Antibodies that bind to ILT4

Abstract

The present invention provides antibodies that specifically bind to immunoglobulin-like transcript 4 (ILT4), also known as LILRB2, LIR2, MIR10, and CD85d, as well as corresponding nucleic acids, host cells, compositions, and uses. [Solution] An isolated antibody is provided that specifically binds to human ILT4 (hILT4 or ILT4), specifically binding to human ILT4 but not significantly binding to ILT2, ILT3, or ILT5, or other members of the LILRA or LILRB family.

Description

[Technical Field]

[0001] This application relates to an antibody that specifically binds to and inhibits ILT4, and to its use in cancer treatment. [Background technology]

[0002] ILT4 (immunoglobulin-like transcript 4; also known as LILRB2, LIR2, MIR10, and CD85d) is expressed on myeloid cells such as monocytes, macrophages, and dendritic cells. ILT4 is part of a structurally related family of receptors, which also includes the proteins LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, ILT2, ILT3, ILT5, and LIR8.

[0003] The extracellular domains of proteins within this family contain several immunoglobulin-like repeats, while the cytoplasmic tails contain several tyrosine-based inhibitory motifs (ITIMs) that recruit tyrosine phosphatases.

[0004] This application claims priority to a U.S. provisional patent application containing at least one drawing in color derived from the list shown below. Once this international application is published and the provisional application for which it claims priority becomes publicly available, the color drawings will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0005] [Figure 1] Figure 1 shows the nucleotide and amino acid sequences of the heavy chain variable region ("VH") of the anti-hILT5 antibody 9G4, indicating the location of VH Cmdr. [Figure 2] Figure 2 shows the nucleotide and amino acid sequences of the light chain variable region ("VL") of the anti-hILT4 antibody 9G4, indicating the location of the VL CDR. [Figure 3] Figure 3 shows the nucleotide and amino acid sequences of VH in the anti-hILT4 antibody 9C8, indicating the location of the VH CDR. [Figure 4] Figure 4 shows the nucleotide and amino acid sequences of the VL of the anti-hILT4 antibody 9C8, indicating the location of the VL CDR. [Figure 5] Figure 5 shows the nucleotide and amino acid sequences of VH in the anti-hILT4 antibody 2H2, indicating the position of VH CDR. [Figure 6] Figure 6 shows the nucleotide and amino acid sequences of the VL of the anti-hILT4 antibody 2H2, indicating the location of the VL CDR. [Figure 7] Figure 7 shows the nucleotide and amino acid sequences of VH in anti-hILT4 antibody 2E5, indicating the position of VH CDR. [Figure 8] Figure 8 shows the nucleotide and amino acid sequences of the VL of the anti-hILT4 antibody 2E5, indicating the location of the VL CDR. [Figure 9] Figure 9 shows the nucleotide and amino acid sequences of VH in the anti-hILT4 antibody 24E5, indicating the position of VH CDR. [Figure 10] Figure 10 shows the nucleotide and amino acid sequences of the VL of the anti-hILT4 antibody 24E5, indicating the position of the VL CDR. [Figure 11] Figure 11 shows the nucleotide and amino acid sequences of VH in the anti-hILT4 antibody 21D9, indicating the position of VH CDR. [Figure 12] Figure 12 shows the nucleotide and amino acid sequences of the VL of the anti-hILT4 antibody 21D9, indicating the location of the VL CDR. [Figure 13A]Figures 13A and 13C show the amino acid sequences of the variable domains of the heavy chain (VH) (Figure 13A) and light chain (VL) (Figure 13C) of the ILT4 antibody 21D9, respectively, and their alignment with the germline immunoglobulin sequences hIGHV3-23*01 and hIGKV3-20*01. Arrows indicate germline mutations. The AbM VH CDR1 and Kabat VH CDR2, VH CDR3, and VL CDR1-3 sequences are shown in lighter letters. Figure 13B shows the VH amino acid sequences of four germline revertant mutants 21D9.b, 21D9.c, 21D9.d, and 21D9.e, along with the IgG1.3 heavy chain constant region (i.e., 21D9.b-.ehIgG1.3). Amino acid residues shown in capital letters in Figure 13B are germline revertant substitutions. [Figure 13B] Figures 13A and 13C show the amino acid sequences of the variable domains of the heavy chain (VH) (Figure 13A) and light chain (VL) (Figure 13C) of the ILT4 antibody 21D9, respectively, and their alignment with the germline immunoglobulin sequences hIGHV3-23*01 and hIGKV3-20*01. Arrows indicate germline mutations. The AbM VH CDR1 and Kabat VH CDR2, VH CDR3, and VL CDR1-3 sequences are shown in lighter letters. Figure 13B shows the VH amino acid sequences of four germline revertant mutants 21D9.b, 21D9.c, 21D9.d, and 21D9.e, along with the IgG1.3 heavy chain constant region (i.e., 21D9.b-.ehIgG1.3). Amino acid residues shown in capital letters in Figure 13B are germline revertant substitutions. [Figure 13C]Figures 13A and 13C show the amino acid sequences of the variable domains of the heavy chain (VH) (Figure 13A) and light chain (VL) (Figure 13C) of the ILT4 antibody 21D9, respectively, and their alignment with the germline immunoglobulin sequences hIGHV3-23*01 and hIGKV3-20*01. Arrows indicate germline mutations. The AbM VH CDR1 and Kabat VH CDR2, VH CDR3, and VL CDR1-3 sequences are shown in lighter letters. Figure 13B shows the VH amino acid sequences of four germline revertant mutants 21D9.b, 21D9.c, 21D9.d, and 21D9.e, along with the IgG1.3 heavy chain constant region (i.e., 21D9.b-.ehIgG1.3). Amino acid residues shown in capital letters in Figure 13B are germline revertant substitutions. [Figure 14A] Figures 14A and 14B show the amino acid sequences of the variable domains of the heavy and light chains of the ILT4 antibody 21A5, respectively, and their alignment with the germline immunoglobulin sequences hIGHV1-46*01 (Figure 14A) and hIGKV3-20*01 (Figure 14B). The AbM VH CDR1 and Kabat VH CDR2, VH CDR3, and VL CDR1-3 sequences are shown in lighter font. Arrows indicate germline reverse mutations. [Figure 14B] Figures 14A and 14B show the amino acid sequences of the variable domains of the heavy and light chains of the ILT4 antibody 21A5, respectively, and their alignment with the germline immunoglobulin sequences hIGHV1-46*01 (Figure 14A) and hIGKV3-20*01 (Figure 14B). The AbM VH CDR1 and Kabat VH CDR2, VH CDR3, and VL CDR1-3 sequences are shown in lighter font. Arrows indicate germline reverse mutations. [Figure 15]Figure 15 shows the amino acid sequences of the variable domains of the heavy and light chains of the ILT4 antibody 10F10, respectively, and their alignment with the germline immunoglobulin sequences hIGHV3-30*01 (top 3 rows) and hIGKV1-13*02 (bottom 3 rows). The AbM VH CDR1, Kabat VH CDR2, VH CDR3, and VL CDR1-3 sequences are shown in lighter font. Arrows indicate germline reverse mutations. [Figure 16] Figure 16 shows a sensorogram of the binding of the anti-hILT4 antibody to hILT4. A schematic diagram of the method used is shown above the sensorogram panel. [Figure 17] Figure 17 shows the binding of anti-hILT4 antibodies 2H2, 10F10 wild-type (WT, i.e., parent antibody), 21A5.a, 21A5 WT, 21D9 WT, 21D9e, and isotype control hIgG1.3 to hILT4-transfected CHO cells as a function of antibody concentration, and as determined by flow cytometry. EC50 values ​​are provided below the graph of binding data. [Figure 18A] Figure 18A shows the binding of specific anti-hILT4 antibodies and isotype controls to hILT4-transfected CHO cells as a function of antibody concentration, and as determined by flow cytometry. EC50 values ​​are provided to the right of the binding data graph. Figure 18B shows the binding of anti-hILT4 antibodies to human monocytes isolated from the peripheral blood of healthy donors. Antibody species are listed next to the graph, and EC50 values ​​are provided in the table immediately below the graph. [Figure 18B] Figure 18A shows the binding of specific anti-hILT4 antibodies and isotype controls to hILT4-transfected CHO cells as a function of antibody concentration, and as determined by flow cytometry. EC50 values ​​are provided to the right of the binding data graph. Figure 18B shows the binding of anti-hILT4 antibodies to human monocytes isolated from the peripheral blood of healthy donors. Antibody species are listed next to the graph, and EC50 values ​​are provided in the table immediately below the graph. [Figure 19A]Figure 19A shows flow cytometry diagrams of the binding of anti-hILT4 antibodies 21D9, 21D9.e, 21A5, 21A5.a, 10F10, and 2H2 (from left to right) to human LILRA1, human LILRA2, human LILRA3, human LILRA4, human LILRA5, human LILRA6, human ILT2, human ILT3, human ILT4, human ILT5, and human LIR8 (from top to bottom). Figure 19B shows flow cytometry diagrams of the binding of anti-hILT4 antibody 21A5 and germline revertant mutant 21A5.a to human LIRA1, LIRA3, and ILT4. [Figure 19B] Figure 19A shows flow cytometry diagrams of the binding of anti-hILT4 antibodies 21D9, 21D9.e, 21A5, 21A5.a, 10F10, and 2H2 (from left to right) to human LILRA1, human LILRA2, human LILRA3, human LILRA4, human LILRA5, human LILRA6, human ILT2, human ILT3, human ILT4, human ILT5, and human LIR8 (from top to bottom). Figure 19B shows flow cytometry diagrams of the binding of anti-hILT4 antibody 21A5 and germline revertant mutant 21A5.a to human LIRA1, LIRA3, and ILT4. [Figure 20] Figures 20A and 20B show the percentage of maximum binding of specific antibodies 21D9 ("21D9 IgG1.1") and 21D9.e (Figure 20A), and 21A5 ("21A5WT") and 21A5.a (Figure 20B) to specific human LILRA / LILRB family members, LILRA5 and ILT4 in Figure 20A, and LILRA1, LILRA3, and ILT4 in Figure 20B, as a function of antibody concentration, indicating that the anti-ILT4 antibodies 21D9, 21D9.e, 21A5, and 21A5.a exhibit weak cross-reactivity with other LILRA family members. [Figure 21A]Figures 21A–D show the T cell activity of anti-hILT4 antibodies. Figure 21A is a histogram of 3H thymidine uptake of T cells co-cultured with hILT4 and OKT3 transfected CHO cells, incubated with no antibody, isotype control hIgG1.3 antibody, and antibodies 21D9 ("21D9 WT"), 21D9.e, 21A5 ("21A5 WT"), 21A5.a, and 10F10 ("10F10 WT"), as a function of antibody concentration (20, 4, 0.8, 0.16, 0.032, and 0.0064 μg / mL) (from left to right on the x-axis). Figure 21B provides the relevant EC50 values. Figure 21C shows the T cell activity of anti-hILT4 antibodies. Figure 21C is a histogram of IFNγ production in T cells co-cultured with hILT4 and OKT3 transfected CHO cells, incubated with no antibody, isotype control hIgG1.3 antibody, and antibodies 21D9 ("21D9 WT"), 21D9.e, 21A5 ("21A5 WT"), 21A5.a, and 10F10 ("10F10 WT"), as a function of antibody concentration (20, 4, 0.8, 0.16, 0.032, and 0.0064 μg / ml) (from left to right on the x-axis). Figure 21D provides the relevant EC50 values. [Figure 21B]Figures 21A–D show the T cell activity of anti-hILT4 antibodies. Figure 21A is a histogram of 3H thymidine uptake of T cells co-cultured with hILT4 and OKT3 transfected CHO cells, incubated with no antibody, isotype control hIgG1.3 antibody, and antibodies 21D9 ("21D9 WT"), 21D9.e, 21A5 ("21A5 WT"), 21A5.a, and 10F10 ("10F10 WT"), as a function of antibody concentration (20, 4, 0.8, 0.16, 0.032, and 0.0064 μg / mL) (from left to right on the x-axis). Figure 21B provides the relevant EC50 values. Figure 21C shows the T cell activity of anti-hILT4 antibodies. Figure 21C is a histogram of IFNγ production in T cells co-cultured with hILT4 and OKT3 transfected CHO cells, incubated with no antibody, isotype control hIgG1.3 antibody, and antibodies 21D9 ("21D9 WT"), 21D9.e, 21A5 ("21A5 WT"), 21A5.a, and 10F10 ("10F10 WT"), as a function of antibody concentration (20, 4, 0.8, 0.16, 0.032, and 0.0064 μg / ml) (from left to right on the x-axis). Figure 21D provides the relevant EC50 values. [Figure 21C]Figures 21A–D show the T cell activity of anti-hILT4 antibodies. Figure 21A is a histogram of 3H thymidine uptake of T cells co-cultured with hILT4 and OKT3 transfected CHO cells, incubated with no antibody, isotype control hIgG1.3 antibody, and antibodies 21D9 ("21D9 WT"), 21D9.e, 21A5 ("21A5 WT"), 21A5.a, and 10F10 ("10F10 WT"), as a function of antibody concentration (20, 4, 0.8, 0.16, 0.032, and 0.0064 μg / mL) (from left to right on the x-axis). Figure 21B provides the relevant EC50 values. Figure 21C shows the T cell activity of anti-hILT4 antibodies. Figure 21C is a histogram of IFNγ production in T cells co-cultured with hILT4 and OKT3 transfected CHO cells, incubated with no antibody, isotype control hIgG1.3 antibody, and antibodies 21D9 ("21D9 WT"), 21D9.e, 21A5 ("21A5 WT"), 21A5.a, and 10F10 ("10F10 WT"), as a function of antibody concentration (20, 4, 0.8, 0.16, 0.032, and 0.0064 μg / ml) (from left to right on the x-axis). Figure 21D provides the relevant EC50 values. [Figure 21D]Figures 21A–D show the T cell activity of anti-hILT4 antibodies. Figure 21A is a histogram of 3H thymidine uptake of T cells co-cultured with hILT4 and OKT3 transfected CHO cells, incubated with no antibody, isotype control hIgG1.3 antibody, and antibodies 21D9 ("21D9 WT"), 21D9.e, 21A5 ("21A5 WT"), 21A5.a, and 10F10 ("10F10 WT"), as a function of antibody concentration (20, 4, 0.8, 0.16, 0.032, and 0.0064 μg / mL) (from left to right on the x-axis). Figure 21B provides the relevant EC50 values. Figure 21C shows the T cell activity of anti-hILT4 antibodies. Figure 21C is a histogram of IFNγ production in T cells co-cultured with hILT4 and OKT3 transfected CHO cells, incubated with no antibody, isotype control hIgG1.3 antibody, and antibodies 21D9 ("21D9 WT"), 21D9.e, 21A5 ("21A5 WT"), 21A5.a, and 10F10 ("10F10 WT"), as a function of antibody concentration (20, 4, 0.8, 0.16, 0.032, and 0.0064 μg / ml) (from left to right on the x-axis). Figure 21D provides the relevant EC50 values. [Figure 22A] Figures 22A–D show T cell proliferation (Figures 22A and 22C) and IFNγ production (Figures 22B and 22D) in allogeneic MLR (mixed lymphocyte reaction) of monocyte-derived dendritic cells (MoDCs) co-cultured with T cells. During monocyte differentiation into MoDCs, anti-hILT4 antibodies 21A5.a or 21D9.e (Figures 22A and 22B), 2H2 or 21D9 (Figures 22C and 22D), or isotype controls or no antibody ("untreated") were added. No antibodies were added during the allogeneic MLR assay. [Figure 22B]Figures 22A–D show T cell proliferation (Figures 22A and 22C) and IFNγ production (Figures 22B and 22D) in allogeneic MLR (mixed lymphocyte reaction) of monocyte-derived dendritic cells (MoDCs) co-cultured with T cells. During monocyte differentiation into MoDCs, anti-hILT4 antibodies 21A5.a or 21D9.e (Figures 22A and 22B), 2H2 or 21D9 (Figures 22C and 22D), or isotype controls or no antibody ("untreated") were added. No antibodies were added during the allogeneic MLR assay. [Figure 22C] Figures 22A–D show T cell proliferation (Figures 22A and 22C) and IFNγ production (Figures 22B and 22D) in allogeneic MLR (mixed lymphocyte reaction) of monocyte-derived dendritic cells (MoDCs) co-cultured with T cells. During monocyte differentiation into MoDCs, anti-hILT4 antibodies 21A5.a or 21D9.e (Figures 22A and 22B), 2H2 or 21D9 (Figures 22C and 22D), or isotype controls or no antibody ("untreated") were added. No antibodies were added during the allogeneic MLR assay. [Figure 22D] Figures 22A–D show T cell proliferation (Figures 22A and 22C) and IFNγ production (Figures 22B and 22D) in allogeneic MLR (mixed lymphocyte reaction) of monocyte-derived dendritic cells (MoDCs) co-cultured with T cells. During monocyte differentiation into MoDCs, anti-hILT4 antibodies 21A5.a or 21D9.e (Figures 22A and 22B), 2H2 or 21D9 (Figures 22C and 22D), or isotype controls or no antibody ("untreated") were added. No antibodies were added during the allogeneic MLR assay. [Figure 23A] Figures 23A–D show the expression levels of CD83 and CD86 on monocyte-derived dendritic cells (MoDCs). Anti-hILT4 antibodies 21D9.e, 21A5.a (Figure 23A), 21A5, 10F10, 21D9 (Figure 23B), or 2H2 (Figures 23C and 23D), or isotype controls, were incubated during monocyte differentiation into MoDCs, or left untreated (no antibody). [Figure 23B]Figures 23A–D show the expression levels of CD83 and CD86 on monocyte-derived dendritic cells (MoDCs). Anti-hILT4 antibodies 21D9.e, 21A5.a (Figure 23A), 21A5, 10F10, 21D9 (Figure 23B), or 2H2 (Figures 23C and 23D), or isotype controls, were incubated during monocyte differentiation into MoDCs, or left untreated (no antibody). [Figure 23C] Figures 23A–D show the expression levels of CD83 and CD86 on monocyte-derived dendritic cells (MoDCs). Anti-hILT4 antibodies 21D9.e, 21A5.a (Figure 23A), 21A5, 10F10, 21D9 (Figure 23B), or 2H2 (Figures 23C and 23D), or isotype controls, were incubated during monocyte differentiation into MoDCs, or left untreated (no antibody). [Figure 23D] Figures 23A–D show the expression levels of CD83 and CD86 on monocyte-derived dendritic cells (MoDCs). Anti-hILT4 antibodies 21D9.e, 21A5.a (Figure 23A), 21A5, 10F10, 21D9 (Figure 23B), or 2H2 (Figures 23C and 23D), or isotype controls, were incubated during monocyte differentiation into MoDCs, or left untreated (no antibody). [Figure 24] Figures 24A and 24B show the levels of TNF-alpha secreted from in vitro differentiated macrophages activated by lipopolysaccharide (LPS) (Figure 24A) or the STING agonist 2'3'-cGAMP (Figure 24B). Anti-ILT4 antibodies 21D9, 21D9.e, 21A5, 21A5.a, 2H2, and 10F10 or isotype controls were included during activation. [Figure 25]Figures 25A and 25B show the percentage of CD4 T cell proliferation (Figure 25A) and the amount of IFN-gamma (Figure 25B) produced from allogeneic MLRs of CD4 T cells co-cultured with macrophages differentiated in vitro in the presence of anti-ILT4 antibodies 21D9, 21D9.e, 21A5, or 21A5.a, or anti-PD-L1 antibody, or isotype control antibody. Figure 25A shows the percentage of CD4 T cell proliferation by FACS analysis based on CFSE dilution, and Figure 25B shows IFNγ production as measured by Alphalisa, both as a function of ILT4 antibody or control. [Figure 26] Figures 26A and 26B show that the combination of anti-ILT4 antibody 21D9 and anti-PD-L1 antibody clone 29E.2A3 enhances IFNγ production to a higher level in macrophage:CD4 T cell allogeneic MLRs compared to each antibody alone. Figure 26A shows one experiment derived from one macrophage:CD4 T allogeneic pair. Figure 26B shows responses derived from multiple allogeneic pairs, with each line representing one experiment derived from one allogeneic pair. Statistical analysis was performed using nonparametric one-way ANOVA multiple comparisons (Friedman test). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 27A] Figures 27A, 27B, and 27C show the HDX epitope mappings of anti-hILT4 Fab 21D9 and 2H2 Fab. Figure 27A shows pepsin-mediated hILT4 sequence coverage. Figure 27B shows differential HDX for 21D9 and 2H2 Fab. Of the sequence portions depicted in the figures, underlined portions have less HDX protection than ununderlined portions, as noted below the figures. Figure 27C shows the HDX epitopes of the two Fabs mapped to the linear sequence of hILT4. The epitope for antibody 21D9 is indicated by a single underline. The epitope for antibody 2H2 is indicated by a double underline and shaded / shaded underlined portions of the sequence. [Figure 27B]Figures 27A, 27B, and 27C show the HDX epitope mappings of anti-hILT4 Fab 21D9 and 2H2 Fab. Figure 27A shows pepsin-mediated hILT4 sequence coverage. Figure 27B shows differential HDX for 21D9 and 2H2 Fab. Of the sequence portions depicted in the figures, underlined portions have less HDX protection than ununderlined portions, as noted below the figures. Figure 27C shows the HDX epitopes of the two Fabs mapped to the linear sequence of hILT4. The epitope for antibody 21D9 is indicated by a single underline. The epitope for antibody 2H2 is indicated by a double underline and shaded / shaded underlined portions of the sequence. [Figure 27C] Figures 27A, 27B, and 27C show the HDX epitope mappings of anti-hILT4 Fab 21D9 and 2H2 Fab. Figure 27A shows pepsin-mediated hILT4 sequence coverage. Figure 27B shows differential HDX for 21D9 and 2H2 Fab. Of the sequence portions depicted in the figures, underlined portions have less HDX protection than ununderlined portions, as noted below the figures. Figure 27C shows the HDX epitopes of the two Fabs mapped to the linear sequence of hILT4. The epitope for antibody 21D9 is indicated by a single underline. The epitope for antibody 2H2 is indicated by a double underline and shaded / shaded underlined portions of the sequence. [Figure 28] Figures 28A, B, C, and D show the inhibition of hILT4 binding to HLA-A and HLA-B by antibodies 21D9 (Figure 28A), 2H2 (Figure 28B), 10F10 (Figure 28C), and 21A5 (Figure 28D). [Figure 29] Figures 29A-D show that 21D9e.IgG1.3 enhances the secretion of both IFN-γ and TNF-α by CD4+ and CD8+ T cells, as determined by intracellular IFN-γ and TNF-α staining. Figure 29A shows IFN-γ secretion in CD4+ T cells, Figure 29B shows TNF-α secretion in CD4+ T cells, Figure 29C shows IFN-γ secretion in CD8+ T cells, and Figure 29D shows TNF-α secretion in CD8+ T cells. [Figure 30]Figure 30 shows that anti-ILT4 antibodies 21D9, 21A5, and 21D9 Fab enhance IFN-γ secretion upon antigen stimulation in the CMV Lysate Assay. Nivolumab was used as a positive control, and the absence of antibodies ("Ab-none") and isotype controls were used as negative controls. The concentrations of anti-ILT4 antibodies, from left to right, are 0.00128; 0.0064; 0.032; 0.16; 0.8; 4; and 20 μg / mL (bars for each concentration are shown in the histogram). Antibodies and controls are shown from left to right, in the same order as shown on the right side of the histogram (e.g., "Ab-none" corresponds to the left bar, and "Nivo" corresponds to the right bar). [Figure 31] Figure 31 shows that anti-ILT4 antibodies 2H2 and 21D9, as IgG1.1f or Fab, enhance T cell proliferation (measured by 3H uptake) in monocyte:T allogeneic MLR. [Figure 32] Figures 32A-C show that the anti-ILT4 antibodies 21D9e.IgG1.3 (labeled "ILT4.8" in the figure) and 9G4.IgG1.3 (labeled "ILT4.1") (see Table 1 for antibody naming conventions) promote the expression of the costimulatory molecules CD86 (Figure 32A) and CD80 (Figure 32B), as well as the dendritic cell activation marker CD83 (Figure 32C), on cynomolgus monkey monocyte-derived dendritic cells in vitro. Each antibody was added during the differentiation of monocyte-derived dendritic cells (DCs) into cynomolgus monkey monocytes. [Figure 33] Figures 33A and 33B show the levels of TNF-alpha secreted from in vitro differentiated macrophages activated by lipopolysaccharide (LPS) (Figure 33A) or the STING agonist 2'3'cGAMP (Figure 33B). Anti-ILT4 antibodies 21D9.e and 10F10.4 or isotype controls were included during activation. [Figure 34] Figure 34 shows that the levels of TNF-alpha secreted by in vitro differentiated macrophages incubated with 21D9.e in the context of IgG1, IgG4, or IgG1.3 are similar. [Figure 35]Figure 35 shows the percentages of monomers, high molecular weight (HMW) species, and low molecular weight (LMW) species of 21D9.e.IgG1.3 after 0, 1, 2, and 3 months at 25°C or 40°C. Monomers, HMW species, and LMW species correspond to the lower, middle, and upper portions of their respective bars in the histogram. [Figure 36] Figure 36 shows the percentage of activated basophils in a total of eight donors after incubation with 21D9e.IgG1.3 ("ILT4.8" in the figure), 21A5.a.IgG1.3 ("ILT4.9"), isotype control antibody "anti-DT", anti-FcεRI, or N-formylmethionine-leucyl-phenylalanine (fMLP), demonstrating the lack of basophil activation by 21D9e.IgG1.3 (ILT4.8) and 21A5.a.IgG1.3 ("ILT4.9"). [Figures 37A-37B] Figures 37A–D show the results of experiments to determine the epitopes of specific antibodies. Figures 37A and 37B show the results of competitive (or cross-blockage) assays of 2H2 (Figure 37A) and 21D9 (Figure 37B) of hILT4 expressed on the surface of CHO cells with 10F10 and 21A5 antibodies. Figure 37C is a diagram showing a three-dimensional representation of hILT4 including the locations of Ig-like domains 1 and 2, and indicating the regions of hILT4 to which antibodies 21D9, 29A5, 2H2, and 10F10 bind. Figure 37D shows a further three-dimensional representation of hILT4 including the locations of Ig-like domains 1 and 2, and indicating the regions of hILT4 to which antibodies 21D9, 21A5, and 10F10 bind. [Figure 37C]Figures 37A–D show the results of experiments to determine the epitopes of specific antibodies. Figures 37A and 37B show the results of competitive (or cross-blockage) assays of 2H2 (Figure 37A) and 21D9 (Figure 37B) of hILT4 expressed on the surface of CHO cells with 10F10 and 21A5 antibodies. Figure 37C is a diagram showing a three-dimensional representation of hILT4 including the locations of Ig-like domains 1 and 2, and indicating the regions of hILT4 to which antibodies 21D9, 29A5, 2H2, and 10F10 bind. Figure 37D shows a further three-dimensional representation of hILT4 including the locations of Ig-like domains 1 and 2, and indicating the regions of hILT4 to which antibodies 21D9, 21A5, and 10F10 bind. [Figure 37D] Figures 37A–D show the results of experiments to determine the epitopes of specific antibodies. Figures 37A and 37B show the results of competitive (or cross-blockage) assays of 2H2 (Figure 37A) and 21D9 (Figure 37B) of hILT4 expressed on the surface of CHO cells with 10F10 and 21A5 antibodies. Figure 37C is a diagram showing a three-dimensional representation of hILT4 including the locations of Ig-like domains 1 and 2, and indicating the regions of hILT4 to which antibodies 21D9, 29A5, 2H2, and 10F10 bind. Figure 37D shows a further three-dimensional representation of hILT4 including the locations of Ig-like domains 1 and 2, and indicating the regions of hILT4 to which antibodies 21D9, 21A5, and 10F10 bind. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Certain embodiments of this disclosure are summarized in the claims, which are located at the end of this disclosure. For example, this disclosure includes the following embodiments, as well as other embodiments described in further sections of the text and in the drawings and arrangement of this specification. [Means for solving the problem]

[0007] Embodiment 1: An isolated antibody that specifically binds to human ILT4 (hILT4 or ILT4), comprising a heavy chain and a light chain, and having the following characteristics: - For example, 10 -8 M or less, or 10 -9 K below M D Specific binding to hILT4 (e.g., including the amino acid sequences of SEQ ID NOs. 108, 109, 111, 112, or 119); - Lack of specific binding to hILT2, hILT3, and / or hILT5; - Lack of specific binding to one or more members of the LILRA and / or LILRB family; - For example, stimulating T cell activation in a mixed lymphocyte reaction (MLR) assay, as measured by increased T cell proliferation or IFN-gamma secretion, as shown in the assay described in the examples; - For example, stimulating the differentiation or activation of monocytes into macrophages, as shown in the assay described in the examples; for example, stimulating the differentiation of monocytes into inflammatory macrophages; - For example, promoting the expression of CD83 and CD86 on human monocyte-derived immature dendritic cells (Mo-iDCs), as shown in the assay described in the examples; - For example, enhancing IFN-γ secretion upon antigen stimulation in a cytomegalovirus (CMV) lysate assay, as shown in the assay described in the examples; - For example, enhancing IFN-γ and TNF-α secretion by CD4+ and CD8+ T cells in an allogeneic mixed lymphocyte response (MLR) assay upon CD3 stimulation, as shown in the assay described in the examples; - Inhibiting the binding of HLA-A and / or HLA-B to ILT4; - For example, when determined by deuterium exchange (HDX), as shown in the HDX assay described in the examples, 70 ITRIRPELVKNGQFHIPSITWEHTGRYGCQY 100Bind to (Accession No. 122); - Compete with the antibodies described herein for binding to hILT4; - Specifically bind to cynomolgus ILT4 containing Accession No. 118, as shown, for example, in the binding assays described in the Examples; - Inhibit the binding of human ILT4 (hILT4) to ILT4 binding partners such as MHC class I molecules such as HLA-A and HLA-B (e.g., inhibit the binding of hILT4 to both HLA-A and HLA-B); - Have the HLA-A and HLA-B binding profiles shown in FIG. 28; - Promote the inflammatory polarization of macrophages to M1 macrophages; - Have a binding profile as shown in FIG. 27; - Not induce (or elicit) basophil activation; - The following regions of hILT4: (i) 70 ITRIRPEL 77 (Accession No. 120) and / or 78 VKNGQFHIPSITWEHTGRYGCQY 100 (Accession No. 121); (ii) 70 ITRIRPELVKNGQFHIPSITWEHTGRYGCQY 100 (Accession No. 122); or (iii) 154 ILCKEGEEEHPQCLNSQPHARGSSRAIF 181 (Accession No. 123) and / or 425 SSPPPTGPIS 434 (Accession No. 124), [in the sequence, the amino acid numbering of hILT4 is that of immature hILT4 (i.e., ILT4 containing its native signal sequence)] bind, and when binding to (i) or (ii), not significantly bind to other regions of the extracellular domain of ILT4, such as regions or residues located N-terminal to amino acid I70, as necessary; and - For example, as described in the examples, interacting with one or more (or all) of the amino acid residues Lys43, Ile49, Thr50 and Arg51 of mature hILT4 as determined by carbene footprinting, or interacting with one or more (or all) of the amino acid residues Gly117, Val119, Try120, Leu134, Lys136, Gln149, Pro150, Ile159, Ser161, Val162, Gly163, Pro164, Pro167, His173, Try178, Pro183 and Tyr184 of mature hILT4, or interacting with one or more (or all) of the amino acid residues Glu42, Lys43, Gly76, Cys77, Leu88, Pro91, Pro183 and Tyr184 of mature hILT4 An antibody further comprising one or more of the following.

[0008] Embodiment 2: The isolated antibody according to Embodiment 1, comprising a heavy chain and a light chain, wherein the heavy chain comprises VH CDR1, CDR2 and CDR3 of anti-ILT4 antibodies 9G4 (SEQ ID NOs. 125-127), 9C8 (SEQ ID NOs. 131-133), 2H2 (SEQ ID NOs. 137-139), 2E5 (SEQ ID NOs. 143-145), 24E5 (SEQ ID NOs. 149-151), 21D9 (SEQ ID NOs. 155-157), 21A5 (SEQ ID NOs. 161-163), or 10F10 (SEQ ID NOs. 167-169).

[0009] Embodiment 3: An isolated antibody according to Embodiment 1 or 2, comprising a heavy chain and a light chain, wherein the light chain comprises VL CDR1, CDR2, and CDR3 of anti-ILT4 antibodies 9G4 (SEQ ID NOs: 128-130), 9C8 (SEQ ID NOs: 134-136), 2H2 (SEQ ID NOs: 140-142), 2E5 (SEQ ID NOs: 146-148), 24E5 (SEQ ID NOs: 152-154), 21D9 (SEQ ID NOs: 158-160), 21A5 (SEQ ID NOs: 164-166), or 10F10 (SEQ ID NOs: 170-172).

[0010] Embodiment 4: The isolated antibody according to Embodiment 2, wherein the heavy chain comprises VH CDR1, CDR2 and CDR3 and VL CDR1, CDR2 and CDR3 of anti-ILT4 antibody 9G4 (SEQ ID NOs. 143-148), 24E5 (SEQ ID NOs. 149-154), 21D9 (SEQ ID NOs. 155-160), 21A5 (SEQ ID NOs. 161-166), or 10F10 (SEQ ID NOs. 167-172).

[0011] Embodiment 5: - VH containing the amino acid sequences of 9G4's VH CDR1, CDR2, and CDR3 (SEQ ID NOs: 125-127), and VL containing the amino acid sequences of 9G4's VL CDR1, CDR2, and CDR3 (SEQ ID NOs: 128-130); - VH containing the amino acid sequences of 9C8 CDR1, CDR2, and CDR3 (SEQ ID NOs: 131-133), and VL containing the amino acid sequences of 9C8 CDR1, CDR2, and CDR3 (SEQ ID NOs: 134-136); - VH containing the amino acid sequences of 2H2 CDR1, CDR2, and CDR3 (SEQ ID NOs. 137-139), and VL containing the amino acid sequences of 2H2 CDR1, CDR2, and CDR3 (SEQ ID NOs. 140-142); - VH containing the amino acid sequences of 2E5's VH CDR1, CDR2, and CDR3 (SEQ ID NOs. 143-145), and VL containing the amino acid sequences of 2E5's VL CDR1, CDR2, and CDR3 (SEQ ID NOs. 146-148); - VH containing the amino acid sequences of 24E5's VH CDR1, CDR2, and CDR3 (SEQ ID NOs: 149-151), and VL containing the amino acid sequences of 24E5's VL CDR1, CDR2, and CDR3 (SEQ ID NOs: 152-154); - VH containing the amino acid sequences of 21D9's VH CDR1, CDR2, and CDR3 (SEQ ID NOs. 155-157), and VL containing the amino acid sequences of 21D9's VL CDR1, CDR2, and CDR3 (SEQ ID NOs. 158-160); - VH containing the amino acid sequences of 21D9.b's VH CDR1, CDR2, and CDR3 (sequence numbers 155-157), and VL containing the amino acid sequences of 21D9.b's VL CDR1, CDR2, and CDR3 (sequence numbers 158-160); - VH containing the amino acid sequences of 21D9.c's VH CDR1, CDR2, and CDR3 (SEQ ID NOs. 155-157), and VL containing the amino acid sequences of 21D9.c's VL CDR1, CDR2, and CDR3 (SEQ ID NOs. 158-160); - VH containing the amino acid sequences of 21D9.d's VH CDR1, CDR2, and CDR3 (SEQ ID NOs. 155-157), and VL containing the amino acid sequences of 21D9.d's VL CDR1, CDR2, and CDR3 (SEQ ID NOs. 158-160); - VH containing the amino acid sequences of 21D9.e's VH CDR1, CDR2, and CDR3 (sequence numbers 155-157), and VL containing the amino acid sequences of 21D9.e's VL CDR1, CDR2, and CDR3 (sequence numbers 158-160); - VH containing the amino acid sequences of 21A5's VH CDR1, CDR2, and CDR3 (SEQ ID NOs. 161-163), and VL containing the amino acid sequences of 21A5's VL CDR1, CDR2, and CDR3 (SEQ ID NOs. 164-166); - VH containing the amino acid sequences of 21A5.a's VH CDR1, CDR2, and CDR3 (SEQ ID NOs. 161-163), and VL containing the amino acid sequences of 21A5.a's VL CDR1, CDR2, and CDR3 (SEQ ID NOs. 164-166); - VH containing the amino acid sequences of 10F10's VH CDR1, CDR2, and CDR3 (SEQ ID NOs. 167-169), and VL containing the amino acid sequences of 10F10's VL CDR1, CDR2, and CDR3 (SEQ ID NOs. 170-172); - VH containing the amino acid sequences of 10F10.1's VH CDR1, CDR2, and CDR3 (sequence numbers 167-169), and VL containing the amino acid sequences of 10F10.1's VL CDR1, CDR2, and CDR3 (sequence numbers 170-172); - VH containing the amino acid sequences of 10F10.3's VH CDR1, CDR2, and CDR3 (sequence numbers 167-169), and VL containing the amino acid sequences of 10F10.3's VL CDR1, CDR2, and CDR3 (sequence numbers 170-172); or - VH containing the amino acid sequences of 10F10.4's VH CDR1, CDR2, and CDR3 (sequence numbers 167-169), and VL containing the amino acid sequences of 10F10.4's VL CDR1, CDR2, and CDR3 (sequence numbers 170-172). An isolated antibody according to any one of Embodiments 1 to 4, comprising:

[0012] Embodiment 6: An isolated antibody according to any one of Embodiments 1 to 5, wherein the antibody heavy chain comprises a VH having an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of VH of 9G4 (SEQ ID NO: 51, amino acids 20-135), 9C8 (SEQ ID NO: 55), 2H2 (SEQ ID NO: 58), 2E5 (SEQ ID NO: 63), 24E5 (SEQ ID NO: 67), 21D9 (SEQ ID NO: 71), 21D9.b (SEQ ID NO: 74), 21D9.c (SEQ ID NO: 75), 21D9.d (SEQ ID NO: 78), 21D9.e (SEQ ID NO: 80), 21A5 (SEQ ID NO: 83), 21A5.a (SEQ ID NO: 87), 10F10 (SEQ ID NO: 91), 10F10.1 (SEQ ID NO: 91), 10F10.3 (SEQ ID NO: 91), or 10F10.4 (SEQ ID NO: 91).

[0013] Embodiment 7: An isolated antibody according to any one of Embodiments 1 to 6, wherein the antibody light chain comprises a VL having an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the VL of 9G4 (SEQ ID NO: 50, amino acids 19-125), 9C8 (SEQ ID NO: 54), 2H2 (SEQ ID NO: 59), 2E5 (SEQ ID NO: 62), 24E5 (SEQ ID NO: 66), 21D9 (SEQ ID NO: 70), 21D9.b (SEQ ID NO: 70), 21D9.c (SEQ ID NO: 70), 21D9.d (SEQ ID NO: 70), 21D9.e (SEQ ID NO: 70), 21A5 (SEQ ID NO: 82), 21A5.a (SEQ ID NO: 86), 10F10 (SEQ ID NO: 90), 10F10.1 (SEQ ID NO: 94), 10F10.3 (SEQ ID NO: 96), or 10F10.4 (SEQ ID NO: 114).

