0441] According to a particular embodiment, the preparation of the polypeptide comprises solid phase peptide synthesis.

[0442] For solid phase peptide synthesis, overviews of many techniques can be found in JM Stewart and JD Young, Solid Phase Peptide Synthesis, WH Freeman Co. (San Francisco), 1963, and J. Meienhofer, Hormonal Proteins and Peptides, vol.2, p.46, Academic Press (New York), 1973. For classical solution synthesis, see G. Schroder and K. Lupke, The Peptides, vol.1, Academic Press (New York), 1965.

[0443] In general, these methods involve the sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain. Usually, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then be attached to an inert solid support or utilized in solution by addition to the next amino acid in the sequence that has a suitably protected complementary (amino or carboxyl) group under conditions suitable for the formation of an amide bond. The protecting group is then removed from this newly added amino acid residue, and then the next (suitably protected) amino acid is added, and so on. After all the desired amino acids have been coupled in the proper order, any remaining protecting groups (and any solid support) are removed, either sequentially or simultaneously, to obtain the final peptide compound. Simple modifications of this general procedure make it possible to add more than one amino acid at a time to the growing chain, for example, by coupling a protected tripeptide with a suitably protected dipeptide (under conditions that do not racemize the chiral centers) to form a pentapeptide, etc. after deprotection. Further description of peptide synthesis is disclosed in U.S. Pat. No. 6,472,505.

[0444] Large scale peptide synthesis is described by Andersson Biopolymers 2000, 55(3):227-50.

[0445] According to specific embodiments, a polypeptide (eg, a LILRB (eg, LILRB2, LILRB1) polypeptide, composition comprising same, fusion or dimer) is synthesized using an in vitro expression system.

[0446] Thus, any of the polypeptides described herein can be encoded from polynucleotides which may be used per se or may be used in the recombinant production of the polypeptides disclosed herein.

[0447] A "recombinant" polypeptide refers to a polypeptide produced by recombinant DNA techniques, ie, produced from cells transformed with an exogenous DNA construct encoding the desired polypeptide.

[0448] Thus, according to another aspect of the present invention, there is provided a polynucleotide encoding a LILRB (eg, LILRB2, LILRB1) polypeptide, fusion polypeptide or dimer as disclosed herein.

[0449] Non-limiting examples of polynucleotide sequences that can be used in specific embodiments of the present invention are set forth in Table 3 below.

[0450] Non-limiting examples of polynucleotides encoding LILRB2 polypeptides of some embodiments of the invention are provided in SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24.

[0451] Non-limiting examples of polynucleotides encoding LILRB2-Fc fusion polypeptides of some embodiments of the invention are provided in SEQ ID NOs: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 97, and 106.

[0452] Non-limiting examples of polynucleotides encoding SIRPα-Fc fusion polypeptides of some embodiments of the invention are provided in SEQ ID NOs:36, 91, 95 and 99.

[0453] As used herein, the term "polynucleotide" refers to a single- or double-stranded nucleic acid sequence isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence, and / or a composite polynucleotide sequence (e.g., a combination of the above).

[0454] According to specific embodiments, any of the polynucleotides and nucleic acid sequences disclosed herein may contain conservative nucleic acid substitutions. A conservatively modified polynucleotide refers to a nucleic acid that encodes the same or essentially the same amino acid sequence, or, if the nucleic acid does not encode an amino acid sequence, to an essentially identical or related (e.g., naturally contiguous) sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, in all positions where alanine is specified by a codon, this codon can be changed to another of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations", which are a type of conservatively modified polynucleotide. According to specific embodiments, any of the polynucleotides and nucleic acid sequences described herein that encode a polypeptide herein also describe silent variations of that nucleic acid. Those of skill in the art will recognize that, in certain circumstances, each codon in a nucleic acid (except AUG, which is normally the only codon for methionine, and TGG, which is normally the only codon for tryptophan) can be modified to obtain a functionally identical molecule. Accordingly, silent variations of a polynucleotide which encodes a polypeptide are implied in the sequences described for the expression product.

[0455] To express an exogenous polypeptide in a mammalian cell, the polynucleotide sequence encoding the polypeptide is preferably ligated into a nucleic acid construct suitable for mammalian cell expression, such a nucleic acid construct comprising a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.

[0456] Thus, according to one aspect of the present invention there is provided a nucleic acid construct comprising a polynucleotide encoding a LILRB2 polypeptide, fusion polypeptide or dimer and regulatory elements for directing expression of the polynucleotide in a host cell.

[0457] According to a specific embodiment, the regulatory element (promoter) is a heterologous regulatory element.

[0458] The nucleic acid constructs (also referred to herein as "expression vectors") of some embodiments of the present invention contain additional sequences that make the vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). In addition, a typical cloning vector may also contain transcription and translation initiation sequences, transcription and translation terminators, and polyadenylation signals. By way of example, such constructs typically include a 5'LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis, and a 3'LTR or a portion thereof.

[0459] The nucleic acid construct of some embodiments of the invention typically comprises a signal sequence for secretion of the peptide from a host cell into which the construct is introduced. Preferably, the signal sequence for this purpose is a mammalian signal sequence or a signal sequence of a polypeptide variant of some embodiments of the invention. According to specific embodiments, the nucleic acid construct comprises a signal peptide, for example as provided in SEQ ID NOs: 25-26.

[0460] Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and the upstream promoter element. The TATA box is located 25-30 base pairs upstream of the transcription start site and is thought to be involved in directing RNA polymerase to initiate RNA synthesis. The other upstream promoter element determines the rate at which transcription is initiated.

[0461] Preferably, the promoters utilized by the nucleic acid constructs of some embodiments of the present invention are active in the specific cell population transformed. Examples of cell type-specific and / or tissue-specific promoters include liver-specific promoters such as albumin promoters [Pinkert et al., (1987) Genes Dev. 1:268-277], lymphoid-specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275], in particular promoters of T cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulin promoters [Banerji et al. (1983) Cell 33729-740], neuron-specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], pancreatic-specific promoters [Edlunch et al. (1985) Science 230:912-916] or mammary gland-specific promoters, such as the whey promoter (U.S. Pat. No. 4,873,316 and European Patent Publication No. 264,166).

[0462] Enhancer elements can stimulate transcription up to 1,000-fold from a combined homologous or heterologous promoter. Enhancers are active when placed downstream or upstream from the transcription start site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer / promoter combinations suitable for some embodiments of the present invention include those derived from polyoma virus, human or mouse cytomegalovirus (CMV), various retroviruses such as murine leukemia virus, mouse or Rous sarcoma virus, and long terminal repeat sequences from HIV. See Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, NY 1983, which is incorporated herein by reference.

[0463] In constructing an expression vector, a promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural environment, although, as is known in the art, some variation in this distance can be accommodated without loss of promoter function.

[0464] Polyadenylation sequences can also be added to expression vectors to increase the efficiency of mRNA translation. Accurate and efficient polyadenylation requires two distinct sequence elements: a GU- or U-rich sequence located downstream from the polyadenylation site and a highly conserved six-nucleotide sequence, AAUAAA, located 11-30 nucleotides upstream. Suitable termination and polyadenylation signals for some embodiments of the invention include those derived from SV40.

[0465] In addition to the elements already described, the expression vectors of some embodiments of the present invention may contain other specialized elements, typically intended to increase the level of expression of the cloned nucleic acid or to facilitate the identification of cells carrying the recombinant DNA. For example, many animal viruses contain DNA sequences that promote extrachromosomal replication of the viral genome in permissive cell types. Plasmids carrying these viral replicons are replicated episomally as long as the appropriate factors are provided on the plasmid or by the genome of the host cell and the genes carried.

[0466] Vector may or may not contain eukaryotic replicon. If eukaryotic replicon exists, vector can be amplified in eukaryotic cell by using suitable selectable marker. If vector does not contain eukaryotic replicon, episomal amplification is not possible. Instead, recombinant DNA is integrated into the genome of engineered cell, where promoter directs the expression of desired nucleic acid.

[0467] The expression vectors of some embodiments of the invention may further comprise additional polynucleotide sequences, e.g., allowing translation of several proteins from a single mRNA, such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.

[0468] Thus, according to a specific embodiment, both monomers comprised in the heterodimer are expressed from a single construct.

[0469] According to other specific embodiments, each of the monomers comprised in the heterodimer is expressed from a different construct.

[0470] It will be appreciated that individual elements contained within an expression vector may be arranged in a variety of configurations. For example, enhancer elements, promoters, and the like, and even polynucleotide sequence(s) encoding polypeptides arranged in a "head-to-tail" configuration may exist in a complementary configuration, either as reverse complements or as antiparallel strands. While such configurational variations are more likely to occur with non-coding elements of an expression vector, alternative configurations of coding sequences within an expression vector are also envisioned.

[0471] Examples of mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+ / -), pcDNA3.4, pGL3, pZeoSV2(+ / -), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81 available from Invitrogen, pCI available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV available from Strategene, pTRES available from Clontech, and derivatives thereof.

[0472] Expression vectors containing regulatory elements from eukaryotic viruses, such as retroviruses, can also be used. SV40 vectors include pSVT7 and pMT2. Vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein-Barr virus include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector that allows expression of a protein under the direction of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown to be effective for expression in eukaryotic cells.

[0473] As mentioned above, viruses are highly specialized infectious agents that have evolved to often evade the defense mechanisms of the host. Typically, viruses infect and propagate in specific cell types. The targeting specificity of viral vectors utilizes their natural specificity to specifically target a given cell type, thereby introducing recombinant genes into the infected cells. Thus, the type of vector used by some embodiments of the present invention depends on the cell type to be transformed. The ability to select a suitable vector according to the cell type to be transformed is well within the skill of the artisan, and therefore a general description of the selection considerations is not provided herein. For example, bone marrow cells can be targeted using human T-cell leukemia virus type I (HTLV-I), and kidney cells can be targeted using the heterologous promoter present in the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV), as described in Liang CY et al., 2004 (Arch Virol. 149:51-60).

