Antibodies that bind to ILT4

Abstract

This application relates to antibodies that specifically bind to immunoglobulin-like transcript 4 (ILT4), which is also known as LILRB2, LIR2, MIR10, and CD85d; as well as corresponding nucleic acids, host cells, compositions, and uses. In some embodiments, the antibodies specifically bind to human ILT4, but do not significantly bind to ILT2, ILT3, or ILT5 or other members of the LILRA or LILRB family.

Description

Technical Field

[0001] This application relates to antibodies that specifically bind to and inhibit ILT4, and their use in cancer treatment. Background Art

[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 family of structurally related receptors that also includes the proteins LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, ILT2, ILT3, ILT5, and LIR8.

[0003] The extracellular domains of the proteins in this family contain several immunoglobulin-like repeats, while their cytoplasmic tail regions contain several tyrosine-based inhibitory motifs (ITIMs) that recruit tyrosine phosphatases. Summary of the Invention

[0004] Certain embodiments of the invention are outlined in the claims at the end of the invention. For example, the invention includes the following embodiments as well as other embodiments described in other parts of the text herein and in the figures and sequences.

[0005] Embodiment 1: An isolated antibody that specifically binds to human ILT4 (hILT4 or ILT4), the antibody comprising a heavy chain and a light chain, and further comprising one or more of the following characteristics:

[0006] - For example, with a K -8 d of 10 -9 M or lower, or 10 D M or lower, specifically binds to hILT4 (e.g., comprising the amino acid sequence of SEQ ID NO: 108, 109, 111, 112, or 119);

[0007] - Does not specifically bind to hILT2, hILT3, and / or hILT5;

[0008] - Does not specifically bind to one or more members of the LILRA and / or LILRB families;

[0009] - For example, stimulates T cell activation in a mixed lymphocyte reaction (MLR) assay, as measured by increased T cell proliferation or IFN-γ secretion, such as shown in the assays described in the examples;

[0010] - Stimulate the differentiation or activation of monocytes into macrophages, such as stimulating the differentiation of monocytes into pro-inflammatory macrophages, as shown, for example, in the assays described in the examples;

[0011] - Promote the expression of CD83 and CD86 on human monocyte-derived immature dendritic cells (Mo-iDC), as shown, for example, in the assays described in the examples;

[0012] - Enhance IFN-γ secretion after antigen stimulation in a cytomegalovirus (CMV) lysate assay, as shown, for example, in the assays described in the examples;

[0013] - Enhance IFN-γ and TNF-α secretion by CD4+ and CD8+ T cells after CD3 stimulation in an allogeneic mixed lymphocyte reaction (MLR) assay, as shown, for example, in the assays described in the examples;

[0014] - Inhibit the binding of HLA-A and / or HLA-B to ILT4;

[0015] - Bind to 70 ITRIRPELVKNGQFHIPSITWEHTGRYGCQY 100 (SEQ ID NO:122), as determined by hydrogen-deuterium exchange (HDX), as shown, for example, in the HDX assays described in the examples;

[0016] - Compete with the antibodies described herein for binding to hILT4;

[0017] - Specifically bind to cynomolgus macaque ILT4 comprising SEQ ID NO:118, as shown, for example, in the binding assays described in the examples;

[0018] - 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);

[0019] - Have the HLA-A and HLA-B binding profiles shown in Figure 28;

[0020] - Promote the pro-inflammatory polarization of macrophages towards M1 macrophages;

[0021] - Have the binding profiles shown in Figure 27;

[0022] - Do not induce (or trigger) basophil activation;

[0023] - Bind to the following regions of hILT4: (i) 70ITRIRPEL 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), and if it binds to (i) or (ii), optionally does not significantly bind to other regions of the extracellular domain of ILT4, such as the region or residues N-terminal to amino acid 170, where the amino acid numbering of hILT4 is that of immature hILT4 (i.e., ILT4 containing its native signal sequence); and

[0024] - interacts with one or more (or all) amino acid residues Lys43, Ile49, Thr50 and Arg51 of mature hILT4, or 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 with one or more (or all) amino acid residues Glu42, Lys43, Gly76, Cys77, Leu88, Pro91, Pro183 and Tyr184 of mature hILT4, as determined, for example, by carbene footprinting as described in the Examples.

[0025] Embodiment 2: The isolated antibody as described in Embodiment 1, which comprises a heavy chain and a light chain, wherein the heavy chain comprises the VH CDR1, CDR2 and CDR3 of the following anti-ILT4 antibodies: 9G4 (SEQ ID NO: 125-127), 9C8 (SEQ ID NO: 131-133), 2H2 (SEQ ID NO: 137-139), 2E5 (SEQ ID NO: 143-145), 24E5 (SEQ ID NO: 149-151), 21D9 (SEQ ID NO: 155-157), 21A5 (SEQ ID NO: 161-163) or 10F10 (SEQ ID NO: 167-169).

[0026] Embodiment 3: The isolated antibody as described in Embodiment 1 or 2, which comprises a heavy chain and a light chain, wherein the light chain comprises the VL CDR1, CDR2 and CDR3 of the following anti-ILT4 antibodies: 9G4 (SEQ ID NO: 128-130), 9C8 (SEQ ID NO: 134-136), 2H2 (SEQ ID NO: 140-142), 2E5 (SEQ ID NO: 146-148), 24E5 (SEQ ID NO: 152-154), 21D9 (SEQ ID NO: 158-160), 21A5 (SEQ ID NO: 164-166) or 10F10 (SEQ ID NO: 170-172).

[0027] Embodiment 4: The isolated antibody as described in Embodiment 2, wherein the heavy chain comprises the VH CDR1, CDR2 and CDR3 and VL CDR1, CDR2 and CDR3 of the following anti-ILT4 antibodies: 9G4 (SEQ ID NO: 143-148), 24E5 (SEQ ID NO: 149-154), 21D9 (SEQ ID NO: 155-160), 21A5 (SEQ ID NO: 161-166) or 10F10 (SEQ ID NO: 167-172).

[0028] Embodiment 5: The isolated antibody as described in any one of Embodiments 1-4, which comprises:

[0029] - a VH comprising the amino acid sequences (SEQ ID NO: 125-127) of the VH CDR1, CDR2 and CDR3 of 9G4, and a VL comprising the amino acid sequences (SEQ ID NO: 128-130) of the VL CDR1, CDR2 and CDR3 of 9G4;

[0030] - VH containing the amino acid sequences (SEQ ID NOs: 131-133) of 9C8's VH CDR1, CDR2, and CDR3, and VL containing the amino acid sequences (SEQ ID NOs: 134-136) of 9C8's VL CDR1, CDR2, and CDR3;

[0031] - VH containing the amino acid sequences (SEQ ID NOs: 137-139) of 2H2's VH CDR1, CDR2, and CDR3, and VL containing 2H2's VL CDR1, CDR2, and CDR3 (SEQ ID NOs: 140-142);

[0032] - VH containing the amino acid sequences (SEQ ID NOs: 143-145) of 2E5's VH CDR1, CDR2, and CDR3, and VL containing the amino acid sequences (SEQ ID NOs: 146-148) of 2E5's VL CDR1, CDR2, and CDR3;

[0033] - VH containing the amino acid sequences (SEQ ID NOs: 149-151) of 24E5's VH CDR1, CDR2, and CDR3, and VL containing the amino acid sequences (SEQ ID NOs: 152-154) of 24E5's VL CDR1, CDR2, and CDR3;

[0034] - VH containing the amino acid sequences (SEQ ID NOs: 155-157) of 21D9's VH CDR1, CDR2, and CDR3, and VL containing the amino acid sequences (SEQ ID NOs: 158-160) of 21D9's VL CDR1, CDR2, and CDR3;

[0035] - VH containing the amino acid sequences (SEQ ID NOs: 155-157) of 21D9.b's VH CDR1, CDR2, and CDR3, and VL containing the amino acid sequences (SEQ ID NOs: 158-160) of 21D9.b's VL CDR1, CDR2, and CDR3;

[0036] - VH containing the amino acid sequences (SEQ ID NOs: 155-157) of 21D9.c's VH CDR1, CDR2, and CDR3, and VL containing the amino acid sequences (SEQ ID NOs: 158-160) of 21D9.c's VL CDR1, CDR2, and CDR3;

[0037] - A VH containing the amino acid sequences (SEQ ID NO: 155 - 157) of VH CDR1, CDR2, and CDR3 of 21D9.d, and a VL containing the amino acid sequences (SEQ ID NO: 158 - 160) of VL CDR1, CDR2, and CDR3 of 21D9.d;

[0038] - A VH containing the amino acid sequences (SEQ ID NO: 155 - 157) of VH CDR1, CDR2, and CDR3 of 21D9.e, and a VL containing the amino acid sequences (SEQ ID NO: 158 - 160) of VL CDR1, CDR2, and CDR3 of 21D9.e;

[0039] - A VH containing the amino acid sequences (SEQ ID NO: 161 - 163) of VH CDR1, CDR2, and CDR3 of 21A5, and a VL containing the amino acid sequences (SEQ ID NO: 164 - 166) of VL CDR1, CDR2, and CDR3 of 21A5;

[0040] - A VH containing the amino acid sequences (SEQ ID NO: 161 - 163) of VH CDR1, CDR2, and CDR3 of 21A5.a, and a VL containing the amino acid sequences (SEQ ID NO: 164 - 166) of VL CDR1, CDR2, and CDR3 of 21A5.a;

[0041] - A VH containing the amino acid sequences (SEQ ID NO: 167 - 169) of VH CDR1, CDR2, and CDR3 of 10F10, and a VL containing the amino acid sequences (SEQ ID NO: 170 - 172) of VL CDR1, CDR2, and CDR3 of 10F10;

[0042] - A VH containing the amino acid sequences (SEQ ID NO: 167 - 169) of VH CDR1, CDR2, and CDR3 of 10F10.1, and a VL containing the amino acid sequences (SEQ ID NO: 170 - 172) of VL CDR1, CDR2, and CDR3 of 10F10.1;

[0043] - A VH containing the amino acid sequences (SEQ ID NO: 167 - 169) of VH CDR1, CDR2, and CDR3 of 10F10.3, and a VL containing the amino acid sequences (SEQ ID NO: 170 - 172) of VL CDR1, CDR2, and CDR3 of 10F10.3; or

[0044] - A VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of 10F10.4 (SEQ ID NO: 167-169), and a VL comprising the amino acid sequences of VL CDR1, CDR2 and CDR3 of 10F10.4 (SEQ ID NO: 170-172).

[0045] Embodiment 6: The isolated antibody according to any one of Embodiments 1-5, wherein the heavy chain of the antibody 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 the VH of any of the following: 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).

[0046] Embodiment 7: An isolated antibody as described in any one of Embodiments 1-6, 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 any of the following: 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).

[0047] Embodiment 8: An isolated antibody as described in any one of Embodiments 1-7, wherein the antibody heavy chain comprises a VH having an amino acid sequence that contains 1, 2, 3, 4, or 5 amino acid substitutions, conservative substitutions, or back substitutions compared to the amino acid sequence of the VH of any of the following: 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).

[0048] Embodiment 9: The isolated antibody according to any one of Embodiments 1-8, wherein the antibody light chain comprises a VL having an amino acid sequence comprising 1, 2, 3, 4 or 5 amino acid substitutions, conservative substitutions or revertant substitutions compared to the amino acid sequence of the VL of any of the following: 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).

[0049] Embodiment 10: The isolated antibody according to any one of Embodiments 1-9, wherein the heavy chain comprises a VH of the following anti-ILT4 antibodies: 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).

[0050] Embodiment 11: The isolated antibody according to any one of Embodiments 1-10, wherein the light chain comprises the VL of the following anti-ILT4 antibodies: 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).

[0051] Embodiment 12: The isolated antibody according to any one of Embodiments 1-11, which comprises the VH and VL of the following 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.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).

[0052] Embodiment 13: The isolated antibody according to any one of Embodiments 1-11, comprising:

[0053] - a VH comprising the VH CDRs (SEQ ID NOs: 125-127) of the VH of 9G4, and a VL comprising the VL CDRs (SEQ ID NOs: 128-130) of the VL of 9G4, and VH and VL amino acid sequences having 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% identity to the VH and VL of 9G4 (SEQ ID NO: 51, amino acids 20-135 and SEQ ID NO: 50, amino acids 19-125);

[0054] - a VH comprising the VH CDRs (SEQ ID NOs: 131-133) of the VH of 9C8, and a VL comprising the VL CDRs (SEQ ID NOs: 134-136) of the VL of 9C8, and VH and VL amino acid sequences having 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% identity to the VH and VL of 9C8 (SEQ ID NO: 55 and SEQ ID NO: 54);

[0055] - a VH comprising the VH CDRs (SEQ ID NOs: 137-139) of the VH of 2H2, and a VL comprising the VL CDRs (SEQ ID NOs: 140-142) of the VL of 2H2, and VH and VL amino acid sequences having 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% identity to the VH and VL of 2H2 (SEQ ID NO: 58 and SEQ ID NO: 59);

[0056] - a VH comprising the VH CDRs (SEQ ID NOs: 143-145) of the VH of 2E5, and a VL comprising the VL CDRs (SEQ ID NOs: 146-148) of the VL of 2E5, and VH and VL amino acid sequences having 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% identity to the VH and VL of 2E5 (SEQ ID NO: 63 and SEQ ID NO: 62);

[0057] - A VH containing the VH CDRs (SEQ ID NOs: 149 - 151) of VH of 24E5, and a VL containing the VL CDRs (SEQ ID NOs: 152 - 154) of VL of 24E5, and VH and VL amino acid sequences having 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% identity to the VH and VL (SEQ ID NOs: 67 and SEQ ID NO: 66) of 24E5;

[0058] - A VH containing the VH CDRs (SEQ ID NOs: 155 - 157) of VH of 21D9, and a VL containing the VL CDRs (SEQ ID NOs: 158 - 160) of VL of 21D9, and VH and VL amino acid sequences having 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% identity to the VH and VL (SEQ ID NOs: 71 and SEQ ID NO: 70) of 21D9;

[0059] - A VH containing the VH CDRs (SEQ ID NOs: 155 - 157) of VH of 21D9.b, and a VL containing the VL CDRs (SEQ ID NOs: 158 - 160) of VL of 21D9.b, and VH and VL amino acid sequences having 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% identity to the VH and VL (SEQ ID NOs: 74 and SEQ ID NO: 70) of 21D9.b;

[0060] - A VH containing the VH CDRs (SEQ ID NOs: 155 - 157) of VH of 21D9.c, and a VL containing the VL CDRs (SEQ ID NOs: 158 - 160) of VL of 21D9.c, and VH and VL amino acid sequences having 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% identity to the VH and VL (SEQ ID NOs: 75 and SEQ ID NO: 70) of 21D9.c;

[0061] - A VH comprising the VH CDRs (SEQ ID NOs: 155 - 157) of VH of 21D9.d, and a VL comprising the VL CDRs (SEQ ID NOs: 158 - 160) of VL of 21D9.d, and VH and VL amino acid sequences having 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% identity to the VH and VL (SEQ ID NOs: 78 and SEQ ID NO: 70) of 21D9.d;

[0062] - A VH comprising the VH CDRs (SEQ ID NOs: 155 - 157) of VH of 21D9.e, and a VL comprising the VL CDRs (SEQ ID NOs: 158 - 160) of VL of 21D9.e, and VH and VL amino acid sequences having 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% identity to the VH and VL (SEQ ID NOs: 80 and SEQ ID NO: 70) of 21D9.e;

[0063] - A VH comprising the VH CDRs (SEQ ID NOs: 161 - 163) of VH of 21A5, and a VL comprising the VL CDRs (SEQ ID NOs: 164 - 166) of VL of 21A5, and VH and VL amino acid sequences having 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% identity to the VH and VL (SEQ ID NOs: 83 and SEQ ID NO: 82) of 21A5;

[0064] - A VH comprising the VH CDRs (SEQ ID NOs: 161 - 163) of VH of 21A5.a, and a VL comprising the VL CDRs (SEQ ID NOs: 164 - 166) of VL of 21A5.a, and VH and VL amino acid sequences having 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% identity to the VH and VL (SEQ ID NOs: 87 and SEQ ID NO: 86) of 21A5.a;

[0065] - A VH comprising the VH CDRs (SEQ ID NOs: 167 - 169) of 10F10's VH, and a VL comprising the VL CDRs (SEQ ID NOs: 170 - 172) of 10F10's VL, and VH and VL amino acid sequences having 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% identity to the VH and VL of 10F10 (SEQ ID NOs: 91 and 90);

[0066] - A VH comprising the VH CDRs (SEQ ID NOs: 167 - 169) of 10F10.1's VH, and a VL comprising the VL CDRs (SEQ ID NOs: 170 - 172) of 10F10.1's VL, and VH and VL amino acid sequences having 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% identity to the VH and VL of 10F10.1 (SEQ ID NOs: 91 and 94);

[0067] - A VH comprising the VH CDRs (SEQ ID NOs: 167 - 169) of 10F10.3's VH, and a VL comprising the VL CDRs (SEQ ID NOs: 170 - 172) of 10F10.3's VL, and VH and VL amino acid sequences having 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% identity to the VH and VL of 10F10.3 (SEQ ID NOs: 91 and 96); or

[0068] - A VH comprising the VH CDRs (SEQ ID NOs: 167 - 169) of 10F10.4's VH, and a VL comprising the VL CDRs (SEQ ID NOs: 170 - 172) of 10F10.4's VL, and VH and VL amino acid sequences having 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% identity to the VH and VL of 10F10.4 (SEQ ID NOs: 91 and 114).

[0069] Embodiment 14: The isolated antibody according to any one of Embodiments 1 - 13, comprising:

[0070] - A VH comprising the amino acid sequence of the VH of 9G4 (residues 20 - 135 of SEQ ID NO:51) and a VL comprising the amino acid sequence of the VL of 9G4 (residues 19 - 125 of SEQ ID NO:50);

[0071] - A VH comprising the amino acid sequence of the VH of 9C8 and a VL comprising the amino acid sequence of the VL of 9C8 (SEQ ID NO:55 and SEQ ID NO:54);

[0072] - A VH comprising the amino acid sequence of the VH of 2H2 and a VL comprising the amino acid sequence of the VL of 2H2 (SEQ ID NO:58 and SEQ ID NO:59);

[0073] - A VH comprising the amino acid sequence of the VH of 2E5 and a VL comprising the amino acid sequence of the VL of 2E5 (SEQ ID NO:63 and SEQ ID NO:62);

[0074] - A VH comprising the amino acid sequence of the VH of 24E5 and a VL comprising the amino acid sequence of the VL of 24E5 (SEQ ID NO:67 and SEQ ID NO:66);

[0075] - A VH comprising the amino acid sequence of the VH of 21D9 and a VL comprising the amino acid sequence of the VL of 21D9 (SEQ ID NO:71 and SEQ ID NO:70);

[0076] - A VH comprising the amino acid sequence of the VH of 21D9.b and a VL comprising the amino acid sequence of the VL of 21D9.b (SEQ ID NO:74 and SEQ ID NO:70);

[0077] - A VH comprising the amino acid sequence of the VH of 21D9.c and a VL comprising the amino acid sequence of the VL of 21D9.c (SEQ ID NO:75 and SEQ ID NO:70);

[0078] - A VH comprising the amino acid sequence of the VH of 21D9.d and a VL comprising the amino acid sequence of the VL of 21D9.d (SEQ ID NO:78 and SEQ ID NO:70);

[0079] - A VH comprising the amino acid sequence of the VH of 21D9.e and a VL comprising the amino acid sequence of the VL of 21D9.e (SEQ ID NO:80 and SEQ ID NO:70);

[0080] - VH comprising the amino acid sequence of VH of 21A5 and VL comprising the amino acid sequence of VL of 21A5 (SEQ ID NO: 83 and SEQ ID NO: 82);

[0081] - VH comprising the amino acid sequence of VH of 21A5.a and VL comprising the amino acid sequence of VL of 21A5.a (SEQ ID NO: 87 and SEQ ID NO: 86);

[0082] - VH comprising the amino acid sequence of VH of 10F10 and VL comprising the amino acid sequence of VL of 10F10 (SEQ ID NO: 91 and SEQ ID NO: 90);

[0083] - VH comprising the amino acid sequence of VH of 10F10.1 and VL comprising the amino acid sequence of VL of 10F10.1 (SEQ ID NO: 91 and SEQ ID NO: 94);

[0084] - VH comprising the amino acid sequence of VH of 10F10.3 and VL comprising the amino acid sequence of VL of 10F10.3 (SEQ ID NO: 91 and SEQ ID NO: 96); or

[0085] - VH comprising the amino acid sequence of VH of 10F10.4 and VL comprising the amino acid sequence of VL of 10F10.4 (SEQ ID NO: 91 and SEQ ID NO: 114).

[0086] Embodiment 15: The isolated antibody according to any one of Embodiments 1-14, which is an IgG antibody.

[0087] Embodiment 16: The isolated antibody according to Embodiment 15, which is an IgG1, IgG2 or IgG4 antibody, wherein the IgG4 optionally comprises the S228P substitution (EU numbering).

[0088] Embodiment 17: The isolated antibody according to any one of Embodiments 1-16, wherein the antibody is an effectorless antibody.

[0089] Embodiment 18: The isolated antibody according to Embodiment 17, wherein the heavy chain constant region comprises 1, 2, 3, 4 or 5 mutations in another wild-type human heavy chain constant region, and these mutations reduce the effector function of the antibody compared to an antibody having the same amino acid sequence but without the 1, 2, 3, 4 or 5 mutations.

[0090] Embodiment 19: The isolated antibody according to any one of Embodiments 1-18, wherein the heavy chain constant region of the antibody comprises an IgG1.3 heavy chain constant region, an IgG1.1 heavy chain constant region, or an IgG1 heavy chain constant region having a P238K (EU numbering) substitution, or an IgG1 heavy chain constant region comprising the amino acid sequence of any one of SEQ ID NO: 98, 100, 102, 103 or 104.

[0091] Embodiment 20: The isolated antibody according to any one of Embodiments 1-16 or 19, wherein the antibody has effector function.

[0092] Embodiment 21: The isolated antibody according to Embodiment 20, wherein the antibody is a defucosylated antibody (such as a defucosylated IgG1 antibody).

[0093] Embodiment 22: The isolated antibody according to Embodiment 20 or 21, wherein the heavy chain constant region comprises 1, 2, 3, 4 or 5 mutations in another wild-type human heavy chain constant region, and these mutations enhance the effector function of the antibody compared to an antibody having the same amino acid sequence but without the 1, 2, 3, 4 or 5 mutations.

[0094] Embodiment 23: The isolated antibody according to any one of Embodiments 1-22, which comprises the HC of anti-ILT4 antibodies 9G4, 9C8, 2H2, 2E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, .....

[0095] Embodiment 24: The isolated antibody according to Embodiment 23, wherein the antibody comprises the following HC amino acid sequence:

[0096] -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),

[0097] -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),

[0098] -IgG1.3 (such as 9G4.IgG1.3, etc.), 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 SEQ ID NO:100), 10F10.3 (SEQ ID NO:91 and SEQ ID NO:100) or 10F10.4 (SEQ ID NO:91 and SEQ ID NO:100);

[0099] -IgG1.1f (such as 9G4.IgG1.1f, etc.), 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 (SEQ ID NO:87 and SEQ ID NO:103), 10F10 (SEQ ID NO:91 and SEQ ID NO:103), 10F10.1 (SEQ ID NO:91 and SEQ ID NO:103), 10F10.3 (SEQ ID NO:91 and SEQ ID NO:103) or 10F10.4 (SEQ ID NO:91 and SEQ ID NO:103),

[0100] -IgG1fa.P238K (such as 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

[0101] -IgG4 S228P (such as 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).

[0102] Embodiment 25: The isolated antibody according to Embodiment 23, wherein the HC of the antibody has no C-terminal lysine residue.

[0103] Embodiment 26: The isolated antibody according to Embodiment 23, wherein the HC of the antibody comprises the amino acid sequence of the heavy chain constant region of any one of SEQ ID NO:98, 100, 102, 103, 104, or 179.

[0104] Embodiment 27: The isolated antibody according to any one of Embodiments 1 - 26, which comprises the LC 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.

[0105] Embodiment 28: The isolated antibody according to Embodiment 27, wherein the light chain constant region is a human κ light chain constant region.

[0106] Embodiment 29: The isolated antibody as described in Embodiment 27, wherein the LC comprises the following sequences: 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).

[0107] Embodiment 30: The isolated antibody as described in any one of Embodiments 1-29, which comprises the HC and LC of an 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 (such as 9G4.IgG1, etc.), IgG1.3 (such as 9G4.IgG1.3, etc.), IgG1.1f (such as 9G4.IgG1.1f, etc.), IgG4 (such as 9G4.IgG4, etc.), or IgG4 S228P (EU number) (such as 9G4.IgG4_S228P).

[0108] Embodiment 31: The isolated antibody as described in Embodiment 30, which comprises:

[0109] - A heavy chain (HC) comprising the amino acid sequence of the heavy chain of 9G4 ((i) SEQ ID NO:2, or (ii) SEQ ID NO:51, amino acids 20-135, and one of SEQ ID NO:98, 100, 102, 103, 104, or 179), and a light chain (LC) comprising the amino acid sequence of the light chain of 9G4 (SEQ ID NO:1);

[0110] - A heavy chain comprising the amino acid sequence of the heavy chain of 9C8 ((i) SEQ ID NO:4 or (ii) SEQ ID NO:55 and one of SEQ ID NO:98, 100, 102, 103, 104, or 179), and a light chain comprising the amino acid sequence of the light chain of 9C8 (SEQ ID NO:3);

[0111] - A heavy chain comprising the amino acid sequence of the heavy chain containing 2H2 ((i) SEQ ID NO: 6 or (ii) SEQ ID NO: 58 and one of SEQ ID NO: 98, 100, 102, 103, 104 or 179), and a light chain comprising the amino acid sequence of the light chain containing 2H2 (SEQ ID NO: 5);

[0112] - A heavy chain comprising the amino acid sequence of the heavy chain containing 2E5 ((i) SEQ ID NO: 8 or (ii) SEQ ID NO: 63 and one of SEQ ID NO: 98, 100, 102, 103, 104 or 179), and a light chain comprising the amino acid sequence of the light chain containing 2E5 (SEQ ID NO: 7);

[0113] - A heavy chain comprising the amino acid sequence of the heavy chain containing 24E5 ((i) SEQ ID NO: 10 or (ii) SEQ ID NO: 67 and one of SEQ ID NO: 98, 100, 102, 103, 104 or 179), and a light chain comprising the amino acid sequence of the light chain containing 24E5 (SEQ ID NO: 9);

[0114] - A heavy chain comprising the amino acid sequence of the heavy chain containing 21D9 ((i) SEQ ID NO: 12 or 113, or (ii) SEQ ID NO: 71 and one of SEQ ID NO: 98, 100, 102, 103, 104 or 179), and a light chain comprising the amino acid sequence of the light chain containing 21D9 (SEQ ID NO: 11);

[0115] - A heavy chain comprising the amino acid sequence of the heavy chain containing 21D9.b (one of SEQ ID NO: 74 and SEQ ID NO: 98, 100, 102, 103, 104 or 179), and a light chain comprising the amino acid sequence of the light chain containing 21D9.b (SEQ ID NO: 11);

[0116] - A heavy chain comprising the amino acid sequence of the heavy chain containing 21D9.c (one of SEQ ID NO: 75 and SEQ ID NO: 98, 100, 102, 103, 104 or 179), and a light chain comprising the amino acid sequence of the light chain containing 21D9.c (SEQ ID NO: 11);

[0117] - A heavy chain comprising the amino acid sequence of the heavy chain containing 21D9.d (one of SEQ ID NO: 78 and SEQ ID NO: 98, 100, 102, 103, 104 or 179), and a light chain comprising the amino acid sequence of the light chain containing 21D9.d (SEQ ID NO: 11);

[0118] - A heavy chain comprising the amino acid sequence of the heavy chain of 21D9.e ((i) SEQ ID NO: 13, 176, 177, or 178; or (ii) SEQ ID NO: 80 and one of SEQ ID NO: 98, 100, 102, 103, 104, or 179), and a light chain comprising the amino acid sequence of the light chain of 21D9.e (SEQ ID NO: 11);

[0119] - A heavy chain comprising the amino acid sequence of the heavy chain of 21A5 ((i) SEQ ID NO: 15 or (ii) SEQ ID NO: 83 and one of SEQ ID NO: 98, 100, 102, 103, 104, or 179), and a light chain comprising the amino acid sequence of the light chain of 21A5 (SEQ ID NO: 14);

[0120] - A heavy chain comprising 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 NO: 98, 100, 102, 103, 104, or 179), and a light chain comprising the amino acid sequence of the light chain of 21A5.a (SEQ ID NO: 16);

[0121] - A heavy chain comprising the amino acid sequence of the heavy chain of 10F10 ((i) SEQ ID NO: 19 or (ii) SEQ ID NO: 91 and one of SEQ ID NO: 98, 100, 102, 103, 104, or 179), and a light chain comprising the amino acid sequence of the light chain of 10F10 (SEQ ID NO: 18);

[0122] - A heavy chain comprising 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 NO: 98, 100, 102, 103, 104, or 179), and a light chain comprising the amino acid sequence of the light chain of 10F10.1 (SEQ ID NO: 20);

[0123] - A heavy chain comprising 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 NO: 98, 100, 102, 103, 104, or 179), and a light chain comprising the amino acid sequence of the light chain of 10F10.3 (SEQ ID NO: 21); or

[0124] -A heavy chain comprising the amino acid sequence of 10F10.4 heavy chain ((i) SEQ ID NO: 19 or (ii) SEQ ID NO: 91 and one of SEQ ID NO: 98, 100, 102, 103, 104 or 179), and a light chain comprising the amino acid sequence of 10F10.4 light chain (SEQ ID NO: 116).