[0014] Embodiment 8: An isolated antibody according to any one of Embodiments 1 to 7, wherein the antibody heavy chain comprises a VH having an amino acid sequence that includes 1, 2, 3, 4, or 5 amino acid substitutions, conservative substitutions, or reverse substitutions compared to the amino acid sequence of VH of 9G4 (SEQ ID NO: 51, amino acids 20-135), 9C8 (SEQ ID NO: 55), 2H2 (SEQ ID NO: 58), 2E5 (SEQ ID NO: 63), 24E5 (SEQ ID NO: 67), 21D9 (SEQ ID NO: 71), 21D9.b (SEQ ID NO: 74), 21D9.c (SEQ ID NO: 75), 21D9.d (SEQ ID NO: 78), 21D9.e (SEQ ID NO: 80), 21A5 (SEQ ID NO: 83), 21A5.a (SEQ ID NO: 87), 10F10 (SEQ ID NO: 91), 10F10.1 (SEQ ID NO: 91), 10F10.3 (SEQ ID NO: 91), or 10F10.4 (SEQ ID NO: 91).

[0015] Embodiment 9: An isolated antibody according to any one of Embodiments 1 to 8, wherein the antibody light chain comprises a VL having an amino acid sequence that includes 1, 2, 3, 4, or 5 amino acid substitutions, conservative substitutions, or reverse substitutions compared to the amino acid sequence of a VL of 9G4 (SEQ ID NO: 50, amino acids 19-125), 9C8 (SEQ ID NO: 54), 2H2 (SEQ ID NO: 59), 2E5 (SEQ ID NO: 62), 24E5 (SEQ ID NO: 66), 21D9 (SEQ ID NO: 70), 21D9.b (SEQ ID NO: 70), 21D9.c (SEQ ID NO: 70), 21D9.d (SEQ ID NO: 70), 21D9.e (SEQ ID NO: 70), 21A5 (SEQ ID NO: 82), 21A5.a (SEQ ID NO: 86), 10F10 (SEQ ID NO: 90), 10F10.1 (SEQ ID NO: 94), 10F10.3 (SEQ ID NO: 96), or 10F10.4 (SEQ ID NO: 114).

[0016] Embodiment 10: An isolated antibody according to any one of Embodiments 1 to 9, wherein the heavy chain comprises the VH of the anti-ILT4 antibody 9G4 (SEQ ID NO: 51, amino acids 20-135), 9C8 (SEQ ID NO: 55), 2H2 (SEQ ID NO: 58), 2E5 (SEQ ID NO: 63), 24E5 (SEQ ID NO: 67), 21D9 (SEQ ID NO: 71), 21D9.b (SEQ ID NO: 74), 21D9.c (SEQ ID NO: 75), 21D9.d (SEQ ID NO: 78), 21D9.e (SEQ ID NO: 80), 21A5 (SEQ ID NO: 83), 21A5.a (SEQ ID NO: 87), 10F10 (SEQ ID NO: 91), 10F10.1 (SEQ ID NO: 91), 10F10.3 (SEQ ID NO: 91), or 10F10.4 (SEQ ID NO: 91).

[0017] Embodiment 11: An isolated antibody according to any one of Embodiments 1 to 10, wherein the light chain comprises the VL of anti-ILT4 antibody 9G4 (SEQ ID NO: 50, amino acids 19-125), 9C8 (SEQ ID NO: 54), 2H2 (SEQ ID NO: 59), 2E5 (SEQ ID NO: 62), 24E5 (SEQ ID NO: 66), 21D9 (SEQ ID NO: 70), 21D9.b (SEQ ID NO: 70), 21D9.c (SEQ ID NO: 70), 21D9.d (SEQ ID NO: 70), 21D9.e (SEQ ID NO: 70), 21A5 (SEQ ID NO: 82), 21A5.a (SEQ ID NO: 86), 10F10 (SEQ ID NO: 90), 10F10.1 (SEQ ID NO: 94), 10F10.3 (SEQ ID NO: 96), or 10F10.4 (SEQ ID NO: 114).

[0018] Embodiment 12: Anti-ILT4 antibody 9G4 (SEQ ID NO: 51, amino acids 20-135 and SEQ ID NO: 50, amino acids 19-125), 9C8 (SEQ ID NO: 55 and SEQ ID NO: 54), 2H2 (SEQ ID NO: 58 and SEQ ID NO: 59), 2E5 (SEQ ID NO: 63 and SEQ ID NO: 62), 24E5 (SEQ ID NO: 67 and SEQ ID NO: 66), 21D9 (SEQ ID NO: 71 and SEQ ID NO: 70), 21D9.b (SEQ ID NO: 74 and SEQ ID NO: 70), 21D9.c (SEQ ID NO: 75 and SEQ ID NO: 70), 21D9.d (Sequence number The isolated antibody according to any one of Embodiments 1 to 11, comprising VH and VL of (No. 78 and SEQ ID NO. 70), 21D9.e (SEQ ID NO. 80 and SEQ ID NO. 70), 21A5 (SEQ ID NO. 83 and SEQ ID NO. 82), 21A5.a (SEQ ID NO. 87 and SEQ ID NO. 86), 10F10 (SEQ ID NO. 91 and SEQ ID NO. 90), 10F10.1 (SEQ ID NO. 91 and SEQ ID NO. 94), 10F10.3 (SEQ ID NO. 91 and SEQ ID NO. 96), or 10F10.4 (SEQ ID NO. 91 and SEQ ID NO. 114).

[0019] Embodiment 13: - VH containing the VH CDRs of 9G4 VH (SEQ ID NOs: 125-127), VL containing the VL CDRs of 9G4 VH (SEQ ID NOs: 128-130), and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 9G4 (SEQ ID NOs: 51, amino acids 20-135 and SEQ ID NOs: 50, amino acids 19-125); - VH containing the VH CDR of 9C8 VH (SEQ ID NOs. 131-133), VL containing the VL CDR of 9C8 (SEQ ID NOs. 134-136), and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 9C8 (SEQ ID NOs. 55 and 54); - VH containing the VH CDR of 2H2 VH (SEQ ID NOs. 137-139), VL containing the VL CDR of 2H2 (SEQ ID NOs. 140-142), and VH and VL amino acid sequences that are 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%, or at least 99% identical to the VH and VL of 2H2 (SEQ ID NOs. 58 and 59); - VH containing the VH CDR of 2E5 VH (SEQ ID NOs. 143-145), VL containing the VL CDR of 2E5 (SEQ ID NOs. 146-148), and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 2E5 (SEQ ID NOs. 63 and 62); - VH containing the VH CDR of 24E5 VH (SEQ ID NOs. 149-151), VL containing the VL CDR of 24E5 (SEQ ID NOs. 152-154), and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 24E5 (SEQ ID NOs. 67 and 66); - VH containing the VH CDR of 21D9 VH (SEQ ID NOs. 155-157), VL containing the VL CDR of 21D9 (SEQ ID NOs. 158-160), and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 21D9 (SEQ ID NOs. 71 and 70); - VH containing the VH CDR (SEQ ID NOs. 155-157) of 21D9.b, and VL containing the VL CDR (SEQ ID NOs. 158-160) of 21D9.b, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL (SEQ ID NOs. 74 and 70) of 21D9.b; - VH containing the VH CDR (SEQ ID NOs. 155-157) of 21D9.c, and VL containing the VL CDR (SEQ ID NOs. 158-160) of 21D9.c, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL (SEQ ID NOs. 75 and 70) of 21D9.c; - VH containing the VH CDR (SEQ ID NOs. 155-157) of VH from 21D9.d, and VL containing the VL CDR (SEQ ID NOs. 158-160) of VH from 21D9.d, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL (SEQ ID NOs. 78 and 70) from 21D9.d; - VH containing the VH CDR (SEQ ID NOs. 155-157) of VH from 21D9.e, and VL containing the VL CDR (SEQ ID NOs. 158-160) of 21D9.e, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL (SEQ ID NOs. 80 and 70) of 21D9.e; - VH containing the VH CDR of 21A5 VH (SEQ ID NOs: 161-163), VL containing the VL CDR of 21A5 VH (SEQ ID NOs: 164-166), and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 21A5 (SEQ ID NOs: 83 and 82); - VH containing the VH CDR (SEQ ID NOs. 161-163) of VH from 21A5.a, and VL containing the VL CDR (SEQ ID NOs. 164-166) of 21A5.a, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL (SEQ ID NOs. 87 and 86) of 21A5.a; - VH containing the VH CDR of 10F10 VH (SEQ ID NOs. 167-169), VL containing the VL CDR of 10F10 (SEQ ID NOs. 170-172), and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 10F10 (SEQ ID NOs. 91 and 90); - VH containing the VH CDR (SEQ ID NOs. 167-169) of 10F10.1, and VL containing the VL CDR (SEQ ID NOs. 170-172) of 10F10.1, as well as VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL (SEQ ID NOs. 91 and 94) of 10F10.1; - VH containing the VH CDRs of 10F10.3 (SEQ ID NOs. 167-169), VL containing the VL CDRs of 10F10.3 (SEQ ID NOs. 170-172), and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 10F10.3 (SEQ ID NOs. 91 and 96); or - VH containing the VH CDR (SEQ ID NOs. 167-169) of 10F10.4, and VL containing the VL CDR (SEQ ID NOs. 170-172) of 10F10.4, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 10F10.4 (SEQ ID NOs. 91 and 114). An isolated antibody according to any one of embodiments 1 to 11, including the isolated antibody described above.

[0020] Embodiment 14: - VH containing the amino acid sequence of 9G4 VH (residues 20-135 of SEQ ID NO: 51) and VL containing the amino acid sequence of 9G4 VL (residues 19-125 of SEQ ID NO: 50); - VH containing the amino acid sequence of VH at 9C8, and VL containing the amino acid sequence of VL at 9C8 (SEQ ID NO: 55 and SEQ ID NO: 54); - VH containing the amino acid sequence of VH of 2H2, and VL containing the amino acid sequence of VL of 2H2 (SEQ ID NOs. 58 and 59); - VH containing the amino acid sequence of 2E5 VH, and VL containing the amino acid sequence of 2E5 VL (SEQ ID NOs. 63 and 62); - VH containing the amino acid sequence of VH at 24E5, and VL containing the amino acid sequence of VL at 24E5 (SEQ ID NOs. 67 and 66); - VH containing the amino acid sequence of 21D9 VH, and VL containing the amino acid sequence of 21D9 VL (SEQ ID NOs. 71 and 70); - VH containing the amino acid sequence of VH from 21D9.b, and VL containing the amino acid sequence of VL from 21D9.b (SEQ ID NOs. 74 and 70); - VH containing the amino acid sequence of VH from 21D9.c, and VL containing the amino acid sequence of VL from 21D9.c (SEQ ID NOs. 75 and 70); - VH containing the amino acid sequence of VH from 21D9.d, and VL containing the amino acid sequence of VL from 21D9.d (SEQ ID NOs. 78 and 70); - VH containing the amino acid sequence of VH from 21D9.e, and VL containing the amino acid sequence of VL from 21D9.e (SEQ ID NO: 80 and SEQ ID NO: 70); - VH containing the amino acid sequence of 21A5 VH, and VL containing the amino acid sequence of 21A5 VL (SEQ ID NOs. 83 and 82); - VH containing the amino acid sequence of VH from 21A5.a, and VL containing the amino acid sequence of VL from 21A5.a (SEQ ID NOs: 87 and 86); - VH containing the amino acid sequence of 10F10 VH, and VL containing the amino acid sequence of 10F10 VL (SEQ ID NOs. 91 and 90); - VH containing the amino acid sequence of VH of 10F10.1, and VL containing the amino acid sequence of VL of 10F10.1 (SEQ ID NOs. 91 and 94); - VH containing the amino acid sequence of VH of 10F10.3 and VL containing the amino acid sequence of VL of 10F10.3 (SEQ ID NOs. 91 and 96); or - VH containing the amino acid sequence of 10F10.4 VH, and VL containing the amino acid sequence of 10F10.4 VL (SEQ ID NOs. 91 and 114) An isolated antibody according to any one of Embodiments 1 to 13, including the above.

[0021] Embodiment 15: An isolated antibody according to any one of Embodiments 1 to 14, which is an IgG antibody.

[0022] Embodiment 16: The isolated antibody according to Embodiment 15, wherein the antibody is IgG1, IgG2, or IgG4, and IgG4 optionally includes S228P substitution (EU numbering).

[0023] Embodiment 17: An isolated antibody according to any one of Embodiments 1 to 16, which is an antibody without effector function.

[0024] Embodiment 18: The isolated antibody according to Embodiment 17, wherein the heavy chain constant region contains 1, 2, 3, 4, or 5 mutations in the otherwise wild-type human heavy chain constant region, which reduces the effector function of the antibody compared to an antibody having the same amino acid sequence as otherwise.

[0025] Embodiment 19: An isolated antibody according to any one of Embodiments 1 to 18, wherein the heavy chain constant region of the antibody comprises an IgG1.3 heavy chain constant region, an IgG1 heavy chain constant region having a P238K (Eu numbered) substitution, or an IgG1 heavy chain constant region containing the amino acid sequence described in any one of SEQ ID NOs. 98, 100, 102, 103, or 104.

[0026] Embodiment 20: An isolated antibody according to any one of Embodiments 1 to 16 or Embodiment 19, having an effector function.

[0027] Embodiment 21: The isolated antibody according to Embodiment 20, which is defucosylated (e.g., defucosylated IgG1 antibody).

[0028] Embodiment 22: An isolated antibody according to Embodiment 20 or 21, wherein the heavy chain constant region contains one, two, three, four, or five mutations in the otherwise wild-type human heavy chain constant region, which enhances the effector function of the antibody compared to an antibody having the same amino acid sequence as otherwise.

[0029] Embodiment 23: An isolated antibody according to any one of Embodiments 1 to 22, comprising the HC of anti-ILT4 antibody 9G4, 9C8, 2H2, 2E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, or 10F10.3, wherein the constant region of the HC is IgG1 (e.g., 9G4.IgG1), IgG1.3 (e.g., 9G4.IgG1.3), IgG1.1f (e.g., 9G4.IgG1.1f), IgG4 (e.g., 9G4.IgG4), or IgG4 S228P (EU numbering) (e.g., 9G4.IgG4_S228P).

[0030] Embodiment 24: The following, - IgG1, for example, 9G4.IgG1, 9G4 (sequence number 2), 9C8 (sequence number 4), 2H2 (sequence number 6), 2E5 (sequence number 8), 24E5 (sequence number 10), 21D9 (sequence number 12), 21D9.b (sequence numbers 74 and 98), 21D9.c (sequence numbers 75 and 98), 21D9.d (sequence numbers 78 and 98), 21D9.e (sequence numbers 80 and 98), 21A5 (sequence numbers 83 and 98), 21A5.a (sequence numbers 87 and 98), 10F10 (sequence numbers 91 and 98), 10F10.1 (sequence numbers 91 and 98), 10F10.3 (sequence numbers 91 and 98), or 10F10.4 (sequence numbers 91 and 98), - IgG1, e.g., 9G4.IgG1, 9G4 (SEQ ID NO: 51, amino acids 20-135, and SEQ ID NO: 102), 9C8 (SEQ ID NO: 55 and SEQ ID NO: 102), 2H2 (SEQ ID NO: 58 and SEQ ID NO: 102), 2E5 (SEQ ID NO: 63 and SEQ ID NO: 102), 24E5 (SEQ ID NO: 67 and SEQ ID NO: 102), 21D9 (SEQ ID NO: 71 and SEQ ID NO: 102), 21D9.b (SEQ ID NO: 74 and SEQ ID NO: 102), 21D9.c (SEQ ID NO: 75 and SEQ ID NO: 102), 102), 21D9.d (sequences 78 and 102), 21D9.e (sequences 80 and 102), 21A5 (sequences 83 and 102), 21A5.a (sequences 87 and 102), 10F10 (sequences 91 and 102), 10F10.1 (sequences 91 and 102), 10F10.3 (sequences 91 and 102), or 10F10.4 (sequences 91 and 102), - IgG1.3 (e.g., 9G4.IgG1.3), 9G4 (SEQ ID NO: 51, amino acids 20-135, and SEQ ID NO: 100), 9C8 (SEQ ID NO: 55 and SEQ ID NO: 100), 2H2 (SEQ ID NO: 58 and SEQ ID NO: 100), 2E5 (SEQ ID NO: 63 and SEQ ID NO: 100), 24E5 (SEQ ID NO: 67 and SEQ ID NO: 100), 21D9 ((i) SEQ ID NO: 113, or (ii) SEQ ID NO: 71 and SEQ ID NO: 10 0), 21D9.b (sequence number 36), 21D9.c (sequence number 38), 21D9.d (sequence number 40), 21D9.e (sequence number 13), 21A5 (sequence number 15), 21A5.a (sequence number 17), 10F10 (sequence number 19), 10F10.1 (sequence numbers 91 and 100), 10F10.3 (sequence numbers 91 and 100), or 10F10.4 (sequence numbers 91 and 100); - IgG1.1f (e.g., 9G4.IgG1.1f), 9G4 (SEQ ID NO: 51, amino acids 20-135, and SEQ ID NO: 103), 9C8 (SEQ ID NO: 55 and SEQ ID NO: 103), 2H2 (SEQ ID NO: 58 and SEQ ID NO: 103), 2E5 (SEQ ID NO: 63 and SEQ ID NO: 103), 24E5 (SEQ ID NO: 67 and SEQ ID NO: 103), 21D9 (SEQ ID NO: 71 and SEQ ID NO: 103), 21D9.b (SEQ ID NO: 74 and SEQ ID NO: 103), 21D9.c (SEQ ID NO: 75 and 21D9.d (sequences 78 and 103), 21D9.e (sequences 80 and 103), 21A5 (sequences 83 and 103), 21A5.a (sequences 87 and 103), 10F10 (sequences 91 and 103), 10F10.1 (sequences 91 and 103), 10F10.3 (sequences 91 and 103), or 10F10.4 (sequences 91 and 103), - IgG1fa.P238K (e.g., 9G4.IgG1fa.P238K), 9G4 (SEQ ID NO: 51, amino acids 20-135, and SEQ ID NO: 104), 9C8 (SEQ ID NO: 55 and SEQ ID NO: 104), 2H2 (SEQ ID NO: 58 and SEQ ID NO: 104), 2E5 (SEQ ID NO: 63 and SEQ ID NO: 104), 24E5 (SEQ ID NO: 67 and SEQ ID NO: 104), 21D9 (SEQ ID NO: 71 and SEQ ID NO: 104), 21D9.b (SEQ ID NO: 74 and SEQ ID NO: 104), 21D9.c (SEQ ID NO: 75 and SEQ ID NO: 104), 21D9.d (SEQ ID NO: 78 and SEQ ID NO: 104), 21D9.e (SEQ ID NO: 80 and SEQ ID NO: 104), 21A5 (SEQ ID NO: 83 and SEQ ID NO: 104), 21A5.a (SEQ ID NO: 87 and SEQ ID NO: 104), 10F10 (SEQ ID NO: 91 and SEQ ID NO: 104), 10F10.1 (SEQ ID NO: 91 and SEQ ID NO: 104), 10F10.3 (SEQ ID NO: 91 and SEQ ID NO: 104), or 10F10.4 (SEQ ID NO: 91 and SEQ ID NO: 104), or - IgG4 S228P (e.g., 9G4.IgG4 S228P), 9G4 (SEQ ID NO: 51, amino acids 20-135, and SEQ ID NO: 179), 9C8 (SEQ ID NO: 55 and SEQ ID NO: 179), 2H2 (SEQ ID NO: 58 and SEQ ID NO: 179), 2E5 (SEQ ID NO: 63 and SEQ ID NO: 179), 24E5 (SEQ ID NO: 67 and SEQ ID NO: 179), 21D9 (SEQ ID NO: 71 and SEQ ID NO: 179), 21D9.b (SEQ ID NO: 74 and SEQ ID NO: 179), 21D9.c (SEQ ID NO: 75 and SEQ ID NO: 179) ), 21D9.d (sequences 78 and 179), 21D9.e (sequences 80 and 179), 21A5 (sequences 83 and 179), 21A5.a (sequences 87 and 179), 10F10 (sequences 91 and 179), 10F10.1 (sequences 91 and 179), 10F10.3 (sequences 91 and 179), or 10F10.4 (sequences 91 and 179) The isolated antibody according to Embodiment 23, comprising the HC amino acid sequence.

[0031] Embodiment 25: The isolated antibody according to Embodiment 23, wherein the HC of the antibody lacks a C-terminal lysine residue.

[0032] Embodiment 26: The isolated antibody according to Embodiment 23, wherein the HC of the antibody contains one of the heavy chain constant region amino acid sequences of SEQ ID NOs: 98, 100, 102, 103, 104, or 179.

[0033] Embodiment 27: An isolated antibody according to any one of Embodiments 1 to 26, comprising LC of anti-ILT4 antibody 9G4, 9C8, 2H2, 2E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4.

[0034] Embodiment 28: The isolated antibody according to Embodiment 27, wherein the light chain constant region is the human kappa light chain constant region.

[0035] Embodiment 29: The isolated antibody according to Embodiment 27, wherein LC contains the sequence 9G4 (SEQ ID NO: 1), 9C8 (SEQ ID NO: 3), 2H2 (SEQ ID NO: 5), 2E5 (SEQ ID NO: 7), 24E5 (SEQ ID NO: 9), 21D9 (SEQ ID NO: 11), 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5 (SEQ ID NO: 14), 21A5.a (SEQ ID NO: 16), 10F10 (SEQ ID NO: 18), 10F10.1 (SEQ ID NO: 20), 10F10.3 (SEQ ID NO: 21), or 10F10.4 (SEQ ID NO: 116).

[0036] Embodiment 30: An isolated antibody according to any one of Embodiments 1 to 29, comprising HC and LC of anti-ILT4 antibody 9G4, 9C8, 2H2, 2E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4, wherein the constant region of the HC is IgG1 (e.g., 9G4.IgG1), IgG1.3 (e.g., 9G4.IgG1.3), IgG1.1f (e.g., 9G4.IgG1.1f), IgG4 (e.g., 9G4.IgG4), or IgG4 S228P (EU numbering) (e.g., 9G4.IgG4_S228P).

[0037] Embodiment 31 - A heavy chain (HC) containing the amino acid sequence of the 9G4 heavy chain ((i) SEQ ID NO: 2, or (ii) SEQ ID NO: 51, amino acids 20-135, and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain (LC) containing the 9G4 light chain amino acid sequence (SEQ ID NO: 1); - A heavy chain containing the amino acid sequence of the 9C8 heavy chain ((i) SEQ ID NO: 4, or (ii) SEQ ID NO: 55, and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the 9C8 light chain (SEQ ID NO: 3); - A heavy chain containing the amino acid sequence of the 2H2 heavy chain ((i) SEQ ID NO: 6, or (ii) SEQ ID NO: 58, and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the 2H2 light chain (SEQ ID NO: 5); - A heavy chain containing the amino acid sequence of the 2E5 heavy chain ((i) SEQ ID NO: 8, or (ii) SEQ ID NO: 63, and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the 2E5 light chain (SEQ ID NO: 7); - A heavy chain containing the amino acid sequence of the 24E5 heavy chain ((i) SEQ ID NO: 10, or (ii) SEQ ID NO: 67, and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the 24E5 light chain (SEQ ID NO: 9); - A heavy chain containing the amino acid sequence of the 21D9 heavy chain ((i) SEQ ID NO: 12 or 113, or (ii) SEQ ID NO: 71 and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the 21D9 light chain (SEQ ID NO: 11); - A heavy chain containing the amino acid sequence of the heavy chain of 21D9.b (sequence number 74 and one of sequence numbers 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the light chain of 21D9.b (sequence number 11); - A heavy chain containing the amino acid sequence of the heavy chain of 21D9.c (sequence number 75 and one of sequence numbers 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the light chain of 21D9.c (sequence number 11); - A heavy chain containing the amino acid sequence of the heavy chain of 21D9.d (sequence number 78 and one of sequence numbers 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the light chain of 21D9.d (sequence number 11); - A heavy chain containing the amino acid sequence of the heavy chain of 21D9.e ((i) SEQ ID NOs. 13, 176, 177, or 178; or (ii) SEQ ID NOs. 80 and one of SEQ ID NOs. 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the light chain of 21D9.e (SEQ ID NOs. 11); - A heavy chain containing the amino acid sequence of the 21A5 heavy chain ((i) SEQ ID NO: 15 or (ii) SEQ ID NO: 83 and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the 21A5 light chain (SEQ ID NO: 14); - A heavy chain containing the amino acid sequence of the heavy chain of 21A5.a ((i) SEQ ID NO: 17 or (ii) SEQ ID NO: 87 and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the light chain of 21A5.a (SEQ ID NO: 16); - A heavy chain containing the amino acid sequence of the 10F10 heavy chain ((i) SEQ ID NO: 19 or (ii) SEQ ID NO: 91 and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the 10F10 light chain (SEQ ID NO: 18); - A heavy chain containing the amino acid sequence of the 10F10.1 heavy chain ((i) SEQ ID NO: 19 or (ii) SEQ ID NO: 91 and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the 10F10.1 light chain (SEQ ID NO: 20); - A heavy chain containing the amino acid sequence of the heavy chain of 10F10.3 ((i) SEQ ID NO: 19 or (ii) SEQ ID NO: 91 and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179) and a light chain containing the amino acid sequence of the light chain of 10F10.3 (SEQ ID NO: 21); or - A heavy chain containing the amino acid sequence of the 10F10.4 heavy chain ((i) SEQ ID NO: 19 or (ii) SEQ ID NO: 91 and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179) and a light chain containing the amino acid sequence of the 10F10.4 light chain (SEQ ID NO: 116). The isolated antibody according to Embodiment 30, including the aforementioned antibody.

[0038] Embodiment 32: An isolated antibody that specifically binds to human ILT4, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 13 and a light chain containing the light chain amino acid sequence of SEQ ID NO: 11.

[0039] Embodiment 33: An isolated antibody that specifically binds to human ILT4, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 176 and a light chain containing the light chain amino acid sequence of SEQ ID NO: 11.

[0040] Embodiment 34: An isolated antibody that specifically binds to human ILT4, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 177 and a light chain containing the light chain amino acid sequence of SEQ ID NO: 11.

[0041] Embodiment 35: An isolated antibody that specifically binds to human ILT4, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 178 and a light chain containing the light chain amino acid sequence of SEQ ID NO: 11.

[0042] Embodiment 36: An isolated antibody according to any one of Embodiments 32 to 35, having one or more properties of the antibody described in Embodiment 1.

[0043] Embodiment 37: An isolated antibody according to any one of Embodiments 1 to 36, which is a full-length antibody.

[0044] Embodiment 38: An isolated antibody according to any one of Embodiments 1 to 14, which is an antibody fragment.

[0045] Embodiment 39: An isolated antibody according to any one of Embodiments 1 to 38, which is a multimeric (e.g., dimeric or trimer) antibody.

[0046] Embodiment 40: An isolated antibody according to any one of Embodiments 1 to 39, which is linked (e.g., covalently) to another molecule.

[0047] Embodiment 41: The isolated antibody according to Embodiment 40, wherein another molecule is used as a label.

[0048] Embodiment 42: The isolated antibody according to Embodiment 40, wherein the other molecule is a peptide.

[0049] Embodiment 43: An isolated antibody according to Embodiment 40, which is an antibody-drug conjugate (ADC).

[0050] Embodiment 44: An isolated nucleic acid encoding an antibody according to any one of Embodiments 1 to 43.

[0051] Embodiment 45: An isolated nucleic acid encoding the heavy and / or light chain of an antibody according to any one of Embodiments 1 to 43.

[0052] Embodiment 46: A set of at least two isolated nucleic acids encoding the heavy and light chains of an antibody according to any one of Embodiments 1 to 43.

[0053] Embodiment 47: A composition comprising a nucleic acid encoding the heavy chain of an antibody according to any one of Embodiments 1 to 43 and a nucleic acid encoding the light chain of an antibody according to any one of Embodiments 1 to 43.

[0054] Embodiment 48: A cell comprising an isolated nucleic acid according to any one of Embodiments 44 to 46 or the composition according to Embodiment 47.

[0055] Embodiment 49: A method for preparing an antibody, comprising culturing the cells described in Embodiment 48 under conditions in which the antibody is expressed.

[0056] Embodiment 50: A composition comprising an isolated antibody, nucleic acid, composition, or cell according to any one of Embodiments 1 to 49, and a pharmaceutically acceptable carrier.

[0057] Embodiment 51: The composition according to Embodiment 50, comprising a second therapeutic agent.

[0058] Embodiment 52: The composition according to Embodiment 51, wherein the second therapeutic agent is an immunostimulant.

[0059] Embodiment 53: The composition according to Embodiment 52, wherein the immunostimulant is an antagonist of an immunosuppressive molecule, for example, PD-1 / PD-L1, CTLA-4 and LAG-3, or an agonist of an immunostimulant molecule, for example, GITR and OX40.

[0060] Embodiment 54: A method for treating a subject having cancer, comprising administering to the subject a therapeutically effective amount of a composition according to any one of Embodiments 50 to 53 or an isolated antibody according to any one of Embodiments 1 to 43, which stimulates an immune response and / or is an ILT-4 antagonist.

[0061] Embodiment 55: The method according to Embodiment 54, further comprising administering a second therapy.

[0062] Embodiment 56: The method according to Embodiment 55, wherein the second therapy is radiotherapy, surgery, or administration of a second drug.

[0063] Embodiment 57: The method according to Embodiment 55, wherein the second therapy is a second drug, and the second drug is an immunostimulant.

[0064] Embodiment 58: The method according to Embodiment 57, wherein the immunostimulant is an antagonist of an immunosuppressive molecule, e.g., PD-1 / PD-L1, CTLA-4 and LAG-3, or an agonist of an immunostimulant molecule, e.g., GITR and OX40.

[0065] Embodiment 60: A method for treating an infection (e.g., a viral disease) in a subject, comprising administering to the subject a therapeutically effective amount of a composition according to any one of Embodiments 50 to 53 or an isolated antibody according to any one of Embodiments 1 to 43, which stimulates an immune response and / or is an ILT4 antagonist.

[0066] Embodiment 61: A method for detecting ILT4 in a sample, comprising contacting the sample with an ILT4 antibody described in any one of Embodiments 1 to 43.

[0067] Embodiment 62: Features: a. For example, 10 -8 M or less, or 10 -9 K below M D Therefore, it specifically binds to hILT4 (for example, including the amino acid sequences of SEQ ID NOs. 108, 109, 111, 112, or 119); b. For example, stimulating the differentiation or activation of monocytes into macrophages, as shown in the assay described in the Examples, e.g., stimulating the differentiation of monocytes into inflammatory macrophages; c. Having a coupling profile as shown in Figure 27; and The following regions of d.hILT4: (i) 70 ITRIRPEL 77 (Sequence ID 120) and / or 78 VKNGQFHIPSITWEHTGRYGCQY 100 (Sequence ID 121); (ii) 70 ITRIRPELVKNGQFHIPSITWEHTGRYGCQY 100 (Sequence ID 122); or (iii) 154 ILCKEGEEEHPQCLNSQPHARGSSRAIF 181 (Sequence ID 123) and / or 425 SSPPPTGPIS 434 (Sequence ID 124) and other domains that bind to Ig-like domains 1, 2, or 1 and 2 of hILT4, Furthermore, the amino acid number of hILT4 is that of immature hILT4 (i.e., ILT4 containing its native signal sequence), and it does not significantly bind to other regions of the extracellular domain of ILT4, such as the region or residue located at the N-terminus relative to amino acid I70. An isolated antibody according to any one of embodiments 1 to 43, having the characteristics of an isolated antibody.

[0068] Embodiment 63: Features: a. For example, 10 -8 M or less, or 10 -9 K below M D Specific binding to hILT4 (e.g., including the amino acid sequences of SEQ ID NOs. 108, 109, 111, 112, or 119); b. Lack of specific binding to hILT2, hILT3, and / or hILT5; c. Lack of specific binding to one or more members of the LILRA and / or LILRB family; d. Stimulating T cell activation, for example, in a mixed lymphocyte reaction (MLR) assay, when measured by increased T cell proliferation or IFN-gamma secretion, as shown in the assay described in the Examples; e. For example, stimulating the differentiation or activation of monocytes into macrophages, as shown in the assay described in the Examples, e.g., stimulating the differentiation of monocytes into inflammatory macrophages; Inhibiting the binding of f.hILT4 to HLA-A and HLA-B; g. Having a coupling profile as shown in Figure 27; The following regions of h.hILT4: (i) 70 ITRIRPEL 77 (Sequence ID 120) and / or 78 VKNGQFHIPSITWEHTGRYGCQY 100 (Sequence ID 121); (ii) 70 ITRIRPELVKNGQFHIPSITWEHTGRYGCQY 100 (Sequence ID 122) and other domains that bind to Ig-like domains 1, 2, or 1 and 2 of hILT4, The amino acid number of hILT4 is that of immature hILT4 (i.e., ILT4 with its signal sequence), and it does not significantly bind to other regions of the extracellular domain of ILT4, such as the region or residue located at the N-terminus relative to amino acid I70; i. Promoting inflammatory polarization of macrophages to M1 macrophages; j. Not induce (or trigger) basophil activation; and k. After incubation at 25°C for 3 months, it contains less than 5% high and low molecular weight species, and / or after incubation at 40°C for 3 months, it contains less than 10% high and low molecular weight species. An isolated antibody according to any one of embodiments 1 to 43, having the characteristics of an isolated antibody.

[0069] Embodiment 64: Features: a. For example, promoting the expression of CD83 and CD86 on human monocyte-derived immature dendritic cells (Mo-iDCs), as shown in the assay described in the Examples; b. For example, enhancing IFN-γ secretion upon antigen stimulation in a cytomegalovirus (CMV) lysate assay, as shown in the assay described in the Examples; c. For example, enhancing IFN-γ and TNF-α secretion by CD4+ and CD8+ T cells in an allogeneic mixed lymphocyte response (MLR) assay upon CD3 stimulation, as shown in the assay described in the Examples; d. Inhibiting the binding of HLA-A and / or HLA-B to ILT4; e. For example, as shown in the HDX assay described in the examples, when determined by deuterium exchange (HDX), (i) 70 ITRIRPEL 77 (Sequence ID 120) and / or 78 VKNGQFHIPSITWEHTGRYGCQY 100 (Sequence ID 121); (ii) 70 ITRIRPELVKNGQFHIPSITWEHTGRYGCQY 100 (Sequence ID 122); or (iii) 154 ILCKEGEEEHPQCLNSQPHARGSSRAIF 181 (Sequence ID 123) and / or 425 SSPPPTGPIS 434 Binding to Ig-like domains 1, 2, or 1 and 2 of hILT4, such as the region containing (SEQ ID NO: 124); f. For example, as determined by carbene footprinting as described in the examples, interacting with one or more (or all) amino acid residues Lys43, Ile49, Thr50 and Arg51 of mature hILT4, or interacting with one or more (or all) amino acid residues Gly117, Val119, Try120, Leu134, Lys136, Gln149, Pro150, Ile159, Ser161, Val162, Gly163, Pro164, Pro167, His173, Try178, Pro183 and Tyr184 of mature hILT4, or interacting with one or more (or all) amino acid residues Glu42, Lys43, Gly76, Cys77, Leu88, Pro91, Pro183 and Tyr184 of mature hILT4; Regarding binding to g.hILT4, it competes with the antibodies described herein; and h. For example, as shown in the binding assay described in the Examples, it specifically binds to cynomolgus monkey ILT4 containing SEQ ID NO: 118. An isolated antibody according to any one of Examples 1 to 43, having the properties of: [Modes for carrying out the invention]

[0070] Detailed description of a particular embodiment definition Unless otherwise defined, scientific and technical terms used in connection with this invention shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms.

[0071] In this application, the use of “or” means “and / or” unless otherwise stated. In the context of multiple dependent claims, the use of “or” refers only to one or more of the aforementioned independent or dependent claims. The terms “comprising,” “including,” and “having” may be used interchangeably herein. According to the present invention, an “isolated” molecule is a molecule that has been taken out of its natural environment. As such, the term “isolated” does not necessarily reflect the degree to which the molecule has been purified.

[0072] The term “polypeptide” refers to a polymer of amino acid residues, and is not limited to a minimum length. A “protein” may comprise one or more polypeptides. Such polymers of amino acid residues may include, but are not limited to, native or non-native amino acid residues, and include peptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. Both full-length proteins and their fragments are encompassed by definition. The term also includes post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, and phosphorylation. Furthermore, for the purposes of this invention, “polypeptide” or “protein” refers to a polypeptide or protein, respectively, that includes modifications (generally conserved in nature), such as deletions, additions, and substitutions, to the native sequence, as long as the protein maintains the desired activity. These modifications may be planned, such as by site-directed mutagenesis, or accidental, such as by mutations in the host producing the protein or errors in PCR amplification. A protein may comprise two or more polypeptides. The letter “h” preceding a protein name hereinafter signifies a native, human protein, e.g., “hILT4”.

[0073] The terms “ILT4,” “human ILT4,” “hILT4,” “immunoglobulin-like transcript 4,” “Ig-like transcript 4,” “leukocyte immunoglobulin-like receptor B2,” “LIR2,” “LILRB2,” “MIR10,” and “CD85d” are all used interchangeably and refer to native, human ILT4 unless otherwise specifically indicated (e.g., mouse ILT4, cynomolgus monkey ILT4, etc.). This term includes full-length, unprocessed ILT4 as well as any form of ILT4 resulting from processing in cells. This term also includes naturally occurring variants of human ILT4, such as splice variants or allele variants. In some embodiments, ILT4 contains or consists of the amino acid sequence of SEQ ID NO: 107 (precursor, including signal peptide) or SEQ ID NO: 108 (mature, not including signal peptide). In some embodiments, ILT4 contains the amino acid sequence of SEQ ID NO: 110 (precursor, including signal peptide) or SEQ ID NO: 111 (mature, not including signal peptide).

[0074] As used herein, the term “ILT4 fragment” refers to ILT4 in which one or more residues are deleted from the N and / or C-terminus of a full-length ILT4. ILT4 fragments may or may not contain the N-terminal signal peptide but retain the ability to bind to T cells. As used herein, the term “ILT4 variant” refers to ILT4 containing naturally occurring amino acid additions, deletions, and substitutions but retaining the ability to bind to T cells.