[0474] Recombinant viral vectors are useful for in vivo expression of polypeptides because they offer advantages such as horizontal infection and targeting specificity. Horizontal infection, for example, is inherent in the life cycle of retroviruses, and is the process by which a single infected cell produces many progeny virions that bud and infect neighboring cells. This results in rapid infection of a wide area, most of which were not initially infected by the original viral particle. This is in contrast to vertical infection, where the infectious agent is only transmitted via daughter progeny. It is also possible to produce viral vectors that cannot be transmitted horizontally. This feature can be useful when the desired purpose is to introduce a defined gene into only a limited number of targeted cells.

[0475] A variety of methods can be used to introduce the expression vectors of some embodiments of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989); Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich.(1995); Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995); Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et al. [Biotechniques 4(6): 504-512, 1986] and include, for example, stable or transient transfection, polyethylenimine lipofection, electroporation and infection with recombinant viral vectors. In addition, see US Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0476] Introduction of nucleic acids by viral infection offers several advantages over other methods, such as lipofection and electroporation, since higher transfection efficiencies can be obtained due to the infectious nature of viruses.

[0477] Currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, herpes simplex virus type I, or adeno-associated virus (AAV), and lipid-based systems. Useful lipids for lipid-mediated gene transfer are, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al., Cancer Investigation, 14 (1):54-65 (1996)]. The most preferred constructs for use in gene therapy are viruses, most preferably adenovirus, AAV, lentivirus, or retrovirus. Viral constructs, such as retroviral constructs, contain at least one transcription promoter / enhancer or locus-defining element(s), or other elements that control gene expression by other means, such as alternative messenger splicing, nuclear RNA transport, or post-translational modification. Such vector constructs also include a packaging signal, a long terminal repeat (LTR) or a portion thereof, and positive and negative strand primer binding sites appropriate for the virus used, unless already present in the viral construct. In addition, such constructs typically include a signal sequence for secretion of the peptide from the host cell into which the construct is introduced. Preferably, the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of a polypeptide variant of some embodiments of the present invention. Optionally, the construct may also include a signal directing polyadenylation, as well as one or more restriction sites and a translation termination sequence. By way of example, such constructs typically include a 5'LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis, and a 3'LTR or a portion thereof. Other vectors, non-viral, such as cationic lipids, polylysine, and dendrimers, can be used.

[0478] As mentioned above, the expression constructs of some embodiments of the invention, apart from containing elements necessary for the transcription and translation of the inserted coding sequence, may also contain sequences engineered to improve the stability, production, purification, yield or toxicity of the expressed polypeptide. For example, expression of a fusion protein or a cleavable fusion protein comprising a polypeptide of some embodiments of the invention and a heterologous protein can be engineered. Such a fusion protein can be designed such that the fusion protein can be easily isolated by affinity chromatography, e.g., by immobilization on a column specific for the heterologous protein. If a cleavage site is engineered between the polypeptide of some embodiments of the invention and the heterologous protein, the polypeptide can be released from the chromatography column by treatment with an appropriate enzyme or agent that destroys the cleavage site [see, e.g., Booth et al. (1988) Immunol. Lett. 19:65-70, and Gardella et al., (1990) J. Biol. Chem. 265:15854-15859].

[0479] The present invention also contemplates cells comprising the compositions described herein and methods of making and using the same.

[0480] Thus, according to one aspect of the present invention, there is provided a host cell comprising a LILRB (e.g., LILRB2, LILRB1) polypeptide, a fusion polypeptide or dimer comprising same, or a polynucleotide encoding same.

[0481] As mentioned above, various prokaryotic or eukaryotic cells can be used as host expression systems to express the polypeptides of some embodiments of the present invention. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the coding sequence, yeast transformed with recombinant yeast expression vectors containing the coding sequence, and plant cell lines infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmids, containing the coding sequence. Mammalian expression systems can also be used to express the polypeptides of some embodiments of the present invention.

[0482] Examples of bacterial constructs include the pET series of E. coli expression vectors [Studier et al. (1990) Methods in Enzymol. 185:60-89].

[0483] Examples of eukaryotic cells that can be used with the teachings of the present invention include, but are not limited to, a mammalian cell, a fungal cell, a yeast cell, an insect cell, an algae cell, or a plant cell.

[0484] In yeast, a number of vectors containing constitutive or inducible promoters as disclosed in U.S. Patent Application No. 5,932,447 may be used, or vectors which facilitate integration of foreign DNA sequences into the yeast chromosome may be used.

[0485] When a plant expression vector is used, a number of promoters can drive the expression of the coding sequence. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al. (1984) Nature 310:511-514], or the coat protein promoter of TMV [Takamatsu et al. (1987) EMBO J. 6:307-311] can be used. Alternatively, plant promoters such as the small subunit of RUBISCO [Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843] or heat shock promoters such as soybean hsp17.5-E or hsp17.3-B [Gurley et al. (1986) Mol. Cell. Biol. 6:559-565] can be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to those of skill in the art, see, e.g., Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.

[0486] Other expression systems, such as insect and mammalian host cell systems, that are well known in the art can also be used in accordance with some embodiments of the present invention.

[0487] According to a particular embodiment, the cell is a mammalian cell.

[0488] According to a specific embodiment, the cell is a human cell.

[0489] According to a specific embodiment, the cells are not derived from a human embryo.

[0490] According to a specific embodiment, the cell is an isolated cell.

[0491] According to a specific embodiment, the cell is a cell line.

[0492] According to another specific embodiment, the cells are primary cells.

[0493] The cells may be derived from any suitable tissue, including, but not limited to, blood, muscle, nerve, brain, heart, lung, liver, pancreas, spleen, thymus, esophagus, stomach, intestine, kidney, testes, ovaries, hair, skin, bone, breast, uterus, bladder, spinal cord, or various types of bodily fluids. The cells may be derived from any developmental stage, including embryonic, fetal, and adult, and from any developmental origin, i.e., ectodermal, mesodermal, and endodermal origin.

[0494] Non-limiting examples of mammalian cells include SV40 transformed monkey kidney CV1 line (COS, e.g., COS-7, ATCC CRL1651), human embryonic kidney line (HEK293 or HEK293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 1977), baby hamster kidney cells (BHK, ATCC CCL10), mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 1980), monkey kidney cells (CV1 ATCC CCL70), African green monkey kidney cells (Vero-76, ATCC CRL-1587), human cervical carcinoma cells (HeLa, ATCC CCL2), NIH3T3, Jurkat, canine kidney cells (MDCK, ATCC CCL34), buffalo rat liver cells (BRL 3A, ATCC CRL1442), human lung cells (W138, ATCC CCL75), human liver cells (Hep G2, HB8065), mouse mammary tumor (MMT060562, ATCC CCL51), TRI cells (Mather et al., Annals NY Acad. Sci., 383:44-68 1982), MRC5 cells, FS4 cells, as well as human hepatoma lines (Hep G2), PER.C6, K562, and Chinese hamster ovary cells (CHO).

[0495] According to some embodiments of the invention, the mammalian cell is selected from the group consisting of Chinese Hamster Ovary (CHO), HEK293, PER.C6, HT1080, NS0, Sp2 / 0, BHK, Namalwa, COS, HeLa and Vero cells.

[0496] According to some embodiments of the invention, the host cell comprises a Chinese Hamster Ovary (CHO), PER.C6 or 293 (eg, Expi 293F) cell.

[0497] According to another aspect of the present invention there is provided a method of producing a polypeptide comprising introducing a polynucleotide or nucleic acid construct as described herein into a host cell or culturing a cell which expresses a polynucleotide or nucleic acid construct as described herein.

[0498] According to a specific embodiment, the method is an in vitro or ex vivo method.

[0499] According to a specific embodiment, production involves culturing at 32-37° C. and 5-10% CO 2 for 5-13 days.

[0500] Non-limiting examples of production conditions that can be used in specific embodiments of the present invention are disclosed in the Examples section below.

[0501] Thus, for example, an expression vector encoding a polypeptide is introduced into mammalian cells, such as expi 293F, expi CHO cells, CHO-K1, CHO-S, CHO-DUC or CHO-DG44. The transfected cells are then cultured in a cell-specific culture medium at 32-37° C. and 5-10% CO2, and after at least 5 days of culture, the protein is collected from the supernatant and purified.

[0502] According to specific embodiments, the culture is operated in batch, split-batch, fed-batch, or perfusion mode.

[0503] According to a specific embodiment, the culture is operated under fed-batch conditions.

[0504] According to a specific embodiment, the culture is carried out at 36.5°C.

[0505] According to a specific embodiment, the culture is performed at 36.5° C. with a temperature shift to 32° C. This temperature shift can be performed to slow down the cell metabolism before reaching stationary phase.

[0506] According to specific embodiments, the methods include isolating a LILRB (eg, LILRB2, LILRB1) polypeptide, fusion polypeptide, or dimer.

[0507] According to a specific embodiment, the recovery of the recombinant polypeptide is carried out after a suitable period of culture.

[0508] According to specific embodiments, recovery of the recombinant polypeptide refers to collecting the entire culture medium containing the heterodimer and does not necessarily imply additional separation or purification steps. According to specific embodiments, the polypeptides of some embodiments of the present invention can be purified using a variety of standard protein purification techniques, including but not limited to affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, mixed mode chromatography, metal affinity chromatography, lectin affinity chromatography, chromatofocusing, and differential solubilization.

