Antibodies binding to ILT4

Abstract

The present application relates to antibodies that specifically bind to immunoglobulin-like transcript 4 (ILT4), also referred to 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

[0001] This application is a divisional application of the invention application with a filing date of July 8, 2019, Chinese application number 201980057104.7, and invention name “Antibody binding to ILT4”. Technical Field

[0002] The present application relates to antibodies that specifically bind to and inhibit ILT4, and their use in cancer treatment. Background Art

[0003] 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.

[0004] The extracellular domains of the proteins in this family contain several immunoglobulin-like repeats, while their cytoplasmic tails contain several tyrosine-based inhibitory motifs (ITIMs) that recruit tyrosine phosphatases. Summary of the Invention

[0005] Certain embodiments of the invention are summarized in the claims at the end of this invention.For example, the invention includes the following embodiments and other embodiments described in other parts of the text, figures and sequences herein.

[0006] 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:

[0007] - 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);

[0008] - does not specifically bind to hILT2, hILT3 and / or hILT5;

[0009] - does not specifically bind to one or more members of the LILRA and / or LILRB family;

[0010] - stimulates 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;

[0011] - stimulating the differentiation or activation of monocytes into macrophages, e.g., stimulating the differentiation of monocytes into pro-inflammatory macrophages, e.g., as shown in the assays described in the Examples;

[0012] - promoting the expression of CD83 and CD86 on human monocyte-derived immature dendritic cells (Mo-iDCs), e.g., as shown in the assays described in the Examples;

[0013] - enhancing IFN-γ secretion following antigen stimulation in a cytomegalovirus (CMV) lysate assay, e.g., as shown in the assay described in the Examples;

[0014] - 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 assay described in the Examples;

[0015] - Inhibits the binding of HLA-A and / or HLA-B to ILT4;

[0016] - Combined to 70 ITRIRPELVKNGQFHIPSITWEHTGRYGCQY 100 (SEQ ID NO: 122), as determined by hydrogen deuterium exchange (HDX), e.g., as shown in the HDX assay described in the Examples;

[0017] - compete with the antibodies described herein for binding to hILT4;

[0018] - specifically binds to cynomolgus monkey ILT4 comprising SEQ ID NO: 118, e.g., as shown in a binding assay described in the Examples;

[0019] - inhibiting 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., inhibiting the binding of hILT4 to both HLA-A and HLA-B);

[0020] - have the HLA-A and HLA-B binding profiles shown in Figure 28;

[0021] - Promotes the pro-inflammatory polarization of macrophages towards M1 macrophages;

[0022] - have a binding profile as shown in Figure 27;

[0023] -Does not induce (or trigger) basophil activation;

[0024] - Binds to 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 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

[0025] - 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.

[0026] Embodiment 2: The isolated antibody of embodiment 1, comprising 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 NOs: 125-127), 9C8 (SEQ ID NOs: 131-133), 2H2 (SEQ ID NOs: 137-139), 2E5 (SEQ ID NOs: 143-145), 24E5 (SEQ ID NOs: 149-151), 21D9 (SEQ ID NOs: 155-157), 21A5 (SEQ ID NOs: 161-163), or 10F10 (SEQ ID NOs: 167-169).

[0027] Embodiment 3: The isolated antibody of embodiment 1 or 2, comprising 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 NOs: 128-130), 9C8 (SEQ ID NOs: 134-136), 2H2 (SEQ ID NOs: 140-142), 2E5 (SEQ ID NOs: 146-148), 24E5 (SEQ ID NOs: 152-154), 21D9 (SEQ ID NOs: 158-160), 21A5 (SEQ ID NOs: 164-166), or 10F10 (SEQ ID NOs: 170-172).

[0028] Embodiment 4: The isolated antibody of embodiment 2, wherein the heavy chain comprises the VH CDR1, CDR2 and CDR3 and the VL CDR1, CDR2 and CDR3 of the following anti-ILT4 antibodies: 9G4 (SEQ ID NOs: 143-148), 24E5 (SEQ ID NOs: 149-154), 21D9 (SEQ ID NOs: 155-160), 21A5 (SEQ ID NOs: 161-166) or 10F10 (SEQ ID NOs: 167-172).

[0029] Embodiment 5: The isolated antibody of any one of embodiments 1-4, comprising:

[0030] a VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of 9G4 (SEQ ID NOs: 125-127), and a VL comprising the amino acid sequences of VL CDR1, CDR2, and CDR3 of 9G4 (SEQ ID NOs: 128-130);

[0031] a VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of 9C8 (SEQ ID NOs: 131-133), and a VL comprising the amino acid sequences of VL CDR1, CDR2, and CDR3 of 9C8 (SEQ ID NOs: 134-136);

[0032] a VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of 2H2 (SEQ ID NOs: 137-139), and a VL comprising VL CDR1, CDR2, and CDR3 of 2H2 (SEQ ID NOs: 140-142);

[0033] a VH comprising the amino acid sequence of VH CDR1, CDR2, and CDR3 of 2E5 (SEQ ID NOs: 143-145), and a VL comprising the amino acid sequence of VL CDR1, CDR2, and CDR3 of 2E5 (SEQ ID NOs: 146-148);

[0034] a VH comprising the amino acid sequence of VH CDR1, CDR2, and CDR3 of 24E5 (SEQ ID NOs: 149-151), and a VL comprising the amino acid sequence of VL CDR1, CDR2, and CDR3 of 24E5 (SEQ ID NOs: 152-154);

[0035] - a VH comprising the amino acid sequence of VH CDR1, CDR2, and CDR3 of 21D9 (SEQ ID NOs: 155-157), and a VL comprising the amino acid sequence of VL CDR1, CDR2, and CDR3 of 21D9 (SEQ ID NOs: 158-160);

[0036] - a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of 21D9.b (SEQ ID NOs: 155-157), and a VL comprising the amino acid sequence of VL CDR1, CDR2 and CDR3 of 21D9.b (SEQ ID NOs: 158-160);

[0037] a VH comprising the amino acid sequence of VH CDR1, CDR2, and CDR3 of 21D9.c (SEQ ID NOs: 155-157), and a VL comprising the amino acid sequence of VL CDR1, CDR2, and CDR3 of 21D9.c (SEQ ID NOs: 158-160);

[0038] - a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of 21D9.d (SEQ ID NOs: 155-157), and a VL comprising the amino acid sequence of VL CDR1, CDR2 and CDR3 of 21D9.d (SEQ ID NOs: 158-160);

[0039] - a VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of 21D9.e (SEQ ID NOs: 155-157), and a VL comprising the amino acid sequence of VL CDR1, CDR2 and CDR3 of 21D9.e (SEQ ID NOs: 158-160);

[0040] a VH comprising the amino acid sequence of VH CDR1, CDR2, and CDR3 of 21A5 (SEQ ID NOs: 161-163), and a VL comprising the amino acid sequence of VL CDR1, CDR2, and CDR3 of 21A5 (SEQ ID NOs: 164-166);

[0041] a VH comprising the amino acid sequence of VH CDR1, CDR2, and CDR3 of 21A5.a (SEQ ID NOs: 161-163), and a VL comprising the amino acid sequence of VL CDR1, CDR2, and CDR3 of 21A5.a (SEQ ID NOs: 164-166);

[0042] a VH comprising the amino acid sequence of VH CDR1, CDR2, and CDR3 of 10F10 (SEQ ID NOs: 167-169), and a VL comprising the amino acid sequence of VL CDR1, CDR2, and CDR3 of 10F10 (SEQ ID NOs: 170-172);

[0043] a VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of 10F10.1 (SEQ ID NOs: 167-169), and a VL comprising the amino acid sequences of VL CDR1, CDR2, and CDR3 of 10F10.1 (SEQ ID NOs: 170-172);

[0044] a VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of 10F10.3 (SEQ ID NOs: 167-169), and a VL comprising the amino acid sequences of VL CDR1, CDR2, and CDR3 of 10F10.3 (SEQ ID NOs: 170-172); or

[0045] - A VH comprising the amino acid sequence of VH CDR1, CDR2 and CDR3 of 10F10.4 (SEQ ID NOs: 167-169), and a VL comprising the amino acid sequence of VL CDR1, CDR2 and CDR3 of 10F10.4 (SEQ ID NOs: 170-172).

[0046] Embodiment 6: The isolated antibody of any one of embodiments 1-5, wherein the antibody heavy chain comprises a VH having an amino acid sequence 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 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: 92), 10F10.2 (SEQ ID NO: 93), 10F10.3 (SEQ ID NO: 94), 10F10.4 (SEQ ID NO: 95), 10F10.5 (SEQ ID NO: 96), 10F10.6 (SEQ ID NO: 97), 10F10.7 (SEQ ID NO: 98), 10F10.8 (SEQ ID NO: 99), 10F10.9 (SEQ ID NO: NO:91), 10F10.3 (SEQ ID NO:91) or 10F10.4 (SEQ ID NO:91).

[0047] Embodiment 7: The isolated antibody of any one of embodiments 1-6, wherein the antibody light chain comprises a VL having an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the VL of 9G4 (SEQ ID NO: 50, amino acids 19-125), 9C8 (SEQ ID NO: 54), 2H2 (SEQ ID NO: 59), 2E5 (SEQ ID NO: 62), 24E5 (SEQ ID NO: 66), 21D9 (SEQ ID NO: 70), 21D9.b (SEQ ID NO: 70), 21D9.c (SEQ ID NO: 70), 21D9.d (SEQ ID NO: 70), 21D9.e (SEQ ID NO: 70), 21A5 (SEQ ID NO: 82), 21A5.a (SEQ ID NO: 86), 10F10 (SEQ ID NO: 90), 10F10.1 (SEQ ID NO: 91), NO:94), 10F10.3 (SEQ ID NO:96) or 10F10.4 (SEQ ID NO:114).

[0048] Embodiment 8: The isolated antibody of any one of embodiments 1-7, wherein the antibody heavy chain comprises a VH having an amino acid sequence comprising 1, 2, 3, 4, or 5 amino acid substitutions, conservative substitutions, or back substitutions compared to the amino acid sequence of the VH of 9G4 (SEQ ID NO: 51, amino acids 20-135), 9C8 (SEQ ID NO: 55), 2H2 (SEQ ID NO: 58), 2E5 (SEQ ID NO: 63), 24E5 (SEQ ID NO: 67), 21D9 (SEQ ID NO: 71), 21D9.b (SEQ ID NO: 74), 21D9.c (SEQ ID NO: 75), 21D9.d (SEQ ID NO: 78), 21D9.e (SEQ ID NO: 80), 21A5 (SEQ ID NO: 83), 21A5.a (SEQ ID NO: 87), 10F10 (SEQ ID NO: 91), 10F10.1 (SEQ ID NO: 92), NO:91), 10F10.3 (SEQ ID NO:91) or 10F10.4 (SEQ ID NO:91).

[0049] Embodiment 9: The isolated antibody of 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 back substitutions compared to the amino acid sequence of the VL of 9G4 (SEQ ID NO: 50, amino acids 19-125), 9C8 (SEQ ID NO: 54), 2H2 (SEQ ID NO: 59), 2E5 (SEQ ID NO: 62), 24E5 (SEQ ID NO: 66), 21D9 (SEQ ID NO: 70), 21D9.b (SEQ ID NO: 70), 21D9.c (SEQ ID NO: 70), 21D9.d (SEQ ID NO: 70), 21D9.e (SEQ ID NO: 70), 21A5 (SEQ ID NO: 82), 21A5.a (SEQ ID NO: 86), 10F10 (SEQ ID NO: 90), 10F10.1 (SEQ ID NO: 91), NO:94), 10F10.3 (SEQ ID NO:96) or 10F10.4 (SEQ ID NO:114).

[0050] Embodiment 10: The isolated antibody of any one of embodiments 1-9, wherein the heavy chain comprises the VH of the following anti-ILT4 antibody: 9G4 (SEQ ID NO: 51, amino acids 20-135), 9C8 (SEQ ID NO: 55), 2H2 (SEQ ID NO: 58), 2E5 (SEQ ID NO: 63), 24E5 (SEQ ID NO: 67), 21D9 (SEQ ID NO: 71), 21D9.b (SEQ ID NO: 74), 21D9.c (SEQ ID NO: 75), 21D9.d (SEQ ID NO: 78), 21D9.e (SEQ ID NO: 80), 21A5 (SEQ ID NO: 83), 21A5.a (SEQ ID NO: 87), 10F10 (SEQ ID NO: 91), 10F10.1 (SEQ ID NO: 91), 10F10.3 (SEQ ID NO: 91), or 10F10.4 (SEQ ID NO: 91).

[0051] Embodiment 11: The isolated antibody of any one of embodiments 1-10, wherein the light chain comprises the VL of the following anti-ILT4 antibody: 9G4 (SEQ ID NO: 50, amino acids 19-125), 9C8 (SEQ ID NO: 54), 2H2 (SEQ ID NO: 59), 2E5 (SEQ ID NO: 62), 24E5 (SEQ ID NO: 66), 21D9 (SEQ ID NO: 70), 21D9.b (SEQ ID NO: 70), 21D9.c (SEQ ID NO: 70), 21D9.d (SEQ ID NO: 70), 21D9.e (SEQ ID NO: 70), 21A5 (SEQ ID NO: 82), 21A5.a (SEQ ID NO: 86), 10F10 (SEQ ID NO: 90), 10F10.1 (SEQ ID NO: 94), 10F10.3 (SEQ ID NO: 96), or 10F10.4 (SEQ ID NO: 97). NO:114).

[0052] Embodiment 12: The isolated antibody of any one of embodiments 1-11, comprising 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: 81), 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).

[0053] Embodiment 13: The isolated antibody of any one of Embodiments 1-11, comprising:

[0054] a VH comprising the VH CDRs of the VH of 9G4 (SEQ ID NOs: 125-127), and a VL comprising the VL CDRs of 9G4 (SEQ ID NOs: 128-130), and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 9G4 (SEQ ID NO: 51, amino acids 20-135 and SEQ ID NO: 50, amino acids 19-125);

[0055] a VH comprising the VH CDRs of the VH of 9C8 (SEQ ID NOs: 131-133), and a VL comprising the VL CDRs of 9C8 (SEQ ID NOs: 134-136), and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 9C8 (SEQ ID NO: 55 and SEQ ID NO: 54);

[0056] a VH comprising the VH CDRs of the VH of 2H2 (SEQ ID NOs: 137-139), and a VL comprising the VL CDRs of 2H2 (SEQ ID NOs: 140-142), and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 2H2 (SEQ ID NO: 58 and SEQ ID NO: 59);

[0057] a VH comprising the VH CDRs of the VH of 2E5 (SEQ ID NOs: 143-145), and a VL comprising the VL CDRs of 2E5 (SEQ ID NOs: 146-148), and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 2E5 (SEQ ID NO: 63 and SEQ ID NO: 62);

[0058] a VH comprising the VH CDRs of the VH of 24E5 (SEQ ID NOs: 149-151), and a VL comprising the VL CDRs of 24E5 (SEQ ID NOs: 152-154), and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 24E5 (SEQ ID NO: 67 and SEQ ID NO: 66);

[0059] a VH comprising the VH CDRs of the VH of 21D9 (SEQ ID NOs: 155-157), and a VL comprising the VL CDRs of 21D9 (SEQ ID NOs: 158-160), and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 21D9 (SEQ ID NO: 71 and SEQ ID NO: 70);

[0060] a VH comprising the VH CDRs of the VH of 21D9.b (SEQ ID NOs: 155-157), and a VL comprising the VL CDRs of 21D9.b (SEQ ID NOs: 158-160), and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 21D9.b (SEQ ID NO: 74 and SEQ ID NO: 70);

[0061] a VH comprising the VH CDRs of the VH of 21D9.c (SEQ ID NOs: 155-157), and a VL comprising the VL CDRs of 21D9.c (SEQ ID NOs: 158-160), and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 21D9.c (SEQ ID NO: 75 and SEQ ID NO: 70);

[0062] a VH comprising the VH CDRs of the VH of 21D9.d (SEQ ID NOs: 155-157), and a VL comprising the VL CDRs of 21D9.d (SEQ ID NOs: 158-160), and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 21D9.d (SEQ ID NO: 78 and SEQ ID NO: 70);

[0063] a VH comprising the VH CDRs of the VH of 21D9.e (SEQ ID NOs: 155-157), and a VL comprising the VL CDRs of 21D9.e (SEQ ID NOs: 158-160), and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 21D9.e (SEQ ID NO: 80 and SEQ ID NO: 70);

[0064] a VH comprising the VH CDRs of the VH of 21A5 (SEQ ID NOs: 161-163), and a VL comprising the VL CDRs of 21A5 (SEQ ID NOs: 164-166), and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 21A5 (SEQ ID NO: 83 and SEQ ID NO: 82);

[0065] a VH comprising the VH CDRs of the VH of 21A5.a (SEQ ID NOs: 161-163), and a VL comprising the VL CDRs of 21A5.a (SEQ ID NOs: 164-166), and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 21A5.a (SEQ ID NO: 87 and SEQ ID NO: 86);

[0066] a VH comprising the VH CDRs of the VH of 10F10 (SEQ ID NOs: 167-169), and a VL comprising the VL CDRs of 10F10 (SEQ ID NOs: 170-172), and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 10F10 (SEQ ID NO: 91 and SEQ ID NO: 90);

[0067] a VH comprising the VH CDRs of the VH of 10F10.1 (SEQ ID NOs: 167-169), and a VL comprising the VL CDRs of 10F10.1 (SEQ ID NOs: 170-172), and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 10F10.1 (SEQ ID NO: 91 and SEQ ID NO: 94);

[0068] a VH comprising the VH CDRs of the VH of 10F10.3 (SEQ ID NOs: 167-169), and a VL comprising the VL CDRs of 10F10.3 (SEQ ID NOs: 170-172), and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 10F10.3 (SEQ ID NO: 91 and SEQ ID NO: 96); or

[0069] - A VH comprising the VH CDRs of the VH of 10F10.4 (SEQ ID NOs: 167-169), and a VL comprising the VL CDRs of 10F10.4 (SEQ ID NOs: 170-172), and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 10F10.4 (SEQ ID NO: 91 and SEQ ID NO: 114).

[0070] Embodiment 14: The isolated antibody of any one of Embodiments 1-13, comprising:

[0071] a VH comprising the amino acid sequence of the VH of 9G4 (SEQ ID NO: 51 residues 20-135) and a VL comprising the amino acid sequence of the VL of 9G4 (SEQ ID NO: 50 residues 19-125);

[0072] - a VH comprising the amino acid sequence of VH of 9C8 and a VL comprising the amino acid sequence of VL of 9C8 (SEQ ID NO: 55 and SEQ ID NO: 54);

[0073] - a VH comprising the amino acid sequence of VH of 2H2 and a VL comprising the amino acid sequence of VL of 2H2 (SEQ ID NO: 58 and SEQ ID NO: 59);

[0074] - a VH comprising the amino acid sequence of VH of 2E5 and a VL comprising the amino acid sequence of VL of 2E5 (SEQ ID NO: 63 and SEQ ID NO: 62);

[0075] - a VH comprising the amino acid sequence of VH of 24E5 and a VL comprising the amino acid sequence of VL of 24E5 (SEQ ID NO: 67 and SEQ ID NO: 66);

[0076] - a VH comprising the amino acid sequence of VH of 21D9 and a VL comprising the amino acid sequence of VL of 21D9 (SEQ ID NO: 71 and SEQ ID NO: 70);

[0077] - a VH comprising the amino acid sequence of VH of 21D9.b and a VL comprising the amino acid sequence of VL of 21D9.b (SEQ ID NO: 74 and SEQ ID NO: 70);

[0078] - a VH comprising the amino acid sequence of VH of 21D9.c and a VL comprising the amino acid sequence of VL of 21D9.c (SEQ ID NO: 75 and SEQ ID NO: 70);

[0079] - a VH comprising the amino acid sequence of VH of 21D9.d and a VL comprising the amino acid sequence of VL of 21D9.d (SEQ ID NO: 78 and SEQ ID NO: 70);

[0080] - a VH comprising the amino acid sequence of VH of 21D9.e and a VL comprising the amino acid sequence of VL of 21D9.e (SEQ ID NO: 80 and SEQ ID NO: 70);

[0081] - a VH comprising the amino acid sequence of VH of 21A5 and a VL comprising the amino acid sequence of VL of 21A5 (SEQ ID NO: 83 and SEQ ID NO: 82);

[0082] - a VH comprising the amino acid sequence of VH of 21A5.a and a VL comprising the amino acid sequence of VL of 21A5.a (SEQ ID NO: 87 and SEQ ID NO: 86);

[0083] - a VH comprising the amino acid sequence of VH of 10F10 and a VL comprising the amino acid sequence of VL of 10F10 (SEQ ID NO: 91 and SEQ ID NO: 90);

[0084] a VH comprising the amino acid sequence of VH of 10F10.1 and a VL comprising the amino acid sequence of VL of 10F10.1 (SEQ ID NO: 91 and SEQ ID NO: 94);

[0085] - a VH comprising the amino acid sequence of VH of 10F10.3 and a VL comprising the amino acid sequence of VL of 10F10.3 (SEQ ID NO: 91 and SEQ ID NO: 96); or

[0086] - a VH comprising the amino acid sequence of VH of 10F10.4 and a VL comprising the amino acid sequence of VL of 10F10.4 (SEQ ID NO: 91 and SEQ ID NO: 114).

[0087] Embodiment 15: The isolated antibody of any one of Embodiments 1-14, which is an IgG antibody.

[0088] Embodiment 16: The isolated antibody of embodiment 15, which is an IgG1, IgG2 or IgG4 antibody, wherein the IgG4 optionally comprises an S228P substitution (EU numbering).

[0089] Embodiment 17: The isolated antibody of any one of Embodiments 1-16, wherein the antibody is an effector-free antibody.

[0090] Embodiment 18: The isolated antibody of embodiment 17, wherein the heavy chain constant region comprises 1, 2, 3, 4 or 5 mutations in an otherwise wild-type human heavy chain constant region, which mutations reduce the effector function of the antibody compared to an antibody without the 1, 2, 3, 4 or 5 mutations but with the same amino acid sequence.

[0091] Embodiment 19: An isolated antibody as described in 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.

[0092] Embodiment 20: The isolated antibody of any one of Embodiments 1-16 or 19, wherein the antibody has effector function.

[0093] Embodiment 21: The isolated antibody of embodiment 20, wherein the antibody is a defucosylated antibody (eg, a defucosylated IgG1 antibody).

[0094] Embodiment 22: The isolated antibody of embodiment 20 or 21, wherein the heavy chain constant region comprises 1, 2, 3, 4 or 5 mutations in an otherwise wild-type human heavy chain constant region, which mutations enhance the effector function of the antibody compared to an antibody without the 1, 2, 3, 4 or 5 mutations but having the same amino acid sequence.