[0125] Embodiment 32: An isolated antibody that specifically binds to human ILT4, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 13 and a light chain comprising the amino acid sequence of SEQ ID NO: 11.

[0126] Embodiment 33: An isolated antibody that specifically binds to human ILT4, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 176 and a light chain comprising the amino acid sequence of SEQ ID NO: 11.

[0127] Embodiment 34: An isolated antibody that specifically binds to human ILT4, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 177 and a light chain comprising the amino acid sequence of SEQ ID NO: 11.

[0128] Embodiment 35: An isolated antibody that specifically binds to human ILT4, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 178 and a light chain comprising the amino acid sequence of SEQ ID NO: 11.

[0129] Embodiment 36: The isolated antibody according to any one of Embodiments 32 - 35, wherein the antibody has one or more properties of the antibody according to Embodiment ①.

[0130] Embodiment 37: The isolated antibody according to any one of Embodiments 1 - 36, which is a full - length antibody.

[0131] Embodiment 38: The isolated antibody according to any one of Embodiments 1 - 14, which is an antibody fragment.

[0132] Embodiment 39: The isolated antibody according to any one of Embodiments 1 - 38, which is a multimeric (e.g., dimeric or trimeric) antibody.

[0133] Embodiment 40: The isolated antibody according to any one of Embodiments 1 - 39, which is linked (e.g., covalently linked) to another molecule. ]

[0134] Embodiment 41: The isolated antibody according to Embodiment 40, wherein the another molecule is a label.

[0135] Note: There is a reference to "Embodiment ①" in Embodiment 36 which seems to be a mistake and should probably be "Embodiment 1". Also, the " " etc. tags are likely some kind of internal identifiers in the original patent text and are left unchanged as per the instructions.Embodiment 42: The isolated antibody as described in Embodiment 40, wherein the other molecule is a peptide.

[0136] Embodiment 43: The isolated antibody as described in Claim 40, which is an antibody-drug conjugate (ADC).

[0137] Embodiment 44: An isolated nucleic acid encoding the antibody as described in any one of Embodiments 1-43.

[0138] Embodiment 45: An isolated nucleic acid encoding the heavy chain and / or light chain of the antibody as described in any one of Embodiments 1-43.

[0139] Embodiment 46: A collection of at least two isolated nucleic acids encoding the heavy chain and light chain of the antibody as described in any one of Embodiments 1-43.

[0140] Embodiment 47: A composition comprising a nucleic acid encoding the heavy chain of the antibody as described in any one of Embodiments 1-43 and a nucleic acid encoding the light chain of the antibody as described in any one of Embodiments 1-43.

[0141] Embodiment 48: A cell comprising the isolated nucleic acid as described in any one of Embodiments 44-46 or the composition as described in Embodiment 47.

[0142] Embodiment 49: A method for preparing an antibody, which comprises culturing the cell as described in Embodiment 48 under conditions for expressing the antibody.

[0143] Embodiment 50: A composition comprising the isolated antibody, nucleic acid, composition or cell as described in any one of Embodiments 1-49 and a pharmaceutically acceptable carrier.

[0144] Embodiment 51: The composition as described in Embodiment 50, which comprises a second therapeutic agent.

[0145] Embodiment 52: The composition as described in Embodiment 51, wherein the second therapeutic agent is an immune stimulant.

[0146] Embodiment 53: The composition as described in Embodiment 52, wherein the immune stimulant is an antagonist of an immunosuppressive molecule, such as PD-1 / PD-L1, CTLA-4 and LAG-3, or an agonist of an immune stimulatory molecule, such as GITR and OX40.

[0147] Embodiment 54: A method of treating a subject having cancer, comprising administering to the subject a therapeutically effective amount of a composition as described in any one of Embodiments 50-53 or an isolated antibody as described in any one of Embodiments 1-43, which stimulates an immune response and / or is an ILT-4 antagonist.

[0148] Embodiment 55: The method as described in Embodiment 54, wherein the method further comprises administering a second therapy.

[0149] Embodiment 56: The method as described in Embodiment 55, wherein the second therapy is radiotherapy, surgery or administration of a second agent.

[0150] Embodiment 57: The method as described in Embodiment 55, wherein the second therapy is a second agent, and the second agent is an immune stimulant.

[0151] Embodiment 58: The method as described in Embodiment 57, wherein the immune stimulant is an antagonist of an immunosuppressive molecule, such as PD-1 / PD-L1, CTLA-4 and LAG-3, or an agonist of an immune stimulatory molecule, such as GITR and OX40.

[0152] Embodiment 60: A method of treating an infectious disease (e.g., a viral disease) in a subject, comprising administering to the subject a therapeutically effective amount of a composition as described in any one of Embodiments 50-53 or an isolated antibody as described in any one of Embodiments 1-43, which stimulates an immune response and / or is an ILT4 antagonist.

[0153] Embodiment 61: A method of detecting ILT4 in a sample, comprising contacting the sample with an ILT4 antibody as described in any one of Embodiments 1-43.

[0154] Embodiment 62: The isolated antibody as described in any one of Embodiments 1-43, having the following characteristics:

[0155] a. Specifically binds to hILT4 (e.g., an amino acid sequence comprising SEQ ID NO: 108, 109, 111, 112 or 119), for example, with a K -8 of 10 -9 M or lower, or 10 D M or lower;

[0156] b. Stimulates the differentiation or activation of monocytes into macrophages, for example, stimulates the differentiation of monocytes into pro-inflammatory macrophages, as shown in the assays described in the examples;

[0157] c. Has a binding profile as shown in Figure 27; and

[0158] d. Binds to Ig-like domains 1, 2, or 1 and 2 of hILT4, such as comprising 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), and does not significantly bind to other regions of the extracellular domain of ILT4, such as the region or residues N-terminal to amino acid 170, where the amino acid numbering of hILT4 is that of immature hILT4 (i.e., ILT4 containing its native signal sequence).

[0159] Embodiment 63: The isolated antibody of any one of Embodiments 1-43, having the following characteristics:

[0160] a. For example, 10 -8 M or lower, or 10 -9 M or lower K D specifically binds to hILT4 (e.g., an amino acid sequence comprising SEQ ID NO: 108, 109, 111, 112, or 119);

[0161] b. does not specifically bind to hILT2, hILT3 and / or hILT5;

[0162] c. does not specifically bind to one or more members of the LILRA and / or LILRB family;

[0163] d. stimulating T cell activation, e.g., in a mixed lymphocyte reaction (MLR) assay, as measured by increased T cell proliferation or IFN-γ secretion, e.g., as shown in the assays described in the Examples;

[0164] e. stimulating differentiation or activation of monocytes into macrophages, eg, stimulating differentiation of monocytes into pro-inflammatory macrophages, eg, as shown in the assays described in the Examples;

[0165] f. Inhibit the binding of hILT4 to HLA-A and HLA-B;

[0166] g. having a binding profile as shown in Figure 27;

[0167] h. binds to Ig-like domains 1, 2, or 1 and 2 of hILT4, such as comprising 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), and does not significantly bind to other regions of the extracellular domain of ILT4, such as the region or residues N-terminal to amino acid 170; wherein the amino acid numbering of hILT4 is that of immature hILT4 (i.e., ILT4 with its signal sequence);

[0168] i. Promote the pro-inflammatory polarization of macrophages towards M1 macrophages;

[0169] j. does not induce (or trigger) basophil activation; and

[0170] k. Contains less than 5% high and low molecular weight species after incubation at 25°C for 3 months and / or contains less than 10% high and low molecular weight species after incubation at 40°C for 3 months.

[0171] Embodiment 64: The isolated antibody of any one of Embodiments 1-43, having the following characteristics:

[0172] a. promoting the expression of CD83 and CD86 on human monocyte-derived immature dendritic cells (Mo-iDC), for example as shown in the assay described in the Examples;

[0173] b. enhancing IFN-γ secretion following antigen stimulation in a cytomegalovirus (CMV) lysate assay, for example as shown in the assay described in the Examples;

[0174] c. enhancing IFN-γ and TNF-α secretion by CD4+ and CD8+ T cells following CD3 stimulation in an allogeneic mixed lymphocyte reaction (MLR) assay, e.g., as shown in the assays described in the Examples;

[0175] d. Inhibit the binding of HLA-A and / or HLA-B to ILT4;

[0176] e. Binding to Ig-like domains 1, 2, or 1 and 2 of hILT4, such as regions comprising: (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), as determined by hydrogen-deuterium exchange (HDX), e.g., as shown in the HDX assays described in the Examples;

[0177] f. Interacting with one or more (or all) of the amino acid residues Lys43, Ile49, Thr50, and Arg51 of mature hILT4, or 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 with one or more (or all) of the amino acid residues Glu42, Lys43, Gly76, Cys77, Leu88, Pro91, Pro183, and Tyr184 of mature hILT4, as determined by, e.g., the carbene footprinting method described in the Examples;

[0178] g. Competing with the antibodies described herein for binding to hILT4; and

[0179] h. Specifically binding to cynomolgus monkey ILT4 comprising SEQ ID NO:118, e.g., as shown in the binding assays described in the Examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0180] This application claims priority to a U.S. Provisional Patent Application that contains at least one color drawing from the list below. Once this international application is published and its priority provisional application becomes available to the public, it is assumed that these color drawings will be provided by the U.S. Patent and Trademark Office upon request and payment of the required fees.

[0181] Figure 1 Displays the nucleotide and amino acid sequences of the heavy chain variable region (“VH”) of anti-hILT4 antibody 9G4, indicating the positions of VHCmdr.

[0182] Figure 2 Displays the nucleotide and amino acid sequences of the light chain variable region (“VL”) of anti-hILT4 antibody 9G4, indicating the positions of VLCDR.

[0183] Figure 3 Displays the nucleotide and amino acid sequences of the VH of anti-hILT4 antibody 9C8, indicating the positions of VH CDR.

[0184] Figure 4 Displays the nucleotide and amino acid sequences of the VL of anti-hILT4 antibody 9C8, indicating the positions of VL CDR.

[0185] Figure 5 Displays the nucleotide and amino acid sequences of the VH of anti-hILT4 antibody 2H2, indicating the positions of VH CDR.

[0186] Figure 6 Displays the nucleotide and amino acid sequences of the VL of anti-hILT4 antibody 2H2, indicating the positions of VL CDR.

[0187] Figure 7 Displays the nucleotide and amino acid sequences of the VH of anti-hILT4 antibody 2E5, indicating the positions of VH CDR.

[0188] Figure 8 Displays the nucleotide and amino acid sequences of the VL of anti-hILT4 antibody 2E5, indicating the positions of VL CDR.

[0189] Figure 9 Displays the nucleotide and amino acid sequences of the VH of anti-hILT4 antibody 24E5, indicating the positions of VH CDR.

[0190] Figure 10 Displays the nucleotide and amino acid sequences of the VL of anti-hILT4 antibody 24E5, indicating the positions of VL CDR.

[0191] Figure 11 Displays the nucleotide and amino acid sequences of the VH of anti-hILT4 antibody 21D9, indicating the positions of VH CDR.

[0192] Figure 12 Displays the nucleotide and amino acid sequences of the VL of anti-hILT4 antibody 21D9, indicating the positions of the VL CDRs.

[0193] Figure 13A and Figure 13C Displays the alignment of the amino acid sequences of the heavy chain variable domain (VH) ( Figure 13A ) and the light chain variable domain (VH) ( Figure 13C ) of ILT4 antibody 21D9 with the germline immunoglobulin sequences hIGHV3-23*01 and hIGKV3-20*01, respectively. Arrows point to germline mutations. AbM VH CDR1 and Kabat VH CDR2, VH CDR3 and VL CDR1-3 sequences are shown in light text. Figure 13B Displays the VH amino acid sequences of four germline revertant mutants 21D9.b, 21D9.c, 21D9.d, and 21D9.e with the IgG1.3 heavy chain constant region (i.e., 21D9.b-.e hIgG1.3). Figure 13B Amino acid residues shown in capital letters in

[0194] Figure 14A and Figure 14B Displays the alignment of the amino acid sequences of the heavy chain variable domain and the light chain variable domain of ILT4 antibody 21A5 with the germline immunoglobulin sequences hIGHV1-46*01 ( Figure 14A ) and hIGKV3-20*01 ( Figure 14B ), respectively. AbM VH CDR1 and Kabat VH CDR2, VH CDR3 and VL CDR1-3 sequences are shown in light text. Arrows point to germline mutations.

[0195] Figure 15 Displays the alignment of the amino acid sequences of the heavy chain variable domain and the light chain variable domain of ILT4 antibody 10F10 with the germline immunoglobulin sequences hIGHV3-30*01 (top 3 lines) and hIGKV1-13*02 (bottom 3 lines), respectively. AbM VH CDR1 and Kabat VH CDR2, VH CDR3 and VL CDR1-3 sequences are shown in light text. Arrows point to germline mutations.

[0196] Figure 16 Displays the sensorgrams indicating the binding of the indicated anti-hILT4 antibodies to hILT4. A schematic of the method used is shown above the sensorgram panel.

[0197] Figure 17Shown is the binding of anti-hILT4 antibodies 2H2, 10F10 wild type (WT, i.e., parental antibody), 21A5.a, 21A5 WT, 21D9WT, 21D9e, and isotype control hIgG1.3 to hILT4-transfected CHO cells, which varies with antibody concentration and is determined by flow cytometry. EC 50 values are provided below the binding data graph.

[0198] Figure 18A Shown is the binding of the indicated anti-hILT4 antibodies and isotype control to hILT4-transfected CHO cells, which varies with antibody concentration and is determined by flow cytometry. EC 50 values are provided to the right of the binding data graph. Figure 18B Shown is the binding of anti-hILT4 antibodies to human monocytes isolated from peripheral blood of normal healthy donors. The antibody species are listed adjacent to the graph, and EC50 values are provided in the table immediately below the graph.

[0199] Figure 19A Flow cytometry patterns showing 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 Flow cytometry patterns showing the binding of anti-hILT4 antibodies 21A5 and germline revertant 21A5.a to human LIRA1, LIRA3, and ILT4.

[0200] Figure 20A and Figure 20B Shown are the indicated antibodies 21D9 (“21D9 IgG1.1”) and 21D9.e ( Figure 20A ) and 21A5 (“21A5WT”) and 21A5.a ( Figure 20B ) with the indicated human LILRA / LILRB family members ( Figure 20A LILRA5, ILT4 in Figure 20B and LILRA1, LILRA3, and ILT4 in

[0201] Figures 21A to 21D The maximum binding percentages, which vary with antibody concentration, indicating that anti-ILT4 antibodies 21D9, 21D9.e, 21A5, and 21A5.a show weak cross-reactivity with other LILRA family members. Figure 21AFor T cells co-cultured with hILT4- and OKT3-transfected CHO cells 3 Histogram of H thymidine incorporation of T cells co-cultured with hILT4- and OKT3-transfected CHO cells, which T cells (from left to right on the x-axis) were not incubated with antibody, with isotype control hIgG1.3 antibody and antibodies 21D9 (“21D9 WT”), 21D9.e, 21A5 (“21A5 WT”), 21A5.a and 10F10 (“10F10 WT”), and the 3 H thymidine incorporation varied with antibody concentration (20, 4, 0.8, 0.16, 0.032 and 0.0064 μg / ml). Figure 21B Provide the relevant EC50 values. Figure 21C Demonstrate the T cell activity of anti-hILT4 antibodies. Figure 21C Histogram of IFNγ production of T cells co-cultured with hILT4- and OKT3-transfected CHO cells, which T cells (from left to right on the x-axis) were not incubated with antibody, with isotype control hIgG1.3 antibody and antibodies 21D9 (“21D9 WT”), 21D9.e, 21A5 (“21A5 WT”), 21A5.a and 10F10 (“10F10 WT”), and the IFNγ production varied with antibody concentration (20, 4, 0.8, 0.16, 0.032 and 0.0064 μg / ml). Figure 21D Provide the relevant EC50 values.

[0202] Figures 22A to 22D Demonstrate the proliferation ( Figure 22A and Figure 22C ) and IFNγ production ( Figure 22B and Figure 22D ) of T cells in the allogeneic MLR (mixed lymphocyte reaction) of monocyte-derived dendritic cells (MoDCs) co-cultured with T cells. Anti-hILT4 antibodies 21A5.a or 21D9.e ( Figure 22A and Figure 22B ), or 2H2 or 21D9 ( Figure 22C and Figure 22D ) were added during the differentiation of monocytes into MoDCs, or isotype control or no antibody was added (“untreated”), and no antibody was added during the allogeneic MLR assay.

[0203] Figures 23A to 23D Demonstrate 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 ( Figure 23C and Figure 23D) or isotype control or no treatment (no antibody).

[0204] Figure 24A and Figure 24B show the levels of TNF-α secreted from in vitro differentiated macrophages activated by lipopolysaccharide (LPS) ( Figure 24A ) or the STING agonist 2'3'-cGAMP ( Figure 24B ). During activation, it includes anti-ILT4 antibodies 21D9, 21D9.e, 21A5, 21A5.a, 2H2 and 10F10 or isotype control.

[0205] Figure 25A and Figure 25B show the percentage of CD4 T cell proliferation ( Figure 25A ) and the amount of IFN-γ produced from allogeneic MLR of CD4 T cells co-cultured with in vitro differentiated macrophages in the presence of anti-hILT4 antibody 21D9, 21D9.e, 21A5 or 21A5.a or anti-PD-L1 antibody or isotype control antibody ( Figure 25B ). Figure 25A show the percentage of CD4 T cell proliferation obtained by FACS analysis based on CFSE dilution, and Figure 25B show IFNγ production measured by Alphalisa, both varying with ILT4 antibody or control.

[0206] Figure 26A and Figure 26B show that in macrophage:CD4 T cell allogeneic MLR, the combination of anti-ILT4 antibody 21D9 and anti-PD-L1 antibody clone 29E.2A3 enhances IFNγ production to a higher level compared to each antibody alone. Figure 26A show an experiment of one macrophage:CD4 T allogeneic pair. Figure 26B show the responses of multiple allogeneic pairs, where each line represents an experiment of one allogeneic pair. Statistical analysis was performed using non-parametric one-way Anova multiple comparisons (Friedman test; Friedman test). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0207] Figure 27A 、 Figure 27B and Figure 27C show the HDX epitope mapping of anti-hILT4 Ab 21D9 and 2H2 Fab. Figure 27A show the coverage of pepsin on the hILT4 sequence. Figure 27BDifferent HDXs of 21D9 and 2H2 Fab are shown. In the sequence portions indicated in the figure, those underlined have less HDX protection than those not underlined, as also noted below the figure. Figure 27C The HDX epitopes of two Fabs mapped to the linear sequence of hILT4 are shown. The epitope of antibody 21D9 is shown with a single underline. The epitope of antibody 2H2 is shown with a double underline and the shaded / hatched underline portion of the sequence.

[0208] Figure 28A 、 Figure 28B 、 Figure 28C and Figure 28D Display antibody 21D9 ( Figure 28A )、2H2( Figure 28B )、10F10( Figure 28C ) and 21A5( Figure 28D ) Inhibition of hILT4 binding to HLA-A and HLA-B.

[0209] Figures 29A to 29D 21D9e.IgG1.3 was shown to enhance 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 Demonstrate IFN-γ secretion in CD4 + T cells; Figure 29B Demonstrate TNF-α secretion in CD4+ T cells; Figure 29C Demonstrate IFN-γ secretion in CD8+ T cells; and Figure 29D Demonstrates TNF-α secretion in CD8+ T cells.

[0210] Figure 30 Anti-ILT4 antibodies 21D9, 21A5, 21D9 Fab are shown to enhance IFN-g secretion after antigen stimulation in a CMV lysate assay. Nivolumab was used as a positive control, and no antibody ("no Ab") and an isotype control were used as negative controls. The concentrations of anti-ILT4 antibodies were from left to right: 0.00128 μg / ml; 0.0064 μg / ml; 0.032 μg / ml; 0.16 μg / ml; 0.8 μg / ml; 4 μg / ml; 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., "no Ab" corresponds to the bar on the left, and "Nivo" corresponds to the bar on the right).

[0211] Figure 31 Anti-ILT4 antibodies 2H2 and 21D9 displayed in IgG1.1f or Fab format enhanced T cell proliferation in monocyte:T allogeneic MLR (by3 H incorporated measurement

[0212] Figures 32A to 32C Show that anti-ILT4 antibodies 21D9e.IgG1.3 ("ILT4.8" in the figure) and 9G4.IgG1.3 ("ILT4.1") (for the nomenclature of the antibodies, see Table 1) promote in vitro the expression of the costimulatory molecule CD86 ( Figure 32A ) and CD80 ( Figure 32B ) and the dendritic cell activation marker CD83 ( Figure 32C ) on cynomolgus monkey monocyte-derived dendritic cells. Each antibody was added during the differentiation of cynomolgus monkey monocytes into monocyte-derived dendritic cells (DC).

[0213] Figure 33A and Figure 33B Show the levels of TNF-α 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.

[0214] Figure 34 Show that the levels of TNF-α secreted from in vitro differentiated macrophages incubated with 21D9.e are similar in the case of IgG1, IgG4 or IgG1.3.

[0215] Figure 35 Show the percentages of monomeric species, high molecular weight (HMW) species and low molecular weight (LMW) species of 21D9e.IgG1.3 after 0, 1, 2 and 3 months at 25 °C or 40 °C. The monomeric species, HMW species and LMW species correspond to the bottom part, middle part and top part of each bar in the histogram, respectively.

[0216] Figure 36 Show the percentages of activated basophils of a total of 8 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-formylmethionyl-leucyl-phenylalanine (fMLP), showing that 21D9e.IgG1.3 (ILT4.8) and 21A5.a.IgG1.3 ("ILT4.9") do not have basophil activation.

[0217] Figures 37A to 37D Show the results of experiments to determine the epitopes of certain antibodies. Figure 37A and Figure 37BCompetition (or cross - blocking) assay results of antibodies 2H2 ( Figure 37A ) and 21D9 ( Figure 37B ) that bind to hILT4 expressed on the surface of CHO cells. Figure 37C Schematic of a three - dimensional representation of hILT4 showing the positions of Ig - like domains 1 and 2 and indicating the regions of hILT4 to which antibodies 21D9, 29A5, 2H2, and 10F10 bind. Figure 37D Another three - dimensional representation of hILT4 showing the positions of Ig - like domains 1 and 2 and indicating the regions of hILT4 to which antibodies 21D9, 21A5, and 10F10 bind. Detailed Description

[0218] Definitions

[0219] Unless otherwise defined, scientific and technical terms used in connection with the present invention will have the meanings commonly understood by one of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

[0220] In this application, unless otherwise stated, the use of "or" means "and / or". In the case of multiple dependent claims, the use of "or" only refers alternatively to more than one of the foregoing 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 removed from its natural environment. Thus, the term "isolated" does not necessarily reflect the degree to which the molecule has been purified.

[0221] 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 contain natural or non - natural amino acid residues and include (but are not limited to) peptides, oligopeptides, dimers, trimers, and multimers composed of amino acid residues. Both full - length proteins and their fragments are covered by this definition. These terms also include post - translational modifications of polypeptides such as glycosylation, sialylation, acetylation, phosphorylation, and similar modifications. In addition, for the purposes of the present invention, a "polypeptide" or "protein" respectively refers to a polypeptide or protein that includes modifications such as deletions, additions, and substitutions (generally substantially conservative substitutions) of the native sequence, provided that the protein maintains the desired activity. These modifications may be intentional, such as induced by site - directed mutagenesis, or may be accidental, such as due to mutations in the host producing the protein or errors resulting from PCR amplification. A protein may comprise two or more polypeptides. The letter "h" preceding a protein name indicates a native human protein herein, e.g., "hILT4".

[0222] Unless otherwise specifically indicated (e.g., murine ILT4, cynomolgus monkey ILT4, etc.), 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 used interchangeably and refer to native human ILT4. The terms include full-length, unprocessed ILT4 and any form of ILT4 produced by a processing cell. The terms encompass variants of native human ILT4, such as splice variants or allelic variants. In some embodiments, ILT4 comprises or consists of the amino acid sequence of SEQ ID NO:107 (precursor, with signal peptide) or SEQ ID NO:108 (mature, without signal peptide). In some embodiments, ILT4 comprises the amino acid sequence of SEQ ID NO:110 (precursor, with signal peptide) or SEQ ID NO:111 (mature, without signal peptide).

[0223] As used herein, the term “ILT4 fragment” refers to an ILT4 having one or more residues deleted from the N-terminus and / or C-terminus of full-length ILT4. The ILT4 fragment may or may not include the N-terminal signal peptide, but retains the ability to bind to a T cell. As used herein, the term “ILT4 variant” refers to an ILT4 containing naturally occurring amino acid additions, deletions, and substitutions, but retaining the ability to bind to a T cell.

[0224] The term "antagonist" is used in its broadest sense and includes any molecule that partially or completely inhibits or neutralizes the biological activity of a polypeptide such as ILT4. Exemplary antagonist molecules include antagonist antibodies. The term "ILT4 antagonist" refers to a molecule that inhibits or blocks the biological activity of ILT4 by, for example, blocking or inhibiting the interaction between ILT4 and a target cell (e.g., a T cell) and / or a target molecule. Exemplary ILT4 antagonists include antibodies that block the binding of ILT4 to a target cell (e.g., a T cell) and / or a target molecule. An ILT4 antagonist is considered to "block the binding of ILT4 to a target cell or target molecule" when it reduces the detectable amount of binding of ILT4 to at least one of a target cell (e.g., a T cell) and / or a target molecule by at least 50% in a cell binding assay. In some embodiments, the ILT4 antagonist reduces the detectable amount of binding by at least 60%, at least 70%, at least 80%, or at least 90%. In some such embodiments, the antagonist is said to block ligand binding by at least 50%, at least 60%, at least 70%, etc. Blocking of the binding of ILT4 to a target cell (e.g., a T cell) can be demonstrated, for example, by the binding of a cell transfected with ILT4 (e.g., ILT4 ECD and transmembrane domain) or a recombinant ILT4 Fc fusion protein (e.g., a recombinant ILT4 ECD Fc fusion protein) to a cell such as a T cell in the presence or absence of the antagonist.

[0225] The term "inhibit" refers to any reduction, decrease, or cessation of a phenotypic characteristic, or a decrease, reduction, or cessation of the incidence, degree, or likelihood of said characteristic. In some embodiments, "decrease" or "inhibit" means the ability to cause a decrease of 20% or more. In another embodiment, "decrease" or "inhibit" means the ability to cause a decrease of 50% or more. In yet another embodiment, "decrease" or "inhibit" means the ability to cause an overall decrease of 75%, 85%, 90%, 95% or more.

[0226] The term "enhance T cell activity" or "enhancement of T cell activity" refers to enhancing or increasing at least one of T cell activation, cytokine secretion (such as interferon-γ (IFN-γ) secretion), or T cell proliferation in a subject. "Enhancing T cell activity" can be achieved by using an agent that is an agonist of T cell activity and / or an agent 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 an IFN-γ ELISA as described in the examples.