[0509] According to specific embodiments, after production and purification, the therapeutic efficacy of the polypeptide can be assayed either in vivo or in vitro, such methods are known in the art and include, for example, binding, cell viability, survival of transgenic mice, and expression of activation markers.

[0510] The compositions of some embodiments of the present invention (e.g., LILRB (e.g., LILRB2, LILRB1) polypeptides described herein, compositions comprising same, fusion proteins or dimers, polynucleotides encoding same and / or cells) can be administered to an organism on their own or in a pharmaceutical composition in which the composition is mixed with a suitable carrier or excipient.

[0511] Accordingly, the invention, in some embodiments, features a pharmaceutical composition including a therapeutically effective amount of a composition disclosed herein.

[0512] As used herein, the term "active ingredient" refers to a composition responsible for a biological effect (e.g., a LILRB (e.g., LILRB2, LILRB1) polypeptide described herein, a composition comprising same, a fusion protein or dimer, a polynucleotide encoding same and / or a cell).

[0513] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0514] Hereinafter, the terms "physiologically acceptable carrier" and "pharmaceutical acceptable carrier", which may be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the compound being administered. These terms include adjuvants.

[0515] As used herein, "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). The active compound may include one or more pharmaceutically acceptable salts depending on the route of administration. "Pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, for example, Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids, such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like, and non-toxic organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, and the like, and non-toxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0516] Pharmaceutical compositions according to at least some embodiments of the present invention may also include pharma- ceutically acceptable antioxidants, examples of which include (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc., (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc., and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc. Pharmaceutical compositions according to at least some embodiments of the invention may also include additives, such as detergents and solubilizing agents (e.g., Tween 20 (Polysorbate-20), Tween 80 (Polysorbate-80)) and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol).

[0517] Examples of suitable aqueous and non-aqueous carriers that can be employed in pharmaceutical compositions according to at least some embodiments of the present invention include water, various buffer contents (e.g., Tris-HCl, acetate, phosphate), buffered saline solutions of various pH and ionic strengths, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.

[0518] Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0519] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured both by the above-mentioned sterilization procedures and by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc., in the composition. In addition, prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0520] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.The use of such media and agents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical composition according to at least some embodiments of the present invention is contemplated.Auxiliary active compounds can also be incorporated into the composition.

[0521] Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentration. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating, for example, lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols, for example, mannitol, sorbitol, or sodium chloride, in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. Sterile injectable solutions can be prepared by incorporating the required amount of active compound in an appropriate solvent, optionally with one or a combination of ingredients as enumerated above, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0522] Sterile injectable solution can be prepared by incorporating the required amount of active compound in a suitable solvent with one or a combination of the above-listed ingredients as required, followed by sterilization precision filtration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and the other ingredients listed above as required.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying (lyophilization), which produces a powder of active ingredient and any additional desired ingredient from its solution that has been previously sterile-filtered.

[0523] The amount of active ingredient that can be combined with carrier materials to produce a single dosage form varies depending on the subject being treated and the specific mode of administration.The amount of active ingredient that can be combined with carrier materials to produce a single dosage form is generally the amount of the composition that produces a therapeutic effect.Generally, this amount ranges from about 0.01 percent to about 99 percent of active ingredient, preferably about 0.1 percent to about 70 percent, most preferably about 1 percent to about 30 percent of active ingredient in combination with a pharma- ceutically acceptable carrier, out of 100 percent.

[0524] Dosage regimens are adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to a physically distinct unit suitable as a unitary dosage for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect with the required pharmaceutical carrier. The specifications for unit dosage forms according to at least some embodiments of the present invention are determined by and directly depend on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the inherent constraints in the art of compounding such active compounds for the treatment of individual susceptibility.

[0525] Techniques for formulation and administration of drugs can be found in the latest edition of "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, incorporated herein by reference.

[0526] Pharmaceutical compositions of some embodiments of the present invention can be manufactured by processes well known in the art, e.g., by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0527] The compositions of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those of skill in the art, the route and / or mode of administration will vary depending on the desired results. Preferred routes of administration of therapeutic agents according to at least some embodiments of the invention include intravascular delivery (e.g., injection or infusion), intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, oral, enteral, rectal, pulmonary (e.g., inhalation), nasal, topical (including transdermal, buccal and sublingual), intravesical, intravitreal, intraperitoneal, vaginal, brain delivery (e.g., intraventricular, intracerebral, and convection-enhanced diffusion), CNS delivery (e.g., intrathecal, perispinal, and intraspinal) or parenteral (including subcutaneous, intramuscular, intraperitoneal, intravenous (IV) and intradermal), transdermal (either passively or using iontophoresis or electroporation), transmucosal (e.g., sublingual, nasal, vaginal, rectal, or sublingual) administration, or administration via an implant, or other parenteral administration routes, for example, by injection or infusion, or other delivery routes and / or forms of administration known in the art. The phrase "parenteral administration" as used herein means a mode of administration other than enteral and topical administration, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion or the use of bioerodible inserts, and may be formulated in a dosage form appropriate for each administration route. In a specific embodiment, a protein, therapeutic agent or pharmaceutical composition according to at least some embodiments of the present invention may be administered intraperitoneally or intravenously.

[0528] According to a specific embodiment, the compositions disclosed herein are administered by parenteral injection in an aqueous solution. The formulation may be in the form of a suspension or emulsion. In general, a pharmaceutical composition for parenteral injection is provided that comprises an effective amount of the compositions described herein, which optionally comprises pharma- ceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions may optionally contain one or more of the following: diluents, sterile water, various buffer contents (e.g., Tris-HCl, acetate, phosphate), buffered saline of pH and ionic strength, and additives, such as detergents and solubilizers (e.g., Tween 20 (polysorbate-20), Tween 80 (polysorbate-80)), antioxidants (e.g., water-soluble antioxidants, such as ascorbic acid, sodium metabisulfite, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium phosphate ... Examples of non-aqueous solvents or vehicles include ethanol, 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 (lyophilized) or vacuum-dried and redissolved / resuspended immediately before use. The formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition.

[0529] Various compositions (e.g., polypeptides) disclosed herein can be applied locally. Although topical administration does not work well for most peptide formulations, they can be particularly effective when applied to the lungs, nasal cavity, oral cavity (sublingual, buccal), vaginal, or rectal mucosa.

[0530] The compositions of the present invention, when delivered as either aerosols or spray-dried particles with an aerodynamic diameter of less than about 5 microns, can be delivered to the lungs during inhalation and can cross the lung epithelial layer into the bloodstream. A wide range of mechanical devices designed for pulmonary delivery of therapeutic products can be used, 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 are the Ultravent Nebulizer (Mallinckrodt Inc., St. Louis, Missouri), the Acorn II Nebulizer (Marquest Medical Products, Englewood, Colorado), the Ventolin Metered Dose Inhaler (Glaxo Inc., Research Triangle Park, North Carolina), and the Spinhaler Powder Inhaler (Fisons Corp., Bedford, Massachusetts). Nektar, Alkermes, and Mannkind all have approved inhalable insulin powder preparations or are in clinical trials where technology can be applied to the formulations described herein.

[0531] Formulations for administration to mucosa are typically spray-dried drug particles that can be incorporated into tablets, gels, capsules, suspensions or emulsions. Standard pharmaceutical excipients are available from any pharmaceutical manufacturer. Oral formulations can be in the form of chewing gum, gel strips, tablets or lozenges.

[0532] Transdermal formulations can also be prepared. These are typically ointments, lotions, sprays, or patches, all of which can be prepared using standard techniques. Transdermal formulations require the inclusion of a penetration enhancer. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an effective amount of the active ingredient to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without toxicity to the patient. The dosage level selected will depend on various pharmacokinetic factors, including the activity of the particular composition of the present invention used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, disease state, general health and medical history of the patient being treated, and similar factors well known in the medical field.

[0533] According to a specific embodiment, the compositions disclosed herein are administered to a subject in a therapeutically effective amount.As used herein, the term "effective amount" or "therapeutically effective amount" refers to a dosage that is sufficient to treat, inhibit or alleviate one or more symptoms of the disorder being treated, or otherwise provide a desired pharmacological and / or physiological effect.The determination of a therapeutically effective amount is well within the skill of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0534] For any preparation used in the method of the present invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in an animal model to achieve a desired concentration or titer. Such information can be used to more accurately determine a useful dose in humans. The toxicity and therapeutic efficacy of the active ingredients described herein can be determined in vitro, in cell cultures or experimental animals by standard pharmaceutical procedures. The data obtained from these in vitro and cell culture assays and animal tests can be used in formulating a range of dosages for use in humans. Dosages can vary depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration and dosage can be selected by the individual physician in consideration of the patient's condition (see, for example, Fingl, et al., 1975 in "The Pharmacological Basis of Therapeutics", Ch.1 p.1).

[0535] Dosage amount and interval can be adjusted individually to provide a level of active ingredient that is sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC varies for each preparation but can be estimated from in vitro data. The dosage required to achieve the MEC depends on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0536] Depending on the severity and responsiveness of the condition to be treated, dosage may be of single or multiple administrations, with the course of treatment lasting from several days to several weeks, or until a cure is effected or a diminution of the disease is achieved.

[0537] The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0538] In some embodiments, the composition (e.g., a LILRB (e.g., LILRB2, LILRB1) polypeptide described herein, a composition comprising the same, a fusion protein or dimer, a polynucleotide encoding the same, and / or cells) is administered locally, for example, by direct injection into the site to be treated. Typically, the injection causes an increase in the local concentration of the composition that is higher than the concentration that can be achieved by systemic administration. The composition can be combined with a matrix as described above to help create an increased local concentration of the polypeptide composition by reducing passive diffusion of the polypeptide from the site to be treated.