[0095] Embodiment 23: An isolated antibody as described in any one of embodiments 1-22, which comprises the HC of anti-ILT4 antibody 9G4, 9C8, 2H2, 2E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1 or 10F10.3, wherein the constant region of the HC is IgG1 (e.g., 9G4.IgG1, etc.), IgG1.3 (e.g., 9G4.IgG1.3, etc.), IgG1.1f (e.g., 9G4.IgG1.1f, etc.), IgG4 (e.g., 9G4.IgG4, etc.) or IgG4 S228P (EU numbering) (e.g., 9G4.IgG4_S228P).

[0096] Embodiment 24: The isolated antibody of Embodiment 23, wherein the antibody comprises the following HC amino acid sequence:

[0097] -IgG1, e.g., 9G4.IgG1, 9G4 (SEQ ID NO: 2), 9C8 (SEQ ID NO: 4), 2H2 (SEQ ID NO: 6), 2E5 (SEQ ID NO: 8), 24E5 (SEQ ID NO: 10), 21D9 (SEQ ID NO: 12), 21D9.b (SEQ ID NO: 74 and 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), 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),

[0098] -IgG1, e.g., 9G4.IgG1, 9G4 (SEQ ID NO:51, amino acids 20-135, and SEQ ID NO: 102), 9C8 (SEQ ID NO:55 and SEQ ID NO: 102), 2H2 (SEQ ID NO:58 and SEQ ID NO: 102), 2E5 (SEQ ID NO:63 and SEQ ID NO: 102), 24E5 (SEQ ID NO:67 and SEQ ID NO: 102), 21D9 (SEQ ID NO:71 and SEQ ID NO: 102), 21D9.b (SEQ ID NO:74 and SEQ ID NO: 102), 21D9.c (SEQ ID NO:75 and SEQ ID NO: 102), 21D9.d (SEQ ID NO:78 and SEQ ID NO: 102), 21D9.e (SEQ ID NO:80 and SEQ ID NO: 102), 21A5 (SEQ ID NO:83 and SEQ ID NO: 102), 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),

[0099] : -IgG1.3 (e.g., 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 (SEQ ID NO: 20), 10F10 (SEQ ID NO: 21), 10F10 (SEQ ID NO: 22), 10F10 (SEQ ID NO: 23), 10F10 (SEQ ID NO: 24), 10F10 (SEQ ID NO: 25), 10F10 (SEQ ID NO: 26), 10F10 (SEQ ID NO: 27), 10F10 (SEQ ID NO: 28), 10F10 (SEQ ID NO: 29), 10F10 (SEQ ID NO: 30), 10 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);

[0100] : -IgG1.1f (e.g., 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), 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),

[0101] -IgG1fa.P238K (e.g., 9G4.IgG1fa.P238K, etc.) 9G4 (SEQ ID NO:51, amino acids 20-135, and SEQ ID NO: 104), 9C8 (SEQ ID NO:55 and SEQ ID NO: 104), 2H2 (SEQ ID NO:58 and SEQ ID NO: 104), 2E5 (SEQ ID NO:63 and SEQ ID NO: 104), 24E5 (SEQ ID NO:67 and SEQ ID NO: 104), 21D9 (SEQ ID NO:71 and SEQ ID NO: 104), 21D9.b (SEQ ID NO:74 and SEQ ID NO: 104), 21D9.c (SEQ ID NO:75 and SEQ ID NO: 104), 21D9.d (SEQ ID NO:78 and SEQ ID NO: 104), 21D9.e (SEQ ID NO:80 and SEQ ID NO: 104), 21A5 (SEQ ID NO:81 and SEQ ID NO: 104), 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

[0102] : 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: 81 and SEQ ID NO: 179), 21A5 (SEQ ID NO: 82 and SEQ ID NO: 179), 21A5 (SEQ ID NO: 83 and SEQ ID NO: 179), 21A5 (SEQ ID NO: 84 and SEQ ID NO: 179), 21A5 (SEQ ID NO: 85 and SEQ ID NO: 179), 21A5 (SEQ ID NO: 86 and SEQ ID NO: 179), 21A5 (SEQ ID NO: 87 and SEQ ID NO: 179), 21A5 (SEQ ID NO: 88 and SEQ ID NO: 179), 21A5 (SEQ ID NO: 89 and SEQ ID NO: 180), 21A5 (SEQ ID NO: 90 and SEQ ID NO: 181), 21A5 (SEQ ID NO: 91 and SEQ ID NO: 182), 21A5 (SEQ ID NO: 92 and SEQ ID NO: 183), 21A5 (SEQ ID NO: 93 and SEQ ID NO: 184), 21A5 (SEQ ID NO: 94 and SEQ ID NO: 185), 21A5 (SEQ ID NO: 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).

[0103] Embodiment 25: The isolated antibody of Embodiment 23, wherein the HC of the antibody lacks a C-terminal lysine residue.

[0104] Embodiment 26: The isolated antibody of Embodiment 23, wherein the HC of the antibody comprises the heavy chain constant region amino acid sequence of any one of SEQ ID NO: 98, 100, 102, 103, 104 or 179.

[0105] Embodiment 27: The isolated antibody of any one of embodiments 1-26, comprising the LC of anti-ILT4 antibody 9G4, 9C8, 2H2, 2E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4.

[0106] Embodiment 28: The isolated antibody of Embodiment 27, wherein the light chain constant region is a human kappa light chain constant region.

[0107] Embodiment 29: The isolated antibody of Embodiment 27, wherein the LC comprises the sequence of 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).

[0108] Embodiment 30: An isolated antibody as described in any one of embodiments 1-29, which comprises the HC and LC of the anti-ILT4 antibody 9G4, 9C8, 2H2, 2E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3 or 10F10.4, wherein the constant region of the HC is IgG1 (e.g., 9G4.IgG1, etc.), IgG1.3 (e.g., 9G4.IgG1.3, etc.), IgG1.1f (e.g., 9G4.IgG1.1f, etc.), IgG4 (e.g., 9G4.IgG4, etc.) or IgG4 S228P (EU numbering) (e.g., 9G4.IgG4_S228P).

[0109] Embodiment 31: The isolated antibody of embodiment 30, comprising:

[0110] 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 NOs: 98, 100, 102, 103, 104, or 179), and a light chain (LC) comprising the light chain amino acid sequence of 9G4 (SEQ ID NO: 1);

[0111] - a heavy chain comprising the amino acid sequence of the heavy chain of 9C8 ((i) SEQ ID NO: 4 or (ii) one of SEQ ID NO: 55 and SEQ ID NO: 98, 100, 102, 103, 104 or 179), and a light chain comprising the light chain amino acid sequence of 9C8 (SEQ ID NO: 3);

[0112] - a heavy chain comprising the amino acid sequence of the heavy chain of 2H2 ((i) SEQ ID NO: 6 or (ii) one of SEQ ID NO: 58 and SEQ ID NO: 98, 100, 102, 103, 104 or 179), and a light chain comprising the light chain amino acid sequence of 2H2 (SEQ ID NO: 5);

[0113] a heavy chain comprising the amino acid sequence of the heavy chain of 2E5 ((i) SEQ ID NO: 8 or (ii) one of SEQ ID NO: 63 and SEQ ID NO: 98, 100, 102, 103, 104 or 179), and a light chain comprising the amino acid sequence of the light chain of 2E5 (SEQ ID NO: 7);

[0114] a heavy chain comprising the amino acid sequence of the heavy chain of 24E5 ((i) SEQ ID NO: 10 or (ii) one of SEQ ID NO: 67 and SEQ ID NO: 98, 100, 102, 103, 104 or 179), and a light chain comprising the amino acid sequence of the light chain of 24E5 (SEQ ID NO: 9);

[0115] - a heavy chain comprising the amino acid sequence of the heavy chain of 21D9 ((i) SEQ ID NO: 12 or 113, or (ii) one of SEQ ID NO: 71 and SEQ ID NO: 98, 100, 102, 103, 104 or 179), and a light chain comprising the light chain amino acid sequence of 21D9 (SEQ ID NO: 11);

[0116] a heavy chain comprising the amino acid sequence of the heavy chain of 21D9.b (SEQ ID NO: 74 and one of SEQ ID NOs: 98, 100, 102, 103, 104 or 179), and a light chain comprising the amino acid sequence of the light chain of 21D9.b (SEQ ID NO: 11);

[0117] a heavy chain comprising the amino acid sequence of the heavy chain of 21D9.c (SEQ ID NO: 75 and one of SEQ ID NOs: 98, 100, 102, 103, 104 or 179), and a light chain comprising the amino acid sequence of the light chain of 21D9.c (SEQ ID NO: 11);

[0118] a heavy chain comprising the amino acid sequence of the heavy chain of 21D9.d (SEQ ID NO: 78 and one of SEQ ID NOs: 98, 100, 102, 103, 104 or 179), and a light chain comprising the amino acid sequence of the light chain of 21D9.d (SEQ ID NO: 11);

[0119] - 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) one of SEQ ID NO: 80 and SEQ ID NO: 98, 100, 102, 103, 104 or 179), and a light chain comprising the light chain amino acid sequence of 21D9.e (SEQ ID NO: 11);

[0120] a heavy chain comprising the amino acid sequence of the heavy chain of 21A5 ((i) SEQ ID NO: 15 or (ii) one of SEQ ID NO: 83 and 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);

[0121] - a heavy chain comprising the amino acid sequence of the heavy chain of 21A5.a ((i) SEQ ID NO: 17 or (ii) one of SEQ ID NO: 87 and SEQ ID NO: 98, 100, 102, 103, 104 or 179), and a light chain comprising the light chain amino acid sequence of 21A5.a (SEQ ID NO: 16);

[0122] a heavy chain comprising the amino acid sequence of the heavy chain of 10F10 ((i) SEQ ID NO: 19 or (ii) one of SEQ ID NO: 91 and 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);

[0123] - a heavy chain comprising the amino acid sequence of the heavy chain of 10F10.1 ((i) SEQ ID NO: 19 or (ii) one of SEQ ID NO: 91 and SEQ ID NO: 98, 100, 102, 103, 104 or 179), and a light chain comprising the light chain amino acid sequence of 10F10.1 (SEQ ID NO: 20);

[0124] - a heavy chain comprising the amino acid sequence of the heavy chain of 10F10.3 ((i) SEQ ID NO: 19 or (ii) one of SEQ ID NO: 91 and SEQ ID NO: 98, 100, 102, 103, 104 or 179), and a light chain comprising the light chain amino acid sequence of 10F10.3 (SEQ ID NO: 21); or

[0125] - a heavy chain comprising the amino acid sequence of the heavy chain of 10F10.4 ((i) SEQ ID NO: 19 or (ii) one of SEQ ID NO: 91 and SEQ ID NO: 98, 100, 102, 103, 104 or 179), and a light chain comprising the light chain amino acid sequence of 10F10.4 (SEQ ID NO: 116).

[0126] 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 light chain amino acid sequence of SEQ ID NO: 11.

[0127] 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 light chain amino acid sequence of SEQ ID NO: 11.

[0128] 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 light chain amino acid sequence of SEQ ID NO: 11.

[0129] 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 light chain amino acid sequence of SEQ ID NO: 11.

[0130] Embodiment 36: The isolated antibody of any one of Embodiments 32-35, wherein the antibody has one or more properties of the antibody of Embodiment 1.

[0131] Embodiment 37: The isolated antibody of any one of Embodiments 1-36, which is a full-length antibody.

[0132] Embodiment 38: The isolated antibody of any one of Embodiments 1-14, which is an antibody fragment.

[0133] Embodiment 39: The isolated antibody of any one of Embodiments 1-38, which is a multimeric (eg, dimeric or trimeric) antibody.

[0134] Embodiment 40: The isolated antibody of any one of Embodiments 1-39, which is linked (eg, covalently linked) to another molecule.

[0135] Embodiment 41: The isolated antibody of Embodiment 40, wherein the other molecule is a label.

[0136] Embodiment 42: The isolated antibody of Embodiment 40, wherein the other molecule is a peptide.

[0137] Embodiment 43: The isolated antibody of claim 40, which is an antibody drug conjugate (ADC).

[0138] Embodiment 44: An isolated nucleic acid encoding the antibody of any one of embodiments 1-43.

[0139] Embodiment 45: An isolated nucleic acid encoding the heavy chain and / or light chain of the antibody of any one of embodiments 1-43.

[0140] Embodiment 46: A collection of at least two isolated nucleic acids encoding the heavy and light chains of the antibody of any one of embodiments 1-43.

[0141] Embodiment 47: A composition comprising a nucleic acid encoding the heavy chain of the antibody of any one of embodiments 1-43 and a nucleic acid encoding the light chain of the antibody of any one of embodiments 1-43.

[0142] Embodiment 48: A cell comprising the isolated nucleic acid of any one of embodiments 44-46 or the composition of embodiment 47.

[0143] Embodiment 49: A method of producing an antibody, comprising culturing the cell of embodiment 48 under conditions where the antibody is expressed.

[0144] Embodiment 50: A composition comprising the isolated antibody, nucleic acid, composition or cell of any one of embodiments 1-49 and a pharmaceutically acceptable carrier.

[0145] Embodiment 51: The composition of Embodiment 50, comprising a second therapeutic agent.

[0146] Embodiment 52: The composition of Embodiment 51, wherein the second therapeutic agent is an immunostimulatory agent.

[0147] Embodiment 53: The composition of embodiment 52, wherein the immunostimulatory agent is an antagonist of an immunosuppressive molecule, such as PD-1 / PD-L1, CTLA-4, and LAG-3, or an agonist of an immunostimulatory molecule, such as GITR and OX40.

[0148] Embodiment 54: A method of treating a subject having cancer, comprising administering to the subject a therapeutically effective amount of the composition of any one of embodiments 50-53 or the isolated antibody of any one of embodiments 1-43 that stimulates an immune response and / or is an ILT-4 antagonist.

[0149] Embodiment 55: The method of embodiment 54, further comprising administering a second therapy.

[0150] Embodiment 56: The method of embodiment 55, wherein the second therapy is radiation therapy, surgery, or administration of a second dose.

[0151] Embodiment 57: The method of embodiment 55, wherein the second therapy is a second dose, and the second dose is an immunostimulant.

[0152] Embodiment 58: The method of embodiment 57, wherein the immunostimulatory agent is an antagonist of an immunosuppressive molecule, such as PD-1 / PD-L1, CTLA-4, and LAG-3, or an agonist of an immunostimulatory molecule, such as GITR and OX40.

[0153] 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 that stimulates an immune response and / or is an ILT4 antagonist.

[0154] Embodiment 61: A method of detecting ILT4 in a sample, comprising contacting the sample with the ILT4 antibody of any one of embodiments 1-43.

[0155] Embodiment 62: The isolated antibody of any one of Embodiments 1-43, having the following characteristics:

[0156] 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);

[0157] b. 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;

[0158] c. having a binding profile as shown in Figure 27; and

[0159] 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).

[0160] Embodiment 63: The isolated antibody of any one of Embodiments 1-43, having the following characteristics:

[0161] 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);

[0162] b. does not specifically bind to hILT2, hILT3 and / or hILT5;

[0163] c. does not specifically bind to one or more members of the LILRA and / or LILRB family;

[0164] 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;

[0165] 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;

[0166] f. Inhibit the binding of hILT4 to HLA-A and HLA-B;

[0167] g. having a binding profile as shown in Figure 27;

[0168] 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);

[0169] i. Promote the pro-inflammatory polarization of macrophages towards M1 macrophages;

[0170] j. does not induce (or trigger) basophil activation; and

[0171] 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.

[0172] Embodiment 64: The isolated antibody of any one of Embodiments 1-43, having the following characteristics:

[0173] 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;

[0174] 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;

[0175] 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;

[0176] d. Inhibit the binding of HLA-A and / or HLA-B to ILT4;

[0177] e. Binds to Ig-like domains 1, 2, or 1 and 2 of hILT4, such as the region 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 assay described in the Examples;

[0178] f. 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;

[0179] g. compete with the antibodies described herein for binding to hILT4; and

[0180] h. specifically binds to cynomolgus monkey ILT4 comprising SEQ ID NO: 118, e.g., as shown in a binding assay described in the Examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0181] This application claims the benefit of U.S. Provisional Patent Application which contains at least one color drawing from the following list. These color drawings will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee once this international application is published and the priority provisional application becomes available to the public.

[0182] Figure 1 Shown are the nucleotide and amino acid sequences of the heavy chain variable region ("VH") of the anti-hlLT4 antibody 9G4, with the location of the VHC mdr indicated.

[0183] Figure 2 Shown are the nucleotide and amino acid sequences of the light chain variable region ("VL") of anti-hlLT4 antibody 9G4, with the location of the VLCDR indicated.

[0184] Figure 3 The nucleotide and amino acid sequences of the VH of the anti-hlLT4 antibody 9C8 are shown, with the positions of the VH CDRs indicated.

[0185] Figure 4 The nucleotide and amino acid sequences of the VL of the anti-hlLT4 antibody 9C8 are shown, with the positions of the VL CDRs indicated.

[0186] Figure 5 The nucleotide and amino acid sequences of the VH of the anti-hlLT4 antibody 2H2 are shown, with the positions of the VH CDRs indicated.

[0187] Figure 6 The nucleotide and amino acid sequences of the VL of the anti-hlLT4 antibody 2H2 are shown, with the positions of the VL CDRs indicated.

[0188] Figure 7 The nucleotide and amino acid sequences of the VH of the anti-hlLT4 antibody 2E5 are shown, with the positions of the VH CDRs indicated.

[0189] Figure 8 The nucleotide and amino acid sequences of the VL of the anti-hlLT4 antibody 2E5 are shown, with the positions of the VL CDRs indicated.

[0190] Figure 9 The nucleotide and amino acid sequences of the VH of the anti-hlLT4 antibody 24E5 are shown, with the positions of the VH CDRs indicated.

[0191] Figure 10 The nucleotide and amino acid sequences of the VL of the anti-hlLT4 antibody 24E5 are shown, with the positions of the VL CDRs indicated.

[0192] Figure 11 The nucleotide and amino acid sequences of the VH of the anti-hlLT4 antibody 21D9 are shown, with the positions of the VH CDRs indicated.

[0193] Figure 12 The nucleotide and amino acid sequences of the VL of the anti-hlLT4 antibody 21D9 are shown, with the positions of the VL CDRs indicated.

[0194] 13A to 13C : Figure 13A and Figure 13C Displays the heavy chain variable domain (VH) of the ILT4 antibody 21D9 ( Figure 13A ) and light chain variable domain (VH) ( Figure 13C ) amino acid sequences were aligned with the germline immunoglobulin sequences hIGHV3-23*01 and hIGKV3-20*01, respectively. Arrows point to germline mutations. The AbM VH CDR1 and Kabat VH CDR2, VH CDR3, and VLCDR1-3 sequences are shown in light text. Figure 13B Shown are the VH amino acid sequences of four germline revertants 21D9.b, 21D9.c, 21D9.d, and 21D9.e that have an IgG1.3 heavy chain constant region (ie, 21D9.b-.e hIgG1.3). Figure 13B The amino acid residues indicated by capital letters are germline reversion substitutions.

[0195] FIG. 14A to FIG. 14B The amino acid sequences of the heavy chain variable domain and light chain variable domain of the ILT4 antibody 21A5 were respectively identical to the germline immunoglobulin sequence hIGHV1-46*01 ( Figure 14A ) and hIGKV3-20*01( Figure 14B ). AbM VH CDR1 and Kabat VH CDR2, VH CDR3, and VL CDR1-3 sequences are shown in light text. Arrows point to germline backmutations.

[0196] Figure 15 Shown are the amino acid sequences of the heavy and light chain variable domains of the ILT4 antibody 10F10, aligned with the germline immunoglobulin sequences hIGHV3-30*01 (top three bars) and hIGKV1-13*02 (bottom three bars), respectively. The AbM VH CDR1 and Kabat VH CDR2, VH CDR3, and VL CDR1-3 sequences are shown in light text. Arrows point to germline backmutations.

[0197] Figure 16 Sensorgrams showing binding of the indicated anti-hILT4 antibodies to hILT4. A schematic diagram of the method used is shown above the sensorgram panels.

[0198] Figure 17Binding of anti-hILT4 antibodies 2H2, 10F10 wild type (WT, i.e., parental antibody), 21A5.a, 21A5 WT, 21D9 WT, 21D9e, and isotype control hIgG1.3 to hILT4-transfected CHO cells is shown as a function of antibody concentration and as determined by flow cytometry. 50 The values ​​are provided below the combined data graph.

[0199] Figure 18A Binding of the indicated anti-hILT4 antibodies and isotype controls to hILT4-transfected CHO cells is shown as a function of antibody concentration and as determined by flow cytometry. 50 Values ​​are provided to the right of the combined data graph. Figure 18B Binding of anti-hlT4 antibodies to human mononuclear cells isolated from peripheral blood of normal healthy donors is shown. Antibody species are listed next to the graph and EC50 values ​​are provided in the table immediately below the graph.

[0200] Figure 19A Flow cytometry graphs showing binding of anti-hILT4 antibodies 21D9, 21D9.e, 21A5, 21A5.a, 10F10, and 2H2 (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 (top to bottom). Figure 19B Flow cytometry graphs showing binding of anti-hILT4 antibody 21A5 and germline revertant 21A5.a to human LIRA1, LIRA3, and ILT4.

[0201] Figure 20A and Figure 20B Displays designated antibodies 21D9 ("21D9 IgG1.1") and 21D9.e ( Figure 20A ) and 21A5 ("21A5WT") and 21A5.a ( Figure 20B ) and the specified human LILRA / LILRB family members ( Figure 20A LILRA5, ILT4 and Figure 20B The maximum percentage binding of anti-ILT4 antibodies 21D9, 21D9.e, 21A5, and 21A5.a to LILRA family members (LILRA1, LILRA3, and ILT4 in the WT group) varied with antibody concentration, indicating that the anti-ILT4 antibodies 21D9, 21D9.e, 21A5, and 21A5.a displayed weaker cross-reactivity with other LILRA family members.

[0202] 21A to 21D Demonstration of T cell activity of anti-hILT4 antibodies. Figure 21AT cells co-cultured with CHO cells transfected with hILT4 and OKT3 3 Histograms of H thymidine incorporation by T cells (from left to right on the x-axis) incubated with no antibody, with the isotype control hIgG1.3 antibody, and with antibodies 21D9 ("21D9 WT"), 21D9.e, 21A5 ("21A5 WT"), 21A5.a, and 10F10 ("10F10 WT"), 3 H-thymidine incorporation varied with antibody concentration (20, 4, 0.8, 0.16, 0.032, and 0.0064 μg / ml). Figure 21B Relevant EC50 values ​​are provided. Figure 21C Demonstration of T cell activity of anti-hILT4 antibodies. Figure 21C Figure 2 is a histogram of IFNγ production by T cells co-cultured with CHO cells transfected with hILT4 and OKT3, which were incubated (from left to right on the x-axis) with no antibody, with isotype control hIgG1.3 antibody, and with antibodies 21D9 ("21D9 WT"), 21D9.e, 21A5 ("21A5 WT"), 21A5.a, and 10F10 ("10F10 WT"), as a function of antibody concentration (20, 4, 0.8, 0.16, 0.032, and 0.0064 μg / ml). Figure 21D Relevant EC50 values ​​are provided.