[0227] As used herein, the term "ILT4 antibody" or "hILT4 antibody" or "anti-ILT4 antibody" or "anti-hILT4 antibody" or "antibody that binds ILT4" refers to an antibody that binds to ILT4 and optionally inhibits the biological activity of ILT4, such as by blocking or inhibiting the binding of ILT4 to a target cell (such as a T cell) or a target molecule. In some embodiments, such as measured by SPR or in a radioimmunoassay (RIA), the degree of binding of the ILT4 antibody to an unrelated non-ILT4 protein is less than 10% of the binding of the antibody to ILT4. In some embodiments, such as measured by SPR or in a radioimmunoassay (RIA), the degree of binding of the ILT4 antibody to other ILT family proteins (such as LILRA) is less than 20%, less than 10%, less than 5% of the binding of the antibody to ILT4. In some embodiments, the ILT4 antibody binds ILT4 but does not bind at least one protein selected from ILT2, ILT3, and ILT5.

[0228] The term "leader peptide" or "leader sequence" or "signal peptide" or "signal sequence" refers to a peptide or amino acid residue sequence located at the N-terminus of a polypeptide that facilitates the secretion of the polypeptide from a mammalian cell. The leader sequence can be cleaved to form a mature protein upon export of the polypeptide from the mammalian cell. The leader sequence can be natural or synthetic, and it can be heterologous or homologous to the protein to which it is linked.

[0229] The term "antibody" or "Ab" as used herein is used in the broadest sense and encompasses various antibody structures, including (but not limited to) monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. As used herein, the term refers to a molecule that includes 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, wherein the molecule is capable of binding to an antigen. The term antibody also includes (but not limited to) chimeric antibodies, humanized antibodies, human antibodies, and antibodies from various species such as mice, cynomolgus monkeys, etc. The term "antibody fragment" includes (but not limited to) fragments capable of binding an antigen, such as Fv, single-chain Fv (scFv), Fab, Fab', and (Fab')2.

[0230] The term "heavy chain" or "HC" refers to a polypeptide that includes at least the 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 the heavy chain variable region and the 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 the heavy chain variable region and the heavy chain constant region, without a leader sequence and with or without a C-terminal lysine (K).

[0231] The term "heavy chain variable region" or "VH" refers to the region that includes heavy chain complementarity determining region (CDR) 1, framework region (FR) 2, CDR2, FR3, and CDR3 of the heavy chain. In some embodiments, the heavy chain variable region further includes at least a portion of FR1 and / or at least a portion of FR4. In some embodiments, the heavy chain CDRs are specified herein, such as in the Sequence Listing below or Figures 1 to 12 as specified in. As used herein, VH CDR1, CDR2, and CDR3 are Kabat CDRs as provided, for example, in Figures 1 to 12 as provided in.

[0232] The term "light chain" or "LC" refers to a polypeptide that includes at least one 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 and does not have a leader sequence.

[0233] The term "light chain variable region" or "VL" refers to the region that includes light chain CDR1, FR2, HVR2, FR3, and HVR3. In some embodiments, the light chain variable region further includes FR1 and / or FR4. In some embodiments, the light chain CDRs are specified herein, such as in the Sequence Listing below or Figures 1 to 12 as specified in. As used herein, VL CDR1, CDR2, and CDR3 are Kabat CDRs as provided, for example, in Figures 1 to 12 as provided in.

[0234] A "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a particular source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species. In some embodiments, a chimeric antibody refers to an antibody that includes at least one variable region from a first species (such as a mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (such as a 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 variable regions of the chimeric antibody are from a first species and all constant regions of the chimeric antibody are from a second species.

[0235] A "humanized antibody" refers to an antibody in which at least one amino acid in the framework region of a non-human variable region has been replaced by the corresponding amino acid from a human variable region. In some embodiments, a humanized antibody includes at least one human constant region or a fragment thereof. In some embodiments, a humanized antibody is a Fab, scFv, (Fab')2, etc.

[0236] As used herein, "human antibody" refers to an antibody produced in humans, an antibody produced in a non-human animal that contains human immunoglobulin genes, such as and an antibody selected using in vitro methods (such as phage display), wherein the antibody library is based on human immunoglobulin sequences.

[0237] In some embodiments, an antibody herein may contain one or more "conservative substitutions" compared to a specific designated sequence. "Conservative amino acid substitution" herein refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, a predicted non-essential amino acid residue in an antibody herein is replaced with another amino acid residue from the same side chain family (e.g., basic, acidic, β-branched, aromatic, uncharged polar). Methods for identifying nucleotide and amino acid conservative substitutions that do not eliminate antigen binding have been 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)).

[0238] In some embodiments, an antibody herein may contain one or more "reversion substitutions". Examples are depicted in FIGS. 13 to Figure 15 herein. A reversion substitution is a mutation that returns to the germline amino acid sequence from which the antibody heavy or light chain was derived.

[0239] The "K D " or "dissociation constant" of the binding of an antibody to a protein (e.g., ILT4) is a measure of the affinity or specific binding of the antibody to the protein (e.g., ILT4). Compared to a higher K D a lower K D indicates improved binding or affinity. K D is determined by the "dissociation rate" or k of the antibody and the polypeptideoff or k d and the "association rate", or k on or k a constitute the ratio therebetween.

[0240] The term "specific binding" or a similar term refers to the K D between two polypeptides such as an antibody and its polypeptide target, which is less than that between two random polypeptides present under the same conditions. In other words, K D is less than that due to non-specific aggregation of polypeptides in the system.

[0241] As used herein, the "tumor model" refers to an in vivo preclinical assay that can be used to study the biological activity of an ILT4 antibody and includes xenograft or native mouse tumor assay systems. In some cases, the tumor model may allow tracking of tumor size or growth upon treatment with the antibody, and / or tracking of the presence of immune cells such as specific types of T cells or NK cells in the tumor to determine whether the antibody triggers or enhances an immune response.

[0242] As used herein, the term "immunostimulant" refers to a molecule that stimulates the immune system by acting as an agonist of an immune stimulatory molecule (including a co-stimulatory molecule) or as an antagonist of an immune inhibitory molecule (including a co-inhibitory molecule). The immune stimulatory molecule or immune inhibitory molecule can be an immune checkpoint modulator, such as a checkpoint inhibitor or a checkpoint stimulator. The immunostimulant can be a biological agent, such as an antibody or an antibody fragment, another protein or a vaccine, or can be a small molecule drug. "Immune stimulatory molecule" includes a receptor or ligand that enhances, stimulates, induces or otherwise "turns on" an immune response. The immune stimulatory molecule as defined herein includes co-stimulatory molecules. "Immune inhibitory molecule" includes a receptor or ligand that reduces, inhibits, suppresses or otherwise "turns off" an immune response. The immune inhibitory molecule as defined herein includes co-inhibitory molecules. Such immune stimulatory and immune inhibitory molecules can be, for example, receptors or ligands found on immune cells such as T cells or on cells involved in innate immunity such as NK cells.

[0243] "Percent amino acid sequence identity (%)", "identity %", "percent amino acid sequence homology", and "homology %" relative to a peptide, polypeptide, or antibody sequence means the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a specific peptide or polypeptide sequence after aligning the sequences, introducing gaps if necessary to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purposes of determining percent amino acid sequence identity can be achieved in various ways within the skill in the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or MEGALIGNTM (DNASTAR) software. Those of skill in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to achieve the maximum alignment within the full length of the sequences being compared.

[0244] "Treatment" as used herein encompasses any administration or application of a therapeutic agent for a disease in a human and includes inhibiting the disease progression of a disease or one or more symptoms of a disease, slowing a disease or its progression or one or more of its symptoms, arresting its development, partially or completely alleviating one or more of the disease or its symptoms, or preventing recurrence of one or more symptoms of a disease.

[0245] Unless otherwise specifically stated, the terms "subject" and "patient" are used interchangeably herein to refer to a human.

[0246] The term "effective amount" or "therapeutically effective amount" means an amount of a drug that is effective in treating a disease or disorder in a subject, such as to partially or completely alleviate one or more symptoms. In some embodiments, an effective amount means an amount that is effective upon administration and persists for a necessary period of time to achieve the desired therapeutic or prophylactic outcome.

[0247] The term "cancer" is used herein to refer to a population of cells that exhibits an abnormally high level of proliferation and growth. Cancer can be benign (also referred to as a benign tumor), pre-cancerous, or malignant. Cancer cells can be solid cancer cells or leukemia cancer cells. The term "tumor growth" is used herein to refer to the proliferation or growth of one or more cells containing cancer that results in a corresponding increase in the size or extent of the cancer.

[0248] Examples of cancers amenable to the treatment methods described herein include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific non-limiting examples of such cancers include squamous cell carcinoma, small cell lung cancer, pituitary carcinoma, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous cell non-small cell lung cancer), lung adenocarcinoma, pulmonary squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland carcinoma, kidney cancer, renal cell carcinoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, brain cancer, endometrial cancer, testicular cancer, cholangiocarcinoma, gallbladder carcinoma, stomach cancer, melanoma, and various types of head and neck cancers (including head and neck squamous cell carcinoma).

[0249] Administration "in combination" with one or more other therapeutic agents includes simultaneous (concurrent) and sequential (in series) administration in any order.

[0250] "Pharmaceutically acceptable carrier" refers to conventional non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials, formulation aids or carriers in the art, which are used together with a therapeutic agent to form a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosages and concentrations employed and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation employed. For example, if the therapeutic agent is intended for oral administration, the carrier may be a gelatin capsule. If the therapeutic agent is intended for subcutaneous administration, the carrier is desirably non-irritating to the skin and does not cause injection site reactions.

[0251] "Chemotherapeutic agent" is a compound suitable for treating cancer. Examples of chemotherapeutic agents that may be administered in the methods described herein include, but are not limited to: alkylating agents such as thiotepa and Cyclophosphamide; alkyl sulfonates such as busulfan, prosulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; polyacetyls (especially bullatacin and bullatacinone); camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;Nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ωI1 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A, bisphosphonates, such as clodronate; esperamicin, and neocarzinostatin chromophore and related pigment protein enediyne antibiotic chromophores, aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine; Doxorubicin (including morpholino-doxorubicin, cyano-morpholino-doxorubicin, pyrrolino-doxorubicin, and daunorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as leucovorin; acetylglucuronide; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;Norpseudoephedrine (defofamine); demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoid, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; Polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichloroethylamine; trichothecene (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoid, such as Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), Albumin-engineered nanoparticle formulation of paclitaxel without polysorbate 80 (American Pharmaceutical Partners, Schaumberg, Illinois) and Docetaxel( Rorer, Antony, France); Chlorambucil; Gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analogs such as cisplatin, oxaliplatin and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Vinorelbine; Mitoxantrone; Teniposide; Edatrexate; Daunorubicin; Aminopterin; Xeloda; Ibandronate; Irinotecan ((Irinotecan)(Camptosar, CPT-11)(including treatment regimens of irinotecan with 5-FU and leucovorin)); Topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoids such as retinoic acid; Capecitabine; Combretastatin; Leucovorin (LV); Oxaliplatin, including oxaliplatin treatment regimens (FOLFOX); PKC-α, Raf, H-Ras, EGFR (such as erlotinib )) and VEGF-A inhibitors; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0252] Other non-limiting exemplary chemotherapeutic agents that can be administered in the methods herein include antihormonal agents for modulating or inhibiting the hormonal effects on cancer, such as antiestrogens and selective estrogen receptor modulators (SERM), including for example tamoxifen (including tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene; Aromatase inhibitors that inhibit aromatase, which modulates estrogen production in the adrenal gland, such as 4(5)-imidazole, aminoglutethimide, megestrol acetate, Exemestane, formestanie, fadrozole, Vorozole, Letrozole and Anastrozole; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (1,3 - dioxolane nucleoside cytosine analogue); antisense oligonucleotides, particularly those that inhibit gene expression in signal transduction pathways involved in abnormal cell proliferation, such as PKC-α, Ralf, and H-Ras; ribonucleases such as VEGF expression inhibitors (e.g., ribonuclease) and HER2 expression inhibitors; vaccines such as gene therapy vaccines, e.g., vaccine, vaccine, and vaccine; rIL-2; Topoisomerase 1 inhibitor; rmRH; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0253] "Angiogenesis inhibitor" or "anti-angiogenic agent" refers to small molecular weight substances, polynucleotides (including, for example, inhibitory RNA (RNAi or siRNA)), polypeptides, isolated proteins, recombinant proteins, antibodies, or conjugates or fusion proteins thereof that directly or indirectly inhibit angiogenesis, vasculogenesis, or unwanted vascular permeability. It is understood that angiogenesis inhibitors include those agents that bind and block the angiogenic activity of angiogenic factors or their receptors. For example, angiogenesis inhibitors that can be administered in the methods herein may include antibodies or other antagonists of angiogenic agents, such as an antibody to VEGF-A (e.g., bevacizumab ) or an antibody to a VEGF-A receptor (e.g., the KDR receptor or the Flt-1 receptor); anti-PDGFR inhibitors such as (Imatinib Mesylate); small molecules that block VEGF receptor signaling (e.g., PTK787 / ZK2284, SU6668, / SU11248 (sunitinib malate), AMG706 or small molecules such as those described in International Patent Application WO 2004 / 113304). Anti-angiogenic agents also include natural angiogenesis inhibitors such as angiostatin, endostatin, 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 for malignant melanoma); Ferrara and 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).

[0254] As used herein, "growth inhibitor" refers to a compound or composition that inhibits the growth of cells (such as cells expressing VEGF) in vitro or in vivo. Thus, a growth inhibitor that can be administered in the methods herein can be a growth inhibitor that significantly reduces the percentage of cells (such as cells expressing VEGF) in S phase. Examples of growth inhibitors include (but are not limited to) agents that block cell cycle progression (at times other than S phase), such as agents that induce G1 arrest and M phase arrest. Classical M phase blockers include vincas (vincristine and vinblastine), taxane, and type II topoisomerase inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 also extend to S phase arrest, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Other information can be found in Mendelsohn and Israel eds., The Molecular Basis of Cancer, Chapter 1, titled "Cell cycle regulation, oncogenes, and antineoplastic drugs", Murakami et al. (W.B. Saunders, Philadelphia, 1995), for example, page 13. Taxanes (paclitaxel and docetaxel) are anti-cancer drugs both derived from the yew tree. Docetaxel ( Rhone - Poulenc Rorer) is derived from European yew and is a semi - synthetic analogue of paclitaxel from the Pacific Ocean ( Bristol - Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which causes inhibition of mitosis in cells.

[0255] The term "anti - neoplastic composition" refers to a composition containing at least one active therapeutic agent that can be used to treat cancer. Examples of therapeutic agents include (but are not limited to) for example chemotherapeutic agents, growth inhibitors, cytotoxic agents, agents for radiotherapy, anti - angiogenic agents, cancer immunotherapeutic agents, apoptosis agents, anti - tubulin agents, and other agents for treating cancer, such as anti - HER - 2 antibodies, anti - CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (such as tyrosine kinase inhibitors), HER1 / EGFR inhibitors (such as erlotinib Platelet - derived growth factor inhibitors (such as (imatinib mesylate)), COX - 2 inhibitors (such as celecoxib), interferons, CTLA4 inhibitors (such as the anti - CTLA antibody ipilimumab ), PD - 1 or PD - L1 inhibitors (such as ), TIM3 inhibitors (such as anti - TIM3 antibodies), LAG - 3 inhibitors, cytokines, antagonists (such as neutralizing antibodies) that bind to one or more of the following targets ErbB2, ErbB3, ErbB4, PDGFR - β, BlyS, APRIL, BCMA, CTLA4, TIM3, or one or more VEGF receptors, TRAIL / Apo2, and other bioactive and organic chemical agents, etc. Combinations thereof are also included in the present invention.

[0256] Antibodies that specifically bind to ILT4

[0257] The following sequence listings respectively provide the amino acid sequences of human ILT4 with or without a signal peptide (see SEQ ID NO: 107 - 108 and 110 - 111). The extracellular domain (ECD) contains the amino acid residues shown in SEQ ID NO: 109 and 112. An exemplary ECD coupled to a His - Avi tag contains the amino acid sequence of SEQ ID NO: 119.

[0258] Anti - ITL4 antibodies (Ab) can specifically bind to ILT4 - ECD or its fragments (see SEQ ID NO: 109 and 112 and 119).

[0259] This document provides an Ab that binds to ILT4 with a K of 10 -6 M or lower, 10 -7 M or lower, 10 -8 M or lower, 10 -9 M or lower, or 10 -10 or lower, as measured, for example, at 25 °C or at 37 °C. D The extent of Ab binding to the ILT4 protein can be determined using several different methods. For example, it can be determined by surface plasmon resonance (SPR), such as by

[0260] the assays described. Exemplary SPR assays include capturing one or several antibodies on a CM4 sensor chip with immobilized capture reagent (e.g., using a human Fc capture kit, GE Healthcare catalog number BR-1008-39, or a mouse capture kit, GE Healthcare catalog number BR-1008-39), and flowing the ILT4 antigen (e.g., ILT4 ECD) as an analyte in a concentration series to determine the binding kinetics and affinity in the running buffer. In one embodiment, ILT4 is injected at two to five concentrations in the range of 0.1 nM to 500 nM (e.g., 0.1 nM, 1 nM, 10 nM, 100 nM, 500 nM) at a flow rate of 30 μL / min for an association time of up to four minutes and a dissociation time of up to ten minutes. Between binding cycles, the capture surface is regenerated following the manufacturer's instructions for the corresponding capture kit. All data are double-referenced using a reference flow cell and a blank injection. The data with simple 1:1 kinetics are fit to the Langmuir binding model using the mass transport with the T200 evaluation software. The SPR methods described in the examples can also be used. The affinity of the Ab for the ILT4 ECD polypeptide can be determined using cells expressing the ILT4 polypeptide on their surface, and the method includes flow cytometry. Exemplary flow cytometry assays include the following: Resuspending T cells or other cells ectopically expressing ILT4 in a buffer in which the Ab has been serially diluted from approximately 20 μg / mL and incubating with the resuspended cells at 4 °C for 30 minutes. The cells are then washed twice with the same buffer, maintaining the desired buffer conditions, and incubated with a secondary antibody conjugated to a fluorophore that recognizes the primary antibody (e.g., human IgG). The cells are then washed as described above and acquired immediately without fixation on a BD

[0261] or other flow cytometer. The affinity of the Ab for the ILT4 polypeptide can be determined as described in the examples.

[0262] In certain embodiments, an Ab that binds to ILT4 blocks the binding of ILT4 to target cells such as T cells. The inhibition or blocking can be 100% or at least 99%, 95%, 90%, 85%, 80%, 75% or 50%.

[0263] Inhibition of the binding between ILT4 and ILT4-binding cells such as T cells can be determined by measuring the inhibition of binding of cells bound by ILT4 in the presence and absence of the antibody. An exemplary experiment that can be used to determine whether an antibody inhibits the binding of ILT4 to ILT4-binding cells is a flow cytometry assay, for example an assay that includes the following: Human peripheral blood mononuclear cells, buffy coat or leukopak from donor blood are resuspended in a buffer consisting of HBSS + 1% BSA. The cells are then incubated for 30 minutes at 4 °C with, for example, 20 μg / mL of ILT4 ECD or an ILT4 ECD fusion protein fused to human IgG1 Fc and a candidate ILT4-blocking antibody or control antibody at varying concentrations. The cells are then washed twice in the same buffer and reincubated for 30 minutes at 4 °C with a secondary antibody conjugated to a fluorophore that recognizes ILT4 instead of the candidate blocking antibody or control antibody. The cells are then washed as described above and acquired immediately without fixation on a BD or other flow cytometer. Binding inhibition can also be determined, for example, as described in the Examples.

[0264] Exemplary ILT4-binding Ab

[0265] Provided herein are Abs that specifically bind to ILT4, such as ILT4 ECD.

[0266] In certain embodiments, the ILT4 Ab comprises a heavy chain variable region (“VH”) that comprises VH CDR1, CDR2, and / or CDR3 of any of the ILT4 Abs provided herein. Unless otherwise specified, VH CDRs herein are as described herein Figure 1 、 Figure 3 、 Figure 5 、 Figure 7 、 Figure 9 and Figure 11The Kabat CDRs shown in, e.g., AbM CDR1, etc. In certain embodiments, the ILT4 Ab comprises a VH comprising VH CDR1, CDR2, and / or CDR3 of any one of the ILT4 Abs provided herein. In certain embodiments, the ILT4 Ab comprises a VH comprising VH CDR1, CDR2, and / or CDR3 of any 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 (SEQ ID NO:125 - 127), 9C8 (SEQ ID NO:131 - 133), 2H2 (SEQ ID NO:137 - 139), 2E5 (SEQ ID NO:143 - 145), 24E5 (SEQ ID NO:149 - 151), 21D9 (SEQ ID NO:155 - 157), 21D9.b (SEQ ID NO:155 - 157), 21D9.c (SEQ ID NO:155 - 157), 21D9.d (SEQ ID NO:155 - 157), 21D9.e (SEQ ID NO:155 - 157), 21A5 (SEQ ID NO:161 - 163), 10F10 (SEQ ID NO:167 - 169), 10F10.1 (SEQ ID NO:167 - 169), 10F10.3 (SEQ ID NO:167 - 169), or 10F10.4 (SEQ ID NO:167 - 169)).

[0267] In certain embodiments, the ILT4 Ab comprises a VH comprising VH CDR1, CDR2, and CDR3 of any one of the ILT4 Abs provided herein. In certain embodiments, the ILT4 Ab comprises a VH comprising VH CDR1, CDR2, and / or CDR3 of 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.

[0268] In certain embodiments, the ILT4 Ab comprises a VH comprising VH CDR1, CDR2, and / or CDR3 of any one of the ILT4 Abs provided herein and a VL comprising CDR1, CDR2, and / or CDR3 of any one of the ILT4 Abs provided herein. Unless otherwise specified, the VL CDRs herein are as described herein Figure 2, Figure 4 , Figure 6 , Figure 8 , Figure and ​ the Kabat CDRs shown in, e.g., AbM CDR1, etc. In certain embodiments, an ILT4 Ab comprises a VH comprising the VH CDR1, CDR2, and CDR3 of any one of the ILT4 Abs provided herein and a VL comprising the CDR1, CDR2, and CDR3 of any one of the ILT4 Abs provided herein. In certain embodiments, an ILT4 Ab comprises a VH comprising the VH CDR1, CDR2, and / or CDR3 of 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 and a VL comprising the VL CDR1, CDR2, and / or CDR3 of 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: 128 - 130), 9C8 (SEQ ID NO: 134 - 136), 2H2 (SEQ ID NO: 140 - 142), 2E5 (SEQ ID NO: 146 - 148), 24E5 (SEQ ID NO: 152 - 154), 21D9 (SEQ ID NO: 158 - 160), 21D9.b (SEQ ID NO: 158 - 160), 21D9.c (SEQ ID NO: 158 - 160), 21D9.d (SEQ ID NO: 158 - 160), 21D9.e (SEQ ID NO: 158 - 160), 21A5 (SEQ ID NO: 164 - 166), 10F10 (SEQ ID NO: 170 - 172), 10F10.1 (SEQ ID NO: 170 - 172), 10F10.3 (SEQ ID NO: 170 - 172), or 10F10.4 (SEQ ID NO: 170 - 172)).

[0269] In some embodiments, an ILT4 Ab may comprise:

[0270] (a) a VH comprising the amino acid sequence of the VH CDR1, CDR2, and CDR3 of 9G4, and a VL comprising the VL CDR1, CDR2, and CDR3 of 9G4;

[0271] (b) A VH comprising the amino acid sequences of the VH CDR1, CDR2, and CDR3 of 9C8, and a VL comprising the VL CDR1, CDR2, and CDR3 of 9C8;

[0272] (c) A VH comprising the amino acid sequences of the VH CDR1, CDR2, and CDR3 of 2H2, and a VL comprising the VL CDR1, CDR2, and CDR3 of 2H2;

[0273] (d) A VH comprising the amino acid sequences of the VH CDR1, CDR2, and CDR3 of 25E5, and a VL comprising the VL CDR1, CDR2, and CDR3 of 25E5;

[0274] (e) A VH comprising the amino acid sequences of the VH CDR1, CDR2, and CDR3 of 24E5, and a VL comprising the VL CDR1, CDR2, and CDR3 of 24E5;

[0275] (f) A VH comprising the amino acid sequences of the VH CDR1, CDR2, and CDR3 of 21D9, and a VL comprising the VL CDR1, CDR2, and CDR3 of 21D9;

[0276] (g) A VH comprising the amino acid sequences of the VH CDR1, CDR2, and CDR3 of 21D9.b, and a VL comprising the VL CDR1, CDR2, and CDR3 of 21D9.b;

[0277] (h) A VH comprising the amino acid sequences of the VH CDR1, CDR2, and CDR3 of 21D9.c, and a VL comprising the VL CDR1, CDR2, and CDR3 of 21D9.c;

[0278] (i) A VH comprising the amino acid sequences of the VH CDR1, CDR2, and CDR3 of 21D9.d, and a VL comprising the VL CDR1, CDR2, and CDR3 of 21D9.d;

[0279] (j) A VH comprising the amino acid sequences of the VH CDR1, CDR2, and CDR3 of 21D9.e, and a VL comprising the VL CDR1, CDR2, and CDR3 of 21D9.e;

[0280] (k) A VH comprising the amino acid sequences of the VH CDR1, CDR2, and CDR3 of 21A5, and a VL comprising the VL CDR1, CDR2, and CDR3 of 21A5;

[0281] (l) A VH comprising the amino acid sequences of the VH CDR1, CDR2, and CDR3 of 21A5.a, and a VL comprising the VL CDR1, CDR2, and CDR3 of 21A5.a;

[0282] (m) A VH comprising the amino acid sequences of the VH CDR1, CDR2 and CDR3 of 10F10, and a VL comprising the VL CDR1, CDR2 and CDR3 of 10F10;

[0283] (n) A VH comprising the amino acid sequences of the VH CDR1, CDR2 and CDR3 of 10F10.1, and a VL comprising the VL CDR1, CDR2 and CDR3 of 10F10.1;

[0284] (o) A VH comprising the amino acid sequences of the VH CDR1, CDR2 and CDR3 of 10F10.3, and a VL comprising the VL CDR1, CDR2 and CDR3 of 10F10.3; or

[0285] (p) A VH comprising the amino acid sequences of the VH CDR1, CDR2 and CDR3 of 10F10.4, and a VL comprising the VL CDR1, CDR2 and CDR3 of 10F10.4.

[0286] Again, the Sequence Listing below provides the heavy and light chain variable region sequences and the full-length heavy and light chain sequences of the antibodies listed above.

[0287] In certain embodiments, the ILT4 Ab comprises a VH comprising the amino acid sequence of the VH of any of the ILT4 Abs provided herein. The individual VH sequences of specific antibody species provided by the present invention are listed in the sequence listing. In certain embodiments, the ILT4 Ab comprises a VH comprising the amino acid sequence of the VH of any 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., any 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)).

[0288] In some embodiments, the ILT4 Ab comprises a VH of any of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 2\1D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4, but having 1, 2, 3, 4, or 5 amino acid substitutions (such as 1, 2, 3, 4, or 5 conservative substitutions) in the framework region of the VH sequence. In some embodiments, the ILT4 Ab comprises a VH of any 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 having 1, 2, 3, 4, or 5 back substitutions in the framework region of the VH sequence.

[0289] In some embodiments, the ILT4 Ab comprises VH CDRs and VL CDRs of any one of the ILT4 Abs described herein, wherein among all the CDRs, these CDRs contain 1, 2, or 3 amino acid additions, substitutions (such as conservative substitutions), or deletions.

[0290] In certain embodiments, the ILT4 Ab comprises VH CDR1, CDR2, and CDR3 having amino acid sequences that comprise the VH CDRs of any one of the ILT4 Abs provided herein and a VH having 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% identity to the VH of any one of the ILT4 Abs provided herein. In certain embodiments, the ILT4 Ab comprises a VH having an amino acid sequence having 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% identity to the amino acid sequence of the VH of any one of the following: 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 the VH of the VH sequence shown in the sequence listing by 1, 2, 3, 4, or 5 amino acid substitutions (such as 1, 2, 3, 4, or 5 conservative substitutions) in the framework region of the VH sequence. In certain embodiments, the VH of the antibody differs from the VH of the VH sequence shown in the sequence listing by 1, 2, 3, 4, or 5 back substitutions in the framework region of the VH sequence.

[0291] In certain embodiments, the ILT4 Ab comprises a VH consisting of the amino acid sequence of the VH of any one of the ILT4 Abs provided herein. In certain embodiments, the ILT4 Ab comprises a VH consisting of the amino acid sequence of the VH of 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.