[0539] The pharmaceutical composition of the present invention can be administered using medical devices known in the art.For example, in any embodiment, the pharmaceutical composition according to at least some embodiments of the present invention can be administered using needle-type hypodermic injection device, such as the device disclosed in U.S. Patent No. 5,399,163, U.S. Patent No. 5,383,851, U.S. Patent No. 5,312,335, U.S. Patent No. 5,064,413, U.S. Patent No. 4,941,880, U.S. Patent No. 4,790,824 or U.S. Patent No. 4,596,556. Examples of well-known implants and modules useful in the present invention include U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing medicine at a controlled rate, U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medicine through the skin, U.S. Patent No. 4,447,233, which discloses a medicine infusion pump for delivering medicine at a precise infusion rate, U.S. Patent No. 4,447,224, which discloses a variable flow rate implantable infusion device for continuous drug delivery, U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with a multi-chamber compartment, and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0540] Active compound can be prepared with carrier that protects compound from rapid release, for example, controlled release formulations including implants, transdermal patches, and microencapsulated delivery systems.Biodegradable biocompatible polymers can be used, for example, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Many methods for preparing such formulations are patented or generally known to those skilled in the art.For example, see Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0541] Controlled release polymeric devices can be made for long-term systemic release after implantation of the polymeric device (rods, cylinders, films, disks) or injection (microparticles). The matrix can be in the form of microparticles, e.g., microspheres in which the peptide is dispersed within a solid polymeric matrix, or microcapsules in which the peptide is dispersed or suspended in a core that is of a different material than the polymeric shell and can be substantially liquid or solid. Unless specifically defined herein, microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, the polymer can be cast as thin slabs or films ranging from nanometers to 4 centimeters, can be a powder produced by grinding or other standard techniques, or even can be a gel, e.g., a hydrogel.

[0542] Either non-biodegradable or biodegradable matrices can be used to deliver the active agents disclosed herein, but biodegradable matrices are preferred. They can be natural or synthetic polymers, but synthetic polymers are preferred due to better characterization of the degradation and release profiles. The polymer is selected based on the period of time over which release is desired. In some cases, linear release may be most useful, while in other cases, pulsed or "bulk release" may provide more effective results. The polymer may be in the form of a hydrogel (typically absorbing up to about 90% water by weight), and may be optionally crosslinked with multivalent ions or polymers.

[0543] The matrices can be formed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art. Bioerodible microspheres can be prepared using any of the methods developed for the fabrication of microspheres for drug delivery, e.g., as 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 ScL, 35:755-774 (1988).

[0544] The devices can be formulated for local release to treat the area of ​​implantation or injection, typically delivering a dosage significantly smaller than that for whole body treatment, or can be formulated for systemic delivery: they can be implanted or injected subcutaneously, into muscle, into fat, or swallowed.

[0545] In some embodiments, to ensure that therapeutic compounds according to at least some embodiments of the present invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of producing liposomes, see, for example, U.S. Patent Nos. 4,522,811, 5,374,548, and 5,399,331. Liposomes can contain one or more moieties that selectively transport into specific cells or organs, thus improving targeted drug delivery (see, for example, VV Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al.), mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038), antibodies (PG Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180), surfactant protein A receptor (Briscoe et al. (1995) Am. J Physiol. 1233: 134), p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090), and the like. 346:123, see also JJ Killion, IJ Fidler (1994) Immunomethods 4:273.

[0546] The compositions of some embodiments of the present invention can be provided as a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient, if desired. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also contain a notice associated with the container in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects the approval by that agency of the form of the composition or its administration to humans or animals. Such notice may, for example, be that of the labeling approved by the U.S. Food and Drug Administration for prescription drugs or an approved product insert. A composition comprising the preparation of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in a suitable container, and further labeled for the treatment of an indicated condition as detailed above.

[0547] According to specific embodiments, any of the genes, polynucleotides, proteins, polypeptides and / or proteinaceous portions described herein may have the sequence of a human gene, polynucleotide, protein, polypeptide and / or proteinaceous portion, or a functional fragment or homolog thereof that exhibits a desired activity as described herein.

[0548] According to specific embodiments, the genes, polynucleotides, proteins, polypeptides and / or proteinaceous portions are of human origin.

[0549] According to other specific embodiments, the genes, polynucleotides, proteins, polypeptides and / or proteinaceous moieties are homologues of human genes, polynucleotides, proteins, polypeptides and / or proteinaceous moieties. Such homologues may be, for example, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical or homologous to the human sequence.

[0550] As used herein, the term "about" refers to ±10%.

[0551] The terms "comprises," "comprising," "include," "including," "having" and their conjugations mean "including, but not limited to."

[0552] The term "consisting of" means "including and limited to."

[0553] The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or moieties, but only if the additional components, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0554] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" includes a plurality of compounds, and may also include mixtures thereof.

[0555] Throughout this specification, various embodiments of the present invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity, and should not be construed as an inflexible limitation of the scope of the present invention. Thus, the description of a range should be considered to specifically disclose all possible subranges and individual numerical values ​​within that range. For example, the description of a range, e.g., 1-6, should be considered to specifically disclose subranges, e.g., 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0556] Whenever a numerical range is given herein, it is meant to include any recited number (fractional or integer) within the range given. The phrases "ranging between" a first recited number and a second recited number and "ranging from" a first recited number to a second recited number are used interchangeably herein and are meant to include the first and second recited numbers and all fractional and integer numerical values ​​therebetween.

[0557] As used herein, the term "method" refers to manners, means, techniques and procedures for accomplishing a given task, including but not limited to manners, means, techniques and procedures known to or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0558] Where reference is made to a particular sequence listing, such reference should be understood to also encompass sequences that substantially correspond to complementary sequences containing minor sequence variations resulting, for example, from sequencing errors, cloning errors, or other alterations resulting in base substitutions, deletions, or additions, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively less than 1 in 100 nucleotides, alternatively less than 1 in 200 nucleotides, alternatively less than 1 in 500 nucleotides, alternatively less than 1 in 1000 nucleotides, alternatively less than 1 in 5,000 nucleotides, or alternatively less than 1 in 10,000 nucleotides.

[0559] It is recognized that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination or as preferred in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiment is inoperable without those elements.

[0560] Various embodiments and aspects of the present invention as delineated above and as claimed in the claims section below are found to be experimentally supported in the following examples. EXAMPLES

[0561] Reference is now made to the following examples, which together with the above detailed description illustrate certain embodiments of the present invention in a non-limiting manner.

[0562] Example 1 Design of LILRB2 variants Materials and Methods: A structural analysis of LILRB2 (SEQ ID NO: 1, also referred to herein as "WT LILRB2") was performed to identify amino acid substitutions that could optimize and increase its stability and binding affinity to HLA-G (SEQ ID NO: 3), which included the following steps:

[0563] 1. Preparation of both LILRB2 and HLAG complex structures using Biobia Discovery Studio "Protein Preparation" Protocol 1 (Dassault Systems). Preparation included charge fixing and side chain deletion, and energy minimization.

[0564] 2. Structural analysis to identify potential interaction points between LILRB2 and HLA-G (FIG. 1A-B, PDB ID: 2DYP is the high-resolution complex structure used for this purpose).

[0565] 3. Sequence comparison among HLA homologues: HLA-A, HLA-B, HLA-C and HLA-G to identify the LILRB2 interaction site specific for LILRB2-HLA-G interaction (FIG. 1B).

[0566] 4. Identification of amino acids in the LILRB2 interface that interact with a specific amino acid or amino acids of HLA-G identified in step 3 above, i.e., that are not present in HLA-A, HLA-B, or HLA-C (Figure 1D).

[0567] 5. In silico sampling of each possible mutation at the subset of residues defined in steps 3-4 above on LILRB2. This process included: computationally introducing specific mutations at each position separately, optimizing the structure to best accommodate the mutations, and predicting the energy difference (ΔG) between the LILRB2 wild-type sequence (SEQ ID NO: 1) and any mutations for both overall protein stability and binding energy (ΔG relative to WT) using CHARMM-based force field energy calculations. The actual energy terms calculated were the effect of the mutations on the potential energy of binding and stability, not just ΔG. However, ΔG is dominant, and therefore the hint concerns the effect on ΔG.

[0568] 6. The results of the binding / stability energy calculations were compiled into a ranked list and specific point mutations (substitutions) on the LILRB2 sequence (SEQ ID NO: 1) that were predicted to be beneficial for both binding and stability considerations were selected.

[0569] 7. The selected substitutions were used in a second round of mutational analysis, this time introducing combinations of two substitutions each time, and repeating the process of energy evaluation for both binding to HLA-G and stability.

[0570] The final list of suggested surrogates for further testing was selected based on several parameters considered during this analysis. The energetic contribution (ΔG) of the substitution to the stability of the apoprotein (a protein lacking a characteristic prosthetic group or metal), The energy contribution of the substitution to the stability of the complex structure and the interaction energy (ΔG), Conformational minimization of the protein (e.g., by introducing substitutions that are disparate in terms of size, shape and chemical properties compared to the WT LILRB2 sequence), Shape complementarity within protein complex interfaces.

[0571] result: Structural analysis of the HLA-G (SEQ ID NO: 3)-LILRB2 (SEQ ID NO: 1) interaction interface and sequence comparison between HLA homologs revealed only a single dominant difference between HLA-G and its homologs, i.e., Phe195 in HLA-G was replaced by Ser in the other homologs (Figure 1C). Therefore, closely interacting residues on LILRB2 were primarily considered in the analysis.