[0203] 22A to 22D Demonstrates the proliferation of T cells in allogeneic MLR (mixed lymphocyte reaction) of monocyte-derived dendritic cells (MoDC) co-cultured with T cells ( Figure 22A and Figure 22C ) and IFNγ production ( Figure 22B and Figure 22D ). Anti-hILT4 antibodies 21A5.a or 21D9.e were added during the differentiation of monocytes into MoDCs ( Figure 22A and Figure 22B ), or 2H2 or 21D9( Figure 22C and Figure 22D ), or isotype control or no antibody added ("untreated"), and no respective antibody was added during the allogeneic MLR assay.

[0204] 23A to 23D The expression of CD83 and CD86 on monocyte-derived dendritic cells (MoDCs) is shown. Anti-hILT4 antibodies 21D9.e and 21A5.a ( Figure 23A )、21A5、10F10、21D9( Figure 23B ) or 2H2( Figure 23C and Figure 23D) or isotype control or no treatment (no antibody).

[0205] Figure 24A and Figure 24B Displayed by lipopolysaccharide (LPS) ( Figure 24A ) or the STING agonist 2'3'-cGAMP ( Figure 24B ) Levels of TNF-α secreted by activated in vitro differentiated macrophages. Anti-ILT4 antibodies 21D9, 21D9.e, 21A5, 21A5.a, 2H2, and 10F10 or an isotype control were included during activation.

[0206] Figure 25A and Figure 25B The percentage of CD4 T cell proliferation in the presence of anti-hILT4 antibodies 21D9, 21D9.e, 21A5 or 21A5.a or anti-PD-L1 antibody or isotype control antibody is shown ( Figure 25A ) and the amount of IFN-γ produced by allogeneic MLR of CD4 T cells co-cultured with in vitro differentiated macrophages ( Figure 25B ). Figure 25A The percentage of CD4 T cell proliferation based on CFSE dilution by FACS analysis is shown, and Figure 25B IFNγ production measured by Alphalisa is shown, both as a function of ILT4 antibody or control.

[0207] Figure 26A and Figure 26B demonstrated that in a macrophage:CD4 T cell allogeneic MLR, the combination of the anti-ILT4 antibody 21D9 and the anti-PD-L1 antibody clone 29E.2A3 enhanced IFNγ production to higher levels compared to each antibody alone. Figure 26A An experiment showing a macrophage:CD4 T allogeneic pair. Figure 26B Responses of multiple allogeneic pairs are shown, with each line representing one experiment for one allogeneic pair. Statistical analysis was performed using a nonparametric one-way Anova multiple comparison test (Friedman test). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0208] Figure 27A 、 Figure 27B and Figure 27C HDX epitope mapping of anti-hILT4 Ab 21D9 and 2H2 Fab is shown. Figure 27A The coverage of hILT4 sequence by pepsin is shown. 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.

[0209] 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.

[0210] 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.

[0211] 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).

[0212] 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 into the measurement).

[0213] Figures 32A to 32C The 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 the in vitro expression of the co-stimulatory molecule CD86 ( Figure 32A ) and CD80( Figure 32B ) and 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 (DCs).

[0214] Figure 33A and Figure 33B Displayed by lipopolysaccharide (LPS) ( Figure 33A ) or the STING agonist 2'3'cGAMP ( Figure 33B ) Levels of TNF-α secreted by activated in vitro differentiated macrophages. Anti-ILT4 antibodies 21D9.e and 10F10.4 or an isotype control were included during activation.

[0215] Figure 34 It was shown that similar levels of TNF-α were secreted by in vitro differentiated macrophages incubated with 21D9.e in the presence of IgG1, IgG4 or IgG1.3.

[0216] Figure 35 The percentages of monomeric, high molecular weight (HMW), and low molecular weight (LMW) species of 21D9.e.IgG1.3 are shown after 0, 1, 2, and 3 months at 25° C. or 40° C. The monomeric, HMW, and LMW species correspond to the bottom, middle, and top portions of each bar in the histogram, respectively.

[0217] Figure 36 Shown are the percentages of activated basophils for 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-formylmethionine-leucyl-phenylalanine (fMLP), demonstrating that 21D9e.IgG1.3 (ILT4.8) and 21A5.a.IgG1.3 ("ILT4.9") do not have basophil activation.

[0218] Figures 37A to 37D Presents the results of experiments used to determine the antigenic determinants of certain antibodies. Figure 37A and Figure 37B2H2 ( Figure 37A ) and 21D9( Figure 37B ) competition (or cross-blocking) assay results. Figure 37C Diagram showing a three-dimensional representation of hILT4 with the locations of Ig-like domains 1 and 2 and indicating the regions of hILT4 bound by antibodies 21D9, 29A5, 2H2, and 10F10. Figure 37D An additional three-dimensional representation of hILT4 is shown with the locations of Ig-like domains 1 and 2 and indicating the regions of hILT4 to which antibodies 21D9, 21A5, and 10F10 bind. DETAILED DESCRIPTION

[0219] definition

[0220] Unless otherwise defined, scientific and technical terms used in conjunction with the present invention will have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular.

[0221] In this application, unless otherwise indicated, the use of "or" means "and / or". In the case of multiple dependent items, the use of "or" merely refers back to more than one of the preceding independent items or dependent items in an alternative manner. The terms "comprising", "including" and "having" are used interchangeably herein. According to the present invention, an "isolated" molecule is a molecule that has been removed from its natural environment. Therefore, the term "isolated" does not necessarily reflect the degree to which a molecule has been purified.

[0222] 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. Full-length proteins and fragments thereof are encompassed by this definition. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and similar modifications. Furthermore, for the purposes of the present invention, "polypeptide" or "protein" refers to a polypeptide or protein, respectively, that includes modifications of the native sequence, such as deletions, additions, and substitutions (generally conservative substitutions), as long as the protein maintains the desired activity. These modifications may be intentional, such as through site-directed mutagenesis, or may be accidental, such as through mutations in the host producing the protein or errors caused by PCR amplification. A protein may comprise two or more polypeptides. The letter "h" preceding a protein name indicates a native human protein herein, for example, "hILT4."

[0223] Unless otherwise specifically indicated (e.g., mouse 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 all used interchangeably and refer to native human ILT4. The terms include full-length unprocessed ILT4 as well as any form of ILT4 produced by treated cells. The terms encompass naturally occurring variants of 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).

[0224] As used herein, the term "ILT4 fragment" refers to ILT4 that has 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 an N-terminal signal peptide but retains the ability to bind to T cells. As used herein, the term "ILT4 variant" refers to ILT4 that contains naturally occurring amino acid additions, deletions, and substitutions but retains the ability to bind to T cells.

[0225] The term "antagonist" is used in the 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., T cell) and / or target molecule. Exemplary ILT4 antagonists include antibodies that block the binding of ILT4 to a target cell (e.g., T cell) and / or target molecule. An ILT4 antagonist is considered to "block the binding of ILT4 to a target cell or target molecule" when the ILT4 antagonist reduces the amount of detectable binding of ILT4 to at least one of a target cell (e.g., T cell) and / or target molecule in a cell binding assay by at least 50%. In some embodiments, the ILT4 antagonist reduces the amount of detectable 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. Blockade of ILT4 binding to target cells (e.g., T cells) can be demonstrated, for example, by binding of cells transfected with ILT4 (e.g., ILT4 ECD and transmembrane domain) or recombinant ILT4 Fc fusion protein (e.g., recombinant ILT4 ECD Fc fusion protein) on cells such as T cells in the presence or absence of the antagonist.

[0226] The term "inhibit" refers to a decrease, reduction, or cessation of any phenotypic characteristic, or a decrease, reduction, or cessation of the incidence, extent, or likelihood of said characteristic. In some embodiments, "reduce" or "inhibit" means the ability to cause a decrease of 20% or more. In another embodiment, "reduce" or "inhibit" means the ability to cause a decrease of 50% or more. In yet another embodiment, "reduce" or "inhibit" means the ability to cause an overall decrease of 75%, 85%, 90%, 95% or more.

[0227] The term "enhancing 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 IFN-γ ELISA, as described in the Examples.

[0228] As used herein, the term "ILT4 antibody" or "hILT4 antibody" or "anti-ILT4 antibody" or "antibody that binds to 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 by SPR In some embodiments, the binding of an ILT4 antibody to an unrelated, non-ILT4 protein is less than 10% of the binding of the antibody to ILT4, as measured, for example, by SPR or in a radioimmunoassay (RIA). In some embodiments, the binding of an ILT4 antibody to other ILT family proteins (such as LILRA) is less than 20%, less than 10%, or less than 5% of the binding of the antibody to ILT4, as measured, for example, by SPR or in a radioimmunoassay (RIA). In some embodiments, the ILT4 antibody binds to ILT4 but does not bind to at least one protein selected from ILT2, ILT3, and ILT5.

[0229] 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 secretion of the polypeptide from mammalian cells. The leader sequence is cleaved to form the mature protein during export of the polypeptide from mammalian cells. The leader sequence may be natural or synthetic and may be heterologous or homologous to the protein to which it is linked.

[0230] The term "antibody" or "Ab" 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, as long as they exhibit the desired antigen-binding activity. As used herein, the term refers to a molecule comprising at least the complementarity determining regions (CDRs) 1, CDR2, and CDR3 of a heavy chain and at least CDR1, CDR2, and CDR3 of a light chain, wherein the molecule is capable of binding to an antigen. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, human antibodies, and antibodies of various species such as mouse and cynomolgus monkey. The term "antibody fragment" includes, but is not limited to, fragments capable of binding to an antigen, such as Fv, single-chain Fv (scFv), Fab, Fab', and (Fab')2.

[0231] The term "heavy chain" or "HC" refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain comprises at least a portion of a heavy chain constant region. The term "full-length heavy chain" refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence and with or without a C-terminal lysine (K). The term "mature full-length heavy chain" refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, without a leader sequence and with or without a C-terminal lysine (K).

[0232] The term "heavy chain variable region" or "VH" refers to the region comprising the 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 comprises at least a portion of FR1 and / or at least a portion of FR4. In some embodiments, the heavy chain CDRs are as described herein, such as in the sequence listing below or Figures 1 to 12 As used herein, VH CDR1, CDR2 and CDR3 are as specified in Figures 1 to 12 The Kabat CDRs provided in .

[0233] The term "light chain" or "LC" refers to a polypeptide comprising at least one light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region. The term "full-length light chain" refers to a polypeptide comprising 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 comprising a light chain variable region and a light chain constant region, without a leader sequence.

[0234] The term "light chain variable region" or "VL" refers to the region comprising the light chain CDR1, FR2, HVR2, FR3, and HVR3. In some embodiments, the light chain variable region further comprises FR1 and / or FR4. In some embodiments, the light chain CDRs are as described herein, such as in the sequence listing below or Figures 1 to 12 As used herein, VL CDR1, CDR2 and CDR3 are as specified in Figures 1 to 12 The Kabat CDRs provided in .

[0235] "Chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a specific source or species, while the rest 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 comprising 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 comprises at least one mouse variable region and at least one human constant region. In some embodiments, a chimeric antibody comprises at least one cynomolgus monkey variable region and at least one human constant region. In some embodiments, all variable regions of a chimeric antibody are from a first species and all constant regions of a chimeric antibody are from a second species.

[0236] 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 comprises at least one human constant region or a fragment thereof. In some embodiments, the humanized antibody is a Fab, scFv, (Fab')2, etc.

[0237] As used herein, "human antibodies" refer to antibodies produced in humans, antibodies produced in non-human animals, such as and antibodies selected using in vitro methods, such as phage display, in which the antibody repertoire is based on human immunoglobulin sequences.

[0238] In some embodiments, the antibodies herein may contain one or more "conservative substitutions" compared to a specific specified sequence. "Conservative amino acid substitutions" herein refer to replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, the predicted non-essential amino acid residues in the antibodies herein are replaced by another amino acid residue from the same side chain family (e.g., basic, acidic, β-branched, aromatic, uncharged polar). Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate antigen binding are described, for example, in Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997).

[0239] In some embodiments, the antibodies herein may contain one or more "reversion substitutions." Examples are depicted in Figures 13 to 14 herein. Figure 15 A back substitution is a mutation that returns to the germline amino acid sequence from which the antibody heavy or light chain was derived.

[0240] The "K" of the binding of an antibody to a protein (e.g., ILT4) D " or "dissociation constant" is a measure of the affinity or specific binding of the antibody to the protein (e.g., ILT4). D Compared with the lower K D Indicating improved binding or affinity. K D The “off-rate” or k of antibodies and peptidesoff or k d and the “association rate” or k on or k a The ratio between them.

[0241] The term "specific binding" or similar terms refers to the specific binding of two polypeptides, such as an antibody and its polypeptide target. D is smaller than that between two random polypeptides under the same conditions. In other words, K D Smaller than that caused by nonspecific aggregation of peptides in the system.

[0242] As used herein, "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 natural mouse tumor assay systems. In some cases, a tumor model can allow tracking of tumor size or growth when treated with an antibody, and / or tracking 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.

[0243] As used herein, the term "immunostimulator" refers to a molecule that stimulates the immune system by acting as an agonist of an immunostimulatory molecule (including a costimulatory molecule) or as an antagonist of an immunosuppressive molecule (including a co-inhibitory molecule). An immunostimulatory molecule or an immunosuppressive molecule can be an immune checkpoint regulator, such as a checkpoint inhibitor or a checkpoint stimulator. An immunostimulatory agent can be a biologic, such as an antibody or antibody fragment, other proteins or a vaccine, or can be a small molecule drug. An "immunostimulatory molecule" includes a receptor or ligand for enhancing, stimulating, inducing or otherwise "opening" an immune response. As defined herein, immunostimulatory molecules include costimulatory molecules. An "immunosuppressive molecule" includes a receptor or ligand for reducing, inhibiting, suppressing or otherwise "closing" an immune response. As defined herein, immunosuppressive molecules include co-inhibitory molecules. Such immunostimulatory and immunosuppressive molecules can be, for example, receptors or ligands found on immune cells (such as T cells) or cells related to innate immunity (such as NK cells).

[0244] "Percent (%) amino acid sequence identity," "% identity," "percent amino acid sequence homology," and "% homology" with respect 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 particular peptide or polypeptide sequence, after aligning the sequences and 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 purpose of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm needed to achieve maximum alignment over the full length of the sequences being compared.

[0245] As used herein, "treatment" encompasses any administration or application of a therapeutic agent for a disease in a human, and includes inhibiting disease progression or one or more disease symptoms of a disease, slowing the disease or its progression or one or more of its symptoms, arresting its development, partially or completely relieving the disease or one or more of its symptoms, or preventing recurrence of one or more symptoms of a disease.

[0246] Unless specifically stated otherwise, the terms "subject" and "patient" are used interchangeably herein to refer to a human.

[0247] The term "effective amount" or "therapeutically effective amount" refers to an amount of a drug that is effective to treat a disease or condition in a subject, such as to partially or completely alleviate one or more symptoms. In some embodiments, an effective amount refers to an amount that is effective when administered and lasts for a necessary period of time to achieve the desired therapeutic or preventive result.

[0248] 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 known as a benign tumor), precancerous, or malignant. Cancer cells can be solid cancer cells or leukemic cancer cells. The term "tumor growth" is used herein to refer to the proliferation or growth of one or more cells comprising a cancer that results in a corresponding increase in the size or extent of the cancer.

[0249] Examples of cancers suitable for the methods of treatment 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 cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous cell non-small cell lung cancer), lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, 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 cancer, brain cancer, endometrial cancer, testicular cancer, bile duct cancer, gallbladder carcinoma, stomach cancer, melanoma, and various types of head and neck cancer (including head and neck squamous cell carcinoma).

[0250] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and consecutive (sequential) administration in any order.

[0251] A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation aid or vehicle conventional in the art, which is used with the therapeutic agent to form a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration used and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation used. For example, if the therapeutic agent is intended for oral administration, the carrier can be a gel capsule. If the therapeutic agent is intended for subcutaneous administration, the carrier is ideally non-irritating to the skin and does not cause injection site reactions.

[0252] A "chemotherapeutic agent" is a compound useful in the treatment of cancer. Examples of chemotherapeutic agents that can be administered in the methods herein include, but are not limited to, alkylating agents such as thiotepa and Cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; polyacetyl groups (especially bullatacin and bullatacinone); one); camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin); cryptophycin (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard mustard, such as chlorambucil, chlornaphazine, clofosamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard;Nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma and calicheamicin omega (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994); dynemicins, including dynemicin A, bisphosphonates such as clodronate; esperamicins and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, 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 deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, 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, e), 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, propionate, epitiostanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; acetylglucosuronate; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid acid); 2-ethylhydrazide; procarbazine; Polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermane; tenuazonic acid; triazoquinone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, A) and anguidine); urethanes; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), Cetearyl alcohol albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois) and Docetaxel Rorer, Antony, France); chloranbucil; 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 (Camptosar, CPT-11) (including regimens of irinotecan with 5-FU and leucovorin); the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including oxaliplatin regimens (FOLFOX); inhibitors that reduce cell proliferation such as PKC-α, Raf, H-Ras, and EGFR (e.g., erlotinib); ) and VEGF-A inhibitors; and pharmaceutically acceptable salts, acids or derivatives of any one of the above.

[0253] Other non-limiting exemplary chemotherapeutic agents that can be administered in the methods herein include anti-hormonal agents used to modulate or inhibit the effects of hormones on cancer, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and Toremifene; aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, Megestrol acetate, exemestane, formestane, fadrozole, Vorozole, Letrozole and anastrozole; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as PKC-α, Ralf, and H-Ras; ribonucleases, such as inhibitors of VEGF expression (e.g., RNA) and HER2 expression inhibitors; vaccines, such as gene therapy vaccines, e.g. vaccine, Vaccines and vaccine; rIL-2; topoisomerase 1 inhibitors; rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0254] "Anti-angiogenic agent" or "angiogenesis inhibitor" refers to a small molecular weight substance, polynucleotide (including, for example, inhibitory RNA (RNAi or siRNA)), polypeptide, isolated protein, recombinant protein, antibody, or conjugate or fusion protein thereof that directly or indirectly inhibits angiogenesis, vasculogenesis, or undesirable vascular permeability. It is understood that anti-angiogenic agents include those that bind to and block the angiogenic activity of angiogenic factors or their receptors. For example, anti-angiogenic agents that can be administered in the methods herein may include antibodies or other antagonists of angiogenic agents, such as antibodies to VEGF-A (e.g., bevacizumab) or VEGF-A receptors. ) or VEGF-A receptors (e.g., KDR receptor or 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, and the like. See, e.g., Klagsbrun and D'Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22:3172-3179 (e.g., Table 3 listing antiangiogenic 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 antiangiogenic factors); and Sato (2003) Int. J. Clin. Oncol. 8:200-206 (e.g., Table 1 listing antiangiogenic agents used in clinical trials).

[0255] As used herein, a "growth inhibitory agent" 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 inhibitory agent that can be administered in the methods herein can be one that significantly reduces the percentage of cells (such as cells expressing VEGF) in S phase. Examples of growth inhibitory agents include, but are not limited to, agents that block cell cycle progression (other than during S phase), such as agents that induce G1 arrest and M phase arrest. Classical M phase blockers include the vincas (vincristine and vinblastine), taxanes, and type II topoisomerase inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Agents that block G1 also extend into S phase arrest, such as DNA alkylating agents, such as tamoxifen, prednisone, dacarbazine, nitrogen mustard, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs", Murakami et al. (WB Saunders, Philadelphia, 1995), e.g., page 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs that are both derived from the yew tree. Docetaxel ( Rhone-PoulencRorer) is derived from European yew and is a paclitaxel ( Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which leads to the inhibition of mitosis in cells.

[0256] The term "anti-neoplastic composition" refers to a composition comprising at least one active therapeutic agent that can be used to treat cancer. Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used for radiation therapy, anti-angiogenic agents, cancer immunotherapeutic agents, apoptotic 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 (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib), and other agents for treating cancer. Platelet-derived growth factor inhibitors (e.g. (imatinib mesylate), COX-2 inhibitors (such as celecoxib), interferons, CTLA4 inhibitors (such as the anti-CTLA antibody ipilimumab) ), PD-1 or PD-L1 inhibitors (e.g. ), TIM3 inhibitors (e.g., anti-TIM3 antibodies), LAG-3 inhibitors, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets ErbB2, ErbB3, ErbB4, PDGFR-β, BlyS, APRIL, BCMA, CTLA4, TIM3, or one or more VEGF receptors, TRAIL / Apo2, and other biologically active and organic chemical agents, etc. Combinations thereof are also included in the present invention.

[0257] Antibodies that specifically bind to ILT4

[0258] The following sequence listing provides the amino acid sequence of human ILT4 with or without a signal peptide (see SEQ ID NOs: 107-108 and 110-111, respectively). The extracellular domain (ECD) comprises the amino acid residues shown in SEQ ID NOs: 109 and 112. An exemplary ECD coupled to a His-Avi tag comprises the amino acid sequence of SEQ ID NO: 119.

[0259] The anti-ITL4 antibody (Ab) can specifically bind to ILT4-ECD or a fragment thereof (see SEQ ID NOs: 109, 112, and 119).

[0260] Provided herein are values ​​of 10 as measured, for example, at 25°C or at 37°C. -6 M or lower, 10 -7 M or lower, 10 -8 M or lower, 10 -9 M or lower or 10 -10 or lower K D Ab that binds to ILT4.

[0261] Determination of the extent of Ab binding to ILT4 protein can be performed using several different methods. For example, by surface plasmon resonance (SPR), such as by An exemplary SPR assay involves capturing one or several antibodies on a CM4 sensor chip with an immobilized capture reagent (e.g., using Anti-Human Fc Capture Kit, GE Healthcare catalog number BR-1008-39, or Anti-mouse capture kit, GE Healthcare catalog number BR-1008-39), and ILT4 antigen (e.g., ILT4 ECD) as the analyte is flowed in a concentration series to determine binding kinetics and affinity in running buffer. In one embodiment, ILT4 is injected at two to five concentrations ranging from 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 up to four minutes of association time and up to ten minutes of dissociation time. 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 T200 evaluation software fits the data with simple 1:1 kinetics to a Langmuir binding model using mass shift. The SPR method described in the Examples can also be used.

[0262] The affinity of Ab for ILT4 ECD polypeptide can be determined using cells expressing ILT4 polypeptide on their surface, the method including flow cytometry. An exemplary flow cytometry assay comprises the following: T cells or other cells ectopically expressing ILT4 are resuspended in a buffer in which the Ab has been serially diluted from approximately 20 μg / mL and incubated 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 immediately incubated on a BD Biosciences 5000 without fixation. The affinity of Ab for ILT4 polypeptide can be determined as described in the Examples.