[0075] The term “antagonist” is used in its broadest sense to include any molecule that partially or completely inhibits or neutralizes the biological activity of a polypeptide such as ILT4. Exemplary agonist molecules include antagonist antibodies. The term “ILT4 antagonist” refers to a molecule that inhibits or blocks the biological activity of ILT4, for example, by blocking or inhibiting the interaction between ILT4 and target cells, e.g., T cells, and / or target molecules. Exemplary ILT4 antagonists include antibodies that block the binding of ILT4 to target cells, e.g., T cells, and / or target molecules. An ILT4 antagonist is considered to “block the binding of ILT4 to target cells or target molecules” if it reduces the amount of detectable binding of at least one ILT4 to target cells, e.g., T cells, and / or target molecules by at least 50% in a cell binding assay. In some embodiments, ILT4 antagonists reduce the amount of detectable binding by at least 60%, at least 70%, at least 80%, or at least 90%. In some such embodiments, the antagonists are said to block ligand binding by at least 50%, at least 60%, at least 70%, etc. Blocking of ILT4 binding to target cells, e.g., T cells, can be demonstrated by the binding of ILT4, e.g., ILT4 ECD and transmembrane domain, or recombinant ILT4 Fc fusion protein, e.g., recombinant ILT4 ECD Fc fusion protein, to cells such as T cells, e.g., in or out of the presence of an antagonist.

[0076] The terms “inhibit” or “inhibit” refer to a reduction, reduction, or cessation of any phenotypic trait, or a reduction, reduction, or cessation of the occurrence, degree, or possibility of such trait. In some embodiments, “reduce” or “inhibit” means the ability to cause a reduction of 20% or more. In other embodiments, “reduce” or “inhibit” means the ability to cause a reduction of 50% or more. In yet another embodiment, “reduce” or “inhibit” means the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or more.

[0077] The terms "enhancing T cell activity" or "enhancing T cell activity" refer to the enhancement or increase of at least one of the following in a subject: T cell activation, cytokine secretion such as interferon-gamma (IFN-γ) secretion, or T cell proliferation. "Enhancing T cell activity" may be achieved by using a drug that is an agonist of T cell activity and / or a drug that is an antagonist (i.e., inhibits or blocks) of a mechanism that inhibits T cell activity. Changes in T cell activity can be measured, for example, by a T cell proliferation assay or IFN-γ ELISA, as described in the examples.

[0078] As used herein, the terms “ILT4 antibody,” “hILT4 antibody,” “anti-ILT4 antibody,” “anti-hILT4 antibody,” or “antibody that binds to ILT4” refer to antibodies that bind to ILT4 and, if necessary, inhibit the biological activity of ILT4 by blocking or inhibiting its binding to target cells such as T cells or target molecules. In some embodiments, the degree of binding of the ILT4 antibody to unrelated non-ILT4 proteins is less than 10% of the antibody's binding to ILT4, as measured, for example, by SPR (Biacore®) or by radioimmunoassay (RIA). In some embodiments, the degree of binding of the ILT4 antibody to other ILT family proteins such as LILRA is less than 20%, less than 10%, or less than 5% of the antibody's binding to ILT4, as measured, for example, by SPR or by radioimmunoassay (RIA). In some embodiments, the ILT4 antibody binds to ILT4 but not to at least one protein selected from ILT2, ILT3, and ILT5.

[0079] The terms "leader peptide," "leader sequence," "signal peptide," or "signal sequence" refer to a sequence of peptides or amino acid residues located at the N-terminus of a polypeptide that facilitates the secretion of the polypeptide from mammalian cells. Leader sequences can be cleaved during polypeptide efflux from mammalian cells to form mature proteins. Leader sequences may be native or synthetic, and they may be heterologous or homologous to the proteins to which they are attached.

[0080] The terms “antibody” or “Ab” are used herein in a broad sense and include, but are not limited to, a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments insofar as they exhibit desired antigen-binding activity. As used herein, the term refers to a molecule comprising at least complementarity-determining regions (CDRs) 1, 2, and 3 of the heavy chain and at least CDRs 1, 2, and 3 of the light chain, the molecule being capable of binding to an antigen. The term “antibody” also includes, but is not limited to, chimeric antibodies, humanized antibodies, human antibodies, and antibodies of various species, such as mouse and cynomolgus monkey. The term “antibody fragment” includes, but is not limited to, fragments capable of binding to an antigen, such as Fv, single-chain Fv(scFv), Fab, Fab', and (Fab')2.

[0081] The terms “heavy chain” or “HC” refer to a polypeptide that includes at least a heavy chain variable region, with or without a leader sequence. In some embodiments, the heavy chain includes at least a portion of the heavy chain constant region. The term “full-length heavy chain” refers to a polypeptide that includes a heavy chain variable region and a heavy chain constant region, with or without a leader sequence, and with or without a C-terminal lysine (K). The term “mature full-length heavy chain” refers to a polypeptide that includes a heavy chain variable region and a heavy chain constant region, but does not include a leader sequence, and may or may not include a C-terminal lysine (K).

[0082] The term “heavy chain variable region” or “VH” refers to the region of the heavy chain comprising the heavy chain complementarity determining region (CDR) 1, framework region (FR) 2, CDR2, FR3, and CDR3. In some embodiments, the heavy chain variable region also includes at least a portion of FR1 and / or at least a portion of FR4. In some embodiments, the heavy chain CDR is as identified herein in the following sequence listing or Figures 1-12, etc. As used herein, VH CDR1, CDR2, and CDR3 are Kabat CDRs, for example, as provided in Figures 1-12.

[0083] The terms “light chain” or “LC” refer to a polypeptide that includes at least a light chain variable region, with or without a leader sequence. In some embodiments, the light chain includes at least a portion of the light chain constant region. The term “full-length light chain” refers to a polypeptide that includes a light chain variable region and a light chain constant region, with or without a leader sequence. The term “mature full-length light chain” refers to a polypeptide that includes a light chain variable region and a light chain constant region, but does not include a leader sequence.

[0084] The term “light chain variable region” or “VL” refers to the region comprising the light chain CDR1, FR2, HVR2, FR3, and HVR3. In some embodiments, the light chain variable region also includes FR1 and / or FR4. In some embodiments, the light chain CDR is as identified herein in the sequence listing or in Figures 1-12, etc. As used herein, VL CDR1, CDR2, and CDR3 are Kabat CDRs, for example, as provided in Figures 1-12.

[0085] A "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species. In some embodiments, a chimeric antibody refers to an antibody that includes at least one variable region originating from a first species (e.g., mouse, rat, cynomolgus monkey, etc.) and at least one constant region originating from a second species (e.g., human, cynomolgus monkey, etc.). In some embodiments, a chimeric antibody includes at least one mouse variable region and at least one human constant region. In some embodiments, a chimeric antibody includes at least one cynomolgus monkey variable region and at least one human constant region. In some embodiments, all of the variable regions of a chimeric antibody originate from the first species, and all of the constant regions of the chimeric antibody originate from the second species.

[0086] A "humanized antibody" refers to an antibody in which at least one amino acid in the framework region of a non-human variable region is replaced with a corresponding amino acid derived from a human variable region. In some embodiments, the humanized antibody includes at least one human constant region or a fragment thereof. In some embodiments, the humanized antibody is Fab, scFv, (Fab')2, etc.

[0087] "Human antibody" as used herein refers to antibodies produced in humans, antibodies produced in non-human animals containing human immunoglobulin genes, and antibodies selected using, for example, XenoMouse® and in vitro methods, such as phage display where the antibody repertoire is based on human immunoglobulin sequences.

[0088] In some embodiments, the antibodies described herein may contain one or more “conservative substitutions” compared to a particularly specified sequence. “Conservative amino acid substitution” as used herein means the substitution of an amino acid residue with an amino acid residue having a similar side chain. The family of amino acid residues having similar side chains includes amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, the predicted non-essential amino acid residues in the antibodies herein are replaced with other amino acid residues derived from the same side-chain family (e.g., basic, acidic, beta-branched, aromatic, uncharged). Methods for identifying conserved nucleotide and amino acid substitutions that do not eliminate antigen binding are described, for example, in Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997).

[0089] In some embodiments, the antibodies described herein may contain one or more "reverse substitutions." Examples are illustrated in Figures 13-15 herein. A reverse substitution is a mutation that returns the antibody heavy chain or light chain to the germline amino acid sequence from which it originated.

[0090] The binding of antibody proteins, for example, ILT4, is called "K". D The "dissociation constant" is a measure of the affinity or specific binding of an antibody to a protein, such as ILT4. D is high K DIt exhibits improved binding or affinity that surpasses K. D This refers to the "off-rate" of antibodies and polypeptides. off or k d and "on-rate" or k on or k a It is composed of proportions between [the two values].

[0091] The terms "specific binding" or "specifically binding" or similar terms refer to the binding of two polypeptides, such as an antibody and its polypeptide target. D However, this shows that it is smaller than what would be the case between two random polypeptides under identical conditions. In other words, K D This is less than that caused by nonspecific aggregation of polypeptides in the system.

[0092] "Tumor model" as used herein refers to in vivo preclinical assays that may be used to study the biological activity of ILT4 antibodies, including xenograft or natural mouse tumor assay systems. In some cases, tumor models allow tracking the size or growth of a tumor and / or the presence of immune cells in the tumor, such as specific types of T cells or NK cells, when treated with the antibody, and to determine whether the antibody triggered or enhanced an immune response.

[0093] The term “immunostimulant” as used herein refers to a molecule that stimulates the immune system either by acting as an agonist of an immunostimulant molecule, including co-stimulatory molecules, or by acting as an antagonist of an immunoinhibitory molecule, including co-inhibitory molecules. Immunostimulant or immunoinhibitory molecules may also be immune checkpoint modifiers, such as checkpoint inhibitors or checkpoint stimulants. Immunostimulants may be biologics, e.g., antibodies or antibody fragments, other proteins or vaccines, or small molecule drugs. “Immunostimulant molecules” include receptors or ligands that act to enhance, stimulate, induce or otherwise “turn on” an immune response. Co-stimulatory molecules are examples of immunostimulant molecules as defined herein. “Immunoshibitory molecules” include receptors or ligands that act to inhibit, suppress or otherwise “turn off” an immune response. Co-inhibitory molecules are examples of immunoinhibitory molecules as defined herein. Such immunostimulant and immunoinhibitory molecules may be receptors or ligands found, for example, on immune cells such as T cells, or on cells involved in innate immunity such as NK cells.

[0094] With respect to peptide, polypeptide, or antibody sequences, “amino acid sequence identity percentage (%)”, “identity %”, “amino acid sequence homology percentage”, and “homology %” refer to the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence, after the sequences have been aligned to achieve the maximum sequence identity percentage, gaps have been introduced as necessary, and no conservative substitutions have been considered as part of the sequence identity. Alignment aimed at determining the amino acid sequence identity percentage can be achieved by various methods within the scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment across the sequences being compared.

[0095] "Treatment" as used herein refers to any administration or application of a therapeutic agent for a disease in a human being, including inhibiting the progression of the disease or one or more disease symptoms, slowing the progression of the disease or one or more of its symptoms, stopping its onset, partially or completely alleviating the disease or one or more of its symptoms, or preventing the recurrence of one or more of the symptoms of the disease.

[0096] The terms "subject" and "patient" are used interchangeably in this specification and refer to human beings unless otherwise specifically stated.

[0097] The terms “effective dose” or “therapeutic effective dose” refer to the amount of drug that is effective in treating a disease or disorder in a control to partially or completely alleviate one or more symptoms. In some embodiments, an effective dose refers to the amount that is effective in terms of dosage and duration to achieve the desired therapeutic or preventive outcome.

[0098] The term “cancer” is used herein to refer to a group of cells exhibiting abnormally high levels of proliferation and growth. Cancer can be benign (also referred to as a benign tumor), premalignant, or malignant. Cancer cells can be solid cancer cells or leukemic cancer cells. The term “tumor growth” is used herein to refer to proliferation or growth by cancer-containing cells(s) that lead to a corresponding increase in the size or extent of the cancer.

[0099] Examples of cancers to which the treatment methods described herein are applicable include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specifically, examples of such cancers that are not limited to squamous cell carcinoma include squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous non-small cell lung cancer), lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland cancer, kidney cancer, renal cell carcinoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, brain cancer, endometrial cancer, testicular cancer, bile duct cancer, gallbladder carcinoma, stomach cancer, melanoma, and various types of head and neck cancers (including squamous cell carcinoma of the head and neck).

[0100] Administration "in combination" with one or more additional therapeutic agents includes simultaneous (concurrent) and sequential (sequential) administration in any order.

[0101] A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid expander, diluent, encapsulating material, formulation adjuvant, or conventional carrier in the art, intended for use with a therapeutic agent containing a "pharmaceutical composition" to be administered to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration used and is compatible with the other components of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation in which it is used. For example, if the therapeutic agent is to be administered orally, the carrier may be a gel capsule. If the therapeutic agent is to be administered subcutaneously, the carrier should ideally not be irritating to the skin and should not cause an injection site reaction.

[0102] "Chemotherapy agents" are compounds useful in the treatment of cancer. Examples of chemotherapy agents that may be administered in the methods described herein include, but are not limited to, alkylating agents, e.g., thiotepa and cytoxane® cyclosphosphamide; alkyl sulfonates, e.g., busulfan, improsulfan and piposulfan; aziridines, e.g., benzodopa, carbocon, meturedopa and uredopa; methylamelamine, including ethyleneimine and altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomellamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including its synthetic analog topotecan); bryostatin; calistatin; CC-1065 (its adzeresin, karzeresin and bizerecin). Including resin synthetic analogs); cryptophycin (especially cryptophycin 1 and cryptophycin 8); drastatin; duocalmycin (including synthetic analogs, KW-2189 and CB1-TM1); eryuterobin; pancratistatin; sarcodictiin; spongistatin; nitrogen mustard, e.g., chlorambucil, chlornafadin, colophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine hydrochloride oxy D, melphalan, nobembicin, fenestrine, prednimustine, trophosphamide, uracil mustard; nitrosureas, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics, e.g., enegyoin antibiotics (e.g., calicheamicin, in particular calicheamicin gamma 1I and calicheamicin omega I1 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dinemycin including dinemycin A; bisphosphonates, e.g., clodronate; esperamycin;Furthermore, neocartinostatin chromophore and related pigment proteins (eneggioin antibiotic chromophore), acrasinomycin, actinomycin, autramycin, azaserin, bleomycin, kakutinomycin, carabicin, carminomycin, cardinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, adriamycin®, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, for example, mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potfiromycin. , puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folate analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioglycolate Anine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, phloxuridine; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenals, e.g., aminoglutethimide, mitotane, trilostane; folic acid replenishers, e.g., folinic acid; acegraton; aldofamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisantren; edatraxate; defofamine; demecolsin; diaziquan; elfornithine; eriptinium acetate; epotilone;Etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; mytansinoids, e.g., mytansine and anthamitosine; mitogwazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS natural product, Eugene, OR); razoxane; lyzoxin; Schizophyllan; spirogermanium; tenuazonic acid; triadicone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, beraclin A, loridine A and anguidin); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitractol; pipobromane; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., Taxol® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, New Jersey), Abraxane® cremofol-free, albumin-modified nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumburg, Illinois), and Taxotere® doxetaxel (Rhone-Poulenc Rorer, France, Antony); Chloranbucil; Gemcitabine (Gemzar®); 6-Thiogunine; Mercaptopurine; Methotrexate; Platinum analogs, e.g., cisplatin, oxaliplatin, and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Vinorelbine (Navelbine®); Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopterin; Xeloda; Ibandronate; Irinotecan (Camptosar, CPT-11) (including treatment regimens of irinotecan with 5-FU and leucovorin); Topoisomerase inhibitor RFS2000; Difluoromethylornithine (DMFO);Examples include retinoids, such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatins, including oxaliplatin-treated regimens (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation, as well as any pharmaceutically acceptable salts, acids, or derivatives of the above.

[0103] Further, but not limited to, exemplary chemotherapeutic agents that may be administered in the methods herein include anti-hormone agents that act to modulate or inhibit the hormonal effects on cancer, such as tamoxifen (including Nolvadex® tamoxifen), raloxifene, doroxifene, 4-hydroxytamoxifen, trioxyfen, keoxifene, LY117018, onapristone, and Fareston® toremifene, for example, anti-estrogen and selective estrogen receptor modulators (SERMs); aromatase inhibitors that inhibit aromatase, an enzyme that regulates estrogen production in the adrenal gland, such as 4(5)-imidazole, aminoglutethimide, Megase® megestrol acetate, Aromasin® exemestane, formestanie, fadrozol, Rivisor® borozole, Femara® letrozole, and Arimi Examples include Dex® anastrozole; and antiandrogens, e.g., flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, in particular those that inhibit gene expression in signaling pathways involved in adherent cell proliferation, e.g., PKC-alpha, Ralf, and H-Ras; ribozymes such as VEGF expression inhibitors (e.g., Angiozyme® ribozymes) and HER2 expression inhibitors; gene therapy vaccines, e.g., vaccines such as Allovectin® vaccine, Leuvectin® vaccine, and Vaxid® vaccine; Proleukin® rIL-2; Lulutotecan® topoisomerase 1 inhibitor; Abarelix® rmRH; and any pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0104] "Anti-angiogenic agents" or "angiogenic inhibitors" refer to low molecular weight substances, polynucleotides [e.g., including inhibitory RNA (RNAi or siRNA)], polypeptides, isolated proteins, recombinant proteins, antibodies or their conjugates or fusion proteins that directly or indirectly inhibit angiogenesis, vascular formation, or undesirable vascular permeability. It should be understood that anti-angiogenic agents include substances that bind to and block the angiogenic vitality of angiogenic factors or their receptors. For example, anti-angiogenic agents that can be administered in the methods described herein include antibodies against angiogenic agents or other antagonists, e.g., antibodies against VEGF-A [e.g., bevacizumab (Avastin®)] or antibodies against the VEGF-A receptor (e.g., KDR receptor or Flt-1 receptor), anti-PDGFR inhibitors, e.g., Gleevec® (imatinib mesylate), small molecules that block VEGF receptor signaling (e.g., PTK787 / ZK2284, SU6668, Sutent® / SU11248 (sunitinib malate), AMG706, or, for example, those described in international patent application WO2004 / 113304). Anti-angiogenic agents also include natural angiogenesis inhibitors, e.g., angiostatins, endostatins, etc. See, for example, Klagsbrun and D'Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22:3172-3179 (e.g., Table 3 listing anti-angiogenic therapies in malignant melanoma); Ferrara & Alitalo (1999) Nature Medicine 5(12):1359-1364; Tonini et al. (2003) Oncogene 22:6549-6556 (e.g., Table 2 listing known anti-angiogenic factors); and Sato (2003) Int. J. Clin. Oncol. 8:200-206 (e.g., Table 1 listing anti-angiogenic agents used in clinical trials).

[0105] "Growth inhibitor" means a compound or composition that inhibits the growth of cells (e.g., cells expressing VEGF) in vitro or in vivo, as used herein. Therefore, growth inhibitors that can be administered in the methods herein may significantly reduce the percentage of S-phase cells (e.g., cells expressing VEGF). Examples of growth inhibitors, but not limited to them, include substances that block cell cycle progression (at locations other than the S phase), such as substances that induce G1 arrest and M-phase arrest. Classical M-phase blockers include vinca (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Substances that stop G1 also have an effect on S-phase arrest, such as DNA alkylating agents, such as tamoxifen, prednisone, dacarbazine, mechloretamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found, for example, on page 13, in Mendelsohn and Israel, eds., *The Molecular Basis of Cancer*, Chapter 1 (WB Saunders, Philadelphia, 1995), entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. Taxanes (paclitaxel and docetaxel) are both anticancer drugs derived from the yew tree. Docetaxel (Taxotere®, Rhone-Poulenc Rorer), derived from the European yew, is a semi-synthetic analog of paclitaxel (Taxol®, Bristol-Myers Squibb). Paclitaxel and docetaxel stabilize microtubules by promoting microtubule assembly from tubulin dimers and preventing depolymerization, which leads to the inhibition of mitosis in cells.

[0106] The term "antineoplastic composition" refers to a composition useful for treating cancer that contains at least one active therapeutic agent. Examples of therapeutic agents include, but are not limited to, chemotherapy drugs, growth inhibitors, cytotoxic agents, substances used in radiotherapy, anti-angiogenic agents, cancer immunotherapies, apoptotic agents, antitubulins, and other agents for treating cancer, such as anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (Tarceva®)), platelet-derived growth factor inhibitors (e.g., Gleevec® (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, and CTLA4 inhibitors (e.g., anti-CTLA antibody ipilim). Examples include mab (YERVOY®), PD-1 or (ore)PD-L1 inhibitors (e.g., Opdivo®, Keytruda®, Tecentriq®, Bavencio®, IMFINZI®), TIM3 inhibitors (e.g., anti-TIM3 antibodies), LAG-3 inhibitors, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets: ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA, CTLA4, TIM3, or VEGF receptors, TRAIL / Apo2, and other bioactive and organic chemicals. Combinations thereof are also included in this disclosure.

[0107] Antibodies that specifically bind to ILT4 The following sequence listings provide amino acid sequences of human ILT4, with or without the signal peptide (see SEQ ID NOs. 107-108 and 110-111). The extracellular domain (ECD) contains the amino acid residues shown in SEQ ID NOs. 109 and 112. An exemplary ECD bound to the His-Avi tag contains the amino acid sequence of SEQ ID NO. 119.

[0108] Anti-ILT4 antibodies (Ab) may specifically bind to ILT4-ECD or its fragments (see SEQ ID NOs. 109 and 112, and SEQ ID NOs. 119).

[0109] For example, if measured at 25°C or 37°C, 10 -6 M or less, 10 -7 M or less, 10 -8 The following 10 -9 M or less or 10 -10 K below M D Ab that binds to ILT4 is provided herein.

[0110] Determining how well Ab binds to the ILT4 protein can be carried out using several different methods, for example, by surface plasmon resonance (SPR), for example, by the BIACORE® assay. An exemplary SPR assay involves capturing one or more antibodies on a CM4 sensor chip using an immobilized capture reagent (e.g., using the Biacore® anti-human Fc capture kit, GE Healthcare catalog no. BR-1008-39 or the Biacore® anti-mouse capture kit, GE Healthcare catalog no. BR-1008-39) and running the ILT4 antigen (e.g., ILT4 ECD) as the analyte in a concentration series to determine the binding kinetics and affinity in the running buffer. In one embodiment, ILT4 is injected at a flow rate of 30 μL / min at 2 to 5 concentrations ranging from 0.1 nM to 500 nM (e.g., 0.1 nM, 1 nM, 10 nM, 100 nM, 500 nM) for a maximum association time of 4 minutes and a maximum dissociation time of 10 minutes. During the binding cycle, the capture surface is regenerated according to the manufacturer's instructions for use for each capture kit. All data are double-referenced using a reference flow cell and blank injection. Data with simple 1:1 kinetics are fitted to a Langmuir binding model with mass transfer using Biacore® T200 evaluation software. The SPR method described in the examples may also be used.

[0111] The affinity of Ab to the ILT4 ECD polypeptide can be determined using cells expressing the ILT4 polypeptide on their surface, a method comprising flow cytometry. An exemplary flow cytometry assay includes: resuspending T cells or other cells ectopically expressing ILT4 in buffer, and incubating Ab serially diluted from approximately 20 μg / mL with the resuspended cells at 4°C for 30 minutes. Then, while maintaining the desired buffer conditions, the cells are washed twice with the same buffer and incubated with a secondary antibody conjugated with a fluorophore that recognizes a primary antibody (e.g., human IgG). Then, the cells are washed as before and immediately acquired without fixation using a BD Fortessa® or other flow cytometer. The affinity of Ab to the ILT4 polypeptide can be determined as described in the examples.

[0112] In certain embodiments, Ab, which binds to ILT4, blocks the binding of ILT4 to target cells such as T cells. The inhibition or blockade may be 100% or at least 99%, 95%, 90%, 85%, 80%, 75%, or 50%.

[0113] The inhibition of ILT4 binding to ILT4-binding cells, such as T cells, can be determined by measuring the inhibition of ILT4 binding to cells in the presence and absence of the antibody. An exemplary experiment that can be used to investigate whether an antibody inhibits the binding of ILT4 to ILT4-binding cells is a flow cytometry assay, e.g., an assay including: human peripheral blood mononuclear cells obtained from donor blood, buffy coat, or leukopak are resuspended in a buffer consisting of HBSS + 1% BSA. The cells are then incubated at 4°C for 30 minutes with 20 μg / mL of, for example, an ILT4 ECD fusion protein fused to human IgG1 Fc, and with varying concentrations of a candidate ILT4 blocking antibody or a control antibody. The cells are then washed twice with the same buffer and incubated for a further 30 minutes at 4°C with a fluorophore-conjugated secondary antibody that recognizes ILT4 but not the candidate blocking antibody or control antibody. The cells are then washed as before, and acquired immediately using a BD Fortessa® or other flow cytometer without fixation. Binding inhibition can also be determined, for example, as described in the examples.

[0114] Exemplary ILT4-bound Ab ILT4, for example, an Ab that specifically binds to ILT4 ECD, is provided herein.

[0115] In certain embodiments, ILT4 Ab includes a heavy-chain variable region ("VH") comprising any of the VH CDR1, CDR2 and / or CDR3 of the ILT4 Ab provided herein. The VH CDRs herein are Kabat CDRs shown in Figures 1, 3, 5, 7, 9, and 11 unless otherwise specified (e.g., AbM CDR1). In certain embodiments, ILT4 Ab includes a VH comprising any of the VH CDR1, CDR2, and CDR3 of the ILT4 Ab provided herein. In certain embodiments, ILT4 Ab is one of 9G4, 9C8, 2H2, 2E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4 (i.e., 9G4 (sequence numbers 125-127), 9C8 (sequence numbers 131-133), 2H2 (sequence numbers 137-139), 2E5 (sequence numbers 143-145), 24E5 (sequence numbers 149-151), 21D9 ( VH containing CDR1, CDR2 and / or CDR3 of any one of the following: (sequence numbers 155-157), 21D9.b (sequence numbers 155-157), 21D9.c (sequence numbers 155-157), 21D9.d (sequence numbers 155-157), 21D9.e (sequence numbers 155-157), 21A5 (sequence numbers 161-163), 10F10 (sequence numbers 167-169), 10F10.1 (sequence numbers 167-169), 10F10.3 (sequence numbers 167-169), or 10F10.4 (sequence numbers 167-169).

[0116] In certain embodiments, ILT4 Ab includes a VH comprising any VH CDR1, CDR2, and CDR3 of ILT4 Ab provided herein. In certain embodiments, ILT4 Ab includes a VH comprising any one VH CDR1, CDR2, and CDR3 of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4.

[0117] In certain embodiments, ILT4 Ab includes a VH comprising any VH CDR1, CDR2 and / or CDR3 of ILT4 Ab provided herein, and a VL comprising any CDR1, CDR2 and / or CDR3 of ILT4 Ab provided herein. VL CDRs herein are Kabat CDRs shown in Figures 2, 4, 6, 8, 10 and 12 herein, unless otherwise specified (e.g., AbM CDR1). In certain embodiments, ILT4 Ab includes a VH comprising any VH CDR1, CDR2 and CDR3 of ILT4 Ab provided herein, and a VL comprising any CDR1, CDR2 and CDR3 of ILT4 Ab provided herein. In a particular embodiment, ILT4 Ab is one of the following VHs: 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4 VH including CDR1, CDR2 and / or CDR3, and one of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4 (i.e., 9G4 (sequences 128-130), 9C8 (sequences 134-136), 2H2 (sequences 140-142), 2E5 (sequences 146-148), 24E5 (sequence 15 VLs including CDR1, CDR2, and / or CDR3 of any of the following: 2-154), 21D9 (sequences 158-160), 21D9.b (sequences 158-160), 21D9.c (sequences 158-160), 21D9.d (sequences 158-160), 21D9.e (sequences 158-160), 21A5 (sequences 164-166), 10F10 (sequences 170-172), 10F10.1 (sequences 170-172), 10F10.3 (sequences 170-172), or 10F10.4 (sequences 170-172).

[0118] In some embodiments, ILT4 Ab is (a) VH containing the amino acid sequences of 9G4's VH CDR1, CDR2, and CDR3, and VL containing 9G4's VL CDR1, CDR2, and CDR3; (b) VH containing the amino acid sequences of 9C8 CDR1, CDR2 and CDR3, and VL containing 9C8 CDR1, CDR2 and CDR3; (c) VH of 2H2 containing the amino acid sequences of CDR1, CDR2, and CDR3, and VL of 2H2 containing CDR1, CDR2, and CDR3; (d) VH containing the amino acid sequences of 25E5 CDR1, CDR2, and CDR3, and VL containing the amino acid sequences of 25E5 CDR1, CDR2, and CDR3; (e) VH containing the amino acid sequences of 24E5 CDR1, CDR2 and CDR3, and VL containing 24E5 CDR1, CDR2 and CDR3; (f) VH containing the amino acid sequences of 21D9 CDR1, CDR2 and CDR3, and VL containing 21D9 CDR1, CDR2 and CDR3; (g) VH containing the amino acid sequences of CDR1, CDR2, and CDR3 of 21D9.b, and VL containing CDR1, CDR2, and CDR3 of 21D9.b; (h) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of 21D9.c, and VL containing CDR1, CDR2 and CDR3 of 21D9.c; (i) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of 21D9.d, and VL containing CDR1, CDR2 and CDR3 of 21D9.d; (j) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of 21D9.e, and VL containing CDR1, CDR2 and CDR3 of 21D9.e; (k) VH containing the amino acid sequences of 21A5 CDR1, CDR2, and CDR3, and VL containing 21A5 CDR1, CDR2, and CDR3; (l) VH containing the amino acid sequences of CDR1, CDR2, and CDR3 of 21A5.a, and VL containing CDR1, CDR2, and CDR3 of 21A5.a; (m)10F10 VH containing the amino acid sequences of CDR1, CDR2 and CDR3, and VL containing CDR1, CDR2 and CDR3 of 10F10; (n) VH containing the amino acid sequences of CDR1, CDR2, and CDR3 of 10F10.1, and VL containing CDR1, CDR2, and CDR3 of 10F10.1; (o) VH containing the amino acid sequences of 10F10.3 VH CDR1, CDR2 and CDR3, and VL containing 10F10.3 VL CDR1, CDR2 and CDR3; or (p) VH containing the amino acid sequences of CDR1, CDR2 and CDR3 of 10F10.4, and VL containing CDR1, CDR2 and CDR3 of 10F10.4 It may include.

[0119] Again, the following sequence listings provide the heavy chain and light chain variable region sequences and the full-length heavy chain and light chain sequences of the antibodies listed above.

[0120] In certain embodiments, ILT4 Ab comprises a VH containing the amino acid sequence of any of the VHs of ILT4 Ab provided herein. The individual VH sequences of the particular antibody species provided herein are listed in the sequence listing. In certain embodiments, ILT4 Ab comprises one of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4 (i.e., 9G4 (SEQ ID NO: 51, amino acids 20-135), 9C8 (SEQ ID NO: 55), 2H2 (SEQ ID NO: 58), 2E5 (SEQ ID NO: 63), 24E5 (SEQ ID NO: 67), 21 Contains a VH containing the amino acid sequence of any one of the following: D9 (sequence number 71), 21D9.b (sequence number 74), 21D9.c (sequence number 75), 21D9.d (sequence number 78), 21D9.e (sequence number 80), 21A5 (sequence number 83), 21A5.a (sequence number 87), 10F10 (sequence number 91), 10F10.1 (sequence number 91), 10F10.3 (sequence number 91), or 10F10.4 (sequence number 91).

[0121] In some embodiments, ILT4 Ab contains one of the following VH sequences: 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4, but includes 1, 2, 3, 4, or 5 amino acid substitutions within the framework region of the VH sequence, such as 1, 2, 3, 4, or 5 conservative substitutions. In some embodiments, ILT4 Ab contains one of the following VH sequences: 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4, but with 1, 2, 3, 4, or 5 return substitutions within the framework region of the VH sequence.

[0122] In some embodiments, ILT4 Ab comprises any VH and VL CDR of any ILT4 Ab described herein, wherein the CDR contains one, two, or three amino acid additions, substitutions (e.g., conservative substitutions), or deletions in all CDRs.

[0123] In certain embodiments, ILT4 Ab comprises VH CDR1, CDR2, and CDR3, which include the amino acid sequence of any VH CDR of ILT4 Ab provided herein, and VH which is 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%, or at least 99% identical to any VH of any ILT4 Ab provided herein. In certain embodiments, ILT4 Ab includes a VH having an amino acid sequence that is 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%, or at least 99% identical to the amino acid sequence of any one of the following VHs: 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4. In certain embodiments, the VH of the antibody differs from that of the VH sequence shown in the sequence listing due to 1, 2, 3, 4, or 5 amino acid substitutions in the framework region of the VH sequence, such as 1, 2, 3, 4, or 5 conservative substitutions. In certain embodiments, the VH of the antibody differs from that of the VH sequence shown in the sequence listing due to one, two, three, four, or five return substitutions in the framework region of the VH sequence.

[0124] In certain embodiments, ILT4 Ab comprises a VH consisting of the amino acid sequence of any of the VHs provided herein. In certain embodiments, ILT4 Ab comprises a VH consisting of the amino acid sequence of any one of the VHs: 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4.

[0125] In certain embodiments, ILT4 Ab comprises a VL consisting of the amino acid sequence of any VL of ILT4 Ab provided herein. In certain embodiments, ILT4 Ab is any one of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4 (i.e., 9G4 (SEQ ID NO: 50, amino acids 19-125), 9C8 (SEQ ID NO: 54), 2H2 (SEQ ID NO: 59), 2E5 (SEQ ID NO: 62), 24E5 (SEQ ID NO: 66) Includes VLs containing the amino acid sequences of 21D9 (sequence number 70), 21D9.b (sequence number 70), 21D9.c (sequence number 70), 21D9.d (sequence number 70), 21D9.e (sequence number 70), 21A5 (sequence number 82), 21A5.a (sequence number 86), 10F10 (sequence number 90), 10F10.1 (sequence number 94), 10F10.3 (sequence number 96), or 10F10.4 (sequence number 114). In certain embodiments, ILT4 Ab comprises VL CDR1, CDR2, and CDR3, which include the amino acid sequence of any VL CDR of ILT4 Ab provided herein, and which are 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%, or at least 99% identical to any VL of ILT4 Ab provided herein. In a particular embodiment, ILT4 Ab includes a VL having an amino acid sequence that is 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%, or at least 99% identical to the amino acid sequence of any one VL of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4.In certain embodiments, the VL of the antibody differs from that of the VL sequence shown in the sequence listing due to one, two, three, four, or five amino acid substitutions in the framework region of the VL sequence, such as one, two, three, four, or five conservative substitutions. In certain embodiments, the VL of the antibody differs from that of the VL sequence shown in the sequence listing due to one, two, three, four, or five return substitutions.

[0126] In certain embodiments, ILT4 Ab comprises a VL consisting of the amino acid sequence of any VL of ILT4 Ab provided herein. In certain embodiments, ILT4 Ab comprises a VL consisting of the amino acid sequence of any one VL of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4.

[0127] In certain embodiments, ILT4 Ab comprises a VH containing the amino acid sequence of any VH of ILT4 Ab provided herein, and a VL containing the amino acid sequence of any VL of ILT4 Ab provided herein. In certain embodiments of these, ILT4 Ab comprises a VH containing the amino acid sequence of any one of the following VHs: 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4, and a VL containing the amino acid sequence of any one of the following VLs: 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4.

[0128] However, in certain embodiments, the VH of the antibody is one of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4, but containing 1, 2, 3, 4, or 5 amino acid substitutions in the framework region of the VH sequence, such as 1, 2, 3, 4, or 5 conserved substitutions, and the VL is one of the same species derived from the list above. However, in certain embodiments, the VH of the antibody is one of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4, but contains 1, 2, 3, 4, or 5 return substitutions in the framework region of the VH sequence.

[0129] In a particular embodiment, ILT4 Ab includes VH and VL containing the amino acid sequences of one of the following VH and VL: 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4.

[0130] In certain embodiments, ILT4 Ab includes VH CDR1, CDR2, and CDR3 containing the amino acid sequence of any ILT4 Ab VH CDR provided herein, and VL CDR1, CDR2, and CDR3 containing the amino acid sequence of any ILT4 Ab VL CDR provided herein, and also includes VH and VL which are 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%, or at least 99% identical to the corresponding VH and VL of any ILT4 Ab provided herein. In certain embodiments, the VH and VL of the antibody differ from the VH and VL sequences shown in the sequence listing due to 1, 2, 3, 4, or 5 amino acid substitutions in the framing region of the sequence, such as 1, 2, 3, 4, or 5 conservative substitutions, or 1, 2, 3, 4, or 5 return substitutions.

[0131] In certain embodiments, ILT4 Ab comprises VH and VL consisting of the amino acid sequences of any of the VH and VL of ILT4 Ab provided herein. In certain embodiments, ILT4 Ab comprises VH and VL each consisting of the amino acid sequences of any one of the following VH and VL: 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4.

[0132] ILT4 Ab is (a) VH containing the amino acid sequence of 9G4 VH, and VL containing the amino acid sequence of 9G4 VL; (b) VH containing the amino acid sequence of VH at 9C8, and VL containing the amino acid sequence of VL at 9C8; (c) VH containing the amino acid sequence of VH of 2H2, and VL containing the amino acid sequence of VL of 2H2; (d) VH containing the amino acid sequence of VH of 25E5, and VL containing the amino acid sequence of VL of 25E5; (e) VH containing the amino acid sequence of VH at 24E5, and VL containing the amino acid sequence of VL at 24E5; (f) VH containing the amino acid sequence of VH of 21D9, and VL containing the amino acid sequence of VL of 21D9; (g) VH containing the amino acid sequence of VH from 21D9.b, and VL containing the amino acid sequence of VL from 21D9.b; (h) VH containing the amino acid sequence of VH in 21D9.c, and VL containing the amino acid sequence of VL in 21D9.c; (i) VH containing the amino acid sequence of VH from 21D9.d, and VL containing the amino acid sequence of VL from 21D9.d; (j) VH containing the amino acid sequence of VH in 21D9.e, and VL containing the amino acid sequence of VL in 21D9.e; (k) VH containing the amino acid sequence of VH of 21A5, and VL containing the amino acid sequence of VL of 21A5; (l) VH containing the amino acid sequence of VH from 21A5.a, and VL containing the amino acid sequence of VL from 21A5.a; VH containing the amino acid sequence of (m)10F10 VH, and VL containing the amino acid sequence of 10F10 VL; (n) VH containing the amino acid sequence of VH of 10F10.1, and VL containing the amino acid sequence of VL of 10F10.1; (o) VH containing the amino acid sequence of VH of 10F10.3 and VL containing the amino acid sequence of VL of 10F10.3; or (p) VH containing the amino acid sequence of VH of 10F10.4 and VL containing the amino acid sequence of VL of 10F10.4 It may include.