[0572] In silico sampling of each possible mutation in a subset of amino acid residues that define the interface between LILRB2 and HLA-G Phe195 interacting residues, i.e., LILRB2 (SEQ ID NO: 1) residues Ser45, Ile49, Thr50 and Val57 (FIG. 1D), was found to have a significant positive impact on the binding energy, i.e., lower ΔG. In addition, the calculated binding / stability energy results of each possible mutation in the four amino acids, Ser45, Ile49, Thr50 and Val57, were compiled into a ranked list. Of course, the ranking was done by the in silico predicted contribution of the mutations to both the individual protein stability and the binding free energy (affinity).

[0573] Table 1 below shows the top ranked single substitutions and Table 2 below shows the top ranked double substitutions.

[0574] Specific single substitutions and combinations of two amino acid substitutions were then selected for further analysis, as further described in the Examples below.

[0575] [Table 3]

[0576] [Table 4]

[0577] Example 2 Generation and characterization of LILRB2 variants Materials and Methods: Reagents - ExcelBand™ 3-Color High Range Protein Marker or ExcelBand™ 3-Color Extra Range Protein Marker (SMOBIO Catalog #PM2600 or PM2800 respectively), Sample Buffer (GenScript Catalog #M00676), Polyacrylamide Gel 8% or 4-20% GenScript Catalog #M00662 or M00656 respectively), Fugene® HD Transfection Reagent (Promega Catalog #TM328), ECL Plus Western Blotting Substrate (Pierce Catalog #32132).

[0578] Antibodies - APC anti-human HLA-G (from US Patent Publication No. 2020 / 0102390A1), APC human IgG4 (Biolegend, catalog #403706), APC anti-human IgG1 (Southern Biotech, catalog #9052-31), APC mouse IgG1k (Biolegend, catalog #400120), APC anti-human CD47 antibody (Biolegend, catalog #323124), CD47 blocker Ab (from WO 2011 / 143624A2), rabbit anti-human LILRB2 (Abclonal, catalog #A10135), biotinylated rabbit anti-human SIRPα LsBio catalog (#LS-C370337), goat anti-rabbit IgG(H+L)-HRP conjugate (R&D Systems, catalog #170-6515), and HRP-conjugated streptavidin (Pierce, catalog #TS21126).

[0579] Cell lines - Expi 293F (Gibco, Cat #A14257), HT1080 cell line (ATCC, CCL-121), HT1080-HLA-G, THP1 cell line (ATCC, TIB-202), THP1-EV and THP1-HLA-G. Cells were generated by viral infection with HLA-G expression plasmid or empty vector (EV). Briefly, 293T cells were transfected with lentiviral plasmids (containing HLA-G constructs or empty vector and reporter reagent GFP) after 48 hours at 37°C, 5% CO2, supernatants were collected and used to transduce HT1080 or THP1 cells. Cells were transduced for 24 hours. HLA-G cells are GFP positive.

[0580] Media and tissue culture reagents - Expi 269 medium (Gibco, catalog #A-14351-01), RPMI 1640 (Biological Industries, catalog #01-100-1A), EMEM (Biological Industries, catalog #01-040-1A), DMEM (Biological Industries, catalog #01-055-1A), DMEM / F-12 (Gibco, catalog #31330095), FCS (Gibco, catalog #12657-029), 10% BSA (Biological Industries, catalog #12657-029), 10% PBS ... Industries, catalog #03-010-1B), EDTA (Sigma, catalog #E7889), Tryp LE Express (Gibco, catalog #12604-13), Glutamax (Gibco, catalog #35050-038), and Penicillin-Streptomycin (Gibco, catalog #151140-122).

[0581] Equipment - FACS device (Stratadigm Cytometry S1000EXI).

[0582] Recombinant Protein Production - For comparative functional analysis and production evaluation, several recombinant proteins containing wild-type (WT) LILRB2 domain (SEQ ID NO: 1) or mutant LILRB2 domain (referred to herein as "LILRB2 variants") were produced (see Table 3 below). Production was performed in Expi 293F cells transfected with pcDNA3.4 expression vectors cloned with coding sequences for the desired Fc fusion proteins. Restriction enzymes, e.g., EcoRI and HindIII or XbaI and EcoRV, were used to clone sequences into the vector, adding a Kozak sequence and a stop codon and an artificial signal peptide (MESPAQLLFLLLLWLPDGVHA, SEQ ID NO: 25). Proteins were collected from cell culture supernatants, and in some cases, proteins were purified by one-step purification using Protein A (PA) Polosumab Capture A resin.

[0583] [Table 5]

[0584] SDS-PAGE analysis - 35 μl of cell culture supernatant or 3 μg of purified protein from each recombinant protein sample was mixed with loading buffer with or without β-mercaptoethanol (reducing and non-reducing conditions, respectively), heated at 95° C. for 5 min, and separated on 8% gel electrophoresis SDS-PAGE. Protein migration on the gel was visualized by e-stain instrument (GenScript) according to the manufacturer's instructions.

[0585] Western blot analysis - Samples (50-500 ng per lane) containing the produced heterodimers were treated under reducing or non-reducing conditions (in loading buffer with or without β-mercaptoethanol, respectively), heated at 95°C for 5 min, and separated on 8% or 4-20% gradient SDS-PAGE gels. Proteins were subsequently transferred onto PVDF membranes and incubated with primary antibodies for 1 h or overnight, followed by incubation with HRP-conjugated secondary antibodies for 1 h. Signals were detected after ECL development.

[0586] Flow cytometry - To evaluate the binding of recombinant proteins to HLA-G expressed on the surface of cells, HT1080-WT or THP-1-EV cells, or HT1080 or THP-1 cells overexpressing human HLA-G, were incubated with serial dilutions of the produced recombinant proteins for 30 min at 4°C, followed by immunostaining with fluorescently labeled antibodies specific for the IgG1 backbone and flow cytometry analysis. To test the binding specificity, cells were subjected to preincubation with a blocker antibody against human HLA-G at a concentration of 5 μg / ml for 1 h at 37°C before incubation with recombinant proteins. Binding curve graphs were generated using GraphPad Prism software) using the MFI values.

[0587] result: Several heterodimeric proteins containing either WT LILRB2 (SEQ ID NO: 1) or mutant LILRB2 (referred to herein as "LILRB2 variants")-Fc fusions and SIRPα-Fc fusions were produced and analyzed. The heterodimer containing WT LILRB2 is referred to herein as "DSP216-V5," while the heterodimers containing LILRB2 variants are referred to herein as "DSP216-V11," "DSP216-V12," "DSP216-V13," "DSP216-V14," "DSP216-V15," "DSP216-V16," "DSP216-V17," "DSP216-V18," "DSP216-V19," and "DSP216-V20." In addition, two LILRB2 homodimer proteins were produced and analyzed: a homodimer containing WT LILRB2, referred to herein as "LILRB-V5-Fc," and a homodimer containing a LILRB2 variant, referred to herein as "LILRB2-V12-Fc." A complete description of each heterodimer / homodimer is provided in Table 3 above.

[0588] As demonstrated in FIG. 2A, a high proportion of the protein in the predicted heterodimeric molecular weight form was observed under non-reducing conditions, and expression of the two subunits was confirmed under reducing conditions.

[0589] Subsequently, binding of the produced heterodimers to HLA-G was determined by flow cytometry analysis using HT1080-WT and HT1080 overexpressing HLA-G (HT1080-HLA-G) and THP1 mock transduced with empty vector (THP1-EV) or THP1 overexpressing HLA-G (THP1-HLA-G) cell lines and anti-hIgG1 as detection antibody (Figure 3A-F). Significantly higher binding to HLA-G overexpressing cells compared to WT or EV cells expressing hCD47 only was observed (Figure 3A-B). Furthermore, binding of heterodimers containing LILRB2 variant domains to HLA-G expressing cells was significantly higher than that of heterodimers containing WT LILRB2 domains (DSP216-V5) (Figure 3C).

[0590] Because HT1080-HLA-G cells express both HLA-G and CD47 compared to HT1080 cells expressing only CD47 (Figure 3A), the specific binding of the generated heterodimers to HLA-G was further tested using an anti-HLA-G blocking antibody. The levels of anti-HLA-G binding inhibition for heterodimers containing LILRB2 variants were higher compared to the inhibition for heterodimers containing WT LILRB2, indicating their superiority in specific binding to HLA-G (Figure 3D-F).

[0591] Binding of LILRB2-V5 or LILRB2-V12 homodimers to HLA-G was determined by flow cytometry analysis using HT1080-WT and HT1080 overexpressing HLA-G (HT1080-HLA-G) and anti-hIgG1 as detection antibody (Figure 4). Significantly higher binding of LILRB2-V12 homodimers to HLA-G overexpressing cells was observed compared to WT cells expressing hCD47 only (Figure 4). Furthermore, binding of homodimers containing the LILRB2-V12 variant domain to HLA-G expressing cells was significantly higher than that of homodimers containing the WT LILRB2 domain (LILRB2-V5, Figure 4). Furthermore, the levels of anti-HLA-G binding inhibition for homodimers containing the LILRB2-V12 variant were higher compared to that for homodimers containing WT LILRB2, indicating their superiority in specific binding to HLA-G (Figure 4).

[0592] Additional heterodimeric proteins were produced and analyzed, including fusions of LILRB2 variants fused to the Fc domain with or without the LALA mutation (SEQ ID NOs: 29 and 85, respectively) and fusions of SIRPα (SEQ ID NOs: 27 or 88) fused to Fc with or without the LALA mutation (SEQ ID NOs: 31 and 86, respectively). A full description of each heterodimer is provided in Table 3 above.