[0263] In certain embodiments, Abs that bind to ILT4 block the binding of ILT4 to target cells, such as T cells. The inhibition or blockade can be 100% or at least 99%, 95%, 90%, 85%, 80%, 75% or 50%.

[0264] The inhibition of binding between ILT4 and ILT4-binding cells (such as T cells) can be determined by measuring the inhibition of binding to 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 comprising 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 20 μg / mL of, for example, ILT4 ECD or ILT4 ECD fusion protein fused to human IgG1 Fc and varying concentrations of candidate ILT4 blocking antibodies or control antibodies. The cells are then washed twice in the same buffer and incubated for another 30 minutes at 4°C with a secondary antibody conjugated to a fluorophore that recognizes ILT4, but not the candidate blocking antibody or control antibody. The cells are then washed as described above and immediately incubated on a BD Biosciences 500 kit without fixation. Or other flow cytometers. Binding inhibition can also be determined, for example, as described in the Examples.

[0265] Exemplary ILT4-binding Abs

[0266] Provided herein are Abs that specifically bind to ILT4, eg, ILT4 ECD.

[0267] In certain embodiments, the ILT4 Ab comprises a heavy chain variable region ("VH") comprising the VH CDR1, CDR2, and / or CDR3 of any of the ILT4 Abs provided herein. Unless otherwise specified, the VH CDRs herein are as described herein. Figure 1 、 Figure 3 、 Figure 5 、 Figure 7 、 Figure 9 and Figure 11In certain embodiments, the ILT4Ab comprises a VH comprising the VH CDR1, CDR2, and / or CDR3 of any one of the ILT4Abs provided herein. In certain embodiments, the ILT4 Ab comprises a VH CDR1, CDR2, and / or CDR3 comprising 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 NOs: 125-127), 9C8 (SEQ ID NOs: 131-133), 2H2 (SEQ ID NOs: 137-139), 2E5 (SEQ ID NOs: 143-145), 24E5 (SEQ ID NOs: 149-151), 21D9 (SEQ ID NOs: 155-157), 21D9.b (SEQ ID NOs: 158-159), 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)).

[0268] In certain embodiments, the ILT4 Ab comprises a VH comprising the VH CDR1, CDR2, and CDR3 of any one of the ILT4 Abs provided herein. In certain embodiments, the ILT4 Ab comprises a VH comprising the VH CDR1, CDR2, and 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.

[0269] In certain embodiments, the ILT4 Ab comprises a VH comprising the VH CDR1, CDR2 and / or CDR3 of any ILT4 Ab provided herein and a VL comprising the CDR1, CDR2 and / or CDR3 of any ILT4 Ab provided herein. Unless otherwise indicated, the VL CDRs herein are as described herein. Figure 2、 Figure 4 、 Figure 6 、 Figure 8 、 Figure 10 and Figure 12 In certain embodiments, the ILT4 Ab comprises a VH comprising the VH CDR1, CDR2 and CDR3 of any ILT4 Ab provided herein and a VL comprising the CDR1, CDR2 and CDR3 of any ILT4 Ab provided herein. In certain embodiments, the ILT4 Ab comprises a VH comprising 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. 10F10.4 (SEQ ID NOs: 128-130), 9C8 (SEQ ID NOs: 134-136), 2H2 (SEQ ID NOs: 140-142), 2E5 (SEQ ID NOs: 146-148), 24E5 (SEQ ID NOs: 152-154), 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4) 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)).

[0270] In some embodiments, the ILT4 Ab may comprise:

[0271] (a) a VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of 9G4, and a VL comprising VL CDR1, CDR2, and CDR3 of 9G4;

[0272] (b) a VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of 9C8, and a VL comprising VL CDR1, CDR2, and CDR3 of 9C8;

[0273] (c) a VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of 2H2, and a VL comprising VL CDR1, CDR2, and CDR3 of 2H2;

[0274] (d) a VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of 25E5, and a VL comprising VL CDR1, CDR2, and CDR3 of 25E5;

[0275] (e) a VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of 24E5, and a VL comprising VL CDR1, CDR2, and CDR3 of 24E5;

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

[0277] (g) a VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of 21D9.b, and a VL comprising VLCDR1, CDR2, and CDR3 of 21D9.b;

[0278] (h) a VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of 21D9.c, and a VL comprising VLCDR1, CDR2, and CDR3 of 21D9.c;

[0279] (i) a VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of 21D9.d, and a VL comprising VLCDR1, CDR2, and CDR3 of 21D9.d;

[0280] (j) a VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of 21D9.e, and a VL comprising VLCDR1, CDR2, and CDR3 of 21D9.e;

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

[0282] (1) a VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of 21A5.a, and a VL comprising VLCDR1, CDR2, and CDR3 of 21A5.a;

[0283] (m) a VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of 10F10, and a VL comprising VLCDR1, CDR2, and CDR3 of 10F10;

[0284] (n) a VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of 10F10.1, and a VL comprising VLCDR1, CDR2, and CDR3 of 10F10.1;

[0285] (o) a VH comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 of 10F10.3, and a VL comprising VLCDR1, CDR2, and CDR3 of 10F10.3; or

[0286] (p) VH comprising the amino acid sequences of VH CDR1, CDR2 and CDR3 of 10F10.4, and VL comprising VLCDR1, CDR2 and CDR3 of 10F10.4.

[0287] 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.

[0288] 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. Individual VH sequences of specific antibody species provided herein are listed in the sequence listing. In certain embodiments, the ILT4 Ab comprises an amino acid sequence comprising 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., 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: 79), 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).

[0289] 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 amino acid substitutions (such as 1, 2, 3, 4, or 5 conservative substitutions) in the framework regions of the VH sequence. In some embodiments, the ILT4 Ab comprises a VH comprising 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 with 1, 2, 3, 4, or 5 back substitutions in the framework regions of the VH sequence.

[0290] In some embodiments, the ILT4 Ab comprises the VH CDRs and VLCDRs of any one of the ILT4 Abs described herein, wherein the CDRs comprise 1, 2, or 3 amino acid additions, substitutions (eg, conservative substitutions), or deletions among all CDRs.

[0291] In certain embodiments, the ILT4 Ab comprises a VH CDR1, CDR2, and CDR3 comprising the amino acid sequence of the VH CDRs of any ILT4 Ab provided herein and comprises a VH that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH of any ILT4 Ab provided herein. In certain embodiments, the ILT4 Ab comprises a VH comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of any of the following VHs: 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4. In certain embodiments, the VH of the antibody differs from 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 regions of the VH sequence. In certain embodiments, the VH of the antibody differs from the VH sequence shown in the sequence listing by 1, 2, 3, 4, or 5 back substitutions in the framework regions of the VH sequence.

[0292] 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.

[0293] 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 an amino acid sequence comprising a VL 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., 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: 71). 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 ILT4Ab comprises a VL CDR1, CDR2, and CDR3 comprising the amino acid sequence of the VL CDRs of any of the ILT4 Abs provided herein and comprises a VL that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VL of any of the ILT4Abs provided herein. In certain embodiments, the ILT4 Ab comprises a VL comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of any of the following VLs: 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4. 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 regions 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 back substitutions.

[0294] 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.

[0295] In certain embodiments, the ILT4 Ab comprises a VH comprising the amino acid sequence of the VH of any ILT4 Ab provided herein and comprises a VL comprising the amino acid sequence of the VL of any ILT4 Ab provided herein. In certain 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.

[0296] However, in certain embodiments, the VH of the antibody is that 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 with 1, 2, 3, 4, or 5 amino acid substitutions (such as 1, 2, 3, 4, or 5 conservative substitutions) in the framework regions of the VH sequence, and the VL is a VL from any of the same species listed above. However, in certain embodiments, the VH of the antibody is 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 has 1, 2, 3, 4, or 5 back substitutions in the framework regions of the VH sequence.

[0297] In certain embodiments, the ILT4 Ab comprises a VH and a VL comprising the amino acid sequence of the VH and VL 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.

[0298] In certain embodiments, the ILT4 Ab comprises a VH CDR1, CDR2, and CDR3 comprising the amino acid sequence of the VH CDR of any ILT4 Ab provided herein, and a VL CDR1, CDR2, and CDR3 comprising the amino acid sequence of the VL CDR of any ILT4 Ab provided herein, and further comprises a VH and a VL each 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 corresponding VH and VL of any ILT4 Ab provided herein. In certain embodiments, the VH and VL of the antibody differ from the VH and VL sequences shown in the sequence listing by 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 back substitutions) in the framework regions of the sequence.

[0299] 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.

[0300] An ILT4 Ab may contain:

[0301] (a) a VH comprising the amino acid sequence of VH of 9G4 and a VL comprising the amino acid sequence of VL of 9G4;

[0302] (b) a VH comprising the amino acid sequence of VH of 9C8 and a VL comprising the amino acid sequence of VL of 9C8;

[0303] (c) a VH comprising the amino acid sequence of VH of 2H2 and a VL comprising the amino acid sequence of VL of 2H2;

[0304] (d) a VH comprising the amino acid sequence of VH of 25E5 and a VL comprising the amino acid sequence of VL of 25E5;

[0305] (e) a VH comprising the amino acid sequence of VH of 24E5 and a VL comprising the amino acid sequence of VL of 24E5;

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

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

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

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

[0310] (j) a VH comprising the amino acid sequence of VH of 21D9.e and a VL comprising the amino acid sequence of VL of 21D9.e;

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

[0312] (1) a VH comprising the amino acid sequence of VH of 21A5.a and a VL comprising the amino acid sequence of VL of 21A5.a;

[0313] (m) a VH comprising the amino acid sequence of VH of 10F10 and a VL comprising the amino acid sequence of VL of 10F10;

[0314] (n) a VH comprising the amino acid sequence of VH of 10F10.1 and a VL comprising the amino acid sequence of VL of 10F10.1;

[0315] (o) a VH comprising the amino acid sequence of VH of 10F10.3 and a VL comprising the amino acid sequence of VL of 10F10.3; or

[0316] (p) VH comprising the amino acid sequence of VH of 10F10.4 and VL comprising the amino acid sequence of VL of 10F10.4.

[0317] An ILT4 Ab may contain:

[0318] (a) a VH comprising the VH CDRs of the VH of 9G4 and a VL comprising the VL CDRs of 9G4, and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 9G4;

[0319] (b) a VH comprising the VH CDRs of the VH of 9C8 and a VL comprising the VL CDRs of 9C8, and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 9C8;

[0320] (c) a VH comprising the VH CDRs of the VH of 2H2 and a VL comprising the VL CDRs of 2H2, and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 2H2;

[0321] (d) a VH comprising the VH CDRs of the VH of 25E5 and a VL comprising the VL CDRs of 25E5, and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 25E5;

[0322] (e) a VH comprising the VH CDRs of the VH of 24E5 and a VL comprising the VL CDRs of 24E5, and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 24E5;

[0323] (f) a VH comprising the VH CDRs of the VH of 21D9 and a VL comprising the VL CDRs of 21D9, and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 21D9;

[0324] (g) a VH comprising the VH CDRs of the VH of 21D9.b and a VL comprising the VL CDRs of 21D9.b, and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 21D9.b;

[0325] (h) a VH comprising the VH CDRs of the VH of 21D9.c and a VL comprising the VL CDRs of 21D9.c, and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 21D9.c;

[0326] (i) a VH comprising the VH CDRs of the VH of 21D9.d and a VL comprising the VL CDRs of 21D9.d, and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 21D9.d;

[0327] (j) a VH comprising the VH CDRs of the VH of 21D9.e and a VL comprising the VL CDRs of 21D9.e, and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 21D9.e;

[0328] (k) a VH comprising the VH CDRs of the VH of 21A5 and a VL comprising the VL CDRs of 21A5, and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 21A5;

[0329] (l) a VH comprising the VH CDRs of the VH of 21A5.a and a VL comprising the VL CDRs of 21A5.a, and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 21A5.a;

[0330] (m) a VH comprising the VH CDRs of the VH of 10F10 and a VL comprising the VL CDRs of 10F10, and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 10F10;

[0331] (n) a VH comprising the VH CDRs of the VH of 10F10.1 and a VL comprising the VL CDRs of 10F10.1, and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 10F10.1;

[0332] (o) a VH comprising the VH CDRs of the VH of 10F10.3 and a VL comprising the VL CDRs of 10F10.3, and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 10F10.3; or

[0333] (p) a VH comprising the VH CDRs of the VH of 10F10.4 and a VL comprising the VL CDRs of 10F10.4, and VH and VL amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and VL of 10F10.4.

[0334] In some of the above embodiments, VH and / or VL may differ from the sequence of each of species (a) to (p) by the presence of 1, 2, 3, 4, or 5 amino acid substitutions, such as 1, 2, 3, 4, or 5 conservative substitutions. In some embodiments, VH may comprise 1, 2, 3, 4, or 5 back substitutions.

[0335] An ILT4 Ab may contain:

[0336] (a) VH consisting of the amino acid sequence of VH of 9G4 and VL consisting of VL of 9G4;

[0337] (b) VH consisting of the amino acid sequence of VH of 9C8 and VL consisting of VL of 9C8;

[0338] (c) VH consisting of the amino acid sequence of VH of 2H2 and VL consisting of VL of 2H2;

[0339] (d) VH consisting of the amino acid sequence of VH of 25E5 and VL consisting of VL of 25E5;

[0340] (e) VH consisting of the amino acid sequence of VH of 24E5 and VL consisting of VL of 24E5;

[0341] (f) VH consisting of the amino acid sequence of VH of 21D9 and VL consisting of VL of 21D9;

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

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

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

[0345] (j) a VH consisting of the amino acid sequence of VH of 21D9.e and a VL consisting of VL of 21D9.e;

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

[0347] (l) a VH consisting of the amino acid sequence of VH of 21A5.a and a VL consisting of VL of 21A5.a;

[0348] (m) VH consisting of the amino acid sequence of VH of 10F10 and VL consisting of VL of 10F10;

[0349] (n) a VH consisting of the amino acid sequence of VH of 10F10.1 and a VL consisting of VL of 10F10.1;

[0350] (o) a VH consisting of the amino acid sequence of VH of 10F10.3 and a VL consisting of VL of 10F10.3; or

[0351] (p) VH consisting of the amino acid sequence of VH of 10F10.4 and VL consisting of VL of 10F10.4.

[0352] In certain embodiments, the ILT4 Ab comprises any of the variable regions and / or variable region CDRs 1 to 3 of the antibodies described above and elsewhere herein, such as:

[0353] (1) one or more of the following VH CDR1s, CDR2s, and CDR3s: 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] (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;

[0355] (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;

[0356] (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;

[0357] (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;

[0358] (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;

[0359] or

[0360] (7) Exceptions are VL and VH with 1, 2, 3, 4, or 5 back 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 that of IgG1.3 or IgG1.1f, or another constant region described herein, such as IgG1 and IgG1.238K.

[0361] In certain embodiments, the ILT4 Ab comprises any of the variable regions and / or variable region CDRs 1 to 3 of the antibodies described above and elsewhere herein, such as:

[0362] (1) one or more of the following VH CDR1s, CDR2s, and CDR3s: 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] (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;

[0364] (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;

[0365] (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;

[0366] (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;

[0367] (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;

[0368] or

[0369] (7) VL and VH of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4, with the exception of 1, 2, 3, 4, or 5 back or conservative substitutions in VH and / or VL;

[0370] and

[0371] The antibody further comprises one or more of the following features:

[0372] - For example, 10 -8 M, 10 -9 M or lower K D Specific binding to hILT4;

[0373] -Does not specifically bind to hILT2, hILT3, or hILT5;

[0374] - does not specifically bind to one or more members of the LILRA and / or LILRB family;

[0375] - stimulates T cell activation, e.g. in an MLR, as measured by increased T cell proliferation or IFN-g secretion, e.g. as shown in the assays described in the Examples;

[0376] - stimulating the differentiation or activation of monocytes into macrophages, e.g. stimulating the differentiation of monocytes into pro-inflammatory macrophages, e.g. as shown in the assays described in the Examples;

[0377] - promoting the expression of CD83 and CD86 on human monocyte-derived immature dendritic cells (Mo-iDCs), e.g., as shown in the assays described in the Examples;

[0378] - enhancing IFN-γ secretion after antigen stimulation in a CMV lysate assay, e.g. as shown in the assay described in the Examples;

[0379] - enhancing IFN-γ and TNF-α secretion by CD4+ and CD8+ T cells following CD3 stimulation in an allogeneic mixed lymphocyte reaction (allogeneic MLR) assay, e.g., as shown in the assays described in the Examples;

[0380] -Does not induce (or trigger) basophil activation;

[0381] - Inhibits the binding of HLA-A and / or HLA-B to ILT4;

[0382] - With the following figure 27 or Figure 37C The binding profiles to hILT4 are shown in ;

[0383] - binds to Ig-like domain 1, 2, or 1 and 2 of hILT4 (which correspond to amino acids 27-110 and 111-229, respectively, of SEQ ID NO: 107), such as 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 assay described in the Examples;

[0384] - does not significantly bind to other regions of the extracellular domain of ILT4, such as regions or residues located 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);

[0385] - compete with the antibodies described herein for binding to hILT4;

[0386] - specifically binds to cynomolgus monkey ILT4 comprising SEQ ID NO: 118, e.g., as shown in a binding assay described in the Examples;

[0387] - Promotes the pro-inflammatory polarization of macrophages towards M1 macrophages;

[0388] - does not induce (or trigger) basophil activation; and

[0389] - contains less than 5% of high and low molecular weight species after 3 months incubation at 25°C and / or contains less than 10% of high and low molecular weight species after 3 months incubation at 40°C.

[0390] In certain embodiments, the ILT4 Ab comprises a heavy chain (HC) comprising the amino acid sequence of the heavy chain of any ILT4 Ab provided herein. In certain embodiments, the ILT4 Ab comprises a heavy chain comprising the amino acid sequence of any one of the heavy chains of 9G4, 9C8, 2H2, 25E5, 24E5, 21D9, 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5, 21A5.a, 10F10, 10F10.1, 10F10.3, or 10F10.4 as shown below in the sequence listing (i.e., the complete HC sequence as provided in the table, or a composite sequence of the VH sequence of the antibody with the HC constant region sequence, such as SEQ ID NO: 98, 100, 102, 103, 104, or 179 as provided in the table).

[0391] 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 sequence:

[0392] a. IgG1, e.g., 9G4.IgG1, 9G4 (SEQ ID NO: 2), 9C8 (SEQ ID NO: 4), 2H2 (SEQ ID NO: 6), 2E5 (SEQ ID NO: 8), 24E5 (SEQ ID NO: 10), 21D9 (SEQ ID NO: 12), 21D9.b (SEQ ID NO: 74 and 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), 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),

[0393] b. IgG1, e.g., 9G4.IgG1, 9G4 (SEQ ID NO:51, amino acids 20-135, and SEQ ID NO: 102), 9C8 (SEQ ID NO:55 and SEQ ID NO: 102), 2H2 (SEQ ID NO:58 and SEQ ID NO: 102), 2E5 (SEQ ID NO:63 and SEQ ID NO: 102), 24E5 (SEQ ID NO:67 and SEQ ID NO: 102), 21D9 (SEQ ID NO:71 and SEQ ID NO: 102), 21D9.b (SEQ ID NO:74 and SEQ ID NO: 102), 21D9.c (SEQ ID NO:75 and SEQ ID NO: 102), 21D9.d (SEQ ID NO:78 and SEQ ID NO: 102), 21D9.e (SEQ ID NO:80 and SEQ ID NO: 102), 21A5 (SEQ ID NO:83 and SEQ ID NO: 102), 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),

[0394] c.IgG1.3 (e.g., 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: 16), 21A5.c (SEQ ID NO: 17), 21A5.d (SEQ ID NO: 19), 21A5.e (SEQ ID NO: 20), 21A5.f (SEQ ID NO: 21A5.g (SEQ ID NO: 22), 21A5.h (SEQ ID NO: 23), 21A5.h (SEQ ID NO: 24), 21A5.h (SEQ ID NO: 25), 21A5.i (SEQ ID NO: 26), 21A5.j (SEQ ID NO: 27), 21A5.j (SEQ ID NO: 28), 21A5.j (SEQ ID NO: 29), 21A5.j (SEQ ID NO: 30), 21A5.j (SEQ ID NO: 31), 21A5.j (SEQ ID NO: 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);

[0395] d.IgG1.1f (e.g., 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:81 and SEQ ID NO: 103), 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),

[0396] e.IgG1fa.P238K (e.g., 9G4.IgG1fa.P238K, etc.) 9G4 (SEQ ID NO:51, amino acids 20-135, and SEQ ID NO: 104), 9C8 (SEQ ID NO:55 and SEQ ID NO: 104), 2H2 (SEQ ID NO:58 and SEQ ID NO: 104), 2E5 (SEQ ID NO:63 and SEQ ID NO: 104), 24E5 (SEQ ID NO:67 and SEQ ID NO: 104), 21D9 (SEQ ID NO:71 and SEQ ID NO: 104), 21D9.b (SEQ ID NO:74 and SEQ ID NO: 104), 21D9.c (SEQ ID NO:75 and SEQ ID NO: 104), 21D9.d (SEQ ID NO:78 and SEQ ID NO: 104), 21D9.e (SEQ ID NO:80 and SEQ ID NO: 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

[0397] f. IgG4 S228P (e.g., 9G4.IgG4 S228P, etc.), 9G4 (SEQ ID NO:51, amino acids 20-135, and SEQ ID NO: 179), 9C8 (SEQ ID NO:55 and SEQ ID NO: 179), 2H2 (SEQ ID NO:58 and SEQ ID NO: 179), 2E5 (SEQ ID NO:63 and SEQ ID NO: 179), 24E5 (SEQ ID NO:67 and SEQ ID NO: 179), 21D9 (SEQ ID NO:71 and SEQ ID NO: 179), 21D9.b (SEQ ID NO:74 and SEQ ID NO: 179), 21D9.c (SEQ ID NO:75 and SEQ ID NO: 179), 21D9.d (SEQ ID NO:78 and SEQ ID NO: 179), 21D9.e (SEQ ID NO:80 and SEQ ID NO: 179), 21A5 (SEQ ID NO:81 and SEQ ID NO: 179), 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).

[0398] In certain embodiments, the ILT4 Ab comprises: a heavy chain comprising the amino acid sequence of the heavy chain of any ILT4 Ab provided herein, which comprises an IgG1.3 heavy chain constant region; and an amino acid sequence of the light chain of any ILT4 Ab provided herein. In certain embodiments, the 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 an HC constant region (which is represented by the nomenclature 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 kappa light chain comprising the following amino acid sequence: 9G4 (SEQ ID NO: 1), 9C8 (SEQ ID NO: 3), 2H2 (SEQ ID NO: 5), 2E5 (SEQ ID NO: 7), 24E5 (SEQ ID NO: 9), 21D9 (SEQ ID NO: 11), 21D9.b, 21D9.c, 21D9.d, 21D9.e, 21A5 (SEQ ID NO: 14), 21A5.a (SEQ ID NO: 16), 10F10 (SEQ ID NO: 18), 10F10.1 (SEQ ID NO: 20), 10F10.3 (SEQ ID NO: 21), or 10F10.4 (SEQ ID NO: 116).