[0292] In certain embodiments, the ILT4 Ab comprises a VL comprising the amino acid sequence of the VL of any one of the ILT4 Abs provided herein. In certain embodiments, the ILT4 Ab comprises a VL comprising the amino acid sequence of the VL of 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), 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)). In certain embodiments, the ILT4 Ab comprises VL CDR1, CDR2, and CDR3 comprising the amino acid sequences of the VL CDRs of any one of the ILT4 Abs provided herein and comprises a VL having 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% identity to the VL of any one of the ILT4 Abs provided herein. In certain embodiments, the ILT4 Ab comprises a VL comprising an amino acid sequence having 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% identity to the amino acid sequence of the VL of 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. In certain embodiments, the VL of the antibody differs from the VL sequence shown in the sequence listing by 1, 2, 3, 4, or 5 amino acid substitutions (such as 1, 2, 3, 4, or 5 conservative substitutions) in the framework region of the VL sequence.In certain embodiments, the VL of the antibody differs from the VL of the VL sequence shown in the Sequence Listing by 1, 2, 3, 4, or 5 revertant substitutions.

[0293] In certain embodiments, the ILT4 Ab comprises a VL consisting of the amino acid sequence of the VL of any one of the ILT4 Abs provided herein. In certain embodiments, the ILT4 Ab comprises a VL consisting of the amino acid sequence of the VL of 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.

[0294] In certain embodiments, the ILT4 Ab comprises a VH comprising the amino acid sequence of the VH of any one of the ILT4 Abs provided herein and a VL comprising the amino acid sequence of the VL of any one of the ILT4 Abs provided herein. In some of these embodiments, the ILT4 Ab comprises a VH comprising the amino acid sequence of the VH of 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 and a VL comprising the amino acid sequence of the VL of 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.

[0295] However, in certain embodiments, the VH of the antibody 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, but has 1, 2, 3, 4, or 5 amino acid substitutions (such as 1, 2, 3, 4, or 5 conservative substitutions) in the framework region of the VH sequence, and the VL is a VL from any one of the same species in the list above. However, in certain embodiments, the VH of the antibody 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, but has 1, 2, 3, 4, or 5 revertant substitutions in the framework region of the VH sequence.

[0296] In certain embodiments, the ILT4 Ab comprises a VH and a VL comprising the amino acid sequences of a VH and a VL of 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.

[0297] In certain embodiments, the ILT4 Ab comprises VH CDR1, CDR2, and CDR3 comprising the amino acid sequences of the VH CDRs of any one of the ILT4 Abs provided herein and VL CDR1, CDR2, and CDR3 comprising the amino acid sequences of the VL CDRs of any one of the ILT4 Abs provided herein, and further comprises a VH and a VL that each have 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% identity to the corresponding VH and VL of any one of the ILT4 Abs 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 (such as 1, 2, 3, 4, or 5 conservative substitutions or such as 1, 2, 3, 4, or 5 revertant substitutions) in the framework region of the sequence.

[0298] In certain embodiments, the ILT4 Ab comprises a VH and a VL consisting of the amino acid sequences of the VH and VL of any one of the ILT4 Abs provided herein. In certain embodiments, the ILT4 Ab comprises a VH and a VL each consisting of the amino acid sequences of the VH and VL of 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.

[0299] An ILT4 Ab can comprise:

[0300] (a) a VH comprising the amino acid sequence of the VH of 9G4 and a VL comprising the amino acid sequence of the VL of 9G4;

[0301] (b) a VH comprising the amino acid sequence of the VH of 9C8 and a VL comprising the amino acid sequence of the VL of 9C8;

[0302] (c) a VH comprising the amino acid sequence of the VH of 2H2 and a VL comprising the amino acid sequence of the VL of 2H2;

[0303] (d) a VH comprising the amino acid sequence of the VH of 25E5 and a VL comprising the amino acid sequence of the VL of 25E5;

[0304] (e) a VH comprising the amino acid sequence of the VH of 24E5 and a VL comprising the amino acid sequence of the VL of 24E5;

[0305] (f) a VH comprising the amino acid sequence of the VH of 21D9 and a VL comprising the amino acid sequence of the VL of 21D9;

[0306] (g) a VH comprising the amino acid sequence of the VH of 21D9.b and a VL comprising the amino acid sequence of the VL of 21D9.b;

[0307] (h) a VH comprising the amino acid sequence of the VH of 21D9.c and a VL comprising the amino acid sequence of the VL of 21D9.c;

[0308] (i) a VH comprising the amino acid sequence of the VH of 21D9.d and a VL comprising the amino acid sequence of the VL of 21D9.d;

[0309] (j) a VH comprising the amino acid sequence of the VH of 21D9.e and a VL comprising the amino acid sequence of the VL of 21D9.e;

[0310] (k) a VH comprising the amino acid sequence of the VH of 21A5 and a VL comprising the amino acid sequence of the VL of 21A5;

[0311] (l) A VH comprising the amino acid sequence of the VH of 21A5.a and a VL comprising the amino acid sequence of the VL of 21A5.a;

[0312] (m) A VH comprising the amino acid sequence of the VH of 10F10 and a VL comprising the amino acid sequence of the VL of 10F10;

[0313] (n) A VH comprising the amino acid sequence of the VH of 10F10.1 and a VL comprising the amino acid sequence of the VL of 10F10.1;

[0314] (o) A VH comprising the amino acid sequence of the VH of 10F10.3 and a VL comprising the amino acid sequence of the VL of 10F10.3; or

[0315] (p) A VH comprising the amino acid sequence of the VH of 10F10.4 and a VL comprising the amino acid sequence of the VL of 10F10.4.

[0316] An ILT4 Ab may comprise:

[0317] (a) A VH comprising the VH CDR of the VH of 9G4 and a VL comprising the VL CDR of 9G4, and VH and VL amino acid sequences having 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% identity to the VH and VL of 9G4;

[0318] (b) A VH comprising the VH CDR of the VH of 9C8 and a VL comprising the VL CDR of 9C8, and VH and VL amino acid sequences having 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% identity to the VH and VL of 9C8;

[0319] (c) A VH comprising the VH CDR of the VH of 2H2 and a VL comprising the VL CDR of 2H2, and VH and VL amino acid sequences having 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% identity to the VH and VL of 2H2;

[0320] (d) A VH comprising the VH CDR of the VH of 25E5 and a VL comprising the VL CDR of 25E5, and VH and VL amino acid sequences having 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% identity to the VH and VL of 25E5;

[0321] (e) A VH having the VH CDR of the VH containing 24E5 and a VL having the VL CDR of the VL containing 24E5, and VH and VL amino acid sequences having 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% identity to the VH and VL of 24E5;

[0322] (f) A VH having the VH CDR of the VH containing 21D9 and a VL having the VL CDR of the VL containing 21D9, and VH and VL amino acid sequences having 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% identity to the VH and VL of 21D9;

[0323] (g) A VH having the VH CDR of the VH containing 21D9.b and a VL having the VL CDR of the VL containing 21D9.b, and VH and VL amino acid sequences having 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% identity to the VH and VL of 21D9.b;

[0324] (h) A VH having the VH CDR of the VH containing 21D9.c and a VL having the VL CDR of the VL containing 21D9.c, and VH and VL amino acid sequences having 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% identity to the VH and VL of 21D9.c;

[0325] (i) A VH having the VH CDR of the VH containing 21D9.d and a VL having the VL CDR of the VL containing 21D9.d, and VH and VL amino acid sequences having 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% identity to the VH and VL of 21D9.d;

[0326] (j) A VH having the VH CDR of the VH containing 21D9.e and a VL having the VL CDR of the VL containing 21D9.e, and VH and VL amino acid sequences having 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% identity to the VH and VL of 21D9.e;

[0327] (k) a VH comprising the VH CDR of the VH of 21A5 and a VL comprising the VL CDR of the VL of 21A5, and VH and VL amino acid sequences having 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% identity to the VH and VL of 21A5;

[0328] (l) a VH comprising the VH CDR of the VH of 21A5.a and a VL comprising the VL CDR of the VL of 21A5.a, and VH and VL amino acid sequences having 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% identity to the VH and VL of 21A5.a;

[0329] (m) a VH comprising the VH CDR of the VH of 10F10 and a VL comprising the VL CDR of the VL of 10F10, and VH and VL amino acid sequences having 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% identity to the VH and VL of 10F10;

[0330] (n) a VH comprising the VH CDR of the VH of 10F10.1 and a VL comprising the VL CDR of the VL of 10F10.1, and VH and VL amino acid sequences having 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% identity to the VH and VL of 10F10.1;

[0331] (o) a VH comprising the VH CDR of the VH of 10F10.3 and a VL comprising the VL CDR of the VL of 10F10.3, and VH and VL amino acid sequences having 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% identity to the VH and VL of 10F10.3; or

[0332] (p) a VH comprising the VH CDR of the VH of 10F10.4 and a VL comprising the VL CDR of the VL of 10F10.4, and VH and VL amino acid sequences having 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% identity to the VH and VL of 10F10.4.

[0333] In some of the above embodiments, the VH and / or VL may differ from the sequences of each of species (a) to (p) due to the presence of 1, 2, 3, 4, or 5 amino acid substitutions (such as 1, 2, 3, 4, or 5 conservative substitutions). In some embodiments, the VH may contain 1, 2, 3, 4, or 5 revertant substitutions.

[0334] An ILT4 Ab may comprise:

[0335] (a) a VH consisting of the amino acid sequence of the VH of 9G4 and a VL consisting of the VL of 9G4;

[0336] (b) a VH consisting of the amino acid sequence of the VH of 9C8 and a VL consisting of the VL of 9C8;

[0337] (c) a VH consisting of the amino acid sequence of the VH of 2H2 and a VL consisting of the VL of 2H2;

[0338] (d) a VH consisting of the amino acid sequence of the VH of 25E5 and a VL consisting of the VL of 25E5;

[0339] (e) a VH consisting of the amino acid sequence of the VH of 24E5 and a VL consisting of the VL of 24E5;

[0340] (f) a VH consisting of the amino acid sequence of the VH of 21D9 and a VL consisting of the VL of 21D9;

[0341] (g) a VH consisting of the amino acid sequence of the VH of 21D9.b and a VL consisting of the VL of 21D9.b;

[0342] (h) a VH consisting of the amino acid sequence of the VH of 21D9.c and a VL consisting of the VL of 21D9.c;

[0343] (i) a VH consisting of the amino acid sequence of the VH of 21D9.d and a VL consisting of the VL of 21D9.d;

[0344] (j) a VH consisting of the amino acid sequence of the VH of 21D9.e and a VL consisting of the VL of 21D9.e;

[0345] (k) a VH consisting of the amino acid sequence of the VH of 21A5 and a VL consisting of the VL of 21A5;

[0346] (l) a VH consisting of the amino acid sequence of the VH of 21A5.a and a VL consisting of the VL of 21A5.a;

[0347] (m) a VH consisting of the amino acid sequence of the VH of 10F10 and a VL consisting of the VL of 10F10;

[0348] (n) A VH consisting of the amino acid sequence of VH of 10F10.1 and a VL consisting of VL of 10F10.1;

[0349] (o) A VH consisting of the amino acid sequence of VH of 10F10.3 and a VL consisting of VL of 10F10.3; or

[0350] (p) A VH consisting of the amino acid sequence of VH of 10F10.4 and a VL consisting of VL of 10F10.4.

[0351] In certain embodiments, the ILT4 Ab comprises the variable region of the antibodies described above and elsewhere herein and / or any one of the variable region CDRs 1 to 3, such as:

[0352] (1) One or more of the following VH CDR1, CDR2, and CDR3: 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;

[0353] (2) The following VH CDR1, CDR2, and CDR3: 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;

[0354] (3) The following VH: 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;

[0355] (4) One or more of the following VH CDR1, CDR2, and CDR3 and one or more of the following VL CDR1, CDR2, and CDR3: 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;

[0356] (5) The following VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3: 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;

[0357] (6) 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;

[0358] or

[0359] (7) Exceptions are VL and VH having 1, 2, 3, 4, or 5 replacements or conservative substitutions in the following VH and / or 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; and the ILT4 Ab is also an IgG antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody or a modified form thereof as described in the following sections. In some embodiments, the constant region has effector function, and in some embodiments, the constant region has no effector function. In certain embodiments, the constant region is the constant region of IgG1.3 or IgG1.1f or another constant region described herein, such as IgG1 and IgG1.238K.

[0360] In certain embodiments, the ILT4 Ab comprises a variable region of the antibody described above and elsewhere herein and / or any one of variable region CDRs 1 to 3, such as:

[0361] (1) One or more of the following VH CDR1, CDR2, and CDR3: 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;

[0362] (2) The following VH CDR1, CDR2, and CDR3: 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;

[0363] (3) The following VH: 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 2A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4;

[0364] (4) One or more of the following VH CDR1, CDR2, and CDR3 and one or more of the following VL CDR1, CDR2, and CDR3: 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;

[0365] (5) The following VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3: 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;

[0366] (6) 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;

[0367] or

[0368] (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, with the exception of having 1, 2, 3, 4, or 5 replacements or conservative substitutions in the VH and / or VL;

[0369] and

[0370] The antibody further comprises one or more of the following features:

[0371] - For example, at 10 -8 M, 10 -9 M or lower K D specifically binds to hILT4;

[0372] - Does not specifically bind to hILT2, hILT3, hILT5;

[0373] - Does not specifically bind to one or more members of the LILRA and / or LILRB family;

[0374] - For example, stimulates T cell activation in the MLR, as measured by increased T cell proliferation or IFN-γ secretion, such as shown in the assays described in the examples;

[0375] - Stimulates the differentiation or activation of monocytes into macrophages, for example, stimulates the differentiation of monocytes into pro-inflammatory macrophages, such as shown in the assays described in the examples;

[0376] - Promotes the expression of CD83 and CD86 on human monocyte-derived immature dendritic cells (Mo-iDCs), such as shown in the assays described in the examples;

[0377] - Enhances IFN-γ secretion after antigen stimulation in the CMV lysis assay, such as shown in the assays described in the examples;

[0378] - Enhances IFN-γ and TNF-α secretion after CD3 stimulation by CD4+ and CD8+ T cells in the allogeneic mixed lymphocyte reaction (allogeneic MLR) assay, such as shown in the assays described in the examples;

[0379] - Does not induce (or trigger) basophil activation;

[0380] - Inhibits the binding of HLA-A and / or HLA-B to ILT4;

[0381] - Has a binding profile to hILT4 as shown in Figure 27 or ​ as shown therein;

[0382] - Binds to Ig-like domains 1, 2 or 1 and 2 of hILT4 (which correspond to amino acids 27-110 and 111-229 of SEQ ID NO: 107, respectively), such as binds to a region comprising: (i) 70 ITRIRPEL 77 (SEQ ID NO: 120) and / or 78 VKNGQFHIPSIT WEHTGRYGCQY 100(SEQ ID NO:121); (ii) 70 ITRIRPELVKNGQFHIPSITWEHTGRYGCQY 100 (SEQ ID NO:122); or (iii) 154 ILCKEGE EEHPQCLNSQPHARGSSRAIF 181 (SEQ ID NO:123) and / or 425 SSPPPT GPIS 434 (SEQ ID NO:124), as determined by hydrogen-deuterium exchange (HDX), e.g., as shown in the HDX assays described in the Examples;

[0383] - does not significantly bind to other regions of the extracellular domain of ILT4, such as regions or residues located N-terminal to amino acid I70; wherein the amino acid numbering of hILT4 is the amino acid numbering of immature hILT4 (i.e., ILT4 with its signal sequence);

[0384] - competes with the antibodies described herein for binding to hILT4;

[0385] - specifically binds to cynomolgus monkey ILT4 comprising SEQ ID NO:118, e.g., as shown in the binding assays described in the Examples;

[0386] - promotes the pro-inflammatory polarization of macrophages towards M1 macrophages;

[0387] - does not induce (or trigger) basophil activation; and

[0388] - contains less than 5% high molecular weight material and low molecular weight material after incubation at 25 °C for 3 months, and / or contains less than 10% high molecular weight material and low molecular weight material after incubation at 40 °C for 3 months.

[0389] In certain embodiments, the ILT4 Ab comprises a heavy chain (HC) comprising the amino acid sequence of the heavy chain of any of the ILT4 Abs provided herein. In certain embodiments, the ILT4 Ab comprises a heavy chain comprising the amino acid sequence of the heavy chain of any 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 sequence listing below (i.e., the complete HC sequence as provided in the said table, or a composite sequence of the VH sequence of the antibody and the HC constant region sequence, such as SEQ ID NO:98, 100, 102, 103, 104, or 179 as provided in the said table).

[0390] Thus, for example, in some embodiments, the antibody may comprise an HC amino acid sequence of one of the following types, comprising the following amino acid sequences:

[0391] 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),

[0392] 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),

[0393] c. IgG1.3 (such as 9G4.IgG1.3, etc.), 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 SEQ ID NO:100), 10F10.3 (SEQ ID NO:91 and SEQ ID NO:100) or 10F10.4 (SEQ ID NO:91 and SEQ ID NO:100);

[0394] d. IgG1.1f (such as 9G4.IgG1.1f, etc.), 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 (SEQ ID NO:87 and SEQ ID NO:103), 10F10 (SEQ ID NO:91 and SEQ ID NO:103), 10F10.1 (SEQ ID NO:91 and SEQ ID NO:103), 10F10.3 (SEQ ID NO:91 and SEQ ID NO:103) or 10F10.4 (SEQ ID NO:91 and SEQ ID NO:103),

[0395] e. IgG1fa.P238K (such as 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

[0396] f. IgG4 S228P (such as 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).

[0397] In certain embodiments, an ILT4 Ab comprises: a heavy chain comprising the amino acid sequence of the heavy chain of any one of the ILT4 Abs provided herein, which comprises an IgG1.3 heavy chain constant region; and the amino acid sequence of the light chain of any one of the ILT4 Abs provided herein. In certain embodiments, an ILT4 Ab comprises: a heavy chain comprising the amino acid sequence of the VH of 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, which comprises an IgG1.3 HC constant region (designated as 9G4.IgG1.3, etc.); and a light chain comprising the amino acid sequence of the light chain of 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. In some embodiments, the light chain is a κ light chain comprising the following amino acid sequences: 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).

[0398] An ILT4 Ab may comprise:

[0399] (a) a heavy chain comprising the amino acid sequence of the heavy chain of 9G4 and a light chain comprising the amino acid sequence of the light chain of 9G4;

[0400] (b) a heavy chain comprising the amino acid sequence of the heavy chain of 9C8 and a light chain comprising the amino acid sequence of the light chain of 9C8;

[0401] (c) a heavy chain comprising the amino acid sequence of the heavy chain of 2H2 and a light chain comprising the amino acid sequence of the light chain of 2H2;

[0402] (d) a heavy chain comprising the amino acid sequence of the heavy chain of 25E5 and a light chain comprising the amino acid sequence of the light chain of 25E5;

[0403] (e) A heavy chain comprising the amino acid sequence of the heavy chain of 24E5 and a light chain comprising the amino acid sequence of the light chain of 24E5;

[0404] (f) A heavy chain comprising the amino acid sequence of the heavy chain of 9G4 and a light chain comprising the amino acid sequence of the light chain of 21D9;

[0405] (g) A heavy chain comprising the amino acid sequence of the heavy chain of 21D9.b and a light chain comprising the amino acid sequence of the light chain of 21D9.b;

[0406] (h) A heavy chain comprising the amino acid sequence of the heavy chain of 21D9.c and a light chain comprising the amino acid sequence of the light chain of 21D9.c;

[0407] (i) A heavy chain comprising the amino acid sequence of the heavy chain of 21D9.d and a light chain comprising the amino acid sequence of the light chain of 21D9.d;

[0408] (j) A heavy chain comprising the amino acid sequence of the heavy chain of 21D9.e and a light chain comprising the amino acid sequence of the light chain of 21D9.e;

[0409] (k) A heavy chain comprising the amino acid sequence of the heavy chain of 21A5 and a light chain comprising the amino acid sequence of the light chain of 21A5;

[0410] (l) A heavy chain comprising the amino acid sequence of the heavy chain of 21A5.a and a light chain comprising the amino acid sequence of the light chain of 21A5.a;

[0411] (m) A heavy chain comprising the amino acid sequence of the heavy chain of 10F10 and a light chain comprising the amino acid sequence of the light chain of 10F10;

[0412] (n) A heavy chain comprising the amino acid sequence of the heavy chain of 10F10.1 and a light chain comprising the amino acid sequence of the light chain of 10F10.1;

[0413] (o) A heavy chain comprising the amino acid sequence of the heavy chain of 10F10.3 and a light chain comprising the amino acid sequence of the light chain of 10F10.3; or

[0414] (p) A heavy chain comprising the amino acid sequence of the heavy chain of 10F10.4 and a light chain comprising the amino acid sequence of the light chain of 10F10.4.

[0415] An ILT4 Ab may comprise:

[0416] (a) The heavy chain (HC) of the HC CDR of the HC containing 9G4 and the light chain (LC) of the LC CDR of the LC containing 9G4, and HC and LC amino acid sequences that have 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% identity to the HC and LC of 9G4, respectively;

[0417] (b) The HC of the HC CDR of the HC containing 9C8 and the light chain (LC) of the LC CDR of the LC containing 9C8, and HC and LC amino acid sequences that have 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% identity to the HC and LC of 9C8, respectively;

[0418] (c) The HC of the HC CDR of the HC containing 2H2 and the light chain (LC) of the LC CDR of the LC containing 2H2, and HC and LC amino acid sequences that have 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% identity to the HC and LC of 2H2, respectively;

[0419] (d) The HC of the HC CDR of the HC containing 25E5 and the light chain (LC) of the LC CDR of the LC containing 25E5, and HC and LC amino acid sequences that have 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% identity to the HC and LC of 25E5, respectively;

[0420] (e) The HC of the HC CDR of the HC containing 24E5 and the light chain (LC) of the LC CDR of the LC containing 24E5, and HC and LC amino acid sequences that have 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% identity to the HC and LC of 24E5, respectively;

[0421] (f) The HC of the HC CDR of the HC containing 21D9 and the light chain (LC) of the LC CDR of the LC containing 21D9, and HC and LC amino acid sequences that have 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% identity to the HC and LC of 21D9, respectively;

[0422] (g) An HC of the HC CDR of an HC containing 21D9.b and a light chain (LC) of the LC CDR of an LC containing 21D9.b, and HC and LC amino acid sequences that have 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% identity to the HC and LC of 21D9.b, respectively;

[0423] (h) An HC of the HC CDR of an HC containing 21D9.c and a light chain (LC) of the LC CDR of an LC containing 21D9.c, and HC and LC amino acid sequences that have 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% identity to the HC and LC of 21D9.c, respectively;

[0424] (i) An HC of the HC CDR of an HC containing 21D9.d and a light chain (LC) of the LC CDR of an LC containing 21D9.d, and HC and LC amino acid sequences that have 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% identity to the HC and LC of 21D9.d, respectively;

[0425] (j) An HC of the HC CDR of an HC containing 21D9.e and a light chain (LC) of the LC CDR of an LC containing 21D9.e, and HC and LC amino acid sequences that have 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% identity to the HC and LC of 21D9.e, respectively;

[0426] (k) An HC of the HC CDR of an HC containing 21A5 and a light chain (LC) of the LC CDR of an LC containing 21A5, and HC and LC amino acid sequences that have 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% identity to the HC and LC of 21A5, respectively;

[0427] (l) An HC of the HC CDR of an HC containing 21A5.a and a light chain (LC) of the LC CDR of an LC containing 21A5.a, and HC and LC amino acid sequences that have 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% identity to the HC and LC of 21A5.a, respectively;

[0428] (m) An HC of the HC CDR of an HC containing 10F10 and a light chain (LC) of the LC CDR of an LC containing 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;

[0429] (n) An HC of the HC CDR of an HC containing 10F10.1 and a light chain (LC) of the LC CDR of an LC containing 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;

[0430] (o) An HC of the HC CDR of an HC containing 10F10.3 and a light chain (LC) of the LC CDR of an LC containing 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

[0431] (p) An HC of the HC CDR of an HC containing 10F10.3 and a light chain (LC) of the LC CDR of an LC containing 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.

[0432] In some of the above embodiments, the HC and / or LC may differ from the sequences of each of species (a) to (p) due to the presence of 1, 2, 3, 4, or 5 amino acid substitutions (such as 1, 2, 3, 4, or 5 conservative substitutions). In some of the above embodiments, the HC and / or LC may differ from the sequences of each of species (a) to (p) due to the presence of 1, 2, 3, 4, or 5 amino acid substitutions (such as 1, 2, 3, 4, or 5 back substitutions).

[0433] In some embodiments, the ILT4 Ab may comprise:

[0434] (a) A heavy chain consisting of the amino acid sequence of the heavy chain of 9G4 and a light chain consisting of the amino acid sequence of the light chain of 9G4;

[0435] (b) A heavy chain consisting of the amino acid sequence of the heavy chain of 9C8 and a light chain consisting of the amino acid sequence of the light chain of 9C8;

[0436] (c) A heavy chain consisting of the amino acid sequence of the heavy chain of 2H2 and a light chain consisting of the amino acid sequence of the light chain of 2H2;

[0437] (d) A heavy chain consisting of the amino acid sequence of the heavy chain of 25E5 and a light chain consisting of the amino acid sequence of the light chain of 25E5;

[0438] (e) A heavy chain consisting of the amino acid sequence of the heavy chain of 24E5 and a light chain consisting of the amino acid sequence of the light chain of 24E5;

[0439] (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;

[0440] (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;

[0441] (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;

[0442] (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;

[0443] (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;

[0444] (k) A heavy chain consisting of the amino acid sequence of the heavy chain of 21A5 and a light chain consisting of the amino acid sequence of the light chain of 21A5;

[0445] (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;

[0446] (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;

[0447] (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;

[0448] (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

[0449] (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.

[0450] In some embodiments, the ILT4 Ab can comprise a heavy chain amino acid sequence that includes the VH amino acid sequence of the antibody species herein, rather than the IgG1.3 heavy chain constant region as provided in the HC sequence in the sequence listing herein (and see SEQ ID NO:100), and the antibody can comprise different heavy chain constant region sequences, such as the human wild-type IgG1 constant region, such as those provided in SEQ ID NO:98 and 102 - 104, or the IgG4 heavy chain constant region, or the IgG4 constant region with an S228P substitution (EU numbering), or any of the other heavy chain constant regions described in the following sections regarding constant regions.

[0451] In some embodiments, such modified ILT4 Abs have one or more of the following characteristics:

[0452] - For example, with a K -8 of 10 -9 M or lower, or 10 D specifically binds to hILT4;

[0453] - Does not specifically bind to one, two, or all three of hILT2, hILT3, hILT5;

[0454] - Does not specifically bind to one or more members of the LILRA and / or LILRB family;

[0455] - For example, stimulates T cell activation in an MLR assay, as measured by increased T cell proliferation or IFN-γ secretion; and

[0456] stimulates monocyte differentiation or activation into macrophages.

[0457] In some embodiments, the antibody binds to Ig-like domain 1, 2, or 1 and 2 of hILT4, which corresponds to amino acids 27 - 110 (domain 1) on sequence SEQ ID NO:107 and amino acids 111 - 229 (domain 2) on said sequence, and contains 70 ITRIRPEL 77 (SEQ ID NO:120) and / or 78 VKNGQFHIPSITWEHTGRYGCQY 100 (SEQID 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), as determined by hydrogen-deuterium exchange (HDX), such as shown in the HDX assays described in the Examples. (See Figure 27.)

[0458] Exemplary antibody constant regions

[0459] In some embodiments, the antibodies described herein comprise one or more human constant regions. In some embodiments, the human heavy chain constant regions are of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant regions are of an isotype selected from κ and λ. In some embodiments, the antibodies described herein comprise a human IgG constant region, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibodies described herein comprise a human IgG4 heavy chain constant region. In some such embodiments, the antibodies described herein comprise the S241P mutation in the human IgG4 constant region. In some embodiments, the antibodies described herein comprise a human IgG4 constant region and a human κ light chain.

[0460] The choice of heavy chain constant region can determine whether the antibody will have in vivo effector functions. In some embodiments, such effector functions include antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), and can result in killing of the cells to which the antibody binds. In some therapeutic methods, including methods for treating some cancers, for example when the antibody binds to cells that support the maintenance or growth of a tumor, it may be desirable to kill the cells. Exemplary cells that can support the maintenance or growth of a tumor include (but are not limited to) the tumor cells themselves, cells that help recruit blood vessels to the tumor, and cells that provide support or promote tumor growth or tumor survival ligands, growth factors, or counter-receptors. In some embodiments, when effector functions are desired, antibodies comprising a human IgG1 heavy chain or a human IgG3 heavy chain are selected.