[0593] A high proportion of the protein in the predicted heterodimeric molecular weight form was observed under non-reducing conditions, and expression of the two subunits was confirmed under reducing conditions, as demonstrated in Figure 9. Furthermore, Western blot analysis confirmed that the produced DSP216-V12, DSP216-V12 short chain, DSP216-V21 and DSP216-V21 short chain contained the domains of SIRPα and LILRB2, as demonstrated in Figure 10.

[0594] Binding of DSP216-WT, DSP216-V3, DSP216-V6, DSP216-V12 short chain, DSP216-V21 and DSP216-V21 short chain heterodimer to HLA-G was determined by flow cytometry analysis using HT1080-WT and HT1080 overexpressing HLA-G (HT1080-HLA-G) and anti-hIgG1 as detection antibody. As shown in Figure 11, significantly higher binding of all tested DSP216 heterodimers to HLA-G overexpressing cells was observed compared to WT cells expressing only hCD47. Furthermore, binding of DSP216 variants containing the V57R substitution in the LILRB2 sequence (DSP216-V21, DSP216-V21 short chain and DSP216-V12 short chain) to HLA-G expressing cells was significantly higher compared to the binding of heterodimers containing the WT LILRB2 domain (DSP216-WT, DSP216-V3 and DSP216-V6). Furthermore, significant binding inhibition was demonstrated by anti-HLA-G blocker antibodies, indicating specific binding to HLA-G.

[0595] Example 3 Binding of LILRB2 variants to human HLA-G To further confirm the superiority of the LILRB2 variants, the binding affinity of the LILRB2 variants to human HLA-G and competition with WT LILRB2 are determined by surface plasmon resonance (SPR) assay.

[0596] Example 4 LILRB2 variants block natural LILRB2-HLA-G binding Endogenous LILRB2 is expressed on the cell surface of human monocytes, B cells, and at lower levels on myeloid and plasmacytoid dendritic cells (Katz HR. Adv Immunol. (2006) 91:251-272; Kang X, et al. Cell Cycle. (2016) 15(1): 25-40).

[0597] Endogenous LILRB1 is expressed on various dendritic cell subsets, including human macrophages, some T cells, NK cells, B cells, monocytes, myeloid, plasmacytoid and tolerogenic DCs (Katz HR. Adv Immunol. (2006) 91:251-272; Kang X, et al. Cell Cycle. (2016) 15(1):25-40).

[0598] Endogenous HLA-G is expressed primarily on cellular trophoblasts in the placenta; however, cells associated with several pathological conditions (e.g., cancer, viral infections, autoimmune and inflammatory diseases, GvHD) have been shown to express HLA-G (Contini P, et al., Front Immunol. (2020) 11:1613, PMID:32983083, Morandi F, et al., J Immunol Res. (2016) 2016:4326495, PMID:27652273).

[0599] The interaction of LILRB2 and LILRB1 expressed on immune cells with their natural ligand HLA-G initiates a signaling pathway that inhibits the activity of immune cells. Proteins containing LILRB2 are designed to block the interaction of endogenous LILRB2 and LILRB1 expressed on immune cells with HLA-G expressed on, for example, tumor cells.

[0600] The effectiveness of the produced proteins containing LILRB2 variants as blockers of their interaction is evaluated by ELISA assay and compared with WT LILRB2. For this purpose, ELISA plates are coated with recombinant human HLA-G. The plates are then washed and incubated with different concentrations of the produced proteins containing LILRB2 or a positive control anti-HLA-G blocker antibody for 1 hour. LILRB2-Fc (mIgG) or LILRB1-Fc (mIgG) is added, followed by additional incubation, and then the plates are washed and blotted with anti-mouse IgG-HRP and TMB substrate according to standard ELISA protocols. The plates are analyzed at 450 nm with a reference of 620 nm using a plate reader (Thermo Scientific, Multiscan FC).

[0601] Example 5 Ligand-binding ELISA The binding of LILRB2 to its counterpart HLA-G is tested by a binding ELISA assay.

[0602] hHLA-G is bound to the surface of a plastic plate, and LILRB2 is added and allowed to bind to the immobilized hHLA-G. After washing, HRP-anti-hIgG1 is added and allowed to bind to the Fc backbone of the molecule. Detection is performed at 450 nm with a reference of 540 nm using a plate reader (Thermo Scientific, Multiscan FC) following standard ELISA protocols with TMB substrate.

[0603] Example 6 In vivo antitumor effects of LILRB2 variants Three different in vivo mouse models are used to test the efficacy of the generated heterodimers containing LILRB2 variants in the treatment of cancer: 1. NSG mice inoculated with human stem cells or human PBMCs or fixed human PBMCs and human tumor cells expressing CD47 and HLA-G. 2. Nude SCID mice inoculated with human tumor cells expressing CD47 and HLA-G. 3. Syngeneic mouse tumor model using mice inoculated with a mouse cancer cell line expressing CD47 and HLA-G and a surrogate mouse protein for the test heterodimer.

[0604] In all models, mice are inoculated with tumor cells intravenously (IV), intraperitoneally (IP), subcutaneously (SC) or orthotopically. 3 ), mice are treated IV, IP, SC or orthotopically with different doses and different regimens of the produced heterodimer.

[0605] Mice are followed for body weight and clinical signs. Tumors are measured by caliper several times a week and tumor volume is calculated according to the following formula: V=length×width. 2 / 2. Mouse body weights are routinely measured. Tumor growth and survival are monitored throughout the experiment.

[0606] Immune cell infiltration and subtyping in tumors is examined by tumor or draining lymph node resection, digestion, and immunophenotyping using specific antibody staining and flow cytometry analysis. Additionally or alternatively, immune cell infiltration or tumor necrosis grade is determined by tumor resection, paraffin embedding, and sectioning for immunohistochemical staining with specific antibodies.

[0607] At sacrifice, mouse organs are harvested and embedded in paraffin blocks for H&E and IHC staining.

[0608] Blood samples are taken from mice at different time points following general procedures for the following tests: PK analysis, cytokine measurements in plasma, FACS profiling of circulating blood cell subpopulations, hematological tests, serum chemical tests, anti-drug antibody (ADA) analysis and neutralizing antibody analysis (NAB).

[0609] Example 7 Effect of LILRB2 variants on M-CSF-dependent macrophage maturation The LILRB2 domain of the produced protein is designed to block the immunosuppressive signals induced by HLA-G expressed on tumor or immune cells toward endogenous LILRB1 and LILRB2 expressed on antigen presenting cells (APCs), such as macrophages and dendritic cells, by competing and blocking their interaction. It has been reported that M1 macrophages exhibit antitumor activity, whereas M2 macrophages promote tumor progression. M-CSF is known to drive the differentiation of monocytes into naive M0 (M2-like) macrophages, which can be subsequently polarized into pro-inflammatory (M1 macrophages) or anti-inflammatory (M2 macrophages) phenotypes by different activation stimuli (Chistiakov DA, et al., J. Cell. Mol. Med. (2015) 19(6):1163-1173 PMID:25973901). Blockade of LILRB2 with an antagonist antibody during M-CSF-dependent macrophage maturation was shown to result in a rounder, more tightly adherent M1 (anti-tumor) phenotype with lower expression of CD14 and CD163 (Chen HM, et al., J Clin Invest. (2018)128(12):5647-5662. PMID:30352428). After stimulation of the generated macrophages with LPS, enhanced secretion of the pro-inflammatory cytokine TNFα and reduced secretion of the anti-inflammatory IL-10 were detected (Chen HM et al.).

[0610] The effect of produced recombinant proteins containing LILRB2 variants on M-CSF-dependent macrophage maturation was assessed using flow cytometry-based detection of M1 / M2 markers and by measuring TNFα and IL-6 (known M1-associated cytokines) release following stimulation of LPS-pretreated macrophages.

[0611] material: Reagents - DSP216-V12, DSP216-V12 short chain, fresh buffy coat samples from healthy donors (Hadassah Blood Bank), Ficoll-Paque™ Plus 1.077 (Cytiva, Catalog #GE-17-1440-03), Human Trastain FcX (Biolegend, Catalog #422302), PBS (Sartorius, Catalog #20-023-1A), EDTA (Sigma, Catalog #E7889), Sodium Azide (Sigma, Catalog #S2002), 10% BSA (Sartorius, Catalog #03-010-1B or Biological Industries, Catalog #03-010-1B), BD Cytometric Bead Array (CBA) (BD, Catalog #551809), RPMI 1640 (Biological Industries, Catalog #10001), 10% PBS (Sartorius, Catalog #03-010-1B or Bi ... DMEM (Biological Industries, Catalog #01-100-1A), DMEM (Biological Industries, Catalog #01-055-1A), FBS (Gibco, Catalog #12657-029), TrypLE Express (Gibco, Catalog #12604-13), Glutamax (Gibco, Catalog #35050-038), Penicillin-Streptomycin (Gibco, Catalog #151140-122), and human recombinant M-CSF (R&D, Catalog #216-MC-100).

[0612] Antibodies - anti-HLA-G Tizona-like (described in US Patent Publication No. 20200102390), BV785 anti-human CD11b (Biolegend, Catalog #301346), APC anti-human CD163 (Biolegend, Catalog #333610), APC anti-human CD206 (Biolegend, Catalog #321110), APC anti-human HLA-DR (Biolegend, Catalog #307610), APC mouse IgG1 (Biolegend, Catalog #40120) isotype control for CD163 and CD206, APC mouse IgG2a (Biolegend, Catalog #400220) isotype control for HLA-DR.

[0613] Human monocytes - PBMCs were purified from two fresh buffy coat samples by Ficoll gradient according to the manufacturer's instructions. Cells were seeded (1 × 10 per flask) in T75 flasks with RPMI medium. 8 cells) and incubated at 37°C and 5% CO2. After 2 hours of incubation, the medium was replaced with fresh RPMI supplemented with 50ng / mL M-CSF. After 6 days of incubation, the cells are defined as M0 macrophages (Tarique AA, et al., American J of Respiratory Cell and Molecular Biology (2015) 53(5): 676-688, PMID:25870903).