[0399] An ILT4 Ab may contain:

[0400] (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;

[0401] (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;

[0402] (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;

[0403] (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;

[0404] (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;

[0405] (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;

[0406] (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;

[0407] (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;

[0408] (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;

[0409] (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;

[0410] (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;

[0411] (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;

[0412] (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;

[0413] (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;

[0414] (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

[0415] (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.

[0416] An ILT4 Ab may contain:

[0417] (a) a heavy chain (HC) comprising the HC CDRs of the HC of 9G4 and a light chain (LC) comprising the LC CDRs of 9G4, 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 9G4, respectively;

[0418] (b) a HC comprising the HC CDRs of the HC of 9C8 and a light chain (LC) comprising the LC CDRs of 9C8, and HC and LC amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the HC and LC of 9C8, respectively;

[0419] (c) a HC comprising the HC CDRs of the HC of 2H2 and a light chain (LC) comprising the LC CDRs of 2H2, and HC and LC amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the HC and LC of 2H2, respectively;

[0420] (d) a HC comprising the HC CDRs of the HC of 25E5 and a light chain (LC) comprising the LC CDRs of 25E5, and HC and LC amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the HC and LC of 25E5, respectively;

[0421] (e) a HC comprising the HC CDRs of the HC of 24E5 and a light chain (LC) comprising the LC CDRs of 24E5, 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 24E5, respectively;

[0422] (f) a HC comprising the HC CDRs of the HC of 21D9 and a light chain (LC) comprising the LC CDRs of 21D9, and HC and LC amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the HC and LC of 21D9, respectively;

[0423] (g) a HC comprising the HC CDRs of the HC of 21D9.b and a light chain (LC) comprising the LC CDRs of 21D9.b, and HC and LC amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the HC and LC of 21D9.b, respectively;

[0424] (h) a HC comprising the HC CDRs of the HC of 21D9.c and a light chain (LC) comprising the LC CDRs of 21D9.c, and HC and LC amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the HC and LC of 21D9.c, respectively;

[0425] (i) a HC comprising the HC CDRs of the HC of 21D9.d and a light chain (LC) comprising the LC CDRs of 21D9.d, and HC and LC amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the HC and LC of 21D9.d, respectively;

[0426] (j) a HC comprising the HC CDRs of the HC of 21D9.e and a light chain (LC) comprising the LC CDRs of 21D9.e, and HC and LC amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the HC and LC of 21D9.e, respectively;

[0427] (k) a HC comprising the HC CDRs of the HC of 21A5 and a light chain (LC) comprising the LC CDRs of 21A5, and HC and LC amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the HC and LC of 21A5, respectively;

[0428] (l) a HC comprising the HC CDRs of the HC of 21A5.a and a light chain (LC) comprising the LC CDRs of 21A5.a, and HC and LC amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the HC and LC of 21A5.a, respectively;

[0429] (m) a HC comprising the HC CDRs of the HC of 10F10 and a light chain (LC) comprising the LC CDRs of 10F10, and HC and LC amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the HC and LC of 10F10, respectively;

[0430] (n) a HC comprising the HC CDRs of the HC of 10F10.1 and a light chain (LC) comprising the LC CDRs of 10F10.1, and HC and LC amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the HC and LC of 10F10.1, respectively;

[0431] (o) a HC comprising the HC CDRs of the HC of 10F10.3 and a light chain (LC) comprising the LC CDRs of 10F10.3, and HC and LC amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the HC and LC of 10F10.3, respectively; or

[0432] (p) a HC comprising the HC CDRs of the HC of 10F10.3 and a light chain (LC) comprising the LC CDRs of 10F10.4, and HC and LC amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the HC and LC of 10F10.4, respectively.

[0433] In some of the above embodiments, the HC and / or LC may differ from the sequence of each of species (a) to (p) by the presence of 1, 2, 3, 4, or 5 amino acid substitutions (such as 1, 2, 3, 4, or 5 conservative substitutions). In some of the above embodiments, the HC and / or LC may differ from the sequence of each of species (a) to (p) by the presence of 1, 2, 3, 4, or 5 amino acid substitutions (such as 1, 2, 3, 4, or 5 back substitutions).

[0434] In some embodiments, the ILT4 Ab may comprise:

[0435] (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;

[0436] (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;

[0437] (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;

[0438] (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;

[0439] (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;

[0440] (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;

[0441] (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;

[0442] (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;

[0443] (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;

[0444] (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;

[0445] (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;

[0446] (1) 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;

[0447] (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;

[0448] (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;

[0449] (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

[0450] (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.

[0451] In some embodiments, the ILT4 Ab may comprise a heavy chain amino acid sequence comprising the VH amino acid sequence of the antibody species herein, rather than an IgG1.3 heavy chain constant region as provided in the HC sequence in the sequence listing herein (and see SEQ ID NO: 100), the antibody may comprise a different heavy chain constant region sequence, such as a human wild-type IgG1 constant region, such as those provided in SEQ ID NOs: 98 and 102-104, or an IgG4 heavy chain constant region, or an IgG4 constant region with an S228P substitution (EU numbering), or any of the other heavy chain constant regions described in the section below on constant regions.

[0452] In some embodiments, such modified ILT4 Abs have one or more of the following characteristics:

[0453] - For example, 10 -8 M or lower, or 10 -9 M or lower K D Specific binding to hILT4;

[0454] - does not specifically bind to one, two, or all three of hILT2, hILT3, and hILT5;

[0455] - does not specifically bind to one or more members of the LILRA and / or LILRB family;

[0456] - stimulates T cell activation, e.g., in an MLR assay, as measured by increased T cell proliferation or IFN-γ secretion; and

[0457] Stimulates monocyte differentiation or activation into macrophages.

[0458] 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) and amino acids 111-229 (domain 2) of SEQ ID NO: 107, and contains 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 assay described in the Examples. (See FIG27 .)

[0459] Exemplary Antibody Constant Regions

[0460] In some embodiments, the antibodies described herein comprise one or more human constant regions. In some embodiments, the human heavy chain constant region belongs to an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region belongs to 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 an S241P mutation in a human IgG4 constant region. In some embodiments, the antibodies described herein comprise a human IgG4 constant region and a human κ light chain.

[0461] The choice of heavy chain constant region can determine whether the antibody will have effector functions in vivo. In some embodiments, such effector functions include antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), and can result in killing cells to which the antibody is bound. In some therapeutic methods, including methods for treating some cancers, for example, when an antibody binds to cells that support the maintenance or growth of a tumor, it may be necessary to kill the cells. Exemplary cells that can support the maintenance or growth of a tumor include, but are not limited to, tumor cells themselves, cells that help recruit blood vessels to the tumor, and cells that provide ligands, growth factors, or counter-receptors that support or promote tumor growth or tumor survival. In some embodiments, when effector functions are needed, an antibody comprising a human IgG1 heavy chain or a human IgG3 heavy chain is selected.

[0462] In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of antibody glycosylation. Adding glycosylation sites to an antibody or deleting glycosylation sites from an antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0463] In the case where the antibody comprises an Fc region, the carbohydrate attached thereto can be changed. Natural antibodies produced by mammalian cells typically comprise branched biantennary oligosaccharides, which are typically attached to the Asn297 of the CH2 domain of the Fc region via an N-link. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to the GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the present invention can be modified to produce antibodies with certain improved properties. For example, in some embodiments, antibodies can be defucosylated, for example, by mutating residues such as Asn297 that are typically glycosylated with fucose or glycosylated via other means. In some embodiments, the antibodies herein can comprise defucosylated human IgG1 constant regions.

[0464] Also provided are antibodies with bisected oligosaccharides, for example, biantennary oligosaccharides bound to the Fc region of the antibody bisected by GlcNAc. Such antibodies may have reduced fucosylation and / or improved ADCC function. Examples of such antibodies are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Also provided are antibodies in which at least one galactose residue in the oligosaccharide is bound to the Fc region. Such antibodies may have improved CDC function. Such antibodies are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0465] Antibodies with amino-terminal leader sequence extensions 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 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 light chains of the antibody.

[0466] The in vivo or serum half-life of a human FcRn high affinity binding polypeptide can be determined, for example, in transgenic mice, humans, or non-human primates administered with polypeptides having variant Fc regions. See also, for example, Petkova et al., International Immunology 18(12):1759-1769 (2006).

[0467] In some embodiments of the invention, the defucosylated antibody mediates ADCC more efficiently in the presence of human effector cells than the parent antibody comprising fucose. Typically, ADCC activity can be determined using an in vitro ADCC assay as disclosed herein, but other assays or methods for determining ADCC activity, such as in animal models, are contemplated.

[0468] In certain embodiments, the Fc region is changed by replacing at least one amino acid residue with a different amino acid residue to change 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 can be replaced with different amino acid residues to change the affinity of the antibody for the effector ligand, but retain the antigen binding ability of the parent antibody. The effector ligand with changed affinity can be, for example, an Fc receptor or the C1 component of complement. This method is also described in detail in both U.S. Patents Nos. 5,624,821 and 5,648,260 to Winter et al.

[0469] In some embodiments, one or more amino acids selected from amino acid residues 329, 331, and 322 can be substituted with a different amino acid residue such that the antibody has altered C1q binding and / or reduced or abolished complement-dependent cytotoxicity (CDC). This approach is also described in detail in U.S. Patent No. 6,194,551 to Idusogie et al.

[0470] In some embodiments, one or more amino acid residues within amino acid positions 231 and 239 are altered to alter the ability of the antibody to fix complement. This approach 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 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, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312 90, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438, or 439. Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Exemplary variants include 239D / 332E, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T, and 267E / 268F / 324T. Other Fc modifications that can be made to Fcs are those that reduce or eliminate binding to FcγRs 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), where 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), where numbering is according to the EU index. The Fc variant may comprise 236R / 328R.Other modifications used to reduce the interaction of FcγRs with complement include substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P, and 234V, as well as removal of glycosylation at position 297 by mutation or enzymatic means or by production in an organism that does not glycosylate proteins, 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 a P238K substitution. IgG1.1f contains L234A, L235E, G237A, A330S and P331S substitutions.

[0471] Fc variants that enhance affinity for 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 a selectively enhanced affinity for FcγRIIb relative to one or more activating receptors. Modifications for altering 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 affinity for FcγRllb 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 enhanced binding to FcyRIIb include 235Y / 267E, 236D / 267E, 239D / 268D, 239D / 267E, 267E / 268D, 267E / 268E, and 267E / 328F.

[0472] Other modifications used to enhance the interaction of FcγRs with complement include, but are not limited to, substitutions 298A, 333A, 334A, 326A, 2471, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 3051, 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, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331 6, 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, wherein the numbering of the Fc region residues is that of the EU index as in patent publication No. WO 00 / 42072.

[0473] Optionally, the Fc region may comprise non-naturally occurring amino acid residues at additional and / or alternative positions known to those skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821, 6,277,375, 6,737,056, 6,194,551, 7,317,091, 8,101,720; PCX patent publications WO 00 / 42072, WO 01 / 58957, WO 02 / 06919, WO 04 / 016750, WO 04 / 029207, WO 04 / 035752, WO 04 / 074455, WO 04 / 099249, WO 04 / 063351, WO 05 / 070963, WO 05 / 040217, WO 05 / 092925, and WO 06 / 020114).

[0474] The affinity and binding properties of the Fc region for its ligand can be determined by a variety of in vitro assays known in the art (biochemical or immunological based assays), including but not limited to equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetics (e.g., BIACORE analysis) and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize labels on one or more of the components being assayed and / or employ a variety of detection methods, including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinity and kinetics can be found in Paul, WE, ed., Fundamental Immunology, 4th Edition, Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

[0475] In certain embodiments, antibodies are modified to extend their biological half-life. Various approaches are possible. 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 biological half-life, antibodies can be modified within the CH1 or CL regions to contain salvage receptor binding epitopes derived from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patent 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 binding to FcRn include: 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al., 2004, J. Biol. Chem. 279(8):6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 311A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields et al., Journal of Biological Chemistry 2006:114-116). Chemistry,2001,276(9):6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 311S, 433R, 433S, 4331, 433P, 433Q, 434H, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H, 308T / 309P / 311S(Dall Acqua et al., Journal of Immunology, 2002, 169:5171-5180, Dall'Acqua et al., 2006, Journal of Biological Chemistry 281: 23514-23524). Other modifications for modulating FcRn binding are described in Yeung et al., 2010, J Immunol, 182: 7663-7671.

[0476] In certain embodiments, hybrid IgG isotypes with specific biological characteristics can be used. For example, IgG1 / IgG3 hybrid variants can be constructed by replacing the IgG1 position in CH2 and / or CH3 district with the amino acid substitution of IgG3 from the different positions of two isotypes. Therefore, hybrid variant IgG antibodies comprising one or more substitutions, such as 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R and 436F can be constructed. In some embodiments described herein, IgG1 / IgG2 hybrid variants can be constructed by replacing the IgG2 position in CH2 and / or CH3 district with the amino acid substitution of IgG1 from the different positions of two isotypes. Therefore, hybrid variant IgG antibodies comprising one or more substitutions, such as one or more of the following amino acid substitutions: 233E, 234L, 235L, -236G (referring to insertion of glycine at position 236) and 327A can be constructed.

[0477] In addition, the binding sites for FcγRI, FcγRII, FcγRIII, and FcRn on human IgG1 have been mapped and variants with improved binding have been described (see Shields, RL et al. (2001), J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcγRIII. In addition, the following combination 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). The introduction of triple mutations into antibodies such as alemtuzumab (CD52 specific), trastuzumab (HER2 / neu specific), rituximab (CD20 specific), and cetuximab (EGFR specific) has been translated into greatly enhanced ADCC activity in vitro, and the S239D / I332E variant has been shown to enhance the 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 that 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.

[0478] In certain embodiments, an Fc is selected that has reduced binding to FcγRs. An exemplary Fc with reduced FcγR binding (eg, IgG1 Fc) comprises the following three amino acid substitutions: L234A, L235E, and G237A.

[0479] In certain embodiments, an Fc is selected that has reduced complement binding. An exemplary Fc with reduced complement binding (eg, an IgG1 Fc) has the following two amino acid substitutions: A330S and P331S.

[0480] In certain embodiments, an Fc is selected that has substantially no effector function, i.e., reduced binding to FcγRs and reduced complement binding. An exemplary Fc with no effector function (e.g., IgG1 Fc) comprises the following five mutations: L234A, L235E, G237A, A330S, and P331S.

[0481] When an IgG4 constant domain is used, it may include the substitution S228P, which mimics the hinge sequence in IgG1 and thereby stabilizes the IgG4 molecule.

[0482] Fc modifications described in WO 2017 / 087678 or WO2016081746 may also be used.

[0483] In certain embodiments, the glycosylation of the antibody is modified. For example, aglycosylated antibodies (i.e., antibodies lacking glycosylation) can be produced. Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be achieved, for example, by changing 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 the sites. Such aglycosylation can increase the affinity of the antibody for the antigen. This approach is also described in detail in U.S. Patents Nos. 5,714,350 and 6,350,861 to Co et al.

[0484] Glycosylation of the constant region at N297 can be prevented by mutating the N297 residue to another residue, such as N297A, and / or by mutating the adjacent amino acid, such as 298, thereby reducing glycosylation at N297.

[0485] Additionally or alternatively, antibodies with altered glycosylation types can be produced, 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 a host cell with an altered glycosylation mechanism. Cells with altered glycosylation mechanisms have been described in the art and can be used as host cells for expressing the recombinant antibodies described herein to thereby produce antibodies with altered glycosylation. For example, EP 1,176,195 by Hanai et al. describes a cell line in which the FUT8 gene encoding the fucosyltransferase is functionally disrupted, such that antibodies expressed in such a cell line exhibit hypofucosylation. PCT Publication WO 03 / 035835 by Presta describes a variant CHO cell line, Led 3 cells, which has a reduced ability to attach fucose to carbohydrates attached to Asn(297), and also results in hypofucosylation of antibodies expressed in the host cells (see also Shields, RL et al. (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99 / 54342 by Umana et al. describes a cell line that has been engineered to express a glycoprotein-modifying glycosyltransferase {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).

[0486] Another modification of the antibodies described herein is pegylation. Antibodies can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or its fragment is typically reacted with polyethylene glycol (PEG) (such as a reactive ester or aldehyde derivative of PEG) under conditions where one or more PEG groups are attached 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 similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any PEG form that has been used to derive other proteins, such as single (CI-CIO) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is a deglycosylated antibody. The method for protein pegylation is 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.

[0487] Specific exemplary antibody embodiments herein are further listed in the following sections.

[0488] Embodiments related to antibody 21D9.e:

[0489] 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 a VH CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 155, 156, and 157, respectively, and the light chain comprises a 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.

[0490] 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 a VH CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 155, 156, and 157, respectively, and the light chain comprises a 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 stimulates T cell activation (or potentiates a T cell response), e.g., (i) in a mixed lymphocyte reaction (MLR) assay, as demonstrated, e.g., 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).

[0491] 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 a VH CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 155, 156, and 157, respectively, and the light chain comprises a 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 T cell activation in a monocyte:T cell allogeneic MLR.

[0492] 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 VH comprising SEQ ID NO: 80 and the light chain comprises VL comprising SEQ ID NO: 70, wherein the Ab enhances IFN-γ secretion from PBMCs following antigen stimulation in a cytomegalovirus (CMV) lysate assay.

[0493] 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 VH comprising SEQ ID NO: 80 and the light chain comprises VL comprising SEQ ID NO: 70, wherein the antibody enhances TNF-α secretion from macrophages, e.g., differentiated macrophages.

[0494] 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 VH comprising SEQ ID NO: 80 and the light chain comprises VL comprising SEQ ID NO: 70, wherein the antibody increases expression of CD83 and / or CD86 on monocyte-derived dendritic cells (MoDCs).

[0495] 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 VH comprising SEQ ID NO: 80 and the light chain comprises VL comprising SEQ ID NO: 70, wherein the antibody inhibits binding of hILT4 to T cells and / or binding partners (e.g., HLA-A and / or HLA-B).

[0496] 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 VH comprising SEQ ID NO: 80 and the light chain comprises VL comprising SEQ ID NO: 70, wherein the Ab (a) binds to the Ig-like domain 1 of hILT4 and / or the VL 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) amino acid residues Lys43, Ile49, Thr50, and Arg51 of mature hILT4, as determined by carbene footprinting.

[0497] 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 VH comprising SEQ ID NO: 80 and the light chain comprises VL comprising SEQ ID NO: 70, wherein the Ab inhibits binding of (or competes with) an antibody comprising a HC comprising SEQ ID NO: 12 or 13 and a light chain comprising SEQ ID NO: 11 to hILT4 and / or inhibits binding of an antibody comprising VH and VL of 21D9.IgG1.1f and / or 21D9.e.IgG1.3 to hILT4.

[0498] 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: 155, 156, and 157, respectively, and the light chain comprises a 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 is expressed in 10 -8 M or 10 -9 Binds to hILT4 with a KD of M or lower.

[0499] 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: 155, 156, and 157, respectively, and the light chain comprises a 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 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.

[0500] 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: 155, 156, and 157, respectively, and the light chain comprises a 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 the human proteins LILRA1, LILRA2, LILRA3, LILRA4, LILRA6, ILT2, ILT3, ILT5, or LIR8.

[0501] 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 VH comprising SEQ ID NO: 80 and the light chain comprises VL comprising SEQ ID NO: 70, wherein the Ab promotes pro-inflammatory polarization of macrophages to M1 macrophages.

[0502] 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 VH comprising SEQ ID NO: 80 and the light chain comprises VL comprising SEQ ID NO: 70, wherein the Ab does not induce (or trigger) basophil activation.

[0503] 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 VH comprising SEQ ID NO: 80 and the light chain comprises VL comprising SEQ ID NO: 70, wherein a composition comprising the Ab comprises less than 5% high and low molecular weight species after incubation at 25°C for 3 months, and / or comprises less than 10% high and low molecular weight species after incubation at 40°C for 3 months.

[0504] 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 a VH CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 155, 156, and 157, respectively, and the light chain comprises a 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, e.g., 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).

[0505] 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.

[0506] Embodiment 18: An isolated antibody that binds to hILT4, wherein the Ab comprises: two heavy chains, each comprising SEQ ID NO: 13; and two light chains, each comprising SEQ ID NO: 11, wherein optionally, one or both of the heavy chains comprises a C-terminal lysine.

[0507] 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 connecting the heavy chains.

[0508] 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.

[0509] 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.

[0510] 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.

[0511] Embodiment 23: An isolated nucleic acid or collection of nucleic acids encoding the antibody of any one of embodiments 1-22.

[0512] Embodiment 24: A host cell for producing any one of the antibodies of embodiments 1-22, eg, comprising the isolated nucleic acid or nucleic acid collection of embodiment 23.

[0513] Embodiment 25: A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective dose of the antibody of 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 a VH CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 155, 156, and 157, respectively, and the light chain comprises a 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.

[0514] Embodiment 26: A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of:

[0515] (A) (1) the antibody of 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 a VH CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 155, 156, and 157, respectively, and the light chain comprises a 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

[0516] (B) Antagonists of PD-1 or PD-L1, such as antagonist antibodies that bind to human PD-1 or human PD-L1, for example, nivolumab, pembrolizumab, cemiplimab, toripalimab, sintilimab, atezolizumab, durvalumab, or avelumab.

[0517] Embodiment 27: A composition comprising:

[0518] (A) (1) the antibody of 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 a VH CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 155, 156, and 157, respectively, and the light chain comprises a 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

[0519] (B) Antagonists of PD-1 or PD-L1, such as antagonist antibodies that bind to human PD-1 or human PD-L1, for example, nivolumab, pembrolizumab, simvastatin, toripalimab, sintilimab, atezolizumab, durvalumab, or avelumab.

[0520] Embodiments related to antibody 21D5.a:

[0521] Embodiment 1: 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 VH comprising SEQ ID NO: 87 and the light chain comprises VL comprising SEQ ID NO: 86.

[0522] Embodiment 2: 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 stimulates T cell activation (or enhances a T cell response), e.g., (i) in a mixed lymphocyte reaction (MLR) assay, as demonstrated, e.g., 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).

[0523] 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: 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.

[0524] 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 VH comprising SEQ ID NO: 87 and the light chain comprises VL comprising SEQ ID NO: 86, wherein the Ab enhances IFN-γ secretion from PBMCs following antigen stimulation in a CMV lysate assay.

[0525] 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 VH comprising SEQ ID NO: 87 and the light chain comprises VL comprising SEQ ID NO: 86, wherein the antibody enhances TNF-α secretion from macrophages, e.g., differentiated macrophages.

[0526] 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 VH comprising SEQ ID NO: 87 and the light chain comprises VL comprising SEQ ID NO: 86, wherein the antibody increases expression of CD83 and / or CD86 on monocyte-derived dendritic cells (Mo-DCs).