[0133] ILT4 Ab is (a) VH containing the VH CDR of 9G4 VH and VL containing the VL CDR of 9G4, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 9G4, (b) VH containing the VH CDR of 9C8 and VL containing the VL CDR of 9C8, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 9C8, (c) VH containing VH CDR of 2H2 and VL containing VL CDR of 2H2, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 2H2. (d) VH containing the VH CDR of VH of 25E5 and VL containing the VL CDR of VH of 25E5, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 25E5. (e) VH containing the VH CDR of VH of 24E5 and VL containing the VL CDR of 24E5, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 24E5. (f) VH containing the VH CDR of VH of 21D9 and VL containing the VL CDR of 21D9, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 21D9. (g) VH containing the VH CDR of VH of 21D9.b and VL containing the VL CDR of 21D9.b, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 21D9.b. (h)VH containing the VH CDR of VH of 21D9.c and VL containing the VL CDR of 21D9.c and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 21D9.c. (i) VH containing the VH CDR of VH of 21D9.d and VL containing the VL CDR of 21D9.d, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 21D9.d. (j)VH containing the VH CDR of VH of 21D9.e and VL containing the VL CDR of 21D9.e and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 21D9.e. (k) VH containing the VH CDR of 21A5 and VL containing the VL CDR of 21A5, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 21A5, (l) VH containing the VH CDR of VH of 21A5.a and VL containing the VL CDR of 21A5.a, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 21A5.a. (m)VH containing VH CDR of VH of 10F10 and VL containing VL CDR of 10F10, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 10F10, (n) VH containing the VH CDR of VH of 10F10.1 and VL containing the VL CDR of 10F10.1, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 10F10.1. (o) VH containing the VH CDR of VH of 10F10.3 and VL containing the VL CDR of 10F10.3, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 10F10.3, or (p)VH containing the VH CDR of VH of 10F10.3 and VL containing the VL CDR of 10F10.4, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 10F10.4 It may include. In some of the embodiments described above, VH and / or VL may differ from the respective sequences of species (a) to (p) by the presence of one, two, three, four, or five amino acid substitutions, such as one, two, three, four, or five conservative substitutions. In some embodiments, VH may include one, two, three, four, or five return substitutions.

[0134] ILT4 Ab is (a) VH consisting of the amino acid sequence of VH of 9G4 and VL consisting of VL of 9G4, (b) VH consisting of the amino acid sequence of VH at 9C8 and VL consisting of VL at 9C8, (c) VH consisting of the amino acid sequence of VH of 2H2 and VL consisting of VL of 2H2, (d) VH consisting of the amino acid sequence of VH of 25E5 and VL consisting of VL of 25E5, (e) VH consisting of the amino acid sequence of VH of 24E5 and VL consisting of VL of 24E5, (f) VH consisting of the amino acid sequence of VH of 21D9 and VL consisting of VL of 21D9, (g) VH consisting of the amino acid sequence of VH of 21D9.b and VL consisting of VL of 21D9.b, (h) VH consisting of the amino acid sequence of VH of 21D9.c and VL consisting of VL of 21D9.c, (i) VH consisting of the amino acid sequence of VH of 21D9.d and VL consisting of VL of 21D9.d, (j) VH consisting of the amino acid sequence of VH from 21D9.e and VL consisting of VL from 21D9.e, (k) VH consisting of the amino acid sequence of VH of 21A5 and VL consisting of VL of 21A5, (l) VH consisting of the amino acid sequence of VH of 21A5.a and VL consisting of VL of 21A5.a, VH consisting of the amino acid sequence of (m)10F10 VH and VL consisting of 10F10 VL, (n) VH consisting of the amino acid sequence of VH of 10F10.1 and VL consisting of VL of 10F10.1, (o) VH consisting of the amino acid sequence of VH of 10F10.3 and VL consisting of VL of 10F10.3, or (p) VH consisting of the amino acid sequence of VH of 10F10.4 and VL consisting of VL of 10F10.4 It may include.

[0135] In certain embodiments, ILT4 Ab is one of the variable regions and / or variable regions CDR1-3 of the antibody described above and elsewhere in this specification, for example, (1) One or more VH CDR1, CDR2, and CDR3 of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4; (2) VH CDR1, CDR2 and CDR3 of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4; (3) VH of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4; (4) One or more VH CDR1, CDR2, and CDR3 of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4, and one or more VL CDR1, CDR2, and CDR3; (5) VH CDR1, CDR2 and CDR3 and VL CDR1, CDR2 and CDR3 of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4; (6) VH and VL of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4; or (7) The VL and VH of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4 may be included, excluding 1, 2, 3, 4, or 5 reversion or conservative substitutions in the VH and / or VL; and ILT4 Ab may also be an IgG antibody such as IgG1, IgG2, IgG3, or IgG4 antibody or a modification thereof as described in the following sections. In some embodiments, the constant region has effector function, and in some embodiments, the constant region does not have effector function. In certain embodiments, the constant region is IgG1.3 or IgG1.1f, or another constant region as described herein, e.g., IgG1 and IgG1.238K.

[0136] In certain embodiments, ILT4 Ab is one of the variable regions and / or variable regions CDR1-3 of the antibody described above and elsewhere in this specification, for example, (1) One or more VH CDR1, CDR2, and CDR3 of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4; (2) VH CDR1, CDR2 and CDR3 of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4; (3) VH of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4; (4) One or more VH CDR1, CDR2, and CDR3 of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4, and one or more VL CDR1, CDR2, and CDR3; (5) VH CDR1, CDR2 and CDR3 and VL CDR1, CDR2 and CDR3 of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4; (6) VH and VL of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4; or (7) VL and VH of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4, excluding one, two, three, four, or five reversion or conservative substitutions in VH and / or VL; Furthermore Antibodies further include one or more of the following characteristics: - For example, 10 -8 M, 10 -9 K below M D Specific binding to hILT4; - Lack of specific binding to hILT2, hILT3, and hILT5; - Lack of specific binding to one or more members of the LILRA and / or LILRB family; - For example, stimulating T cell activation in the MLR, as measured by increased T cell proliferation or IFN-g secretion, as shown in the assay described in the examples; - For example, stimulating the differentiation or activation of monocytes into macrophages, as shown in the assay described in the examples; for example, stimulating the differentiation of monocytes into inflammatory macrophages; - For example, promoting the expression of CD83 and CD86 on human monocyte-derived immature dendritic cells (Mo-iDCs), as shown in the assay described in the examples; - For example, enhancing IFN-γ secretion upon antigen stimulation in a CMV lysate assay, as shown in the assay described in the examples; - For example, enhancing IFN-γ and TNF-α secretion by CD4+ and CD8+ T cells in an allogeneic mixed lymphocyte response (allogeneic MLR) assay upon CD3 stimulation, as shown in the assay described in the examples; - Do not induce (or trigger) basophil activation; - Inhibiting the binding of HLA-A and / or HLA-B to ILT4; - Having a binding profile to hILT4, as shown in Figure 27 or Figure 37C; - For example, as shown in the HDX assay described in the examples, when determined by deuterium exchange (HDX), (i) 70 ITRIRPEL 77 (Sequence ID 120) and / or 78 VKNGQFHIPSITWEHTGRYGCQY 100 (Sequence ID 121); (ii) 70 ITRIRPELVKNGQFHIPSITWEHTGRYGCQY 100 (Sequence ID 122); or (iii) 154 ILCKEGEEEHPQCLNSQPHARGSSRAIF 181 (Sequence ID 123) and / or425 SSPPPTGPIS 434 Binding to the Ig-like domains 1, 2, or 1 and 2 of hILT4 (corresponding to amino acids 27-110 and 111-229 of SEQ ID NO: 107, respectively), such as the region containing (SEQ ID NO: 124); - The amino acid number of hILT4 is that of immature hILT4 (i.e., ILT4 with its signal sequence), and it does not significantly bind to other regions of the extracellular domain of ILT4, such as the region or residue located at the N-terminus relative to amino acid I70; - Competing with the antibodies described herein for binding to hILT4; - For example, specifically binding to cynomolgus monkey ILT4 containing SEQ ID NO: 118, as shown in the binding assay described in the examples; - Promoting inflammatory polarization of macrophages to M1 macrophages; - Not induce (or trigger) basophil activation; and - After incubation at 25°C for 3 months, it must contain less than 5% high and low molecular weight species, and / or less than 10% high and low molecular weight species after incubation at 40°C for 3 months.

[0137] In certain embodiments, ILT4 Ab comprises a heavy chain (HC) containing the amino acid sequence of any of the heavy chains of ILT4 Ab provided herein. In certain embodiments, ILT4 Ab comprises a heavy chain containing the amino acid sequence of any one of the heavy chains of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4, as shown in the following sequence listing (i.e., the complete HC sequence provided in the table, or a composite of the VH sequence of the antibody and an HC constant region sequence such as SEQ ID NOs. 98, 100, 102, 103, 104, or 179, as shown in the table).

[0138] Thus, for example, in some embodiments, the antibody may comprise one HC amino acid sequence of the following types, comprising the following amino acid sequences: a. IgG1, such as 9G4.IgG1, 9G4 (SEQ ID NO: 2), 9C8 (SEQ ID NO: 4), 2H2 (SEQ ID NO: 6), 2E5 (SEQ ID NO: 8), 24E5 (SEQ ID NO: 10), 21D9 (SEQ ID NO: 12), 21D9.b (SEQ ID NO: 74 and SEQ ID NO: 98), 21D9.c (SEQ ID NO: 75 and SEQ ID NO: 98), 21D9.d (SEQ ID NO: 78 and SEQ ID NO: 98), 21D9.e (SEQ ID NO: 80 and SEQ ID NO: 98), 21A5 (SEQ ID NO: 83 and SEQ ID NO: 98), 21A5.a (SEQ ID NO: 87 and SEQ ID NO: 98), 10F10 (SEQ ID NO: 91 and SEQ ID NO: 98), 10F10.1 (SEQ ID NO: 91 and SEQ ID NO: 98), 10F10.3 (SEQ ID NO: 91 and SEQ ID NO: 98), or 10F10.4 (SEQ ID NO: 91 and SEQ ID NO: 98), b. IgG1, such as 9G4.IgG1, 9G4 (SEQ ID NO: 51, amino acids 20 - 135, and SEQ ID NO: 102), 9C8 (SEQ ID NO: 55 and SEQ ID NO: 102), 2H2 (SEQ ID NO: 58 and SEQ ID NO: 102), 2E5 (SEQ ID NO: 63 and SEQ ID NO: 102), 24E5 (SEQ ID NO: 67 and SEQ ID NO: 102), 21D9 (SEQ ID NO: 71 and SEQ ID NO: 102), 21D9.b (SEQ ID NO: 74 and SEQ ID NO: 102), 21D9.c (SEQ ID NO: 75 and SEQ ID NO: 102), 21D9.d (SEQ ID NO: 78 and SEQ ID NO: 102), 21D9.e (SEQ ID NO: 80 and SEQ ID NO: 102), 21A5 (SEQ ID NO: 83 and SEQ ID NO: 102), 21A5.a (SEQ ID NO: 87 and SEQ ID NO: 102), 10F10 (SEQ ID NO: 91 and SEQ ID NO: 102), 10F10.1 (SEQ ID NO: 91 and SEQ ID NO: 102), 10F10.3 (SEQ ID NO: 91 and SEQ ID NO: 102), or 10F10.4 (SEQ ID NO: 91 and SEQ ID NO: 102),[[ID=⑤]] c.IgG1.3 (e.g., 9G4.IgG1.3), 9G4 (SEQ ID NO: 51, amino acids 20-135, and SEQ ID NO: 100), 9C8 (SEQ ID NO: 55 and SEQ ID NO: 100), 2H2 (SEQ ID NO: 58 and SEQ ID NO: 100), 2E5 (SEQ ID NO: 63 and SEQ ID NO: 100), 24E5 (SEQ ID NO: 67 and SEQ ID NO: 100), 21D9 ((i) SEQ ID NO: 113, or (ii) SEQ ID NO: 71 and SEQ ID NO: 1 00), 21D9.b (sequence number 36), 21D9.c (sequence number 38), 21D9.d (sequence number 40), 21D9.e (sequence number 13), 21A5 (sequence number 15), 21A5.a (sequence number 17), 10F10 (sequence number 19), 10F10.1 (sequence numbers 91 and 100), 10F10.3 (sequence numbers 91 and 100), or 10F10.4 (sequence numbers 91 and 100); d.IgG1.1f (e.g., 9G4.IgG1.1f), 9G4 (SEQ ID NO: 51, amino acids 20-135, and SEQ ID NO: 103), 9C8 (SEQ ID NO: 55 and SEQ ID NO: 103), 2H2 (SEQ ID NO: 58 and SEQ ID NO: 103), 2E5 (SEQ ID NO: 63 and SEQ ID NO: 103), 24E5 (SEQ ID NO: 67 and SEQ ID NO: 103), 21D9 (SEQ ID NO: 71 and SEQ ID NO: 103), 21D9.b (SEQ ID NO: 74 and SEQ ID NO: 103), 21D9.c (SEQ ID NO: 75) (and sequence number 103), 21D9.d (sequence number 78 and sequence number 103), 21D9.e (sequence number 80 and sequence number 103), 21A5 (sequence number 83 and sequence number 103), 21A5.a (sequence number 87 and sequence number 103), 10F10 (sequence number 91 and sequence number 103), 10F10.1 (sequence number 91 and sequence number 103), 10F10.3 (sequence number 91 and sequence number 103), or 10F10.4 (sequence number 91 and sequence number 103), e. IgG1fa.P238K (e.g., 9G4.IgG1fa.P238K, etc.), 9G4 (SEQ ID NO: 51, amino acids 20 - 135, and SEQ ID NO: 104), 9C8 (SEQ ID NO: 55 and SEQ ID NO: 104), 2H2 (SEQ ID NO: 58 and SEQ ID NO: 104), 2E5 (SEQ ID NO: 63 and SEQ ID NO: 104), 24E5 (SEQ ID NO: 67 and SEQ ID NO: 104), 21D9 (SEQ ID NO: 71 and SEQ ID NO: 104), 21D9.b (SEQ ID NO: 74 and SEQ ID NO: 104), 21D9.c (SEQ ID NO: 75 and SEQ ID NO: 104), 21D9.d (SEQ ID NO: 78 and SEQ ID NO: 104), 21D9.e (SEQ ID NO: 80 and SEQ ID NO: 104), 21A5 (SEQ ID NO: 83 and SEQ ID NO: 104), 21A5.a (SEQ ID NO: 87 and SEQ ID NO: 104), 10F10 (SEQ ID NO: 91 and SEQ ID NO: 104), 10F10.1 (SEQ ID NO: 91 and SEQ ID NO: 104), 10F10.3 (SEQ ID NO: 91 and SEQ ID NO: 104), or 10F10.4 (SEQ ID NO: 91 and SEQ ID NO: 104), or f. IgG4 S228P (e.g., 9G4.IgG4 S228P, etc.), 9G4 (SEQ ID NO: 51, amino acids 20 - 135, and SEQ ID NO: 179), 9C8 (SEQ ID NO: 55 and SEQ ID NO: 179), 2H2 (SEQ ID NO: 58 and SEQ ID NO: 179), 2E5 (SEQ ID NO: 63 and SEQ ID NO: 179), 24E5 (SEQ ID NO: 67 and SEQ ID NO: 179), 21D9 (SEQ ID NO: 71 and SEQ ID NO: 179), 21D9.b (SEQ ID NO: 74 and SEQ ID NO: 179), 21D9.c (SEQ ID NO: 75 and SEQ ID NO: 179), 21D9.d (SEQ ID NO: 78 and SEQ ID NO: 179), 21D9.e (SEQ ID NO: 80 and SEQ ID NO: 179), 21A5 (SEQ ID NO: 83 and SEQ ID NO: 179), 21A5.a (SEQ ID NO: 87 and SEQ ID NO: 179), 10F10 (SEQ ID NO: 91 and SEQ ID NO: 179), 10F10.1 (SEQ ID NO: 91 and SEQ ID NO: 179), 10F10.3 (SEQ ID NO: 91 and SEQ ID NO: 179), or 10F10.4 (SEQ ID NO: 91 and SEQ ID NO: 179).

[0139] In certain embodiments, ILT4 Ab comprises a heavy chain containing the amino acid sequence of any of the heavy chains of ILT4 Ab provided herein, including the IgG1.3 heavy chain constant region, and an amino acid sequence of any of the light chains of ILT4 Ab provided herein. In certain embodiments, ILT4 Ab comprises IgG1.3 It comprises a heavy chain containing the amino acid sequence of one of the following VHs: 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4, and a light chain containing the amino acid sequence of one of the following light chains: 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4. In some embodiments, the light chain is a kappa light chain containing the following amino acid sequence: 9G4 (SEQ ID NO: 1), 9C8 (SEQ ID NO: 3), 2H2 (SEQ ID NO: 5), 2E5 (SEQ ID NO: 7), 24E5 (SEQ ID NO: 9), 21D9 (SEQ ID NO: 11), 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5 (SEQ ID NO: 14), 21A5.a (SEQ ID NO: 16), 10F10 (SEQ ID NO: 18), 10F10.1 (SEQ ID NO: 20), 10F10.3 (SEQ ID NO: 21), or 10F10.4 (SEQ ID NO: 116).

[0140] ILT4 Ab is (a) A heavy chain containing the amino acid sequence of the 9G4 heavy chain and a light chain containing the amino acid sequence of the 9G4 light chain, (b) A heavy chain containing the amino acid sequence of the 9C8 heavy chain and a light chain containing the amino acid sequence of the 9C8 light chain, (c) Heavy chain containing the amino acid sequence of the 2H2 heavy chain and light chain containing the amino acid sequence of the 2H2 light chain, (d) A heavy chain containing the amino acid sequence of the 25E5 heavy chain and a light chain containing the amino acid sequence of the 25E5 light chain, (e) A heavy chain containing the amino acid sequence of the 24E5 heavy chain and a light chain containing the amino acid sequence of the 24E5 light chain, (f) A heavy chain containing the amino acid sequence of the 9G4 heavy chain and a light chain containing the amino acid sequence of the 21D9 light chain, (g) Heavy chain containing the amino acid sequence of the heavy chain of 21D9.b and light chain containing the amino acid sequence of the light chain of 21D9.b, (h) Heavy chain containing the amino acid sequence of the heavy chain of 21D9.c and light chain containing the amino acid sequence of the light chain of 21D9.c, (i) A heavy chain containing the amino acid sequence of the heavy chain of 21D9.d and a light chain containing the amino acid sequence of the light chain of 21D9.d, (j) Heavy chain containing the amino acid sequence of the heavy chain of 21D9.e and light chain containing the amino acid sequence of the light chain of 21D9.e, (k) A heavy chain containing the amino acid sequence of the 21A5 heavy chain and a light chain containing the amino acid sequence of the 21A5 light chain, (l) A heavy chain containing the amino acid sequence of the heavy chain of 21A5.a and a light chain containing the amino acid sequence of the light chain of 21A5.a, (m) A heavy chain containing the amino acid sequence of the heavy chain of 10F10 and a light chain containing the amino acid sequence of the light chain of 10F10, (n) A heavy chain containing the amino acid sequence of the heavy chain of 10F10.1 and a light chain containing the amino acid sequence of the light chain of 10F10.1, (o) A heavy chain containing the amino acid sequence of the heavy chain of 10F10.3 and a light chain containing the amino acid sequence of the light chain of 10F10.3, or (p) Heavy chain containing the amino acid sequence of the heavy chain of 10F10.4 and light chain containing the amino acid sequence of the light chain of 10F10.4 It may include.

[0141] ILT4 Ab is (a) A heavy chain (HC) containing an HC CDR of 9G4 and a light chain (LC) containing an LC CDR of 9G4, and the HC and LC amino acid sequences that are 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%, or at least 99% identical to the HC and LC of 9G4, respectively. (b) HC containing an HC CDR of 9C8 and a light chain (LC) containing an LC CDR of 9C8, and HC and LC amino acid sequences that are 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%, or at least 99% identical to the HC and LC of 9C8, respectively. (c) HC containing an HC CDR of 2H2 and a light chain (LC) containing an LC CDR of 2H2, and HC and LC amino acid sequences that are 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%, or at least 99% identical to the HC and LC of 2H2, respectively. (d) HC containing an HC CDR of 25E5 and a light chain (LC) containing an LC CDR of 25E5, and HC and LC amino acid sequences that are 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%, or at least 99% identical to HC and LC of 25E5, respectively. (e) an HC containing an HC CDR of 24E5 and a light chain (LC) containing an LC CDR of 24E5, and the HC and LC amino acid sequences being 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%, or at least 99% identical to the HC and LC of 24E5, respectively. (f) HC containing the HC CDR of 21D9 and light chain (LC) containing the LC CDR of 21D9, and HC and LC amino acid sequences that are 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%, or at least 99% identical to the HC and LC of 21D9, respectively. (g) HC containing the HC CDR of 21D9.b and light chain (LC) containing the LC CDR of 21D9.b, and HC and LC amino acid sequences that are 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%, or at least 99% identical to the HC and LC of 21D9.b, respectively. (h) HC containing the HC CDR of 21D9.c and light chain (LC) containing the LC CDR of 21D9.c, and HC and LC amino acid sequences that are 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%, or at least 99% identical to the HC and LC of 21D9.c, respectively. (i) an HC containing an HC CDR of 21D9.d and a light chain (LC) containing an LC CDR of 21D9.d, and the HC and LC amino acid sequences that are 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%, or at least 99% identical to the HC and LC of 21D9.d, respectively. (j) HC containing the HC CDR of 21D9.e and light chain (LC) containing the LC CDR of 21D9.e, and HC and LC amino acid sequences that are 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%, or at least 99% identical to the HC and LC of 21D9.e, respectively. (k) HC containing the HC CDR of 21A5 and light chain (LC) containing the LC CDR of 21A5, and HC and LC amino acid sequences that are 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%, or at least 99% identical to the HC and LC of 21A5, respectively. (l) HC containing the HC CDR of 21A5.a and light chain (LC) containing the LC CDR of 21A5.a, and HC and LC amino acid sequences that are 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%, or at least 99% identical to the HC and LC of 21A5.a, respectively. (m) HC containing the HC CDR of 10F10 and light chain (LC) containing the LC CDR of 10F10, and HC and LC amino acid sequences that are 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%, or at least 99% identical to the HC and LC of 10F10, respectively. (n) HC containing the HC CDR of 10F10.1 and light chain (LC) containing the LC CDR of 10F10.1, and HC and LC amino acid sequences that are 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%, or at least 99% identical to the HC and LC of 10F10.1, respectively. (o) HC containing the HC CDR of 10F10.3 and light chain (LC) containing the LC CDR of 10F10.3, and HC and LC amino acid sequences that are 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%, or at least 99% identical to the HC and LC of 10F10.3, respectively, or (p) HC containing the HC CDR of HC of 10F10.3 and light chain (LC) containing the LC CDR of 10F10.4, and HC and LC amino acid sequences that are 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%, or at least 99% identical to the HC and LC of 10F10.4, respectively. It may include.

[0142] In some of the embodiments described above, HC and / or LC may differ from the respective sequences of species (a) to (p) by the presence of 1, 2, 3, 4, or 5 amino acid substitutions, such as 1, 2, 3, 4, or 5 conservative substitutions.

[0143] In some embodiments, ILT4 Ab is (a) A heavy chain consisting of the amino acid sequence of the 9G4 heavy chain and a light chain consisting of the amino acid sequence of the 9G4 light chain, (b) A heavy chain consisting of the amino acid sequence of the 9C8 heavy chain and a light chain consisting of the amino acid sequence of the 9C8 light chain, (c) Heavy chain consisting of the amino acid sequence of the 2H2 heavy chain and light chain consisting of the amino acid sequence of the 2H2 light chain, (d) A heavy chain consisting of the amino acid sequence of the 25E5 heavy chain and a light chain consisting of the amino acid sequence of the 25E5 light chain, (e) A heavy chain consisting of the amino acid sequence of the 24E5 heavy chain and a light chain consisting of the amino acid sequence of the 24E5 light chain, (f) A heavy chain consisting of the amino acid sequence of the heavy chain of 21D9 and a light chain consisting of the amino acid sequence of the light chain of 21D9, (g) A heavy chain consisting of the amino acid sequence of the heavy chain of 21D9.b and a light chain consisting of the amino acid sequence of the light chain of 21D9.b, (h) A heavy chain consisting of the amino acid sequence of the heavy chain of 21D9.c and a light chain consisting of the amino acid sequence of the light chain of 21D9.c, (i) A heavy chain consisting of the amino acid sequence of the heavy chain of 21D9.d and a light chain consisting of the amino acid sequence of the light chain of 21D9.d, (j) A heavy chain consisting of the amino acid sequence of the heavy chain of 21D9.e and a light chain consisting of the amino acid sequence of the light chain of 21D9.e, (k) A heavy chain consisting of the amino acid sequence of the 21A5 heavy chain and a light chain consisting of the amino acid sequence of the 21A5 light chain, (l)A heavy chain consisting of the amino acid sequence of the heavy chain of 21A5.a and a light chain consisting of the amino acid sequence of the light chain of 21A5.a, (m)A heavy chain consisting of the amino acid sequence of the heavy chain of 10F10 and a light chain consisting of the amino acid sequence of the light chain of 10F10, (n)A heavy chain consisting of the amino acid sequence of the heavy chain of 10F10.1 and a light chain consisting of the amino acid sequence of the light chain of 10F10.1, (o)A heavy chain consisting of the amino acid sequence of the heavy chain of 10F10.3 and a light chain consisting of the amino acid sequence of the light chain of 10F10.3, or (p)A heavy chain consisting of the amino acid sequence of the heavy chain of 10F10.4 and a light chain consisting of the amino acid sequence of the light chain of 10F10.4 may be included.

[0144] In some embodiments, the ILT4 Ab may comprise a heavy chain amino acid sequence comprising the VH amino acid sequence of the antibody species herein, rather than the IgG1.3 heavy chain constant region, as provided in the HC sequences in the Sequence Listing herein (see also SEQ ID NO: 100), and the antibody may be a human wild-type IgG1 constant region such as those provided in SEQ ID NOs: 98 and 102-104, or an IgG4 heavy chain constant region, or an IgG4 constant region having an S228P substitution (EU numbering), or any other heavy chain constant region sequence described in the section regarding constant regions below.

[0145] In some embodiments, such modified ILT4 Abs have one or more of the following characteristics: - For example, 10 -8 M or less, or 10 -9 M or less of K D specific binding to hILT4; - Lack of specific binding to one, two, or all three of hILT2, hILT3, hILT5; - Lack of specific binding to one or more members of the LILRA and / or LILRB family; - Stimulating T cell activation, as measured by increased T cell proliferation or IFN-gamma secretion, for example in an MLR assay; and To stimulate the differentiation or activation of monocytes into macrophages.

[0146] In some embodiments, the antibody is determined by deuterium exchange (HDX), for example, as shown in the HDX assay described in the examples. 70 ITRIRPEL 77 (Sequence ID 120) and / or 78 VKNGQFHIPSITWEHTGRYGCQY 100 (Sequence ID 121); (ii) 70 ITRIRPELVKNGQFHIPSITWEHTGRYGCQY 100 (Sequence ID 122); or (iii) 154 ILCKEGEEEHPQCLNSQPHARGSSRAIF 181 (Sequence ID 123) and / or 425 SSPPPTGPIS 434 It binds to the Ig-like domains 1, 2, or 1 and 2 of hILT4 at the position containing (SEQ ID NO: 124) (corresponding to amino acids 27-110 (domain 1) and amino acids 111-229 (domain 2) on the sequence of SEQ ID NO: 107) (see Figure 27).

[0147] Exemplary antibody constant region In some embodiments, the antibodies described herein include one or more human constant regions. In some embodiments, the human heavy chain constant region is an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is an isotype selected from κ and λ. In some embodiments, the antibodies described herein include a human IgG constant region, e.g., IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibodies described herein include a human IgG4 heavy chain constant region. In some such embodiments, the antibodies described herein include an S241P mutation in the human IgG4 constant region. In some embodiments, the antibodies described herein include a human IgG4 constant region and a human κ light chain.

[0148] The selection of the heavy chain constant region can determine whether the antibody has effector function in vivo. Such effector function, in some embodiments, may include antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), which can result in the death of cells to which the antibody binds. In some methods of treatment, including methods for treating certain cancers, cell death may be desirable, for example, if the antibody binds to cells that support tumor maintenance or growth. Exemplary cells that may support tumor maintenance or growth include, but are not limited to, tumor cells themselves, cells that assist in the recruitment of vascular structures to the tumor, and cells that provide ligands, growth factors, or counterreceptors that support or promote tumor growth or tumor survival. In some embodiments, when effector function is desired, antibodies containing human IgG1 heavy chain or human IgG3 heavy chain are selected.

[0149] In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. The addition or deletion of glycosylation sites to an antibody can conveniently be achieved by altering the amino acid sequence so that one or more glycosylation sites are created or removed.

[0150] If the antibody contains an Fc region, the carbohydrate to which it is attached may also be modified. Natural antibodies produced by mammalian cells typically contain branched oligosaccharides attached by an N-bond to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides may include various carbohydrates attached to GlcNAc in the “stem” of the branched oligosaccharide structure, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and fucose. In some embodiments, the oligosaccharides in the antibodies of the present invention may be modified to produce antibodies with certain improved properties. For example, in some embodiments, the antibody may be defucosylated by mutating a residue such as Asn297, which is normally glycosylated by fucose-containing glycosylation. In some embodiments, the antibodies described herein may contain a defucosylated human IgG1 constant region.

[0151] The antibody is further provided with a bifid oligosaccharide, for example, a branched oligosaccharide attached to the Fc region of the antibody is bifid by GlcNAc. Such antibodies may have reduced fucosylation and / or improved ADCC function. Examples of such antibodies are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and U.S.2005 / 0123546 (Umana et al.). Antibodies having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibodies may have improved CDC function. Such antibodies are described, for example, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0152] Antibodies are also given an amino-terminal leader extension. For example, one or more amino acid residues of an amino-terminal leader sequence are present at the amino-terminus of any one or more heavy or light chains of the antibody. An exemplary amino-terminal leader extension includes or consists of three amino acid residues, a VHS, present on one or both of the light chains of the antibody.

[0153] The in vivo or serum half-lives of human FcRn high-affinity binding polypeptides can be assayed in humans or non-human primates, for example, in transgenic mice administered with polypeptides containing mutant Fc regions. See, for example, Petkova et al. International Immunology 18(12):1759-1769 (2006).

[0154] In some embodiments of the present invention, defucosylated antibodies mediate ADCC more efficiently in the presence of human effector cells than parental antibodies containing fucose, and ADCC activity can generally be determined using in vitro ADCC assays as disclosed herein, but other assays or methods for determining ADCC activity, such as in animal models, are also conceived.

[0155] In certain embodiments, the Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, 322, 330, and / or 331 may be replaced with a different amino acid residue so that the antibody has a modified affinity for the effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand whose affinity is modified may be, for example, the Fc receptor or C1 component of complement. This approach is described in more detail in U.S. Patents 5,624,821 and 5,648,260, both by Winter et al.

[0156] In some cases, one or more amino acids selected from amino acid residues 329, 331, and 322 may be replaced with different amino acid residues so that the antibody has altered C1q binding and / or reduced or absent complement-dependent cell-mediated cytotoxicity (CDC). This approach is described in more detail in U.S. Patent No. 6,194,551 by Idusogie et al.

[0157] In some cases, one or more amino acid residues within amino acid positions 231 and 239 are modified, thereby altering the antibody's ability to fix complement. This approach is further described in PCT publication WO94 / 29351 by Bodmer et al. In some cases, the Fc region may be modified to reduce antibody-dependent cell-mediated cytotoxicity (ADCC) and / or reduce affinity for the Fcγ receptor by modifying one or more amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 28 0, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438 or 439. Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Exemplary variants include 239D / 332E, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T, and 267E / 268F / 324T. Other Fc modifications that can be performed on Fc include reducing or cleaving binding to FcγR and / or complement proteins, thereby reducing or cleaving Fc-mediated effector functions, such as ADCC, ADCP, and CDC. Exemplary modifications include, but are not limited to, substitutions, insertions, and deletions of positions 234, 235, 236, 237, 267, 269, 325, 328, 330, and / or 331 (e.g., 330 and 331), and numbering follows the EU index.Exemplary substitutions, but not limited to them, include 234A, 235E, 236R, 237A, 267R, 269R, 325L, 328R, 330S, and 331S (e.g., 330S and 331S), numbered according to the EU index. Fc variants may include 236R / 328R. Other modifications to reduce FcγR and complement interactions include substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P, and 234V, as well as the removal of glycosylation at position 297 by mutation, enzymatic means, or production in organisms such as non-glycosylating bacteria. These and other modifications are outlined in Strohl, 2009, Current Opinion in Biotechnology 20:685-691. For example, the human IgG1.3 Fc constant region contains L234A, L235E, and G237A substitutions. IgG1fa.P238K (or IgG1.P238K) contains the P238K substitution. IgG1.1f contains L234A, L235E, G237A, A330S, and P331S substitutions.

[0158] Fc variants that enhance affinity for the inhibitory receptor FcγRIIb may also be used. Such variants may provide an Fc fusion protein having immunomodulatory activity associated with FcγRIIb cells, including, for example, B cells and monocytes. In one embodiment, the Fc variant selectively provides enhanced affinity for FcγRIIb associated with one or more activating receptors. Modifications to alter binding to FcγRIIb include one or more modifications at positions selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, 330, 331, and 332 according to the EU index. Examples of substitutions to enhance FcγRllb affinity include, but are not limited to, 234A, 234D, 234E, 234F, 234W, 235D, 235E, 235F, 235R, 235Y, 236D, 236N, 237A, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, 330S, 331S, and 332E. Examples of substitutions include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Other Fc variants that enhance binding to FcγRIIb include 235Y / 267E, 236D / 267E, 239D / 268D, 239D / 267E, 267E / 268D, 267E / 268E, and 267E / 328F.

[0159] Other modifications to enhance FcγR and complement interactions include, but are not limited to, substitutions of 298A, 333A, 334A, 326A, 2471, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 3051, and 396L. These and other modifications are outlined in Strohl, 2009, Current Opinion in Biotechnology 20:685-691. Fc modifications that increase binding to the Fcγ receptor are located at amino acid positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 324, 32 Examples of amino acid modifications include any one or more of 7, 329, 330, 335, 337, 338, 340, 360, 373, 376, 379, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439, where the numbering of residues in the Fc region is that of the EU index, as in Patent Publication WO00 / 42072.

[0160] The Fc region may optionally include amino acid residues that do not exist in nature at further and / or alternative positions known to those skilled in the art (e.g., U.S. Patents 5,624,821, 6,277,375, 6,737,056, 6,194,551, 7,317,091, 8,101,720, PCX Patent Publication WO00 / 42072, See WO01 / 58957, WO02 / 06919, WO04 / 016750, WO04 / 029207, WO04 / 035752, WO04 / 074455, WO04 / 099249, WO04 / 063351, WO05 / 070963, WO05 / 040217, WO05 / 092925 and WO06 / 020114.

[0161] The affinity and binding properties of the Fc region to its ligand can be determined by various in vitro assay methods (biochemical or immunological assays) known in the art, including, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetics (e.g., BIACORE analysis) and other methods, such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods can utilize labels on one or more of the components being investigated and / or can use various detection methods, including chromogenic, fluorescent, luminescent, or isotopic labeling. A detailed description of binding affinity and kinetics can be found in Paul, WE, ed., Fundamental immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

[0162] In certain embodiments, antibodies are modified to increase their biological half-life. Various approaches are possible. For example, this can be done by increasing the binding affinity of the Fc region to FcRn. For example, one or more of the following residues may be mutated, as described in U.S. Patent No. 6,277,375: 252, 254, 256, 433, 435, 436. Specific exemplary substitutions include one or more of the following: T252L, T254S, and / or T256F. Alternatively, to increase the biological half-life, as described in U.S. Patents No. 5,869,046 and 6,121,022 by Presta et al., antibodies may be modified within the CH1 or CL region to contain a salvage receptor-binding epitope taken from two loops of the CH2 domain of the Fc region of IgG. Other exemplary variants that increase binding to FcRn and / or improve pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434, such as 2591, 308F, 428L, 428M, 434S, 4341, 1.434F, 434Y, and 434X1.Other mutants that increase Fc coupling with FcRn include: 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al. 2004, J. Biol. Chem. 279(8):6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 311A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields et al., Journal of Biological Chemistry, 2001, 276(9):6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 311S, 433 R, 433S, 4331, 433P, 433Q, 434H, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H, 308T / 309P / 311S (Dall Acqua et al. Journal of Immunology, 2002, 169:5171-5180, Dall'Acqua et al., 2006, Journal of Biological Chemistry 281:23514-23524). Other modifications for regulating FcRn binding are described in Yeung et al., 2010, J Immunol, 182:7663-7671.

[0163] In certain embodiments, hybrid IgG isotypes having specific biological characteristics may be used. For example, IgG1 / IgG3 hybrid variants can be constructed by substituting IgG1 positions in the CH2 and / or CH3 regions with amino acids derived from IgG3, with the two isotypes at different positions. Thus, hybrid variant IgG antibodies containing one or more substitutions, e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 422I, 435R, and 436F, can be constructed. In some embodiments described herein, IgG1 / IgG2 hybrid variants can be constructed by substituting IgG2 positions in the CH2 and / or CH3 regions with amino acids derived from IgG1, with the two isotypes at different positions. Therefore, hybrid mutant IgG antibodies can be constructed that contain one or more substitutions, for example, one or more of the following amino acid substitutions: 233E, 234L, 235L, -236G (called a glycine insertion at position 236), and 327A.

[0164] Furthermore, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn have been mapped, and mutants with improved binding have been described (see Shields, RL et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcγRIII. Additionally, the following combination mutants have been shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A (Shields et al., 2001), which have been shown to exhibit enhanced FcγRIIIa binding and ADCC activity. Other IgG1 mutants with strongly enhanced binding to FcγRIIIa have been identified, including mutants with the S239D / I332E and S239D / I332E / A330L mutations, which showed the greatest increase in affinity for FcγRIIIa, decreased FcγRIIb binding, and potent cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006). By introducing triple mutations into antibodies such as alemtuzumab (CD52-specific), trastuzumab (HER2 / neu-specific), rituximab (CD20-specific), and cetuximab (EGFR-specific), they were converted to significantly enhanced ADCC activity in vitro, and the S239D / I332E mutant showed an enhanced ability to deplete B cells in monkeys (Lazar et al., 2006). Furthermore, IgG1 mutants containing L235V, F243L, R292P, Y300L, and P396L mutations have been identified in transgenic mice expressing human FcγRIIIa in B-cell malignancies and breast cancer models, showing enhanced FcγRIIIa binding and simultaneously enhanced ADCC activity (Stavenhagen et al., 2007; Nordstrom et al., 2011).Other Fc mutants that may be used include S298A / E333A / L334A, S239D / I332E, S239D / I332E / A330L, L235V / F243L / R292P / Y300L / P396L, and M428L / N434S.