[0614] Cell line - HT1080 cells overexpressing HLA-G (HT1080-HLA-G) as described above in Example 2. Of course, HT1080 cells overexpressing HLA-G are GFP positive.

[0615] method: The effect of DSP216-V12 on M0 macrophage polarization was evaluated by co-culturing macrophages with HT1080HLA-G cells. After 6 days of incubation with M-CSF, M0 macrophages were seeded (250,000 cells per well) in 6-well plates with RPMI and 50 ng / ml M-CSF for overnight incubation. The next day, HT1080-HLA-G cells were incubated with 2, 4 or 10 μg / ml DSP216-V12 or 1.5 μg / ml anti-human HLA-G (equimolar to 2 μg / ml DSP216-V12) as a positive control for 1 h at 37° C. and then seeded on top of M0 macrophages at a 2:1 E:T ratio (250,000 M0 macrophages:125,000 HT1080-HLA-G cells). After 24 h of incubation, cells were harvested and expression of established polarization markers was determined by flow cytometry (Cytoflex B53000 Cytoflex B5-R3-V5). Specifically, macrophages were gated as CD11b positive cells and surface expression of M2 markers CD163 and CD206, as well as M1 marker HLA-DR, was assessed. In addition, supernatants were collected for determination of cytokine secretion. FACS data were analyzed using MFI values ​​(FlowJo v10.8.1 software) and CBA data of cytokine levels in the supernatants were analyzed using FCAP Array v3.0 software.

[0616] result: After treatment with DSP216-V12 or DSP216-V12 short chain (heterodimeric protein containing LILRB2 variant-Fc fusion and SIRPα-Fc fusion, see Table 3 for a complete description), the expression of M2-associated markers CD163 and CD206 in M-CSF-polarized monocytes co-cultured with HT1080HLA-G cells was downregulated in a dose-dependent manner compared to untreated or anti-HLA-G control groups (Figures 5A-B and 12A-B), whereas the expression of M1-associated marker HLA-DR was upregulated (Figures 5C and 12C). Furthermore, treatment with DSP216-V12 induced the secretion of M1-associated cytokines, TNFα and IL-6 (Figures 6A-B).

[0617] Taken together, the results demonstrated that DSP216-V12 and DSP216-V12 short chain could convert M0 (M2-like) macrophages into M1 macrophages.

[0618] Example 8 Effects of LILRB2 variants on macrophages and polymorphonuclear cells The LILRB2 domain of the protein is designed to block the immunosuppressive signals induced by HLA-G expressed on tumor or immune cells toward endogenous LILRB1 and LILRB2 expressed on APCs, such as macrophages and dendritic cells, by competing and blocking their interaction. This blockade of the "don't eat me signal" of HLA-G induces tumor cell phagocytosis and interferes with the inhibitory HLA-G-LILRB1 / 2 signaling between cancer and immune cells, which in turn enhances phagocytosis.

[0619] The effect of the produced recombinant protein containing the LILRB2 variant on tumor cell phagocytosis can be evaluated by mixing macrophages with CFSE cells or cell tracing violet (CTV) labeled cancer cells preincubated in the presence of different concentrations of the produced recombinant protein. After various incubation times (e.g., 3 hours), the macrophages are stained with anti-CD11b antibody and the phagocytic uptake of the stained cancer cells is evaluated by microscopy or flow cytometry analysis.

[0620] material: Reagents - DSP216-V12, fresh buffy coat samples from healthy donors (Sanquin Blood Bank), Ficoll Lymhoprep (Serumwerk Bernburg AG, Alere Technologies AS, Oslo, Norway, 04-03-9391 / 02), Triple LE Express (gibco, Cat# 12604021), FC receptor blocking solution (Nanogen), PBS (in-house, umcg pharmacy), EDTA (sigma Aldrich, Cat# E9884-1KG), sodium azide (BDH Chemical LTD), BSA (BSA Fraction V, Roche, Catalog #10735094001), RPMI1640 (Gibco, 52400-025), FBS (Gibco), human recombinant M-CSF (Immunotools, Catalog #11343115), human recombinant IL-10 (Immunotools, Catalog #1134107), CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher Scientific, Catalog #C34557).

[0621] Antibody - Anti-HLA-G Tizona (described in US Patent Publication No. 20200102390), APC anti-human CD47 (Biolegend, Catalog #323124), APC anti-human CD11b (Biolegend, Catalog #301310), APC anti-human CD163 (e-Bioscience, Catalog #17-1639-41), APC anti-human LILRB2 (Biolegend, Catalog #338708), APC mouse IgG4 (Biolegend, Catalog #403706) isotype control for HLA-G, APC mouse IgG1 (Biolegend, Catalog #40120) isotype control for CD47, CD11b and CD163, APC rat IgG2a (Biolegend, Catalog #400512) isotype control for LILRB2, CD47 blocking Ab Like Inhibrix (described in U.S. Patent Application Publication No. 2015 / 0183874A1).

[0622] Human monocytes - Samples were obtained from fresh buffy coat samples by purification on a Ficoll gradient according to the manufacturer's instructions. Cells were seeded (5 × 10 per well in 2 mL) in 6-well plates with RPMI medium supplemented with 10% FCS and 50 ng / mL M-CSF. 6 Cells) and incubated at 37°C and 5% CO2. After overnight incubation, the medium was replaced with fresh RPMI supplemented with 10% FCS and 50 ng / mL M-CSF. After 6 days of incubation, the medium was replaced with RPMI supplemented with 10% FCS and 50 ng / mL IL-10. After 48 hours of incubation, the cells were defined as M2c macrophages.

[0623] Cell line - 721.221 cells overexpressing HLA-G (Shimizu, Y., and R. DeMars, J. Immunol. (1989) 142:3320; Markel G. et al., J Immunol (2002) 168(6): 2803-2810) (721.221-HLA-G). Cells were generated by viral infection with plasmids expressing HLA-G and GFP or GFP alone (empty vector-EV).

[0624] method: The effect of DSP216-V12 on M2c macrophage phagocytosis was evaluated by co-culture of macrophages with 721.221-HLA-G or 721.221-EV cells. 721.221-HLA-G and 721.221-EV cells were stained with CTV, and 250,000 cells were seeded in FACS tubes and incubated with medium containing 2.5, 5 or 10 μg / mL DSP216-V12 or 6 μg / mL CD47 blocking antibody for 20 min at 4°C. 50,000 M2c macrophages were added to each tube containing cancer cells. After 3 h of incubation, macrophages were stained with APC-CD11b antibody and the percentage of phagocytosis was determined by staining with CD11b positive for CTV. + The percentage of cells with CD47 and HLA-G expression was determined in parallel by examination of their expression on the surface of 721.221-HLA-G and -EV cells using FACS, and CD11b, CD163 and LILRB2 expression levels were examined on M2c macrophages. FACS data were analyzed with FlowJo v10.8.1 software and statistical analysis was performed with GraphPad software (Prism 9 for Mac OS, version 9.4.1).

[0625] result: Following treatment with DSP216-V12 (a heterodimeric protein containing a LILRB2 variant-Fc fusion and a SIRPα-Fc fusion), phagocytosis of 721.221EV and 721.221-HLA-G cells by M2c macrophages was increased in a dose-dependent manner. + HLA-G- For 721.221-EV cells, the increase in phagocytosis was not significant at any DSP216-V12 concentration, whereas a CD47 blocking antibody significantly increased phagocytosis ( ** p=0.0028) (Figure 13A). + HLA-G + For 721.221-HLA-G cells, the increase in phagocytosis was significant at 10 μg / mL of DSP216-V12 ( * p=0.0465), whereas CD47 blocking antibodies did not significantly increase phagocytosis (p=0.1654) (Figure 13B).

[0626] Taken together, our results suggest that DSP216-V12 inhibits CD47 uptake by M2c macrophages. + / HLA-G + It was shown that it can significantly increase phagocytosis of cells.

[0627] This effect results from blocking the CD47 / SIRPα axis together with the HLA-G / LILRB1 / 2 axis, since blocking only the CD47 / SIRPα axis with a CD47 blocking antibody does not inhibit CD47 + / HLA-G + This is because 721.221-HLA-G has a lower effect on phagocytosis.

[0628] Example 9 Cytotoxic activation of NK cells by LILRB2 variants Natural killer (NK) cells induce direct cytotoxicity or secretion of cytokines / chemokines without the recognition of specific antigens as B and T cells. NK cytotoxicity plays an important role in the immune response against infected cells, malignant tumors and stressed cells and is involved in the pathological processes of various diseases.

[0629] A number of assays known in the art can be used to determine the effect of produced recombinant proteins containing LILRB2 variants on NK activation, including, but not limited to, the following:

[0630] Cytotoxicity assay - killing of target cells by NK cells (effector cells) in a co-culture assay. The % killing is analyzed by flow cytometric analysis (FACS). Target cells are plated in 96-well plates and incubated with pre-labeled primary NK cells at various effector-target (E:T) ratios in the presence of different concentrations of the produced recombinant protein. NK cells are cultured with 1000 U / mL IL-2 for 48 hours and then assayed. Cells are harvested after 4 and 24 hours and assayed by flow cytometry. The number of target cells recovered from cultures without NK cells is used as a reference.

[0631] Cytotoxicity assay - killing of target cells by NK cells (effector cells) in a co-culture assay in the presence of different concentrations of the produced recombinant protein. The % killing is determined using labeled target cells and a caspase-sensitive fluorescent substrate with an Incucyte instrument.