[0527] 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 VH comprising SEQ ID NO: 87 and the light chain comprises VL comprising SEQ ID NO: 86, wherein the antibody inhibits binding of hILT4 to T cells and / or binding partners (e.g., HLA-A and / or HLA-B).

[0528] 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 VH comprising SEQ ID NO: 87 and the light chain comprises VL comprising SEQ ID NO: 86, wherein the Ab (a) binds to Ig-like domain 2 of hILT4 and / or hILT4 comprises: 154 ILCKEGEEEHPQCLNSQPHARGSSRAIF 181 (SEQ ID NO: 123) and / or 425 SSPPPTGPIS 434 (SEQ ID NO: 124), as determined by HDX; and / or (b) interacts 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, as determined by carbene footprinting.

[0529] 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: 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, wherein the Ab inhibits (or competes with) binding of an antibody comprising a HC comprising SEQ ID NO: 15 or 17 and a light chain comprising SEQ ID NO: 14 or 16, respectively, to hILT4 and / or inhibits binding of an antibody comprising the VH and VL of 21D5 or 21D5.a to hILT4.

[0530] 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: 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, wherein the Ab is expressed in 10 -8 M or 10 -9 Binds to hILT4 with a KD of M or lower.

[0531] 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: 161, 162, and 163, respectively, and the light chain comprises a 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 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.

[0532] 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: 161, 162, and 163, respectively, and the light chain comprises a 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 the human proteins LILRA1, LILRA2, LILRA4, LILRA5, LILRA6, ILT2, ILT3, ILT5, or LIR8.

[0533] 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 VH comprising SEQ ID NO: 87 and the light chain comprises VL comprising SEQ ID NO: 86, wherein the Ab promotes pro-inflammatory polarization of macrophages to M1 macrophages.

[0534] 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 VH comprising SEQ ID NO: 87 and the light chain comprises VL comprising SEQ ID NO: 86, wherein the Ab does not induce (or trigger) basophil activation.

[0535] 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 VH comprising SEQ ID NO: 87 and the light chain comprises VL comprising SEQ ID NO: 86, wherein a composition comprising the Ab comprises less than 5% high and low molecular weight species after incubation at 25°C for 3 months, and / or comprises less than 10% high and low molecular weight species after incubation at 40°C for 3 months.

[0536] 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 a VH CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 161, 162, and 163, respectively, and the light chain comprises a 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, e.g., 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).

[0537] 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.

[0538] Embodiment 18: An isolated antibody that binds to hILT4, wherein the Ab comprises: two heavy chains, each comprising SEQ ID NO: 17; and two light chains, each comprising SEQ ID NO: 16, wherein optionally, one or both of the heavy chains comprises a C-terminal lysine.

[0539] 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 connecting the heavy chains.

[0540] 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.

[0541] 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.

[0542] 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.

[0543] Embodiment 23: An isolated nucleic acid or collection of nucleic acids encoding the antibody of any one of embodiments 1-22.

[0544] Embodiment 24: A host cell for producing any one of the antibodies of embodiments 1-22, eg, comprising the isolated nucleic acid or nucleic acid collection of embodiment 23.

[0545] Embodiment 25: 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: 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.

[0546] 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 a VH CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 161, 162, and 163, respectively, and the light chain comprises a 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, and / or (iii) the heavy chain comprises SEQ ID NO: 17 and the light chain comprises SEQ ID NO: 18 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, simvastatin, toripalimab, sintilimab, atezolizumab, durvalumab, or avelumab.

[0547] 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 a VH CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 161, 162, and 163, respectively, and the light chain comprises a 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, and / or (iii) the heavy chain comprises SEQ ID NO: 17 and the light chain comprises SEQ ID NO: 18 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, simvastatin, toripalimab, sintilimab, atezolizumab, durvalumab, or avelumab.

[0548] Embodiments related to antibodies 10F10, 10F10.1, 10F10.3, and 10F10.4:

[0549] Embodiment 1: 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.

[0550] Embodiment 2: 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 antibody stimulates T cell activation (or potentiates a T cell response), e.g., (i) in a mixed lymphocyte reaction (MLR) assay, as demonstrated, e.g., 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).

[0551] 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 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 enhances T cell activation in a monocyte:T cell allogeneic MLR.

[0552] 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 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 enhances IFN-γ secretion from PBMCs following antigen stimulation in a CMV lysate assay.

[0553] 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 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 inhibits binding of hILT4 to T cells and / or to binding partners (e.g., HLA-A and / or HLA-B).

[0554] 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 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 antibody enhances TNF-α secretion from macrophages, e.g., differentiated macrophages.

[0555] 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 VH comprising SEQ ID NO: 91 and the light chain comprises VL comprising SEQ ID NOs: 90, 94, 96, or 114, wherein the antibody increases expression of CD83 and / or CD86 on monocyte-derived dendritic cells (MoDCs).

[0556] 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 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 CDR1 comprising SEQ ID NO: 92. NO: 90, 94, 96 or 114 VL, wherein the Ab (i) binds to Ig-like domain 2 of hILT4 proximal to Ig-like domain 1, and / or (ii) interacts with one or more (or all) amino acid residues Glu42, Lys43, Gly76, Cys77, Leu88, Pro91, Pro183 and Tyr184 of mature hILT4, as determined by carbene footprinting.

[0557] 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 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 binds to a heavy chain comprising a HC comprising SEQ ID NO: 19 and a light chain comprising SEQ ID NOs: 171, 172, or 174, respectively. NO: 18, 20, 21 or 116 light chain (or competes therewith) and / or inhibits the binding of an antibody comprising the VH and VL of 10F10, 10F10.1, 10F10.3 and / or 10F10.4 to hILT4.

[0558] 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.

[0559] 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.

[0560] 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.

[0561] 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 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 does not induce (or trigger) basophil activation.

[0562] 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 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 a composition comprising the Ab comprises less than 5% high and low molecular weight species after incubation at 25°C for 3 months, and / or comprises less than 10% high and low molecular weight species after incubation at 40°C for 3 months.

[0563] 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 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 does not significantly bind to the human proteins LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, ILT2, ILT3, ILT5, or LIR8.

[0564] 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 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 antibody comprises an IgG heavy chain constant region, e.g., 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).

[0565] 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.

[0566] 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.

[0567] 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 connecting the heavy chains.

[0568] 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.

[0569] 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.

[0570] 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.

[0571] 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.

[0572] Embodiment 24: An isolated nucleic acid or collection of nucleic acids encoding the antibody of any one of embodiments 1-23.

[0573] Embodiment 25: A host cell for producing any one of the antibodies of embodiments 1-23, eg, comprising the isolated nucleic acid or nucleic acid collection of embodiment 24.

[0574] Embodiment 26: 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 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, and / or (iii) the heavy chain comprises SEQ ID NO: 19 and the light chain comprises SEQ ID NO: 18, 20, 21, or 116.

[0575] 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 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, and / or (iii) the heavy chain comprises SEQ ID NO: 19 and the light chain comprises SEQ ID NO: 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, simvastatin, toripalimab, sintilimab, atezolizumab, durvalumab or avelumab.

[0576] 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 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, and / or (iii) the heavy chain comprises SEQ ID NO: 19 and the light chain comprises SEQ ID NO: 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, simvastatin, toripalimab, sintilimab, atezolizumab, durvalumab or avelumab.

[0577] Nucleic acids and host cells

[0578] Also provided are nucleic acids encoding antibodies or their heavy or light chains or portions thereof. Exemplary nucleic acids are provided in the sequence listing. Contemplated 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 contemplated are compositions comprising nucleic acids encoding the antibodies provided herein, as well as cells comprising these compositions and methods for preparing the antibodies, comprising culturing cells transformed with nucleic acids encoding an ILT4 antibody and isolating the antibody from the culture medium or cells.

[0579] Treatment methods using ILT4-binding Abs and related pharmaceutical compositions

[0580] The antibodies described herein can be used, for example, to treat cancer. In some embodiments, a method of treating cancer is provided, comprising administering to a patient an effective amount of an antibody described herein. In some embodiments, the Ab can trigger or enhance a 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 a patient.

[0581] Provided herein are methods for treating a subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of an ILT4 antibody as 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.

[0582] The cancer may be a solid tumor or a hematological malignancy (liquid tumor).

[0583] 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, kidney cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone refractory 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 (e.g., 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 cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors of childhood, ureteral cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain cancer, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma cancer), squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos), virus-related cancers or cancers of viral origin (e.g., human papilloma virus (HPV-related or HPV-induced tumors)), and hematologic malignancies derived from either of the two major blood cell lineages, i.e., the myeloid lineage (which produces granulocytes, erythrocytes, platelets, macrophages, and mast cells) or the lymphoid lineage (which produces B, T, NK, and plasma cells), such as all types of leukemias, lymphomas, and myeloma, e.g., acute, chronic, lymphocytic and / or myeloid leukemias, such as acute 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 (PML), and myeloma. leukemia) (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant [M4E] with eosinophilia), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma and chloroma; lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma;NHL), B-cell malignancies (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, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma 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 neoplasms of the lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma largecell lymphoma), precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma;myeloma, such as IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also known as indolent myeloma), solitary plasmacytoma and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma, and leukemia. hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; seminomas, teratocarcinomas, central and peripheral nerve tumors (including astrocytomas, schwannomas); tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma, and teratothelial carcinoma; hematopoietic tumors of lymphoid lineage, such as T-cell and B-cell tumors, including but not limited to T-cell disorders such as pre-T lymphocytic leukemia (T-PLL), including small cell and brain cell types; T-cell large granular lymphocyte leukemia (TLLL), including leukemia of the thyroid gland, ... 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 previous immunotherapy, such as with antibodies that block CTLA-4 or PD-1), and / or recurrent cancer.

[0584] In certain embodiments, the antibodies described herein are administered to patients with cancers that have not responded sufficiently to previous treatments or have progressed, such as previous treatments using immuno-oncology or immunotherapy drugs. In some embodiments, the cancer is refractory or resistant to previous treatments, inherently refractory or resistant (e.g., difficult to treat with PD-1 pathway antagonists), or acquires resistance or refractory states. For example, the antibodies described herein may be administered to subjects who have progressed after treatment with an anti-PD-1 pathway antagonist therapy, such as alone or in combination with another therapy (e.g., with an anti-PD-1 pathway antagonist therapy). In other embodiments, the antibodies described herein are administered to patients who have not previously received a tumor immunotherapy agent (e.g., a PD-1 pathway antagonist) (i.e., treated with it).

[0585] Combination with immunostimulants

[0586] 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 may be infused together or injected at approximately 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 the at least one immunostimulatory agent such that the two therapeutic agents are administered 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 5 days, 7 days, or two weeks apart.

[0587] In some embodiments, before administering at least one immunostimulant, at least one dose, at least two doses, at least three doses, at least five doses or at least ten doses of an antibody are administered. In some embodiments, before administering the antibody, 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 immunostimulant are administered. In some embodiments, before administering the first dose of an antibody, the last dose of an immunostimulant is administered for 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. In some embodiments, before administering the first dose of at least one immunostimulant, the last dose of an antibody is administered for 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. In some embodiments, the subject has received or is receiving therapy using at least one immunostimulant, and an ILT4 antibody is added to the treatment regimen.

[0588] In some embodiments, at least one immunostimulatory agent comprises an antagonist of an activation inhibitor of an immune cell, such as a T cell, and in some embodiments, at least one immunostimulatory agent comprises an agonist of an activation stimulator of an immune cell, such as a T cell. In some embodiments, at least one immunostimulatory agent 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 immunostimulatory agent includes 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, IFN α, an agonist of STING, or a Toll-like receptor agonist, such as a TLR2 / 4 agonist. In some embodiments, at least one immunostimulatory agent includes an agent that is bound 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 immunostimulatory agent comprises an agent that binds to a TNF receptor family member, such as CD40, CD40L, OX40, OX40L, GITR, GITRL, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, T RAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR / Fn14, TWEAK, BAFFR, EDA, 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 immunostimulatory agent comprises an agent that antagonizes or inhibits cytokines that inhibit T cell activation, such as IL-6, IL-10, TGFβ, VEGF.In some embodiments, at least one immunostimulatory agent comprises an agonist of a cytokine that stimulates T cell activation (such as IL-2, IL-7, IL-12, IL-15, IL-21, and IFNα). In some embodiments, at least one immunostimulatory agent comprises an antagonist of a chemokine (such as CXCR2, CXCR4, CCR2, or CCR4). In some embodiments, at least one immunostimulatory agent comprises an antibody. In some embodiments, at least one immunostimulatory agent may comprise a vaccine, such as a mesothelin-targeted vaccine or an attenuated Listeria cancer vaccine, such as CRS-207.

[0589] For example, an ILT4 antibody described herein can be administered with one or more of the following agents:

[0590] (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.

[0591] Exemplary agents that can be used in combination with the ILT4 antibodies described herein to treat cancer include: (ipilimumab) or tremelimumab (targeting CTLA-4), galiximab (targeting B7.1), BMS-936558 (targeting PD-1), MK-3475 (targeting PD-1), atezolizumab Avelumab, durvalumab, AMP224 (targeting B7DC), BMS-936559 (targeting B7-H1), MPDL3280A (targeting B7-H1), MEDI-570 (targeting ICOS), AMG557 (targeting B7H2), MGA271 (targeting B7H3), IMP321 (targeting LAG-3), BMS-663513 (targeting CD137), PF-05082566 (targeting CD137), CDX-1127 (targeting CD27), anti-OX40 (Providence Health Services), huMAbOX40L (directed to OX40L), Atacicept (directed to TACI), CP-870893 (directed to CD40), Lucatumumab (directed to CD40), Dacetuzumab (directed to CD40), Muromonab-CD3 (directed to CD3); anti-GITR antibodies MK4166, TRX518, Medi1873, INBRX-110, LK2-145, GWN-323, GITRL-Fc, or any combination thereof.

[0592] Other molecules that can be used in combination with ILT4 antibodies to treat cancer include antagonists of inhibitory receptors on NK cells, or agonists of activating receptors on NK cells, such as antagonists of KIR (eg, lirilumab).

[0593] T cell activation can also be regulated by soluble cytokines. In some embodiments, ILT4 antibodies can be used in combination with antagonists for suppressing cytokines for T cell activation or agonists for cytokines that stimulate T cell activation. For example, ILT4 antibodies can be used in combination with: (i) antagonists (or inhibitors or blockers) of proteins of the IgSF family or B7 family or TNF family that suppress T cell activation, or antagonists of cytokines (such as IL-6, IL-10, TGF-β, VEGF; "immunosuppressive cytokines") that suppress T cell activation and / or (ii) stimulating receptors of the IgSF family, B7 family or TNF family or agonists of cytokines that stimulate T cell activation.

[0594] Other agents for use in 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, WO13 / 87699, WO13 / 119716, WO13 / 132044) or FPA-008 (WO11 / 140249, WO13169264, WO14 / 036357).

[0595] ILT4 antibodies can also be administered with agents that inhibit TGF-β signaling.

[0596] Additional agents that can be combined with ILT4 antibodies 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).

[0597] Other therapies that can be combined with ILT4 antibodies include therapies that deplete or block Treg cells, such as agents that specifically bind to CD25.

[0598] Another therapy that can be combined with an ILT4 antibody is one that inhibits metabolic enzymes such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase.

[0599] Another class of agents that can be used with ILT4 antibodies include agents that inhibit adenosine formation, such as CD73 inhibitors, or agents that inhibit adenosine A2A receptors.

[0600] Other therapies that can be used in combination with ILT4 antibodies to treat cancer include therapies that reverse / prevent T cell anergy or exhaustion and therapies that trigger innate immune activation and / or inflammation at tumor sites.

[0601] Other therapies that can be used in combination with ILT4 antibodies to treat cancer include, for example, the use of Therapies that block IL-8.

[0602] ILT4 antibodies can be combined with more than one tumor immunotherapy agent, and can be combined, for example, with a combination approach for targeting multiple elements of the immune pathway, such as one or more of the following: therapy that enhances tumor antigen presentation (e.g., dendritic cell vaccines, GM-CSF-secreting cell vaccines, CpG oligonucleotides, imiquimod); therapy that inhibits negative immune regulation, for example, by inhibiting CTLA-4 and / or PD1 / PD-L1 / PD-L2 pathways and / or depleting or blocking Treg or other immunosuppressive cells; therapy that stimulates positive immune regulation, for example, using agonists that stimulate CD-137, OX-40 and / or CD40 or GITR pathways and / or stimulates T cell effector function; therapy that systemically increases the appearance of anti-tumor T cells; for example Therapies that deplete or suppress Tregs (such as Tregs in tumors) using antagonists of CD25 (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion; therapies that affect the function of suppressor myeloid cells in tumors; therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines); adaptive 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), dioxygenases, 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.

[0603] The ILT4 antibodies described herein can be used with one or more of the following: agonists that engage positive co-stimulatory receptors; blockers that attenuate signaling through inhibitory receptors; antagonists; and one or more agents that systemically increase the appearance 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 suppressing 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.

[0604] In certain embodiments, if the subject is BRAF V600 mutation positive, the subject is administered an ILT4 antibody along with a BRAF inhibitor.

[0605] PD-1 antagonists suitable for use in 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.

[0606] Exemplary anti-PD-1 antibodies are nivolumab (BMS-936558) or antibodies comprising the CDRs or variable regions of one of antibodies 17D8, 2D3, 4H1, 5C4, 7D3, 5F4, and 4A11 as described in WO 2006 / 121168. In certain embodiments, the anti-PD-1 antibody is MK-3475 (Lambrolizumab) as described in WO 2012 / 145493; AMP-514 as described in WO 2012 / 145493; or PDR001. Other known PD-1 antibodies and other PD-1 inhibitors include those described in WO 2009 / 014708, WO 03 / 099196, WO 2009 / 114335, WO 2011 / 066389, WO 2011 / 161699, WO 2012 / 145493, U.S. Patent Nos. 7,635,757 and 8,217,149, and U.S. Patent Publication No. 2009 / 0317368. Any of the anti-PD-1 antibodies disclosed in WO 2013 / 173223 may also be used. Anti-PD-L1 antibodies that compete with one of these antibodies for binding and / or bind to the same epitope on PD-1 as one of these antibodies may also be used in combination therapy.

[0607] In some embodiments, the anti-PD-L1 antibody useful in 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 No. WO 07 / 005874 and U.S. Patent No. 7,943,743. In a certain 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; WO 2013 / 79174), or rHigM12B7. Any of the anti-PD-L1 antibodies disclosed in WO2013 / 173223, WO2011 / 066389, WO2012 / 145493, U.S. Patent Nos. 7,635,757 and 8,217,149, and U.S. Publication No. 2009 / 145493 may also be used. Anti-PD-L1 antibodies that compete with and / or bind to the same antigenic determinant as any of these antibodies may also be used in combination therapy.

[0608] In certain embodiments, the ILT4 antibodies of the present invention can be used together with a CTLA-4 antagonist (e.g., 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 described in any of the following publications, or an anti-CTLA-4 antibody: WO 98 / 42752; WO 00 / 37504; U.S. Pat. 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 may also be used.

[0609] In some embodiments, the ILT4 antibodies of the present invention are used in combination with LAG3 antagonists. Examples of anti-LAG3 antibodies include antibodies comprising the CDRs or variable regions of antibodies 25F7, 26H10, 25E3, 8B7, 11F2, or 17E5, described in U.S. Patent Publication Nos. US 2011 / 0150892, WO 10 / 19570, and WO 2014 / 008218. In one embodiment, the anti-LAG-3 antibody is BMS-986016. Other art-recognized anti-LAG-3 antibodies that may be used include IMP731 and IMP-321, described in US 2011 / 007023, WO 08 / 132601, and WO 09 / 44273. Anti-LAG-3 antibodies that compete with and / or bind to the same antigenic determinant as any of these antibodies may also be used in combination therapies.

[0610] In some embodiments, the ILT4 antibodies of the present invention can be administered in combination with a CD137 (4-1BB) agonist (such as an agonist CD137 antibody). Suitable CD137 antibodies include, for example, urelumab and PF-05082566 (WO12 / 32433).

[0611] In some embodiments, an ILT4 antibody may 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).

[0612] In one embodiment, an ILT4 antibody is administered in combination with a CD40 agonist (such as an agonist CD40 antibody). In certain embodiments, the tumor immunotherapy agent is a CD40 antagonist, such as an antagonist CD40 antibody. Suitable CD40 antibodies include, for example, rukatumomab (HCD122), daclizumab (SGN-40), CP-870,893, or Chi Lob 7 / 4.

[0613] 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).

[0614] In certain embodiments, an ILT4 antibody is administered with an anti-GITR antibody, e.g., an antibody having the CDR sequences of 6C8, e.g., a humanized antibody having the CDRs of 6C8, as described, e.g., 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 referred to herein.

[0615] In some embodiments, an ILT4 antibody is administered in combination with MGA271 (directed against B7H3) (WO 11 / 109400).

[0616] In some embodiments, an ILT4 antibody is administered in combination with a KIR antagonist, such as lirelumab.

[0617] In some embodiments, an 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.

[0618] In some embodiments, an ILT4 antibody is administered in combination with a Toll-like receptor agonist, e.g., 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).

[0619] In one embodiment, ILT4 is administered in combination with a TGF-β inhibitor (eg, GC1008, LY2157299, TEW7197, or IMC-TR1).

[0620] Additional combination therapy

[0621] Ab herein can also be provided before, substantially simultaneously or after other modes of treatment (e.g., surgery, chemotherapy, radiotherapy) or administration of a biological agent (such as another therapeutic antibody). In some embodiments, cancer recurs or progresses after a therapy selected from surgery, chemotherapy and radiotherapy or a combination thereof. For example, when there is a risk of micrometastasis and / or in order to reduce the risk of recurrence, an ILT4 antibody as described herein can be administered in adjuvant therapy form.

[0622] For cancer treatment, the combination can be administered in conjunction with one or more additional anti-cancer agents, such as chemotherapeutic agents, growth inhibitory agents, anti-cancer vaccines (such as gene therapy vaccines), anti-angiogenic agents, and / or anti-neoplastic compositions. Non-limiting examples of chemotherapeutic agents, growth inhibitory agents, anti-cancer vaccines, anti-angiogenic agents, and anti-neoplastic compositions that can be used in combination with the antibodies of the invention are provided herein under "Definitions."

[0623] In some embodiments, anti-inflammatory drugs, such as steroids or nonsteroidal anti-inflammatory drugs (NSAIDs), may be administered with the combination. In cases where it is necessary to combine therapy with the ILT4 antibodies described herein or to render abnormally proliferating cells dormant prior to therapy with the antibodies, hormones and steroids (including synthetic analogs) such as 17a-ethinylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testolactone, megestrolacetate, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisone, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide, toremifene, When employing the methods or compositions described herein, other agents used in the clinical setting to modulate tumor growth or metastasis, such as antiemetics, may also be administered as needed.

[0624] The antibodies described herein can 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 can 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).