[0461] In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of antibody glycosylation. Adding glycosylation sites to or removing glycosylation sites from the antibody can be readily achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0462] In the case where an antibody comprises an Fc region, the carbohydrates attached thereto can be altered. Native antibodies produced by mammalian cells typically comprise branched-chain biantennary oligosaccharides that are generally attached via an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention can be modified to produce antibodies having certain improved properties. For example, in some embodiments, an antibody can be defucosylated, e.g., by mutating a residue such as Asn297 that is normally glycosylated with fucose-containing sugars or glycosylated via other means. In some embodiments, the antibodies herein can comprise a defucosylated human IgG1 constant region.

[0463] Antibodies having equianalyte oligosaccharides are also provided, e.g., wherein the biantennary oligosaccharides attached to the Fc region of the antibody are equianalyzed by GlcNAc. Such antibodies can have reduced fucosylation and / or improved ADCC function. Examples of such antibodies are described, e.g., in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.). Antibodies are also provided in which at least one galactose residue in the oligosaccharide is attached to the Fc region. Such antibodies can have improved CDC function. Such antibodies are described, e.g., in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0464] Antibodies having an amino-terminal leader sequence extension are also provided. For example, one or more amino acid residues of the amino-terminal leader sequence are present at the amino terminus of any one or more of the heavy or light chains of the antibody. Exemplary amino-terminal leader sequence extensions comprise or consist of three amino acid residues VHS, which are present on one or both of the light chains of the antibody.

[0465] The in vivo or serum half-life of a human FcRn high-affinity binding polypeptide can be determined, e.g., in transgenic mice, humans, or non-human primates administered a polypeptide having a variant Fc region. See also, e.g., Petkova et al., International Immunology 18(12):1759-1769 (2006).

[0466] In some embodiments of the invention, the afucosylated antibody more effectively mediates ADCC in the presence of human effector cells as compared to the parental antibody that contains fucose. Generally, ADCC activity can be measured using an in vitro ADCC assay as disclosed herein, but other assays or methods for measuring ADCC activity, such as in animal models and the like, are encompassed.

[0467] In certain embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter one or more effector functions 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 different amino acid residues such that the affinity of the antibody for an effector ligand is altered while retaining the antigen-binding ability of the parental antibody. The effector ligand with altered affinity may be, for example, an Fc receptor or the C1 component of complement. This method is also described in detail in both U.S. Patent Nos. 5,624,821 and 5,648,260 to Winter et al.

[0468] In some embodiments, one or more amino acids selected from amino acid residues 329, 331, and 322 are replaced with different amino acid residues such that the antibody has altered C1q binding and / or reduced or eliminated complement-dependent cytotoxicity (CDC). This method is also described in detail in U.S. Patent No. 6,194,551 to Idusogie et al.

[0469] In some embodiments, one or more amino acid residues within amino acid positions 231 and 239 are altered, thereby altering the ability of the antibody to fix complement. This method is also described, for example, in PCT Publication WO 94 / 29351 by Bodmer et al. In some embodiments, the Fc region can be modified to reduce antibody-dependent cellular cytotoxicity (ADCC) and / or reduce the affinity for Fcγ receptors 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, 280, 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, 2,67E, 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 made to Fcs are those that reduce or eliminate binding to FcγR and / or complement proteins, thereby reducing or eliminating Fc-mediated effector functions such as ADCC, ADCP and CDC. Exemplary modifications include (but are not limited to) substitutions, insertions and deletions at positions 234, 235, 236, 237, 267, 269, 325, 328, 330 and / or 331 (e.g., 330 and 331), wherein numbering is according to the EU index. Exemplary substitutions include (but are not limited to) 234A, 235E, 236R, 237A, 267R, 269R, 325L, 328R, 330S and 331S (e.g., 330S and 331S), wherein numbering is according to the EU index. The Fc variant can comprise 236R / 328R.Other modifications for reducing the interaction of FcγR with complement include substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P, and 234V, and the glycosylation at position 297 is removed by mutation or enzymatically or by production in an organism that does not glycosylate the protein, such as bacteria. These and other modifications are reviewed 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.

[0470] Fc variants that enhance the affinity for the inhibitory receptor FcγRIIb can also be used. Such variants can provide Fc fusion proteins with immunomodulatory activity associated with FcγRIIb cells, including, for example, B cells and monocytes. In one embodiment, the Fc variant provides selectively enhanced affinity for FcγRIIb relative to one or more activating receptors. Modifications for altering the 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. Exemplary substitutions for enhancing 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. Exemplary substitutions include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Other Fc variants for enhancing the binding to FcγRIIb include 235Y / 267E, 236D / 267E, 239D / 268D, 239D / 267E, 267E / 268D, 267E / 268E, and 267E / 328F.

[0471] Other modifications for enhancing the interaction of FcγR with complement include (but are not limited to) substitutions 298A, 333A, 334A, 326A, 247I, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 305I, and 396L. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691. Fc modifications that increase binding to Fcγ receptors include amino acid modifications at any one or more of the following amino acid positions in the Fc region: 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, 327, 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 residues in the Fc region are numbered as in the EU index of Patent Publication No. WO 00 / 42072.

[0472] Optionally, the Fc region may contain non-naturally occurring amino acid residues at additional and / or alternative positions known to those of skill in the art (see, for example, U.S. Patent Nos. 5,624,821, 6,277,375, 6,737,056, 6,194,551, 7,317,091, 8,101,720; PCT Patent Publications WO 00 / 42072, WO 01 / 58957, WO02 / 06919, WO 04 / / 016750, WO 04 / 029207, WO 04 / 035752, WO04 / 074455, WO 04 / 099249, WO04 / 063351, WO 05 / 070963, WO05 / 040217, WO 05 / 092925, and WO 06 / 020114).

[0473] The affinity and binding characteristics of the Fc region for its ligand can be determined by a variety of in vitro assays (biochemical or immunology-based assays) known in the art, including (but not limited to) equilibrium methods (such as enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetics (such as BIACORE analysis) and other methods, such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (such as gel filtration). These and other methods can utilize labels on one or more of the components being tested and / or employ a variety of detection methods, including (but not limited to) chromogenic, fluorescent, luminescent, or isotopic labeling. A detailed description of binding affinity and kinetics can be found in Paul, W. E. (ed.), Fundamental immunology, 4th ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

[0474] In certain embodiments, the antibody is modified to extend its biological half-life. A variety of methods are available. For example, this can be achieved by increasing the binding affinity of the Fc region for FcRn. For example, one or more of the following residues can be mutated: 252, 254, 256, 433, 435, 436, as described in U.S. Patent No. 6,277,375. Specific exemplary substitutions include one or more of the following: T252L, T254S, and / or T256F. Alternatively, to increase the biological half-life, the antibody can be altered in the CH1 or CL region to contain the salvage receptor-binding epitope of two loops of the CH2 domain of the Fc region obtained from IgG, as described in U.S. Patents Nos. 5,869,046 and 6,121,022 to Presta et al. Other exemplary variants that increase binding to FcRn and / or improve pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434, including, for example, 259I, 308F, 428L, 428M, 434S, 434I, 434F, 434Y, and 434X1. Other variants that increase Fc-FcRn binding 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, 433R, 433S, 433I, 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 modulating FcRn binding are described in Yeung et al., 2010, J Immunol, 182:7663-7671.

[0475] In certain embodiments, hybrid IgG isotypes with specific biological characteristics can be used. For example, an IgG1 / IgG3 hybrid variant can be constructed by replacing IgG1 positions in the CH2 and / or CH3 regions with amino acids from IgG3 at positions that are different between the two isotypes. Thus, hybrid variant IgG antibodies can be constructed that contain one or more substitutions, such as 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 422I, 435R, and 436F. In some embodiments described herein, an IgG1 / IgG2 hybrid variant can be constructed by replacing IgG2 positions in the CH2 and / or CH3 regions with amino acids from IgG1 at positions that are different between the two isotypes. Thus, hybrid variant IgG antibodies can be constructed that contain one or more substitutions, such as one or more of the following amino acid substitutions: 233E, 234L, 235L, -236G (which refers to the insertion of glycine at position 236), and 327A.

[0476] In addition, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn have been mapped and variants with improved binding have been described (see Shields, R.L. 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. In addition, the following combinatorial mutants have been shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A, which have been shown to exhibit enhanced FcγRIIIa binding and ADCC activity (Shields et al., 2001). Other IgG1 variants that strongly enhance binding to FcγRIIIa have been identified, including variants with S239D / I332E and S239D / I332E / A330L mutations, which show the greatest increase in affinity for FcγRIIIa, a decrease in FcγRIIb binding, and strong cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006). Introduction of triple mutations into antibodies such as alemtuzumab (CD52-specific), trastuzumab (HER2 / neu-specific), rituximab (CD20-specific), and cetuximab (EGFR-specific) converts them into antibodies with greatly enhanced ADCC activity in vitro, and the S239D / I332E variant shows enhanced ability to deplete B cells in monkeys (Lazar et al., 2006). In addition, IgG1 mutants containing L235V, F243L, R292P, Y300L, and P396L mutations have been identified in transgenic mice expressing human FcγRIIIa in models of B cell malignancies and breast cancer, which exhibit enhanced binding to FcγRIIIa and concomitant enhanced ADCC activity (Stavenhagen et al., 2007; Nordstrom et al., 2011). Other Fc mutants that can be used include: S298A / E333A / L334A, S239D / I332E, S239D / I332E / A330L, L235V / F243L / R292P / Y300L / P396L, and M428L / N434S.

[0477] In certain embodiments, an Fc with reduced binding to FcγRs is selected. Exemplary Fcs (e.g., IgG1 Fc) with reduced FcγR binding contain the following three amino acid substitutions: L234A, L235E, and G237A.

[0478] In certain embodiments, an Fc with reduced complement binding is selected. Exemplary Fcs (such as IgG1 Fc) with reduced complement binding have the following two amino acid substitutions: A330S and P331S.

[0479] In certain embodiments, an Fc that is substantially devoid of effector functions is selected, i.e., its binding to FcγR is reduced and complement binding is reduced. Exemplary Fcs without effector functions (such as IgG1 Fc) contain the following five mutations: L234A, L235E, G237A, A330S and P331S.

[0480] When using the IgG4 constant domain, it may include the substitution S228P, which mimics the hinge sequence in IgG1 and thereby stabilizes the IgG4 molecule.

[0481] Fc modifications described in WO 2017 / 087678 or WO2016081746 may also be used.

[0482] In certain embodiments, the glycosylation of the antibody is modified. For example, an aglycosylated antibody (i.e., an antibody lacking glycosylation) can be produced. The glycosylation can be altered, for example, to increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made to exclude one or more variable region framework glycosylation sites, thereby eliminating glycosylation at said sites. Such aglycosylation can increase the affinity of the antibody for the antigen. This method is also described in detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 to Co et al.

[0483] Glycosylation of the constant region at N297 can be blocked by mutating the N297 residue to another residue such as N297A, and / or by mutating an adjacent amino acid such as 298, thereby reducing glycosylation at N297.

[0484] Additionally or alternatively, antibodies with altered glycosylation patterns can be generated, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in host cells with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells for expressing the recombinant antibodies described herein to thereby generate antibodies with glycosylation alterations. For example, EP 1,176,195 by Hanai et al. describes a cell line in which the FUT8 gene encoding fucosyltransferase is functionally disrupted such that antibodies expressed in this cell line exhibit hypofucosylation. PCT publication WO 03 / 035835 by Presta describes a mutant CHO cell line, Led 3 cells, which have a reduced ability to attach fucose to the carbohydrate linked to Asn(297), also resulting in hypofucosylation of antibodies expressed in the host cells (see also Shields, R.L. et al., (2002) J. Biol. Chem. 277:26733-26740). PCT publication WO 99 / 54342 by Umana et al. describes a cell line engineered to express a glycosyltransferase that modifies glycoproteins {e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).

[0485] Another modification of the antibodies described herein is pegylation. Antibodies can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG) (such as a reactive ester or aldehyde derivative of PEG) under conditions that result in the attachment of one or more PEG groups to the antibody or antibody fragment. In some embodiments, pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono(C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is a deglycosylated antibody. Methods for protein pegylation are known in the art and can be applied to the antibodies described herein. See, for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

[0486] Specific exemplary antibody embodiments herein are further enumerated in the following sections.

[0487] ​

[0488] Embodiment 1: An isolated antibody (“Ab”) that binds to human ILT4 (hILT4), wherein the 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 NO: 80 and the light chain comprises VL comprising SEQ ID NO: 70.

[0489] Embodiment 2: An isolated antibody that binds to human ILT4, wherein the 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 NO: 80 and the light chain comprises VL comprising SEQ ID NO: 70, wherein the antibody, for example, stimulates T cell activation (or enhances the T cell response), (i) in a mixed lymphocyte reaction (MLR) assay, as demonstrated, for example, by enhanced T cell proliferation or IFN-γ secretion or TNF-α production, or (ii) in a T cell:CHO-OKT3-ILT4 assay (e.g., as described in Example 5).

[0490] Embodiment 3: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 NO: 80 and the light chain comprises VL comprising SEQ ID NO: 70, wherein the Ab enhances T cell activation in a monocyte:T cell allogeneic MLR.

[0491] Embodiment 4: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 80 and the light chain comprises a VL comprising SEQ ID NO: 70, wherein the Ab enhances IFN-γ secretion from PBMCs after antigen stimulation in a cytomegalovirus (CMV) lysate assay.

[0492] Embodiment 5: An isolated antibody that binds to human ILT4, wherein the 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 a VH comprising SEQ ID NO: 80 and the light chain comprises a VL comprising SEQ ID NO: 70, wherein the antibody enhances TNF-α secretion from macrophages, such as differentiated macrophages.

[0493] Embodiment 6: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 80 and the light chain comprises a VL comprising SEQ ID NO: 70, wherein the antibody increases the expression of CD83 and / or CD86 on monocyte-derived dendritic cells (MoDCs).

[0494] Embodiment 7: An isolated antibody that binds to human ILT4, wherein the 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 a VH comprising SEQ ID NO: 80 and the light chain comprises a VL comprising SEQ ID NO: 70, wherein the antibody inhibits the binding of hILT4 to T cells and / or binding partners (such as HLA-A and / or HLA-B).

[0495] Embodiment 8: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 80 and the light chain comprises a VL comprising SEQ ID NO: 70, wherein the Ab (a) binds to the Ig-like domain 1 of hILT4 and / or the region of hILT4 comprising 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), as determined by HDX; and / or (b) interacts with one or more (or all) of the amino acid residues Lys43, Ile49, Thr50, and Arg51 of mature hILT4, as determined by carbene footprinting.

[0496] Embodiment 9: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 80 and the light chain comprises a VL comprising SEQ ID NO: 70, wherein the Ab inhibits the binding of an antibody (or competes therewith) comprising an HC comprising SEQ ID NO: 12 or 13 and a light chain comprising SEQ ID NO: 11 to hILT4 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.

[0497] Embodiment 10: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 80 and the light chain comprises a VL comprising SEQ ID NO: 70, wherein the Ab binds to hILT4 with a KD of 10 -8 M or 10 -9 M or lower.

[0498] Embodiment 11: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 80 and the light chain comprises a VL comprising SEQ ID NO: 70, wherein the Ab binds to cynomolgus macaque ILT4 with a KD of 10 -7 M or 10 -8 M or lower and / or promotes the expression of CD80, CD83, and / or CD86 on cynomolgus macaque monocyte-derived dendritic cells.

[0499] Embodiment 12: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 80 and the light chain comprises a VL comprising SEQ ID NO: 70, wherein the Ab does not significantly bind to human proteins LILRA1, LILRA2, LILRA3, LILRA4, LILRA6, ILT2, ILT3, ILT5, or LIR8.

[0500] Embodiment 13: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 80 and the light chain comprises a VL comprising SEQ ID NO: 70, wherein the Ab promotes the pro-inflammatory polarization of macrophages into M1 macrophages.

[0501] Embodiment 14: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 80 and the light chain comprises a VL comprising SEQ ID NO: 70, wherein the Ab does not induce (or trigger) basophil activation.

[0502] Embodiment 15: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 80 and the light chain comprises a VL comprising SEQ ID NO: 70, wherein the composition comprising the Ab comprises less than 5% of high molecular weight material and low molecular weight material after incubation at 25 °C for 3 months, and / or comprises less than 10% of high molecular weight material and low molecular weight material after incubation at 40 °C for 3 months.

[0503] Embodiment 16: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 80 and the light chain comprises a VL comprising SEQ ID NO: 70, wherein the Ab comprises an IgG heavy chain constant region, such as IgG1 (e.g., SEQ ID NO: 98 or 102), IgG1.1f (SEQ ID NO: 103), IgG1.3 (SEQ ID NO: 100), IgGP238K (SEQ ID NO: 104), IgG4, or IgG4.S228P (SEQ ID NO: 179).

[0504] Embodiment 17: An isolated antibody that binds to hILT4, wherein the Ab comprises a heavy chain comprising SEQ ID NO: 13 and a light chain comprising SEQ ID NO: 11, wherein optionally, the heavy chain comprises a C-terminal lysine.

[0505] Embodiment 18: An isolated antibody that binds to hILT4, wherein the Ab comprises: two heavy chains, each heavy chain comprising SEQ ID NO: 13; and two light chains, each light chain comprising SEQ ID NO: 11, wherein optionally, one or both of the heavy chains comprise a C-terminal lysine.

[0506] Embodiment 19: An isolated antibody that binds to hILT4, wherein the Ab comprises a heavy chain comprising SEQ ID NO:13 and a light chain comprising SEQ ID NO:11, wherein optionally, the heavy chain comprises a C-terminal lysine, and wherein the Ab comprises at least one disulfide bond linking the heavy chains.

[0507] Embodiment 20: An isolated antibody that binds to hILT4, wherein the Ab comprises a heavy chain comprising SEQ ID NO:176 and a light chain comprising SEQ ID NO:11, wherein optionally, the heavy chain comprises a C-terminal lysine.

[0508] Embodiment 21: An isolated antibody that binds to hILT4, wherein the Ab comprises a heavy chain comprising SEQ ID NO:177 and a light chain comprising SEQ ID NO:11, wherein optionally, the heavy chain comprises a C-terminal lysine.

[0509] Embodiment 22: An isolated antibody that binds to hILT4, wherein the Ab comprises a heavy chain comprising SEQ ID NO:178 and a light chain comprising SEQ ID NO:11, wherein optionally, the heavy chain comprises a C-terminal lysine.

[0510] Embodiment 23: An isolated nucleic acid or nucleic acid set encoding an antibody as described in any one of Embodiments 1-22.

[0511] Embodiment 24: A host cell for producing any one of the antibodies described in Embodiments 1-22, for example, which comprises the isolated nucleic acid or nucleic acid set as described in Embodiment 23.

[0512] Embodiment 25: A method of treating cancer in a subject, which comprises administering to the subject a therapeutically effective dose of an antibody as described in any one of Embodiments 1-22 or an antibody that binds to human ILT4 (hILT4), wherein the 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; (ii) the heavy chain comprises VH comprising SEQ ID NO:80 and the light chain comprises VL comprising SEQ ID NO:70; and / or (iii) the heavy chain comprises SEQ ID NO:13 and the light chain comprises SEQ ID NO:11.

[0513] Embodiment 26: A method of treating cancer in a subject, which comprises administering to the subject a therapeutically effective dose of:

[0514] (A) (1) An antibody as described in any one of embodiments 1-19 or (2) an antibody that binds to human ILT4 (hILT4), wherein the 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; (ii) the heavy chain comprises a VH comprising SEQ ID NO: 80 and the light chain comprises a VL comprising SEQ ID NO: 70; and / or (iii) the heavy chain comprises SEQ ID NO: 13 and the light chain comprises SEQ ID NO: 11, and

[0515] (B) An antagonist of PD-1 or PD-L1, such as an antagonist antibody that binds to human PD-1 or human PD-L1, for example nivolumab, pembrolizumab, cemiplimab, toripalimab, sintilimab, atezolizumab, durvalumab, or avelumab.

[0516] Embodiment 27: A composition comprising:

[0517] (A) (1) An antibody as described in any one of embodiments 1-19 or (2) an antibody that binds to human ILT4 (hILT4), wherein the 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; (ii) the heavy chain comprises a VH comprising SEQ ID NO: 80 and the light chain comprises a VL comprising SEQ ID NO: 70; and / or (iii) the heavy chain comprises SEQ ID NO: 13 and the light chain comprises SEQ ID NO: 11, and

[0518] (B) An antagonist of PD-1 or PD-L1, such as an antagonist antibody that binds to human PD-1 or human PD-L1, for example nivolumab, pembrolizumab, cemiplimab, toripalimab, sintilimab, atezolizumab, durvalumab, or avelumab.

[0519] ​

[0520] Embodiment 1: An isolated antibody that binds to human ILT4 (hILT4), wherein said 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 a VH comprising SEQ ID NO: 87 and the light chain comprises a VL comprising SEQ ID NO: 86.

[0521] Embodiment 2: An isolated antibody that binds to human ILT4 (hILT4), wherein said 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 a VH comprising SEQ ID NO: 87 and the light chain comprises a VL comprising SEQ ID NO: 86, wherein the antibody stimulates T cell activation (or enhances the T cell response), for example (i) in a mixed lymphocyte reaction (MLR) assay, as demonstrated, for example, by enhanced T cell proliferation or IFN-γ secretion or TNF-α production, or (ii) in a T cell:CHO-OKT3-ILT4 assay (such as described in Example 5).

[0522] Embodiment 3: An isolated antibody that binds to human ILT4 (hILT4), wherein said 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 a VH comprising SEQ ID NO: 87 and the light chain comprises a VL comprising SEQ ID NO: 86, wherein the Ab enhances T cell activation in a monocyte:T cell allogeneic MLR.

[0523] Embodiment 4: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 87 and the light chain comprises a VL comprising SEQ ID NO: 86, wherein the Ab enhances IFN-γ secretion from PBMCs after antigen stimulation in a CMV lysate assay.

[0524] Embodiment 5: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 87 and the light chain comprises a VL comprising SEQ ID NO: 86, wherein the antibody enhances TNF-α secretion from macrophages, such as differentiated macrophages.

[0525] Embodiment 6: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 87 and the light chain comprises a VL comprising SEQ ID NO: 86, wherein the antibody increases the expression of CD83 and / or CD86 on monocyte-derived dendritic cells (Mo-DCs).

[0526] Embodiment 7: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 87 and the light chain comprises a VL comprising SEQ ID NO: 86, wherein the antibody inhibits the binding of hILT4 to T cells and / or binding partners (such as HLA-A and / or HLA-B).

[0527] Embodiment 8: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 87 and the light chain comprises a VL comprising SEQ ID NO: 86, wherein the Ab (a) binds to the Ig-like domain 2 of hILT4 and / or the region of hILT4 comprising: 154 ILCKEGEEEHPQCLNSQPHARGSSRAIF 181 (SEQ IDNO:123) and / or 425 SSPPPTGPIS 434 (SEQ ID NO:124) as determined by HDX; and / or (b) interacts 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 as determined by carbene footprinting.

[0528] Embodiment 9: An isolated antibody that binds to human ILT4 (hILT4), wherein said 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 a VH comprising SEQ ID NO: 87 and the light chain comprises a VL comprising SEQ ID NO: 86, wherein said Ab inhibits the binding of an antibody (or competes therewith) comprising an HC comprising SEQ ID NO: 15 or 17 and a light chain comprising SEQ ID NO: 14 or 16 to hILT4 and / or inhibits the binding of an antibody comprising the VH and VL of 21D5 or 21D5.a to hILT4.

[0529] Embodiment 10: An isolated antibody that binds to human ILT4 (hILT4), wherein said 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 a VH comprising SEQ ID NO: 87 and the light chain comprises a VL comprising SEQ ID NO: 86, wherein said Ab binds to hILT4 with a KD of 10 -8 M or 10 -9 M or lower.

[0530] Embodiment 11: An isolated antibody that binds to human ILT4 (hILT4), wherein said 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 a VH comprising SEQ ID NO: 87 and the light chain comprises a VL comprising SEQ ID NO: 86, wherein said Ab binds to cynomolgus monkey ILT4 with a KD of 10 -7 M or 10 -8 M or lower and / or promotes the expression of CD80, CD83, and / or CD86 on cynomolgus monkey monocyte-derived dendritic cells.

[0531] Embodiment 12: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 87 and the light chain comprises a VL comprising SEQ ID NO: 86, wherein the Ab does not significantly bind to human proteins LILRA1, LILRA2, LILRA4, LILRA5, LILRA6, ILT2, ILT3, ILT5, or LIR8.

[0532] Embodiment 13: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 87 and the light chain comprises a VL comprising SEQ ID NO: 86, wherein the Ab promotes the pro-inflammatory polarization of macrophages into M1 macrophages.

[0533] Embodiment 14: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 87 and the light chain comprises a VL comprising SEQ ID NO: 86, wherein the Ab does not induce (or trigger) basophil activation.

[0534] Embodiment 15: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 87 and the light chain comprises a VL comprising SEQ ID NO: 86, wherein the composition comprising the Ab comprises less than 5% of high molecular weight material and low molecular weight material after incubation at 25°C for 3 months, and / or comprises less than 10% of high molecular weight material and low molecular weight material after incubation at 40°C for 3 months.

[0535] Embodiment 16: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 87 and the light chain comprises a VL comprising SEQ ID NO: 86, wherein the Ab comprises an IgG heavy chain constant region, such as IgG1 (e.g., SEQ ID NO: 98 or 102), IgG1.1f (SEQ ID NO: 103), IgG1.3 (SEQ ID NO: 100), IgGP238K (SEQ ID NO: 104), IgG4 or IgG4.S228P (SEQ ID NO: 179).

[0536] Embodiment 17: An isolated antibody that binds to hILT4, wherein the Ab comprises a heavy chain comprising SEQ ID NO: 17 and a light chain comprising SEQ ID NO: 16, wherein optionally, the heavy chain comprises a C-terminal lysine.

[0537] Embodiment 18: An isolated antibody that binds to hILT4, wherein the Ab comprises: two heavy chains, each heavy chain comprising SEQ ID NO: 17; and two light chains, each light chain comprising SEQ ID NO: 16, wherein optionally, one or both of the heavy chains comprises a C-terminal lysine.

[0538] Embodiment 19: An isolated antibody that binds to hILT4, wherein the Ab comprises a heavy chain comprising SEQ ID NO:17 and a light chain comprising SEQ ID NO:16, wherein optionally, the heavy chain comprises a C-terminal lysine, and wherein the Ab comprises at least one disulfide bond linking the heavy chains.

[0539] Embodiment 20: An isolated antibody that binds to hILT4, wherein the Ab comprises a heavy chain comprising SEQ ID NO:15 and a light chain comprising SEQ ID NO:14, wherein optionally, the heavy chain comprises a C-terminal lysine.

[0540] Embodiment 21: An isolated antibody that binds to hILT4, wherein the Ab comprises a heavy chain comprising SEQ ID NO:17 and a light chain comprising SEQ ID NO:16, wherein optionally, the heavy chain comprises a C-terminal lysine.

[0541] Embodiment 22: An isolated antibody that binds to hILT4, wherein the Ab comprises a heavy chain comprising SEQ ID NO:15 or 17 and a light chain comprising SEQ ID NO:14 or 16, wherein optionally, the heavy chain comprises a C-terminal lysine.

[0542] Embodiment 23: An isolated nucleic acid or nucleic acid set encoding an antibody according to any one of Embodiments 1-22.

[0543] Embodiment 24: A host cell for producing any one of the antibodies described in Embodiments 1-22, for example, which comprises the isolated nucleic acid or nucleic acid set according to Embodiment 23.

[0544] Embodiment 25: A method of treating cancer in a subject, which comprises administering to the subject a therapeutically effective dose of an antibody that binds to human ILT4 (hILT4), wherein the 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 NO: 87 and the light chain comprises VL comprising SEQ ID NO: 86, and / or (iii) the heavy chain comprises SEQ ID NO: 17 and the light chain comprises SEQ ID NO: 16.

[0545] Embodiment 26: A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective dose of: (A) an antibody that binds to human ILT4 (hILT4), wherein the 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 NO: 87 and the light chain comprises VL comprising SEQ ID NO: 86, and / or (iii) the heavy chain comprises SEQ ID NO: 17 and the light chain comprises SEQ ID NO: 16, and (B) an antagonist of PD-1 or PD-L1, such as an antagonist antibody that binds to human PD-1 or human PD-L1, for example, nivolumab, pembrolizumab, sintilimab, toripalimab, sintilimab, atezolizumab, durvalumab, or avelumab.