[0165] In certain embodiments, Fc having reduced FcγR binding is selected. Exemplary Fc having reduced FcγR binding, for example, IgG1 Fc, includes the following three amino acid substitutions: L234A, L235E, and G237A.

[0166] In certain embodiments, Fc having reduced complement binding is selected. An exemplary Fc having reduced complement binding, for example, IgG1 Fc, has the following two amino acid substitutions: A330S and P331S.

[0167] In certain embodiments, an Fc that is essentially without effector function is selected, i.e., it has reduced binding to FcγR and reduced complement binding. Exemplary Fc without effector function, e.g., IgG1 Fc, includes the following five mutations: L234A, L235E, G237A, A330S, and P331S.

[0168] When using the IgG4 constant domain, it can contain the substitution S228P, which mimics the hinge sequence in IgG1, thereby stabilizing the IgG4 molecule.

[0169] The Fc modification described in WO2017 / 087678 or WO2016081746 may also be used.

[0170] In certain embodiments, the glycosylation of an antibody is modified. For example, a non-glycosylated antibody may be made (i.e., the antibody lacks glycosylation). Glycosylation may be modified, for example, to increase the antibody's affinity for an antigen. Such carbohydrate modification may be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions may be made to eliminate one or more variable region framework glycosylation sites, thereby resulting in the elimination of glycosylation at those sites. Such nonglycosylation may increase the antibody's affinity for an antigen. Such approaches are described in more detail in U.S. Patents 5,714,350 and 6,350,861 by Co et al.

[0171] Constant region glycosylation at N297 can be prevented by mutating the N297 residue to another residue, e.g., N297A, and / or by mutating an adjacent amino acid, e.g., 298, thereby reducing glycosylation at N297.

[0172] Furthermore, antibodies with altered glycosylation types can be produced, for example, low-fucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bipartite GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC capacity of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing antibodies in host cells having an altered glycosylation mechanism. Cells having an altered glycosylation mechanism are described in the Art and can be used as host cells for expressing recombinant antibodies described herein and thereby producing antibodies with altered glycosylation. For example, Hanai et al., in EP1,176,195, describe a cell line having a functionally disrupted FUT8 gene encoding fucosyltransferase, and as a result, antibodies expressed in such cell lines exhibit low fucosylation. Presta's PCT publication WO03 / 035835 describes the Led 3 cell line, a mutant CHO cell line that has reduced ability to attach fucose to Asn(297)-linked carbohydrates, resulting in reduced fucosylation of antibodies expressed in its host cells (see also Shields, RL et al. (2002) J. Biol. Chem. 277:26733-26740). Umana et al.'s PCT publication WO99 / 54342 describes a cell line engineered to express a glycoprotein-modified glycosyltransferase (e.g., beta(l,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that the antibody expressed in the engineered cell line exhibits an enlarged bifid GlcNac structure, which results in the antibody's increased ADCC activity (see also Umana et al. (1999) Nat. Biotech. 17: 176-180).

[0173] Another modification of antibodies described herein is pegylation. Antibodies may be pegylated, for example, to increase their biological (e.g., serum) half-life. To pegylate an antibody, the antibody or a fragment thereof is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups are attached to the antibody or antibody fragment. In some embodiments, pegylation is carried out by an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). In this specification, the term “polyethylene glycol” is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono(CI-CIO)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is a non-glycosylated antibody. Methods for pegyling proteins are known in the art and may be applied to the antibodies described herein. For example, see EP0154316 by Nishimura et al. and EP0401384 by Ishikawa et al.

[0174] Specific exemplary embodiments of antibodies described herein are further listed in the following sections.

[0175] Embodiments relating to antibody 21D9.e: Embodiment 1: An isolated antibody that conjugates to human ILT4 (hILT4), comprising a heavy chain and a light chain, wherein (i) the heavy chain comprises VH CDR1, CDR2 and CDR3, each containing SEQ ID NOs. 155, 156 and 157, and the light chain comprises VL CDR1, CDR2 and CDR3, each containing SEQ ID NOs. 158, 159 and 160, and / or (ii) the heavy chain comprises VH, each containing SEQ ID NOs. 80, and the light chain comprises VL, each containing SEQ ID NOs. 70.

[0176] Embodiment 2: Ab comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 155, 156 and 157, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 158, 159 and 160, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 80, and the light chain comprises VL comprising SEQ ID NOs. 70, and the antibody is an isolated antibody that binds to human ILT4, for example, (i) in a mixed lymphocyte reaction (MLR) assay as demonstrated, for example, by enhanced T cell proliferation or IFN-gamma secretion or TNF-alpha production, or (ii) in a T cell:CHO-OKT3-ILT4 assay (for example, as described in Example 5).

[0177] Embodiment 3: Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 155, 156 and 157, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 158, 159 and 160, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 80, and the light chain comprises VL comprising SEQ ID NOs. 70, and Ab is an isolated antibody that conjugates to human ILT4 (hILT4) and enhances T cell activation in monocyte:T cell allogeneic MLR.

[0178] Embodiment 4: Ab comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 155, 156 and 157, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 158, 159 and 160, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 80, and the light chain comprises VL comprising SEQ ID NOs. 70, and Ab is an isolated antibody that conjugates to human ILT4 (hILT4) and enhances IFN-gamma secretion from PBMCs upon antigen stimulation in a cytomegalovirus (CMV) lysate assay.

[0179] Embodiment 5: Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 155, 156 and 157, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 158, 159 and 160, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 80, and the light chain comprises VL comprising SEQ ID NOs. 70, wherein the antibody is an isolated antibody that binds to human ILT4, which enhances TNF-alpha secretion from macrophages, e.g., differentiated macrophages.

[0180] Embodiment 6: An isolated antibody that conjugates to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 155, 156 and 157, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 158, 159 and 160, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 80, and the light chain comprises VL comprising SEQ ID NOs. 70, and the antibody increases the expression of CD83 and / or CD86 on monocyte-derived dendritic cells (MoDCs).

[0181] Embodiment 7: Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 155, 156 and 157, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 158, 159 and 160, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 80, and the light chain comprises VL comprising SEQ ID NOs. 70, and the antibody is an isolated antibody that binds to human ILT4, inhibiting the binding of hILT4 to T cells and / or binding partners, such as HLA-A and / or HLA-B.

[0182] Embodiment 8: Ab comprises a heavy chain and a light chain, and (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 each comprising SEQ ID NO: 155, 156, and 157, and the light chain comprises VL CDR1, CDR2 and CDR3 each comprising SEQ ID NO: 158, 159 and 160, and / or (ii) the heavy chain comprises a VH comprising SEQ ID NO: 80, the light chain comprises a VL comprising SEQ ID NO: 70, and Ab, when determined by (a) HDX, 70 ITRIRPEL 77 (SEQ ID NO: 120) and / or 78 VKNGQFHIPSITWEHTGRYGCQY 100 (SEQ ID NO: 121) and / or 70 ITRIRPELVKNGQFHIPSITWEHTGRYGCQY 100 (SEQ ID NO: 122) binds to the Ig-like domain of hILT4 and / or a region of hILT4; and / or (b) when determined by carbene footprinting, binds to human ILT4 (hILT4) that interacts with one or more (or all) of the amino acid residues Lys43, Ile49, Thr50 and Arg51 of mature hILT4. <00,01095>

[0183] Embodiment 9: Ab comprises a heavy chain and a light chain, and (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 each comprising SEQ ID NO: 155, 156, and 157, and the light chain comprises VL CDR1, CDR2 and CDR3 each comprising SEQ ID NO: 158, 159 and 160, and / or (ii) the heavy chain comprises a VH comprising SEQ ID NO: 80, the light chain comprises a VL comprising SEQ ID NO: 70, and Ab inhibits (or competes with) the binding of an antibody comprising a HC comprising SEQ ID NO: 12 or 13 and a light chain comprising SEQ ID NO: 11, and / or inhibits the binding of an antibody comprising the VH and VL of 21D9.IgG1.1f and / or 21D9.e.IgG1.3 to hILT4, an isolated antibody that binds to human ILT4 (hILT4).

[0184] Embodiment 10: Ab comprises a heavy chain and a light chain, and (i) the heavy chain comprises VH CDR1, CDR2, and CDR3 each containing SEQ ID NO: 155, 156, and 157, and the light chain comprises VL CDR1, CDR2, and CDR3 each containing SEQ ID NO: 158, 159, and 160, and / or (ii) the heavy chain comprises a VH containing SEQ ID NO: 80, the light chain comprises a VL containing SEQ ID NO: 70, and Ab is 10 -8 M or 10 -9 An isolated antibody that binds to human ILT4 (hILT4) with a KD of M or less than 10

[0185] Embodiment 11: Ab comprises a heavy chain and a light chain, and (i) the heavy chain comprises VH CDR1, CDR2, and CDR3 each containing SEQ ID NO: 155, 156, and 157, and the light chain comprises VL CDR1, CDR2, and CDR3 each containing SEQ ID NO: 158, 159, and 160, and / or (ii) the heavy chain comprises a VH containing SEQ ID NO: 80, the light chain comprises a VL containing SEQ ID NO: 70, and Ab is 10 -7 M or 10 -8 An isolated antibody that binds to cynomolgus ILT4 with a KD of M or less than 10 and / or promotes the expression of CD80, CD83, and / or CD86 on cynomolgus monocyte-derived dendritic cells, and binds to human ILT4 (hILT4).

[0186] Embodiment 12: Ab comprises a heavy chain and a light chain, and (i) the heavy chain comprises VH CDR1, CDR2, and CDR3 each containing SEQ ID NO: 155, 156, and 157, and the light chain comprises VL CDR1, CDR2, and CDR3 each containing SEQ ID NO: 158, 159, and 160, and / or (ii) the heavy chain comprises a VH containing SEQ ID NO: 80, the light chain comprises a VL containing SEQ ID NO: 70, and Ab does not significantly bind to human proteins LILRA1, LILRA2, LILRA3, LILRA4, LILRA6, ILT2, ILT3, ILT5, or LIR8, and is an isolated antibody that binds to human ILT4 (hILT4).

[0187] Embodiment 13: Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 155, 156 and 157, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 158, 159 and 160, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 80, and the light chain comprises VL comprising SEQ ID NOs. 70, wherein Ab is an isolated antibody that conjugates to human ILT4 (hILT4) and promotes inflammatory polarization of macrophages toward M1 macrophages.

[0188] Embodiment 14: An isolated antibody that binds to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 155, 156 and 157, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 158, 159 and 160, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 80, and the light chain comprises VL comprising SEQ ID NOs. 70, and Ab does not induce (or trigger) basophil activation.

[0189] Embodiment 15: An isolated antibody that conjugates to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 155, 156 and 157, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 158, 159 and 160, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 80, and the light chain comprises VL comprising SEQ ID NOs. 70, and the composition comprising Ab comprises less than 5% high and low molecular weight species after incubation at 25°C for 3 months, and / or less than 10% high and low molecular weight species after incubation at 40°C for 3 months.

[0190] Embodiment 16: An isolated antibody that binds to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 155, 156 and 157, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 158, 159 and 160, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 80, and the light chain comprises VL comprising SEQ ID NOs. 70, and Ab comprises the constant region of the IgG heavy chain, e.g., IgG1 (e.g., SEQ ID NOs. 98 or 102), IgG1.1f (SEQ ID NOs. 103), IgG1.3 (SEQ ID NOs. 100), IgGP238K (SEQ ID NOs. 104), IgG4 or IgG4.S228P (SEQ ID NOs. 179).

[0191] Embodiment 17: An isolated antibody that binds to hILT4, wherein Ab comprises a heavy chain containing SEQ ID NO: 13 and a light chain containing SEQ ID NO: 11, and optionally the heavy chain contains C-terminal lysine.

[0192] Embodiment 18: An isolated antibody that binds to hILT4, wherein Ab comprises two heavy chains, each containing SEQ ID NO: 13, and two light chains, each containing SEQ ID NO: 11, and optionally one or both heavy chains containing C-terminal lysine.

[0193] Embodiment 19: An isolated antibody that binds to hILT4, wherein Ab comprises a heavy chain containing SEQ ID NO: 13 and a light chain containing SEQ ID NO: 11, and optionally the heavy chain contains C-terminal lysine, and Ab contains at least one disulfide bond linking the heavy chain.

[0194] Embodiment 20: An isolated antibody that binds to hILT4, wherein Ab comprises a heavy chain containing SEQ ID NO: 176 and a light chain containing SEQ ID NO: 11, and optionally the heavy chain contains C-terminal lysine.

[0195] Embodiment 21: An isolated antibody that binds to hILT4, wherein Ab comprises a heavy chain containing SEQ ID NO: 177 and a light chain containing SEQ ID NO: 11, and optionally the heavy chain contains C-terminal lysine.

[0196] Embodiment 22: An isolated antibody that binds to hILT4, wherein Ab comprises a heavy chain containing SEQ ID NO: 178 and a light chain containing SEQ ID NO: 11, and optionally the heavy chain contains C-terminal lysine.

[0197] Embodiment 23: An isolated nucleic acid or set of nucleic acids encoding an antibody according to any one of Embodiments 1 to 22.

[0198] Embodiment 24: A host cell for producing any one of the antibodies described in Embodiments 1 to 22, for example, comprising the isolated nucleic acid or set of nucleic acids described in Embodiment 23.

[0199] Embodiment 25: A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective dose of an antibody according to any one of Embodiments 1 to 22, or an antibody conjugated to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, (i) the heavy chain comprising VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 155, 156 and 157, respectively, and the light chain comprising VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 158, 159 and 160, respectively; (ii) the heavy chain comprising VH comprising SEQ ID NOs. 80, and the light chain comprising VL comprising SEQ ID NOs. 70, and / or (iii) the heavy chain comprising SEQ ID NOs. 13, and the light chain comprising SEQ ID NOs. 11.

[0200] Embodiment 26: A method for treating cancer in a subject, wherein the subject is given a therapeutically effective dose, (A)(1) an antibody according to any one of Embodiments 1 to 19, or (2) an antibody that binds to human ILT4 (hILT4), comprising a heavy chain and a light chain, (i) the heavy chain comprising VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 155, 156 and 157, respectively, and the light chain comprising VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 158, 159 and 160, respectively; (ii) the heavy chain comprising VH comprising SEQ ID NOs. 80, and the light chain comprising VL comprising SEQ ID NOs. 70, and / or (iii) the heavy chain comprising Ab comprising SEQ ID NOs. 13, and the light chain comprising SEQ ID NOs. 11, (B) Antagonist antibodies that bind to human PD-1 or human PD-L1, such as nivolumab, pembrolizumab, semiprimab, tripalimab, cintilimab, atezolizumab, durvalumab, or avelumab, which are antagonists of PD-1 or PD-L1. A method including administering [a substance].

[0201] Embodiment 27: (A) (1) an antibody according to any one of Embodiments 1 to 19, or (2) an antibody that binds to human ILT4 (hILT4), comprising a heavy chain and a light chain, (i) the heavy chain comprising VH CDR1, CDR2 and CDR3 including SEQ ID NOs. 155, 156 and 157, respectively, and the light chain comprising VL CDR1, CDR2 and CDR3 including SEQ ID NOs. 158, 159 and 160, respectively. A composition comprising (ii) Ab, whose heavy chain comprises CDR1, CDR2 and CDR3; (ii) Ab, whose heavy chain comprises VH, which comprises SEQ ID NO: 80, and whose light chain comprises VL, which comprises SEQ ID NO: 70, and / or (iii) Ab, whose heavy chain comprises SEQ ID NO: 13, and whose light chain comprises SEQ ID NO: 11; and (B) an antagonist antibody that binds to human PD-1 or human PD-L1, such as nivolumab, pembrolizumab, semiprimab, tripalimab, cintilimab, atezolizumab, durvalumab, or avelumab, which is an antagonist of PD-1 or PD-L1.

[0202] Embodiments relating to antibody 21D5.a: Embodiment 1: An isolated antibody that conjugates to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 161, 162 and 163, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 164, 165 and 166, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 87, and the light chain comprises VL comprising SEQ ID NOs. 86.

[0203] Embodiment 2: Ab comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 161, 162 and 163, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 164, 165 and 166, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 87, and the light chain comprises VL comprising SEQ ID NOs. 86, and the antibody is an isolated antibody that binds to human ILT4 (hILT4) and stimulates T cell activation (or enhances the T cell response) in a mixed lymphocyte reaction (MLR) assay, for example, as demonstrated by (i) enhancement of T cell proliferation or IFN-gamma secretion or TNF-alpha production, for example, or in a T cell:CHO-OKT3-ILT4 assay (for example, as described in Example 5).

[0204] Embodiment 3: Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 161, 162 and 163, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 164, 165 and 166, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 87, and the light chain comprises VL comprising SEQ ID NOs. 86, and Ab is an isolated antibody that conjugates to human ILT4 (hILT4) and enhances T cell activation in monocyte:T cell allogeneic MLR.

[0205] Embodiment 4: Ab comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 161, 162 and 163, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 164, 165 and 166, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 87, and the light chain comprises VL comprising SEQ ID NOs. 86, and Ab is an isolated antibody that conjugates to human ILT4 (hILT4) and enhances IFN-gamma secretion from PBMCs upon antigen stimulation in a CMV lysate assay.

[0206] Embodiment 5: Ab comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 161, 162 and 163, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 164, 165 and 166, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 87, and the light chain comprises VL comprising SEQ ID NOs. 86, and the antibody is an isolated antibody that conjugates to human ILT4 (hILT4) to enhance TNF-alpha secretion from macrophages, e.g., differentiated macrophages.

[0207] Embodiment 6: An isolated antibody that conjugates to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 161, 162 and 163, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 164, 165 and 166, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 87, and the light chain comprises VL comprising SEQ ID NOs. 86, and the antibody increases the expression of CD83 and / or CD86 on monocyte-derived dendritic cells (Mo-DCs).

[0208] Embodiment 7: Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 161, 162 and 163, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 164, 165 and 166, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 87, and the light chain comprises VL comprising SEQ ID NOs. 86, and the antibody is an isolated antibody that binds to human ILT4 (hILT4) and inhibits the binding of hILT4 to T cells and / or binding partners, such as HLA-A and / or HLA-B.

[0209] Embodiment 8: Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3, each including SEQ ID NOs: 161, 162 and 163, and the light chain comprises VL CDR1, CDR2 and CDR3, each including SEQ ID NOs: 164, 165 and 166, and / or (ii) the heavy chain comprises VH, each including SEQ ID NOs: 87, and the light chain comprises VL, each including SEQ ID NOs: 86, and Ab is determined by (a) HDX, 154 ILCKEGEEEHPQCLNSQPHARGSSRAIF 181 (Sequence ID 123) and / or 425 SSPPPTGPIS 434 (b) an isolated antibody that binds to human ILT4 (hILT4), including (SEQ ID NO: 124), the Ig-like 2 domain of hILT4 and / or the region of hILT4; and / or (b) interacts with one or more (or all) of the amino acid residues of mature hILT4, Gly117, Val119, Try120, Leu134, Lys136, Gln149, Pro150, Ile159, Ser161, Val162, Gly163, Pro164, Pro167, His173, Try178, Pro183, and Tyr184.

[0210] Embodiment 9: An isolated antibody that binds to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, and (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 161, 162 and 163, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 164, 165 and 166, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 87, and the light chain comprises VL comprising SEQ ID NOs. 86, and Ab comprises HC comprising SEQ ID NOs. 15 or 17 and a light chain comprising SEQ ID NOs. 14 or 16, respectively, inhibiting (or competing with) the binding of antibodies comprising VH and VL of 21D5 or 21D5.a to hILT4.

[0211] Embodiment 10: Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3, each containing SEQ ID NOs: 161, 162 and 163, and the light chain comprises VL CDR1, CDR2 and CDR3, each containing SEQ ID NOs: 164, 165 and 166, and / or (ii) the heavy chain comprises VH, each containing SEQ ID NOs: 87, and the light chain comprises VL, each containing SEQ ID NOs: 86, and Ab comprises 10 -8 M or 10 -9 Isolated antibodies that bind to human ILT4 (hILT4) with a KD of M or less.

[0212] Embodiment 11: Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3, each containing SEQ ID NOs. 161, 162 and 163, and the light chain comprises VL CDR1, CDR2 and CDR3, each containing SEQ ID NOs. 164, 165 and 166, and / or (ii) the heavy chain comprises VH, each containing SEQ ID NOs. 87, and the light chain comprises VL, each containing SEQ ID NOs. 86, and Ab comprises 10 -7 M or 10 -8 An isolated antibody that binds to cynomolgus monkey ILT4 with a KD of M or less, and / or promotes the expression of CD80, CD83 and / or CD86 on cynomolgus monkey monocyte-derived dendritic cells, and binds to human ILT4 (hILT4).

[0213] Embodiment 12: An isolated antibody that binds to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, and (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 161, 162 and 163, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 164, 165 and 166, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 87, and the light chain comprises VL comprising SEQ ID NOs. 86, and Ab is an isolated antibody that binds to human ILT4 (hILT4), which does not significantly bind to human proteins LILRA1, LILRA2, LILRA4, LILRA5, LILRA6, ILT2, ILT3, ILT5 or LIR8.

[0214] Embodiment 13: Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 161, 162 and 163, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 164, 165 and 166, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 87, and the light chain comprises VL comprising SEQ ID NOs. 86, and Ab is an isolated antibody that conjugates to human ILT4 (hILT4) and promotes inflammatory polarization of macrophages toward M1 macrophages.

[0215] Embodiment 14: An isolated antibody that binds to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 161, 162 and 163, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 164, 165 and 166, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 87, and the light chain comprises VL comprising SEQ ID NOs. 86, and Ab is an isolated antibody that binds to human ILT4 (hILT4) and does not induce (or trigger) basophil activation.

[0216] Embodiment 15: An isolated antibody that conjugates to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 161, 162 and 163, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 164, 165 and 166, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 87, and the light chain comprises VL comprising SEQ ID NOs. 86, and the composition comprising Ab comprises less than 5% high and low molecular weight species after incubation at 25°C for 3 months, and / or less than 10% high and low molecular weight species after incubation at 40°C for 3 months.

[0217] Embodiment 16: An isolated antibody that binds to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 161, 162 and 163, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 164, 165 and 166, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 87, and the light chain comprises VL comprising SEQ ID NOs. 86, and Ab comprises the constant region of the IgG heavy chain, e.g., IgG1 (e.g., SEQ ID NOs. 98 or 102), IgG1.1f (SEQ ID NOs. 103), IgG1.3 (SEQ ID NOs. 100), IgGP238K (SEQ ID NOs. 104), IgG4 or IgG4.S228P (SEQ ID NOs. 179).

[0218] Embodiment 17: An isolated antibody that binds to hILT4, wherein Ab comprises a heavy chain containing SEQ ID NO: 17 and a light chain containing SEQ ID NO: 16, and optionally the heavy chain contains C-terminal lysine.

[0219] Embodiment 18: An isolated antibody that binds to hILT4, wherein Ab comprises two heavy chains, each containing SEQ ID NO: 17, and two light chains, each containing SEQ ID NO: 16, and optionally one or both heavy chains containing C-terminal lysine.

[0220] Embodiment 19: An isolated antibody that binds to hILT4, wherein Ab comprises a heavy chain containing SEQ ID NO: 17 and a light chain containing SEQ ID NO: 16, and optionally the heavy chain contains C-terminal lysine, and Ab contains at least one disulfide bond linking the heavy chain.

[0221] Embodiment 20: An isolated antibody that binds to hILT4, wherein Ab comprises a heavy chain containing SEQ ID NO: 15 and a light chain containing SEQ ID NO: 14, and optionally the heavy chain contains C-terminal lysine.

[0222] Embodiment 21: An isolated antibody that binds to hILT4, wherein Ab comprises a heavy chain containing SEQ ID NO: 17 and a light chain containing SEQ ID NO: 16, and optionally the heavy chain contains C-terminal lysine.

[0223] Embodiment 22: An isolated antibody that binds to hILT4, wherein Ab comprises a heavy chain containing SEQ ID NO: 15 or 17 and a light chain containing SEQ ID NO: 14 or 16, and optionally the heavy chain contains C-terminal lysine.

[0224] Embodiment 23: An isolated nucleic acid or set of nucleic acids encoding the antibody described in any one of Embodiments 1 to 22.

[0225] Embodiment 24: A host cell for producing any one of the antibodies described in Embodiments 1 to 22, for example, comprising the isolated nucleic acid or set of nucleic acids described in Embodiment 23.

[0226] Embodiment 25: A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective dose of an antibody that binds to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, and (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 161, 162 and 163, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 164, 165 and 166, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 87, and the light chain comprises VL comprising SEQ ID NOs. 86, and / or (iii) the heavy chain comprises SEQ ID NOs. 17, and the light chain comprises SEQ ID NOs. 16.

[0227] Embodiment 26: A method for treating cancer in a subject, wherein the subject is given a therapeutically effective dose of (A) an antibody that binds to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, and (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 containing SEQ ID NOs. 161, 162 and 163, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 containing SEQ ID NOs. 164, 165 and 166, respectively. A method comprising administering (b) an antibody comprising CDR1, CDR2 and CDR3, and / or (ii) a heavy chain comprising VH comprising SEQ ID NO: 87, a light chain comprising VL comprising SEQ ID NO: 86, and / or (iii) a heavy chain comprising SEQ ID NO: 17, a light chain comprising SEQ ID NO: 16, and (b) an antagonist antibody that binds to human PD-1 or human PD-L1, such as nivolumab, pembrolizumab, semiprimab, tripalimab, cintilimab, atezolizumab, durvalumab or avelumab, which is an antagonist of PD-1 or PD-L1.

[0228] Embodiment 27: (A) An antibody that binds to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3, each containing SEQ ID NOs. 161, 162 and 163, and the light chain comprises VL, each containing SEQ ID NOs. 164, 165 and 166. A composition comprising (b) an antibody comprising (ii) a heavy chain comprising VH comprising SEQ ID NO: 87 and a light chain comprising VL comprising SEQ ID NO: 86 and / or (iii) a heavy chain comprising SEQ ID NO: 17 and a light chain comprising SEQ ID NO: 16, and (b) an antagonist antibody that binds to human PD-1 or human PD-L1, such as a PD-1 or PD-L1 antagonist such as nivolumab, pembrolizumab, semiprimab, tripalimab, cintilimab, atezolizumab, durvalumab or avelumab.

[0229] Embodiments relating to antibodies 10F10, 10F10.1, 10F10.3, and 10F10.4: Embodiment 1: An isolated antibody that conjugates to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3, each containing SEQ ID NOs. 167, 168 and 169, and the light chain comprises VL CDR1, CDR2 and CDR3, each containing SEQ ID NOs. 170, 171 and 172, and / or (ii) the heavy chain comprises VH, each containing SEQ ID NOs. 91, and the light chain comprises VL, each containing SEQ ID NOs. 90, 94, 96 or 114.

[0230] Embodiment 2: Ab comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 167, 168 and 169, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 170, 171 and 172, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 91, and the light chain comprises VL comprising SEQ ID NOs. 90, 94, 96 or 114, and the antibody is an isolated antibody that binds to human ILT4 (hILT4) and stimulates T cell activation (or enhances the T cell response) in a mixed lymphocyte reaction (MLR) assay, for example, as demonstrated by (i) enhancement of T cell proliferation or IFN-gamma secretion or TNF-alpha production, for example, or in a T cell:CHO-OKT3-ILT4 assay (for example, as described in Example 5).

[0231] Embodiment 3: Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 167, 168 and 169, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 170, 171 and 172, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 91, and the light chain comprises VL comprising SEQ ID NOs. 90, 94, 96 or 114, and Ab is an isolated antibody that conjugates to human ILT4 (hILT4) and enhances T cell activation in monocyte:T cell allogeneic MLR.

[0232] Embodiment 4: Ab comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 167, 168 and 169, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 170, 171 and 172, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 91, and the light chain comprises VL comprising SEQ ID NOs. 90, 94, 96 or 114, and Ab is an isolated antibody that conjugates to human ILT4 (hILT4) and enhances IFN-gamma secretion from PBMCs upon antigen stimulation in a CMV lysate assay.

[0233] Embodiment 5: Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 167, 168 and 169, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 170, 171 and 172, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 91, and the light chain comprises VL comprising SEQ ID NOs. 90, 94, 96 or 114, and Ab is an isolated antibody that binds to human ILT4 (hILT4) and inhibits the binding of hILT4 to T cells and / or binding partners, such as HLA-A and / or HLA-B.

[0234] Embodiment 6: Ab comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 167, 168 and 169, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 170, 171 and 172, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 91, and the light chain comprises VL comprising SEQ ID NOs. 90, 94, 96 or 114, and the antibody is an isolated antibody that binds to human ILT4 (hILT4) to enhance TNF-alpha secretion from macrophages, e.g., differentiated macrophages.

[0235] Embodiment 7: An isolated antibody that conjugates to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 167, 168 and 169, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 170, 171 and 172, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 91, and the light chain comprises VL comprising SEQ ID NOs. 90, 94, 96 or 114, and the antibody increases the expression of CD83 and / or CD86 on monocyte-derived dendritic cells (MoDCs).

[0236] Embodiment 8: An isolated antibody that binds to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 167, 168 and 169, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 170, 171 and 172, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 91, and the light chain comprises VL comprising SEQ ID NOs. 90, 94, 96 or 114, and Ab (i) binds to Ig-like domain 2 of hILT4 near Ig-like domain 1, and / or (ii) interacts with one or more (or all) amino acid residues Glu42, Lys43, Glu76, Cys77, Leu88, Pro91, Pro183 and Tyr184 of mature hILT4, as determined by carbene footprinting.

[0237] Embodiment 9: An isolated antibody that binds to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, and (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 167, 168 and 169, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 170, 171 and 172, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 91, and the light chain comprises VL comprising SEQ ID NOs. 90, 94, 96 or 114, and Ab comprises HC comprising SEQ ID NOs. 19 and a light chain comprising SEQ ID NOs. 18, 20, 21 or 116, respectively, inhibits (or competes with) the binding of antibodies comprising VH and VL of 10F10, 10F10.1, 10F10.3 and / or 10F10.4 to hILT4.

[0238] Embodiment 10: Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3, each containing SEQ ID NOs. 167, 168 and 169, and the light chain comprises VL CDR1, CDR2 and CDR3, each containing SEQ ID NOs. 170, 171 and 172, and / or (ii) the heavy chain comprises VH, each containing SEQ ID NOs. 91, and the light chain comprises VL, each containing SEQ ID NOs. 90, 94, 96 or 114, and Ab comprises 10 -8 M or 10 -9 Isolated antibodies that bind to human ILT4 (hILT4) with a KD of M or less.

[0239] Embodiment 11: Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3, each containing SEQ ID NOs. 167, 168 and 169, and the light chain comprises VL CDR1, CDR2 and CDR3, each containing SEQ ID NOs. 170, 171 and 172, and / or (ii) the heavy chain comprises VH, each containing SEQ ID NOs. 91, and the light chain comprises VL, each containing SEQ ID NOs. 90, 94, 96 or 114, and Ab comprises 10 -7 M or 10 -8An isolated antibody that binds to cynomolgus monkey ILT4 with a KD of M or less, and / or promotes the expression of CD80, CD83 and / or CD86 on cynomolgus monkey monocyte-derived dendritic cells, and binds to human ILT4 (hILT4).

[0240] Embodiment 12: Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 167, 168 and 169, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 170, 171 and 172, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 91, and the light chain comprises VL comprising SEQ ID NOs. 90, 94, 96 or 114, and Ab is an isolated antibody that binds to human ILT4 (hILT4) and promotes inflammatory polarization of macrophages toward M1 macrophages.

[0241] Embodiment 13: An isolated antibody that binds to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 167, 168 and 169, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 170, 171 and 172, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 91, and the light chain comprises VL comprising SEQ ID NOs. 90, 94, 96 or 114, and Ab does not induce (or trigger) basophil activation.

[0242] Embodiment 14: An isolated antibody that conjugates to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 167, 168 and 169, respectively, and the light chain comprises VL CDR1, CDR2 and CDR3 comprising SEQ ID NOs. 170, 171 and 172, respectively, and / or (ii) the heavy chain comprises VH comprising SEQ ID NOs. 91, and the light chain comprises VL comprising SEQ ID NOs. 90, 94, 96 or 114, and the composition comprising Ab comprises less than 5% high and low molecular weight species after incubation at 25°C for 3 months, and / or less than 10% high and low molecular weight species after incubation at 40°C for 3 months.

[0243] Embodiment 15: An isolated antibody that binds to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, and (i) the heavy chain comprises VH CDR1, CDR2 and CDR3, each containing SEQ ID NOs. 167, 168 and 169, and the light chain comprises VL CDR1, CDR2 and CDR3, each containing SEQ ID NOs. 170, 171 and 172, and / or (ii) the heavy chain comprises VH, each containing SEQ ID NOs. 91, and the light chain comprises VL, each containing SEQ ID NOs. 90, 94, 96 or 114, and Ab does not significantly bind to human proteins LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, ILT2, ILT3, ILT5 or LIR8.

[0244] Embodiment 16: An isolated antibody that binds to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3, each containing SEQ ID NOs. 167, 168 and 169, and the light chain comprises VL CDR1, CDR2 and CDR3, each containing SEQ ID NOs. 170, 171 and 172, and / or (ii) the heavy chain comprises VH, each containing SEQ ID NOs. 91, and the light chain comprises VL, each containing SEQ ID NOs. 90, 94, 96 or 114, and Ab comprises the constant region of the IgG heavy chain, e.g., IgG1 (e.g., SEQ ID NOs. 98 or 102), IgG1.1f (SEQ ID NOs. 103), IgG1.3 (SEQ ID NOs. 100), IgGP238K (SEQ ID NOs. 104), IgG4 or IgG4.S228P (SEQ ID NOs. 179).

[0245] Embodiment 17: An isolated antibody that binds to hILT4, wherein Ab comprises a heavy chain containing SEQ ID NO: 19 and a light chain containing SEQ ID NOs: 18, 20, 21, or 116, and optionally the heavy chain contains C-terminal lysine.

[0246] Embodiment 18: An isolated antibody that binds to hILT4, wherein Ab comprises a heavy chain containing SEQ ID NO: 19 and a light chain containing SEQ ID NOs: 18, 20, 21, or 116, and optionally one or both heavy chains containing C-terminal lysine.

[0247] Embodiment 19: An isolated antibody that binds to hILT4, wherein Ab comprises a heavy chain containing SEQ ID NO: 19 and a light chain containing SEQ ID NOs: 18, 20, 21, or 116, and optionally the heavy chain contains C-terminal lysine, and Ab contains at least one disulfide bond linking the heavy chain.

[0248] Embodiment 20: An isolated antibody that binds to hILT4, wherein Ab comprises a heavy chain containing SEQ ID NO: 19 and a light chain containing SEQ ID NO: 18, and optionally the heavy chain contains C-terminal lysine.

[0249] Embodiment 21: An isolated antibody that binds to hILT4, wherein Ab comprises a heavy chain containing SEQ ID NO: 19 and a light chain containing SEQ ID NO: 20, and optionally the heavy chain contains C-terminal lysine.

[0250] Embodiment 22: An isolated antibody that binds to hILT4, wherein Ab comprises a heavy chain containing SEQ ID NO: 19 and a light chain containing SEQ ID NO: 21, and optionally the heavy chain contains C-terminal lysine.

[0251] Embodiment 23: An isolated antibody that binds to hILT4, wherein Ab comprises a heavy chain containing SEQ ID NO: 19 and a light chain containing SEQ ID NO: 116, and optionally the heavy chain contains C-terminal lysine.

[0252] Embodiment 24: An isolated nucleic acid or set of nucleic acids encoding an antibody according to any one of Embodiments 1 to 23.

[0253] Embodiment 25: A host cell for producing any one of the antibodies described in Embodiments 1 to 23, for example, comprising the isolated nucleic acid or set of nucleic acids described in Embodiment 24.

[0254] Embodiment 26: A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective dose of an antibody that binds to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, and (i) the heavy chain comprises VH CDR1, CDR2 and CDR3, each containing SEQ ID NOs. 167, 168 and 169, and the light chain comprises VL CDR1, CDR2 and CDR3, each containing SEQ ID NOs. 170, 171 and 172, and / or (ii) the heavy chain comprises VH, each containing SEQ ID NOs. 91, and the light chain comprises VL, each containing SEQ ID NOs. 90, 94, 96 or 114, and / or (iii) the heavy chain comprises SEQ ID NOs. 19, and the light chain comprises SEQ ID NOs. 18, 20, 21 or 116.

[0255] Embodiment 27: A method for treating cancer in a subject, wherein the subject is given a therapeutically effective dose of (A) an antibody that binds to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, and (i) the heavy chain comprises VH CDR1, CDR2 and CDR3, each containing SEQ ID NOs. 167, 168 and 169, and the light chain comprises VL, each containing SEQ ID NOs. 170, 171 and 172. A method comprising administering an antibody comprising CDR1, CDR2 and CDR3, and / or (ii) a heavy chain comprising VH comprising SEQ ID NO: 91, and a light chain comprising VL comprising SEQ ID NO: 90, 94, 96 or 114, and / or (iii) a heavy chain comprising SEQ ID NO: 19, and a light chain comprising SEQ ID NO: 18, 20, 21 or 116, and (B) an antagonist antibody that binds to human PD-1 or human PD-L1, such as nivolumab, pembrolizumab, semiprimab, tripalimab, cintilimab, atezolizumab, durvalumab or avelumab, which is an antagonist of PD-1 or PD-L1.

[0256] Embodiment 28: (A) An antibody that binds to human ILT4 (hILT4), wherein Ab comprises a heavy chain and a light chain, (i) the heavy chain comprises VH CDR1, CDR2 and CDR3, each containing SEQ ID NOs. 167, 168 and 169, and the light chain comprises VL, each containing SEQ ID NOs. 170, 171 and 172. A composition comprising (b) an antibody comprising (ii) a heavy chain comprising VH comprising SEQ ID NO: 91, and a light chain comprising VL comprising SEQ ID NO: 90, 94, 96, or 114, and / or (iii) a heavy chain comprising SEQ ID NO: 19, and a light chain comprising SEQ ID NO: 18, 20, 21, or 116, and (b) an antagonist antibody that binds to human PD-1 or human PD-L1, such as a PD-1 or PD-L1 antagonist such as nivolumab, pembrolizumab, semiprimab, tripalimab, cintilimab, atezolizumab, durvalumab, or avelumab.

[0257] nucleic acids and host cells Nucleic acids encoding antibodies or their heavy chain or light chain or a portion thereof are also provided. Exemplary nucleic acids are provided in the sequence listing. Any nucleic acids comprising at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the nucleic acids in the sequence listing are included herein. Compositions comprising nucleic acids encoding antibodies provided herein are also included, as are methods for preparing antibodies comprising culturing cells comprising such nucleic acids and cells transformed with nucleic acids encoding ILT4 antibodies, and isolating antibodies from the culture medium or cells.