[0625] ILT4 antibodies described herein can also be combined with vaccination protocols. Many experimental strategies for vaccination against tumors have been designed (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; also see Restifo, N. and Sznol, M., Cancer Vaccines, Chapter 61, pp. 3023-3043, DeVita, V. et al. (eds.), 1997, Cancer: Principles and Practice of Oncology. Fifth Edition). In one of these strategies, autologous or allogeneic tumor cells are used to prepare vaccines. These cellular 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. USA 90:3539-43).

[0626] Studies of gene expression and large-scale gene expression patterns in various tumors have led to the definition of so-called tumor-specific antigens (Rosenberg, SA (1999) Immunity 10: 281-7). In many cases, these tumor-specific antigens are differentiation antigens expressed in tumors and in cells from which tumors arise, such as the melanocyte antigen gp100, MAGE antigens, and Trp-2. More importantly, it can be shown that many of these antigens are targets of tumor-specific T cells found in the host. ILT4 inhibition can be used in conjunction with a panel of recombinant proteins and / or peptides expressed in tumors to generate an immune response against these proteins. These proteins are typically observed by the immune system as self-antigens and are therefore tolerant to them. Tumor antigens may include the protein telomerase, which is required for the synthesis of telomeres of chromosomes and is expressed in more than 85% of human cancers and is only expressed 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 create a fusion protein between two unrelated sequences (i.e., bcr-abl in the Philadelphia chromosome) or are idiotypes from B-cell tumors.

[0627] Other tumor vaccines may include proteins from viruses implicated in human cancers, such as human papillomavirus (HPV), hepatitis viruses (HBV and HCV), and Kaposi's herpes sarcoma virus (KHSV). Another form of tumor-specific antigen that can be used in conjunction with ILT4 Ab is purified heat shock proteins (HSPs) 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 elicit tumor immunity (Suot and Srivastava (1995) Science 269:1585-1588; Tamura et al. (1997) Science 278:117-120).

[0628] Dendritic cells (DCs) are powerful antigen-presenting cells that can be used to elicit antigen-specific responses. DCs can be produced in vitro and loaded with various protein and peptide antigens and tumor cell extracts (Nestle et al. (1998) Nature Medicine 4:328-332). DCs can also be transduced by genetic methods to express these tumor antigens. DCs have also been directly fused with tumor cells for the purpose of immunity (Kugler et al., (2000) Nature Medicine 6:332-336). As a vaccination method, DC immunity can be combined with ILT4 inhibition to activate a more powerful anti-tumor response.

[0629] Infectious disease treatment

[0630] The methods described herein can also be used to treat patients who have been exposed to specific toxins or pathogens. Therefore, the present invention also encompasses methods for treating infectious diseases in subjects, comprising administering antibodies as described herein, such as ILT4 antibodies, to subjects to treat infectious diseases in subjects. Similar to its application for tumors as discussed above, antibody-mediated ILT4 inhibition can be used alone or in adjuvant form in combination with vaccines to stimulate immune responses to pathogens, toxins, and self-antigens. Examples of pathogens for which this treatment method may be particularly useful include pathogens for which there are currently no effective vaccines, or pathogens 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 lamblia (Giardia), malaria, Leishmania (Leishmania), Staphylococcus aureus (Staphylococcusaureus), Pseudomonas aeruginosa (Pseudomonas Aeruginosa). ILT4 inhibition can be used to resist infections determined by agents such as HIV that present altered antigens during infection.

[0631] Some examples of pathogenic viruses that cause infections that can be treated by the methods described herein include HIV, hepatitis (A, B, or C), herpes viruses (e.g., 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, virus), poliovirus, rabiesvirus, JC virus, and arboviral encephalitis virus.

[0632] 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.

[0633] Some examples of pathogenic fungi that cause infections treatable by the methods described herein include Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Mucorales (mucor, absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0634] 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.

[0635] In all of the above approaches, ILT4 inhibition can be combined with other forms of immunotherapy, e.g., 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).

[0636] Administration routes and carriers

[0637] In various embodiments, the antibodies can be administered in vivo by a variety of routes, including, but not limited to, intravenous (iv), subcutaneous, oral, intraarterial, parenteral, intranasal, intramuscular, intracardial, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or alternatively by implantation or inhalation. The compositions of the present invention can be formulated into preparations in solid, semisolid, liquid, or gaseous form; including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and sprays. Nucleic acid molecules encoding antibodies can be coated onto gold microparticles and delivered intradermally by particle bombardment devices or "gene guns" as described in the literature (see, for example, Tang et al., Nature 356:152-154 (1992)). The appropriate formulation and route of administration can be selected according to the intended application.

[0638] In various embodiments, the compositions comprising the antibodies are provided in formulations together with a variety of pharmaceutically acceptable carriers (see, for example, 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)). Various pharmaceutically acceptable carriers, including vehicles, adjuvants, and diluents, can be used. In addition, various pharmaceutically acceptable auxiliary substances, such as pH adjustment and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like, can also be used. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0639] In various embodiments, compositions comprising antibodies can be formulated for injection (including subcutaneous administration) by dissolving, suspending or emulsifying in aqueous or non-aqueous solvents, such as vegetable oils or other oils, synthetic aliphatic acid glycerides, esters of higher carbon number aliphatic acids, or propylene glycol; and conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives are optionally used. 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 acid-glycolic acid polymers. Non-limiting exemplary non-biodegradable formulations include polyglycerol fatty acid esters. Certain methods for preparing such formulations are described in, for example, EP 1 125 584A1.

[0640] Also provided are pharmaceutical packages and kits comprising one or more containers, each containing one or more doses of an antibody or antibody combination. In some embodiments, a unit dosage form is 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 prefilled syringes for injection. In various embodiments, the composition contained in the unit dosage form may comprise saline, sucrose, or an analog thereof; a buffer, such as phosphate or an analog thereof; 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 an appropriate 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 composition of the present invention comprises heparin and / or proteoglycans.

[0641] The pharmaceutical composition is administered in an amount effective to treat or prevent a specific indication. The therapeutically effective amount generally depends on the weight of the treated subject, its physiology or health status, the extension of the treated condition, or the age of the treated subject. Generally speaking, the antibody can be administered in an amount within 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 within 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 within 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 within 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 within the range of about 0.5 mg / kg body weight to about 20 mg / kg body weight per administration.

[0642] When needed, the antibody composition can be administered to the subject. The frequency of administration can be determined by those skilled in the art, such as an attending physician, based on the considerations of 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 antibody is administered to the subject one or more times. In various embodiments, an effective dose of 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 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 antibody is administered once every 1, 2, 3, 4, or 5 weeks. In some embodiments, an effective dose of antibody is administered twice or three times a week. An effective dose of antibody is administered to the subject at least once. In some embodiments, an effective dose of antibody can be administered multiple times, including a time period that lasts at least one month, at least six months, or at least one year.

[0643] In certain embodiments, the combination of an ILT4 antibody and a second agent discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as a separate composition containing an ILT4 antibody and a second agent in a pharmaceutically acceptable carrier. In one embodiment, the combination of an ILT4 antibody and a second agent can be administered sequentially. The administration of the two agents can be started, 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 started, 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.

[0644] Example

[0645] The examples discussed below are intended only to illustrate the present invention and should not be construed as limiting the present invention in any way. These examples are not intended to represent that the following experiments are all or only experiments performed. Efforts have been made to ensure accuracy with respect to the quantities (e.g., amounts, temperatures, etc.) used, but some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0646] Example 1: Generation of antibodies that bind to human ILT4

[0647] Transgenic mice expressing human antibody genes were used to generate anti-human ILT4 monoclonal antibodies (ILT4 antibodies or hILT4 antibodies). Fully human monoclonal antibodies against human ILT4 were generated by immunizing two types of human immunoglobulin transgenic animals. KM [M / K] mice were immunized with hILT4-mFc recombinant protein consisting of the extracellular portion of hILT4 and a C-terminal mouse Fc tag. The antigen was mixed with Ribi adjuvant in a 1:1 ratio, and the mice were immunized intraperitoneally and subcutaneously every other week. The HCo42:01 [J / K] strain was immunized with hILT4-his tagged recombinant protein consisting of the extracellular portion of hILT4 and a C-terminal his tag. The antigen was mixed with Ribi adjuvant in a 1:1 ratio, and the mice were immunized in the paw pad every other week. Serum titers of both types of mice were monitored after four and six injections. Mice received a final booster immunization before the final harvest. Depending on the immunization route, draining lymph nodes and spleens were harvested for subsequent fusion.

[0648] Mouse lymphocytes were isolated from immune mice. Fusion tumors were produced by fusion with mouse myeloma fusion partners using Cyto Pulse Hybrimmune large chamber cell fusion electroporator (BTX / Harvard Apparatus) by electric fusion based on electric field. The single cell suspension of lymphocytes from immune mice was fused with equivalent P3X63 Ag8.6.53 (ATCC) non-secretory mouse myeloma cells (fusion numbering 4865 and 6951). The resulting cells were plated in a flat microtiter plate in the culture medium E (StemCell Technologies) supplemented with aminopterin to select fusoma (Sigma).

[0649] After 10 to 12 days of culture, individual wells were screened for the presence of human IgG / human kappa light chain (hIgG / hκ) antibodies using a homogenous HTRF assay. Fusion tumor supernatants from wells positive for hIgG / hκ were tested by FACS or FMAT for binding to hILT4-transfected cells or control CHO cells. The following antibodies that specifically interacted with hILT4 were further characterized: 9C8.A6, 24E5.A7, 2H2.H3, 21D9.H11, 2E5.A11, and 9G4. Antibodies 21A5 and 10F10 were isolated from later immunizations.

[0650] The antibodies from the hybridomas were sequenced using NGS high-throughput sequencing methods. Briefly, for each hybridoma clone in the wells of a 96-well plate, PCR amplification of the VH and VL regions was performed using a unique DNA barcode to identify each well. The 5' PCR primer was hybridized to the leader region to obtain the entire variable region sequence. The specific hybridoma antibody sequence was identified by matching the barcode with the position of the clone on the plate.

[0651] 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 Figures 1 to 12 The position of each CDR is provided in the Figures and indicated by underlining in the variable region sequences in the Sequence Listing, and the CDRs are also included as individual SEQ ID NOs near the end of the Sequence Listing.

[0652] Analysis of the amino acid sequences of the variable domains of antibodies 21D9, 21A5, 2H2 and 10F10 indicated that certain framework amino acid residues are not present in any human germline. To reduce potential immunogenicity issues when these antibodies are administered to humans, germline revertants of these ILT4 antibodies were generated.

[0653] For antibody 21D9, identify four framework mutations (V2G, G10D, A24T and V48I) relative to germline sequence in the variable region of heavy chain and be shown in Figure 13.In the various combinations of these 4 back mutations, produce four kinds of back mutants (21D9.b, 21D9.c, 21D9.d and 21D9.e) (referring to Figure 13) and for being tested in combination with hILT4.All 4 back mutants all comprise N end EGQ to EVQ (or G2V) sudden change, and it is unlikely to affect the combination with ILT4.Except this replacement, mutant 21D9.b, 21D9.c and 21D9.d each comprise one kind (D10G in the additional replacement shown in Figure 13; I24A; And / or I48V), and 21D9.e comprises all three replacements (referring to Figure 13). Four mutants were generated in the context of an IgG1.3 heavy chain constant region containing an IgG1 constant region with amino acid substitutions L234A, L235E, and G237A to reduce effector function. These mutants were referred to as 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 comprise the light chain of 21D9. Germline reversion was not performed on the light chain.

[0654] 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 reversion in the kappa light chain shown at A3V is unlikely to affect binding, a mutant (21A5.a with the 21A5.1 light chain) containing both the heavy chain reversion mutation (I70T) and the light chain reversion mutation (A3V) was generated and tested in the context of IgG1.3 (21A5.a.IgG1.3, also referred to as "21A5.VH-I70T.VK-A3V.IgG1.3").

[0655] For antibody 10F10, two framework mutations (Y36F and S63T) were identified in the light chain variable region and are shown in Figure 15 No framework mutations were found in the heavy chain variable region. Since the F to Y reversion at position 36 was a minor difference, a double mutant (10F10.3) comprising both the light chain reversion mutations F36Y and T63S was generated and tested in the context of IgG1.3 (10F10.3.IgG1.3, also referred to as "10F10.VK-F36Y / T63S.IgG1.3"). A mutant having only the reversion mutation F36Y was also generated and tested in the context of IgG1.3 (10F10.1.IgG1.3, also referred to as "10F10.VK-F36Y.IgG1.3"). Both reversion mutant Abs comprised the heavy chain of 10F10. Table 1 lists alternative names for the recombinant antibodies described herein.

[0656] Table 1: Alternative names for recombinant anti-hILT4 antibodies

[0657] 9G4.IgG1.1 ILT4.1.IgG1.1 9C8.IgG1.1 ILT4.2.IgG1.1 2H2.IgG1.1 ILT4.3.IgG1.1 2E5.IgG1.1 ILT4.4.IgG1.1 24E5.IgG1.1 ILT4.5.IgG1.1 21D9.IgG1.1 ILT4.6.IgG1.1 21D9.IgG1.3 ILT4.6.IgG1.3 21D9.e.IgG1.3 ILT4.8.IgG1.3 21A5.a.IgG1.3 ILT4.9.IgG1.3 10F10.IgG1.3 ILT4.10.IgG1.3 10F10.3.IgG1.3 ILT4.11.IgG1.3

[0658] Example 2: Binding of anti-hILT4 antibodies to soluble hILT4

[0659] use The binding kinetics of the parental antibodies and germline revertant anti-hILT4 antibodies (in the case of IgG1.3) were measured using a T200 instrument via surface plasmon resonance (SPR). The assay 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) with pre-immobilized 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 analysis: for 10F10.1 and 10F10.3a, a 3-fold dilution series with a maximum hILT4 concentration of 1.4 μM was analyzed. For 10F10, two independent hILT4 dilution series were analyzed: a maximum hILT4 concentration of 1.4 μM, a 5-fold dilution, and a 3-fold dilution of 470 nM. For all other antibodies, a maximum hILT4 concentration of 94 nM and a 5-fold dilution series were used. The hILT4 used in this experiment, comprising the extracellular region of hILT4 linked to a His-Avi tag, consists of the following amino acid sequence: QTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEEEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPGVSKKPSLSVQPGPVVAPGESLTLQCVS DVGYDRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSESSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAGAADAPLRLRSIHEYPKYQAEFPMS PVTSAHAGTYRCYGSLNSDPYLLSHPSEPLELVVSSGPSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGSPGGGSGGGSEQKLISEEDLGHHHHHHGLNDIFEAQKIEWHE(hILT4-His-Avi tag; SEQ ID NO:119).

[0660] The results are shown in Table 2 and Figure 16 middle.

[0661] Table 2: Binding kinetics of anti-hILT4 antibodies to hILT4

[0662]

[0663]

[0664] The results showed that the four revertants of 21D9 had similar kinetics and affinities. 21A5 and its revertant 21A5.a also had similar kinetics and affinities. However, the two 10F10 revertants 10F10.1 and 10F10.3 had slightly faster off-rates than their parent 10F10, resulting in approximately a 3-fold loss of overall affinity. The off-rates of 10F10.1 and 10F10.3 were too fast to be determined with high confidence.

[0665] Example 3: Binding of anti-hILT4 antibodies to hILT4-expressing cells

[0666] This example describes the binding characteristics of anti-hILT4 antibodies to hILT4-transfected CHO cells and to hILT4-expressing human monocytes.

[0667] 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, followed by treatment with a PE-conjugated secondary anti-human IgG. EC was determined using FACS analysis for geometric mean fluorescence intensity (GMFI). 50 .

[0668] Binding curves and EC values ​​are shown in Figure 17 and Figure 18A middle.

[0669] exist Figure 18B In Figure 2, mononuclear cells from peripheral blood of normal healthy donors were stained with individual anti-ILT4 antibodies conjugated with Alexa 67. Antibodies were titrated from 20 μg / mL by 3-fold serial dilution, and GMFI was determined by FACS analysis for calculation of EC50 (shown in the table below the graph).

[0670] Example 4: Selectivity of anti-ILT4 antibodies against LILRA / B family members

[0671] ILT4 is a member of a family of related receptors also known as the LILRA and LILRB families. In this example, binding of hILT4 antibodies to various LILRA / LILRB family members was measured.

[0672] Individual ILT family members were overexpressed in 293T cells. Each transfectant was stained with 20 μg / ml of anti-ILT4 antibody followed by 1 μg / ml of anti-human IgG secondary mAb. Antibody binding to each transfectant was measured by flow cytometry. In a second experiment, LILRA1, LILRA3, and ILT4 transfectants were incubated with various concentrations of 21A5 and 21A5.a.

[0673] Shown in Figure 19A and Figure 19B As shown in Table 3, the anti-hILT4 antibodies selectively bind primarily to hILT4 relative to other LILRA and LILRB family members. LILRA3 is the only secreted protein in the LILRA / B family. More specifically, 21D9 and 21D9.e do not significantly bind to LILRA1, LILRA2, LILRA3, LILRA4, LILRA6, ILT2, ILT3, ILT5, and LIR8, and only weakly bind to LILRA5.

[0674] Table 3: Selectivity profile of anti-ILT4 antibodies at 20 μg / ml

[0675] LILRA1 LILRA2 LILRA3 LILRA4 LILRA5 LILRA6 ILT2 ILT3 ILT4 ILT5 LIR8 Parents 21D9 + +++ 21D9.e + +++ 21A5 +++ +++ 21A5.a ++ +++ 2H2 +++ 10F10 +++

[0676] "Parental" in Table 3 refers to the parental cell line expressing the LILRA or LILRB molecule.

[0677] Cross-reactivity against other family members of LILRA was further tested as follows. Antibody titration flow cytometric binding assays were performed to determine the binding potency of 21D9, 21D9.e, 21A5, and 21A5.a to hILT4 and their corresponding cross-reactive ILT molecules. EC determination using the nonlinear regression formula of the software 50 .

[0678] The results presented in Table 4 and Figure 20 show that the anti-ILT4 antibodies 21D9, 21D9.e, 21A5 and 21A5.a showed weaker cross-reactivity with other family members of LILRA: 21D9 and 21D9.e with LILRA5, and 21A5 and 21A5.a with LILRA1 and LILRA3.

[0679] Table 4 - ECs of anti-ILT4 antibodies cross-reacting with ILT 50 Summary of Binding Potency (μg / ml)

[0680] 21D9 21D9.e 21A5 21A5.a ILT4 0.0460 0.039 0.091 0.085 LILRA5 ～29964 ～312.6 No binding No binding LILRA1 No binding No binding 12.53 49.82 LILRA3 No binding No binding 9.18 52.94

[0681] 21D9 and 21D9.e bind to hILT4 with high affinity, but do not bind significantly 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.

[0682] Example 5: Anti-hILT4 Antibodies Enhance T Cell Responses

[0683] The effects of anti-hILT4 antibodies on T cell responses, particularly T cell proliferation and interferon gamma (IFNγ) T cell secretion, were determined as follows. Cytoplasmic truncated hILT4 was transfected into CHO cells (CHO-OKT3 cells) expressing low levels of single-chain variable fragments of anti-CD3 antibodies. The transfected cell line was called 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. The supernatant was harvested to assess IFNγ levels by ELISA, and the culture plates were plated with 3 H-thymidine pulses were performed overnight to assess proliferation by thymidine incorporation.

[0684] Shown in 21A to 21D The results in indicate that the anti-ILT4 antibodies 21D9, 21D9.e, 21A5, 21A5.a and 10F10 stimulate T cell proliferation and IFNγ secretion in a dose-dependent manner. No significant differences in activity were found between these antibodies in this assay.

[0685] Example 6: Pretreatment of monocytes with anti-ILT4 antibodies during differentiation generates monocyte-derived dendritic cells (MoDCs) that are more stimulatory to allogeneic T cells

[0686] use 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.

[0687] 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. Figure 22A and Figure 22C ) and IFNγ secretion ( Figure 22B and Figure 22D ) enhancement. Monocytes from two different donors were used in separate assays.

[0688] 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

[0689] 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.

[0690] The results shown in Figure 23 indicate that when anti-ILT4 antibodies are present during monocyte differentiation into Mo-iDC, the expression of CD86 and CD83 (co-stimulatory and maturation molecules, respectively) on Mo-iDC is increased. These results, together with the results of Example 6, show that treatment of monocytes with anti-hILT4 antibodies during monocyte differentiation into MoDC promotes pro-inflammatory ( 23A to 23D ), which thus triggers T cell proliferation and IFNγ production from allogeneic T cells in an allogeneic MLR assay ( FIG. 22 ). By enhancing T cell function, treatment with anti-hILT4 antibodies can provide an enhanced anti-cancer immune response.

[0691] Example 8: Anti-ILT4 antibodies enhance TNFα secretion from in vitro differentiated macrophages

[0692] Monocytes isolated from human PBMCs were differentiated into macrophages in the presence of M-CSF for 5 days. Figure 24A ) or 5 μg / ml of 2'3'-cGAMP STING agonist ( Figure 24B Macrophages were treated with the indicated concentrations (next to their names and in ng / ml) of anti-ILT4 antibodies 21D9, 21D9.e, 21A5, 21A5.a, 2H2, and 10F10, or an isotype control, in the presence of TNFα. TNFα production was measured by ELISA 24 hours after treatment, and the EC50 values ​​in the presence of each antibody are indicated next to the curves.

[0693] 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 by in vitro differentiated macrophages in the presence of LPS or a STING agonist. Thus, immunosuppressive macrophages exhibit a shift toward a more proinflammatory phenotype when ILT4 is blocked, potentially providing an enhanced anti-cancer immune response. These results suggest that anti-ILT4 antibodies promote proinflammatory polarization toward M1 macrophages.

[0694] Example 9: Anti-ILT4 Antibodies Promote CD4 T Cell Responses in Macrophage:CD4+ T Cell Allogeneic MLR

[0695] Monocytes isolated from peripheral PBMCs were treated with M-CSF for 5 days to differentiate into macrophages. Macrophages were then isolated from PBMCs from different donors and co-cultured with allogeneic CD4+T cells labeled with CFSE dye. 1 μg / ml of indicator antibodies (isotype control, anti-ILT4 or anti-PDL1) were included in the allogeneic MLR co-culture. On the 6th day, supernatants were collected for analysis of IFNγ and D4+T cell proliferation and assessed by FACS based on CFSE dilution.

[0696] Shown in FIG. 25A to FIG. 25B The results in Figure 4 indicate that the presence of anti-ILT4 antibodies 21D9, 21D9.e, 21A5, or 21A5.a in an allogeneic MLR of in vitro differentiated macrophages and CD4+ T cells promotes CD4+ T cell proliferation ( Figure 25A ) and IFNγ production ( Figure 25B These results suggest that anti-ILT4 antibodies promote T cell activation by modulating ILT4 inhibitory signaling in macrophages or blocking its direct inhibitory activity in T cells.