[0546] Embodiment 27: A composition comprising (A) an antibody that binds to human ILT4 (hILT4), wherein the 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 NO: 87 and the light chain comprises VL comprising SEQ ID NO: 86, and / or (iii) the heavy chain comprises SEQ ID NO: 17 and the light chain comprises SEQ ID NO: 16, and (B) an antagonist of PD-1 or PD-L1, such as an antagonist antibody that binds to human PD-1 or human PD-L1, for example, nivolumab, pembrolizumab, sintilimab, toripalimab, sintilimab, atezolizumab, durvalumab, or avelumab.

[0547] ​

[0548] Embodiment 1: An isolated antibody that binds to human ILT4 (hILT4), wherein said 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 a VH comprising SEQ ID NO: 91 and the light chain comprises a VL comprising SEQ ID NO: 90, 94, 96, or 114.

[0549] Embodiment 2: An isolated antibody that binds to human ILT4 (hILT4), wherein said 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 a VH comprising SEQ ID NO: 91 and the light chain comprises a VL comprising SEQ ID NO: 90, 94, 96, or 114, wherein said antibody stimulates T cell activation (or enhances the T cell response), e.g., (i) in a mixed lymphocyte reaction (MLR) assay, as demonstrated, for example, by enhanced T cell proliferation or IFN-γ secretion or TNF-α production, or (ii) in a T cell:CHO-OKT3-ILT4 assay (e.g., as described in Example 5).

[0550] Embodiment 3: An isolated antibody that binds to human ILT4 (hILT4), wherein said 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 a VH comprising SEQ ID NO: 91 and the light chain comprises a VL comprising SEQ ID NO: 90, 94, 96, or 114, wherein said Ab enhances T cell activation in a monocyte:T cell allogeneic MLR.

[0551] Embodiment 4: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 91 and the light chain comprises a VL comprising SEQ ID NO: 90, 94, 96, or 114, wherein the Ab enhances IFN-γ secretion from PBMCs after antigen stimulation in a CMV lysate assay.

[0552] Embodiment 5: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 91 and the light chain comprises a VL comprising SEQ ID NO: 90, 94, 96, or 114, wherein the Ab inhibits the binding of hILT4 to T cells and / or to a binding partner (such as HLA-A and / or HLA-B).

[0553] Embodiment 6: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 91 and the light chain comprises a VL comprising SEQ ID NO: 90, 94, 96, or 114, wherein the antibody enhances TNF-α secretion from macrophages, such as differentiated macrophages.

[0554] Embodiment 7: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 91 and the light chain comprises a VL comprising SEQ ID NO: 90, 94, 96, or 114, wherein the antibody increases the expression of CD83 and / or CD86 on monocyte-derived dendritic cells (MoDCs).

[0555] Embodiment 8: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 91 and the light chain comprises a VL comprising SEQ ID NO: 90, 94, 96, or 114, wherein the 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) of the amino acid residues Glu42, Lys43, Gly76, Cys77, Leu88, Pro91, Pro183, and Tyr184 of mature hILT4, as determined by carbene footprinting.

[0556] Embodiment 9: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 91 and the light chain comprises a VL comprising SEQ ID NO: 90, 94, 96, or 114, wherein the Ab binds to (or competes with) an antibody comprising an HC comprising SEQ ID NO: 19 and a light chain comprising SEQ ID NOs: 18, 20, 21, or 116, respectively, for binding to hILT4 and / or inhibits the binding of an antibody comprising a VH and VL of 10F10, 10F10.1, 10F10.3, and / or 10F10.4 to hILT4.

[0557] Embodiment 10: An isolated antibody that binds to human ILT4 (hILT4), wherein the Ab comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises a VH CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 167, 168, and 169, respectively, and the light chain comprises a VL CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 170, 171, and 172, respectively, and / or (ii) the heavy chain comprises a VH comprising SEQ ID NO: 91 and the light chain comprises a VL comprising SEQ ID NOs: 90, 94, 96, or 114, wherein the Ab is expressed in 10 -8 M or 10 -9 Binds to hILT4 with a KD of M or lower.

[0558] Embodiment 11: An isolated antibody that binds to human ILT4 (hILT4), wherein the Ab comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises a VH CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 167, 168, and 169, respectively, and the light chain comprises a VL CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 170, 171, and 172, respectively, and / or (ii) the heavy chain comprises a VH comprising SEQ ID NO: 91 and the light chain comprises a VL comprising SEQ ID NOs: 90, 94, 96, or 114, wherein the Ab is expressed in 10 -7 M or 10 -8 The invention relates to a novel cynomolgus monkey ILT4 antibody having a KD of 5 M or less and / or promoting the expression of CD80, CD83 and / or CD86 on cynomolgus monkey monocyte-derived dendritic cells.

[0559] Embodiment 12: An isolated antibody that binds to human ILT4 (hILT4), wherein the Ab comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises a VH CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 167, 168, and 169, respectively, and the light chain comprises a VL CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 170, 171, and 172, respectively, and / or (ii) the heavy chain comprises a VH comprising SEQ ID NO: 91 and the light chain comprises a VL comprising SEQ ID NOs: 90, 94, 96, or 114, wherein the Ab promotes proinflammatory polarization of macrophages to M1 macrophages.

[0560] Embodiment 13: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 91 and the light chain comprises a VL comprising SEQ ID NO: 90, 94, 96, or 114, wherein the Ab does not induce (or trigger) basophil activation.

[0561] Embodiment 14: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 91 and the light chain comprises a VL comprising SEQ ID NO: 90, 94, 96, or 114, wherein the composition comprising the Ab comprises less than 5% high molecular weight material and low molecular weight material after incubation at 25°C for 3 months and / or comprises less than 10% high molecular weight material and low molecular weight material after incubation at 40°C for 3 months.

[0562] Embodiment 15: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 91 and the light chain comprises a VL comprising SEQ ID NO: 90, 94, 96, or 114, wherein the Ab does not significantly bind to human proteins LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, ILT2, ILT3, ILT5, or LIR8.

[0563] Embodiment 16: An isolated antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 91 and the light chain comprises a VL comprising SEQ ID NO: 90, 94, 96, or 114, wherein the Ab comprises an IgG heavy chain constant region, such as IgG1 (e.g., SEQ ID NO: 98 or 102), IgG1.1f (SEQ ID NO: 103), IgG1.3 (SEQ ID NO: 100), IgGP238K (SEQ ID NO: 104), IgG4, or IgG4.S228P (SEQ ID NO: 179).

[0564] Embodiment 17: An isolated antibody that binds to hILT4, wherein the Ab comprises a heavy chain comprising SEQ ID NO: 19 and a light chain comprising SEQ ID NO: 18, 20, 21, or 116, wherein optionally, the heavy chain comprises a C-terminal lysine.

[0565] Embodiment 18: An isolated antibody that binds to hILT4, wherein the Ab comprises a heavy chain comprising SEQ ID NO: 19 and a light chain comprising SEQ ID NO: 18, 20, 21, or 116, wherein optionally, one or both of the heavy chains comprise a C-terminal lysine.

[0566] Embodiment 19: An isolated antibody that binds to hILT4, wherein the Ab comprises a heavy chain comprising SEQ ID NO: 19 and a light chain comprising SEQ ID NO: 18, 20, 21, or 116, wherein optionally, the heavy chain comprises a C-terminal lysine, and wherein the Ab comprises at least one disulfide bond linking the heavy chains.

[0567] Embodiment 20: An isolated antibody that binds to hILT4, wherein the Ab comprises a heavy chain comprising SEQ ID NO: 19 and a light chain comprising SEQ ID NO: 18, wherein optionally, the heavy chain comprises a C-terminal lysine.

[0568] Embodiment 21: An isolated antibody that binds to hILT4, wherein the Ab comprises a heavy chain comprising SEQ ID NO: 19 and a light chain comprising SEQ ID NO: 20, wherein optionally, the heavy chain comprises a C-terminal lysine.

[0569] Embodiment 22: An isolated antibody that binds to hILT4, wherein the Ab comprises a heavy chain comprising SEQ ID NO:19 and a light chain comprising SEQ ID NO:21, wherein optionally, the heavy chain comprises a C-terminal lysine.

[0570] Embodiment 23: An isolated antibody that binds to hILT4, wherein the Ab comprises a heavy chain comprising SEQ ID NO:19 and a light chain comprising SEQ ID NO:116, wherein optionally, the heavy chain comprises a C-terminal lysine.

[0571] Embodiment 24: An isolated nucleic acid or nucleic acid collection encoding an antibody as described in any one of Embodiments 1-23.

[0572] Embodiment 25: A host cell for producing any one of the antibodies as described in Embodiments 1-23, for example, which comprises the isolated nucleic acid or nucleic acid collection as described in Embodiment 24.

[0573] Embodiment 26: A method of treating cancer in a subject, which comprises administering to the subject a therapeutically effective dose of an antibody that binds to human ILT4 (hILT4), wherein the Ab comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises VH CDR1, CDR2, and CDR3 respectively comprising SEQ ID NO:167, 168, and 169, and the light chain comprises VL CDR1, CDR2, and CDR3 respectively comprising SEQ ID NO:170, 171, and 172, and / or (ii) the heavy chain comprises VH comprising SEQ ID NO:91 and the light chain comprises VL comprising SEQ ID NO:90, 94, 96, or 114, and / or (iii) the heavy chain comprises SEQ ID NO:19 and the light chain comprises SEQ ID NO:18, 20, 21, or 116.

[0574] Embodiment 27: A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective dose of an antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 91 and the light chain comprises a VL comprising SEQ ID NO: 90, 94, 96, or 114, and / or (iii) the heavy chain comprises SEQ ID NO: 19 and the light chain comprises SEQ ID NO: 18, 20, 21, or 116, and (B) an antagonist of PD-1 or PD-L1, such as an antagonist antibody that binds to human PD-1 or human PD-L1, for example, nivolumab, pembrolizumab, cemiplimab, toripalimab, sintilimab, atezolizumab, durvalumab, or avelumab.

[0575] Embodiment 28: A composition, comprising (A) an antibody that binds to human ILT4 (hILT4), wherein the 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 a VH comprising SEQ ID NO: 91 and the light chain comprises a VL comprising SEQ ID NO: 90, 94, 96, or 114, and / or (iii) the heavy chain comprises SEQ ID NO: 19 and the light chain comprises SEQ ID NO: 18, 20, 21, or 116, and (B) an antagonist of PD-1 or PD-L1, such as an antagonist antibody that binds to human PD-1 or human PD-L1, for example, nivolumab, pembrolizumab, cemiplimab, toripalimab, sintilimab, atezolizumab, durvalumab, or avelumab.

[0576] Nucleic Acids and Host Cells

[0577] Also provided are nucleic acids encoding an antibody or its heavy or light chain or a portion thereof. Exemplary nucleic acids are provided in the sequence listing. Also covered herein are any nucleic acids having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the nucleic acids in the sequence listing. Also covered are compositions comprising nucleic acids encoding the antibodies provided herein, cells comprising such compositions, and methods for preparing antibodies, which methods include culturing cells transformed with nucleic acids encoding an ILT4 antibody and isolating the antibody from the culture medium or the cells.

[0578] Therapeutic methods and related pharmaceutical compositions using ILT4-binding Abs

[0579] The antibodies described herein can be used, for example, to treat cancer. In some embodiments, provided are methods of treating cancer that include administering to a patient an effective amount of an antibody described herein. In some embodiments, the Ab can trigger or enhance the patient's immune response, such as an antigen-specific immune response. In some embodiments, the Ab can stimulate T cell activity. In some embodiments, the Ab can inhibit the growth of at least one tumor in the patient.

[0580] Provided herein are methods of treating a subject having cancer that include administering to the subject a therapeutically effective amount of an ILT4 antibody described herein, thereby treating the subject. The ILT4 antibody can be used alone. Alternatively, the ILT4 antibody can be used in combination with another agent as further described below.

[0581] The cancer can be a cancer having a solid tumor or a hematological malignancy (liquid tumor).

[0582] Non-limiting examples of cancers treated 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, renal cancer (such as clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, renal cancer (such as renal cell carcinoma; RCC), prostate cancer (such as hormone refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, stomach cancer, bladder cancer, liver tumors, breast cancer, colon cancer, and head and neck cancer (or carcinoma);Gastric cancer, germ cell tumors, pediatric sarcoma, sinonasal natural killer, melanoma (such as metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, fallopian tube cancer, endometrial cancer, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, ureteral cancer, renal pelvic carcinoma, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain cancer, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell carcinoma, T cell lymphoma, environmentally induced cancers (including those induced by asbestos), virus-related cancers or cancers of viral origin (such as human papillomavirus (HPV)-associated or HPV-induced tumors), and hematological malignancies derived from either of two major blood cell lineages, namely, the myeloid lineage (which gives rise to granulocytes, erythrocytes, platelets, macrophages, and mast cells) or the lymphoid lineage (which gives rise to B, T, NK, and plasma cells), such as all types of leukemia, lymphoma, and myeloma, for example, acute, chronic, lymphocytic, and / or myeloid leukemia, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), undifferentiated AML (MO), myeloblastic leukemia (M1), myeloid leukemia (M2; 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 chloroma; lymphoma, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma;NHL), B-cell malignancies of hematologic origin (e.g., B-cell lymphoma), T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) large-cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angio immunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplantation lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), hematopoietic tumors of lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma;CTLC (also known as mycosis fungoides or Sézary syndrome) and lymphoplasmacytic lymphoma (LPL) with Waldenström's macroglobulinemia; myelomas such as IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also known as indolent myeloma), solitary plasmocytoma, and multiple myeloma; chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors of mesenchymal cell origin, including fibrosarcoma and rhabdomyosarcoma; seminoma, teratocarcinoma, central and peripheral nerve tumors (including astrocytoma, schwannomas); tumors of mesenchymal cell origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma, and teratocarcinoma; hematopoietic tumors of lymphoid lineage, such as T cell and B cell tumors, including (but not limited to) T cell disorders such as T-prolymphocytic leukemia (T-PLL), including small cell and brain cell types; T cell large granular lymphocyte leukemia (LGL); a / d T-NHL hepatosplenic lymphoma; peripheral / post-thymic T cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T cell lymphoma; head and neck cancer, kidney cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma; and any combination of these cancers. The methods described herein can also be used to treat metastatic cancer, unresectable refractory cancer (e.g., cancer that is difficult to treat with prior immunotherapy, such as cancer that is difficult to treat with blocking CTLA-4 or PD-1 antibodies) and / or recurrent cancer.;

[0583] In certain embodiments, the antibodies described herein are administered to a patient having cancer that exhibits an inadequate response to a prior treatment or has progressed, such as a prior treatment using immuno-oncology or immunotherapy agents. In some embodiments, the cancer is refractory or resistant to a prior treatment, inherently refractory or resistant (e.g., difficult to treat with a PD-1 pathway antagonist), or has acquired a resistant or refractory state. For example, the antibodies described herein can be administered to a subject who is non-responsive or inadequately responsive to a first therapy or has disease progression following treatment with an anti-PD-1 pathway antagonist, such as alone or in combination with another therapy (e.g., in combination with anti-PD-1 pathway antagonist therapy). In other embodiments, the antibodies described herein are administered to a patient who has not previously received a tumor immunomodulator (e.g., a PD-1 pathway antagonist) (i.e., has not been treated therewith).

[0584] Combination with an immunostimulatory agent

[0585] In some embodiments, an antibody as described herein (e.g., an ILT4 antibody as described herein) is administered in combination with at least one immunostimulatory agent. For example, the therapeutic agents can be co-infused or injected at substantially the same time. In some embodiments, the antibody and at least one immunostimulatory agent are administered sequentially. For example, in some embodiments, the antibody is administered sequentially before or after at least one immunostimulatory agent such that the two therapeutic agents are administered separated by 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 two weeks.

[0586] In some embodiments, at least one dose, at least two doses, at least three doses, at least five doses, or at least ten doses of the antibody are administered before administering at least one immunostimulatory agent. In some embodiments, at least one dose, at least two doses, at least three doses, at least five doses, or at least ten doses of at least one immunostimulatory agent are administered before administering the antibody. In some embodiments, the last dose of the immunostimulatory agent is administered at least one day, two days, three days, five days, or ten days, or one week, two weeks, three weeks, five weeks, twelve weeks, or twenty-four weeks before the first dose of the antibody is administered. In some embodiments, the last dose of the antibody is administered at least one day, two days, three days, five days, or ten days, or one week, two weeks, three weeks, five weeks, twelve weeks, or twenty-four weeks before the first dose of at least one immunostimulatory agent is administered. In some embodiments, the subject has received or is receiving therapy with at least one immunostimulatory agent and the ILT4 antibody is added to the treatment regimen.

[0587] In some embodiments, at least one immunostimulant comprises an antagonist of an inhibitor of activation of immune cells such as T cells, while in some embodiments, at least one immunostimulant comprises an agonist of a stimulator of 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, PI3Kδ or IDO. In some embodiments, at least one immunostimulant comprises an 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 Toll-like receptor agonist such as a TLR2 / 4 agonist. In some embodiments, at least one immunostimulant comprises an agent that binds to another member of the membrane-bound protein B7 family (such as B7-1, B7-2, B7-H2 (ICOS-L), B7-H3, B7-H4, and B7-H6). In some embodiments, at least one immunostimulant comprises an agent that binds to a TNF receptor family member or a costimulatory or coinhibitory molecule that binds to a TNF receptor family member, where the TNF receptor family members are 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 comprises an agent that antagonizes or inhibits cytokines (such as IL-6, IL-10, TGFβ, VEGF) that inhibit T cell activation.In some embodiments, at least one immunostimulant comprises an agonist of cytokines (such as IL-2, IL-7, IL-12, IL-15, IL-21, and IFNα) that stimulate T cell activation. In some embodiments, at least one immunostimulant comprises an antagonist of chemokines (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.

[0588] For example, the ILT4 antibody described herein can be administered in combination with one or more of the following agents:

[0589] (1) antagonists (inhibitors or blockers) of proteins that inhibit T cell activation (e.g., immune checkpoint inhibitors), such as 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, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, GITR, ICOS, ICOS-L, OX40, OX40L, CD70, CD27, CD40, DR3, and CD28H.

[0590] Exemplary agents that can be combined with the ILT4 antibody described herein for the treatment of cancer include: (ipilimumab) or tremelimumab (against CTLA-4), galiximab (against B7.1), BMS-936558 (against PD-1), MK-3475 (against PD-1), atezolizumab Avelumab, durvalumab, AMP224 (against B7DC), 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 Health Services), huMAbOX40L (against OX40L), atacicept (against TACI), CP-870893 (against CD40), lucatumumab (against CD40), dacetuzumab (against CD40), muromonab-CD3 (against CD3); anti-GITR antibodies MK4166, TRX518, Medi1873, INBRX-110, LK2-145, GWN-323, GITRL-Fc or any combination thereof.

[0591] Other molecules that can be used in combination with an ILT4 antibody for treating cancer include antagonists of inhibitory receptors on NK cells, or agonists of activating receptors on NK cells, such as antagonists of KIR (e.g., lirilumab).

[0592] T cell activation can also be regulated by soluble cytokines. In some embodiments, the ILT4 antibody can be administered in combination with an antagonist of a cytokine that inhibits T cell activation or an agonist of a cytokine that stimulates T cell activation. For example, the ILT4 antibody can be used in combination with: (i) an antagonist (or inhibitor or blocker) of a protein of the IgSF family or B7 family or TNF family that inhibits T cell activation, or an antagonist of a cytokine that inhibits T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF; “immunosuppressive cytokines”) and / or (ii) an agonist of a stimulating receptor of the IgSF family, B7 family or TNF family or a cytokine that stimulates T cell activation.

[0593] Other agents for combination therapy include agents that inhibit or deplete macrophages or monocytes, including (but not limited to): CSF-1R antagonists such as CSF-1R antagonist antibodies, including RG7155 (WO11 / 70024, WO11 / 107553, WO11 / 131407, W013 / 87699, W013 / 119716, WO13 / 132044) or FPA-008 (WO11 / 140249, W013169264, WO14 / 036357).

[0594] The ILT4 antibody can also be administered together with an agent that inhibits TGF-β signaling.

[0595] Additional agents that can be combined with the ILT4 antibody include agents that enhance tumor antigen presentation, such as dendritic cell vaccines, GM-CSF secreting cell vaccines, CpG oligonucleotides, and imiquimod, or therapies that enhance the immunogenicity of tumor cells (such as anthracyclines).

[0596] Other therapies that can be combined with the ILT4 antibody include therapies that deplete or block Treg cells, such as agents that specifically bind to CD25.

[0597] Another therapy that can be combined with the ILT4 antibody is a therapy that inhibits metabolic enzymes such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase.

[0598] Another class of agents that can be used together with the ILT4 antibody includes agents that inhibit adenosine formation, such as CD73 inhibitors, or agents that inhibit the adenosine A2A receptor.

[0599] Other therapies that can be combined with the ILT4 antibody for the treatment of cancer include therapies that reverse / prevent T cell anergy or exhaustion and therapies that trigger innate immune activation and / or inflammation at the tumor site.

[0600] Other therapies that can be combined with the ILT4 antibody for the treatment of cancer include, for example, using a therapy that blocks IL-8.

[0601] The ILT4 antibody can be combined with more than one tumor immunotherapeutic agent and can be combined, for example, with combination methods for targeting 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 secreting cell vaccines, CpG oligonucleotides, imiquimod); therapies that inhibit negative immune regulation, e.g., by inhibiting the CTLA-4 and / or PD1 / PD-L1 / PD-L2 pathways and / or depleting or blocking Tregs or other immunosuppressive cells; therapies that stimulate positive immune regulation, e.g., using agonists that stimulate the CD-137, OX-40, and / or CD40 or GITR pathways and / or stimulate T cell effector functions; therapies that systemically increase the prevalence of anti-tumor T cells; therapies that deplete or inhibit Tregs (such as Tregs in tumors), e.g., using antagonists of CD25 (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion; therapies that affect the function of myeloid cells, which are inhibitory factors in tumors; therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines); adoptive T cell or NK cell transfer, including genetically modified cells, such as cells modified by chimeric antigen receptors (CAR-T therapy); therapies that inhibit metabolic enzymes such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase; therapies that reverse / prevent T cell anergy or exhaustion; therapies that trigger innate immune activation and / or inflammation at the tumor site; administration of immunostimulatory cytokines; or blockade of immunosuppressive cytokines.

[0602] The ILT4 antibodies described herein can be used in combination with one or more of the following: agonists that engage positive co-stimulatory receptors; blockers that attenuate signal transduction via inhibitory receptors; antagonists; and one or more agents that systemically increase the prevalence of anti-tumor T cells; agents that overcome different immunosuppressive pathways within the tumor microenvironment (e.g., blocking inhibitory receptor engagement (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 such as IDO, or reversing / preventing T cell anergy or exhaustion); and agents that trigger innate immune activation and / or inflammation at the tumor site.

[0603] In certain embodiments, if the subject is positive for the BRAF V600 mutation, the subject is administered an ILT4 antibody and a BRAF inhibitor.

[0604] PD-1 antagonists suitable for the combination therapies described herein include, but are not limited to, ligands, antibodies (e.g., monoclonal antibodies and bispecific antibodies), and multivalent agents. In one embodiment, the PD-1 antagonist is a fusion protein, e.g., an Fc fusion protein such as AMP-244. In one embodiment, the PD-1 antagonist is an anti-PD-1 or anti-PD-L1 antibody.

[0605] Exemplary anti-PD-1 antibodies are nivolumab (BMS-936558) or an antibody comprising the CDR or variable region of one of antibodies 17D8, 2D3, 4H1, 5C4, 7D3, 5F4, and 4A11 described in WO2006 / 121168. In certain embodiments, the anti-PD-1 antibody is MK-3475 (lambrolizumab) described in WO2012 / 145493; AMP-514 described in WO2012 / 145493; or PDR001. Other known PD-1 antibodies and other PD-1 inhibitors include PD-1 antibodies and PD-1 inhibitors described in WO2009 / 014708, WO 03 / 099196, WO 2009 / 114335, WO 2011 / 066389, WO 2011 / 161699, WO2012 / 145493, U.S. Patent Nos. 7,635,757 and 8,217,149, and U.S. Patent Publication No. 2009 / 0317368. Any anti-PD-1 antibody disclosed in WO2013 / 173223 can also be used. Anti-PD-L1 antibodies that competitively bind to and / or bind to the same epitope on PD-1 as one of these antibodies can also be used in the combination therapy.

[0606] In some embodiments, the anti-PD-L1 antibody useful for combination therapy is BMS-936559 (referred to as 12A4 in WO 2007 / 005874 and U.S. Patent No. 7,943,743), or an antibody comprising the CDRs or variable regions of 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4, which are described in PCT Publication WO07 / 005874 and U.S. Patent No. 7,943,743. In one embodiment, 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 can also be used. Anti-PD-L1 antibodies that compete with and / or bind to the same epitope as any of these antibodies can also be used in combination therapy.

[0607] In certain embodiments, the ILT4 antibody of the invention can be used in combination with a CTLA-4 antagonist (such as an anti-CTLA-4 antibody). In one embodiment, the anti-CTLA-4 antibody is an antibody selected from the group consisting of: (ipilimumab or antibody 10D1, described in PCT Publication WO 01 / 14424), tremelimumab (formerly ticilimumab, CP-675,206), a monoclonal antibody or anti-CTLA-4 antibody described in any of the following publications: WO 98 / 42752; WO 00 / 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. 58:5301-5304. Any of the anti-CTLA-4 antibodies disclosed in WO2013 / 173223 can also be used.

[0608] In some embodiments, the ILT4 antibodies of the invention are used in combination with an LAG3 antagonist. Examples of anti-LAG3 antibodies include antibodies comprising the CDRs or variable regions of the antibodies 25F7, 26H10, 25E3, 8B7, 11F2 or 17E5 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 anti-LAG-3 antibodies recognized in the art that can be used include IMP731 and IMP-321 described in US 2011 / 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 can also be used in combination therapy.

[0609] In some embodiments, the ILT4 antibodies of the invention can be administered in combination with a CD137 (4-1BB) agonist, such as an agonistic CD137 antibody. Suitable CD137 antibodies include, for example, urelumab and PF-05082566 (W012 / 32433).

[0610] In some embodiments, the ILT4 antibody can be administered in combination with an OX40 agonist, such as an agonistic OX40 antibody. Suitable OX40 antibodies include, for example, MEDI-6383, MEDI-6469 or MOXR0916 (RG7888; WO06 / 029879).

[0611] In one embodiment, the ILT4 antibody is administered in combination with a CD40 agonist, such as an agonistic CD40 antibody. In certain embodiments, the tumor immunomodulator is a CD40 antagonist, such as an antagonistic CD40 antibody. Suitable CD40 antibodies include, for example, lucatumumab (HCD122), daratumumab (SGN-40), CP-870,893 or Chi Lob 7 / 4.

[0612] In one embodiment, the ILT4 antibody is administered in combination with a CD27 agonist, such as an agonistic CD27 antibody. Suitable CD27 antibodies include, for example, varlilumab (CDX-1127).

[0613] In certain embodiments, the ILT4 antibody is administered in combination with an anti-GITR antibody, such as an antibody having the CDR sequences of 6C8, e.g., a humanized antibody having the CDRs of 6C8, as described, for example, in WO2006 / 105021; an antibody comprising the CDRs of an anti-GITR antibody as described in WO2011 / 028683; an antibody comprising the CDRs of an anti-GITR antibody as described in JP2008278814; an antibody comprising the CDRs 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 mentioned herein.

[0614] In some embodiments, the ILT4 antibody is administered in combination with MGA271 (against B7H3) (WO11 / 109400).

[0615] In some embodiments, the ILT4 antibody is administered in combination with a KIR antagonist, such as lirilumab.

[0616] 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.

[0617] 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-Guerin); a TLR7 agonist (e.g., Hiltonol or imiquimod); a TLR7 / 8 agonist (e.g., Resiquimod); or a TLR9 agonist (e.g., CpG7909).

[0618] In one embodiment, ILT4 is administered in combination with a TGF-β inhibitor, such as GC1008, LY2157299, TEW7197, or IMC-TR1.