[0258] Treatment method using ILT4-bound Ab and related pharmaceutical compositions The antibodies described herein may be used, for example, to treat cancer. In some embodiments, a method for treating cancer is provided, comprising administering an effective amount of the antibodies described herein to a patient. In some embodiments, Ab may induce or enhance an immune response, such as an antigen-specific immune response, in a patient. In some embodiments, Ab may stimulate T cell activity. In some embodiments, Ab may inhibit the growth of at least one type of tumor in a patient.

[0259] A method for treating a subject having cancer is provided herein, comprising administering to the subject a therapeutically effective amount of the ILT4 antibody described herein, thereby treating the subject. The ILT4 antibody may be used alone, or it may be used in combination with another substance, as further described below.

[0260] Cancer may be a solid tumor or a hematological malignancy (liquid tumor).

[0261] Some examples of cancers for treatment include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, glioma, gastrointestinal cancer, kidney cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory adenocarcinoma of the prostate), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma pleomorphism), cervical cancer, stomach cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer and head and neck cancer (or carcinoma), and gastric cancer. Cancer, germ cell tumors, pediatric sarcomas, sinus natural killer tumors, melanoma (e.g., metastatic melanoma, e.g., cutaneous or intraocular melanoma), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcomas, urethral cancer, penile cancer, pediatric solid tumors, ureteral cancer, renal pelvis carcinoma, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axial tumors, brain cancer, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, and environmentally induced cancers including those induced by asbestos. Cancers induced by viruses, virus-associated cancers, or cancers of viral origin (e.g., human papillomavirus (HPV-associated or HPV-derived tumors)) and hematological malignancies originating from either of two major hematological cell lineages, namely, myeloid cell lines (producing granulocytes, erythrocytes, platelets, macrophages, and mast cells) or lymphocyte cell lines (producing B, T, NK, and plasma cells), e.g., all types of leukemia, lymphoma, and myeloma, e.g., acute, chronic, lymphocytic, and / or myeloid leukemia, e.g., acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), anaplastic AML (MO), myeloblastic leukemia (ML), myeloblastic leukemia (M2);(with cell maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma and chloroplasma; lymphoma, e.g., Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), B-cell hematological malignancies, e.g., B-cell lymphoma, T-cell lymphoma, lymphoplasmacytic lymphoma, monocytic B-cell lymphoma, mucosa-associated lymphocyte tissue (MALT) lymphoma, undifferentiated (e.g., Ki 1+) Large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, hematoimmunoblastic T-cell lymphoma, hematocentral lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic lymphoma; and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, histiocytic lymphoma, primary central nervous system Lymphomas of the lymphoid lineage, primary exudative lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), hematopoietic malignancies of the lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-cell lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also known as mycosis fungoides or Sézary syndrome), and Walden's disease. Lymphoplasmacytic lymphoma (LPL) with Ström's hypergammaglobulinemia; myeloma, e.g., IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called low-grade myeloma), solitary plasmacytoma and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; myeloid hematopoietic malignancies, mesenchymal tumors including fibrosarcoma and rhabdomyosarcoma; seminoma, teratoma, astrocytoma, sh Tumors of the central and peripheral nervous system, including Won's tumor; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma, and teratocarcinoma; hematopoietic malignancies of the lymphoid lineage, for example, but not limited to these; T-cell disorders, including T-cell and B-cell tumors, such as pre-T lymphocytic leukemia (T-PLL), including small cell and gyrus cell types;Examples of T-cell types include large granular lymphocytic leukemia (LGL); a / d T-NHL hepatosplenic lymphoma; peripheral / postthymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); hematocentral (nasal) T-cell lymphoma; cancers of the head or neck, kidney cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma; and any combination of the aforementioned cancers. The methods described herein may also be used for the treatment of metastatic, unresectable, refractory cancers (e.g., cancers refractory to conventional immunotherapy using, for example, blockade CTLA-4 or PD-1 antibodies) and / or recurrent cancers.

[0262] In certain embodiments, the antibodies described herein are administered to patients with cancer that has shown an inadequate response to, or has progressed as a result of, treatment prior to immuno-oncology or immunotherapy. In some embodiments, the cancer is either endogenously refractory or resistant (e.g., refractory to PD-1 pathway antagonists) or refractory or resistant to prior treatment, either by acquiring a resistant or refractory state. For example, the antibodies described herein may be administered to subjects or treatments that are unresponsive or insufficiently responsive to a first therapy, such as subjects with disease progression after anti-PD-1 pathway antagonist treatment alone or in combination with another therapy (e.g., using anti-PD-1 pathway antagonist therapy). In other embodiments, the antibodies described herein are administered to patients who have not previously received (i.e., have not been treated with) immuno-oncology substances, such as PD-1 pathway antagonists.

[0263] Combination with immunostimulants In some embodiments, an antibody as described herein, for example, the ILT4 antibody described herein, is administered in combination with at least one immunostimulant. For example, the therapeutic agents may be injected together or almost simultaneously. In some embodiments, the antibody and at least one immunostimulant are administered sequentially. For example, in some embodiments, the antibody is administered sequentially before or after at least one immunostimulant, resulting in the two therapeutic agents being administered 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 5 days, 7 days, or 2 weeks apart.

[0264] In some embodiments, at least one, at least two, at least three, at least five, or at least ten doses of the antibody are administered before the administration of at least one immunostimulant. In some embodiments, at least one, at least two, at least three, at least five, or at least ten doses of at least one immunostimulant are administered before the administration of the antibody. In some embodiments, the final dose of the immunostimulant is administered at least one, two, three, five, or ten days or one, two, three, five, twelve, or twenty-four weeks before the first dose of the antibody. In some embodiments, the final dose of the antibody is administered at least one, two, three, five, or ten days or one, two, three, five, twelve, or twenty-four weeks before the first dose of at least one immunostimulant. In some embodiments, the subject has received or is receiving therapy with at least one immunostimulant, and the ILT4 antibody is added to the treatment regimen.

[0265] In some embodiments, at least one immunostimulant comprises an antagonist of an inhibitor of the activation of immune cells such as T cells, while in some embodiments, at least one immunostimulant comprises an agonist of an agonist of an stimulant of the activation of immune cells such as T cells. In some embodiments, at least one immunostimulant comprises an antagonist of CTLA4, LAG-3, PD-1, PD-L1, galectin 1, galectin 9, CEACAM-1, BTLA, CD25, CD69, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM1, TIM3, TIM4, IL-6, IL-10, TGFβ, VEGF, KIR, adenosine A2A receptor, PI3K delta, or IDO. In some embodiments, at least one immunostimulant includes an agonist or Toll-like receptor agonist of B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD27, CD40, CD40L, DR3, CD28H, IL-2, IL-7, IL-12, IL-15, IL-21, IFNα, STING, or a TLR2 / 4 agonist. In some embodiments, at least one immunostimulant includes a substance that binds to another member of the B7 family of membrane-bound proteins, such as B7-1, B7-2, B7-H2 (ICOS-L), B7-H3, B7-H4, and B7-H6.In some embodiments, at least one immunostimulant is a substance that binds to a member of the TNF receptor family or a co-stimulatory or co-inhibitory molecule that binds to a member of the TNF receptor family, such as CD40, CD40L, OX40, OX40L, GITR, GITRL, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137(4-1BB), TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR / Fn14, TWEAK, BAFFR, EDAR, XEDAR, EDA1, EDA2, TACI, APRIL, BCMA, LTβR, LIGHT, DeR3, HVEM, VEGL / TL1A, TRAMP / DR3, TNFR1, TNFβ, TNFR2, TNFα, 1β2, FAS, FASL, RELT, DR6, TROY, or NGFβ. In some embodiments, at least one immunostimulant includes a substance that antagonists or inhibits cytokines that inhibit T cell activation, e.g., IL-6, IL-10, TGFβ, VEGF. In some embodiments, at least one immunostimulant includes an agonist of cytokines that stimulate T cell activation, e.g., IL-2, IL-7, IL-12, IL-15, IL-21, and IFNα. In some embodiments, at least one immunostimulant comprises a chemokine antagonist, such as CXCR2, CXCR4, CCR2, or CCR4. In some embodiments, at least one immunostimulant comprises an antibody. In some embodiments, at least one immunostimulant may comprise a vaccine, such as a mesothelin-targeted vaccine or an attenuated listeria cancer vaccine, such as CRS-207.

[0266] For example, the ILT4 antibody described herein may be administered together with one or more of the following substances:

[0267] (1) Antagonists (inhibitors or blockers) of proteins that inhibit T cell activation (e.g., immune checkpoint inhibitors), e.g., CTLA-4, PD-1, PD-L1, PD-L2 and LAG-3, galectin-9, CEACAM-1, BTLA, CD69, galectin-1, TIGIT, CD113, GPR56, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, TIM-3 and TIM-4; and / or (2) Agonists of proteins that stimulate T cell activation, e.g., B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, GITR, ICOS, ICOS-L, OX40, OX40L, CD70, CD27, CD40, DR3 and CD28H.

[0268] Exemplary substances that can be combined with the ILT4 antibodies described herein to treat cancer include YERVOY® (ipilimumab) or tremelimumab (for CTLA-4), galiximab (for B7.1), BMS-936558 (for PD-1), MK-3475 (for PD-1), atezolizumab (Tecentriq®), avelumab, durvalumab, and AMP224 (for B7DC). (against), BMS-936559 (against B7-H1), MPDL3280A (against B7-H1), MEDI-570 (against ICOS), AMG557 (against B7H2), MGA271 (against B7H3), IMP321 (against LAG-3), BMS-663513 (against CD137), PF-05082566 (against CD137), CDX-1127 (against CD27), Anti-OX40 (Providence Examples include: Health Services), huMAbOX40L (against OX40L), atacicept (against TACI), CP-870893 (against CD40), lucatumumab (against CD40), dasetuzumab (against CD40), muromonab-CD3 (against CD3); anti-GITR antibodies MK4166, TRX518, Medi1873, INBRX-110, LK2-145, GWN-323, GITRL-Fc, or any combination thereof.

[0269] Other molecules that can be combined with ILT4 antibodies for cancer treatment include inhibitory receptor antagonists or activating receptor agonists on NK cells, such as KIR antagonists (e.g., lirilumab).

[0270] T cell activation can also be regulated by soluble cytokines. In some embodiments, ILT4 antibodies may be administered in combination with cytokine antagonists intended to inhibit T cell activation or cytokine agonists that stimulate T cell activation. For example, ILT4 antibodies may be used in combination with (i) antagonists (or inhibitors or blockers) of proteins of the IgSF family, B7 family, or TNF family that inhibit T cell activation or with cytokine antagonists (e.g., IL-6, IL-10, TGF-β, VEGF; “immunosuppressive cytokines”) and / or (ii) agonists of stimulating receptors of cytokines of the IgSF family, B7 family, or TNF family that stimulate T cell activation.

[0271] Other agents for combination therapy include, but are not limited to, agents that inhibit or deplete macrophages or monocytes, as well as CSF-1R antagonist antibodies, such as RG7155 (WO11 / 70024, WO11 / 107553, WO11 / 131407, W013 / 87699, W013 / 119716, WO13 / 132044) or FPA-008 (WO11 / 140249, W013169264, WO14 / 036357).

[0272] ILT4 antibodies can also be administered together with substances that inhibit TGF-β signaling.

[0273] Further substances that can be combined with ILT4 antibodies include substances that enhance tumor antigen presentation, such as dendritic cell vaccines, GM-CSF secretory cell vaccines, CpG oligonucleotides and imiquimod, or therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines).

[0274] Other therapies that can be combined with ILT4 antibodies include therapies that deplete or block Treg cells, such as substances that specifically bind to CD25.

[0275] Another therapy that can be combined with ILT4 antibodies is therapies that inhibit metabolic enzymes such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthetase.

[0276] Another class of substances that can be used with ILT4 antibodies include substances that inhibit adenosine formation, such as CD73 inhibitors or adenosine A2A receptor inhibitors.

[0277] Other therapies that can be combined with ILT4 antibodies to treat cancer include therapies that reverse / prevent T-cell anergy or depletion, as well as therapies that induce innate immune activation and / or inflammation at the tumor site.

[0278] Other therapies that can be combined with ILT4 antibodies to treat cancer include IL-8 blocking therapies, such as those using HuMax®-IL8.

[0279] ILT4 antibodies can be combined with two or more immuno-oncological substances, for example, in combinatorial approaches intended to target multiple elements of the immune pathway, such as one or more of the following: therapies that enhance tumor antigen presentation (e.g., dendritic cell vaccines, GM-CSF secretory cell vaccines, CpG oligonucleotides, imiquimod); therapies that inhibit negative immunomodulation by inhibiting the CTLA-4 and / or PD1 / PD-L1 / PD-L2 pathways and / or depleting or blocking Treg or other immunosuppressive cells; therapies that stimulate positive immunomodulation using agonists that stimulate the CD-137, OX-40 and / or CD40 or GITR pathways, and / or T cell effector function; therapies that systemically increase the frequency of anti-tumor T cells; for example, using a CD25 antagonist (e.g., daclizumab), or ex Therapies that deplete or inhibit Tregs, e.g., Tregs in tumors, by vivo anti-CD25 bead depletion; therapies that affect the function of suppressor myeloid cells in tumors; therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines); adoptive T cell or NK cell transplantation, including genetically modified cells, e.g., cells modified by chimeric antigen receptors (CAR-T therapy); therapies that inhibit metabolic enzymes, e.g., indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthetase; therapies that reverse / prevent T cell anergy or depletion; therapies that induce innate immune activation and / or inflammation at the tumor site; administration of immunostimulant cytokines; or blockade of immunosuppressive cytokines.

[0280] The ILT4 antibodies described herein may be used in conjunction with one or more of the following: agonists that link positively co-stimulated receptors, blockers that attenuate signaling via inhibitory receptors, antagonists and one or more substances that systemically increase the frequency of antitumor T cells, substances that overcome their respective immunosuppressive pathways within the tumor microenvironment (e.g., blocking inhibitory receptor association (e.g., PD-L1 / PD-1 interaction), depleting or inhibiting Tregs (e.g., using anti-CD25 monoclonal antibodies (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion), inhibiting metabolic enzymes, e.g., IDO, or reversing / preventing T cell anergy or depletion), and substances that induce innate immune activation and / or inflammation at the tumor site.

[0281] In a particular embodiment, the ILT4 antibody is administered to the subject together with a BRAF inhibitor if the subject is positive for the BRAF V600 mutation.

[0282] Suitable PD-1 antagonists for use in the combination therapies described herein include, but are not limited to, ligands, antibodies (e.g., monoclonal antibodies and bispecific antibodies), and polyvalent substances. In one embodiment, the PD-1 antagonist is a fusion protein, e.g., an Fc fusion protein, e.g., AMP-244. In one embodiment, the PD-1 antagonist is an anti-PD-1 or anti-PD-L1 antibody.

[0283] Exemplary anti-PD-1 antibodies are nivolumab (BMS-936558) or antibodies containing one of the CDRs or variable regions of antibodies 17D8, 2D3, 4H1, 5C4, 7D3, 5F4, and 4A11 as described in WO2006 / 121168. In certain embodiments, the anti-PD-1 antibody is MK-3475 (lambrolizumab) as described in WO2012 / 145493, AMP-514 as described in WO2012 / 145493, or PDR001 as described in WO2012 / 145493. Furthermore, known PD-1 antibodies and other PD-1 inhibitors include those described in WO2009 / 014708, WO03 / 099196, WO2009 / 114335, WO2011 / 066389, WO2011 / 161699, WO2012 / 145493, U.S. Patents 7,635,757 and 8,217,149, and U.S. Patent Publication 2009 / 0317368. Any of the anti-PD-1 antibodies disclosed in WO2013 / 173223 may also be used. Among these antibodies, anti-PD-1 antibodies that compete for and / or bind to the same epitope on PD-1 may also be used in combination treatment.

[0284] In some embodiments, the anti-PD-L1 antibody useful for combination therapy is BMS-936559 (referred to as 12A4 in WO2007 / 005874 and U.S. Patent No. 7,943,743) or an antibody containing the CDR or variable region of 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4 as described in PCT Publication WO07 / 005874 and U.S. Patent No. 7,943,743. In certain embodiments, the anti-PD-L1 antibody is MEDI4736 (also known as durvalumab and anti-B7-H1), MPDL3280A (also known as atezolizumab and RG7446), MSB0010718C (also known as avelumab; WO2013 / 79174), or rHigM12B7. Any of the anti-PD-L1 antibodies disclosed in WO2013 / 173223, WO2011 / 066389, WO2012 / 145493, U.S. Patent Nos. 7,635,757 and 8,217,149, and U.S. Publication No. 2009 / 145493 may also be used. Anti-PD-L1 antibodies that compete with and / or bind to the same epitopes as any of these antibodies may also be used in combination treatment.

[0285] In certain embodiments, the ILT4 antibody of this disclosure may be used in conjunction with a CTLA-4 antagonist, such as an anti-CTLA-4 antibody. In one embodiment, the anti-CTLA-4 antibody is Yervoy® (ipilimumab or antibody 10D1, as described in PCT Publication WO01 / 14424), tremelimumab (formerly tisilimumab, CP-675,206), a monoclonal or anti-CTLA-4 antibody as described in any of the following publications: WO98 / 42752; WO00 / 37504; U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Pro. Natl. Acad. Sci. USA 95(17): 10067-10071; Camacho et al. (2004) J. Clin. Oncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res. The antibody is selected from the group 58:5301-5304. Any of the anti-CTLA-4 antibodies disclosed in WO2013 / 173223 may also be used.

[0286] In some embodiments, the ILT4 antibody of this disclosure is used in combination with a LAG3 antagonist. Examples of anti-LAG3 antibodies include antibodies containing the CDR or variable region of antibodies 25F7, 26H10, 25E3, 8B7, 11F2, or 17E5, as described in U.S. Patent Publication Nos. US2011 / 0150892, WO10 / 19570, and WO2014 / 008218. In one embodiment, the anti-LAG-3 antibody is BMS-986016. Other art-recognized anti-LAG-3 antibodies that can be used include IMP731 and IMP-321, as described in U.S. Patent Publication Nos. US2011 / 007023, WO08 / 132601, and WO09 / 44273. Anti-LAG-3 antibodies that compete with and / or bind to the same epitope as any of these antibodies may also be used in combination treatment.

[0287] In some embodiments, the ILT4 antibody of this disclosure may be administered in combination with a CD137(4-1BB) agonist, such as an agonist CD137 antibody. Suitable CD137 antibodies include, for example, urelumab or PF-05082566(W012 / 32433).

[0288] In some embodiments, the ILT4 antibody may be administered in combination with an OX40 agonist, such as an agonist OX40 antibody. Suitable OX40 antibodies include, for example, MEDI-6383, MEDI-6469, or MOXR0916 (RG7888;WO06 / 029879).

[0289] In one embodiment, the ILT4 antibody is administered in combination with a CD40 agonist, such as an agonist CD40 antibody. In a particular embodiment, the immuno-oncological substance is a CD40 antagonist, such as an antagonist CD40 antibody. Suitable CD40 antibodies include, for example, lucatumumab (HCD122), dacetuzumab (SGN-40), CP-870, 893, or Chi Lob 7 / 4.

[0290] In one embodiment, the ILT4 antibody is administered in combination with a CD27 agonist, such as an agonist CD27 antibody. A suitable CD27 antibody is, for example, varlilumab (CDX-1127).

[0291] In certain embodiments, the ILT4 antibody is administered together with an anti-GITR antibody, for example, an antibody having a 6C8 CDR sequence, such as the one described in WO2006 / 105021, for example, a humanized antibody having a 6C8 CDR; an antibody containing a CDR of an anti-GITR antibody as described in WO2011 / 028683; an antibody containing a CDR of an anti-GITR antibody as described in JP2008278814, an antibody containing a CDR of an anti-GITR antibody as described in WO2015 / 031667, WO2015 / 187835, WO2015 / 184099, WO2016 / 054638, WO2016 / 057841 or WO2016 / 057846, or other anti-GITR antibodies described or referenced herein.

[0292] In some embodiments, the ILT4 antibody is administered in combination with MGA271 (against B7H3) (WO11 / 109400).

[0293] In some embodiments, the ILT4 antibody is administered in combination with a KIR antagonist, such as lirilumab.

[0294] In some embodiments, the ILT4 antibody is administered in combination with an IDO antagonist. Suitable IDO antagonists include, for example, INCB-024360 (WO2006 / 122150, WO07 / 75598, WO08 / 36653, WO08 / 36642), indoximod, NLG-919 (WO09 / 73620, WO09 / 1156652, WO11 / 56652, WO12 / 142237), or F001287.

[0295] In some embodiments, the ILT4 antibody is administered in combination with a Toll-like receptor agonist, such as a TLR2 / 4 agonist (e.g., Bacillus calmette-Guérin), a TLR7 agonist (e.g., Hiltonol or imiquimod), a TLR7 / 8 agonist (e.g., reciquimod), or a TLR9 agonist (e.g., CpG7909).

[0296] In one embodiment, ILT4 is administered in combination with a TGF-β inhibitor, such as GC1008, LY2157299, TEW7197, or IMC-TR1.

[0297] Further combination therapy Ab in this specification may also be provided before, substantially simultaneously with, or after other forms of treatment, such as surgery, chemotherapy, radiotherapy, or the administration of a biologic, such as another therapeutic antibody. In some embodiments, the cancer had recurred or progressed after a therapy selected from surgery, chemotherapy, and radiotherapy or a combination thereof. For example, an ILT4 antibody, such as those described herein, may be administered as adjuvant therapy when there is a risk of micrometastasis and / or to reduce the risk of recurrence.

[0298] For the treatment of cancer, the combination may be administered with one or more further anticancer agents, such as chemotherapeutic agents, growth inhibitors, anticancer vaccines, such as gene therapy vaccines, anti-angiogenic agents, and / or antitumor, antineoplastic compositions. Examples of chemotherapeutic agents, growth inhibitors, anticancer vaccines, anti-angiogenic agents, and antitumor, antineoplastic compositions that can be used in combination with the antibodies of the present invention are provided herein under "Definitions".

[0299] In some embodiments, anti-inflammatory agents such as steroids or non-steroidal anti-inflammatory drugs (NSAIDs) may be administered in combination. If it is desirable to quiescently tranquilize abnormally proliferating cells along with, or prior to, the treatment using the ILT4 antibodies described herein, hormones and steroids (including synthetic analogs), such as 17α-ethinylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testactone, megestrolacetate, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianicene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide, toremifene, and Zoladex® may also be administered to the patient. When using the methods or compositions described herein, other substances used in clinical settings for tumor growth or metastasis, such as antimimetics, may also be administered as desired.

[0300] The antibodies described herein may also be combined with immunogens, e.g., cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immunostimulatory cytokines (He et al., (2004) J. Immunol. 173:4919-28). Not limited examples of tumor vaccines that can be used include melanoma antigen peptides, e.g., gp100 peptide, MAGE antigen, Trp-2, MART1, and / or tumor cells transfected to express tyrosinase or cytokine GM-CSF (discussed further below).

[0301] The ILT4 antibodies described herein can also be combined with vaccination protocols. Numerous experimental strategies for tumor vaccination have been devised (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C, 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; also see Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition). In one of these strategies, the vaccine is prepared using autologous or allogeneic tumor cells. These cellular vaccines have been shown to be most effective when tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci USA 90: 3539-43).

[0302] Studies of gene expression and large-scale gene expression patterns in various tumors have led to the definition of so-called tumor-specific antigens (Rosenberg, SA (1999) Immunity 10: 281-7). In many cases, these tumor-specific antigens are differentiation antigens expressed in tumors and in tumor-derived cells, e.g., melanocyte antigen gp100, MAGE antigen, and Trp-2. More importantly, many of these antigens may be shown to be targets of tumor-specific T cells found in the host. ILT4 inhibition can be used with recombinant protein and / or peptide assortments expressed in tumors to induce an immune response against these proteins. These proteins are usually considered autoantigens by the immune system and are therefore tolerant to them. Tumor antigens may include telomerase, a protein required for chromosome telomere synthesis and expressed in over 85% of human cancers and only in a limited number of somatic tissues (Kim et al. (1994) Science 266: 2011-2013). Tumor antigens can also be “neoantigens” expressed in cancer cells due to somatic mutations or idiotypes derived from B-cell tumors that alter protein sequences or create fusion proteins between two unrelated sequences (i.e., bcr-abl in the Philadelphia chromosome).

[0303] Other tumor vaccines may contain proteins derived from viruses involved in human cancer, such as human papillomavirus (HPV), hepatitis viruses (HBV and HCV), and Kaposi's herpes sarcoma virus (KHSV). Another form of tumor-specific antigen that can be used with ILT4 Ab is purified heat shock proteins (HSPs) isolated from tumor tissue itself. These heat shock proteins contain fragments of proteins derived from tumor cells, and these HSPs are highly effective in delivery to antigen-presenting cells to induce tumor immunity (Suot & Srivastava (1995) Science 269: 1585-1588; Tamura et al. (1997) Science 278: 117-120).

[0304] Dendritic cells (DCs) are potent antigen-presenting cells that can be used to stimulate antigen-specific responses. DCs can be produced ex vivo and loaded with various protein and peptide antigens as well as tumor cell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs can also be transduced by genetic means to express these tumor antigens. DCs have also been directly fused to tumor cells for immunotherapy (Kugler et al. (2000) Nature Medicine 6: 332-336). As a method of vaccination, DC immunotherapy can be combined with ILT4 inhibition to activate a more potent antitumor response.

[0305] Treatment for infectious diseases The methods described herein may also be used to treat patients exposed to certain toxins or pathogens. Accordingly, this disclosure envisions a method for treating an infectious disease in a subject, comprising administering to the subject an antibody, such as an ILT4 antibody, as described herein, resulting in the subject being treated for the infectious disease. Similar to its application to tumors as discussed above, antibody-mediated ILT4 inhibition may be used alone, as an adjuvant, or in combination with a vaccine to stimulate an immune response to pathogens, toxins, and autoantigens. Examples of pathogens for which this therapeutic approach would be particularly useful include pathogens for which there are currently no effective vaccines or for which conventional vaccines are never completely effective. These include, but are not limited to, HIV, hepatitis (A, B & C), influenza, herpes, giardia, malaria, leishmania, Staphylococcus aureus, and Pseudomonas aeruginosa. ILT4 inhibition may be useful against established infections caused by pathogens such as HIV that exhibit modified antigens throughout the course of infection.

[0306] Some examples of pathogenic viruses that cause infections that may be treatable by the methods described herein include HIV, hepatitis (A, B, or C), herpesviruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenoviruses, influenza viruses, flaviviruses, echoviruses, rhinoviruses, coxsackieviruses, coronaviruses, respiratory syncytial viruses, mumps viruses, rotaviruses, measles viruses, rubella viruses, parvoviruses, vaccinia viruses, HTLV viruses, dengue viruses, papillomaviruses, molluscum contagiosum viruses, polioviruses, rabies viruses, JC viruses, and arbovirus encephalitis viruses.

[0307] Some examples of pathogenic bacteria that cause infections that may be treatable by the methods described herein include Chlamydia, Rickettsia, Mycobacteria, Staphylococcus, Streptococcus, Pneumococcus pneumoniae, Neisseria meningitidis and Neisseria gonorrhoeae, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, botulism, Anthrax, Plague, Leptospirosis and Lyme disease.

[0308] Some examples of pathogenic fungi that cause infections that may be treatable by the methods described herein include Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, and Coccidioides imitis. Examples include *Immitis* and *Histoplasma capsulatum*.

[0309] Some examples of pathogenic parasites that cause infections that may be treatable by the methods described herein include Entamoeba histolytica, Balantidium colonis, Naegleria fowleri, species of the genus Acanthamoeba, Giardia lambia, species of the genus Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, and Toxoplasma gondii. Examples include *Nippostrongylus gondii* and the Brazilian hookworm (*Nippostrongylus brasiliensis*).

[0310] In all of the above methods, ILT4 inhibition may be combined with other forms of immunotherapy, such as those described herein, including cytokine treatment (e.g., interferon, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy that can provide enhanced presentation of tumor antigens (see, for example, Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2: 1121-1123).

[0311] Route of administration and carrier In various embodiments, antibodies may be administered in vivo by various routes, including, but not limited to, intravenous (iv), subcutaneous, oral, intra-arterial, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, percutaneous, and subarachnoid, or otherwise by implantation or inhalation. The composition may be formulated in solid, semi-solid, liquid, or gaseous forms, including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols. Nucleic acid molecules encoding antibodies may be coated on gold microparticles and delivered intradermally by microparticle gun devices or "gene guns," as described in the literature (see, e.g., Tang et al., Nature 356:152-154 (1992)). Appropriate formulations and routes of administration may be selected according to the intended application.

[0312] In various embodiments, the antibody-containing composition is provided in formulation with various pharmaceutically acceptable carriers (e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 2020). th ed. (2003), Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7 th ed., Lippencott Williams and Wilkins (2004), Kibbe et al., Handbook of Pharmaceutical Excipients, 3 rdSee ed., Pharmaceutical Press (2000). A variety of pharmaceutically acceptable carriers, including media, adjuvants, and diluents, are available. In addition, a variety of pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, tension adjusters, stabilizers, and wetting agents, are also available. Exemplary carriers, not limited to these, include physiological saline, buffered physiological saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0313] In various embodiments, compositions containing antibodies may be formulated for injection, including subcutaneous administration, by dissolving, suspending, or emulsifying them in aqueous or non-aqueous solvents, such as vegetable oils or other oils, synthetic fatty acid glycerides, esters of high fatty acids, or propylene glycol, and optionally with conventional additives, such as solvents, isotonic substances, suspending agents, emulsifiers, stabilizers, and preservatives. In various embodiments, compositions may be formulated for inhalation, for example, using pressurized, acceptable sprays, such as dichlorodifluoromethane, propane, or nitrogen. In various embodiments, compositions may also be formulated into sustained-release microencapsulated formulations, for example, using biodegradable or non-biodegradable polymers. Exemplary biodegradable formulations, not limited to these, include polylactic acid-glycolic acid polymers. Exemplary non-biodegradable formulations, not limited to these, include polyglycerol fatty acid esters. Certain methods for producing such formulations are described, for example, in EP1125584A1.

[0314] Pharmaceutical packs and kits are also provided, each comprising one or more containers containing doses of one or more antibodies or combinations of antibodies. In some embodiments, unit doses are provided, each containing a predetermined amount of a composition comprising an antibody or combination of antibodies with or without one or more further substances. In some embodiments, such unit doses are supplied in single-use, pre-filled syringes for injection. In various embodiments, the composition contained in the unit dose may include saline, sucrose, etc.; buffers, e.g., phosphoric acid, etc.; and / or be formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder that can be reconstituted upon addition of a suitable liquid, e.g., sterile water. In some embodiments, the composition includes, but is not limited to, one or more substances that inhibit protein aggregation, including sucrose and arginine. In some embodiments, the composition of the present invention includes heparin and / or proteoglycans.

[0315] Pharmaceutical compositions may be administered in amounts effective for the treatment or prevention of specific indications. The therapeutically effective dose usually varies depending on the weight of the subject being treated, their physical condition or health status, the extent of the condition to be treated, or the age of the subject being treated. Generally, antibodies may be administered in amounts ranging from about 10 μg / kg of body weight to about 100 mg / kg of body weight per dose. In some embodiments, antibodies may be administered in amounts ranging from about 50 μg / kg of body weight to about 5 mg / kg of body weight per dose. In some embodiments, antibodies may be administered in amounts ranging from about 109 μg / kg of body weight to about 10 mg / kg of body weight per dose. In some embodiments, antibodies may be administered in amounts ranging from about 100 μg / kg of body weight to about 20 mg / kg of body weight per dose. In some embodiments, antibodies may be administered in amounts ranging from about 0.5 mg / kg of body weight to about 20 mg / kg of body weight per dose.

[0316] The antibody composition may be administered to the subject as needed. The frequency of administration may be determined by a person skilled in the art, such as the attending physician, based on considerations such as the condition being treated, the age of the subject being treated, the severity of the condition being treated, and the overall health status of the subject being treated. In some embodiments, the effective dose of the antibody is administered to the subject once or multiple times. In various embodiments, the effective dose of the antibody is administered to the subject once a month, less than once a month, for example, every two months or every three months. In other embodiments, the effective dose of the antibody is administered more than once a month, for example, every three weeks, every two weeks or weekly. In some embodiments, the effective dose of the antibody is administered once every 1, 2, 3, 4 or 5 weeks. In some embodiments, the effective dose of the antibody is administered two or three times a week. The effective dose of the antibody is administered to the subject at least once. In some embodiments, the effective dose of the antibody may be administered multiple times, including a period of at least one month, at least six months or at least one year.

[0317] In certain embodiments, the combination of the ILT4 antibody and the second substance discussed herein may be administered in combination as a single composition in a pharmaceutically acceptable carrier, or as separate compositions having the ILT4 antibody and the second substance in a pharmaceutically acceptable carrier. In one embodiment, the combination of the ILT4 antibody and the second substance may be administered sequentially. The administration of the two substances may begin, for example, at intervals of 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 5 days, 7 days, or more than a week, or the administration of the second substance may begin, for example, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 5 days, 7 days, or more than a week after the administration of the first substance. [Examples]

[0318] The examples discussed below are intended to be purely illustrative of the invention and should not be considered in any way as limiting the invention. The examples are not intended to represent that the following experiments are all or only experiments performed. While efforts have been made to ensure accuracy with respect to the figures used (e.g., quantities, temperatures, etc.), some experimental errors and deviations should be taken into consideration. Unless otherwise indicated, parts are by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure. [Examples]

[0319] Production of antibodies that bind to human ILT4 Anti-human ILT4 monoclonal antibodies (ILT4 antibodies or hILT4 antibodies) were generated using transgenic mice expressing human antibody genes. Fully human monoclonal antibodies against human ILT4 were generated by immunizing two types of human immunoglobulin transgenic animals. KM[M / K] mice were immunized with hILT4-mFc recombinant protein, which consists of the extracellular portion of hILT4 with a C-terminal mouse Fc tag. The antigen was mixed with Ribi adjuvant in a 1:1 ratio, and mice were immunized intraperitoneally and subcutaneously at weekly intervals. The HCo42:01[J / K] strain was immunized with hILT4-his tagged recombinant protein, which consists of the extracellular portion of hILT4 with a C-terminal his tag. The antigen was mixed with Ribi adjuvant in a 1:1 ratio, and mice were immunized on the soles of their feet at weekly intervals. Serum titers in both types of mice were monitored after 4 and 6 injections. The mice received a final booster immunization before the final harvest. Depending on the immunization pathway, the aspiration area lymph nodes and spleen were harvested for subsequent fusion.

[0320] Mouse lymphocytes were isolated from immunized mice. Hybridomas were generated by fusion with mouse myeloma fusion partners via electric field-based electrofusion using a Cyto Pulse Hybrimmune large chamber cell fusion electroporator (BTX / Harvard Apparatus). Single-cell suspensions of lymphocytes derived from immunized mice were fused to an equal number of P3X63 Ag8.6.53 (ATCC) non-secreting mouse myeloma cells (fusion numbers 4865 and 6951). The resulting cells were seeded in E medium (StemCell Technologies) supplemented with aminopterin (Sigma) for hybridoma selection in flat-bottom microtiter plates.

[0321] Individual wells were screened for the presence of human IgG / human kappa light chain (hIgG / hK) antibodies using a homogeneous HTRF assay 10–12 days after culture. Hybridoma supernatants from wells positive for hIgG / hK were tested by FACS or FMAT for binding to hILT4-transfected cells or control CHO cells. The following antibodies, which specifically interact with hILT4, were further characterized: 9C8.A6, 24E5.A7, 2H2.H3, 21D9.H11, 2E5.A11, and 9G4. Antibodies 21A5 and 10F10 were isolated from later immunization.

[0322] Antibodies derived from hybridomas were sequenced using NGS high-efficiency sequencing. Briefly, each well was identified by performing PCR amplification of the VH and VL regions for each hybridoma clone in a 96-well plate using a unique DNA barcode. 5' PCR primers were hybridized to the leader region to obtain the entire variable region sequence. Specific hybridoma antibody sequences were identified by matching the barcode with the position of the clone on the plate.

[0323] The anti-ILT4 antibodies 9G4, 9C8, 2H2, 2E5, 24E5, 21D9, 21A5, and 10F10 are recombinantly expressed in the context of the IgG1, IgG1.1, or IgG1.3 heavy chain constant regions and are also named as provided in Table 1. The amino acid sequences of HC, LC, VH, and VL, and the nucleotide sequences encoding them, are provided in Figures 1–12 and the following sequence listings. The location of each CDR is provided in the figures and is indicated by underlining in the variable region sequences of the sequence listings, and the CDRs are also included as separate sequence numbers towards the end of the sequence listings.

[0324] Analysis of the amino acid sequences of the variable domains of antibodies 21D9, 21A5, 2H2, and 10F10 revealed that certain framework amino acid residues are absent in the human germline. To mitigate potential immunogenicity issues when these antibodies are administered to humans, germline revert mutants of these ILT4 antibodies were generated.

[0325] For antibody 21D9, four framework mutations in the heavy chain variable region relative to the germline sequence were identified (V2G, G10D, A24T, and V48I), shown in Figure 13. From various combinations of these four revertant mutations, four revertant mutants (21D9.b, 21D9.c, 21D9.d, and 21D9.e) were generated (see Figure 13) and tested for binding to hILT4. All four revertant mutants contain an N-terminal EGQ to EVQ (or G2V) mutation, which is unlikely to affect binding to ILT4. In addition to this substitution, mutants 21D9.b, 21D9.c, and 21D9.d each contain one of the further substitutions shown in Figure 13 (D10G; I24A; and / or I48V), and 21D9.e contains all three substitutions (see Figure 13). To reduce effector function, four mutants were generated in the context of the IgG1.3 heavy chain constant region, which is the IgG1 constant region containing amino acid substitutions L234A, L235E, and G237A. These mutants are called 21D9.b.hIgG1.3 ("21D9.VH-G2V / D10G8V.IgG1.3"), 21D9.c.hIgG1.3 ("21D9.VH-G2V / T24A.IgG1.3"), 21D9.d.hIgG1.3 ("21D9.VH-G2V / I48V"), and 21D9.e.hIgG1.3 ("21D9.VH-G2V / D10G / T24A / I48V.IgG1.3"), and contain the 21D9 light chain. No germline reversion was performed on the light chain.

[0326] For antibody 21A5, one framework mutation was identified in the variable regions of both the heavy chain (T70I) and light chain (V3A), as shown in Figure 14. Since the N-terminal return in the kappa light chain, as shown in A3V, is unlikely to affect binding, a single mutant (21A5.a with the 21A5.1 light chain) containing return mutations in both the heavy chain (I70T) and light chain (A3V) was generated and tested in the context of IgG1.3 (21A5.a.IgG1.3, also referred to as "21A5.VH-I70T.VK-A3V.IgG1.3").