[0697] Example 10: Combination Effect of Anti-ILT4 and Anti-PD-L1 in Macrophage:CD4 T Cell Allogeneic MLR

[0698] 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. Allogeneic MLR co-culture included anti-ILT4 antibody 21D9 and anti-PD-L1 antibodies (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, supernatants were collected for analysis of IFNγ.

[0699] The results shown in FIG26 indicate that the combination of anti-ILT4 antibody 21D9 and anti-PD-L1 antibody stimulates enhanced production of IFNγ from macrophage:CD4+ T cell allogeneic MLR relative to each antibody alone.

[0700] Example 11: HDX epitope mapping of anti-hILT4 Ab 21D9 and 2H2 Fab

[0701] HDX-MS was used to identify the regions of hILT4 to which the anti-hILT4 antibodies 21D9 and 2H2 bound.

[0702] HDX-MS probes protein conformation and conformational dynamics in solution by monitoring the rate and extent 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 level of HDX depends on the solvent accessibility of backbone amide hydrogen atoms and protein hydrogen bonding. The mass increase of proteins after HDX can be precisely measured by MS. When this technique is paired with enzymatic digestion, structural features can be resolved at the peptide level, distinguishing surface-exposed peptides from those that are internally folded or sequestered at the interface of protein-protein complexes. Typically, deuterium labeling and subsequent quenching experiments are performed, followed by enzymatic digestion, peptide separation, and MS analysis.

[0703] Prior to the epitope mapping experiments, deuterated experiments were performed to generate a list of consensus peptides for recombinant human ILT4 (15 μM) and protein complexes of ILT4 with the Fabs 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. Reactions were run for different time periods: 1 min, 10 min, and 240 min. At the end of each labeling reaction period, the reaction was 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 a Waters HDX-MS system for analysis. Deuterium uptake of the common peptic peptides was monitored in the absence and presence of the Fabs 21D9 and 2H2.

[0704] Human ILT4 (hILT4) was histidine-tagged and mixed with 21D9 or 2H2 Fab at a 1:1 ratio and subjected to HDX for 1 minute, 10 minutes, or 4 hours. Sequence coverage of hILT4 by pepsin is shown in Figure 27A The region of hILT4 (or hILT4 peptide) that binds to each Fab is shown in Figure 27B and Figure 27C Specifically, HDX-MS experiments provided 85% sequence coverage of human ILT4.

[0705] Analysis of HDX-MS data of 21D9 and 2H2 in human ILT4 indicated that the antigenic determinant of 21D9 is composed of one region of human ILT4 (residue numbers correspond to the native human ILT4 sequence): a region in Ig domain 2: 70ITRIRPELVKNGQFHIPSITWEHT GRYGCQY 100 (SEQ ID NO: 122). (See Figure 27B , above.)

[0706] Analysis of HDX-MS data indicated that the epitope of 2H2 is composed of five regions of human ILT4, with regions 2 and 5 being the major epitopes:

[0707] Region 1: 127 SAQPSPVVTSGGRVTL 142 (SEQ ID NO: 173)

[0708] Region 2: 154 ILCKEGEEEHPQCLNSQPHARGSSRAIF 181 (SEQ ID NO: 123)

[0709] Region 3: 182 SVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDL 213 (SEQ ID NO: 174)

[0710] Region 4: 378 QAEFPMSPVTSAHAG 392 (SEQ ID NO: 175)

[0711] Region 5: 425 SSPPPTGPIS 434 (SEQ ID NO: 124)

[0712] (See Figure 27B , below.)

[0713] Example 12: Anti-ILT4 antibodies block the binding of ILT4 to class I MHC molecules

[0714] A flow cytometry-based antibody blocking assay was performed to determine the ability of anti-ILT4 antibodies to block rhILT4-Fc binding to CHO cells overexpressing HLA-A or HLA-B. An rhILT4-Fc fusion protein containing a murine Fc tail was generated. Anti-hILT4 antibodies 21D9, 2H2, 10F10, and 21A5 were titrated in the presence of rhILT4-Fc at a fixed concentration of 30 μg / ml. 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 bound rhILT4-Fc.

[0715] Shown in 28A to 28D The results in indicate that antibodies 21D9, 2H2, 10F10, and 21A5 inhibited the binding of hILT4 to HLA-A and HLA-B, respectively.

[0716] Example 13: 21D9e.IgG1.3T enhances both IFN-γ and TNF-α secretion by T cells

[0717] This example demonstrates that 21D9e.IgG1.3 enhances IFN-γ and TNF-α secretion by T cells in an autologous mixed lymphocyte reaction (MLR) of monocytes and T cells.

[0718] T cells and monocytes were isolated from peripheral blood mononuclear cells (PBMCs) 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 ug / 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 The cells were acquired using a cell counter (Beckman Coulter) and analyzed using FlowJo TM Software (Tree Star, Inc, Ashland, OR) was used for analysis.

[0719] Shown in 29A to 29D The results in indicate that 21D9e.IgG1.3 enhances both IFN-γ and TNF-α secretion by CD4+ and CD8+ T cells after anti-CD3 stimulation in autologous allogeneic MLR.

[0720] Example 14: Anti-ILT4 Antibodies Enhance IFN-γ Secretion Following Antigen Stimulation in a CMV Lysate Assay

[0721] Peripheral blood mononuclear cells (PBMCs) from CMV reactive donors were plated at 200,000 cells / well in the presence of 0.3 μg / mL 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. Supernatant from each sample was harvested and IFN-γ levels were measured by ELISA (human IFN-γ ELISA kit, BD OptEIA Cat. 555142) after 6 days of incubation.

[0722] Shown in Figure 30 The results in indicate that the anti-ILT4 antibodies 21D9.IgG1.3, 21D9-Fab, and 21A5.IgG1.3 enhance IFN-γ secretion after antigen stimulation in a CMV lysate assay.

[0723] Example 15: ILT4 antagonism enhances T cell activation in monocyte:T allogeneic MLR

[0724] T cells (100,000) from one donor and allogeneic monocytes were co-cultured in a 96-well U-bottom culture plate at a ratio of 2:1 per well. Anti-ILT4 antibodies 2H2.IgG1.1f, 2H2 Fab, 21D9.IgG1.1f, and 21D9 Fab were added to a 4-fold, 7-point titration starting at a concentration of 30 μg / mL for full-length antibodies or 80 μg / mL for Fab antibodies. Anti-KLH-g1.1f (anti-hemocyanin antibody) was added at 30 μg / mL as an isotype control. The cell cultures were incubated for 6 days. During the final 16 hours of culture, the cells were incubated with 1% paraformaldehyde (PNA) for 24 h. 3 Cell proliferation was assessed by H-thymidine incorporation.

[0725] Shown in Figure 31 The results in indicate that both the full-length anti-ILT4 antibodies 21D9.IgG1.1f and 2H2.IgG1.1f and their corresponding Fab fragments induced T cell proliferation.

[0726] Example 16: Binding of anti-hILT4 antibodies to cynomolgus monkey ILT4

[0727] Several ILT family members were obtained from cynomolgus monkeys via PCR. Since sequence analysis of these proteins did not clearly indicate a close match to hILT4, expression profiling and functional assays were performed. These assays identified a clone named "9152" as the closest match to hILT4 in terms of expression profiling and functional properties. The amino acid sequence of the extracellular region of 9152, including the His-Avi tag, is as follows:

[0728] QAGILPKPMLWAEPDRVITQGSPVTLRCQGNLEARGYHLYRERKSASWITLIRPELVKKGQFPIPSITWEDAGRYRCQYYSHSWWSEHSDPLELVVTGAYRKPTLSALPSPVVASGGNVTLQCDSRVALDGFILCKEGEDEHSQRLNSQPRTRGSSRAVFSVGPVSPSRRWSYRCYGYELHSRYVWS LPSDLLELLVPGVSKKPSLSVQPGPVVAGGDKLTLQCGSDAGYDRFALYKEGERDFLQRPGQQLQAGLAQANFTLDPVRGSHGGQYRCYGAHNLSSEWSAPSDPLDILISAGPHSGLRRECDPAVSVTGMDGHFLSDQGGSSSPGGGSGGGSEQKLISEEDLGHHHHHHGLNDIFEAQKIEWHE(SEQ ID NO:118). For binding to hILT4, the extracellular domain (ECD) of the hILT4-His-Avi tag described in Example 2 was used; SEQ ID NO: 119.

[0729] The kinetics of the 21D9.e antibody binding to cynomolgus monkey and human ILT4 using the above ILT4 constructs were analyzed in a comparative SPR assay. The antibody was amine-coupled to a GLC sensor chip in 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) using 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.

[0730] The results shown in Table 5 show that 21D9.e binds to cynomolgus monkey ILT4 9152 with approximately 11-fold weaker affinity than human ILT4 (K D The value is 3.5 nM, and the K D The value was 0.33 nM). Similarly, the parent antibody 21D9 bound to cynomolgus monkey ILT4 with approximately 12-fold weaker affinity than human ILT4. The difference was caused by the faster dissociation rate constant of cynomolgus monkey ILT4 (K D The value is 4.0 nM, and the K D value is 0.32 nM).

[0731] Table 5

[0732]

[0733] Example 17: ILT4 Antibody Promotes the Expression of Costimulatory Molecules and Activation Markers on Cynomolgus Monkey Monocyte-Derived Dendritic Cells in Vitro

[0734] This example shows that the ILT4 antibodies 21D9.e.IgG1.3 (ILT4.8 in FIG. 32 ; see Table 1 ) and 9G4.hIgG1.3 (ILT4.1 in FIG. 32 ; see Table 1 ) stimulate the expression of CD80, CD83, and CD86 on monocyte-derived dendritic cells.

[0735] Monocytes isolated from cynomolgus macaque PBMC (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). Anti-ILT4 antibody was added to the culture at 10 ug / mL when the culture was set up. 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 taken at 4 °C for 24 h. The cells were acquired using a cell counter (Beckman Coulter) and analyzed using FlowJo TM Software (Tree Star, Inc, Ashland, OR) was used for analysis.

[0736] Shown in Figures 32A to 32CThe results in indicate that ILT4 antibodies 21D9.e.IgG1.3 (also known as ILT4.8.IgG1.3; "ILT4.8" in the figure) and 9G4.IgG1.3 (also known as ILT4.1.IgG1.3; "ILT4.1" in the figure) promote the expression of co-stimulatory molecules CD80 and CD86 and the dendritic cell maturation marker CD83 in monocyte-derived DCs, as demonstrated by upregulation of CD80, CD83 and CD86 on dendritic cells.

[0737] Example 18: Modified anti-ILT4 antibody 10F10.4

[0738] The tyrosine in F36Y in the light chains of 10F10.1 and 10F10.3 (see Example 2) is located near the heavy chain CDR3 in the modeling of hILT4 and 10F10 antibodies. Therefore, replacing F36 with Y can cause interference with the isoleucine in the heavy chain CDR3, as demonstrated by the faster dissociation rates 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 therefore the heavy chain of 10F10. This modified antibody is designated 10F10.4, and the amino acid sequence of its light chain is listed in the sequence listing as SEQ ID NO: 116.

[0739] Use at 37°C Binding kinetics of 10F10.4 were determined on a T200 instrument. The running buffer consisted of 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 pre-immobilized anti-human Fc capture antibody from Southern Biotech (Cat. No. 2081-01). Human ILT4 (hILT4-His-Avi tag described in Example 2) was injected at 1.2 μM (for 10F10, 10F10.4, and one replicate of 1E5 ILT4.15), 405 nM, 81 nM (in duplicate), 16 nM, 3 nM, and 0.6 nM. All data were double-referenced and fitted to a 1:1 Langmuir model using mass transport using Biacore T200 Evaluation Software version 3.1.

[0740] The results shown in Table 6 indicate that the binding kinetics of 10F10.4 are very similar to those of 10F10. 21A5, 21A5a, 21D9, and 21D9.e were included as controls. The absolute values ​​differ slightly from those shown in Table 2 due to the use of different capture reagents in the assay.

[0741] Table 6

[0742]

[0743] 10F10.4 was shown to be functionally active by enhancing TNFα secretion from in vitro differentiated macrophages. This was confirmed by incubating 10F10.4 with macrophages differentiated from PBMC monocytes as described in Example 8. 21D9.e and isotype controls were also included. Figure 33A and Figure 33B The results in indicate that the presence of the ILT4 antibody 10F10.4 enhances TNFα secretion during in vitro differentiation of monocytes into macrophages by LPS or a STING agonist.

[0744] Example 19: 21D9.e has similar potency in the context of IgG1, IgG4, and IgG1.3

[0745] This example demonstrates that the potency of 21D9.e is similar in the context of IgG1, IgG4(S228P), and IgG1.3 heavy chains.

[0746] The 21D9.e antibody was cloned in the context of IgGl, IgG4 (with S228P), or IgGl.3 (SEQ ID NOs: 176, 178, and 13, respectively) and used in an assay measuring TNFa secretion as follows.

[0747] Shown in Figure 34 The results in indicate that similar amounts of TNFα are secreted for 21D9.e.IgG1, 21D9.e.IgG4.S228P, and 21D9.e.IgG1.3. Thus, the 21D9.e variable region has similar functional potency in the context of these different Fc regions. DT-1D12 is an isotype control.

[0748] Example 20: 21D9e Binding to the hILT4 Allele

[0749] This example describes an assay used to determine whether 21D9.e binds to alternative alleles of hILT4.

[0750] Only those alleles with an occurrence rate exceeding 1% were considered. Further consideration was given to 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 the 3D modeling showed that the two amino acid residues were not close to the binding interface with class I MHC, it was expected that the amino acid changes at these residues would 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 tested for binding to 21D9.e.IgG1.3 and 21A5.a.IgG1.3 (ILT4.9.IgG1.3; see Table 1) via Octet BLI. It was found that both proteins bound similarly to hILT4 E161D / R103H and hILT4 E161 / R103.

[0751] Example 21: 21D9e.IgG1.3 does not significantly trigger basophil activation

[0752] The assay for detecting basophil activation by anti-ILT4 antibodies involves the following steps. The assay was performed using the Buhlmann Laboratory AG method in the basophil activation test. Human basophils derived from donor blood were detected using anti-Ccr3. CD63 was used as an indicator of basophil activation. Stimulation buffer and antibody were added to whole blood. Following incubation, a staining reagent was added to the blood; and following antibody incubation, red blood cells were lysed and the samples were centrifuged, washed, and analyzed on a BD FACS Canto II.

[0753] Based on this assay, Ccr3+CD63+ activated basophils from 8 donors were incubated 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 and measured by FACS. Positive controls included anti-FcεRI and fMLP (N-formylmethionine-leucyl-phenylalanine). Negative controls were DT1DT12-IgG1.3, which targets an unrelated antigen, diphtheria toxin.

[0754] Shown in Figure 36The results in indicate 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 conditions tested. The lack of basophil activation represents an important safety feature.

[0755] Example 22: Anti-ILT4 antibody 21D9.e.IgG1.3 is stable

[0756] This example describes a 3-month stability study conducted with the anti-ILT4 antibody 21D9.e.IgG1.3 (also known as ILT4.8.IgG1.3), the data of which show that the antibody is stable for at least 3 months.

[0757] Samples of 21D9.e.IgG1.3 were incubated at a concentration of 150 mg / mL at either 25°C or 40°C. The samples were diluted to 1 mg / mL with a mobile phase (40 mM NaH2PO4, 60 mM Na2HPO4, 0.1 M Na2SO4, pH 6.8) and 40 microliters were analyzed using an Agilent 1200 Series HPLC system (Agilent Technologies, Santa Clara, CA) using a TSKgel Super3000SW column (4.6 mm ID x 30 cm, 4 μm, Tosoh Bioscience, King of Prussia, PA) with an isocratic gradient (0.1 M sodium phosphate, 0.1 M sodium sulfate, pH 6.8, at 0.2 mL / min). UV detection was set at 280 nM.

[0758] Shown in Figure 35 The results in indicate that the 21D9.e.IgG1.3(ILT4.8) antibody composition contained less than 5% of high and low molecular weight species after 3 months incubation at 25°C and less than 10% of high and low molecular weight species after 3 months incubation at 40°C.

[0759] Example 23: Cross-blocking of 2H2 and 21D9 by anti-ILT4 monoclonal antibodies

[0760] 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.

[0761] The results are shown in Figure 37A (2H2 cross-blocking) and Figure 37B (21D9 cross-blocks). The results indicate that 10F10 and 21A5 cross-block (or compete with) 2H2 binding to hILT4 ( Figure 37A ), and 10F10, but not 21A5, cross-blocks (or competes with) 21D9 binding to hILT4 ( Figure 37B 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.

[0762] A diagram showing the hILT4 binding region of the antibody on the crystal structure of hILT4 is shown in Figure 37C 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. Figure 37CThe 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 of Ig-like domain 2 that is located near Ig-like domain 1 and can compete with antibodies that bind to either Ig-like domain 1 or Ig-like domain 2. Antibodies that differ only from the antibodies used in these experiments due to germline mutations (e.g., 21D9.e, 29A5.a, and 10F10.3 and 10F10.4) ...

Claims

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 features: - 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); - does not specifically bind to hILT2, hILT3 and / or hILT5; - does not specifically bind to one or more members of the LILRA and / or LILRB family; - stimulate 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 assay described in the Examples; - stimulating differentiation or activation of monocytes into macrophages, e.g. stimulating differentiation of monocytes into pro-inflammatory macrophages, e.g. as shown in the assays described in the Examples; - promoting the expression of CD83 and CD86 on human monocyte-derived immature dendritic cells (Mo-iDCs), e.g. as shown in the assays described in the Examples; - enhancing IFN-γ secretion after antigen stimulation in a cytomegalovirus (CMV) lysate assay, e.g. as shown in the assay described in the Examples; - enhancing IFN-γ and TNF-α secretion by CD4+ and CD8+ T cells after CD3 stimulation in an allogeneic mixed lymphocyte reaction (MLR) assay, e.g. as shown in the assay described in the Examples; - Inhibit the binding of HLA-A and / or HLA-B to ILT4; - Combined to 70 ITRIRPELVKNGQFHIPSITWEHTGRYGCQY 100 (SEQ ID NO: 122), as determined by hydrogen deuterium exchange (HDX), e.g., as shown in the HDX assay described in the Examples; - compete with the antibodies described herein for binding to hILT4; - specifically binds to cynomolgus monkey ILT4 comprising SEQ ID NO: 118, e.g. as shown in a binding assay described in the Examples; - inhibiting 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., inhibiting the binding of hILT4 to both HLA-A and HLA-B); - have the HLA-A and HLA-B binding profiles shown in Figure 28; - has a binding profile as shown in Figure 27; - Binds to 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 if bound 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, wherein the amino acid numbering of hILT4 is that of immature hILT4 (i.e., ILT4 containing its native signal sequence); and - 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 by, for example, the carbene footprinting method described in the Examples.

2. The isolated antibody of claim 1, comprising 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 NOs: 125-127), 9C8 (SEQ ID NOs: 131-133), 2H2 (SEQ ID NOs: 137-139), 2E5 (SEQ ID NOs: 143-145), 24E5 (SEQ ID NOs: 149-151), 21D9 (SEQ ID NOs: 155-157), 21A5 (SEQ ID NOs: 161-163) or 10F10 (SEQ ID NOs: 167-169).

3. The isolated antibody of claim 1 or 2, comprising 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 NOs: 128-130), 9C8 (SEQ ID NOs: 134-136), 2H2 (SEQ ID NOs: 140-142), 2E5 (SEQ ID NOs: 146-148), 24E5 (SEQ ID NOs: 152-154), 21D9 (SEQ ID NOs: 158-160), 21A5 (SEQ ID NOs: 164-166) or 10F10 (SEQ ID NOs: 170-172).

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

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

6. The isolated antibody of any one of claims 1-5, wherein the antibody heavy chain comprises a VH having an amino acid sequence 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 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:92). NO:91), 10F10.3 (SEQ ID NO:91) or 10F10.4 (SEQ ID NO:91).

7. The isolated antibody of any one of claims 1-6, wherein the antibody light chain comprises a VL having an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the VL of 9G4 (SEQ ID NO:50, amino acids 19-125), 9C8 (SEQ ID NO:54), 2H2 (SEQ ID NO:59), 2E5 (SEQ ID NO:62), 24E5 (SEQ ID NO:66), 21D9 (SEQ ID NO:70), 21D9.b (SEQ ID NO:70), 21D9.c (SEQ ID NO:70), 21D9.d (SEQ ID NO:70), 21D9.e (SEQ ID NO:70), 21A5 (SEQ ID NO:82), 21A5.a (SEQ ID NO:86), 10F10 (SEQ ID NO:90), 10F10.1 (SEQ ID NO:91), 10F10.2 (SEQ ID NO:93), 10F10.3 (SEQ ID NO:94), 10F10.4 (SEQ ID NO:95), 10F10.5 (SEQ ID NO:96), 10F10.6 (SEQ ID NO:97), 10F10.7 (SEQ ID NO:98), 10F10.8 (SEQ ID NO:99), 10F10.9 (SEQ ID NO: NO:94), 10F10.3 (SEQ ID NO:96) or 10F10.4 (SEQ ID NO:114).

8. The isolated antibody of any one of claims 1-7, wherein the antibody heavy chain comprises a VH having an amino acid sequence comprising 1, 2, 3, 4 or 5 amino acid substitutions, conservative substitutions or back substitutions compared to the amino acid sequence of the VH of 9G4 (SEQ ID NO:51, amino acids 20-135), 9C8 (SEQ ID NO:55), 2H2 (SEQ ID NO:58), 2E5 (SEQ ID NO:63), 24E5 (SEQ ID NO:67), 21D9 (SEQ ID NO:71), 21D9.b (SEQ ID NO:74), 21D9.c (SEQ ID NO:75), 21D9.d (SEQ ID NO:78), 21D9.e (SEQ ID NO:80), 21A5 (SEQ ID NO:83), 21A5.a (SEQ ID NO:87), 10F10 (SEQ ID NO:91), 10F10.1 (SEQ ID NO:92). NO:91), 10F10.3 (SEQ ID NO:91) or 10F10.4 (SEQ ID NO:91).

9. The isolated antibody of any one of claims 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 back substitutions compared to the amino acid sequence of the VL of 9G4 (SEQ ID NO: 50, amino acids 19-125), 9C8 (SEQ ID NO: 54), 2H2 (SEQ ID NO: 59), 2E5 (SEQ ID NO: 62), 24E5 (SEQ ID NO: 66), 21D9 (SEQ ID NO: 70), 21D9.b (SEQ ID NO: 70), 21D9.c (SEQ ID NO: 70), 21D9.d (SEQ ID NO: 70), 21D9.e (SEQ ID NO: 70), 21A5 (SEQ ID NO: 82), 21A5.a (SEQ ID NO: 86), 10F10 (SEQ ID NO: 90), 10F10.1 (SEQ ID NO: 91). NO:94), 10F10.3 (SEQ ID NO:96) or 10F10.4 (SEQ ID NO:114).

10. The isolated antibody of any one of claims 1-9, wherein the heavy chain comprises the 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).

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