[0619] Additional combination therapies

[0620] The Ab in the present text can also be provided before, substantially simultaneously with, or after other modes of treatment (such as surgery, chemotherapy, radiotherapy) or the administration of biological agents (such as another therapeutic antibody). In some embodiments, the cancer recurs or progresses after a therapy selected from surgery, chemotherapy, radiotherapy, or a combination thereof. For example, when there may be a risk of micrometastases and / or to reduce the risk of recurrence, the ILT4 antibody as described herein can be administered in an adjuvant therapy form.

[0621] For cancer treatment, the combination can be administered in combination with one or more additional anti-cancer agents (such as chemotherapeutic agents, growth inhibitors, anti-cancer vaccines (such as gene therapy vaccines), anti-angiogenic agents, and / or anti-neoplastic compositions). Non-limiting examples of chemotherapeutic agents, growth inhibitors, anti-cancer vaccines, anti-angiogenic agents, and anti-neoplastic compositions that can be used in combination with the antibodies of the present invention are provided herein below in "Definitions".

[0622] In some embodiments, anti-inflammatory drugs (such as steroids or non-steroidal anti-inflammatory drugs (NSAIDs)) can be administered together with the combination. In cases where a treatment that requires the use of the ILT4 antibody described herein or to dormancy abnormal proliferating cells before the treatment with the antibody, hormones and steroids (including synthetic analogs) can also be administered to the patient, such as 17α-ethinylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testolactone, megestrol acetate, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprorelin, flutamide, toremifene, When the methods or compositions described herein are employed, other agents used to modulate tumor growth or metastasis in a clinical setting, such as antiemetics, can also be administered as needed.

[0623] The antibodies described herein may also be combined with immunogenic agents such as 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). Non-limiting examples of tumor vaccines that may be used include peptides of melanoma antigens such as gp100, MAGE antigens, Trp-2, MART1 and / or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF (discussed further below).

[0624] The ILT4 antibodies described herein may also be combined with vaccination protocols. Many experimental strategies have been designed for vaccination against tumors (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; see also Restifo, N. and Sznol, M., Cancer Vaccines, Chapter 61, pp. 3023-3043, DeVita, V. et al. (eds), 1997, Cancer: Principles and Practice of Oncology. 5th Edition). In one of these strategies, vaccines are prepared using autologous or allogeneic tumor cells. These cell vaccines have been shown to be most effective when the 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. U.S.A. 90:3539-43).

[0625] 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, S A (1999) Immunity 10:281-7). In many cases, these tumor-specific antigens are differentiation antigens expressed in tumors and in the cells in which the tumors arise, such as the melanocyte antigen gp100, the MAGE antigens, and Trp-2. More importantly, many of these antigens can be shown to be targets of tumor-specific T cells found in the host. ILT4 inhibition can be used in combination with a group of recombinant proteins and / or peptides expressed in tumors to generate an immune response against these proteins. These proteins are generally observed by the immune system as self-antigens and are thus tolerated. Tumor antigens can include the protein telomerase, which is required for the synthesis of the telomeres of chromosomes and is expressed in greater than 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 that alter the protein sequence or generate a fusion protein between two unrelated sequences (i.e., bcr-abl in the Philadelphia chromosome), or be the idiotypes from B cell tumors.

[0626] Other tumor vaccines can include the proteins of viruses involved in human cancers, such as human papillomavirus (HPV), hepatitis viruses (HBV and HCV), and Kaposi's sarcoma herpesvirus (KHSV). Another form of tumor-specific antigen that can be used in combination with ILT4 Ab is purified heat shock protein (HSP) isolated from the tumor tissue itself. These heat shock proteins contain protein fragments from tumor cells and these HSPs are efficiently delivered to antigen-presenting cells to initiate tumor immunity (Suot and Srivastava (1995) Science 269:1585-1588; Tamura et al. (1997) Science 278:117-120).

[0627] Dendritic cells (DCs) are powerful antigen-presenting cells that can be used to initiate antigen-specific responses. DCs can be generated 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 methods to also express these tumor antigens. DCs have also been directly fused with tumor cells for immunization purposes (Kugler et al., (2000) Nature Medicine 6:332-336). As a vaccination method, DC immunization can be combined with ILT4 inhibition to activate a more powerful anti-tumor response.

[0628] Treatment of infectious diseases

[0629] The methods described herein can also be used to treat patients who have been exposed to specific toxins or pathogens. Accordingly, the present invention also encompasses methods of treating an infectious disease in a subject, which comprise administering to the subject an antibody as described herein, such as an ILT4 antibody, thereby treating the infectious disease in the subject. Similar to its use for tumors as discussed above, antibody-mediated ILT4 inhibition can be used alone or in combination with a vaccine as an adjuvant to stimulate an immune response against pathogens, toxins, and autoantigens. Examples of pathogens for which such a treatment method may be particularly useful include pathogens for which there is currently no effective vaccine, or for which conventional vaccines are not fully effective. These pathogens include (but are not limited to) HIV, hepatitis (A, B, and C), influenza, herpes, Giardia, malaria, Leishmania, Staphylococcus aureus, Pseudomonas Aeruginosa. ILT4 inhibition can be used to combat infections by agents such as HIV that present altered antigens during the course of infection.

[0630] Some examples of pathogenic viruses that cause infections treatable by the methods described herein include HIV, hepatitis (A, B, or C), herpesviruses (such as VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

[0631] Some examples of pathogenic bacteria that cause infections treatable by the methods described herein include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci, and conococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lymes disease bacteria.

[0632] Some examples of pathogenic fungi that cause infections treatable by the methods described herein include Candida (such as Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, etc.), Cryptococcus neoformans, Aspergillus (such as Aspergillus fumigatus, Aspergillus niger, etc.), Mucorales (such as Mucor, Absidia, Rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0633] Some examples of pathogenic parasites that cause infections treatable by the methods described herein include Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondi, and Nippostrongylus brasiliensis.

[0634] In all of the above methods, ILT4 inhibition can be combined with other forms of immunotherapy, such as those described herein, such as cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy, which can provide enhanced tumor antigen presentation (see, e.g., Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2:1121-1123).

[0635] Route of administration and carrier

[0636] In various embodiments, the antibody can be administered in vivo by a variety of routes, including (but not limited to) intravenous (i.v.), subcutaneous, oral, intraarterial, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or alternatively by implantation or inhalation. The compositions of the invention can be formulated into preparations in solid, semi-solid, liquid, or gaseous form; including (but not limited to) lozenges, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and sprays. Nucleic acid molecules encoding the antibody can be coated onto gold microparticles and delivered intradermally by a particle bombardment device or "gene gun" as described in the literature (see, e.g., Tang et al., Nature 356:152-154 (1992)). The appropriate formulation and route of administration can be selected according to the intended application.

[0637] In various embodiments, an antibody-containing composition is provided in a formulation together with a variety of pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000)). A variety of pharmaceutically acceptable carriers can be used, including vehicles, adjuvants, and diluents. In addition, a variety of pharmaceutically acceptable auxiliary substances can also be used, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0638] In various embodiments, an antibody-containing composition can be formulated for injection (including subcutaneous administration) by dissolving, suspending, or emulsifying it in an aqueous or non-aqueous solvent, such as vegetable oil or other oils, synthetic glycerides of fatty acids, esters of higher aliphatic acids, or propylene glycol; and conventional additives, such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers, and preservatives, can be used as needed. In various embodiments, the composition can be formulated for inhalation, for example, using an acceptable pressurized propellant, such as dichlorodifluoromethane, propane, nitrogen, and the like. In various embodiments, the composition can also be formulated into sustained-release microcapsules, such as using biodegradable or non-biodegradable polymers. Non-limiting exemplary biodegradable formulations include polylactic-co-glycolic acid polymers. Non-limiting exemplary non-biodegradable formulations include polyglyceryl fatty acid esters. Some methods for preparing such formulations are described, for example, in EP 1 125 584 A1.

[0639] Also provided are pharmaceutical packages and kits that include one or more containers, each containing one or more doses of an antibody or antibody combination. In some embodiments, unit dosage forms are provided, wherein the unit dosage form contains a predetermined amount of a composition comprising an antibody or antibody combination, with or without one or more additional agents. In some embodiments, such unit dosage forms are supplied in single-use drug syringes for injection. In various embodiments, the composition contained in the unit dosage form may comprise saline, sucrose, or the like; buffers such as phosphate or the like; and / or be formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided in the form of a lyophilized powder that can be reconstituted upon addition of a suitable liquid (e.g., sterile water). In some embodiments, the composition comprises one or more substances that inhibit protein aggregation, including (but not limited to) sucrose and arginine. In some embodiments, the compositions of the present invention comprise heparin and / or proteoglycans.

[0640] The pharmaceutical composition is administered in an amount effective to treat or prevent a specific indication. A therapeutically effective amount generally depends on the body weight of the subject being treated, its physiological or health condition, the extent of the condition being treated, or the age of the subject being treated. Generally, the antibody can be administered in an amount in the range of about 10 μg / kg body weight to about 100 mg / kg body weight per administration. In some embodiments, the antibody can be administered in an amount in the range of about 50 μg / kg body weight to about 5 mg / kg body weight per administration. In some embodiments, the antibody can be administered in an amount in the range of about 100 μg / kg body weight to about 10 mg / kg body weight per administration. In some embodiments, the antibody can be administered in an amount in the range of about 100 μg / kg body weight to about 20 mg / kg body weight per administration. In some embodiments, the antibody can be administered in an amount in the range of about 0.5 mg / kg body weight to about 20 mg / kg body weight per administration.

[0641] When needed, an antibody composition can be administered to a subject. The frequency of administration can be determined by those skilled in the art, such as an 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, the general health of the subject being treated, and similar factors. In some embodiments, an effective dose of the antibody is administered to the subject one or more times. In various embodiments, an effective dose of the antibody is administered to the subject once a month, less than once a month (such as once every two months or once every three months). In other embodiments, an effective dose of the antibody is administered more than once a month (such as once every three weeks, once every two weeks, or once a week). In some embodiments, an effective dose of the antibody is administered once every 1, 2, 3, 4, or 5 weeks. In some embodiments, an effective dose of the antibody is administered two or three times a week. An effective dose of the antibody is administered to the subject at least once. In some embodiments, an effective dose of the antibody can be administered multiple times, including for a period of at least one month, at least six months, or at least one year.

[0642] In certain embodiments, the combination of the ILT4 antibody described herein with a second agent can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions containing the ILT4 antibody and the second agent in a pharmaceutically acceptable carrier. In one embodiment, the combination of the ILT4 antibody and the second agent can be administered sequentially. The administration of the two agents can be initiated, 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 one or more weeks apart, or the administration of the second agent can be initiated, 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 one or more weeks after the administration of the first agent.

[0643] Examples

[0644] The examples discussed below are only intended to illustrate the invention and should not be construed as limiting the invention in any way. These examples are not intended to represent that the following experiments are all the experiments conducted or the only experiments. Efforts have been made to ensure the accuracy of the quantities used (such as amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. 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.

[0645] Example 1: Generation of Antibodies That Bind to Human ILT4

[0646] Transgenic mice expressing human antibody genes are used to generate anti-human ILT4 monoclonal antibodies (ILT4 antibodies or hILT4 antibodies). Pan-human monoclonal antibodies against human ILT4 are generated by immunizing two types of human immunoglobulin transgenic animals. KM[M / K] mice are immunized with an hILT4-mFc recombinant protein consisting of the extracellular portion of hILT4 and a C-terminal murine Fc tag. The antigen is mixed with Ribi adjuvant at a ratio of 1:1, and the mice are immunized intraperitoneally and subcutaneously every other week. HCo42:01[J / K] strain mice are immunized with an hILT4-his tagged recombinant protein consisting of the extracellular portion of hILT4 and a C-terminal his tag. The antigen is mixed with Ribi adjuvant at a ratio of 1:1, and the mice are immunized in the footpad every other week. The serum titers of both types of mice are monitored after four and six injections. The mice receive a final boost immunization before the final harvest. Depending on the immunization route, draining lymph nodes and spleens are harvested for subsequent fusion.

[0647] Mouse lymphocytes are isolated from immunized mice. Hybridomas are generated by fusing with a murine myeloma fusion partner by electrofusion based on an electric field using a Cyto Pulse Hybrimmune large chamber cell fusion electroporator (BTX / Harvard Apparatus). A single cell suspension of lymphocytes from immunized mice is fused with an equal amount of P3X63 Ag8.6.53 (ATCC) non-secreting murine myeloma cells (fusion numbers 4865 and 6951). The resulting cells are plated in flat-bottom microtiter plates in medium E (StemCell Technologies) supplemented with aminopterin for selection of hybridomas (Sigma).

[0648] After culturing for 10 to 12 days, individual wells are screened for the presence of antibodies against human IgG / human κ light chain (hIgG / hK) using a homogeneous HTRF assay. Hybridoma supernatants from wells positive for hIgG / hK are tested by FACS or by FMAT for binding to hILT4-transfected cells or control CHO cells. The following antibodies that interact specifically with hILT4 are further characterized: 9C8.A6, 24E5.A7, 2H2.H3, 21D9.H11, 2E5.A11, and 9G4. Antibodies 21A5 and 10F10 are isolated from a later immunization.

[0649] Antibodies from hybridomas are sequenced using the NGS high-throughput sequencing method. Briefly, for each hybridoma clone in the wells of a 96-well plate, PCR amplification of the VH and VL regions is performed using unique DNA barcodes to identify each well. The 5' PCR primer hybridizes to the leader region to obtain the entire variable region sequence. Specific hybridoma antibody sequences are identified by matching the barcode and the position of the clone on the plate.

[0650] The anti-ILT4 antibodies 9G4, 9C8, 2H2, 2E5, 24E5, 21D9, 21A5, and 10F10 were recombinantly expressed in the context of IgG1, IgG1.1, or IgG1.3 heavy chain constant regions and are also provided in Table 1. The amino acid sequences of HC, LC, VH, and VL and the nucleotide sequences encoding them are provided in ​ and the Sequence Listing below. The positions of each CDR are provided in the figures and indicated by underlining in the variable region sequences of the Sequence Listing, and also include the CDRs in the form of individual SEQ ID NOs near the end of the Sequence Listing.

[0651] Analysis of the amino acid sequences of the variable domains of antibodies 21D9, 21A5, 2H2, and 10F10 indicated certain framework amino acid residues that are not present in any human germline. To reduce potential immunogenicity issues upon administration of these antibodies to humans, germline revertant mutants of these ILT4 antibodies were generated.

[0652] For antibody 21D9, four framework mutations (V2G, G10D, A24T, and V48I) relative to the germline sequence in the heavy chain variable region were identified and are shown in Figure 13. Among various combinations of these 4 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 4 revertant mutants contain the N-terminal EGQ to EVQ (or G2V) mutation, which is not likely to affect binding to ILT4. In addition to this substitution, mutants 21D9.b, 21D9.c, and 21D9.d each contain one of the additional substitutions shown in Figure 13 (D10G; I24A; and / or I48V), and 21D9.e contains all three substitutions (see Figure 13). Four mutants were generated in the context of the IgG1.3 heavy chain constant region to reduce effector function, the IgG1.3 heavy chain constant region being the IgG1 constant region containing the amino acid substitutions L234A, L235E, and G237A. These mutants are designated 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 light chain of 21D9. The light chain was not germline reverted.

[0653] For antibody 21A5, one framework mutation was identified in each of the heavy chain (T70I) and light chain (V3A) variable regions, as shown in Figure 14. Since the N-terminal revertant at A3V in the κ light chain is unlikely to affect binding, a mutant containing both the heavy chain revertant mutation (I70T) and the light chain revertant mutation (A3V) (21A5.a with 21A5.1 light chain) was generated and tested in the case of IgG1.3 (21A5.a.IgG1.3, also referred to as "21A5.VH-I70T.VK-A3V.IgG1.3").

[0654] For antibody 10F10, two framework mutations (Y36F and S63T) were identified in the light chain variable region and are shown in ​ . No framework mutations were found in the heavy chain variable region. Since the F to Y revertant at position 36 is a minor difference, a double mutant (10F10.3) containing both the light chain revertant mutations F36Y and T63S was generated and tested in the case of IgG1.3 (10F10.3.IgG1.3, also referred to as "10F10.VK-F36Y / T63S.IgG1.3"). A mutant having only the revertant mutation F36Y was also generated and tested in the case of IgG1.3 (10F10.1.IgG1.3, also referred to as "10F10.VK-F36Y.IgG1.3"). Both revertant mutant Abs contain the heavy chain of 10F10. Table 1 lists the alternative names of the recombinant antibodies described herein.

[0655] ​

[0656] Example 2: Binding of Anti-hILT4 Antibodies to Soluble hILT4

[0657] Using The T200 instrument measures the binding kinetics of the parental antibody and the germline revertant anti-hILT4 antibody (in the case of IgG1.3) via surface plasmon resonance (SPR). The measurement temperature was 37 °C, and the running buffer was PBS pH 7.4 supplemented with 0.05% Tween-20. All anti-hILT4 antibodies were captured on a CM4 chip (Pierce ThermoScientific catalog number 21186) pre-fixed with Protein A / G. Three different batches of 21D9 were used as controls. Monomeric human ILT4 was injected as the analyte. Appropriate concentrations were used for the analysis: for 10F10.1 and 10F10.3 a, a 3-fold dilution series of the highest concentration of hILT4 at 1.4 μM was analyzed. For 10F10, two independent hILT4 dilution series were analyzed: the highest concentration of 1.4 μM, 5-fold dilution and 470 nM, 3-fold dilution. For all other antibodies, the highest concentration of hILT4 at 94 nM and a 5-fold dilution series were used. The hILT4 comprising the extracellular region of hILT4 linked to a His-Avi tag used for this experiment consisted of the following amino acid sequence: QTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEEEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPGVSKKPSLSVQPGPVVAPGESLTLQCVSDVGYDRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSESSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAGAADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPYLLSHPSEPLELVVSGPSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGSPGGGSGGGSEQKLISEEDLGHHHHHHGLNDIFEAQKIEWHE (hILT4-His-Avi tag; SEQ ID NO:119).

[0658] The results are shown in Table 2 and ​ in

[0659] ​

[0660]

[0661]

[0662] The results showed that four revertant mutants of 21D9 had similar kinetics and affinity. 21A5 and its revertant mutant 21A5.a also had similar kinetics and affinity. However, two 10F10 revertant mutants, 10F10.1 and 10F10.3, had a slightly faster dissociation rate than their parent 10F10, resulting in an approximately 3-fold loss of overall affinity. The dissociation rates of 10F10.1 and 10F10.3 were too fast to be measured with high confidence.

[0663] Example 3: Binding of anti-hILT4 antibodies to hILT4-expressing cells

[0664] This example describes the binding characteristics of anti-hILT4 antibodies to hILT4-transfected CHO cells and to human monocytes expressing hILT4.

[0665] 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) titrated from 20 μg / ml by 3-fold serial dilution, and then treated with PE-conjugated secondary anti-human IgG. EC was determined using FACS analysis for geometric mean fluorescence intensity (GMFI). 50 。

[0666] The binding curves and EC values are shown in ​ and ​ respectively.

[0667] In ​ monocytes from peripheral blood selected from normal healthy donors were stained with individual anti-ILT4 antibodies conjugated to Alexa67. The antibodies were titrated from 20 μg / mL by 3-fold serial dilution, and GMFI was determined by FACS analysis for calculating EC50 (shown in the table below the graph).

[0668] Example 4: Selectivity of anti-ILT4 antibodies for LILRA / B family members

[0669] 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 LILRA / LILRB family members was measured.

[0670] Individual ILT family members are overexpressed in 293T cells. Each transfection was stained with 20 μg / ml of anti-ILT4 antibody and subsequent 1 μg / ml anti-human IgG secondary mAb. Binding of the antibody to each transfection was measured by flow cytometry. In a second experiment, LILRA1, LILRA3, and ILT4 transfections were incubated with various concentrations of 21A5 and 21A5.a.

[0671] Shown in ​ and ​ and the results in Table 3 indicate that, relative to other LILRA and LILRB family members, the anti-hILT4 antibody binds predominantly and selectively to hILT4. LILRA3 is the only secreted protein in the LILRA / B family. More specifically, 21D9 and 21D9.e did not bind significantly to LILRA1, LILRA2, LILRA3, LILRA4, LILRA6, ILT2, ILT3, ILT5, and LIR8, and bound only weakly to LILRA5.

[0672] ​

[0673] ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 21D9 + +++ ​ + +++ 21A5 +++ +++ ​ ++ +++ 2H2 +++ 10F10 +++

[0674] "Parent" in Table 3 refers to the parental cell line expressing the LILRA or LILRB molecule.

[0675] Cross-reactivity against other family members of LILRA was further tested as follows. Antibody titration flow cytometry binding assays were performed to determine the binding potency of 21D9, 21D9.e, 21A5, and 21A5.a to hILT4 and its corresponding cross-reactive ILT molecules. The EC was determined using a non-linear regression formula from GraphPad 50 software.

[0676] The results shown in Table 4 and Figure 20 show that the anti-ILT4 antibodies 21D9, 21D9.e, 21A5, and 21A5.a show weak cross-reactivity with other family members of LILRA: weak cross-reactivity of 21D9 and 21D9.e with LILRA5 and of 21A5 and 21A5.a with LILRA1 and LILRA3.

[0677] Table 4 - EC of anti-ILT4 antibody cross-reacting with ILT 50 Summary of binding potency (μg / ml)

[0678] 21D9 ​ 21A5 ​ ​ 0.0460 0.039 0.091 0.085 ​ ～29964 ～312.6 ​ ​ ​ ​ ​ 12.53 49.82 ​ ​ ​ 9.18 52.94

[0679] 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 only weakly bind to hLILRA5. Thus, compared to other anti-ILT4 antibodies that cross-react with ILT5 (LILRB3) and LILRA6, such as clone 287219 from R&D Systems, 21D9 and 21D9.e bind to hILT4 with much greater specificity relative to other hLILRA and hLIRB family members.

[0680] Example 5: Anti-hILT4 Antibodies Potentiate T Cell Responses

[0681] The effects of anti-hILT4 antibodies on T cell responses, particularly T cell proliferation and interferon γ (IFNγ) T cell secretion, were determined as follows. Cytoplasmic truncated hILT4 was transfected into CHO cells expressing a low level of the single chain variable fragment of an anti-CD3 antibody (CHO-OKT3 cells). The transfected cell line was designated CHO-OKT3-ILT4. All T cells isolated from human PBMCs were co-cultured with irradiated (growth-arrested) CHO-OKT3-ILT4 cells at 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 dilution and incubated at 37 °C for 3 days. Supernatants were harvested to assess IFNγ levels by ELISA, and the culture plates were pulsed with 3 H thymidine overnight to assess proliferation by thymidine incorporation.

[0682] Shown in ​ The results indicate that 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 between these antibodies in this assay.

[0683] Example 6: Monocyte-Derived Dendritic Cells (MoDCs) Generated by Pretreating Monocytes with Anti-ILT4 Antibodies during Differentiation Are More Stimulatory to Allogeneic T Cells

[0684] Using The Human Monocyte Isolation Kit isolates CD14+ monocytes from human PBMCs, plates them at 1,000,000 cells / ml, and differentiates them into monocyte-derived immature DCs (Mo-iDCs) for six or seven days in RPMI medium supplemented with 50 ng / mL GM-CSF and 100 ng / mL IL-4. During differentiation, these cells are incubated with anti-ILT4 or isotype antibodies or remain untreated. After the cells were differentiated into Mo-iDCs, they were washed to remove anti-ILT4 antibodies, GM-CSF and IL-4, and further activated into mature dendritic cells (DCs (Mo-mDCs)) in the presence of 50 ng / mL of CD40 agonistic antibodies. Mature DCs (Mo-mDCs) were prepared for allogeneic mixed leukocyte reaction assays (allogeneic MLRs) by co-culturing them with T cells at a ratio of 1:10 (Mo-mDC:T) for 5 days. Cell supernatants were harvested at the end of the allogeneic MLR for measurement of IFNγ by ELISA, and the cells were cultured for a final 16-hour period. 3 T cell proliferation was assessed by H-thymidine incorporation.

[0685] The results of separate assays shown in FIG22 indicate that CD4+ T cell proliferation ( ) in the allogeneic MLR and MoDC assays was significantly increased when MoDCs were differentiated from monocytes in the presence of anti-ILT4 antibodies 21A5.a or 21D9.e. ​ and ​ ) and IFNγ secretion ( ​ and ​ ) enhancement. Monocytes from two different donors were used in separate assays.

[0686] Example 7: Monocyte-derived dendritic cells (MoDCs) differentiated from monocytes pre-coated with anti-ILT4 antibodies increase the expression of co-stimulatory or maturation molecules

[0687] use Human monocyte isolation kits were used to isolate CD14+ monocytes from human PBMCs, plated at 1,000,000 cells / ml, and differentiated into monocyte-derived immature DCs (Mo-iDCs) in RPMI 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 for six or seven days, or left untreated. Mo-iDCs were then harvested and stained for cell surface markers CD86 and CD83.

[0688] The results shown in Figure 23 indicate that, when anti-ILT4 antibody is present during the differentiation of monocytes into Mo-iDC, the expression of CD86 and CD83 (co-stimulatory and maturation molecules) on Mo-iDC increases, respectively. Together with the results of Example 6, these results show that treatment of monocytes with anti-hILT4 antibody during the differentiation of monocytes into MoDC promotes pro-inflammatory ( ​ ) of MoDC as shown by upregulation of CD83 and CD86, which thus triggers 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 can provide an enhanced anti-cancer immune response.

[0689] Example 8: Anti-ILT4 antibody enhances TNFα secretion of autologous ex vivo-differentiated macrophages

[0690] Monocytes isolated from human PBMC were differentiated into macrophages in the presence of M-CSF for 5 days. Macrophages were treated with indicated concentrations (next to their names and in ng / ml) of anti-ILT4 antibodies 21D9, 21D9.e, 21A5, 21A5.a, 2H2 and 10F10 or isotype control in the presence of 10 ng / ml LPS ( ​ ) or 5 μg / ml 2'3'-cGAMP STING agonist ( ​ ). TNFα production was measured by ELISA 24 hours after treatment, and the EC50 value in the presence of each antibody is labeled next to the curve.

[0691] The results shown in Figure 24 indicate that the presence of ILT4 antibodies 21D9, 21D9.e, 21A5, 21A5.a, 2H2 and 10F10 enhances TNFα secretion of ex vivo-differentiated macrophages by LPS or STING agonist. Thus, immunosuppressive macrophages show a shift towards a more pro-inflammatory phenotype when ILT4 is blocked, which can potentially provide an enhanced anti-cancer immune response. These results suggest that anti-ILT4 antibodies promote pro-inflammatory polarization towards M1 macrophages.

[0692] Example 9: Anti-ILT4 antibody promotes CD4 T cell response in macrophage:CD4+ T cell allogeneic MLR

[0693] Monocytes isolated from peripheral PBMCs were treated with M-CSF for 5 days to differentiate into macrophages. The macrophages were then co-cultured with allogeneic CD4+ T cells isolated from PBMCs of different donors and labeled with CFSE dye. The allogeneic MLR co-culture included 1 μg / ml of the indicated antibodies (isotype control, anti-ILT4, or anti-PDL1). On day 6, the supernatants were collected for analysis of IFNγ and CD4+ T cell proliferation, and evaluated by FACS based on CFSE dilution.

[0694] The results shown in ​ indicate that the presence of anti-ILT4 antibodies 21D9, 21D9.e, 21A5, or 21A5.a in the allogeneic MLR of in vitro-differentiated macrophages and CD4+ T cells promotes CD4+ T cell proliferation ( ​ ) and IFNγ production ( ​ ). These results suggest that anti-ILT4 antibodies promote T cell activation by regulating ILT4 inhibitory signaling in macrophages or blocking its direct inhibitory activity in T cells.

[0695] Example 10: Combinatorial effect of anti-ILT4 and anti-PD-L1 in macrophage:CD4 T cell allogeneic MLR

[0696] Monocytes isolated from peripheral PBMCs were treated with M-CSF for 5 days to differentiate into macrophages, which were then co-cultured with allogeneic CD4+ T cells isolated from PBMCs of allogeneic donors. The allogeneic MLR co-culture included anti-ILT4 antibody 21D9 and anti-PD-L1 antibody (isotype control, anti-ILT4, anti-PD-L1, or combined anti-ILT4 and anti-PD-L1) added alone or together at 10 μg / ml. On day 6, the supernatants were collected for analysis of IFNγ.

[0697] The results shown in Figure 26 indicate that the combination of anti-ILT4 antibody 21D9 and anti-PD-L1 antibody stimulates enhanced production of IFNγ from the macrophage:CD4+ T cell allogeneic MLR relative to each antibody alone.