[0327] For antibody 10F10, two framework mutations were identified in the light chain variable region (Y36F and S63T), shown in Figure 15. No framework mutations were found in the heavy chain variable region. Since the reversion from F to Y at position 36 is a small difference, a double mutant (10F10.3) containing both light chain reversion mutations F36Y and T63S was generated and tested in the context of IgG1.3 (also referred to as "10F10.VK-F36Y / T63S.IgG1.3", 10F10.3.IgG1.3). A mutant containing only the reversion mutation F36Y was also generated and tested in the context of IgG1.3 (also referred to as "10F10.VK-F36Y.IgG1.3", 10F10.1.IgG1.3). Both reversion mutant Abs contain the heavy chain of 10F10. Table 1 lists alternative names for the recombinant antibodies described herein.

[0328] [Table 1] [Examples]

[0329] Binding of anti-hILT4 antibody to soluble hILT4 The binding kinetics of parental and germline revertant anti-hILT4 antibodies (in the context of IgG1.3) were measured by surface plasmon resonance (SPR) using a Biacore® T200 instrument. The assay temperature was 37°C, and the electrophoresis buffer was PBS pH 7.4 with 0.05% Tween-20 added. All anti-hILT4 antibodies were captured on a CM4 chip containing pre-immobilized protein A / G (Pierce Thermo Scientific catalog no. 21186). Three different lots of 21D9 were used as controls. Human ILT4 monomers were injected as analytes. Appropriate concentrations were used for analysis: for 10F10.1 and 10F10.3a, a 3-fold dilution series using the maximum hILT4 concentration of 1.4 μM was analyzed. For 10F10, two independent hILT4 dilution series were analyzed: the maximum concentration of 1.4 μM, a 5-fold dilution, and 470 nM, a 3-fold dilution. For all other antibodies, the maximum hILT4 concentration of 94 nM and a 5-fold dilution series were used.The hILT4 used in this experiment, including the extracellular domain of hILT4 linked to the His-Avi tag, has the following amino acid sequence: QTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEEEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPGVSKKPSLSVQPGPVVVAPGESLTLQCVSD This consists of VGYDRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSESSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAGAADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPYLLSHPSEPLELVVSGPSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGSPGGGSGGGSEQKLISEEDLGHHHHHHGLNDIFEAQKIEWHE (hILT4-His-Avi tag; sequence number 119).

[0330] The results are shown in Table 2 and Figure 16.

[0331] [Table 2]

[0332] The results show that the four revertant mutants of 21D9 have similar dynamics and affinity. 21A5 and its revertant mutant 21A5.a also have similar dynamics and affinity. However, two 10F10 revertant mutants, 10F10.1 and 10F10.3, have slightly faster dissociation rates than their parent 10F10, resulting in a loss of approximately three times the overall affinity. The dissociation rates of 10F10.1 and 10F10.3 are too fast to determine with high reliability. [Examples]

[0333] The binding of anti-hILT4 antibodies to hILT4-expressing cells. This example describes the binding properties of an anti-hILT4 antibody to hILT4-transfected CHO cells and human monocytes expressing hILT4.

[0334] CHO cells were transfected with hILT4 and stained with individual anti-hILT4 antibodies 2H2, 10F10 ("10F10 WT"), 21A5 ("21A5 WT"), 21A5.a, 21D9 ("21D9 WT"), and 21D9e (all in the context of IgG1.3). The cells were titrated by 3-fold serial dilutions starting from 20 μg / mL and then subjected to PE-conjugated secondary anti-human IgG. FACS analysis for geometric mean fluorescence intensity (GMFI) was used to determine the EC (European Chromium) levels. 50 We made that decision.

[0335] The bond curves and EC values ​​are shown in Figures 17 and 18A.

[0336] In Figure 18B, monocytes selected from the peripheral blood of healthy donors were stained with individual anti-ILT4 antibodies conjugated with Alexa67. The antibodies were titrated from 20 μg / mL by 3-fold serial dilutions, and GMFI was determined by FACS analysis to calculate EC50 (shown in the table below the graph). [Examples]

[0337] Selectivity of anti-ILT4 antibodies against members of the LILRA / B family ILT4 is a member of a related receptor family, also known as the LILRA and LILRB families. In this example, the binding of hILT4 antibodies to various members of the LILRA / LILRB family was measured.

[0338] Individual ILT family members were overexpressed in 293T cells. Each transfectant was stained with 20 μg / mL anti-ILT4 antibody, followed by 1 μg / mL anti-human IgG secondary mAb. The binding of the antibodies to each transfectant was measured by flow cytometry. In a second experiment, LILRA1, LILRA3, and ILT4 transfectants were incubated with various concentrations of 21A5 and 21A5.a.

[0339] The results shown in Figures 19A and B and Table 3 demonstrate that the anti-hILT4 antibody binds most selectively to hILT4 compared to other LILRA and LILRB family members. LILRA3 is the only secreted protein in the LILRA / B family. More specifically, 21D9 and 21D9.e do not bind significantly to LILRA1, LILRA2, LILRA3, LILRA4, LILRA6, ILT2, ILT3, ILT5, and LIL8, and show only weak binding to LILRA5.

[0340] [Table 3]

[0341] In Table 3, "parent" refers to the parental cell line expressing the LILRA or LILRB molecule.

[0342] The cross-reactivity of LILRA to other family members was further investigated as follows: Antibody titration flow cytometry binding assays were performed to determine the binding efficacy of 21D9, 21D9.e, 21A5, and 21A5.a to hILT4 and their corresponding cross-reactive ILT molecules. Nonlinear regression equations from GraphPad Prism® software were used to determine EC 50 We made that decision.

[0343] The results shown in Table 4 and Figure 20 indicate that the anti-ILT4 antibodies 21D9, 21D9.e, 21A5, and 21A5.a exhibit weak cross-reactivity with other LILRA family members: 21D9 and 21D9.e against LILRA5, and 21A5 and 21A5.a against LILRA1 and LILRA3.

[0344] [Table 4]

[0345] 21D9 and 21D9.e bind to hILT4 with high affinity, but do not significantly bind to hLILRA1, hLILRA2, hLILRA3, hLILRA4, hLILRA6, hILT2, hILT3, hILT5, and hLIR8, and show only weak binding to hLILRA5. Thus, 21D9 and 21D9.e are far more specific in binding to hILT4 compared to other hLILRA and hLIRB family members than other anti-ILT4 antibodies, such as clone 287219 from R&D Systems, which cross-react with ILT5 (LILRB3) and LILRA6. [Examples]

[0346] Anti-hILT4 antibodies enhance the T cell response. The effects of anti-hILT4 antibodies on T cell responses, particularly T cell proliferation and interferon-gamma (IFNγ) T cell secretion, were determined as follows: Cytoplasmic truncated hILT4 cells were transfected into CHO cells (CHO-OKT3 cells) expressing low levels of single-stranded variable fragments of anti-CD3 antibody. The transfected cell line was named CHO-OKT3-ILT4. Whole T cells isolated from human PBMCs were co-cultured with irradiated (proliferation-arrested) CHO-OKT3-ILT4 cells in a 4:1 ratio. Anti-ILT4 antibodies 21D9 ("21D9 WT"), 21D9.e, 21A5 ("21A5 WT"), 21A5.a, and 10F10 ("10F10 WT") were titrated from 20 μg / mL by 5-fold serial dilutions and incubated at 37°C for 3 days. The supernatant was collected, and the IFNγ level was evaluated by ELISA. 3 The system was pulsed overnight with H-thymidine, and the amplification was evaluated based on thymidine uptake.

[0347] The results shown in Figures 21A-D demonstrate that the anti-ILT4 antibodies 21D9, 21D9.e, 21A5, 21A5.a, and 10F10 stimulate T cell proliferation and IFNγ secretion in a dose-dependent manner. No significant differences in activity were found among these antibodies in this assay. [Examples]

[0348] Pre-treatment of differentiating monocytes with anti-ILT4 antibodies yields monocyte-derived dendritic cells (MoDCs), which are more stimulating to allogeneic T cells. CD14+ monocytes were isolated from human PBMCs using the StemCell EasySep® Human Monocyte Isolation Kit, seeded at 1 million cells per mL, and differentiated into monocyte-derived immature dendritic cells (Mo-iDCs) for 6 or 7 days in RPMI culture medium supplemented with 50 ng / mL GM-CSF and 100 ng / mL IL-4. During differentiation, cells were incubated with anti-ILT4 or isotype antibodies, or left untreated. After differentiation into Mo-iDCs, the cells were washed to remove anti-ILT4 antibody, GM-CSF, and IL-4, and further activated into mature dendritic cells (DCs (Mo-mDCs)) in the presence of 50 ng / mL CD40 agonist antibody. Mature DCs (Mo-mDCs) were set up for allogeneic mixed leukocyte reaction assays (allogeneic MLR) by co-culturing them with T cells in a 1:10 ratio (Mo-mDC:T) for 5 days. Cell supernatant was harvested at the end of allogeneic MLR to measure IFNγ by ELISA, and T cell proliferation was measured during the last 16 hours. 3 Evaluation was performed by H-thymidine uptake.

[0349] Results from another assay, shown in Figure 22, demonstrate that CD4+ T cell proliferation (Figures 22A and C) and IFNγ secretion (Figures 22B and D) in allogeneic MLRs are enhanced when MoDCs are differentiated from monocytes in the presence of anti-ILT4 antibodies 21A5.a or 21D9.e, compared to the MoDC assay. Monocytes from two different donors were used in the other assay. [Examples]

[0350] Monocyte-derived dendritic cells (MoDCs) differentiated from monocytes primed with anti-ILT4 antibodies show increased expression of co-stimulatory or maturation molecules. CD14+ monocytes were isolated from human PBMCs using the StemCell EasySep® Human Monocyte Isolation Kit, seeded at 1 million cells per mL, and differentiated into monocyte-derived immature DCs (Mo-iDCs) for 6 or 7 days in RPMI culture medium supplemented with 50 ng / mL GM-CSF and 100 ng / mL IL-4 in the presence of anti-ILT4 antibodies 21D9.e, 21A5.a, 21A5, 10F10, 21D9, 2H2, or isotype antibodies, or left untreated. The Mo-iDCs were then harvested for staining with cell surface markers CD86 and CD83.

[0351] The results shown in Figure 23 indicate increased expression of CD86 and CD83, costimulatory molecules, and maturation molecules, respectively, on Mo-iDCs when anti-ILT4 antibody was present during monocyte differentiation to Mo-iDCs. Taken together with the results of Example 6, these results suggest that treatment of monocytes with anti-hILT4 antibody during monocyte differentiation to MoDCs promotes inflammation in MoDCs, as indicated by the upregulation of CD83 and CD86 (Figures 23A-D), and consequently induces enhanced T cell proliferation and IFNγ production from allogeneic T cells in the allogeneic MLR assay (Figure 22). By enhancing T cell function, treatment with anti-hILT4 antibody may provide an enhancement of the anti-cancer immune response. [Examples]

[0352] Anti-ILT4 antibodies enhance TNFα secretion from differentiated macrophages in vitro. Monocytes isolated from human PBMCs were differentiated into macrophages for 5 days in the presence of M-CSF. Macrophages were treated with anti-ILT4 antibodies 21D9, 21D9.e, 21A5, 21A5.a, 2H2, and 10F10 or isotype controls at the indicated concentrations (as ng / mL next to their names) in the presence of 10 ng / mL of LPS (Figure 24A) or 5 μg / mL of 2'3'-cGAMP STING agonist (Figure 24B). TNFα production was measured by ELISA 24 hours after treatment, and the EC50 values ​​in the presence of each antibody are noted next to the curves.

[0353] The results shown in Figure 24 indicate that the presence of ILT4 antibodies 21D9, 21D9.e, 21A5, 21A5.a, 2H2, and 10F10 enhanced TNFα secretion in macrophages differentiated in vitro by LPS or STING agonists. Therefore, immunosuppressive macrophages exhibit a shift to a more inflammatory phenotype when ILT4 is blocked, potentially providing an enhanced anti-cancer immune response. These results suggest that anti-ILT4 antibodies promote inflammatory polarization toward M1 macrophages. [Examples]

[0354] Anti-ILT4 antibodies promote the CD4 T cell response in macrophage:CD4+ T cell allogeneic MLRs. Monocytes isolated from peripheral PBMCs were treated with M-CSF for 5 days to differentiate into macrophages. The macrophages were then cultured with allogeneic CD4+ T cells isolated from PBMCs of different donors and labeled with CFSE dye. A 1 μg / mL antibody (isotype control, anti-ILT4, or anti-PDL1) was added to the allogeneic MLR co-culture. On day 6, the supernatant was collected to analyze IFNγ and CD4+ T cell proliferation and evaluated by FACS based on CFSE dilution.

[0355] The results shown in Figures 25A-B demonstrate that the presence of anti-ILT4 antibodies 21D9, 21D9.e, 21A5, or 21A5.a in allogeneic MLRs of differentiated macrophages and CD4+ T cells in vitro promotes CD4+ T cell proliferation (Figure 25A) and IFNγ production (Figure 25B). These results indicate that anti-ILT4 antibodies promote T cell activation by modulating ILT4 repressive signaling in macrophages or by blocking its direct repressive activity in T cells. [Examples]

[0356] Macrophage: Combined effect of anti-ILT4 and anti-PD-L1 in CD4 T cell allogeneic MLR Monocytes isolated from peripheral PBMCs were treated with M-CSF for 5 days to differentiate into macrophages, and then co-cultured with allogeneic CD4+ T cells isolated from allogeneic donor PBMCs. Anti-ILT4 antibody 21D9 and anti-PD-L1 antibody were added individually or together at 10 μg / mL (isotype control, anti-ILT4, anti-PD-L1, or a combination of anti-ILT4 and anti-PD-L1) and included in the allogeneic MLR co-culture. On day 6, the supernatant was collected for IFNγ analysis.

[0357] The results shown in Figure 26 indicate that the combination of the anti-ILT4 antibody 21D9 and the anti-PD-L1 antibody stimulated enhanced IFNγ production from macrophage:CD4+ T cell allogeneic MLRs compared to each antibody alone. [Examples]

[0358] HDX epitope mapping of anti-hILT4 Ab 21D9 and 2H2 Fab Using HDX-MS, we identified the hILT4 regions to which the anti-hILT4 antibodies 21D9 and 2H2 bind.

[0359] HDX-MS explores protein conformation and conformational dynamics in solution by monitoring the rate and extent of deuterium exchange of back-chain amide hydrogen atoms (Huang and Chen (2014) Analytical and Bioanalytical Chem. 406:6541 and Wei et al. (2014) Drug Discovery Today 19:95). The level of HDX varies depending on the solvent reachability of back-chain amide hydrogen atoms and protein hydrogen bonding. The mass increase of the protein during HDX can be accurately measured by MS. When this technique is paired with enzymatic digestion, it is possible to elucidate structural features at the peptide level and distinguish peptides exposed on the surface from those folded internally or sequestered at the interface of protein-protein complexes. Typically, deuterium labeling and subsequent quenching experiments are performed, followed by enzymatic digestion, peptide separation, and MS analysis.

[0360] Prior to the epitope mapping experiment, deuterated experiments were performed to generate a list of common peptides for recombinant human ILT4 (15 μM) and the ILT4-Fab 21D9 and 2H2 protein complexes (1:1 molar ratio). In the HDX-MS experiment, 5 μL of each sample (ILT4 or ILT4 and Fab) was diluted in 55 μL of D2O buffer (10 mM phosphate buffer, D2O, pH 7.0) to initiate the labeling of the reactants. The reaction was carried out for various durations: 1 minute, 10 minutes, and 240 minutes. At the end of each reactant labeling, the reactants were quenched by adding quenching buffer (6 M urea, 1 M TCEP, pH 2.5, 1:1, v / v), and 50 μL of the quenched sample was injected into the Waters HDX-MS system for analysis. Deuterium uptake levels of common pepsin peptides were monitored in the absence and presence of Fab 21D9 and 2H2.

[0361] Human ILT4 (hILT4) was histidine-tagged and mixed with 21D9 or 2H2 Fab in a 1:1 ratio, then subjected to HDX for 1 minute, 10 minutes, or 4 hours. Pepsin-mediated sequence coverage of hILT4 is shown in Figure 27A. The regions of hILT4 (or hILT4 peptide) bound by each Fab are shown in Figures 27B and C. Specifically, HDX-MS experiments provided 85% sequence coverage for human ILT4.

[0362] HDX-MS data analysis of 21D9 and 2H2 in human ILT4 revealed that the 21D9 epitope corresponds to one region in human ILT4 (residue number corresponding to the natural human ILT4 sequence): region in Ig domain 2: 70 ITRIRPELVKNGQFHIPSITWEHTGRYGCQY 100 This indicates that it consists of (Sequence No. 122) (see Figure 27B, upper panel).

[0363] HDX-MS data analysis revealed that the 2H2 epitope is located in five regions of human ILT4, with regions 2 and 5 being the primary epitopes. Area 1: 127 SAQPSPVVTSGGRVTL 142 (Sequence ID 175) Area 2: 154 ILCKEGEEEHPQCLNSQPHARGSSRAIF 181 (Sequence ID 123) Area 3: 182 SVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDL 213 (Sequence ID 176) Area 4: 378 QAEFPMSPVTSAHAG 392 (Sequence ID 177) Area 5: 425 SSPPPTGPIS 434 (Sequence ID 124) This shows that it is composed of (see Figure 27B, lower panel). [Examples]

[0364] Anti-ILT4 antibodies block ILT4 binding to MHC class I molecules. The ability of anti-ILT4 antibodies to block rhILT4-Fc binding to HLA-A or HLA-B overexpressing CHO cells was determined by performing antibody blockade assays based on flow cytometry. rhILT4-Fc fusion proteins containing mouse Fc tails were produced. Anti-hILT4 antibodies 21D9, 2H2, 10F10, and 21A5 were titrated in the presence of immobilized rhILT4-Fc at a concentration of 30 μg / mL. After incubation of the antibody and rhILT4-Fc mixture on ice for 30 minutes, it was added to CHO cells engineered to overexpress HLA-A or HLA-B. The CHO cells were stained on ice for 30 minutes, washed, and then stained with an anti-mouse Ig secondary antibody to detect bound rhILT4-Fc.

[0365] The results shown in Figures 28A-D indicate that antibodies 21D9, 2H2, 10F10, and 21A5 inhibit the binding of hILT4 to HLA-A and HLA-B, respectively. [Examples]

[0366] 21D9e.IgG1.3 enhances the secretion of both IFN-γ and TNF-α by T cells. This example demonstrates that 21D9e.IgG1.3 enhances the secretion of IFN-gamma and TNF-alpha by T cells in the autologous mixed lymphocyte reaction (MLR) between monocytes and T cells.

[0367] T cells and monocytes were isolated from peripheral blood mononuclear cells (PBMCs) and co-cultured in a 1:1 ratio in the presence of 20 ng / mL of anti-CD3 antibody (clone: ​​OKT3, BioLegend). 21D9e.IgG1.3 and isotype control (DT-1D12-g1.3f) were added at 10 μg / mL. After an 88-hour incubation period using fluorescent dye-conjugated IFN-γ antibody (clone number B27, BioLegend) and TNF-α antibody (clone number MAb11, BioLegend), intracellular staining was performed to determine the frequency of IFN-γ and TNF-α-expressing T cells in each sample. Samples were acquired on a Cytoflex® cytometer (Beckman Coulter) and analyzed using FlowJo® software (Tree Star, Inc., Ashland, OR).

[0368] The results shown in Figures 29A-D demonstrate that 21D9e.IgG1.3 enhances the secretion of both IFN-γ and TNF-α by CD4+ and CD8+ T cells in autologous MLRs upon anti-CD3 stimulation. [Examples]

[0369] Anti-ILT4 antibodies enhance IFN-γ secretion upon antigen stimulation in CMV lysate assays. Peripheral blood mononuclear cells (PBMCs) derived from cytomegalovirus (CMV)-responsive donors were seeded at a rate of 200,000 cells per well in the presence of 0.3 μg / mL CMV lysate (CMV antigen 2, Microbix catalog number EL-02-01-001). Cells were treated with anti-ILT4 antibodies 21D9.IgG1.3 and 21A5.IgG1.3, starting at 20 μg / mL, in 8-point, 5-fold titrations. Nivolumab was added at 1 μg / mL as a positive control. 21D9-Fab and DT-1D12-g1.3 were added at 20 μg / mL. After 6 days of incubation, supernatants from each sample were collected and IFN-γ levels were measured by ELISA (Human IFN-γ ELISA Kit, BD OptEIA catalog number 555142).

[0370] The results shown in Figure 30 demonstrate that the anti-ILT4 antibodies 21D9.IgG1.3, 21D9-Fab, and 21A5.IgG1.3 enhance IFN-γ secretion upon antigen stimulation in the CMV lysate assay. [Examples]

[0371] ILT4 antagonism enhances T cell activation in monocyte:T allogeneic MLR. T cells (100,000 cells) derived from one donor and allogeneic monocytes were co-cultured in a 96-well U-bottom plate at a ratio of 2:1 per well. Anti-ILT4 antibodies 2H2.IgG1.1f, 2H2 Fab, 21D9.IgG1.1f, and 21D9 Fab were added in a 4-fold, 7-stop titration, starting at a concentration of 30 μg / mL for full-length antibodies or 80 μg / mL for Fab antibodies. Anti-KLH-g1.1f (anti-hemocyanin antibody) was added at 30 μg / mL as an isotype control. The cell cultures were incubated for 6 days. During the last 16 hours of culture... 3 Cell proliferation was evaluated by H-thymidine uptake.

[0372] The results shown in Figure 31 demonstrate that both full-length anti-ILT4 antibodies, 21D9.IgG1.1f and 2H2.IgG1.1f, as well as their corresponding Fab fragments, induce T cell proliferation. [Examples]

[0373] Binding of anti-hILT4 antibody to cynomolgus monkey ILT4 Several ILT family members were obtained from cynomolgus monkeys by PCR. Sequence analysis of these proteins did not clearly indicate which was the closest match to hILT4, so expression profiling and functional assays were performed. These assays identified clone name "9152" as the best match to hILT4 in terms of expression profiling and functional characteristics. The amino acid sequence of the extracellular region of 9152, including the His-Avi tag, is as follows: QAGILPKPMLWAEPDRVITQGSPVTLRCQGNLEARGYHLYRERKSASWITLIRPELVKKGQFPIPSITWEDAGRYRCQYYSHSWWSEHSDPLELVVTGAYRKPTLSALPSPVVASGGNVTLQCDSRVALDGFILCKEGEDEHSQRLNSQPRTRGSSRAVFSVGPVSPSRRWSYRCYGYELHSRYVWSLPSDLLELLVPGVSK For binding to hILT4, the extracellular region (ECD) of the hILT4-His-Avi tag described in Example 2; SEQ ID NO: 119 was used (Sequence ID NO: 118).

[0374] The dynamics of 21D9.e antibodies constituting cynomolgus monkey and human ILT4 using the above ILT4 construct were analyzed in a comparative SPR assay. The antibodies were amine-coupled to a GLC sensor tip in a Proteon XPR36 instrument (BioRad). Human ILT4 and cynomolgus monkey ILT4 9152 were injected as analytes in a 5-membered, 5-fold dilution series with a maximum concentration of 1 μM (minimum concentration of 1.6 nM). All data were double-referenced, exported, and analyzed using BIAevaluation Software 4.1.1 with the dynamic titration model described by Karlsson et al. (Karlsson et al. (2005) Analytical Biochem. 349:136). The electrophoresis buffer consisted of 10 mM HEPES pH 7.4, 150 mM NaCl, and 0.05% Tween-20. The assay temperature was 37°C.

[0375] The results shown in Table 5 indicate that 21D9.e bound to cynomolgus monkey ILT4 9152 with approximately 11 times weaker affinity than human ILT4 (3.5 nM for cynomolgus monkeys and 0.33 nM for human ILT4). D (Value). Similarly, the parent antibody 21D9 bound to cynomolgus monkeys with approximately 12 times weaker affinity than human ILT4. This difference was attributed to the faster dissociation rate constant for cynomolgus monkey ILT4 (4.0 nM for cynomolgus monkeys and 0.32 nM for human ILT4). D value).

[0376] [Table 5] [Examples]

[0377] ILT4 antibodies promote the expression of costimulatory molecules and activating markers in vitro on cynomolgus monkey monocyte-derived dendritic cells. This example demonstrates that the ILT4 antibodies 21D9.e.IgG1.3 (ILT4.8 in Figure 32; see Table 1) and 9G4.hIgG1.3 (ILT4.1 in Figure 32; see Table 1) stimulated the expression of CD80, CD83, and CD86 on monocyte-derived dendritic cells.

[0378] Monocytes (non-human primate CD14 microbeads, Miltenyi) isolated from cynomolgus monkey PBMCs were cultured for 5 days in the presence of 62.5 U / mL recombinant human GM-CSF (Peprotech) and 125 U / mL recombinant human IL-4 (Peprotech). At the time of culture setup, 10 μg / mL of anti-ILT4 antibody was added to the culture. Cells were stained for the cell surface expression levels of CD86, CD80, and CD83 using anti-CD86 (clone number IT2.2, BioLegend), anti-CD80 (clone number L307), and anti-CD83 (clone number HIB15e). Samples were acquired on a Cytoflex® cytometer (Beckman Coulter) and analyzed using FlowJo® software (Tree Star, Inc., Ashland, OR).

[0379] The results shown in Figures 32A-C demonstrate that the ILT4 antibodies 21D9.e.IgG1.3 (also known as ILT4.8.IgG1.3; "ILT4.8" in the figure) and 9G4.IgG1.3 (also known as ILT4.1.IgG1.3; "ILT4.1" in the figure) promote the expression of the co-stimulatory molecules CD80 and CD86, as well as the dendritic cell maturation marker CD83, in monocyte-derived DCs, as evidenced by the upregulation of CD80, CD83, and CD86 on dendritic cells. [Examples]

[0380] Modified anti-ILT4 antibody 10F10.4 The tyrosine at F36Y in the light chains of 10F10.1 and 10F10.3 (see Example 2) is located near the heavy chain CDR3 in the modeling of the hILT4 and 10F10 antibodies. Therefore, the substitution of F36 with Y can cause collision with isoleucine in the heavy chain CDR3, as evidenced by the faster dissociation rate (off-rate) of 10F10.1 and 10F10.3 compared to 10F10. Further mutants were created in which the F36 of the light chain was not mutated, and the only light chain framework region mutation produced in 10F10 was T63S. The heavy chain was not mutated and is therefore that of 10F10. This modified antibody is called 10F10.4, and the amino acid sequence of its light chain is listed in the sequence listing as SEQ ID NO: 116.

[0381] The binding kinetics of 10F10.4 were determined at 37°C using a Biacore® T200 instrument. The electrophoresis buffer was 10 mM HEPES pH 7.4 with 0.05% Tween-20 and 1 g / L BSA added. All anti-ILT4 antibodies were captured on CM4 chips pre-immobilized with anti-human Fc capture antibody from Southern Biotech (catalog no. 2081-01). Human ILT4 (hILT4-His-Avi tag as described in Example 2) was injected at 1.2 μM (for 10F10, 10F10.4 only, and one repeat for 1E5 ILT4.15), 405 nM, 81 nM (two repeats), 16 nM, 3 nM, and 0.6 nM. All data were double-referenced and mass transported using Biacore T200 Evaluation Software version 3.1 to fit into a 1:1 Langmuir model.

[0382] The results shown in Table 6 indicate that the coupling dynamics of 10F10.4 are very similar to those of 10F10. 21A5, 21A5a, 21D9, and 21D9.e were included as controls. The absolute values ​​differ slightly from those shown in Table 2, which is due to the different capture reagents used in this assay.

[0383] [Table 6]

[0384] 10F10.4 was shown to be functionally active by enhancing TNFα secretion from differentiated macrophages in vitro. This was demonstrated by incubating 10F10.4 with macrophages differentiated from PBMC monocytes as described in Example 8, 21D9.e, as well as including isotype controls. The results shown in Figures 33A and 33B demonstrate that the presence of the ILT4 antibody 10F10.4 enhanced TNFα secretion during in vitro differentiation of monocytes into macrophages by LPS or STING agonists. [Examples]

[0385] 21D9.e has similar potency in the context of IgG1, IgG4, and IgG1.3. This embodiment demonstrates that the efficacy of 21D9.e is similar in the context of IgG1, IgG4(S228P), and IgG1.3 heavy chains.

[0386] The 21D9.e antibody was cloned in the context of IgG1, IgG4 (containing S228P), or IgG1.3 (SEQ ID NOs. 176, 178, and 13, respectively) and used in assays to measure TNFα secretion, as described below.

[0387] The results shown in Figure 34 indicate that 21D9.e.IgG1, 21D9.e.IgG4.S228P, and 21D9.e.IgG1.3 secrete similar amounts of TNFα. Thus, the 21D9.e variable region has similar functional efficacy in the context of these different Fc regions. DT-1D12 is an isotype control. [Examples]

[0388] 21D9e binds to the hILT4 allele. This example describes an assay for determining whether 21D9.e binds to a mutant allele of hILT4.

[0389] Only these alleles with a frequency of more than 1% were considered. Two alleles with amino acid changes in regions D1 and D2: E161D and R103H (SEQ ID NO: 107, i.e., numbered by ILT4 including the signal sequence) were further considered. Three-dimensional modeling showed that both amino acid residues were not near the binding interface with MHC class I, so it was not expected that the amino acid changes at these residues would affect antibody binding to hILT4. To confirm this, hILT4 proteins with substitutions E161D and R103H (hILT4 E161D / R103H) were constructed and tested for binding to 21D9.e.IgG1.3 and 21A5.a.IgG1.3 (ILT4.9.IgG1.3; see Table 1) by Octet BLI. Both proteins were found to bind similarly to hILT4 E161D / R103H and hILT4 E161 / R103. [Examples]

[0390] 21D9e.IgG1.3 does not significantly induce basophil activation. The assay used to detect basophil activation by anti-ILT4 antibody included the following steps. Generally, this assay was performed using the Buhlmann Laboratories AG method used in the Flow CAST (copyright) Basophil Activation Test, as follows: Human basophils derived from donor blood were detected using anti-Ccr3. CD63 was used as an indicator of basophil activation. Stimulation buffer and antibody were added to whole blood. After incubation, staining reagents were added to the blood, and after antibody incubation, red blood cells were lysed, the sample was centrifuged, washed, and analyzed on a BD FACS Canto II.

[0391] Based on this assay, Ccr+CD63+ activated basophils from eight donors were measured by FACS after incubation for 180 minutes at 4.5, 45.45, or 454.55 μg / mL with 21D9.e.IgG1.3 (ILT4.8.IgG1.3; see Table 1), 21.5Aa.IgG1.3 (ILT4.9.IgG1.3), or anti-DT. Positive controls included anti-FcεRI and fMLP (N-formylmethionine-leucyl-phenylalanine). Negative controls were unrelated antigens, targeting diphtheria toxin, and DT1DT12-IgG1.3.

[0392] The results shown in Figure 36 indicate that 21D9.e.IgG1.3 (ILT4.8.IgG1.3) and 21.5Aa.IgG1.3 (ILT4.9.IgG1.3) do not induce basophil activation under the tested conditions. The ab...

Claims

1. An isolated antibody that specifically binds to human ILT4 (hILT4 or ILT4), comprising a heavy chain and a light chain, and having the following characteristics: - For example, 10 －８ M or less, or 10 －９ K below M Ｄ Specific binding to hILT4 (e.g., including the amino acid sequences of SEQ ID NOs. 108, 109, 111, 112, or 119); - Lack of specific binding to hILT2, hILT3, and / or hILT5; - Lack of specific binding to one or more members of the LILRA and / or LILRB family; - For example, stimulating T cell activation in a mixed lymphocyte reaction (MLR) assay, as measured by increased T cell proliferation or IFN-gamma secretion, as shown in the assay described in the examples; - For example, stimulating the differentiation or activation of monocytes into macrophages, as shown in the assay described in the examples, e.g., stimulating the differentiation of monocytes into inflammatory macrophages; - For example, promoting the expression of CD83 and CD86 on human monocyte-derived immature dendritic cells (Mo-iDCs), as shown in the assay described in the examples; - For example, enhancing IFN-γ secretion upon antigen stimulation in a cytomegalovirus (CMV) lysate assay, as shown in the assay described in the examples; - For example, enhancing IFN-γ and TNF-α secretion by CD4+ and CD8+ T cells in an allogeneic mixed lymphocyte response (MLR) assay upon CD3 stimulation, as shown in the assay described in the examples; - Inhibiting the binding of HLA-A and / or HLA-B to ILT4; - For example, when determined by deuterium exchange (HDX), as shown in the HDX assay described in the examples, ７０ ITRIRPELVKNGQFHIPSITWEHTGRYGCQY １００ To combine with (SEQ ID NO: 122); - Competing with the antibodies described herein for binding to hILT4; - For example, as shown in the binding assay described in the examples, it specifically binds to cynomolgus monkey ILT4 containing SEQ ID NO: 118; - Inhibiting the binding of human ILT4 (hILT4) to ILT4-binding partners such as MHC class I molecules, including HLA-A and HLA-B (for example, inhibiting the binding of hILT4 to both HLA-A and HLA-B); - Having the HLA-A and HLA-B binding profiles shown in Figure 28; - Having a coupling profile as shown in Figure 27; - The following regions of hILT4: (i) 70 ITRIRPEL 77 (SEQ ID NO: 120) and / or 78 VKNGQFHIPSITWEHTGRYGCQY 100 (SEQ ID NO: 121); (ii) 70 ITRIRPELVKNGQFHIPSITWEHTGRYGCQY 100 (SEQ ID NO: 122); or (iii) 154 ILCKEGEEEHPQCLNSQPHARGSSRAIF 181 (SEQ ID NO: 123) and / or 425 SSPPPTGPIS 434 (SEQ ID NO: 124), [In the sequence, the amino acid numbering of hILT4 is that of immature hILT4 (i.e., ILT4 including its native signal sequence)], binds to (i) or (ii), and when binding to (i) or (ii), does not significantly bind to other regions of the extracellular domain of ILT4, such as regions or residues located at the N-terminus relative to amino acid I70; and - For example, as described in the examples, one or more (or all) of the amino acid residues Lys43, Ile49, Thr50 and Arg51 of mature hILT4, as determined by carbene footprinting, or the amino acid residues Gly117, Val119, Try120, Leu134, Lys136, Gln149, Pro150, Ile159, S Interacting with one or more (or all) of the following amino acid residues of mature hILT4: er161, Val162, Gly163, Pro164, Pro167, His173, Try178, Pro183, and Tyr184; or interacting with one or more (or all) of the following amino acid residues of mature hILT4: Glu42, Lys43, Gly76, Cys77, Leu88, Pro91, Pro183, and Tyr184. An antibody further comprising one or more of the following.

2. The isolated antibody according to claim 1, comprising a heavy chain and a light chain, wherein the heavy chain comprises VH CDR1, CDR2, and CDR3 of the anti-ILT4 antibody 9G4 (SEQ ID NOs: 125-127), 9C8 (SEQ ID NOs: 131-133), 2H2 (SEQ ID NOs: 137-139), 2E5 (SEQ ID NOs: 143-145), 24E5 (SEQ ID NOs: 149-151), 21D9 (SEQ ID NOs: 155-157), 21A5 (SEQ ID NOs: 161-163), or 10F10 (SEQ ID NOs: 167-169).

3. The isolated antibody according to claim 1 or 2, comprising a heavy chain and a light chain, wherein the light chain comprises VL CDR1, CDR2, and CDR3 of the anti-ILT4 antibody 9G4 (SEQ ID NOs: 128-130), 9C8 (SEQ ID NOs: 134-136), 2H2 (SEQ ID NOs: 140-142), 2E5 (SEQ ID NOs: 146-148), 24E5 (SEQ ID NOs: 152-154), 21D9 (SEQ ID NOs: 158-160), 21A5 (SEQ ID NOs: 164-166), or 10F10 (SEQ ID NOs: 170-172).

4. The isolated antibody according to claim 2, wherein the heavy chain comprises VH CDR1, CDR2 and CDR3 and VL CDR1, CDR2 and CDR3 of the anti-ILT4 antibody 9G4 (SEQ ID NOs. 125-130), 9C8 (SEQ ID NOs. 131-136), 2H2 (SEQ ID NOs. 137-142), 2E5 (SEQ ID NOs. 143-148), 24E5 (SEQ ID NOs. 149-154), 21D9 (SEQ ID NOs. 155-160), 21A5 (SEQ ID NOs. 161-166), or 10F10 (SEQ ID NOs. 167-172).