[0632] Secretion of inflammatory cytokines - Primary NK cells are stimulated with various target cells in various ratios in the presence of different concentrations of the produced recombinant protein for 24 hours. Levels of interferon gamma (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in cell-free culture supernatants are determined using ELISA or cytometric bead array (CBA).

[0633] Example 10 Specific binding of heterodimers containing LILRB2 variants and SIRPα to cells expressing both HLA-G and CD47

[0634] Materials and Methods: Reagents - Alexa Fluor 647-labeled DSP216-V12, fresh buffy coat samples from four healthy donors (Hadassah Blood Bank), fresh whole blood from healthy donors (Hadassah Blood Bank), Ficoll-Paque™ Plus 1.077 (Cytiva, Catalog #GE-17-1440-03), human Trastain FcX (Biolegend, Catalog #422302), RBC Lysis Buffer (Invitrogen, Catalog #00-4333-57), PBS (Sartorius, Catalog #20-023-1A), EDTA (Sigma, Catalog #E7889), sodium azide (Sigma, Catalog #S2002), 10% BSA (Sartorius, Catalog #03-010-1B or Biological Invitrogen Catalog #03-010-1B), Alexa Fluor 647 Microscale Protein Labeling Kit (Invitrogen Catalog #A30009), RPMI 1640 (Biological Industries Catalog #01-100-1A), DMEM (Biological Industries Catalog #01-055-1A), FCS (Gibco Catalog #12657-029), Tryp LE Express (Gibco Catalog #12604-13), Glutamax (Gibco Catalog #35050-038), and Penicillin-Streptomycin (Gibco Catalog #151140-122).

[0635] Antibodies - BV421 anti-human CD45 (Biolegend, catalog #304032), APC mouse IgG1k (Biolegend, catalog #400120), APC anti-human CD47 antibody (Biolegend, catalog #323124).

[0636] Human PBMCs and RBCs - PBMCs were purified from four fresh buffy coat samples by Ficoll gradient according to the manufacturer's instructions. Whole blood from four healthy donors was diluted 1:500 in PBS and considered as RBCs.

[0637] Cell lines - as described above in Example 2. Note that HT1080 cells that overexpress HLA-G are GFP positive.

[0638] CD47 expression - RBC, PBMC or HT1080HLA-G cells were incubated with anti-human CD47 antibody and analyzed by flow cytometry (Cytoflex B53000 Cytoflex B5-R3-V5). Representative graphs were generated using GraphPad Prism software using MFI values.

[0639] Binding assay - 100 μL / well whole blood sample (approximately 1 × 10 6 The wells of purified PBMCs (considered as RBC samples containing 100 RBCs) were mixed with 25,000–50,000 cells / well of purified PBMCs and 25,0000–50,000 cells / well of HT1080 cells overexpressing human HLA-G (HT1080-HLA-G). The mixed cells were incubated with serial dilutions of Alexa Fluor 647-labeled DSP216-V12 for 30 min at 4°C. After incubation, the cells were washed, immunostained with BV421 anti-human CD45 antibody, and analyzed by flow cytometry (Cytoflex B53000 Cytoflex B5-R3-V5). The MFI values ​​were used to generate binding curve graphs using GraphPad Prism software.

[0640] result: CD47 was highly expressed on the surface of HT1080-HLA-G cells (Figure 7). Low expression levels of CD47 were also detected on the surface of PBMCs and RBCs from healthy donors (Figure 7). Binding of Alexa Fluor 647-labeled DSP216-V12 was determined by flow cytometry using a mix of RBCs, PBMCs, and HT1080-HLA-G cells. Anti-hCD45 antibodies were used to gate on RBCs (CD45-negative cells) and PBMCs (CD45-positive cells), and GFP was used to gate on HT1080-HLA-G cells. DSP216-V12 bound to HT1080-HLA-G cells in a dose-dependent manner, while only slight binding to PBMCs was demonstrated and no binding to RBCs was observed (Figures 8A-B).

[0641] Example 11 Design of LILRB1 variants Materials and Methods: Homology analysis was performed comparing human LILRB2 (Uniprot number Q8N423) and LILRB1 (Uniprot number Q8NHL6) using the alignment algorithm BLAST.

[0642] Subsequently, structural analysis of WT LILRB1 (SEQ ID NO: 102) was performed to identify amino acid substitutions that could optimize and increase its stability and binding affinity to HLA-G (SEQ ID NO: 3), which included the following steps: 1. Preparation of complex structures of both LILRB1 and HLAG using PDB ID: 2DYP, 3D2U complex structure. 2. LILRB1 and HLA-G (Figure 14) PDB ID: Structural analysis to identify potential interaction points between 2DYP (HLA-G) and 3D2U (LILRB1) complex structures. 3. Identification of the amino acid in the LILRB1 interface that interacts with Phe195 of HLA-G [identified as specific for HLA-G, i.e., not present in HLA-A, HLA-B, or HLA-C (Figure 1D)]. 4. In silico evaluation of the effect of the V55 mutation in the LILRB1 sequence shown in SEQ ID NO: 102.

[0643] result: We identified high sequence homology between human LILRB2 (Uniprot No. Q8N423) and LILRB1 (Uniprot No. Q8NHL6), especially in the D1 (Ig-like C2 type 1) domain (SEQ ID NOs: 104 and 105, respectively) (Figure 14). Interestingly, extremely high homology was detected in the region containing the amino acid residues identified in Example 1 above as defining the interface between LILRB2 and HLA-G Phe195 interacting residues (residues S45, I49, T50 and V57 of SEQ ID NO: 1). This prompted us to evaluate whether mutations of the corresponding amino acids of LILRB1 (i.e., T43, I47, T50 and V55 of SEQ ID NO: 102) also had a significant positive effect, i.e., a lower ΔG on binding energy.

[0644] For this purpose, a structural analysis of the HLA-G (SEQ ID NO: 3)-LILRB1 (SEQ ID NO: 102) interaction interface, particularly the interaction interface with Phe195 in HLA-G, was performed (FIG. 15A-B). Subsequently, in silico analysis showed that substitution of residue V55 of LILRB1 (SEQ ID NO: 102) with, for example, arginine (R) is likely to stabilize the LILRB1 / HLA-G interaction (FIG. 15C-D).

[0645] While the present invention has been described in terms of specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0646] It is the intention of the applicants that all publications, patents, and patent applications referenced herein be incorporated herein by reference in their entirety, as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, any citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority documents of this application are incorporated herein by reference in their entirety.

Claims

1. A LILRB polypeptide capable of binding to the HLA-G polypeptide set forth in SEQ ID NO: 3 and having at least one mutation located within amino acids 40 to 60 of the D1 domain of the LILRB polypeptide, wherein the LILRB polypeptide has increased stability and / or increased affinity for the HLA-G polypeptide compared to a LILRB polypeptide of the same length and having a sequence that does not contain the at least one mutation.

2. The LILRB polypeptide of claim 1, wherein the LILRB polypeptide is a LILRB2 polypeptide, and the at least one mutation is at a position corresponding to an amino acid selected from the group consisting of S45, I49, T50, and V57 of SEQ ID NO:

1.

3. 3. The LILRB polypeptide of claim 2, wherein the mutation in S45 comprises a S45R, S45N, S45Q, S45H, S45L, S45K, S45M, S45F, S45W, or S45Y mutation; the mutation in I49 comprises a I49R, I49K, I49F, or I49Y mutation; the mutation in T50 comprises a T50R, T50N, T50L, T50K, T50F, T50W, or T50Y mutation; and / or the mutation in V57 comprises a V57R, V57K, V57F, or V57W mutation.

4. 3. The LILRB polypeptide of claim 2, wherein the mutation in S45 comprises an S45Q mutation, the mutation in I49 comprises an I49K mutation, the mutation in T50 comprises a T50F mutation, and / or the mutation in V57 comprises a V57R mutation.

5. The LILRB polypeptide of claim 2 , wherein the at least one mutation comprises at least two mutations.

6. 3. The LILRB polypeptide of claim 2, wherein the at least one mutation comprises a mutation at S45 and additional mutations at I49, T50 and / or V57.

7. 7. The LILRB polypeptide of claim 6, comprising an S45N and T50R mutation, an S45Y and T50K mutation, an S45R and I49F mutation, an S45Q and V57R mutation, an S45Q and I49K mutation, or an S45Y and T50N mutation.

8. The LILRB polypeptide of claim 2, wherein the amino acid sequence of the LILRB2 polypeptide is set forth in SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23.

9. The LILRB polypeptide of claim 1, wherein the LILRB polypeptide is a LILRB1 polypeptide, and the at least one mutation is at a position corresponding to an amino acid selected from the group consisting of T43, I47, T48, and V55 of SEQ ID NO:

102.

10. 10. The LILRB polypeptide of claim 9, wherein the mutation in T43 comprises a T43R, T43N, T43Q, T43H, T43L, T43K, T43M, T43F, T43W, or T43Y mutation; the mutation in I47 comprises a I47R, I47K, I47F, or I47Y mutation; the mutation in T48 comprises a T48R, T48N, T48L, T48K, T48F, T48W, or T48Y mutation; and / or the mutation in V55 comprises a V55R, V55K, V55F, or V55W mutation.

11. 10. The LILRB polypeptide of claim 9, wherein the mutation at V55 comprises a V55R mutation.

12. A polynucleotide encoding the LILRB polypeptide described in any one of claims 1 to 11.

13. A host cell comprising the polynucleotide described in claim 12.

14. 14. A method for producing a polypeptide, comprising culturing the cell of claim 13.

15. 12. The LILRB polypeptide of any one of claims 1 to 11 for use in treating a disease associated with pathological cells expressing HLA-G in a subject in need of such treatment.

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