[0698] Example 11: HDX epitope mapping of anti-hILT4 Abs 21D9 and 2H2 Fab

[0699] HDX-MS was used to identify the regions of hILT4 bound by anti-hILT4 antibodies 21D9 and 2H2.

[0700] HDX-MS probes protein conformation and conformational dynamics in solution by monitoring the rates and extents of deuterium exchange of backbone amide hydrogen atoms (Huang and Chen (2014) Analytical and Bioanalytical Chem. 406:6541 and Wei et al. (2014) Drug Discovery Today 19:95). The levels of HDX depend on the solvent accessibility of backbone amide hydrogen atoms and protein hydrogen bonding. The mass increase of proteins after HDX can be accurately measured by MS. When this technique is paired with enzymatic digestion, structural features can be resolved at the peptide level, differentiating surface-exposed peptides from those folded internally or those chelated at the protein–protein complex interface. Typically, deuterium labeling and subsequent quenching experiments are performed, followed by enzymatic digestion, peptide separation, and MS analysis.

[0701] Prior to the epitope mapping experiments, non-deuterated experiments were performed to generate a list of the consensus peptides for recombinant human ILT4 (15 μM) and the protein complex of ILT4 with Fab 21D9 and 2H2 (1:1 molar ratio). In the HDX-MS experiments, 5 μL of each sample (ILT4 with Fab or ILT4) was diluted into 55 μL of D2O buffer (10 mM phosphate buffer, D2O, pH 7.0) to initiate the labeling reaction. The reactions were carried out for different time periods: 1 min, 10 min, and 240 min. At the end of each labeling reaction period, the reactions 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 of common pepsin-digested peptides was monitored in the absence / presence of Fab 21D9 and 2H2.

[0702] Human ILT4 (hILT4) was histidine-tagged and mixed with 21D9 or 2H2 Fab at a 1:1 ratio, and HDX was performed for 1 minute, 10 minutes, or 4 hours. Sequence coverage of hILT4 by pepsin is shown in ​ The regions of hILT4 (or hILT4 peptides) that bind to each Fab are shown in ​ and ​ Specifically, the HDX-MS experiments provided 85% sequence coverage of human ILT4.

[0703] HDX-MS data analysis of 21D9 and 2H2 in human ILT4 indicated that the epitope of 21D9 consists of a region of human ILT4 (residue numbers corresponding to the native human ILT4 sequence): the region in Ig domain 2: 70ITRIRPELVKNGQFHIPSITWEHT GRYGCQY 100 (SEQ ID NO:122). (See ​ , the figure above.)

[0704] HDX-MS data analysis indicates that the epitope of 2H2 consists of five regions of human ILT4 and regions 2 and 5 as the major epitopes:

[0705] Region 1: 127 SAQPSPVVTSGGRVTL 142 (SEQ ID NO:173)

[0706] Region 2: 154 ILCKEGEEEHPQCLNSQPHARGSSRAIF 181 (SEQ ID NO:123)

[0707] Region 3: 182 SVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDL 213 (SEQ ID NO:174)

[0708] Region 4: 378 QAEFPMSPVTSAHAG 392 (SEQ ID NO:175)

[0709] Region 5: 425 SSPPPTGPIS 434 (SEQ ID NO:124)

[0710] (See ​ , the figure below.)

[0711] Example 12: Anti-ILT4 antibody blocks the binding of ILT4 to class I MHC molecules

[0712] A flow cytometry-based antibody blocking assay was performed to determine the ability of anti-ILT4 antibodies to block the binding of rhILT4-Fc to CHO cells overexpressing HLA-A or HLA-B. An rhILT-Fc fusion protein containing the murine Fc tail region was generated. Anti-hILT4 antibodies 21D9, 2H2, 10F10, and 21A5 were titrated in the presence of a fixed concentration of 30 μg / ml rhILT4-Fc. The antibody and rhILT4-Fc mixture was incubated on ice for 30 minutes and then 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 the bound rhILT4-Fc.

[0713] Shown in ​ the results in indicate that antibodies 21D9, 2H2, 10F10 and 21A5 inhibit the binding of hILT4 to HLA-A and HLA-B, respectively.

[0714] Example 13: 21D9e.IgG1.3 enhances both IFN-γ secretion and TNF-α secretion mediated by T cells

[0715] This example shows that 21D9e.IgG1.3 enhances the IFN-γ and TNF-α secretion of T cells in the autologous mixed lymphocyte reaction (MLR) of monocytes and T cells.

[0716] T cells and monocytes were isolated from peripheral blood mononuclear cells (PBMC) and co-cultured at a 1:1 ratio in the presence of 20 ng / mL anti-CD3 antibody (clone: OKT3, BioLegend). 21D9e.IgG1.3 and isotype control (DT-1D12-g1.3f) were added at 10 μg / mL. Intracellular staining was performed to determine the frequency of T cells expressing IFN-γ and TNF-α in each sample using fluorescent dye-conjugated IFN-γ antibody (clone #B27, BioLegend) and TNF-α antibody (clone #MAb11, BioLegend) after an 88-hour incubation period. Samples were acquired on a cell counter (Beckmen Coulter) and analyzed using FlowJo TM software (Tree Star, Inc, Ashland, OR).

[0717] Shown in ​ the results in indicate that 21D9e.IgG1.3 enhances both IFN-γ secretion and TNF-α secretion mediated by CD4+ and CD8+ T cells after anti-CD3 stimulation in autologous allogeneic MLR.

[0718] Example 14: Anti-ILT4 antibodies enhance IFN-γ secretion after antigen stimulation in a CMV lysate assay

[0719] Peripheral blood mononuclear cells (PBMC) from CMV - reactive donors were plated at 200,000 cells / well in the presence of 0.3 μg / mL of cytomegalovirus (CMV) lysate (CMV Antigen 2, Microbix Cat#EL - 02 - 01 - 001). Cells were treated with 8 - point, 5 - fold titrations of anti - ILT4 antibodies 21D9.IgG1.3 and 21A5.IgG1.3 starting at 20 μg / mL. Nivolumab was added at 1 μg / mL as a positive control. 21D9 - Fab and DT - 1D12 - g1.3 were added at 20 μg / mL. Supernatants were harvested from each sample and IFN - γ levels were measured by ELISA (Human IFN - γ ELISA Kit, BD OptEIA Cat.555142) after 6 days of incubation.

[0720] The results shown in ​ indicate that anti - ILT4 antibodies 21D9.IgG1.3, 21D9 - Fab, and 21A5.IgG1.3 enhance IFN - γ secretion after antigen stimulation in the CMV lysate assay.

[0721] Example 15: ILT4 antagonism enhances T - cell activation in monocyte:T allogeneic MLR

[0722] T cells (100,000) from one donor and allogeneic monocytes were co - cultured in 96 - well U - bottom culture plates at a ratio of 2:1 per well. Anti - ILT4 antibodies 2H2.IgG1.1f, 2H2 Fab, 21D9.IgG1.1f, and 21D9 Fab were added to 4 - fold, 7 - point titrations starting at a concentration of 30 μg / mL for full - length antibodies or 80 μg / mL for Fab antibodies. Anti - KLH - g1.1f (anti - keyhole limpet hemocyanin antibody) was added at 30 μg / mL as an isotype control. The cell cultures were incubated for 6 days. Cell proliferation was evaluated by 3 H - thymidine incorporation during the last 16 hours of culture.

[0723] The results shown in ​ indicate that full - length anti - ILT4 antibodies 21D9.IgG1.1f and 2H2.IgG1.1f and their corresponding Fab fragments all induce T - cell proliferation.

[0724] Example 16: Binding of anti - hILT4 antibodies to cynomolgus monkey ILT4

[0725] Several ILT family members were obtained from cynomolgus monkeys via PCR. Since sequence analysis of these proteins did not clearly indicate their best match to hILT4, expression profiling and functional assays were performed. These assays identified the clone named "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:

[0726] QAGILPKPMLWAEPDRVITQGSPVTLRCQGNLEARGYHLYRERKSASWITLIRPELVKKGQFPIPSITWEDAGRYRCQYYSHSWWSEHSDPLELVVTGAYRKPTLSALPSPVVASGGNVTLQCDSRVALDGFILCKEGEDEHSQRLNSQPRTRGSSRAVFSVGPVSPSRRWSYRCYGYELHSRYVWSLPSDLLELLVPGVSKKPSLSVQPGPVVAGGDKLTLQCGSDAGYDRFALYKEGERDFLQRPGQQLQAGLAQANFTLDPVRGSHGGQYRCYGAHNLSSEWSAPSDPLDILISAGPHSGLRRECDPAVSVTGMDGHFLSDQGGSSSPGGGSGGGSEQKLISEEDLGHHHHHHGLNDIFEAQKIEWHE (SEQ ID NO:118). For binding to hILT4, the extracellular domain (ECD) of hILT4-His-Avi tag described in Example 2 was used; SEQ ID NO:119.

[0727] The kinetics of binding of the above ILT4 constructs to the 21D9.e antibody of cynomolgus monkey and human ILT4 were analyzed in a comparative SPR assay. The antibody was amine-coupled to a GLC sensor chip on a Proteon XPR36 instrument (BioRad). Human ILT4 and cynomolgus monkey ILT4 9152 were injected as analytes in a 5-fold, 5-fold dilution series at a maximum concentration of 1 μM (minimum concentration of 1.6 nM). All data were double-referenced, exported, and analyzed using the kinetic titration model described by Karlsson et al. (Karlsson et al. (2005) Analytical Biochem. 349:136) with BIAevaluation software 4.1.1. The running buffer consisted of 10 mM HEPES pH 7.4, 150 mM NaCl, and 0.05% Tween-20. The assay temperature was 37°C.

[0728] The results shown in Table 5 indicate that 21D9.e binds cynomolgus macaque ILT4 9152 with approximately 11-fold weaker affinity compared to human ILT4 (the K D value for cynomolgus macaque is 3.5 nM and the K D value for human ILT4 is 0.33 nM). Similarly, the parental antibody 21D9 binds cynomolgus macaque ILT4 with approximately 12-fold weaker affinity compared to human ILT4. The difference is caused by the faster dissociation rate constant of cynomolgus macaque ILT4 (the K D value for cynomolgus macaque is 4.0 nM and the K D value for human ILT4 is 0.32 nM).

[0729] ​

[0730]

[0731] Example 17: ILT4 Antibodies Promote the Expression of Costimulatory Molecules and Activation Markers on Cynomolgus Macaque Monocyte-Derived Dendritic Cells In Vitro

[0732] This example shows 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) stimulate the expression of CD80, CD83, and CD86 on monocyte-derived dendritic cells.

[0733] Monocytes isolated from cynomolgus macaque PBMCs (non-human primate CD14 microbeads, Miltenyi) 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 setting up the culture, anti-ILT4 antibodies were added to the culture at 10 μg / mL. Cells were stained with anti-CD86 (clone #IT2.2, BioLegend), anti-CD80 (clone #L307), and anti-CD83 (clone #HIB15e) to determine the expression levels of CD86, CD80, and CD83 on their surface. Samples were acquired on a cell counter (Beckmen Coulter) and analyzed with FlowJo TM software (Tree Star, Inc, Ashland, OR).

[0734] Shown in ​The results in indicate that the ILT4 antibodies 21D9.e.IgG1.3 (also known as ILT4.8.IgG1.3; "ILT4.8" in the figures) and 9G4.IgG1.3 (also known as ILT4.1.IgG1.3; "ILT4.1" in the figures) promote the expression of the costimulatory molecules CD80 and CD86 and the dendritic cell maturation marker CD83 in monocyte-derived DCs, as demonstrated by the upregulation of CD80, CD83, and CD86 on dendritic cells.

[0735] Example 18: Modified anti-ILT4 antibody 10F10.4

[0736] The tyrosine in F36Y in the light chain of 10F10.1 and 10F10.3 (see Example 2) is located near the heavy chain CDR3 in the modeling of hILT4 and the 10F10 antibody. Thus, replacing F36 with Y can cause conflict with the isoleucine in the heavy chain CDR3, as demonstrated by the faster dissociation rate of 10F10.1 and 10F10.3 relative to 10F10 (see Example 2). Another mutant was generated in which F36 of the light chain was not mutated and the only light chain framework region generated in 10F10 was mutated to T63S. The heavy chain was not mutated and was thus the heavy chain 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.

[0737] Using at 37 °C The binding kinetics of 10F10.4 were determined using a T200 instrument. The running buffer was 10 mM HEPES pH 7.4 supplemented with 0.05% Tween-20 and 1 g / L BSA. All anti-ILT4 antibodies were captured on a CM4 chip containing a pre-fixed anti-human Fc capture antibody from Southern Biotech (catalog number 2081-01). Human ILT4 (hILT4-His-Avi tag described in Example 2) was injected at 1.2 uM (only for one replicate of 10F10, 10F10.4, and one 1E5 ILT4.15), 405 nM, 81 nM (in duplicate), 16 nM, 3 nM, and 0.6 nM. All data were double-referenced and fit to a 1:1 Langmuir model using Biacore T200 evaluation software version 3.1 with mass transport.

[0738] The results shown in Table 6 indicate that the binding kinetics of 10F10.4 are extremely similar to those of 10F10. 21A5, 21A5a, 21D9, and 21D9.e were included as controls. The absolute values are slightly different from those shown in Table 2 due to the use of different capture reagents in the assay.

[0739] ​

[0740]

[0741] Exhibit 10F10.4 is functionally active by enhancing TNFα secretion of autologous ex vivo-differentiated macrophages. This was confirmed by incubating 10F10.4 with macrophages differentiated from autologous PBMC monocytes as described in Example 8. Also included were 21D9.e and an isotype control. The results shown in ​ and ​ indicate that the presence of the ILT4 antibody 10F10.4 enhances TNFα secretion during the ex vivo differentiation of monocytes into macrophages by LPS or a STING agonist.

[0742] Example 19: 21D9.e has similar potencies in the case of IgG1, IgG4, and IgG1.3

[0743] This example shows that the potency of 21D9.e is similar in the case of IgG1, IgG4 (S228P), and IgG1.3 heavy chains.

[0744] The 21D9.e antibody was cloned in the case of IgG1, IgG4 (with S228P), or IgG1.3 (SEQ ID NO: 176, 178, and 13, respectively) and used in an assay to measure TNFα secretion conducted as follows.

[0745] The results shown in ​ indicate that similar amounts of TNFα were secreted for 21D9.e.IgG1, 21D9.e.IgG4.S228P, and 21D9.e.IgG1.3. Thus, the 21D9.e variable region has similar functional potencies in the case of these different Fc regions. DT-1D12 was an isotype control.

[0746] Example 20: 21D9e binds to the hILT4 allele

[0747] This example describes an assay for determining whether 21D9.e binds to alternative alleles of hILT4.

[0748] Only consider those alleles with an occurrence rate exceeding 1%. Further consider two alleles with amino acid changes in regions D1 and D2: E161D and R103H (numbered according to SEQ ID NO:107, i.e., ILT4 with a signal sequence). Since 3D modeling shows that the two amino acid residues are not close to the binding interface of class I MHC, it is expected that amino acid changes at these residues do not affect the binding of the antibody to hILT4. To confirm this, hILT4 proteins with substitutions E161D and R103H (hILT4 E161D / R103H) were prepared and their binding to 21D9.e.IgG1.3 and 21A5.a.IgG1.3 (ILT4.9.IgG1.3; see Table 1) was tested via Octet BLI. It was found that the two proteins bound similarly to hILT4 E161D / R103H and hILT4 E161 / R103.

[0749] Example 21: 21D9e.IgG1.3 does not significantly trigger basophil activation

[0750] The assay for detecting basophil activation by anti-ILT4 antibodies involves the following steps. Generally, the assay is performed by using the Buhlmann Laboratories AG method in the basophil activation test. Human basophils from donor blood are detected using anti-Ccr3. CD63 is used as an indicator of basophil activation. Stimulation buffer and antibody are added to whole blood. After incubation, a staining reagent is added to the blood; and after antibody incubation, red blood cells are lysed and the sample is centrifuged, washed, and analyzed on a BD FACS Canto II. Based on this assay, Ccr3+CD63+ activated basophils from 8 donors were measured by FACS after incubation with 21D9.e.IgG1.3 (ILT4.8.IgG1.3; see Table 1), 21.5Aa.IgG1.3 (ILT4.9.IgG1.3), or anti-DT at 4.5 μg / mL, 45.45 μg / mL, or 454.55 μg / mL for 180 minutes. Positive controls included anti-FcεRI and fMLP (N-formylmethionyl-leucyl-phenylalanine). The negative control was DT1DT12-IgG1.3, which targets the unrelated antigen diphtheria toxin.

[0751] Shown in

[0752] Shown in ​The results in indicated that 21D9.e.IgG1.3 (ILT4.8.IgG1.3) and 21.5Aa.IgG1.3 (ILT4.9.IgG1.3) did not induce basophil activation under the tested conditions. The lack of basophil activation represents an important safety feature.

[0753] Example 22: Anti-ILT4 antibody 21D9.e.IgG1.3 is stable

[0754] This example describes a 3-month stability study with anti-ILT4 antibody 21D9.e.IgG1.3 (also referred to as ILT4.8.IgG1.3), the data of which demonstrate that the antibody is stable for at least 3 months.

[0755] Samples of 21D9.e.IgG1.3 were incubated at 25 °C or 40 °C at a concentration of 150 mg / mL. The samples were diluted to 1 mg / mL with mobile phase (40 mM NaH2PO4, 60 mM Na2HPO4, 0.1 M Na2SO4, pH 6.8), and forty microliters were analyzed isocratically (0.1 M sodium phosphate, 0.1 M sodium sulfate, pH 6.8, at 0.2 mL / min) using an Agilent 1200 series HPLC system (Agilent Technologies, Santa Clara, CA) with a TSKgel Super3000SW column (4.6 mm ID × 30 cm, 4 μm, Tosoh Bioscience, King of Prussia, PA). UV detection was set at 280 nM.

[0756] Shown in ​ the results in indicated that the 21D9.e.IgG1.3 (ILT4.8) antibody composition contained less than 5% high molecular weight material and low molecular weight material after incubation at 25 °C for 3 months and less than 10% high molecular weight material and low molecular weight material after incubation at 40 °C for 3 months.

[0757] Example 23: Cross-blocking of 2H2 and 21D9 by anti-ILT4 monoclonal antibodies

[0758] First, CHO cells overexpressing ILT4 were pre-incubated with serial dilutions (3-fold, 12 points, starting at 40 μg / mL) of unconjugated anti-ILT4 antibodies 10F10.IgG1.3 and 21A5.IgG1.3. Anti-KLH-g1.1f (anti-hemocyanin antibody) was added as an isotype control, and unconjugated 2H2.IgG1.1f or 21D9.IgG1.1f was added at 40 μg / mL as a positive control to confirm blocking activity. After a 30-minute incubation period, PE-conjugated 2H2.IgG1.1f or 21D9.IgG1.1f was added at 1.5 μg / mL and incubated with the cells for another 30 minutes. After washing, the samples were analyzed on a FACSCanto TM The cells were acquired using a cell counter (BD Bioscience, San Jose) and analyzed using FlowJo TM Software (Tree Star, Inc, Ashland, OR) was used for analysis.

[0759] The results are shown in ​ (2H2 cross-blocking) and ​ (21D9 cross-blocks). The results indicate that 10F10 and 21A5 cross-block (or compete with) 2H2 binding to hILT4 ( ​ ), and 10F10, but not 21A5, cross-blocks (or competes with) 21D9 binding to hILT4 ( ​ Thus, (i) 21A5 and 2H2 are in the same partition group (i.e., they compete with each other for binding to hILT4); (ii) 21D9 is in a separate partition group from 21A5 / 2H2; and (iii) 10F10 competes with both 21D9 and 2H2 / 21A5 for binding to hILT4.

[0760] A diagram showing the hILT4 binding region of the antibody on the crystal structure of hILT4 is shown in ​ Ig-like domain 1 corresponds to amino acid residues 27-110 of SEQ ID NO: 107, and Ig-like domain 2 corresponds to amino acid residues 111-229 of SEQ ID NO: 107. These two domains are shown in ribbon format. ​In. The results of these experiments indicate that antibody 21D9 binds to Ig-like domain 1, antibodies 2H2 and 21A5 bind to Ig-like domain 2, and antibody 10F10 binds to a region within Ig-like domain 2 that is close to Ig-like domain 1 and that can compete with antibodies that bind to Ig-like domain 1 or Ig-like domain 2. Antibodies that are expected to differ from the antibodies used in these experiments only due to germline mutations (such as 21D9.e, 29A5.a, and 10F10.3 and 10F10.4) bind to the same regions as their parental antibodies (21D9, 29A5, and 10F10, respectively).

[0761] Example 24: Epitope Mapping Using Carbene Chemical Footprinting

[0762] Exchange the reagent buffer for human ILT4 and anti-human ILT4 antibody Fabs 10F10, 21D9, and 21A5 into 100 mM Tris-HCl and 50 mM NaCl, pH 7.5, at 1 mg / ...

Claims

1. An antibody that specifically binds to human ILT4 (hILT4), said antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region (VH) containing: VH complementarity-determining region 1 (CDR1), CDR2, and CDR3 having amino acid sequences as shown in SEQ ID No: 155, 156, and 157 respectively, and wherein the light chain comprises a light chain variable region (VL) containing: VL CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID No: 158, 159, and 160 respectively.

2. The antibody according to claim 1, comprising: a. a VH amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO: 71 and / or a VL amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO: 70; b. a VH amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO: 74 and / or a VL amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO: 70; c. a VH amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO: 75 and / or a VL amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO: 70; d. a VH amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO: 78 and / or a VL amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO: 70; or e. a VH amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO: 80 and / or a VL amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO:

70.

3. The antibody according to claim 1, comprising: a. a VH amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO: 71 and a VL amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO: 70; b. a VH amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO: 74 and a VL amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO: 70; c. a VH amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO: 75 and a VL amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO: 70; d. a VH amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO: 78 and a VL amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO: 70; or e. A VH amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO: 80 and a VL amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO:

70.

4. The antibody according to claim 1, comprising: a. A VH comprising the amino acid sequence shown in SEQ ID NO: 71; b. A VH comprising the amino acid sequence shown in SEQ ID NO: 74; c. A VH comprising the amino acid sequence shown in SEQ ID NO: 75; d. A VH comprising the amino acid sequence shown in SEQ ID NO: 78; or e. A VH comprising the amino acid sequence shown in SEQ ID NO:

80.

5. The antibody according to claim 1, comprising: a VH containing the amino acid sequence shown in SEQ ID NO:

80.

6. The antibody according to claim 1, comprising: a VL containing the amino acid sequence shown in SEQ ID NO:

70.

7. The antibody according to claim 1, comprising: a. A VH comprising the amino acid sequence shown in SEQ ID NO: 71 and a VL comprising the amino acid sequence shown in SEQ ID NO: 70; b. A VH comprising the amino acid sequence shown in SEQ ID NO: 74 and a VL comprising the amino acid sequence shown in SEQ ID NO: 70; c. A VH comprising the amino acid sequence shown in SEQ ID NO: 75 and a VL comprising the amino acid sequence shown in SEQ ID NO: 70; d. A VH comprising the amino acid sequence shown in SEQ ID NO: 78 and a VL comprising the amino acid sequence shown in SEQ ID NO: 70; or e. A VH comprising the amino acid sequence shown in SEQ ID NO: 80 and a VL comprising the amino acid sequence shown in SEQ ID NO:

70.

8. An antibody that specifically binds to human ILT4 (hILT4), the antibody comprising: a VH containing the amino acid sequence shown in SEQ ID NO: 80 and a VL containing the amino acid sequence shown in SEQ ID NO:

70.

9. The antibody according to any one of claims 1-8, having one or more of the following characteristics: - Does not specifically bind to hILT2, hILT3, and / or hILT5; - Does not specifically bind to hILT2, hILT3, and hILT5; - Specifically binds to cynomolgus macaque ILT4 comprising the amino acid sequence shown in SEQ ID NO:

118.

10. The antibody according to any one of claims 1-8, which is an IgG antibody.

11. The antibody according to claim 10, which is an IgG1, IgG2, or IgG4 antibody.

12. The antibody according to claim 11, wherein the antibody is an IgG4 antibody comprising the S228P substitution according to EU numbering.

13. The antibody according to claim 10, wherein the antibody comprises a heavy chain constant region, and the heavy chain constant region comprises 1, 2, 3, 4, or 5 mutations in another wild-type human heavy chain constant region, and the mutations reduce the effector function of the antibody as compared to an antibody having the same amino acid sequence but without the 1, 2, 3, 4, or 5 mutations, or wherein the antibody is an effectorless antibody.

14. The antibody according to claim 10, wherein the heavy chain of the antibody lacks a C-terminal lysine residue.

15. The antibody according to claim 10, which is a full-length antibody, and wherein the heavy chain of the antibody may or may not comprise a C-terminal lysine residue.

16. The antibody according to claim 10, which comprises a heavy chain constant region having an amino acid sequence as shown in any one of SEQ ID NO: 98, 100, 102, 103, 104, or 179.

17. The antibody according to any one of claims 1-7, which comprises a heavy chain constant region having an amino acid sequence as shown in SEQ ID NO:

100.

18. The antibody according to any one of claims 1-8, which comprises a light chain having an amino acid sequence as shown in SEQ ID NO:

11.

19. The antibody according to any one of claims 1-8, which comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO:

13.

20. The antibody according to any one of claims 1-8, which comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 176, SEQ ID NO: 177, or SEQ ID NO:

178.

21. The antibody according to any one of claims 1-8, comprising: (a) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 12 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 11; (b) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 13 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 11; (c) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 36 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 11; (d) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 38 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 11; (e) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 40 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 11; (f) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 176 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 11; (g) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 177 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 11; or (h) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 178 and a light chain comprising the amino acid sequence shown in SEQ ID NO:

11.

22. An antibody that specifically binds to hILT4, the antibody comprising: a VH containing the amino acid sequence shown in SEQ ID NO: 80, a VL containing the amino acid sequence shown in SEQ ID NO: 70, and a heavy chain constant region containing the amino acid sequence shown in SEQ ID NO:

100.

23. An antibody that specifically binds to hILT4, the antibody comprising: a heavy chain containing the amino acid sequence shown in SEQ ID NO: 13, and a light chain containing the amino acid sequence shown in SEQ ID NO:

11.

24. The antibody according to any one of claims 1-8, which is an Fv, single-chain Fv (scFv), Fab, Fab', or (Fab')2.

25. The antibody according to any one of claims 1-8, 22 or 23, which is a multimeric antibody.

26. The antibody according to any one of claims 1-8, 22 or 23, which is linked to a label.

27. A nucleic acid encoding the heavy chain and light chain of the antibody according to any one of claims 1-26.

28. A host cell comprising the nucleic acid according to claim 27.

29. A method for preparing an antibody, comprising culturing the cell according to claim 28 under conditions for expressing the antibody.

30. A collection of at least two nucleic acids, wherein the first nucleic acid encodes the heavy chain of an antibody according to any one of claims 1-26, and the second nucleic acid encodes the light chain of an antibody according to any one of claims 1-26.

31. A host cell comprising the collection of at least two nucleic acids according to claim 30.

32. A method for preparing an antibody, comprising culturing the cell according to claim 31 under conditions for expressing the antibody.

33. A composition comprising a nucleic acid encoding the heavy chain of an antibody according to any one of claims 1-26 and a nucleic acid encoding the light chain of an antibody according to any one of claims 1-26.

34. A composition comprising an antibody according to any one of claims 1-26 and a pharmaceutically acceptable carrier.

35. The composition according to claim 34, comprising a second therapeutic agent.

36. The composition according to claim 35, wherein the second therapeutic agent is an immune stimulant.

37. The composition according to claim 36, wherein the immune stimulant is an antagonist of PD-1 / PD-L1, CTLA-4 or LAG-3, or an agonist of GITR or OX40.

38. Use of an antibody according to any one of claims 1-26 in the preparation of a medicament for treating cancer in a subject, wherein the cancer is a solid tumor.

39. The use according to claim 38, wherein the subject is administered a second therapy.

40. The use according to claim 39, wherein the second therapy is radiotherapy, surgery or administration of a second agent.

41. The use according to claim 39, wherein the second therapy is a second agent, and the second agent is an immune stimulant.

42. The use according to claim 41, wherein the immune stimulant is an antagonist of PD-1 / PD-L1, CTLA-4 or LAG-3, or an agonist of GITR or OX40.

43. Use of an antibody according to any one of claims 1-26 in the preparation of a medicament for treating a viral disease in a subject.

Images