5. (a) VH containing the amino acid sequences of 9G4 CDR1, CDR2, and CDR3 (SEQ ID NOs: 125-127), and VL containing the amino acid sequences of 9G4 CDR1, CDR2, and CDR3 (SEQ ID NOs: 128-130); (b) VH containing the amino acid sequences of 9C8 CDR1, CDR2, and CDR3 (SEQ ID NOs: 131-133), and VL containing the amino acid sequences of 9C8 CDR1, CDR2, and CDR3 (SEQ ID NOs: 134-136); (c) VH of 2H2 containing the amino acid sequences of CDR1, CDR2, and CDR3 (SEQ ID NOs: 137-139), and VL of 2H2 containing the amino acid sequences of CDR1, CDR2, and CDR3 (SEQ ID NOs: 140-142); (d) VH containing the amino acid sequences of 2E5 CDR1, CDR2, and CDR3 (SEQ ID NOs: 143-145), and VL containing the amino acid sequences of 2E5 CDR1, CDR2, and CDR3 (SEQ ID NOs: 146-148); (e) VH containing the amino acid sequences of CDR1, CDR2, and CDR3 of 24E5 (SEQ ID NOs: 149-151), and VL containing the amino acid sequences of CDR1, CDR2, and CDR3 of 24E5 (SEQ ID NOs: 152-154); (f) VH containing the amino acid sequences of CDR1, CDR2, and CDR3 of 21D9 (SEQ ID NOs: 155-157), and VL containing the amino acid sequences of CDR1, CDR2, and CDR3 of 21D9 (SEQ ID NOs: 158-160); (g) VH of 21D9.b containing the amino acid sequences of CDR1, CDR2, and CDR3 (SEQ ID NOs. 155-157), and VL of 21D9.b containing the amino acid sequences of CDR1, CDR2, and CDR3 (SEQ ID NOs. 158-160); (h) VH containing the amino acid sequences of CDR1, CDR2, and CDR3 (SEQ ID NOs. 155-157) of 21D9.c, and VL containing the amino acid sequences of CDR1, CDR2, and CDR3 (SEQ ID NOs. 158-160) of 21D9.c; (i) VH containing the amino acid sequences of CDR1, CDR2, and CDR3 (SEQ ID NOs. 155-157) of 21D9.d, and VL containing the amino acid sequences of CDR1, CDR2, and CDR3 (SEQ ID NOs. 158-160) of 21D9.d; (j) VH of 21D9.e containing the amino acid sequences of CDR1, CDR2, and CDR3 (SEQ ID NOs. 155-157), and VL of 21D9.e containing the amino acid sequences of CDR1, CDR2, and CDR3 (SEQ ID NOs. 158-160); (k) VH containing the amino acid sequences of CDR1, CDR2, and CDR3 of 21A5 (SEQ ID NOs: 161-163), and VL containing the amino acid sequences of CDR1, CDR2, and CDR3 of 21A5 (SEQ ID NOs: 164-166); (l) VH containing the amino acid sequences of CDR1, CDR2, and CDR3 (SEQ ID NOs: 161-163) of 21A5.a, and VL containing the amino acid sequences of CDR1, CDR2, and CDR3 (SEQ ID NOs: 164-166) of 21A5.a; (m) VH containing the amino acid sequences of CDR1, CDR2, and CDR3 of 10F10 (SEQ ID NOs: 167-169), and VL containing the amino acid sequences of CDR1, CDR2, and CDR3 of 10F10 (SEQ ID NOs: 170-172); (n) VH containing the amino acid sequences of CDR1, CDR2, and CDR3 of 10F10.1 (SEQ ID NOs: 167-169), and VL containing the amino acid sequences of CDR1, CDR2, and CDR3 of 10F10.1 (SEQ ID NOs: 170-172); (o) VH containing the amino acid sequences of CDR1, CDR2, and CDR3 of 10F10.3 (SEQ ID NOs: 167-169), and VL containing the amino acid sequences of CDR1, CDR2, and CDR3 of 10F10.3 (SEQ ID NOs: 170-172); or (p) VH containing the amino acid sequences of CDR1, CDR2, and CDR3 of 10F10.4 (SEQ ID NOs: 167-169), and VL containing the amino acid sequences of CDR1, CDR2, and CDR3 of 10F10.4 (SEQ ID NOs: 170-172). An isolated antibody according to any one of claims 1 to 4, comprising:

6. The isolated antibody according to any one of claims 1 to 5, wherein the antibody heavy chain comprises a VH having an amino acid sequence identical to, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of, the amino acid sequence of VH of 9G4 (SEQ ID NO: 51, amino acids 20-135), 9C8 (SEQ ID NO: 55), 2H2 (SEQ ID NO: 58), 2E5 (SEQ ID NO: 63), 24E5 (SEQ ID NO: 67), 21D9 (SEQ ID NO: 71), 21D9.b (SEQ ID NO: 74), 21D9.c (SEQ ID NO: 75), 21D9.d (SEQ ID NO: 78), 21D9.e (SEQ ID NO: 80), 21A5 (SEQ ID NO: 83), 21A5.a (SEQ ID NO: 87), 10F10 (SEQ ID NO: 91), 10F10.1 (SEQ ID NO: 91), 10F10.3 (SEQ ID NO: 91), or 10F10.4 (SEQ ID NO: 91).

7. The isolated antibody according to any one of claims 1 to 6, wherein the antibody light chain comprises a VL having an amino acid sequence identical to, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of, the amino acid sequence of the VL of 9G4 (SEQ ID NO: 50, amino acids 19-125), 9C8 (SEQ ID NO: 54), 2H2 (SEQ ID NO: 59), 2E5 (SEQ ID NO: 62), 24E5 (SEQ ID NO: 66), 21D9 (SEQ ID NO: 70), 21D9.b (SEQ ID NO: 70), 21D9.c (SEQ ID NO: 70), 21D9.e (SEQ ID NO: 70), 21A5 (SEQ ID NO: 82), 21A5.a (SEQ ID NO: 86), 10F10 (SEQ ID NO: 90), 10F10.1 (SEQ ID NO: 94), 10F10.3 (SEQ ID NO: 96), or 10F10.4 (SEQ ID NO: 114).

8. The isolated antibody according to any one of claims 1 to 7, wherein the antibody heavy chain comprises a VH having an amino acid sequence that includes 1, 2, 3, 4, or 5 amino acid substitutions, conservative substitutions, or reverse substitutions compared to the amino acid sequence of VH of 9G4 (SEQ ID NO: 51, amino acids 20-135), 9C8 (SEQ ID NO: 55), 2H2 (SEQ ID NO: 58), 2E5 (SEQ ID NO: 63), 24E5 (SEQ ID NO: 67), 21D9 (SEQ ID NO: 71), 21D9.b (SEQ ID NO: 74), 21D9.c (SEQ ID NO: 75), 21D9.d (SEQ ID NO: 78), 21D9.e (SEQ ID NO: 80), 21A5 (SEQ ID NO: 83), 21A5.a (SEQ ID NO: 87), 10F10 (SEQ ID NO: 91), 10F10.1 (SEQ ID NO: 91), 10F10.3 (SEQ ID NO: 91), or 10F10.4 (SEQ ID NO: 91).

9. The isolated antibody according to any one of claims 1 to 8, wherein the antibody light chain comprises a VL having an amino acid sequence that includes 1, 2, 3, 4, or 5 amino acid substitutions, conservative substitutions, or reverse substitutions compared to the amino acid sequence of VL 9G4 (SEQ ID NO: 50, amino acids 19-125), 9C8 (SEQ ID NO: 54), 2H2 (SEQ ID NO: 59), 2E5 (SEQ ID NO: 62), 24E5 (SEQ ID NO: 66), 21D9 (SEQ ID NO: 70), 21D9.b (SEQ ID NO: 70), 21D9.c (SEQ ID NO: 70), 21D9.e (SEQ ID NO: 70), 21A5 (SEQ ID NO: 82), 21A5.a (SEQ ID NO: 86), 10F10 (SEQ ID NO: 90), 10F10.1 (SEQ ID NO: 94), 10F10.3 (SEQ ID NO: 96), or 10F10.4 (SEQ ID NO: 114).

10. The isolated antibody according to any one of claims 1 to 9, wherein the heavy chain comprises the VH of the anti-ILT4 antibody 9G4 (SEQ ID NO: 51, amino acids 20-135), 9C8 (SEQ ID NO: 55), 2H2 (SEQ ID NO: 58), 2E5 (SEQ ID NO: 63), 24E5 (SEQ ID NO: 67), 21D9 (SEQ ID NO: 71), 21D9.b (SEQ ID NO: 74), 21D9.c (SEQ ID NO: 75), 21D9.d (SEQ ID NO: 78), 21D9.e (SEQ ID NO: 80), 21A5 (SEQ ID NO: 83), 21A5.a (SEQ ID NO: 87), 10F10 (SEQ ID NO: 91), 10F10.1 (SEQ ID NO: 91), 10F10.3 (SEQ ID NO: 91), or 10F10.4 (SEQ ID NO: 91).

11. The isolated antibody according to any one of claims 1 to 10, wherein the light chain comprises the VL of anti-ILT4 antibody 9G4 (SEQ ID NO: 50, amino acids 19-125), 9C8 (SEQ ID NO: 54), 2H2 (SEQ ID NO: 59), 2E5 (SEQ ID NO: 62), 24E5 (SEQ ID NO: 66), 21D9 (SEQ ID NO: 70), 21D9.b (SEQ ID NO: 70), 21D9.c (SEQ ID NO: 70), 21D9.d (SEQ ID NO: 70), 21D9.e (SEQ ID NO: 70), 21A5 (SEQ ID NO: 82), 21A5.a (SEQ ID NO: 86), 10F10 (SEQ ID NO: 90), 10F10.1 (SEQ ID NO: 94), 10F10.3 (SEQ ID NO: 96), or 10F10.4 (SEQ ID NO: 114).

12. Anti-ILT4 antibodies: 9G4 (SEQ ID NO: 51, amino acids 20-135 and SEQ ID NO: 50, amino acids 19-125), 9C8 (SEQ ID NO: 55 and SEQ ID NO: 54), 2H2 (SEQ ID NO: 58 and SEQ ID NO: 59), 2E5 (SEQ ID NO: 63 and SEQ ID NO: 62), 24E5 (SEQ ID NO: 67 and SEQ ID NO: 66), 21D9 (SEQ ID NO: 71 and SEQ ID NO: 70), 21D9. b (SEQ ID NO: 74 and SEQ ID NO: 70), 21D9. c (SEQ ID NO: 75 and SEQ ID NO: 70), 21D9. d (SEQ ID NO: 78 and SEQ ID NO: 70), 21D9. e (SEQ ID NO: 80 and SEQ ID NO: 70), 21A5 (SEQ ID NO: 83 and SEQ ID NO: 82), 21A5. An isolated antibody according to any one of claims 1 to 11, comprising VH and VL of a (SEQ ID NO: 87 and SEQ ID NO: 86), 10F10 (SEQ ID NO: 91 and SEQ ID NO: 90), 10F10.1 (SEQ ID NO: 91 and SEQ ID NO: 94), 10F10.3 (SEQ ID NO: 91 and SEQ ID NO: 96), or 10F10.4 (SEQ ID NO: 91 and SEQ ID NO: 114).

13. a. VH containing the VH CDR of 9G4 VH (SEQ ID NOs: 125-127), VL containing the VL CDR of 9G4 VL (SEQ ID NOs: 128-130), and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 9G4 (SEQ ID NOs: 51, amino acids 20-135 and SEQ ID NOs: 50, amino acids 19-125); b. VH containing the VH CDR of 9C8 VH (SEQ ID NOs: 131-133), VL containing the VL CDR of 9C8 VH (SEQ ID NOs: 134-136), and VH and VL amino acid sequences that are 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%, or at least 99% identical to the VH and VL of 9C8 (SEQ ID NOs: 55 and 54); c. VH containing the VH CDR of 2H2 VH (SEQ ID NOs: 137-139), VL containing the VL CDR of 2H2 (SEQ ID NOs: 140-142), and VH and VL amino acid sequences that are 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%, or at least 99% identical to the VH and VL of 2H2 (SEQ ID NOs: 58 and 59); d. VH containing the VH CDR of 2E5 VH (SEQ ID NOs: 143-145), VL containing the VL CDR of 2E5 (SEQ ID NOs: 146-148), and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 2E5 (SEQ ID NOs: 63 and 62); e. VH containing the VH CDR of 24E5 VH (SEQ ID NOs: 149-151), VL containing the VL CDR of 24E5 (SEQ ID NOs: 152-154), and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 24E5 (SEQ ID NOs: 67 and 66); f. VH containing the VH CDR of 21D9 VH (SEQ ID NOs: 155-157), VL containing the VL CDR of 21D9 (SEQ ID NOs: 158-160), and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 21D9 (SEQ ID NOs: 71 and 70); g. VH containing the VH CDR (SEQ ID NOs. 155-157) of VH of 21D9.b, and VL containing the VL CDR (SEQ ID NOs. 158-160) of VH of 21D9.b, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 21D9.b (SEQ ID NOs. 74 and 70); h. VH containing the VH CDR (SEQ ID NOs. 155-157) of VH of 21D9.c, and VL containing the VL CDR (SEQ ID NOs. 158-160) of VH of 21D9.c, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 21D9.c (SEQ ID NOs. 75 and 70); i. VH containing the VH CDR (SEQ ID NOs. 155-157) of VH of 21D9.d, and VL containing the VL CDR (SEQ ID NOs. 158-160) of VH of 21D9.d, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 21D9.d (SEQ ID NOs. 78 and 70); j. VH containing the VH CDR (SEQ ID NOs. 155-157) of 21D9.e, and VL containing the VL CDR (SEQ ID NOs. 158-160) of 21D9.e, and VH and VL amino acid sequences that are 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%, or at least 99% identical to the VH and VL (SEQ ID NOs. 80 and 70) of 21D9.e; k. VH containing the VH CDR of 21A5 VH (SEQ ID NOs: 161-163), VL containing the VL CDR of 21A5 (SEQ ID NOs: 164-166), and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 21A5 (SEQ ID NOs: 83 and 82); l. VH containing the VH CDR (SEQ ID NOs. 161-163) of 21A5.a, and VL containing the VL CDR (SEQ ID NOs. 164-166) of 21A5.a, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL (SEQ ID NOs. 87 and 86) of 21A5.a; m. VH containing the VH CDR of 10F10 VH (SEQ ID NOs: 167-169), VL containing the VL CDR of 10F10 (SEQ ID NOs: 170-172), and VH and VL amino acid sequences that are 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%, or at least 99% identical to the VH and VL of 10F10 (SEQ ID NOs: 91 and 90); n. VH containing the VH CDR of 10F10.1 (SEQ ID NOs: 167-169), VL containing the VL CDR of 10F10.1 (SEQ ID NOs: 170-172), and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 10F10.1 (SEQ ID NOs: 91 and 94); o. VH containing the VH CDR of 10F10.3 VH (SEQ ID NOs. 167-169), VL containing the VL CDR of 10F10.3 (SEQ ID NOs. 170-172), and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 10F10.3 (SEQ ID NOs. 91 and 96); or p. VH containing the VH CDR (SEQ ID NOs. 167-169) of 10F10.4, VL containing the VL CDR (SEQ ID NOs. 170-172) of 10F10.4, and VH and VL amino acid sequences that are 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%, or at least 99% identical to VH and VL of 10F10.4 (SEQ ID NOs. 91 and 114). An isolated antibody according to any one of claims 1 to 11, comprising:

14. a. VH containing the amino acid sequence of 9G4 VH (residues 20-135 of SEQ ID NO: 51) and VL containing the amino acid sequence of 9G4 VL (residues 19-125 of SEQ ID NO: 50); b. VH containing the amino acid sequence of 9C8 VH and VL containing the amino acid sequence of 9C8 VL (SEQ ID NOs. 55 and 54); c. VH containing the amino acid sequence of 2H2 VH, and VL containing the amino acid sequence of 2H2 VL (SEQ ID NOs. 58 and 59); d. VH containing the amino acid sequence of 2E5 VH, and VL containing the amino acid sequence of 2E5 VL (SEQ ID NO: 63 and SEQ ID NO: 62); e. VH containing the amino acid sequence of 24E5 VH, and VL containing the amino acid sequence of 24E5 VL (SEQ ID NOs. 67 and 66); f. VH containing the amino acid sequence of 21D9 VH, and VL containing the amino acid sequence of 21D9 VL (SEQ ID NOs. 71 and 70); g. VH containing the amino acid sequence of VH of 21D9.b and VL containing the amino acid sequence of VL of 21D9.b (SEQ ID NOs. 74 and 70); h. VH containing the amino acid sequence of VH from 21D9.c and VL containing the amino acid sequence of VL from 21D9.c (SEQ ID NOs. 75 and 70); i. VH containing the amino acid sequence of VH from 21D9.d and VL containing the amino acid sequence of VL from 21D9.d (SEQ ID NOs. 78 and 70); j. VH containing the amino acid sequence of VH from 21D9.e and VL containing the amino acid sequence of VL from 21D9.e (SEQ ID NO: 80 and SEQ ID NO: 70); k. VH containing the amino acid sequence of 21A5 VH, and VL containing the amino acid sequence of 21A5 VL (SEQ ID NOs. 83 and 82); l. VH containing the amino acid sequence of VH of 21A5.a, and VL containing the amino acid sequence of VL of 21A5.a (SEQ ID NOs. 87 and 86); m. VH containing the amino acid sequence of 10F10 VH, and VL containing the amino acid sequence of 10F10 VL (SEQ ID NOs. 91 and 90); n. VH containing the amino acid sequence of VH of 10F10.1 and VL containing the amino acid sequence of VL of 10F10.1 (SEQ ID NOs. 91 and 94); o. VH containing the amino acid sequence of VH of 10F10.3 and VL containing the amino acid sequence of VL of 10F10.3 (SEQ ID NOs. 91 and 96); or p. VH containing the amino acid sequence of 10F10.4 VH, and VL containing the amino acid sequence of 10F10.4 VL (SEQ ID NOs. 91 and 114) An isolated antibody according to any one of claims 1 to 13, comprising:

15. An isolated antibody according to any one of claims 1 to 14, which is an IgG antibody.

16. The isolated antibody according to claim 15, wherein the antibody is an IgG1, IgG2, or IgG4 antibody, and the IgG4 antibody optionally includes an S228P substitution (EU numbering).

17. An isolated antibody according to any one of claims 1 to 16, which is an antibody without effector function.

18. The isolated antibody according to claim 17, wherein the heavy chain constant region contains one, two, three, four, or five mutations in the otherwise wild-type human heavy chain constant region, which reduces the effector function of the antibody compared to an antibody having the same amino acid sequence in other respects.

19. The isolated antibody according to any one of claims 1 to 18, wherein the heavy chain constant region of the antibody comprises an IgG1.3 heavy chain constant region, an IgG1 heavy chain constant region having a P238K (Eu numbering) substitution, or an IgG1 heavy chain constant region containing the amino acid sequence described in any one of SEQ ID NOs. 98, 100, 102, 103, or 104.

20. An isolated antibody according to any one of claims 1 to 16, having an effector function.

21. The isolated antibody according to claim 20, which is defucosylated (for example, a defucosylated IgG1 antibody).

22. The isolated antibody according to claim 20 or 21, wherein the heavy chain constant region contains one, two, three, four, or five mutations in the otherwise wild-type human heavy chain constant region, which enhances the effector function of the antibody compared to an antibody having the same amino acid sequence as otherwise.

23. An isolated antibody according to any one of claims 1 to 22, comprising an anti-ILT4 antibody HC of 9G4, 9C8, 2H2, 2E5, 24E5, 21D9, 21D9, b, 21D9, c, 21D9, d, 21D9, e, 21A5, 21A5, a, 10F10, 10F10.1 or 10F10.3, wherein the constant region of the HC is IgG1 (e.g., 9G4.IgG1, etc.), IgG1.3 (e.g., 9G4.IgG1.3, etc.), IgG1.1f (e.g., 9G4.IgG1.1f, etc.), IgG4 (e.g., 9G4.IgG4, etc.), or IgG4 S228P (EU numbering) (e.g., 9G4.IgG4_S228P).

24. The following, a. IgG1, e.g., 9G4. IgG1, 9G4 (SEQ ID NO: 2), 9C8 (SEQ ID NO: 4), 2H2 (SEQ ID NO: 6), 2E5 (SEQ ID NO: 8), 24E5 (SEQ ID NO: 10), 21D9 (SEQ ID NO: 12), 21D9. b (SEQ ID NO: 74 and 98), 21D9. c (SEQ ID NO: 75 and 98), 21D9. d (SEQ ID NO: 78 and 98), 21D9. e (SEQ ID NO: 80 and 98), 21A5 (SEQ ID NO: 83 and 98), 21A5. a (SEQ ID NO: 87 and 98), 10F10 (SEQ ID NO: 91 and 98), 10F10.1 (SEQ ID NO: 91 and 98), 10F10.3 (SEQ ID NO: 91 and 98), or 10F10.4 (SEQ ID NO: 91 and 98), b. IgG1, e.g., 9G4. IgG1, 9G4 (SEQ ID NO: 51, amino acids 20-135, and SEQ ID NO: 102), 9C8 (SEQ ID NO: 55 and SEQ ID NO: 102), 2H2 (SEQ ID NO: 58 and SEQ ID NO: 102), 2E5 (SEQ ID NO: 63 and SEQ ID NO: 102), 24E5 (SEQ ID NO: 67 and SEQ ID NO: 102), 21D9 (SEQ ID NO: 71 and SEQ ID NO: 102), 21D9. b (SEQ ID NO: 74 and SEQ ID NO: 102), 21D9. c (SEQ ID NO: 75 and SEQ ID NO: 102), 21D9. d (SEQ ID NO: 78 and SEQ ID NO: 102), 21D9. e (SEQ ID NO: 80 and SEQ ID NO: 102), 21A5 (SEQ ID NO: 83 and SEQ ID NO: 102), 21A5. a (sequences 87 and 102), 10F10 (sequences 91 and 102), 10F10.1 (sequences 91 and 102), 10F10.3 (sequences 91 and 102), or 10F10.4 (sequences 91 and 102), c. IgG1.3 (e.g., 9G4.IgG1.3), 9G4 (SEQ ID NO: 51, amino acids 20-135, and SEQ ID NO: 100), 9C8 (SEQ ID NO: 55 and SEQ ID NO: 100), 2H2 (SEQ ID NO: 58 and SEQ ID NO: 100), 2E5 (SEQ ID NO: 63 and SEQ ID NO: 100), 24E5 (SEQ ID NO: 67 and SEQ ID NO: 100), 21D9 ((i) SEQ ID NO: 113, or (ii) SEQ ID NO: 71 and SEQ ID NO: 100), 21D9. b (SEQ ID NO: 36), 21D9. c (SEQ ID NO: 38), 21D9. d (SEQ ID NO: 40), 21D9. e (SEQ ID NO: 13), 21A5 (SEQ ID NO: 15), 21A5. a (SEQ ID NO: 17), 10F10 (SEQ ID NO: 19), 10F10.1 (SEQ ID NO: 91 and 100), 10F10.3 (SEQ ID NO: 91 and 100), or 10F10.4 (SEQ ID NO: 91 and 100); d. IgG1.1f (e.g., 9G4.IgG1.1f), 9G4 (SEQ ID NO: 51, amino acids 20-135, and SEQ ID NO: 103), 9C8 (SEQ ID NO: 55 and SEQ ID NO: 103), 2H2 (SEQ ID NO: 58 and SEQ ID NO: 103), 2E5 (SEQ ID NO: 63 and SEQ ID NO: 103), 24E5 (SEQ ID NO: 67 and SEQ ID NO: 103), 21D9 (SEQ ID NO: 71 and SEQ ID NO: 103), 21D9. b (SEQ ID NO: 74 and SEQ ID NO: 103), 21D9. c (SEQ ID NO: 75 and SEQ ID NO: 103), 21D9. d (SEQ ID NO: 78 and SEQ ID NO: 103), 21D9. e (SEQ ID NO: 80 and SEQ ID NO: 103), 21A5 (SEQ ID NO: 83 and SEQ ID NO: 103), 21A5. a (sequences 87 and 103), 10F10 (sequences 91 and 103), 10F10.1 (sequences 91 and 103), 10F10.3 (sequences 91 and 103), or 10F10.4 (sequences 91 and 103), e. IgG1fa.P238K (e.g., 9G4.IgG1fa.P238K), 9G4 (SEQ ID NO: 51, amino acids 20-135, and SEQ ID NO: 104), 9C8 (SEQ ID NO: 55 and SEQ ID NO: 104), 2H2 (SEQ ID NO: 58 and SEQ ID NO: 104), 2E5 (SEQ ID NO: 63 and SEQ ID NO: 104), 24E5 (SEQ ID NO: 67 and SEQ ID NO: 104), 21D9 (SEQ ID NO: 71 and SEQ ID NO: 104), 21D9. b (SEQ ID NO: 74 and SEQ ID NO: 104), 21D9. c (SEQ ID NO: 75 and SEQ ID NO: 104), 21D9. d (SEQ ID NO: 78 and SEQ ID NO: 104), 21D9. e (SEQ ID NO: 80 and SEQ ID NO: 104), 21A5 (SEQ ID NO: 83 and SEQ ID NO: 104), 21A5. a (sequences 87 and 104), 10F10 (sequences 91 and 104), 10F10.1 (sequences 91 and 104), 10F10.3 (sequences 91 and 104), or 10F10.4 (sequences 91 and 104), or f. IgG4 S228P (e.g., 9G4.IgG4 S228P), 9G4 (SEQ ID NO: 51, amino acids 20-135, and SEQ ID NO: 179), 9C8 (SEQ ID NO: 55 and SEQ ID NO: 179), 2H2 (SEQ ID NO: 58 and SEQ ID NO: 179), 2E5 (SEQ ID NO: 63 and SEQ ID NO: 179), 24E5 (SEQ ID NO: 67 and SEQ ID NO: 179), 21D9 (SEQ ID NO: 71 and SEQ ID NO: 179), 21D9. b (SEQ ID NO: 74 and SEQ ID NO: 179), 21D9. c (SEQ ID NO: 75 and SEQ ID NO: 179), 21D9. d (SEQ ID NO: 78 and SEQ ID NO: 179), 21D9. e (SEQ ID NO: 80 and SEQ ID NO: 179), 21A5 (SEQ ID NO: 83 and SEQ ID NO: 179), 21A5. a (sequences 87 and 179), 10F10 (sequences 91 and 179), 10F10.1 (sequences 91 and 179), 10F10.3 (sequences 91 and 179), or 10F10.4 (sequences 91 and 179) The isolated antibody according to claim 23, comprising the HC amino acid sequence.

25. The isolated antibody according to claim 23, wherein the HC of the antibody lacks a C-terminal lysine residue.

26. The isolated antibody according to claim 23, wherein the HC of the antibody contains one of the heavy chain constant region amino acid sequences of SEQ ID NOs: 98, 100, 102, 103, 104, or 179.

27. An isolated antibody according to any one of claims 1 to 26, comprising LCs of anti-ILT4 antibodies 9G4, 9C8, 2H2, 2E5, 24E5, 21D9, 21D9, b, 21D9, c, 21D9, d, 21D9, e, 21A5, 21A5, a, 10F10, 10F10.1, 10F10.3, or 10F10.

4.

28. The isolated antibody according to claim 27, wherein the light chain constant region is the human kappa light chain constant region.

29. The isolated antibody according to claim 27, wherein LC comprises the sequence 9G4 (SEQ ID NO: 1), 9C8 (SEQ ID NO: 3), 2H2 (SEQ ID NO: 5), 2E5 (SEQ ID NO: 7), 24E5 (SEQ ID NO: 9), 21D9 (SEQ ID NO: 11), 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5 (SEQ ID NO: 14), 21A5.a (SEQ ID NO: 16), 10F10 (SEQ ID NO: 18), 10F10.1 (SEQ ID NO: 20), 10F10.3 (SEQ ID NO: 21), or 10F10.4 (SEQ ID NO: 116).

30. Anti-ILT4 antibodies 9G4, 9C8, 2H2, 2E5, 24E5, 21D9, 21D9. b, 21D9. c, 21D9. d, 21D9. e, 21A5, 21A5. an isolated antibody according to any one of claims 1 to 29, comprising HC and LC of 10F10, 10F10.1, 10F10.3, or 10F10.4, wherein the constant region of the HC is IgG1 (e.g., 9G4.IgG1, etc.), IgG1.3 (e.g., 9G4.IgG1.3, etc.), IgG1.1f (e.g., 9G4.IgG1.1f, etc.), IgG4 (e.g., 9G4.IgG4, etc.), or IgG4 S228P (EU numbering) (e.g., 9G4.IgG4_S228P).

31. a. A heavy chain (HC) containing the amino acid sequence of the 9G4 heavy chain ((i) SEQ ID NO: 2, or (ii) SEQ ID NO: 51, amino acids 20-135, and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain (LC) containing the 9G4 light chain amino acid sequence (SEQ ID NO: 1); b. A heavy chain containing the amino acid sequence of the 9C8 heavy chain ((i) SEQ ID NO: 4, or (ii) SEQ ID NO: 55, and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the 9C8 light chain (SEQ ID NO: 3); c. A heavy chain containing the amino acid sequence of the 2H2 heavy chain ((i) SEQ ID NO: 6, or (ii) SEQ ID NO: 58, and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the 2H2 light chain (SEQ ID NO: 5); d. A heavy chain containing the amino acid sequence of the 2E5 heavy chain ((i) SEQ ID NO: 8, or (ii) SEQ ID NO: 63, and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the 2E5 light chain (SEQ ID NO: 7); e. A heavy chain containing the amino acid sequence of the 24E5 heavy chain ((i) SEQ ID NO: 10, or (ii) SEQ ID NO: 67, and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the 24E5 light chain (SEQ ID NO: 9); f. A heavy chain containing the amino acid sequence of the 21D9 heavy chain ((i) SEQ ID NO: 12 or 113, or (ii) SEQ ID NO: 71, and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the 21D9 light chain (SEQ ID NO: 11); g. A heavy chain containing the amino acid sequence of the heavy chain of 21D9.b (SEQ ID NO: 74 and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the light chain of 21D9.b (SEQ ID NO: 11); h. A heavy chain containing the amino acid sequence of the heavy chain of 21D9.c (SEQ ID NO: 75 and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the light chain of 21D9.c (SEQ ID NO: 11); i. A heavy chain containing the amino acid sequence of the heavy chain of 21D9.d (SEQ ID NO: 78 and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the light chain of 21D9.d (SEQ ID NO: 11); j. A heavy chain containing the amino acid sequence of the heavy chain of 21D9.e ((i) SEQ ID NOs: 13, 176, 177, or 178; or (ii) SEQ ID NOs: 80 and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the light chain of 21D9.e (SEQ ID NO: 11); k. A heavy chain containing the amino acid sequence of the 21A5 heavy chain ((i) SEQ ID NO: 15 or (ii) SEQ ID NO: 83 and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the 21A5 light chain (SEQ ID NO: 14); l. A heavy chain containing the amino acid sequence of the heavy chain of 21A5.a ((i) SEQ ID NO: 17 or (ii) SEQ ID NO: 87 and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the light chain of 21A5.a (SEQ ID NO: 16); m. A heavy chain containing the amino acid sequence of the 10F10 heavy chain ((i) SEQ ID NO: 19 or (ii) SEQ ID NO: 91 and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the 10F10 light chain (SEQ ID NO: 18); n. A heavy chain containing the amino acid sequence of the heavy chain of 10F10.1 ((i) SEQ ID NO: 19 or (ii) SEQ ID NO: 91 and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the light chain of 10F10.1 (SEQ ID NO: 20); o. A heavy chain containing the amino acid sequence of the heavy chain of 10F10.3 ((i) SEQ ID NO: 19 or (ii) SEQ ID NO: 91 and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179), and a light chain containing the amino acid sequence of the light chain of 10F10.3 (SEQ ID NO: 21); or p. A heavy chain containing the amino acid sequence of the heavy chain of 10F10.4 ((i) SEQ ID NO: 19 or (ii) SEQ ID NO: 91 and one of SEQ ID NOs: 98, 100, 102, 103, 104, or 179) and a light chain containing the amino acid sequence of the light chain of 10F10.4 (SEQ ID NO: 116) The isolated antibody according to claim 30, comprising:

32. An isolated antibody that specifically binds to human ILT4, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 13 and a light chain containing the light chain amino acid sequence of SEQ ID NO:

11.

33. An isolated antibody that specifically binds to human ILT4, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 176 and a light chain containing the light chain amino acid sequence of SEQ ID NO:

11.

34. An isolated antibody that specifically binds to human ILT4, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 177 and a light chain containing the light chain amino acid sequence of SEQ ID NO:

11.

35. An isolated antibody that specifically binds to human ILT4, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 178 and a light chain containing the light chain amino acid sequence of SEQ ID NO:

11.

36. An isolated antibody according to any one of claims 32 to 35, having one or more characteristics of the antibody described in claim 1.

37. An isolated antibody according to any one of claims 1 to 36, wherein the antibody heavy chain is a full-length antibody and the antibody heavy chain contains or does not contain a C-terminal lysine residue.

38. An isolated antibody according to any one of claims 1 to 14, which is an antibody fragment.

39. An isolated antibody according to any one of claims 1 to 38, which is a multimeric (e.g., dimeric or trimer) antibody.

40. An isolated antibody according to any one of claims 1 to 39, which is linked to another molecule (for example, by covalent bond).

41. The isolated antibody according to claim 40, wherein another molecule is used as a label.

42. The isolated antibody according to claim 40, wherein the other molecule is a peptide.

43. The isolated antibody according to claim 40, which is an antibody-drug conjugate (ADC).

44. An isolated nucleic acid encoding an antibody according to any one of claims 1 to 43.

45. An isolated nucleic acid encoding the heavy chain and / or light chain of an antibody according to any one of claims 1 to 43.

46. A set of at least two isolated nucleic acids encoding the heavy chain and light chain of an antibody according to any one of claims 1 to 43.

47. A composition comprising a nucleic acid encoding the heavy chain of an antibody according to any one of claims 1 to 43, and a nucleic acid encoding the light chain of an antibody according to any one of claims 1 to 43.

48. A cell comprising an isolated nucleic acid according to any one of claims 44 to 46 or the composition according to claim 47, wherein the nucleic acid comprises a nucleic acid listed in a sequence listing.

49. A method for preparing an antibody, comprising culturing the cells described in claim 48 under conditions in which the antibody is expressed.

50. A composition comprising an isolated antibody, nucleic acid, composition or cell according to any one of claims 1 to 49, and a pharmaceutically acceptable carrier.

51. The composition according to claim 50, comprising a second therapeutic agent.

52. The composition according to claim 51, wherein the second therapeutic agent is an immunostimulant.

53. The composition according to claim 52, wherein the immunostimulant is an antagonist of an immunosuppressive molecule, for example, PD-1 / PD-L1, CTLA-4 and LAG-3, or an agonist of an immunostimulant molecule, for example, GITR and OX40.

54. A method for treating a subject having cancer, comprising administering to the subject a therapeutically effective amount of a composition according to any one of claims 50 to 53 or an isolated antibody according to any one of claims 1 to 43, which stimulates an immune response and / or is an ILT-4 antagonist.

55. The method according to claim 54, further comprising administering a second therapy.

56. The method according to claim 55, wherein the second therapy is radiotherapy, surgery, or administration of a second drug.

57. The method according to claim 55, wherein the second therapy is a second drug, and the second drug is an immunostimulant.

58. The method according to claim 57, wherein the immunostimulant is an antagonist of an immunosuppressive molecule, for example, PD-1 / PD-L1, CTLA-4 and LAG-3, or an agonist of an immunostimulant molecule, for example, GITR and OX40.

59. A method for treating an infectious disease (e.g., a viral disease) in a subject, comprising administering to the subject a therapeutically effective amount of a composition according to any one of claims 50 to 53 or an isolated antibody according to any one of claims 1 to 43, which stimulates an immune response and / or is an ILT4 antagonist.

60. A method for detecting ILT4 in a sample, comprising contacting the sample with an ILT4 antibody according to any one of claims 1 to 43.

61. The following features: a. For example, 10 －８ M or less, or 10 －９ K below M Ｄ And it specifically binds to hILT4 (for example, including the amino acid sequences of SEQ ID NOs. 108, 109, 111, 112, or 119); b. For example, stimulating the differentiation or activation of monocytes into macrophages, as shown in the assay described in the Examples, e.g., stimulating the differentiation of monocytes into inflammatory macrophages; c. Having a coupling profile as shown in Figure 27; and d. The following regions of hILT4: (i) 70 ITRIRPEL 77 (Sequence ID 120) and / or 78 VKNGQFHIPSITWEHTGRYGCQY 100 (Sequence ID 121); (ii) 70 ITRIRPELVKNGQFHIPSITWEHTGRYGCQY 100 (Sequence ID 122); or (iii) 154 ILCKEGEEEHPQCLNSQPHARGSSRAIF 181 (Sequence ID 123) and / or 425 SSPPPTGPIS 434 (Sequence ID 124) and other molecules that bind to Ig-like domains 1, 2, or 1 and 2 of hILT4, Furthermore, the amino acid number of hILT4 is that of immature hILT4 (i.e., ILT4 containing its native signal sequence), and it does not significantly bind to other regions of the extracellular domain of ILT4, such as the region or residue located at the N-terminus relative to amino acid I70. An isolated antibody according to any one of claims 1 to 43, having the characteristics of the isolated antibody.

62. The following features: a. For example, 10 －８ M or less, or 10 －９ K below M Ｄ Specific binding to hILT4 (e.g., including the amino acid sequences of SEQ ID NOs. 108, 109, 111, 112, or 119); b. Lack of specific binding to hILT2, hILT3, and / or hILT5; c. Lack of specific binding to one or more members of the LILRA and / or LILRB family; d. For example, stimulating T cell activation in a mixed lymphocyte reaction (MLR) assay, as measured by increased T cell proliferation or IFN-gamma secretion, as shown in the assay described in the Examples; e. For example, stimulating the differentiation or activation of monocytes into macrophages, as shown in the assay described in the Examples, e.g., stimulating the differentiation of monocytes into inflammatory macrophages; f. Inhibiting the binding of hILT4 to HLA-A and HLA-B; g. Having a coupling profile as shown in Figure 27; h. The following areas of hILT4: (i) 70 ITRIRPEL 77 (Sequence ID 120) and / or 78 VKNGQFHIPSITWEHTGRYGCQY 100 (Sequence ID 121); (ii) 70 ITRIRPELVKNGQFHIPSITWEHTGRYGCQY 100 (Sequence ID 122), etc., binds to Ig-like domains 1, 2, or 1 and 2 of hILT4, The amino acid number of hILT4 is that of immature hILT4 (i.e., ILT4 with its signal sequence), and it does not significantly bind to other regions of the extracellular domain of ILT4, such as the region or residue located at the N-terminus relative to amino acid I70; i. Promoting inflammatory polarization of macrophages to M1 macrophages; j. Not induce (or trigger) basophil activation; and k. After incubation at 25°C for 3 months, it contains less than 5% high and low molecular weight species, and / or after incubation at 40°C for 3 months, it contains less than 10% high and low molecular weight species. An isolated antibody according to any one of claims 1 to 43, having the characteristics of the isolated antibody.

63. The following features: a. For example, promoting the expression of CD83 and CD86 on human monocyte-derived immature dendritic cells (Mo-iDCs), as shown in the assay described in the examples; b. For example, enhancing IFN-γ secretion upon antigen stimulation in a cytomegalovirus (CMV) lysate assay, as shown in the assay described in the Examples; c. For example, enhancing IFN-γ and TNF-α secretion by CD4+ and CD8+ T cells in an allogeneic mixed lymphocyte response (MLR) assay upon CD3 stimulation, as shown in the assay described in the Examples; d. Inhibiting the binding of HLA-A and / or HLA-B to ILT4; e. For example, as shown in the HDX assay described in the Examples, when determined by deuterium exchange (HDX), (i) 70 ITRIRPEL 77 (Sequence ID 120) and / or 78 VKNGQFHIPSITWEHTGRYGCQY 100 (Sequence ID 121); (ii) 70 ITRIRPELVKNGQFHIPSITWEHTGRYGCQY 100 (Sequence ID 122); or (iii) 154 ILCKEGEEEHPQCLNSQPHARGSSRAIF 181 (Sequence ID 123) and / or 425 SSPPPTGPIS 434 Binding to Ig-like domains 1, 2, or 1 and 2 of hILT4, such as the region containing (SEQ ID NO: 124); f. For example, as described in the examples, one or more (or all) of the amino acid residues Lys43, Ile49, Thr50 and Arg51 of mature hILT4, as determined by carbene footprinting, interact with the amino acid residues Gly117, Val119, Try120, Leu134, Lys136, Gln149, Pro150, Ile159, S Interacting with one or more (or all) of the following amino acid residues of mature hILT4: er161, Val162, Gly163, Pro164, Pro167, His173, Try178, Pro183, and Tyr184; or interacting with one or more (or all) of the following amino acid residues of mature hILT4: Glu42, Lys43, Gly76, Cys77, Leu88, Pro91, Pro183, and Tyr184; g. Competition with the antibodies described herein for binding to hILT4; and h. For example, as shown in the binding assay described in the Examples, it specifically binds to cynomolgus monkey ILT4 containing SEQ ID NO:

118. An isolated antibody according to any one of claims 1 to 43, having the characteristics of the isolated antibody.

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